BR112018008081B1 - METHOD FOR T CELL CULTURE, MANUFACTURING ARTICLE AND KIT - Google Patents

Abstract

METHODS FOR CULTURING CELLS AND KITS AND APPARATUS THEREFORE. Provided herein are methods that relate, in some aspects, to incubation or culture, such as to induce stimulation of expansion (proliferation), activation, costimulation, and/or survival, of a composition of cells, such as a population of lymphocytes. In some aspects, provided are methods and reagents for the stimulation, e.g., of expansion (proliferation), survival or persistence, activation, costimulation, or other effect, of cell populations that involve the binding of agents to a molecule on the surface of the cells, thereby providing one or more signals to the cells. In some cases, the reagents are reagents containing a plurality of binding sites for agents, such as multimerization reagents, and thereby the one or more agents are multimerized by reversibly binding to the reagent, e.g., thereby creating a stimulatory reagent (multimerized agent) having stimulatory agents therein. In some aspects, the multimerized agent may provide for expansion or proliferation or other stimulation of a population of cells, and then such stimulatory agents may be removed by reversible binding disruption. Also provided are compositions, apparatus, and methods of use of the (...).

Description

Cross-Reference to Related Requests

[001] This application claims priority from U.S. provisional application No. 62/245,252 filed on October 22, 2015, entitled "Methods for Culturing Cells and Kits and Apparatus for Same," and U.S. provisional application No. 62/245,261 filed on October 22, 2015, entitled "Methods for Culturing Cells and Kits and Apparatus for Same," the contents of each of which are incorporated by reference in their entirety.

Sequence Listing Embedding by Reference

[002] This application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file titled 735042003640SeqList.txt, created on October 20, 2016, which is 132,710 bytes in size. The electronic format information of the Sequence Listing is incorporated by reference in its entirety.

Field

[003] The present disclosure relates in some aspects to incubation or culture, such as to induce stimulation of expansion (proliferation), activation, costimulation and/or survival, of a composition of cells, such as a population of lymphocytes. In some aspects, the disclosure provides methods and reagents for the stimulation, e.g., of expansion (proliferation), survival or persistence, activation, costimulation or other effect, of populations of cells involving the binding of agents to a molecule on the surface of the cells, thereby providing one or more signals to the cells. In some cases, the reagents are reagents containing a plurality of binding sites for agents, such as multimerization reagents, and thus the one or more agents are multimerized by reversible binding to the reagent, e.g., thereby creating a stimulatory reagent (multimerized agent) having multimerized stimulatory agents therein. In some aspects, the multimerized agent can provide for expansion or proliferation or other stimulation of a population of cells and then such stimulatory agents can be removed by reversible binding disruption. Compositions, apparatus, and methods of use thereof are also provided.

Background

[004] Several strategies are available for stimulating T cell populations in vitro, including the expansion of antigen-specific T cells in vitro for use in adoptive cellular immunotherapy or cancer therapy in which infusions of such T cells have been shown to have antitumor reactivity in a tumor-bearing host, or for use in the treatment of viral infections. Improved strategies are needed for expanding cell populations in vitro, including for research, diagnostic, and therapeutic purposes. Provided are reagents, methods, articles of manufacture, and kits that meet such needs.

Summary

[005] Provided herein are methods for incubating cells and compositions thereof, such as for culturing such cells, using reversible reagents to induce or modulate a signal in a target cell. In some embodiments, the cells are T cells.

[006] In some aspects, the method involves incubating a composition containing T cells in the presence of an agent, such as a receptor binding agent, e.g., a stimulatory agent. The receptor binding agent (e.g., stimulatory agent) can be reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the receptor binding agent. In some aspects, the receptor binding agent is capable of specifically binding to a molecule on the surface of cells, e.g., T cells, such as in a manner that induces or modulates a signal in the cells, e.g., the T cells in the composition. In some embodiments, the methods involve aspects, such as particular steps, selection of particular agents, and/or selection of particular reagents, which allow for control or adjustment of the type or strength or duration of the signal received or modulated by the reagent, and/or of properties of the production composition or cell population(s) ultimately generated by the methods. In some embodiments, such aspects are possible due to advantageous properties of the agents and reagents, such as the reversibility of binding of the individual components of the agents or reagents, and thus, the reversibility of binding of the multimerized agents and the cells. Such properties of the reagents can be exploited to achieve control in a variety of ways. For example, in some embodiments, the methods, via reversibility of binding, include exerting temporal control of the signal by controlling the duration of the period in which the cells are in contact with the multimerized agents, and/or the duration of the signaling thereby induced.

[007] In some embodiments, the methods involve controlling, e.g., precisely controlling, the duration of time over which such agents are bound to cells. For example, this can be done by actively reversing such binding at a particular time point, and in some cases while still maintaining the cells for an additional period of time under other culture conditions, such as incubation at a physiological temperature and/or with various nutrients. Thus, unlike other methods that involve simply terminating all or substantially all signals received by the cells, the methods provided in some aspects allow for the specific termination or disruption of signals delivered by particular reagents.

[008] Likewise, in some embodiments, reversibility allows the reagents to be modular in nature, allowing for the replacement of one or more components thereof without engineering or new reagents, e.g., by simply reversing the binding and combining with additional agents or reagents, under conditions where reversible binding is induced. For example, by being able to reversibly bind multiple stimulatory agents to the same multimerization reagent, either at the same time or at different times, the user of the provided methods and compositions can tailor the nature of the particular signal to be released, e.g., by substituting one or more stimulatory agents for another one or more stimulatory agents, such as to induce a stronger or weaker or qualitatively different signal, depending on the desired outcome.

[009] In some embodiments, temporal control and modularity are used in combination, e.g., by incubating cells in the presence of one agent for a period of time, inducing reversal of binding by disruption, followed by incubation in the presence of one or more different agents or reagents. For example, in one embodiment, T cells are initially stimulated with a reagent to release a particular strength or quality of signal, and after a period of time, such signal is disrupted and a qualitatively or quantitatively different signal (e.g., stronger or weaker or activating different signaling pathways or known to be important for different differentiation pathways) is substituted. In some embodiments, such control provides advantages, e.g., by allowing the user to maximize desired outcomes (e.g., expansion or persistence), while avoiding undesirable outcomes, such as exhaustion or anergy.

[0010] In some embodiments, temporal control is achieved by disrupting the reversible binding of the reagents or agents, such as by the addition of a substance. For example, in some embodiments, within a period of time, e.g., within 5 days after the start of the incubation and/or within a certain percentage of the total duration of the incubation, such as within 1/5, %, 1/3, or 1/2 of the time, the reversible binding between the receptor binding agent and the reagent is disrupted. Thus, in some cases, the method results in the generation of cultured T cells.

[0011] In some embodiments, the incubation is carried out under conditions where the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in one or more T cells in the composition.

[0012] In some embodiments, the disruption of the bond between the receptor binding agent and the reagent is effected more than 30 minutes after the start of the incubation. For example, in some aspects, the disruption of the bond between the receptor binding agent and the reagent is effected between 1 hour and 4 days after the start of the incubation, between 6 hours and 3 days after the start of the incubation, between 12 hours and 2 days after the start of the incubation, or between 1 day and 3 days after the start of the incubation. In some cases, the disruption of the binding between the receptor binding agent and the reagent is effected between about 1 hour and about 4 days after the start of the incubation, between about 6 hours and about 3 days after the start of the incubation, between about 12 hours and about 2 days after the start of the incubation, or between about 1 day and about 3 days after the start of the incubation. In some aspects, the disruption is effected greater than or equal to about 1 hour after the start of said incubation and within 1 day, 2 days, 3 days, or 4 days after the start of the incubation. In some embodiments, the binding of the receptor binding agent is capable of initiating or does initiate a signal associated with the TCR/CD3 complex on the T cells. In some aspects, the receptor binding agent specifically binds to an element of a TCR/CD3 complex. In some examples, the receptor binding agent specifically binds to CD3.

[0013] In some embodiments, the molecule (the molecule on the surface of the T cell) is a component of the TCR/CD3 complex or is CD3. In some aspects, the molecule is a first molecule and the receptor binding agent is also capable of specifically binding to a second molecule on the surface of one or more of the T cells. In some cases, the second molecule is capable of inducing or enhancing, decreasing, or modifying a signal delivered through the first molecule on the T cells.

[0014] In some aspects, the receptor binding agent includes a C1 binding partner. In some aspects, the plurality of binding sites contained by the reagent includes two or more binding sites, Z1. In some examples, the two or more Z1 binding sites are each capable of binding to the C1 binding partner to form the reversible bond between the receptor binding agent and the reagent. In some embodiments, disrupting the bond between the receptor binding agent and the reagent includes introducing to the cells a composition containing a substance capable of reversing the bond between the receptor binding agent and the reagent.

[0015] In some embodiments, the receptor binding agent is a first receptor binding agent and the incubation is also carried out in the presence of a second receptor binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells. In some embodiments, the binding of the second receptor binding agent to the second molecule enhances, decreases, or modifies a signal delivered through the first molecule to the T cells.

[0016] In some embodiments, the reagent contains a plurality of binding sites capable of reversibly binding to the second receptor binding agent. In some such cases, the second receptor binding agent is reversibly bound to the reagent. In some aspects, the plurality of binding sites capable of reversibly binding to the first receptor binding agent and the plurality of binding sites capable of reversibly binding to the second receptor binding agent may be the same or may be different.

[0017] In some aspects, the second receptor binding agent includes a C1 or C2 binding partner, which is capable of reversibly binding to the two or more Z1 binding sites. In some such examples, the first and second receptor binding agents are reversibly bound to the reagent via two or more Z1 binding sites. In some cases, the second receptor binding agent contains a C2 binding partner and the reagent also contains a plurality of Z2 binding sites. The plurality of Z2 binding sites may be capable of binding to the C2 binding partner to form the reversible bond between the second receptor binding agent and the reagent. In some aspects, C2 and C1 are the same or substantially the same, or contain the same or substantially the same moiety. In some examples, Z1 and Z2 are the same or substantially the same, or contain the same or substantially the same moiety.

[0018] In some cases, the reagent is a first reagent and the incubation is carried out in the presence of at least one second reagent in which it is reversibly bound to the second receptor binding agent. In some embodiments, the incubation is carried out under conditions in which the second receptor binding agent specifically binds to the second molecule. In some such aspects, the binding of the second receptor binding agent to the second molecule induces or modulates a signal, e.g., that enhances, diminishes, or modifies a signal delivered through the first molecule to the T cells.

[0019] In some embodiments, said disruption includes introducing to the cells a composition containing a substance capable of reversing the binding between the first receptor binding agent and the reagent and/or the second receptor binding agent and the reagent. In some embodiments, said disruption terminates or reduces the signal induced or modulated by the first receptor binding agent and terminates or reduces the signal induced or modulated by the second receptor binding agent.

[0020] Provided herein in some embodiments is a method for culturing T cells that includes incubating a composition containing T cells in the presence of a first receptor binding agent that is capable of specifically binding to a first molecule expressed on the surface of the T cells. In some aspects, binding of the first receptor binding agent to the first molecule induces or modulates a signal associated with the TCR/CD3 complex on the T cell. In some such embodiments, the composition is also incubated in the presence of a second receptor binding agent that is reversibly linked to a reagent containing a plurality of binding sites capable of reversibly binding to the second receptor binding agent. In some aspects, the second receptor binding agent is capable of specifically binding to a second molecule on the surface of the T cells. In some cases, binding of the second receptor binding agent to the second molecule induces or modulates a second signal in the T cell, such as to enhance, diminish, or modify a signal delivered through the first molecule. In some aspects, within 5 days after the start of incubation, the reversible binding between the second receptor binding agent and the reagent is disrupted, thereby generating cultured T cells.

[0021] In some embodiments, the second signal enhances, diminishes, or modifies a signal delivered through the first molecule to the T cells.

[0022] In some embodiments of any of the methods provided herein, the incubation is performed under conditions wherein the first receptor binding agent specifically binds to the first molecule and/or the second receptor binding agent specifically binds to the second molecule, thereby inducing or modulating one or more signals in the T cells. In some embodiments, said disruption is performed more than 30 minutes after the start of said incubation. In some embodiments, said disruption is performed between 1 hour and 4 days after the start of said incubation, between 6 hours and 3 days after the start of said incubation, between 12 hours and 2 days after the start of said incubation, or between 1 day and 3 days after the start of said incubation; or said rupture is accomplished between about 1 hour and about 4 days after the start of said incubation, between about 6 hours and about 3 days after the start of said incubation, between about 12 hours and about 2 days after the start of said incubation, or between about 1 day and about 3 days after the start of said incubation; or said rupture is accomplished more than or equal to about 1 hour after the start of said incubation and within 1 day, 2 days, 3 days, or 4 days after the start of said incubation.

[0023] In some embodiments, the second receptor binding agent includes a binding partner C1. In some such embodiments, the plurality of binding sites contained by the reagent includes two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the second receptor binding agent and the reagent.

[0024] In some embodiments of any of the methods provided herein, the second signal is a signal other than a signal associated with the TCR/CD3 complex; the second signal is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex; or the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family. In some embodiments, the second molecule is selected from CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. In some embodiments, the second molecule is CD28.

[0025] In some embodiments, the second molecule is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule.

[0026] In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2. In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2; and/or the second receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, and TNF, or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-12R, IFN-gammaR, IL-4R, and IL-17R; or the second receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15, and IL-17, or is a biologically active fragment thereof.

[0027] In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain). In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); and/or the second receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); or the second receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0028] In some embodiments, the second receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or the second receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21, and CCL25 or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a chemokine receptor selected from CXCR3, CCR7, CXCR1, and CXCR4; or the second receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0029] In some embodiments, the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1, and VLA-4 or is a biologically active fragment thereof.

[0030] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0031] In some embodiments, said disruption includes introducing to the cells a composition containing a substance capable of reversing the binding between the second receptor binding agent and the reagent. In some embodiments, said disruption terminates or reduces the signal induced or modulated by the second receptor binding agent. For example, in some embodiments, the second molecule is CD28 and the disruption terminates or reduces the CD28 costimulatory signal on the T cells.

[0032] In some embodiments, the incubation is also carried out in the presence of a third receptor binding agent, which is capable of specifically binding to a third molecule on the surface of one or more of the T cells.

[0033] In some embodiments of any of the methods provided herein, the first receptor binding agent specifically binds to a first molecule expressed on the surface of the T cell in a manner that induces or modulates a signal associated with the TCR/CD3 complex on the T cell; the second receptor binding agent specifically binds to the second molecule, thereby inducing or modulating a second signal on the T cell; and a third receptor binding agent specifically binds to a third molecule on the surface of the T cell that induces or modulates another signal on the cell.

[0034] In some embodiments, the first molecule is CD3.

[0035] In some embodiments, the second signal enhances, diminishes, or modifies a signal delivered through the first molecule. In some embodiments, the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family. In some embodiments, the second molecule is selected from CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. In some embodiments, the second molecule is CD28.

[0036] In some embodiments, the third molecule is a cytokine receptor, a chemokine receptor, is or includes an adhesion molecule, or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule. In some embodiments, a third receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2. In some embodiments, a third receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2; and/or a third receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, and TNF, or is a biologically active fragment thereof.

[0037] In some embodiments, a third receptor binding agent specifically binds to a cytokine receptor selected from IL-12R, IFN-gammaR, IL-4R, and IL-17R; or a third receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15, and IL-17 or is a biologically active fragment thereof. In some embodiments, a third receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain). In some embodiments, a third receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); and/or a third receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof. In some embodiments, a third receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); or a third receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof.

[0038] In some embodiments, a third receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. In some embodiments, a third receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or a third receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21, and CCL25 or is a biologically active fragment thereof. In some embodiments, a third receptor binding agent specifically binds to a chemokine receptor selected from CXCR3, CCR7, CXCR1, and CXCR4; or a third receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0039] In some embodiments, the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1, and VLA-4 or is a biologically active fragment thereof.

[0040] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0041] In some embodiments, a third receptor binding agent is reversibly bound to the first reagent or the second reagent; or the incubation is carried out in the presence of another reagent that is reversibly bound to the third receptor binding agent.

[0042] In some embodiments of any of the methods provided herein, the method further includes, after said disruption, also incubating the composition containing the T cells. In some embodiments, the incubation and another incubation are performed in the same vessel; and/or the other incubation is performed in the presence of the substance; and/or the method does not comprise removing the substance, receptor binding agent, second receptor binding agent, and/or reagent from the cell composition prior to the other incubation.

[0043] In some embodiments, the incubation and/or other incubation is(are) carried out at or at about 37°C ± 2°C; and/or the incubation and/or other incubation is(are) carried out in the presence of another agent that is capable of delivering a signal to T cells. In some embodiments, the other agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells, and/or CD8+ T cells. In some embodiments, the other agent is a cytokine selected from IL-2, IL-15, and IL-7. In some embodiments, the other incubation is carried out for a time that is no more than 14 days, no more than 12 days, no more than 10 days, no more than 8 days, or no more than 6 days.

[0044] Provided herein are methods for culturing T cells that include: incubating a composition containing T cells in the presence of a receptor binding agent that specifically binds to a CD28 molecule on the surface of the T cells under conditions to effect signaling through CD28 on the cells; and within 5 days after beginning said incubation, eliminating or reducing the binding of the receptor binding agent and the CD28 molecule, whereby CD28 signaling is terminated or reduced on the cells, thereby generating cultured T cells. In some embodiments, the elimination or reduction is accomplished within 4 days, within 3 days, within 2 days, or within 1 day after beginning the incubation.

[0045] In some examples, the elimination or reduction is effected within 4 days, within 3 days, within 2 days, or within 1 day after the start of incubation. In some cases, the elimination or reduction includes washing the cells, whereby any receptor binding agent that is not specifically bound to CD28 is removed or reduced from the composition. In some aspects, the elimination includes reversing the binding interaction between the receptor binding agent and the CD28 molecule, also including washing the cells to remove or reduce the receptor binding agent from the composition.

[0046] In some aspects, during at least a portion of the incubation and/or subsequent to the incubation, the T cells in the composition are incubated in the presence of an agent that specifically binds to a TCR/CD3 complex molecule, whereby a signal associated with the TCR/CD3 complex is induced or modulated in the cells.

[0047] Provided herein in some aspects is a method for culturing T cells including incubating a composition containing the T cells in the presence of a receptor binding agent. In some embodiments, the receptor binding agent is reversibly linked to a reagent containing a plurality of binding sites capable of reversibly binding to the receptor binding agent. In some cases, the receptor binding agent is capable of specifically binding to a molecule on the surface of the target cells other than CD28, CD3, or CD40 in a manner that induces or modulates a signal in the target cells and/or alters a function of the target cells, thereby generating cultured target cells. In some embodiments, the molecule is not CD137 and/or the receptor binding agent does not specifically bind to CD137.

[0048] In some aspects, the incubation is carried out under conditions where the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in the T cells. In some embodiments, the signal is not a signal associated with the TCR/CD3 complex.

[0049] In some embodiments, the signal is not a signal associated with the TCR/CD3 complex.

[0050] In some embodiments, the receptor binding agent includes a binding partner C1. In some such embodiments, the plurality of reagent binding sites includes two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent and the reagent.

[0051] In some embodiments, the molecule is selected from CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM; and/or the receptor binding agent specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

[0052] In some embodiments, the receptor binding agent is a second receptor binding agent and the molecule is a second molecule, and the incubation is also carried out in the presence of a first receptor binding agent, which is capable of specifically binding to a first molecule on the surface of one or more of the T cells, wherein the first molecule is optionally capable of inducing or modulating a first signal in one or more T cells in the composition. In some embodiments, the first receptor binding agent is reversibly bound to the reagent, said reagent including a plurality of binding sites for the first receptor binding agent and the second receptor binding agent; or the first receptor binding agent is reversibly bound to a second reagent including a plurality of binding sites capable of reversibly binding to the first receptor binding agent.

[0053] In some embodiments, the first receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells. In some aspects, the first receptor binding agent specifically binds to an element of a TCR/CD3 complex. In some cases, the first receptor binding agent specifically binds to CD3.

[0054] In some embodiments, specific binding of the second receptor binding agent to the second molecule is capable of enhancing, diminishing, or modifying a signal delivered through the first molecule. In some cases, specific binding of the second receptor binding agent to the second molecule is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex.

[0055] Provided herein in some embodiments is a method for culturing T cells, wherein the method includes incubating a composition containing T cells in the presence of a first receptor binding agent that is reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the first receptor binding agent. In some cases, the first receptor binding agent is capable of specifically binding to a first molecule on the surface of the T cells, such as to induce or modulate a signal associated with the TCR/CD3 complex on T cells in the composition. In some embodiments, the method includes incubating the composition containing the T cells in the presence of a second receptor binding agent that can be reversibly bound to the reagent also containing a plurality of binding sites for the second receptor binding agent or to a second reagent containing a plurality of binding sites capable of reversibly binding to the second receptor binding agent. In some aspects, the second receptor binding agent is capable of specifically binding to a second molecule on the surface of the T cells such as to induce or modulate a second signal in T cells in the composition. In some aspects, the second molecule is other than CD28. In some embodiments, the incubation is carried out under conditions wherein the signal and/or second signal are induced or modulated in T cells in the composition, thereby generating cultured T cells.

[0056] In some embodiments, the first receptor binding agent specifically binds to an element of a TCR/CD3 complex and/or the first receptor binding agent specifically binds to CD3.

[0057] In some examples, specific binding of the second receptor binding agent to the second molecule is capable of inducing or modulating a signal other than a signal associated with the TCR/CD3 complex. In some aspects, specific binding of the second receptor binding agent to the second molecule is capable of enhancing, diminishing, or modifying a signal delivered through the first molecule. In some embodiments, specific binding of the second receptor binding agent to the second molecule is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex.

[0058] In some embodiments, the molecule, which may be the second molecule, is CD90 (Thy-1), CD95 (Apo-/Fas), CD137, (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some aspects, the receptor binding agent, which may be the second receptor binding agent, specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the molecule, which may be a second molecule, is not CD137 and/or the receptor binding agent, which may be the second receptor binding agent, does not specifically bind to CD137.

[0059] In some aspects, the first receptor binding agent and the second receptor binding agent reversibly bind to the reagent. In some embodiments, the first receptor binding agent and the second receptor binding agent each individually include a C1 binding partner, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner to form the reversible bond between the first and second receptor binding agents and the reagent. In other embodiments, the first receptor binding agent includes a C1 binding partner, the second receptor binding agent includes a C2 binding partner, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner and the C2 binding partner to form the reversible bond between the first and second receptor binding agents and the reagent. In still other embodiments, the first receptor binding agent includes a C1 binding partner, the second receptor binding agent includes a C2 binding partner, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner to form the reversible bond between the first receptor binding agent and the reagent, and two or more binding sites, Z2, each of which is capable of binding to the C2 binding partner to form the reversible bond between the second receptor binding agent and the reagent.

[0060] In some embodiments, the receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2. In some embodiments, the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2; and/or the receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, and TNF, or is a biologically active fragment thereof. In some embodiments, the receptor binding agent specifically binds to a cytokine receptor selected from IL-12R, IFN-gammaR, IL-4R, and IL-17R; or the receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15, and IL-17, or is a biologically active fragment thereof.

[0061] In some embodiments, the receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain). In some embodiments, the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); and/or the receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof. In some embodiments, the receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); or the receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0062] In some embodiments, the receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. In some embodiments, the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21, and CCL25 or is a biologically active fragment thereof. In some embodiments, the receptor binding agent specifically binds to a chemokine receptor selected from CXCR3, CCR7, CXCR1, and CXCR4; or the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0063] In some embodiments, the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1 and VLA-4 or is a biologically active fragment thereof.

[0064] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0065] In some embodiments of any of the methods provided herein, the incubation is carried out under conditions wherein the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in the target cells and/or altering a function in the target cells. In some embodiments, the receptor binding agent includes a binding partner C1, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent and the reagent. In some embodiments, the receptor binding agent is an additional receptor binding agent and the molecule is an additional molecule, and the incubation is also carried out in the presence of a first receptor binding agent, which is capable of specifically binding to a first molecule on the surface of one or more of the T cells, wherein the first molecule is optionally capable of inducing or modulating a first signal in one or more T cells in the composition.

[0066] In some embodiments, the first receptor binding agent is reversibly linked to the reagent, said reagent including a plurality of binding sites for the first receptor binding agent and an additional receptor binding agent; or the first receptor binding agent is reversibly linked to a second reagent including a plurality of binding sites capable of reversibly binding to the first receptor binding agent. In some embodiments, the first receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells; and/or the first receptor binding agent specifically binds to an element of a TCR/CD3 complex; and/or the first receptor binding agent specifically binds to CD3.

[0067] In some embodiments, the first receptor binding agent includes a C2 binding partner, and the plurality of binding sites includes two or more binding sites, Z2, each of which is capable of binding to the C2 binding partner to form the reversible bond between the first receptor binding agent and the reagent.

[0068] In some embodiments, the incubation is also carried out in the presence of a second receptor binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells, which second molecule is optionally capable of inducing or modulating a signal in target cells in the composition to enhance, diminish, or modify a signal delivered through the first molecule.

[0069] In some embodiments, the second signal is a signal other than a signal associated with the TCR/CD3 complex; the second signal is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex; or the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family. In some embodiments, the second molecule is selected from CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM; and/or the second receptor binding agent specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the second molecule is selected from CD28 and CD137. In some embodiments, the second molecule is CD28.

[0070] Provided herein are methods for culturing target cells, which method includes incubating a composition containing target cells in the presence of a receptor binding agent. The receptor binding agent can be reversibly bound to a reagent that is a streptavidin or mutein analog containing a plurality of binding sites capable of reversibly binding to the receptor binding agent. In some cases, the receptor binding agent is capable of specifically binding to a molecule on the surface of target cells, such as to induce or modulate a signal in target cells in the composition. In some aspects, the streptavidin mutein includes a net negative charge or exhibits a lower isoelectric point than the streptavidin mutein comprising the sequence set forth in SEQ ID NO:4 or 6, thereby generating cultured target cells.

[0071] Provided herein in some aspects is a method for culturing target cells, the method including incubating a composition containing target cells in the presence of a receptor binding agent that is reversibly bound to a reagent that is a streptavidin analog or mutein containing a plurality of binding sites capable of reversibly binding to the receptor binding agent. In some embodiments, the receptor binding agent is capable of specifically binding to a molecule on the surface of the target cells in a manner that induces or modulates a signal in target cells in the composition. In some cases, the streptavidin analog or mutein exhibits a higher affinity for a streptavidin-binding peptide containing the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than a streptavidin or mutein containing the amino acid sequence set forth in any of SEQ ID NOS: 1-6, thereby generating cultured target cells.

[0072] In some embodiments, the incubation is carried out under conditions where the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in one or more target cells in the composition.

[0073] In some examples, the plurality of reagent binding sites includes two or more binding sites, Z1. In some cases, the receptor binding agent includes a binding partner C1, which is capable of reversibly binding to the Z1 binding site, wherein the reversible binding between C1 and Z1 effects the reversible binding between the receptor binding agent and the reagent. In some embodiments, the streptavidin analog or mutein includes a plurality of Z1 binding sites, and a plurality of receptor binding agents are reversibly bound to the reagent.

[0074] In some embodiments, the target cells comprise immune cells. For example, in some embodiments, the target cells comprise blood cells; the target cells comprise leukocytes; the target cells comprise lymphocytes; the target cells comprise B cells; the target cells comprise a B cell population; the target cells comprise T cells; the target cells comprise a T cell population; and/or the target cells comprise natural killer (NK) cells. In some embodiments, the target cells comprise antigen-specific T cells or a population thereof, a helper T cell or population thereof, a cytotoxic T cell or population thereof, a memory T cell or population thereof, a regulatory T cell or population thereof, an NK cell or population thereof, antigen-specific B cells or a population thereof, a memory B cell or population thereof, or a regulatory B cell or population thereof. In some embodiments, the target cells are T cells.

[0075] In some embodiments, the molecule is present on the surface of T cells, and the receptor binding agent is capable of inducing or modulating a signal in T cells in the composition. In some aspects, the receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells and/or the receptor binding agent specifically binds to an element of a TCR/CD3 complex. In some cases, the receptor binding agent specifically binds to CD3. In some examples, the molecule is a first molecule and the receptor binding agent is capable of specifically binding to the first molecule and, in some cases, a second molecule on the surface of one or more of the target cells. In some embodiments, binding to the second molecule is capable of enhancing, diminishing, or modifying a signal delivered through the first molecule.

[0076] In some embodiments, the molecule is a first molecule and the receptor binding agent is capable of specifically binding to the first molecule and a second molecule on the surface of one or more of the target cells, wherein binding to the second molecule induces or modulates a signal in the target cells. In some embodiments, binding to the second molecule is optionally capable of enhancing, diminishing, or modifying a signal delivered through the first molecule.

[0077] The receptor binding agent is a first receptor unbinding agent and the incubation is also carried out in the presence of a second receptor binding agent. The second receptor binding agent may be capable of specifically binding to a second molecule on the surface of one or more of the target cells. In some cases, binding of the second receptor binding agent to the second molecule is capable of inducing or modulating a signal to enhance, diminish or modify a signal delivered through the first molecule.

[0078] In some embodiments, the incubation is carried out under conditions wherein the second receptor binding agent specifically binds to the second molecule, thereby inducing or modulating a signal in target cells in the composition to enhance, diminish, or modify a signal delivered through the first molecule. In some embodiments, the streptavidin mutein or analog includes a plurality of binding sites capable of reversibly binding to the second receptor binding agent, whereby the second receptor binding agent is reversibly bound to the streptavidin mutein or analog.

[0079] In some embodiments, the second receptor binding agent includes a C1 or C2 binding partner, wherein it is capable of reversibly binding to the two or more Z2 binding sites present on the mutein or streptavidin analog.

[0080] In some cases, the additional molecule is CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the second receptor binding agent specifically binds to CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some aspects, the additional molecule is CD40 and CD137. In some examples, the second receptor binding agent specifically binds to CD40 or CD137.

[0081] In some embodiments, the second molecule is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule.

[0082] In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2. In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2; and/or the second receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, and TNF, or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-12R, IFN-gammaR, IL-4R, and IL-17R; or the second receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15, and IL-17, or is a biologically active fragment thereof.

[0083] In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain). In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); and/or the second receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); or the second receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0084] In some embodiments, the second receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or the second receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21, and CCL25 or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a chemokine receptor selected from CXCR3, CCR7, CXCR1, and CXCR4; or the second receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0085] In some embodiments, the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1, and VLA-4 or is a biologically active fragment thereof.

[0086] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0087] The second receptor binding agent includes a plurality of different receptor binding agents, each of which is capable of individually binding to the same or a different second molecule on the surface of the T cells in the composition to collectively induce or modulate one or more signals in the cells.

[0088] In some embodiments, the method also includes reversibly disrupting the binding between the first and/or second receptor binding agent and the reagent. In some aspects, the disruption is performed within 14 days after the start of incubation, within 12 days after the start of incubation, within 10 days after the start of incubation, within 8 days after the start of incubation, or within 6 days after the start of incubation.

[0089] In some embodiments, at least a portion of the incubation is carried out in the presence of another agent that is capable of delivering a signal to T cells. In some cases, the other agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells, and/or CD8+ T cells. In some embodiments, the other agent is a cytokine such as IL-2, IL-15, or IL-7. In some examples, the other agent does not specifically bind to CD28 and/or induce CD28 signaling.

[0090] In some embodiments, the T cells or target cells are primary cells from an individual. In some cases, the T cells or target cells are directly isolated from an individual. In some embodiments, the T cells are unfractionated T cells, are enriched or isolated CD3+ T cells, are enriched or isolated CD4+ T cells, or are enriched or isolated CD8+ T cells. In some embodiments, prior to incubation, the T cells are not enriched for CD62L+ cells and/or are not enriched for naïve T cells. In some cases, the T cells or target cells are human cells.

[0091] In some aspects, the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm. In some examples, the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3.

[0092] In some embodiments, the reagent is not, and is not bound to or associated with a solid support, stationary phase, a bead, a microparticle, a magnetic particle, and/or a matrix during said incubation; and/or the reagent is flexible, does not contain a metal or magnetic core, is comprised entirely or primarily of organic multimer, is not spherical, is not substantially spherical or uniform in shape, and/or is not rigid.

[0093] In some embodiments, the reagent is not bound to a support or a solid support during said incubation. In some embodiments, the reagent is bound to a support during at least a portion of the incubation, whereby a plurality of the T cells or target cells are reversibly immobilized on a support during at least a portion of the incubation. In some embodiments, the support is or includes a stationary phase; and/or the support is or includes a solid support.

[0094] In some embodiments, the receptor binding agent, which may be the first receptor binding agent, contains only one binding site, such as B2. In some aspects, the receptor binding agent, which may be the first receptor binding agent, specifically binds to the molecule in a monovalent manner. In some embodiments, the second receptor binding agent contains only one binding site, such as B4. In some cases, the second receptor binding agent specifically binds to the molecule in a monovalent manner. In some embodiments, the binding site, such as B2 or B4, contains an antibody combining site.

[0095] In some aspects, the receptor binding agent, which may be a first receptor binding agent, and/or the second receptor binding agent each individually is an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, or MHC molecule or binding fragments thereof. In some such aspects, the receptor binding agent, which may be a first receptor binding agent, and/or the second receptor binding agent includes an antibody fragment, a Fab fragment, a divalent antibody fragment such as an F(ab')2 fragment, or a divalent single chain Fv (scFv) fragment. In some cases, the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent is a monovalent antibody fragment such as a Fab fragment, an Fv fragment, or an scFv fragment. In some examples, the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent is a proteinaceous binding molecule with antibody-like binding properties, such as an aptamer, mutein based on a lipocalin family polypeptide, glucobody, protein based on an ankyrin scaffold, protein based on a crystalline scaffold, adnectin, or an avimer.

[0096] In some embodiments, the receptor binding agent, which may be the first receptor binding agent, includes an agent that specifically binds to CD3. The agent that specifically binds CD3 may be an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, the second receptor binding agent includes an agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and/or HVEM. The agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and/or HVEM may be an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, an antibody fragment of an anti-CD28 antibody, a CD28 binding proteinaceous molecule with antibody-like binding properties, an anti-CD90 antibody, a divalent antibody fragment of an anti-CD90 antibody, an antibody fragment of an anti-CD90 antibody, a CD90 binding proteinaceous molecule with antibody-like binding properties, an anti-CD95 antibody, a divalent antibody fragment of an anti-CD95 antibody, an antibody fragment of an anti-CD95 antibody, a CD95 binding proteinaceous molecule with antibody-like binding properties, an anti-CD154 antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154 antibody, a proteinaceous CD154 binding molecule with antibody-like binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a proteinaceous CD137 binding molecule with antibody-like binding properties, an anti-ICOS antibody, a divalent antibody fragment of an anti-ICOS antibody, an antibody fragment of an anti-ICOS antibody, a proteinaceous ICOS binding molecule with antibody-like binding properties, an anti-LAT antibody, a divalent antibody fragment of an anti-LAT antibody, an antibody fragment of an anti-LAT antibody, a proteinaceous LAT binding molecule with antibody-like binding properties, an anti-CD27 antibody, a divalent antibody fragment of an anti-CD27 antibody, an antibody fragment of an anti-CD27 antibody, a proteinaceous CD27 binding molecule with antibody-like binding properties binding molecule, an anti-OX40 antibody, a divalent antibody fragment of an anti-OX40 antibody, an antibody fragment of an anti-OX40 antibody, a proteinaceous OX40 binding molecule with antibody-like binding properties, an anti-HVEM antibody, a divalent antibody fragment of an anti-HVEM antibody, an antibody fragment of an anti-HVEM antibody, a proteinaceous HVEM binding molecule with antibody-like binding properties, 4-1BB linker, or any mixture thereof.

[0097] In some embodiments of any of the methods provided herein, the receptor binding agent comprises an antibody fragment; the receptor binding agent comprises a Fab fragment; the receptor binding agent is a divalent antibody fragment selected from an F(ab')2 fragment and a divalent single-chain Fv (scFv) fragment; the receptor binding agent is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment, and an scFv fragment; and/or the receptor binding agent is a proteinaceous binding molecule with antibody-like binding properties selected from aptamers, muteins based on a lipocalin family polypeptide, glucobodies, ankyrin scaffold-based proteins, crystalline scaffold-based proteins, adnectins, and avimers.

[0098] In some embodiments of any of the methods provided herein, the reagent is or contains streptavidin, avidin, a streptavidin analog or mutein that reversibly binds biotin, a biotin analog or a biologically active fragment thereof; an avidin or streptavidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent that includes at least two K chelating groups, wherein the at least two chelating groups are capable of binding a transition metal ion; an agent capable of binding an oligohistidine affinity tag; an agent capable of binding a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding calmodulin-binding peptide (CBP); an agent capable of binding a FLAG peptide; an agent capable of binding an HA tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[0099] In some embodiments, the reagent is or contains a streptavidin analogue or mutein or a mutein or avidin analogue that reversibly binds to biotin or a biologically active fragment; a streptavidin analogue or mutein or a mutein or avidin analogue that reversibly binds to a biotin analogue or a biologically active fragment; and/or a streptavidin analogue or mutein or a mutein or avidin analogue that reversibly binds to a streptavidin-binding peptide.

[00100] In some embodiments, the reagent is an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein that reversibly binds biotin or a biologically active fragment; a streptavidin or avidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent that includes at least two K-chelating groups, wherein the at least two chelating groups are capable of binding a transition metal ion; an agent capable of binding an oligohistidine affinity tag; an agent capable of binding a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding calmodulin-binding peptide (CBP); an agent capable of binding a FLAG peptide; an agent capable of binding an HA tag; an agent capable of binding maltose-binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein. In some embodiments, the reagent includes an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein, or an avidin or mutein analog. In some aspects, individual molecules of the oligomer or polymer are cross-linked by a polysaccharide or a bifunctional linker.

[00101] In some embodiments, the plurality of Z binding sites includes at least 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 72, or more binding sites.

[00102] In some aspects, the streptavidin-binding peptide is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO:17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:18), or Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:19).

[00103] In some embodiments, the reagent includes a streptavidin analog or mutein containing the amino acid sequence Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the streptavidin positions in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the streptavidin analog or mutein includes the amino acid sequence Va144-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the streptavidin positions in the amino acid sequence set forth in SEQ ID NO:1.

[00104] In some embodiments, the streptavidin analog or mutein includes the amino acid sequence set forth in any of SEQ ID NOS: 3-6. In some aspects, the streptavidin analog or mutein includes an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 3-6 and contains the amino acid sequence corresponding to Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47. In some embodiments, the streptavidin analog or mutein reversibly binds biotin or a biologically active form thereof, a biotin analog or mutein or a biologically active fragment thereof, or a streptavidin-binding peptide. In some embodiments, the streptavidin analog or mutein binds a functional fragment of any of the above sequences that reversibly binds biotin or a biologically active form thereof, a biotin analog or mutein or a biologically active fragment thereof, or a streptavidin-binding peptide.

[00105] In some embodiments, the streptavidin analog or mutein also contains an amino acid substitution or substitutions at a position corresponding to 117, 120, and/or 121 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the amino acid substitution or substitutions are selected from Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121, or Phe121. In some embodiments, the amino acid substitution or substitutions are Glu117, Gly120, or Tyr121.

[00106] In some embodiments, the streptavidin analog or mutein contains the amino acid sequence set forth in SEQ ID NO: 27 or 28. In some aspects, the streptavidin analog or mutein contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: 28 and contains the amino acid sequence corresponding to Va144, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121. In some embodiments, the streptavidin analog or mutein reversibly binds biotin or a biologically active fragment thereof, a biotin analog or mutein or a biologically active fragment thereof, or a streptavidin-binding peptide. In some embodiments, the streptavidin analog or mutein reversibly binds a functional fragment to any of the above sequences that reversibly bind biotin or a biologically active fragment thereof, a biotin analog or mutein or a biologically active fragment thereof, or a streptavidin-binding peptide.

[00107] In some embodiments, the C1 binding partner and/or the C2 binding partner, independently, includes a streptavidin-binding peptide such as Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3- Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:18) or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:19);

[00108] In some embodiments, disruption includes introducing to the cells a composition containing a substance capable of reversing the binding between the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent, and the reagent. In some embodiments, the substance is a free binding partner and/or is a competing agent. In some embodiments, the substance in the composition is not detrimental to the T cells or target cells. In some aspects, the addition of the substance does not reduce the percentage of surviving T cells or target cells to less than 90%, 80%, 70%, 60%, or 50%, compared to incubating the T cells or target cells, respectively, under comparable or the same conditions, without the substance. In some embodiments, disruption terminates or reduces the signal induced or modulated by one or both of the receptor binding agent or second receptor binding agent on the T cells or target cells.

[00109] In some embodiments, the reagent is or contains a streptavidin, avidin, a streptavidin analog or mutein, or and an avidin analog or mutein or biologically active fragments thereof. In some embodiments, the substance contains a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analog or biologically active fragment.

[00110] In some embodiments, the substance is a streptavidin-binding peptide such as Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3- Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:18), or Trp-Ser-His-Pro-Gln-Phe-Glu-Lys- (GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19). In some embodiments, the substance is C1 or an analogue thereof or is C2 or an analogue thereof.

[00111] In some embodiments, the dissociation constant (KD) for the reversible binding between the Z1 binding site and the C1 binding partner and/or for the reversible binding between the Z2 binding site and the C2 binding partner is in the range of 10-2 M to 10-13 M.

[00112] In some embodiments, prior to incubation, the cells are contacted with a selection agent that specifically binds to a marker comprised by T cells or target cells of the composition, thereby generating or obtaining the composition containing the T cells or target cells. In some embodiments, at least a portion of the incubation is performed in the presence of a selection agent that specifically binds to a marker comprised by T cells or target cells of the composition, and the cultured T cells are enriched for T cells or target cells containing the marker.

[00113] In some embodiments, the selection agent is reversibly linked to the reagent, and the reagent also contains a plurality of binding sites capable of specifically binding to the selection agent. In some aspects, the selection agent is reversibly linked to a second reagent containing a plurality of binding sites capable of specifically binding to the selection agent.

[00114] In some embodiments, the induction or modulation of the signal effects an increase in expansion (proliferation) and/or activation of the cultured T cells compared to incubating the T cells in the absence of the induction or modulation of the signal. In some embodiments, the increase is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. In some embodiments, the induction or modulation of the additional or second signal increases the expansion (proliferation) and/or activation of the T cells compared to incubating the T cells in the absence of the induction or modulation of the additional or second signal. In some embodiments, the increase is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

[00115] In some embodiments, the method increases the expansion and/or proliferation of T cells in the composition, alters the metabolic profile of T cells in the composition, alters the subset of CD8+ T cells in the composition; and/or increases the percentage of long-lived memory T cells in the composition, each compared to T cells in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling.

[00116] In some embodiments, the methods result in cultured T cells in which the number or percentage of CD3+ T cells, CD4+ T cells, or CD8+ cells is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the number or percentage of CD3+ T cells, CD4+ T cells, or CD8+ T cells, respectively, in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to an analogous incubation but performed in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling. In some embodiments, the methods result in cultured T cells wherein the ratio of CD8+ T cells or the relative or normalized ratio of CD8+ T cells in the composition is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the ratio or the relative or normalized ratio of CD8+ T cells in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to an analogous incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling. In some embodiments, the methods result in cultured T cells wherein the number or percentage of CD62L+, optionally long-lived memory T cells or memory stem cells (TSCM), in the composition is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the number or percentage of the corresponding population of cells, CD62L+, long-lived memory T cells, or TSCM, in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to an analogous incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling.

[00117] In some embodiments, the cultured T cells contain greater than 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of a T cell subset having a phenotype that is surface positive for CD62L (CD62L+) as a percentage of the total T cells in the composition or of the total cells in the composition.

[00118] In some embodiments, the T cell subset also includes a phenotype including CD127+; and/or any one or more of CD45RA+, CD45RO-, CCR7+, and CD27+ and any one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+, and LFA-1+.

[00119] In some embodiments, the T cell subset includes a low level of TCR recombination excision circles (TREC); and/or expresses a proliferation marker, which may be Ki-67; and/or exhibits the ability to proliferate in the presence of a stimulatory agent; and/or exhibits the ability to produce a cytokine such as IFN-gamma, TNF, or IL-2 in the presence of a stimulatory agent.

[00120] In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine such as IL-15 and/or IL-17, or is an agent that is capable of initiating a signal associated with the TCR/CD3 complex on T cells.

[00121] In some embodiments, the T cell subset is or contains long-lived memory T cells. In some embodiments, the T cell subset is or includes T stem memory cells (TSCM).

[00122] In some embodiments, the method also includes introducing a recombinant nucleic acid molecule into T cells or target cells of the population. In some such aspects, the nucleic acid molecule can encode a recombinant protein, whereby the cells express the recombinant protein. In some embodiments, the recombinant receptor is a chimeric antigen receptor or transgenic T cell receptor (TCR). In some aspects, the method is carried out in vitro or ex vivo.

[00123] In some embodiments, the method also includes administering the cultured cells to a subject having a disease or condition.

[00124] Provided herein in some aspects is a composition containing a plurality of the cultured T cells or target cells produced by the methods provided herein, and optionally a pharmaceutically acceptable excipient.

[00125] In some embodiments, after addition of the substance, the cells were not incubated in vitro or ex vivo at a temperature greater than 30 °C for more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours.

[00126] Also provided herein are articles of manufacture that include: a) a reagent that includes a plurality of binding sites capable of binding to a receptor binding agent; and b) the receptor binding agent wherein i) is reversibly bound to the reagent and ii) is capable of specifically binding to a molecule on the surface of T cells in a manner that induces or modulates a signal in T cells. In some embodiments, the molecule is not CD28, CD3, or CD40.

[00127] In some embodiments, binding to the molecule induces or modulates a signal in a T cell other than a signal associated with the TCR/CD3 complex; and/or binding to the molecule enhances or potentiates a signal associated with the TCR/CD3 complex. In some embodiments, the molecule is selected from CD90 (Thy-1), CD95(Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. In some embodiments, the molecule is not CD137.

[00128] In some embodiments, the receptor binding agent specifically binds to a cytokine receptor, specifically binds to a chemokine receptor, is or includes an adhesion molecule, or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule.

[00129] In some embodiments, the receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2. In some embodiments, the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2; and/or the receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, and TNF, or is a biologically active fragment thereof. In some embodiments, the receptor binding agent specifically binds to a cytokine selected from IL-12R, IFN-gammaR, IL-4R, and IL-17R; or the receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15, and IL-17, or is a biologically active fragment thereof.

[00130] In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain). In some embodiments, the second receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); and/or the second receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9, and IL-15, or is a biologically active fragment thereof. In some embodiments, the second receptor binding agent specifically binds to a cytokine receptor selected from IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); or the second receptor binding agent is a ligand selected from IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[00131] In some embodiments, the receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. In some embodiments, the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21, and CCL25 or is a biologically active fragment thereof. In some embodiments, the receptor binding agent specifically binds to a cytokine selected from CXCR3, CCR7, CXCR1, and CXCR4; or the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[00132] In some embodiments, the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106(VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1, and VLA-4 or is a biologically active fragment thereof.

[00133] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[00134] Provided herein are methods for culturing target cells that include incubating a composition containing target cells in the presence of one or more receptor binding agent(s), wherein the receptor binding agent reversibly binds to a reagent that is a streptavidin analog or mutein containing a plurality of binding sites capable of binding to an agent. In some aspects, the streptavidin analog or mutein includes a net negative charge or exhibits a lower isoelectric point than the streptavidin mutein comprising the amino acid sequence set forth in SEQ ID NO: 4 or 6 and/or exhibits a higher affinity for a streptavidin-binding peptide containing the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than a streptavidin or mutein containing the amino acid sequence set forth in any of SEQ ID NOS: 1-6. In some embodiments, the agent is reversibly bound to the reagent and is capable of specifically binding to a molecule on the surface of a cell.

[00135] In some embodiments, the receptor binding agent includes a binding partner C1. In some aspects, the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent or agent and the reagent.

[00136] In some embodiments, the agent is a selection agent or a receptor binding agent.

[00137] In some embodiments, the agent is a receptor binding agent that specifically binds to a molecule that is or includes a member of a TCR/CD3 complex, CD3, a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family, or is or includes an adhesion molecule, or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule.

[00138] In some embodiments, the receptor binding agent is a second receptor binding agent and the molecule is a second molecule, and the reagent also includes: c) a plurality of binding sites capable of reversibly binding to a first receptor binding agent and d) the first receptor binding agent wherein i) is reversibly bound to the reagent and ii) is capable of specifically binding to a first molecule on the surface of a T cell.

[00139] In some embodiments, the agent or the first receptor binding agent specifically binds to an element of a TCR/CD3 complex and/or the first receptor binding agent specifically binds to CD3.

[00140] In some embodiments, the first receptor binding agent and second receptor binding agent each individually contain a C1 binding partner, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner to form the reversible bond between the first and second receptor binding agents and the reagent. In some aspects, the first receptor binding agent contains a C1 binding partner, the second receptor binding agent contains a C2 binding partner, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner and the C2 binding partner to form the reversible bond between the first and second receptor binding agents and the reagent. In some embodiments, the first receptor binding agent contains a C1 binding partner, the second receptor binding agent contains a C2 binding partner, and the plurality of binding sites includes two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner to form the reversible bond between the first receptor binding agent and the reagent, and two or more binding sites, Z2, each of which is capable of binding to the C2 binding partner to form the reversible bond between the second receptor binding agent and the reagent.

[00141] In some embodiments, the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm. In some embodiments, the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3. In some embodiments, the reagent is not bound to a support or solid support. In some examples, the reagent is bound or immobilized on a support. In some cases, the support is a solid support or a stationary phase. In some aspects, the support includes a bead, a particle, a nanoparticle, or a microsphere.

[00142] In some embodiments, the agent is individually selected from an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, a molecule containing Ig domains, and binding fragments thereof.

[00143] In some embodiments, the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent contains an antibody fragment. In some aspects, the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent contains a Fab fragment. In some cases, the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent is a divalent antibody fragment such as an F(ab')2 fragment or a divalent single chain Fv (scFv) fragment. In some embodiments, the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment, and a scFv fragment.

[00144] In some embodiments, the receptor binding agent or first agent includes an agent that specifically binds to CD3. In some embodiments, the agent that specifically binds CD3 is an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, the receptor binding agent or agent, which may be the second receptor binding agent, includes an agent that specifically binds to CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and HVEM, such as an anti-CD90 antibody, a divalent antibody fragment of an anti-CD90 antibody, an antibody fragment of an anti-CD90 antibody, a proteinaceous CD90 binding molecule with antibody-like binding properties, an anti-CD95 antibody, a divalent antibody fragment of an anti-CD95 antibody, an antibody fragment of an anti-CD95 antibody, a proteinaceous CD95 binding molecule with antibody-like binding properties, an anti-CD154 antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154 antibody, a proteinaceous CD154 binding molecule with antibody-like binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a CD137 binding proteinaceous molecule with antibody-like binding properties, an anti-ICOS antibody, a divalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, an ICOS binding proteinaceous molecule with antibody-like binding properties, an anti-LAT antibody, a divalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, a LAT binding proteinaceous molecule with antibody-like binding properties, an anti-CD27 antibody, a divalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, a CD27 binding proteinaceous molecule with antibody-like binding properties, an anti-OX40 antibody, a divalent antibody fragment of an antibody anti-OX40, a monovalent antibody fragment of an anti-OX40 antibody, a proteinaceous OX40 binding molecule with antibody-like binding properties, an anti-HVEM antibody, a divalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, a proteinaceous HVEM binding molecule with antibody-like binding properties, or any mixture thereof.

[00145] In some embodiments, the reagent is or contains streptavidin, avidin, a streptavidin analog or mutein, or and an avidin or mutein analog. In some embodiments, the reagent includes an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein, or an avidin or mutein analog.

[00146] Provided herein in some aspects is a kit, including the reagent described herein and optionally instructions for use. In some embodiments, the kit contains a substance capable of reversing the binding between the receptor binding agent and the reagent. In some embodiments, the kit contains a reagent containing a plurality of binding sites capable of reversibly binding to a receptor binding agent. In some aspects, the receptor binding agent is reversibly bound to the reagent and is capable of specifically binding to a molecule expressed on the surface of target cells. In some examples, binding to the molecule induces or modulates a signal in the target cells. In some cases, the kit contains a substance capable of reversing the binding between the receptor binding agent and the reagent.

[00147] In some embodiments, the target cells are T cells. In some embodiments, the reagent is or includes streptavidin, avidin, a streptavidin analog or mutein, or an avidin or mutein analog. In some embodiments, the reagent includes an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein, or an avidin or mutein analog.

[00148] In some embodiments, the substance includes a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analog or biologically active fragment.

[00149] In some embodiments, the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm. In some embodiments, the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3. In some embodiments, the reagent is not bound to a support or solid support.

[00150] Provided herein in some aspects is a composition including a plurality of T cells genetically modified to express a recombinant receptor that specifically binds to a target antigen. In some cases, greater than 35%, 40%, 50%, 60%, 70%, 80% or 90% of the cells include a T cell subset having a surface phenotype that is CD3+, CD4+ or CD8+ and CD62L+ and one or more of CD127+, CD45RA+, CD45RO-, CCR7+ and CD27+ and one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+ as a percentage of the total T cells in the composition or of the total cells in the composition. In some embodiments, prior to or during genetic engineering, the plurality of T cells containing the T cell subset were not incubated in the presence of a GSK-P inhibitor; were not incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15; or were not enriched for CD62L+ cells. In some embodiments, the composition does not contain a GSK-P inhibitor or a recombinant homeostatic cytokine, optionally IL-7 or IL-15.

[00151] In some embodiments, the T cell subset includes at least 5 X 106, at least 1 x 106, or at least 2 x 106 cells.

[00152] Provided herein in some embodiments is a composition including a plurality of T cells genetically modified to express a recombinant receptor that specifically binds to a target antigen. In some aspects, the genetically modified T cells are derived from transduction of a population of T cells containing a T cell subset having a surface phenotype that is CD3+, CD4+ or CD8+ and CD62L+ and one or more of CD127+, CD45RA+, CD45RO-, CCR7+ and CD27+ and one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population containing primary T cells that were isolated or enriched from a human subject based on the surface expression of one or more markers containing a phenotype. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population of the T cells that were incubated in the presence of a GSK-P inhibitor. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population of the T cells that were incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population of T cells that were stimulated by anti-CD3 and anti-CD8, but wherein the stimulation or activation was for more than 1 day, 2 days, 3 days, 4 days, or 5 days and/or the stimulation was not disrupted in the presence of biotin or a biotin analog.

[00153] In some embodiments, the T cell subset is present in the population at or at about greater than 35%, 40%, 50%, 60%, 70%, 80%, or 90% as a percentage of the total T cells in the population. In some embodiments, the T cell subset includes at least 5 x 10 6 cells, 1 x 10 6 cells, 2 x 10 6 cells, or more.

[00154] In some embodiments, the composition is a pharmaceutical composition.

[00155] Provided herein in some aspects is a method of treatment including administering to a subject having a disease or condition, a composition, e.g., pharmaceutical composition, as described herein.

[00156] In some embodiments, the cells include a recombinant receptor, such as a chimeric antigen receptor (CAR) or TCR. In some aspects, the recombinant receptor, such as a transgenic CAR or TCR specifically binds to an antigen associated with the disease or condition.

[00157] In some embodiments, the disease or condition is a cancer, an autoimmune disease or disorder, or an infectious disease.

Brief Description of the Drawings

[00158] FIG. 1(A-E) provides schematic representations of exemplary embodiments.

[00159] FIG. 1A shows a schematic representation of a reagent (or representative portion thereof) having a plurality of binding sites for reversible binding to agents. In this case, the reagent is shown as capable of reversibly binding to two agents, each of which is capable of specifically binding to a molecule on a cell. The reagent has a plurality of binding sites, including a plurality of binding sites, Z1, each capable of reversibly binding to agents. The first and second agents, which in some cases may be the same, in the schematic representation shown each contain at least one binding partner C1. Binding partner C1 reversibly binds to binding site Z1. The first and second agents each likewise contain a binding site, B2, that can specifically bind to a molecule on the surface of a cell, which in some cases may be on the same cell. Here, the first and second agents are shown specifically binding to molecules on the same cell.

[00160] FIG. 1B shows a schematic representation of a reagent having a plurality of binding sites capable of reversibly binding to a first and second agent, wherein the agents are each capable of specifically binding to a molecule on a first and second cell, respectively. The reagent has a plurality of Z1 binding sites each capable of reversibly binding to an agent. The first and second agents, which in some cases may be the same, each contain a C1 binding partner which reversibly binds to the Z1 binding site. The first and second agents each contain a B2 binding site which can specifically bind to a molecule on the surface of a cell, which in some cases may be on the same cell or a different cell. Here, the first agent is bound to a molecule on the surface of a first cell and the second agent is bound to a molecule on the surface of a second cell.

[00161] FIG. 1C shows a reagent capable of reversibly binding to a first and second agent, wherein the agents are each capable of specifically binding to a molecule on a first and second cell, respectively. The reagent has a plurality of binding sites Z1 and Z2, which may be the same or different, each capable of reversibly binding to one or both agents. The first agent contains a binding partner C1, which reversibly binds to Z1; the second agent contains a binding partner C2, which can reversibly bind to Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent contains a binding site B1, which can specifically bind to a molecule on the surface of a cell, and the second agent contains at least one binding site B3, which can specifically bind to a molecule on the surface of a cell. Binding sites B1 and B3 in some cases bind to two different cell surface molecules, or different epitopes on a single molecule, or the same or different molecules on the surface of different cells. Here, the first agent is shown as being bound, via B1, to a molecule on the surface of a first cell, and the second agent is bound, via B3, to a molecule on the surface of a second cell.

[00162] FIG. 1D shows a reagent capable of reversibly binding to a first and second agent, such as selection agents, which are each capable of specifically binding to a molecule on a cell. The reagent has a plurality of binding sites, including Z1 and Z2, which may be the same or different, each capable of reversibly binding to an agent. The first agent contains a binding partner C1 that can specifically bind to the binding site Z1 and the second agent contains at least one binding partner C2 that can specifically bind to the binding site Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent contains a binding site B1, which can specifically bind to a molecule on the surface of a cell and the second agent contains a binding site B3, which can specifically bind to a molecule on the surface of a cell. In some embodiments, the first agent and second agent may be a selection agent. Binding sites B1 and B3 may bind to the same or different molecules (e.g., receptor) on the surface of a cell, the same or different epitopes on a molecule, or the same or different molecules on the surface of different cells. Here, the first agent is bound to a first molecule on the surface of a cell and the second agent is bound to a second molecule on the surface of the same cell.

[00163] FIG. 1E shows a reagent reversibly linked to a first and second agent, wherein the agents are each capable of specifically binding to a molecule on a cell. The reagent has a plurality of binding sites, including Z1 and Z2, which may be the same or different, each capable of reversibly binding to an agent. The first agent contains a binding partner C1 that can reversibly bind to Z1 of the reagent and the second agent contains a binding partner C2 that can reversibly bind to Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent contains at least one binding site B2, wherein it can specifically bind to a molecule on the surface of a cell and the second agent contains at least one binding site B4, wherein it can specifically bind to a molecule on the surface of a cell. In some embodiments, the first agent and second agent can be stimulatory agents. Binding sites B2 and B4 can bind to the same or different molecules on the surface of a cell, the same or different epitopes on a molecule, or the same or different molecules on the surface of different cells. Here, the first agent is bound to a first molecule on the surface of one cell and the second agent is bound to a second molecule on the surface of the same cell.

[00164] FIG. 2(A-E), which includes FIGS. 2A-2E, provides schematic representations of exemplary embodiments as shown in FIGS. 1A-1E, respectively, except that the reagents described are shown as being immobilized on a support, such as a stationary phase.

[00165] FIG. 3 provides a schematic representation of exemplary embodiments in which oligomeric reagents are used to multimerize stimulatory agents and the resulting complexes incubated with cells to deliver signals to the cells, followed by reversal of binding. Panel A shows an oligomeric reagent 1, which is shown as unbound to any support and as being flexible. Stimulatory agents 2, which are shown here as Fab fragments and are capable of specifically binding to a molecule on the surface of a cell, are combined with the reagent. The agents comprise a binding partner (e.g., binding partner C) that is capable of reversibly binding to a binding site (e.g., binding site Z) on the reagent, multimerizing the agents. Panel B depicts the binding partner reversibly binding to a binding site on the reagent. Cells 3 are added to the system. Panel C depicts multimerized agents (Fab fragments) specifically binding to molecules 4 on the surface of a cell 3. In Panel C, the agents depicted are stimulatory receptor binding agents (e.g., a first receptor binding agent and/or a second receptor binding agent) that can induce or modulate a signal in a cell upon binding of the agent to the molecule on the cell. As shown in Panel D, a substance 5, such as a competitive reagent (e.g., biotin), is added to the composition, which may be a substance that exhibits a higher binding affinity for the binding site on the reagent than for the binding partner on the agent, thereby disrupting the reversible binding between reagent 1 and agent 2. In some cases, the agent, e.g., Fab fragment, may also dissociate from its interaction with molecule 4 on cell 3. In some cases, this may disrupt, reduce, and/or terminate signaling in the cell.

[00166] FIG. 4 provides a schematic representation of exemplary embodiments of a reversible system bound to a support, such as a solid support or a surface, including a stationary phase. Panel A shows a support 6 containing reagent 1. Agents 2, such as Fab fragments, that are capable of specifically binding to a molecule on the surface of a cell are added to the system. Agents 2, such as Fab fragments, comprise a binding partner (e.g., binding partner C) that is capable of reversibly binding to a binding site (e.g., binding site Z) on the reagent. Panel B depicts the binding partner reversibly binding to a binding site on the reagent. Cells 3 are added to the system. Panel C depicts agents 2, e.g., Fab fragments, binding to molecules 4 on the surface of a cell 3. In some embodiments, the scFvs comprise a receptor binding agent or a selection agent. In some embodiments, the agents, e.g., Fab fragments, can be a receptor binding agent or a selection agent. Panel C depicts an exemplary receptor binding agent or agents (e.g., a first receptor binding agent and/or a second receptor binding agent), which can induce or modulate a signal in a cell upon binding of the agent, e.g., Fab fragment, to the molecule on the cell. Substance 5, such as a competitive reagent (e.g., biotin), is added, which can be a substance that exhibits a higher binding affinity for the binding site on the reagent than for the binding partner on the agent, e.g., Fab fragment, thereby disrupting the bond between the reagent and the agent. Panel D depicts disruption of the bond between agent 2, e.g., Fab fragment, and the reagent, thereby resulting in dissociation of the reagent from the agent, and thereby the cell. In some cases, the agent, e.g., Fab fragment, may also dissociate from its interaction with molecule 4 on cell 3. In some cases, this may disrupt, reduce, and/or terminate signaling in the cell.

[00167] FIG. 5(A-C) shows the temporal effect of adding D-Biotin on T cell expansion following stimulation of T cells with anti-CD3/anti-CD28 Fabs that were reversibly linked to an oligomeric mutein streptavidin reagent. FIG. 5A is a schematic representation of the experimental conditions and timeline, including time points of D-Biotin addition and medium exchange during cell culture. FIG. 5B shows total cell counts (degree of expansion) for CD3+, CD4+, and CD8+ cells after stimulation under the indicated conditions, including with the addition of D-Biotin at different time points. For comparison, the dashed horizontal lines indicate the number of expanded CD3+, CD4+, or CD8+ cells in the positive control (anti-CD3/anti-CD28 beads). FIG. 5C shows twofold expansion of CD4+ and CD8+ when normalized to unstimulated control.

[00168] FIG. 6(A-D) shows aspects resulting from short-term activation of CD3+ T cells with anti-CD3/anti-CD28 Fabs that were reversibly bound to an oligomeric mutein streptavidin reagent to which D-Biotin was added one day after the start of stimulation. FIG. 6A is a schematic representation of the experimental conditions and timeline, including time points of D-Biotin addition and medium exchange during cell culture. FIG. 6B shows the cell count (degree of expansion) in cultures at day 7 under the indicated conditions. The dashed horizontal line indicates the number of cells expanded in the positive control anti-CD3/anti-CD28 beads without the addition of D-Biotin. FIG. 6C shows the ratio of CD4+ to CD8+ cells in the cultures of FIG. 6B. The dashed horizontal line indicates the fraction of CD4+ and CD8+ cells in the positive control without the addition of D-Biotin. FIG. 6D shows the number of CD4+ or CD8+ cells normalized to the numbers of CD4+ or CD8+ cells in the unstimulated control. The horizontal line represents the numbers of CD4+ or CD8+ cells expanded in the anti-CD3/anti-CD28 bead-positive control without the addition of D-Biotin. FIG. 6D shows flow cytometric analysis of surface expression of CD62L and CD127 for both CD4+ and CD8+ T cell populations in cultures at day 7 for the indicated conditions. FIG. 6F shows flow cytometric analysis of Ki-67 and T-bet expression for both CD4+ and CD8+ T cell populations in cultures at day 7 for the indicated conditions. ST-MM is a streptavidin mutein multimerized anti-CD3/anti-CD28 Fab.

[00169] FIG. 7(A-C) shows aspects resulting from short-term activation of CD8+ T cells with anti-CD3/anti-CD28 Fabs that were reversibly bound to an oligomeric mutein streptavidin reagent to which D-Biotin was added one day after initiation of stimulation using the experimental conditions and timeline mentioned in FIG. 6A. FIG. 7A shows the CD8+ cell count (degree of expansion) in cultures at day 7 under the indicated conditions. The dashed horizontal line indicates the number of cells expanded in the positive control anti-CD3/anti-CD28 beads without the addition of D-Biotin. FIG. 7B shows flow cytometric analysis of surface expression of CD62L and CD127 in cultures at day 7 for the indicated conditions. FIG. 7C shows flow cytometric analysis of Ki-67 and T-bet expression in cultures at day 7 for the indicated conditions. ST-MM is anti-CD3/anti-CD28 Fab multimerized mutein streptavidin agent.

[00170] FIG. 8(A-F) shows aspects resulting from stimulation of CD3+ T cells with an oligomeric mutein streptavidin reagent reversibly linked by anti-CD3 Fab and various costimulatory molecules. FIG. 8A shows the cell count (degree of expansion) in cultures at day 6 under the indicated conditions. The dashed horizontal line indicates the number of cells expanded in the positive control (anti-CD3/anti-CD28 beads). FIG. 8B shows the number of CD4+ or CD8+ cells normalized to the unstimulated control in cultures at day 6 under the indicated conditions. The resulting numbers of CD4+ or CD8+ cells expanded in the positive control (anti-CD3/anti-CD28 beads) were set at 100% as indicated by the dashed horizontal line. FIGS. 8C and 8D show the normalized numbers of CD62L+/CD4+ (FIG. 8C) or CD62L+/CD8+ (FIG. 8D) cells within the CD3+ cell cultures on day 6 for the indicated conditions. The resulting numbers of CD62L+/CD4+ or CD62L+/CD8+ cells expanded in the positive control (anti-CD3/anti-CD28 beads) were set at 100% as indicated by the horizontal lines. FIG. 8E and FIG. 8F depict flow cytometric analysis of surface expression of CD62L and CD69 or surface expression of CD45RO and CD45RA on CD4+ (FIG. 8E) or CD8+ (FIG. 8F) cells within exemplary CD3+ cells cultured for 6 days under the indicated conditions.

[00171] FIG. 9(A-D) shows aspects resulting from stimulation of CD3+ T cells with an oligomeric mutein streptavidin reagent (Strep-Tactin®; ST-MM A) or an oligomeric mutein streptavidin reagent (Strep-Tactin® XT; ST-MM B), each reversibly bound by anti-CD3 and anti-CD28. FIG. 9A shows the cell count (degree of expansion) in cultures on day 5 under the indicated conditions. The dashed horizontal line indicates the number of expanded cells in the positive control (anti-CD3/anti-CD28 beads). FIG. 9B s shows the proportion of CD4+ and CD8+ cells in the cultures of FIG. 9A. The dashed horizontal line indicates the fraction of CD4+ and CD8+ cells in the positive control (anti-CD3/anti-CD28 beads). FIG. 9C shows the numbers of CD4+ or CD8+ cells normalized to the numbers of CD4+ or CD8+ cells in the unstimulated control. The horizontal line represents the numbers of expanded CD4+ or CD8+ cells in the anti-CD3/anti-CD28-positive bead control. FIG. 9D shows flow cytometric analysis of CD62L and CD69 surface expression in cultures at day 5 for the indicated conditions.

[00172] FIG. 10(A-B) shows aspects resulting from stimulation of CD8+ T cells with an oligomeric mutein streptavidin reagent (Strep-Tactin®; ST-MM A) or an oligomeric mutein streptavidin reagent (Strep-Tactin® XT; ST-MM B), each reversibly bound by anti-CD3 and anti-CD28. FIG. 10A shows cell count (degree of expansion) in cultures at day 6 under the indicated conditions. The dashed horizontal line indicates the number of cells expanded in the positive control (anti-CD3/anti-CD28 beads). FIG. 10B shows flow cytometric analysis of surface expression of CD62L, CD69, and CD8 in cultures at day 6 for the indicated conditions.

[00173] FIG. 11(A-C) shows the results of an experiment in which non-responder CD3+ T cells were proliferated after being stimulated in vitro with αCD3 Fab and αCD28 Fab fragments that were reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®. FIG. 11A is a histogram showing the size distribution (forward scatter) of stimulated cells, FIG. 11B depicts histograms representing the degree of proliferation according to the number of cells per cell division that are indicated at the top of FIG. 11B (0 represents non-dividing cells; 5 represents cells that undergo at least 5 divisions), and FIG. 11C shows an image of the culture dish after 4 days of stimulation.

[00174] FIG. 12(A-D) shows the results of an experiment in which CD3+ T non-responder cells were proliferated after being stimulated in vitro with reversible αCD3/αCD28 Fab fragments that were reversibly immobilized on soluble oligomeric streptavidin mutein acting as a soluble reagent. For the experiments whose results are shown in FIG. 12, 300,000 CD3+ responder T cells (Tresp) were labeled with 2μM carboxyfluorescein succinimidyl ester (CFSE) and stimulated with varying amounts of a soluble oligomeric streptavidin mutein preparation in which a combination of αCD3 Fab fragment and αCD28 Fab both carrying a Streptavidin tag as the streptavidin binding peptide on one heavy chain was immobilized. (“1x” corresponds to 3 μg of oligomeric streptavidin mutein functionalized with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab; numbers indicate duplicate amounts of “1x”). Tresp cells left unstimulated or stimulated with blank oligomeric streptavidin muteins (no Fab) served as a negative control. Tresp cells were plated in duplicate in 48-well plates together with 300,000 CD3-negative autologous feeder cells (irradiated with 30 Gy) in 1 ml of cell culture medium supplemented with 20 U/ml interleukin 2 (IL-2). Cells were incubated at 37°C without media exchange, and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis (Fig. 12B). FIG. 12A shows the size distribution of cells after 5 days in culture. Histograms show live CD3+ cells, while FIG. 12C shows cells after culture, which were released by stimulation reagents after treatment with 1 mM D-biotin and washing. Dissociation and removal of monomeric Fab fragments was analyzed by reprobing with phycoerythrin-labeled oligomeric streptavidin mutein (ST-PE) as a fluorescent marker, and a representative histogram is shown. FIG. 12D shows the absolute number of live cells (trypan blue negative) after 5 days, which were counted using a Neubauer counting chamber and plotted against the respective stimulation condition. Median cell numbers are shown in FIG. 12D; error bars indicate standard deviation (SD). FIG. 12E shows a picture of the culture dish after 5 days of stimulation.

[00175] FIG.13(A-B) shows the proliferation expansion kinetics of purified CD8+ and CD4+ T responder cells (Tresp) that were stimulated in vitro with αCD3/αCD28 Fab fragments or with αCD3/αCD28/αCD8 Fab that were reversibly immobilized on two types of soluble oligomeric streptavidin mutein acting as a soluble reagent. The first type of oligomeric streptavidin mutein was the oligomeric streptavidin mutein fraction (n 3) obtained in Example 3 (also referred to herein as the "conventional" or "smaller" oligomeric streptavidin mutein backbone, illustrated by the downward-pointing triangle symbol in FIG. 13(AB)), the second type of this oligomeric streptavidin mutein used as a soluble reagent was an oligomer that was obtained by reacting the soluble oligomeric streptavidin mutein with biotinylated human serum albumin (HSA). This HSA-based soluble reagent is also referred to herein as the "larger" oligomeric streptavidin mutein backbone). In the experiments of FIG. 13(A-B), expansion was performed without medium exchange. The results for CD4+ T responder cells are shown in FIG. 13A, the results for CD8+ T responder cells are shown in FIG. 13B. In this context, it is noted that the experimentally used soluble reagents that have been functionalized by first reversible binding agents and, optionally, second and third agents are referred to in FIGS. as "multimerized agents."

[00176] FIG. 14(A-B) shows the proliferation expansion kinetics of purified CD4+ and CD8+ T responder (Tresp) cells that were stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized with two types of soluble oligomeric streptavidin mutein acting as the soluble reagent. The first type of oligomeric Strep-tactin® was the oligomeric streptavidin mutein fraction (n>3) obtained in Example 3 (also referred to herein as "conventional oligomeric streptavidin mutein backbone", illustrated by the upward-pointing triangle symbol in FIG. 14(AB)), the second type of this oligomeric streptavidin mutein used as the soluble reagent was the HSA-based soluble reagent, the above-mentioned "large backbone". In the experiments of FIG. 14(A-B) expansion was performed with medium exchange. The results for CD4+ T responder cells are shown in FIG. 14A, the results for CD8+ T responder cells are shown in FIG. 14B.

[00177] FIG. 15(A-B) shows the combined data from the results obtained in Figures 13 and 14 for the proliferation expansion kinetics of purified CD4+ and CD8+ T responder cells, with FIG. 15A representing the results for CD4+ T cells and FIG. 15B representing the results for CD8+ T cells. The straight lines are used for the culture with medium exchange on day 3, while the dashed lines represent the values obtained for the degree of expansion without medium exchange on day 3. The data shown in FIG. 15(AB) are normalized to the input cell number. Only data for Tresp stimulated with the oligomeric streptavidin mutein (n > 3), Tresp stimulated with commercially available anti-CD3/anti-CD28 beads (positive control), and unstimulated T cells (negative control) are shown, however, there is no data on the "large backbone" reagent.

[00178] FIG. 16(A-C) shows the expansion kinetics and phenotype of CD3+ central memory (TCM) T cells (CD3+CD62L+CD45RA- TCM) polyclonally stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric streptavidin mutein (with n > 3) described in Example 3. The graphs shown in FIG. 16(A-C) represent the degree of proliferation according to the number of cells harvested per time point, with FIG. 16A showing proliferation in IL-2-supplemented media alone and FIG. 16B showing proliferation in media supplemented with both IL-2 and IL-15. FIG. 16C shows a flow cytometric analysis of CD62L and CD127 surface expression after 14 days of culture in these varying cytokine media.

[00179] FIG. 17(A-B) shows the yield and expansion phenotype of purified CD8+ T responder cells stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on two types of soluble oligomeric streptavidin muteins acting in a soluble reagent. The first type of oligomeric streptavidin mutein was the oligomeric streptavidin mutein fraction (obtained in Example 3 (conventional backbone), the second type of this oligomeric streptavidin mutein used as a soluble reagent was the soluble oligomer described above and referred to here as the "large backbone". In these experiments, the oligomeric conventional streptavidin mutein fraction (n > 3) was also used as a reagent that was functionalized with single Fab fragments (third bar in FIG. 17A and FIG. 17B) or with a combination of αCD3 and αCD28 Fab fragments. In addition to the combined stimulation with αCD3/αCD28 Fab fragments, also an additional αCD8 Fab fragment (commercially available from IBA GmbH, Gottingen, Germany) was immobilized to test whether it is possible to preferentially stimulate a specific T cell subpopulation. FIG. 17A shows a bar graph representing the degree of proliferation according to the number of cells harvested on day 6 compared to negative controls (unstimulated purified CD8+ T responder cells) and normalized to the positive control (purified CD8+ T responders stimulated with commercially available anti-CD3/anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized). FIG. 17B shows flow cytometric analysis of the surface expression of CD8 and the T cell surface molecule CD45RO (which is indicative of T cell proliferation and activation) after cell culture. The various stimulation conditions were compared using one-way ANOVA, and no significant differences (n.s.) were detected.

[00180] FIG. 18(A-B) shows the yield and phenotype for the expansion of purified CD8+ T responder cells stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric streptavidin mutein acting as a soluble reagent that were functionalized with single Fab fragments or with a combination of Fab fragments (as already described above). In these experiments, the CD8+ T responder cells were stimulated with the soluble reagent (the soluble oligomeric streptavidin mutein (1 mg/ml) of Example 3) that was functionalized with varying amounts of αCD3 and αCD28 Fab fragments, optionally together with the αCD8 Fab fragment described above. The term "1x" corresponds to 1.5μg of oligomeric streptavidin mutein functionalized with 0.5μg of αCD3 Fab fragment only and 1.5μg of oligomeric streptavidin mutein functionalized with 0.5μg of αCD28 Fab only), or 3μL of an oligomeric streptavidin mutein preparation loaded with 0.5μg of αCD3 Fab fragment and 0.5μg of αCD28 Fab, or 4.5μL of an oligomeric streptavidin mutein preparation loaded with 0.5μg of streptavidin-labeled αCD3 Fab, 0.5μg of streptavidin-labeled αCD8 Fab, and 0.5μg of streptavidin-labeled αCD28 Fab. Consequently, the term "2x" corresponds to 3.0 μg of oligomeric streptavidin mutein functionalized with 1 μg of αCD3 Fab fragment only and 3.0 μg of oligomeric streptavidin mutein functionalized with 1 μg of αCD28 Fab only, meaning that twice the amount of immobilized αCD3 Fab fragment was used. Untreated Tresp cells served as a negative control and purified CD8+ T responders stimulated with commercially available anti-CD3/anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) as a positive control. FIG. 18A shows a graph in which the bars represent the degree of proliferation according to the number of cells harvested on day 5 compared to negative controls and normalized to the positive control. FIG.18B shows FACS analysis of CD8 and CD45RO surface expression after cell culture.

[00181] FIG. 19(A-B) shows the expansion of purified CD3+ T responder cells stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric streptavidin mutein of Example 3 that served as a soluble reagent. In one experiment, in addition to the αCD3/αCD28 Fab fragments, a commercially available αCD8 Fab fragment from IBA GmbH, Gottingen, Germany (catalog number 6-8000-203) was also immobilized on the soluble streptavidin mutein oligomer to test whether it is possible to preferentially stimulate in vitro the CD8+ T cell subpopulation within bulk CD3+ culture with a reagent having reversibly immobilized thereon also an αCD8 Fab fragment. In more detail, 500,000 purified CD3+ responder T cells (Tresp) were stimulated with 3 μL of an oligomeric streptavidin mutein preparation (1 mg/mL) loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. As an alternative approach, 4.5 μL of the oligomeric streptavidin mutein was loaded with 0.5 μg of αCD3 Fab, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab described above. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3/anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) served as a positive control. FIG. 19A shows a graph of cell counts (degree of expansion) in cultures under each condition. FIG. 19B shows the proportion of CD4+ and CD8+ cells under each stimulation condition.

[00182] FIG. 20(A-B) shows the results of differential intracellular calcium mobilization in Jurkat cells that are labeled with the αCD3 mAb OKT3 or with OKT3 Fab fragments being multimerized with Strep-tactin® (also referred to as Fab multimers herein). For the experiment in FIG. 20A, Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM and calcium release was activated by injection of αCD3 mAb OKT3 (black squares) or αCD3 OKT3 Fab multimers (derived from the parental OKT3 cell line) with or without prior D-biotin disruption (dark gray triangles and light gray circles, respectively) compared to PBS injection (inverted white triangles). Ionomycin application served as a positive control. Time-resolved changes in intracellular Ca 2+ concentration were monitored by flow cytometry based on changes in the FL6/FL7 ratio. For the experiment in FIG. 20B, Indo-1-AM-labeled Jurkat cells were activated by different αCD3 stimuli as described in Example 11; OKT3: top graph and αCD3 Fab multimer: middle graph) followed by subsequent D-biotin-mediated disruption (t = 140 s) of αCD3 Fab multimer signaling. αCD3 Fab multimer predissociated with D-biotin (bottom graph) and ionomycin served as a negative or positive control. Data are representative of three different experiments.

[00183] FIG. 21 shows the result of reversible labeling of cells by anti-CD3 OKT3 Fab multimers. Freshly isolated PBMCs were labeled with a monoclonal antibody (left dot plot, parental clone for the Fab multimers) or cognate PE-labeled Fab multimers and analyzed before (second dot plot from the left) or after D-biotin treatment (middle dot plot). Remaining Fab monomers were then detected after subsequent washing steps using fresh PE-labeled Strep-Tactin® (second dot plot from the right). Secondary Fab multimer labeling of reversibly labeled cells served as a control (right dot plot). Only live (PI-negative) cells are shown. Numbers in the dot plots indicate the percentage of cells within the gates.

[00184] FIG. 22 shows the isolation of cells by reversible ligation of multimerized anti-CD28 Fab fragments with phycoerythrin-labeled Strep-Tactin® as a fluorescent label. CD28+ cells were selected/isolated by magnetic cell sorting of Fab multimer from freshly isolated PMBCs as described in International Patent Application Publication No. WO2013/011011. Prior to sorting, cells were control labeled with cognate fluorescent aCD28 multimers (left dot plot) or with an antibody directed against the immunoglobulin kappa light chain (second dot plot from the left, α-Ig kappa mAb). After selection, cells were treated with D-biotin and subsequently washed to remove magnetic beads and Fab monomers. Released CD28+ cells were subsequently (re)labeled with αCD28 Fab multimers (second dot plot from the right) or with the α-Ig kappa mAb (right dot plot) to detect potentially remaining Fab monomers. Only live CD3+ cells (PI-negative) are shown. Numbers in the dot plots indicate the percentage of cells within the gates.

[00185] FIG. 23(A-C) shows the formation of early T cell clusters following activation of purified CD4+ and CD8+ T responsive cells stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric streptavidin mutein (n > 3) described in Example 3. FIG. 23A depicts the results for CD4+ T cells and FIG. 23B depicts the results for CD8+ T cells. Data for Tresp stimulated with the soluble multimerization reagent (the oligomeric streptavidin mutein), Tresp stimulated with commercially available anti-CD3/anti-CD28 beads (positive control), and unstimulated T cells (negative control) are presented.

[00186] FIG. 24 (A-F) shows the kinetics of selective antigen-specific (Ag-specific) expansion of a bulk population of purified CD3+CD62L+CD45RA TCM responder cells that were stimulated in vitro equally with a peptide complex: MHC molecule (which acts as the first agent that provides a primary activation signal to the cells) and αCD28 Fab fragment (which acts as the second agent that binds the accessory molecule on the cell surface) and unstimulated T cells (negative control). Both the antigen-specific peptide complex with the MHC molecule and αCD28 Fab fragment were reversibly immobilized on the same soluble oligomeric streptavidin mutein (with n > 3) described in Example 3. The peptide used for antigen-specific expansion in FIG. 24A was the peptide CRVLCCYVL (SEQ ID NO: 38), amino acids 309-317 of the immediate-early protein 1 restricted by the MHC molecule HLA-C702 (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383) representing an HLA-C7/IE-1 epitope that is specific for cytomegalovirus (CMV). The MHC I molecule presenting the peptide carries at its C-terminus of the heavy chain the streptavidin-binding peptide (SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK (SEQ ID NO: 16), which is commercially available as "Twin-Strep-tag®" from IBA GmbH, Gottingen, Germany). FIG. 24A shows an exemplary flow cytometric analysis for the fraction of Ag-specific cells that were proliferated using the peptide:MHC-I complex specific for this HLA-C7/IE-1 epitope as the first agent providing a primary activating signal to cells reversibly immobilized on soluble oligomeric streptavidin mutein. The graphs of FIG. 24B through FIG. 24E illustrate the expansion kinetics of other Ag specificities according to the specific number of peptide:MHCI multimer-positive cells harvested per time point in analogy to FIG. 24A using the distinct complexes of an antigen-specific peptide with the MHC I molecule as the first agent providing a primary activating signal to cells reversibly immobilized on soluble oligomeric streptavidin mutein. In more detail, FIG. 24B shows the expansion of Ag-specific cells that were expanded using the peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF) (SEQ ID NO: 39) restricted by HLA-A2402), FIG. 24C shows the expansion of Ag-specific cells that were expanded using another peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 265-274 (RPHERNGFTV) (SEQ ID NO: 40) restricted by HLA-B702), FIG. 24D shows the expansion of Ag-specific cells that were proliferated using the peptide:MHC-I complex specific for the adenovirus hexon 5 epitope (amino acids 114-124 (CPYSGTAYNSL) (SEQ ID NO: 41) restricted by HLA-B702), FIG. 24E shows the expansion of Ag-specific cells that were proliferated using the peptide:MHC-I complex specific for the CMV HLA-B7/IE-1309-317 epitope (CRVLCCYVL (SEQ ID NO: 38); for exemplary FACS data, see above Figure 24A). All peptide:MHC molecules carrying Twin Strep®-Tag are commercially available from IBA GmbH. In this context, the amino acid sequences of the molecules HLA-A*2402, HLA-B*0702 and HLA-C*0702 carrying "Twin-Strep-tag®" as their C-terminus are shown as SEQ ID NO: 42, 43 and 44 in the accompanying Sequence Listings, while the amino acid sequence of the β2 microglobulin (which forms together with the α chain, which means the HLA-encoded molecules, the respective MHC I molecule) is shown as SEQ ID NO: 45 in the accompanying Sequence Listing. Furthermore, FIG. 24F shows an exemplary flow cytometric analysis of surface expression of CD62L and CD127 after 14 days of culture for the HLA-B7/Hexon5114-124 stimulated/expanded cells of FIG. 24D.

[00187] FIG. 25 shows the kinetics of selective Ag-specific expansion out of purified CD3+CD62L+CD45RA TCM responder cells that were stimulated in vitro with a) antigen-specific MHC I peptide complexes and b) αCD28 Fab fragments that were reversibly immobilized as the first and second agents on soluble oligomeric streptavidin muteins. To this end, 500,000 CD3+CD62L+CD45RA responder TCM cells (Tresp) were specifically Ag stimulated using 3μL of a Streptactin multimerization reagent preparation functionalized with 0.5μg of peptide:MHC class I complexes equipped with a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 (CPYSGTAYNSL, SEQ ID NO: 41) of the HLA-B0702-restricted adenovirus Hexon 5 protein, see above) and 0.5μg of αCD28 Fab. As an alternative, 4.5 μL of a Streptactin multimerization reagent preparation loaded with 0.5 μg of this peptide:MHC class I complex, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab. For comparison, polyclonal stimulation was performed using 3 μL of a Streptactin multimerization reagent preparation (1 mg/mL) or loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. Again as the alternative stimulation condition described above, 4.5 μL of a preparation of Streptactin multimers loaded with 0.5 μg of αCD3 Fab, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab was used. Untreated (unstimulated) Tresp cells served as a negative control and polyclonal Tresp cells stimulated with anti-CD3/anti-CD28 beads as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U/mL IL-2 and 5 ng/mL IL-15. Cells were incubated at 37°C with medium change every 3 days, and cell counts were analyzed after 7 and 14 days. The photographs shown in FIG. 25 represent the degree of cluster formation at day 5 for specific Ag stimulation as exemplified for the adenovirus HLA-B7/Hexon 5 epitope.

[00188] FIG. 26(A-B) shows the activation of intracellular signaling cascades of transduced Jurkat cells that were modified to express an α-CD19 chimeric antigen receptor (CAR) and that were stimulated using the oligomeric Strep-tactin® of Example 3 as a soluble multimerization reagent. The specificity of a CAR is typically derived from an scFv region assembled from the antigen-binding region of a monoclonal antibody (mAb) that specifically binds to a target/tumor-associated antigen such as CD19 and links it to specific T cell signaling (described in Hudecek et al., Clin Cancer Res. 2013 June 15;19(12):3153-3164). In the experiments, the extracellular domain (ECD) of CD19, which contains the natural ligand of the αCD19 CAR, as well as the F(ab)2 fragment of polyclonal αIgG that recognizes the IgG4 spacer (donkey anti-human F(ab)2 is commercially available from Jackson Immuno Research) within the αCD19-CAR were also used in this experiment as the first agent that provides a primary activation signal to Jurkat cells. Reversible immobilization to the soluble oligomeric streptavidin mutein was provided by streptavidin peptide SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 16) that has been fused to the C-terminus of the CD19 ECD or by the biotinylated (Fab)2 fragment of αIgG (since the streptavidin mutein "m2" binds biotin with reduced affinity, this binding is reversible and can, for example, be displaced by the addition of an excess of free biotin). In the control experiment of FIG. 26A , 300,000 Jurkat CD3+ responder (Jresp) cells were stimulated with varying amounts of a mixture of oligomeric Streptactin preparations (1 mg/ml) that was functionalized with αCD3 Fab and αCD28 Fab (“x1” corresponds to 3 μg of multimerized Streptactin functionalized with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab—polyclonal multimerized agent). In the experiment of FIG. 26B, 3 μL of an oligomeric Streptactin preparation was functionalized with 0.5 (x1) or 1 (x2) of the extracellular domain (ECD) of CD19 or with 3 μL of an oligomeric Streptactin preparation loaded with 0.5 (x1) or 1 μg (x2) of αIgG recognizing the IgG4 spacer (which are both CAR-specific multimerizing agents). Jresp stimulated with anti-CD3/anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) or PMA and ionomycin served as positive controls. Jresp cells were seeded in 1.5 mL Eppendorf tubes in 200 μL of cell culture medium supplemented with 30 U/mL IL-2. Cells were incubated at 37°C and placed on ice and lysed after 0 min to 20 min of stimulation. Detection of phosphorylated ERK indicates active MAPK signaling, labeling of housekeeper β-actin indicates loading of equal amounts of total protein per condition and time point.

[00189] FIG. 27 shows the impact of D-Biotin addition on Ca2+ flux in CD3+ T cells following stimulation with αCD3/αCD28 Fabs that were reversibly bound to an oligomeric mutein streptavidin reagent as assessed by flow cytometry. The multimerized reagent was added to the cultures at 60 s (arrow "E"), and the cultures were either left undisrupted (left panel) or further treated by the addition of D-Biotin at 200 s (arrow "B") and the addition of ionomycin at 400 s (arrow "I").

[00190] FIG. 28A shows the cell count (degree of expansion) resulting from activation of CD3+ T cells following stimulation with the multimerized agent reversibly linked to αCD3/αCD28 Fabs (with or without addition of D-Biotin on day 1) or stimulation with control anti-CD3/anti-CD28 beads as assessed on day 0, 3, and 7 in culture. Unstimulated cells were included as a negative control. **: p < 0.05, two-tailed t-test.

[00191] FIG. 28B shows the percentage of annexin V-positive cells (indicating apoptosis) resulting from activation of CD3+ T cells following stimulation with the multimerized agent reversibly linked to αCD3/αCD28 Fabs (with or without addition of D-Biotin on day 1) or stimulation with control anti-CD3/anti-CD28 beads as assessed on day 3 and 7 in culture. Unstimulated cells were included as a negative control. **: p < 0.05, two-tailed t-test.

[00192] FIG. 29 shows cell cycle entry and proliferation resulting from activation of CD3+ T cells following stimulation with the multimerized agent reversibly linked to αCD3/αCD28 Fabs (with or without addition of D-Biotin on day 2) or stimulation with control anti-CD3/anti-CD28 beads analyzed by measuring CFSE dilution on day 3 (d3) and day 7 (d7) in culture. Unstimulated cells were included as a negative control.

[00193] FIG. 30 shows the metabolic activity of purified CD4- or CD8- T cells after stimulation with the multimerized agent reversibly linked to αCD3/αCD28 Fabs (with or without addition of D-Biotin on day 2) or stimulation with control anti-CD3/anti-CD28 beads as assessed by the WST-1 assay at 24 hours and 72 hours in culture. Unstimulated cells were included as a negative control.

[00194] FIG. 31 shows the expansion capacity of CD3+ T cells that were stimulated with the multimerized agent reversibly linked to αCD3/αCD28 Fabs and subsequently cultured (1) after sorting to divide the cells and not further stimulated (sort-purified), (2) after sorting to divide the cells and restimulated with the multimerized agent (sort-purified, restimulation), or (3) without sorting or further restimulation (unsorted). D-Biotin was added on day 1 after further culture for all conditions (dashed line).

Detailed Description

[00195] Provided herein is a method for stimulating, e.g., expanding, inducing survival or persistence of differentiation of a composition of target cells, such as T cells. In some embodiments, the methods relate to reversible reagent systems capable of binding to molecules on the surface of a target cell, such as a receptor binding molecule, thereby providing a signal to the cells, which in some cases may be a primary activation signal. In some embodiments, the methods employ reagents, which may be a multimerization reagent having bound thereto one or more agents, e.g., a first agent, second agent, etc. that provides a signal to the cells, such as a primary activation signal and/or an accessory or costimulatory signal. In some embodiments, the primary activation signal may as such be sufficient to activate the cells to expand/proliferate. This first agent may be bound reversibly or also irreversibly to the multimerization reagent. The multimerization reagent may also be linked to a second agent that stimulates an accessory molecule on the surface of the cells. The second agent, by binding to the accessory molecule on the surface of the cells, may thereby stimulate the activated cells to expand. This second agent may also be linked reversibly or irreversibly to the multimerization reagent. In some aspects, one or more additional agents may also be employed to modulate or induce additional signals in the cells. In some embodiments, the provided methods involve temporarily controlling stimulation of cells by time-controlled disruption of the reversible reagent or reagents.

[00196] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition referred to herein is contrary to or otherwise inconsistent with a definition referred to in the patents, applications, published applications and other publications that are incorporated herein by reference, the definition referred to herein prevails over the definition that is incorporated herein by reference. For example, the contents of PCT International Application No. PCT/EP2015/058339 are incorporated by reference in their entirety.

[00197] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

I. OVERVIEW OF METHODS AND SYSTEMS FOR MODULATING OR STIMULATING CYCLES USING REVERSIBLE REAGENTS

[00198] In some embodiments, the methods involve incubating, such as contacting, a plurality of cells comprising target cells, with one or more multimerization reagents that reversibly bind to an agent. In some embodiments, the multimerization reagent can be immobilized on a solid or soluble support, whereby during at least a portion of the cell incubation, the cells are contacted with the multimerization reagent. In one aspect, the method described herein is a serial expansion of a cell population in which an entire population of lymphocytes is stimulated/expanded, the reagents necessary for the expansion are then removed by chromatography on a suitable stationary phase. In some embodiments, the expanded/stimulated cells, which are the cultured cells, are optionally transfected with, for example, a T cell receptor or a chimeric antigen receptor (CAR), and in some aspects, may undergo a second stimulation expansion with a different stimulatory molecule that binds to the introduced T cell receptor or chimeric antigen receptor.

[00199] Methods of expanding T cell populations in vitro in the absence of exogenous growth factors or low amounts of exogenous growth factors are known in the art (see, e.g., US Patent 6,352,694 B1 and European Patent EP 0 700 430 B1). In general, such methods employ a solid phase surface greater than 1 μM to which various binding agents (e.g., anti-CD3 antibody and/or anti-CD28 antibody) are immobilized. For example, anti-CD3/anti-CD28 beads (Invitrogen) are commercially available reagents for T cell expansion, which are sterile, non-pyrogenic, 4.5 μm superparamagnetic polystyrene beads coated with a mixture of affinity-purified monoclonal antibodies against the cell surface molecules CD3 and CD28 on human T cells. However, in some cases, such magnetic beads are, for example, difficult to integrate into a method for expanding cells under conditions required for clinical trials or therapeutic purposes, since it is necessary to ensure that these magnetic beads are completely removed before administering the T cells to a patient.

[00200] In some embodiments, the methods provided herein address these concerns. In some aspects, the reagents provided are reversible, such that the stimulatory agents can be removed from the cellular composition. Furthermore, in some aspects, the reagent, e.g., multimerization reagent, to which the stimulatory agents are attached is not immobilized on a support, such as not immobilized on a solid support or surface. Thus, in some aspects, the reagent, e.g., multimerization reagent, is flexible and not rigid. In some embodiments, the reagent can adapt or conform to the cell surface. In some embodiments, it is possible to immobilize the reagent on a support, such as a solid support, including a stationary phase. In some embodiments, these methods can be used in conjunction with selection methods using similar selection agents in which one or more target cells can be selected and simultaneously or sequentially exposed to the stimulatory agents. Thus, in some respects, stimulation of particular cells or subsets of cells can be influenced by selection and isolation in conjunction with stimulation.

[00201] In some embodiments, the provided methods involve incubating or culturing, e.g., contacting, a cell composition with a reagent, e.g., multimerization reagent to which is bound one or more receptor binding agents (e.g., stimulatory agents) (see, e.g., FIGS. 3 and 4). In some embodiments, after contacting the cell composition with the multimerization reagent and typically incubating the cell population with the multimerization reagent, the cell population forms complexes/binds to the multimerization agent via the first agent. Other cell populations contained in the initial sample that do not possess the specific cell surface molecule do not bind to the multimerization reagent. In this regard, it is noted that the cell population generally has multiple copies of the cell surface molecule on its surface and binding of these multiple copies is typically necessary for stimulation or activation.

[00202] Thus, the multimerizing agent typically provides more than one binding site, e.g., Z1, wherein in some cases a plurality of agents may be reversibly bound to present the first agent, second agent and/or other agents in a sufficient density to the cell population. In this regard, it is noted that a multimerizing agent may as such have multiple binding sites, e.g., Z1; for example, a streptavidin mutein (being a homotetramer) in its native state has four such binding sites, e.g., Z1, and may further be oligomerized. In some cases, a reagent may have only one binding site, e.g., Z1, for the reversible binding of a binding partner, e.g., C1. One such example is multimeric calmodulin. Calmodulin as such has only one binding site for calmodulin-binding peptides. However, calmodulin can be biotinylated and then reacted with streptavidin oligomers (see also below), thus providing a multimerization reagent in which multiple calmodulin molecules are presented in high density on a "structure", thus providing multimeric calmodulin.

[00203] In some embodiments, after incubation or other suitable time in which stimulation is desired to be disrupted, the bond between the binding partner C, e.g., C1, of a reversibly bound agent, and the binding site Z, e.g., Z1, of the multimerization reagent, is disrupted by disrupting the respective reversible bond. In some cases, disruption can be achieved by adding a competitor to the incubation/reaction mixture containing the cell population to be bound to the multimerization reagent. For competitive disruption (which may be understood as being a competitive elution) of the reversible binding between the binding partner C, e.g. C1, of a reversibly bound agent and the binding site Z, e.g. Z1 of the multimerization reagent, the incubation mixture/cell population may be contacted with a free first binding partner C, e.g. C1, or an analogue of said first binding partner C that is capable of disrupting the binding between the first binding partner and the binding site Z, e.g. Z1. In the example of the binding partner C, e.g. C1, being a streptavidin-binding peptide that binds to the biotin-binding site of streptavidin, the free first partner may be the corresponding free streptavidin-binding peptide or an analogue that binds competitively. Such an analogue may, for example, be biotin or a biotin derivative such as desthiobiotin.

[00204] In some embodiments, the addition of the free partner or its analogue results in the displacement of the binding partner C, e.g., C1, from the multimerization reagent and thus, since the binding partner is comprised in the reversibly bound agent, the displacement of such agent from the multimerization reagent is achieved. This displacement of the agent in turn results in a dissociation of the first agent from the cell surface molecule, in particular if the binding affinity of the bond between the first agent and the cell surface receptor has a dissociation constant (KD) in the range of 10-2 M to 10-13 M and is thus also reversible. Due to this dissociation, in some aspects, the stimulation of the cell population is also terminated.

[00205] In some embodiments, the binding affinity of antibody molecules for their antigen, including for example a cell surface receptor molecule, is typically in the affinity range of a KD of about 10-7 M to about 10-13 M. Thus, conventional monoclonal antibodies can be used as a receptor binding agent (first or second, e.g., stimulatory, or selection agent). In some embodiments, in order to avoid any undesired avidity effects leading to stronger binding, monoclonal antibodies can also be used in the form of their monovalent antibody fragments, such as Fab fragments or single chain Fv fragments.

[00206] The method provided has the advantage that the time period of stimulation or expansion of the cell population can be precisely controlled and thus also the functional state of the cell population can be closely controlled. As described herein, short-term activation of cells, such as T cells, by addition of a substance, such as a competitive agent (e.g., biotin or an analogue) within 5 days of stimulation results in proliferation and stimulation of cells, but alters one or more characteristics or aspects of the cells, such as the percentage of CD4 or CD8 T cells, CD8/CD4 ratio, phenotypic markers and/or differentiation state. For example, the results described herein show that the ability to temporarily control the signal using the embodiments of the reagents provided can result in an increase in less differentiated long-lived population T cells, such as long-lived memory T cells. In some cases, temporal control of the signal, for example by disrupting the binding of the multimerized agent using a competing substance, can be used to tailor or adjust the relative expansion and/or persistence of particular subpopulations compared to others (e.g., CD4+ vs CD8+).

[00207] In some embodiments, due to the dissociation of the reversibly bound agent or agents from the cell surface molecule, the provided method has the additional advantage that the stimulated cell population is free of stimulating agents at the end of the stimulation period. Furthermore, in some embodiments, all other reagents used in the method, namely the agents (e.g., first or second receptor binding agents, e.g., stimulatory agents, or selection agents) as well as the binding partner C competition reagent, e.g., C1, or its analogue can be easily removed from the stimulated cell population via a "removal cartridge" (see, e.g., described in International Application Publication No. WO 2013/124474). In some cases, e.g., where the multimerization reagent is immobilized on a solid support, such as a bioreactor surface or a magnetic bead, it is being retained. Thus, the use of a stripping cartridge for the removal of the free agent and the competing reagent may include loading the elution sample (e.g., sample obtained after reversible bond rupture) onto a second chromatography column.

[00208] In some embodiments, this column chromatography has a suitable stationary phase that is an affinity chromatography matrix and at the same time can act as a gel permeation matrix. In some aspects, this affinity chromatography matrix has an affinity reagent immobilized thereon. In some embodiments, the affinity reagent can, for example, be streptavidin, a streptavidin mutein, avidin, an avidin mutein, or a mixture thereof. In some embodiments, the agent (e.g., first or second receptor binding agents, e.g., stimulatory agents or selection agents), the binding partner C, C1 competition reagent, binds to the affinity reagent, thereby being immobilized on the chromatography matrix. As a result, the elution sample containing the isolated and expanded cell population is being depleted of the agent (e.g., first or second receptor binding agents, e.g., stimulatory agents, or selection agents) and the competing reagent. In some embodiments, the cultured composition is free of any reagents, which in some aspects is advantageous for use in connection with diagnostic applications (e.g., additional FACS™ sorting) or for any cell-based therapeutic application.

[00209] In some embodiments, the ability to remove the reagent and other components of the composition has the additional advantage of being able to avoid any solid support, such as magnetic beads. In some embodiments, this means that there is no risk or minimal risk of contamination of the activated T cells by such magnetic beads. In some embodiments, this also means that a process that is compatible with GMP standards can be more easily established compared to other methods, such as the use of Dynabeads where additional measures have to be taken to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, in some embodiments, the use of a soluble multimerization agent makes it much easier to remove it from the activated cell population (T cells, B cells or also natural killer cells) since the cells can be pelleted by centrifugation and the supernatant, including the soluble multimerization agent, can be discarded. Alternatively, the soluble multimerization agent may be removed from the expanded cell population in a gel permeation matrix of the removal cartridge, as described above (e.g., International Patent Application Publication No. WO 2013/124474). In some embodiments, since no solid phase (e.g., magnetic beads) is present, the present invention also provides an automated closed system for cell expansion that may be integrated into known cell expansion systems, such as the Xuri Cell Expansion System W25 and WAVE Bioreactor 2/10 System, available from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) or the Quantum® Cell Expansion System, available from TerumoBCT Inc. (Lakewood, CO, USA).

[00210] In some aspects, the methods provided herein may include a population of cells that carry at least two specific cell surface molecules. In some embodiments, a first cell surface molecule is involved in a primary activating signal for the cell population, while the second cell surface molecule is an accessory molecule on the cell surface that is involved in providing a stimulus to the cells. In some embodiments, the population of cells is contacted with a multimerization reagent to which is reversibly bound a first agent that provides a primary activating signal to the cells and a second agent that induces or modulates an additional signal, such as stimulating an accessory molecule on the surface of the cells. The population of cells may, for example, be a population of T cells wherein the cell surface molecule is a TCR/CD3 complex and the cell surface molecule is the accessory molecule CD28. Furthermore, as described herein, targeting of other accessory molecules is also contemplated, such as one or more of CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. In some aspects, stimulation by these other accessory molecules can result in an increase in less differentiated T cells and, in some cases, long-lived T cell populations such as long-lived memory T cells compared to conventional stimulation through CD28. In some embodiments, binding of both the TCR/CD3 complex and the primary activation signal and binding of the accessory molecule (e.g., CD28 or another accessory molecule) can be necessary for T cell expansion/proliferation.

[00211] In some embodiments as described, the cell population may alternatively or additionally be stimulated in the presence of a multimerization reagent that is reversibly linked to an agent that targets or binds to a molecule on the cell surface to provide an additional signal that modulates survival, proliferation, and/or one or more other properties of the cells. Such targeting molecules include, but are not limited to, cytokine receptors, chemokine receptors, or adhesion molecules. In some aspects, stimulation by such additional molecules may result in one or other improved aspects of the stimulated or cultured cells, including, for example, increased persistence, survival, and/or activity, or less exhausted phenotype.

[00212] In some embodiments, the methods provided herein can also be further combined to include at least one selection agent reversibly linked to the same reagent, e.g., same multimerization reagent, as the receptor binding agent, e.g., either or both of the first or second, and/or additional receptor binding agent (e.g., stimulatory agent). In some cases, it is possible to enhance or increase one or more aspects resulting from the incubation or culture, such as stimulation of expansion (proliferation), activation, costimulation, and/or survival, in a subset of T cells that can be reversibly selected in the presence of at least one or more selection agents in an incubation or culture that also occurs in the presence of one or more stimulatory agents. For example, as shown in the examples herein, the degree of expansion in a T cell composition was selectively increased in CD8+ cells when such cells were incubated with a multimerized agent to which was reversibly linked an anti-CD8 antibody in addition to the anti-CD3 antibody and anti-CD28 antibody stimulatory agents. In some embodiments, one or more aspects resulting from the incubation or culture, such as stimulation of expansion (proliferation), activation, costimulation, and/or survival, can be increased at least 1.5-fold, at least 2.0-fold, at least 3.0-fold, at least 4.0-fold, at least 5.0-fold, at least 6.0-fold, at least 7.0-fold, at least 8.0-fold, at least 9.0-fold, at least 10.0-fold or more in a subset of T cells in the cultured composition that are positive for a selection marker when incubated in the presence of the one or more stimulatory agents and the selection agent specifically binding to the selection marker as compared to incubation only in the presence of the one or more stimulatory agents, but not the selection agent. This polarization or selectivity of the cell, such as T cells, allows one to control the endpoint aspects of specific subsets or populations of T cells. In some embodiments, the selection marker can be any selection marker as described herein. In some embodiments, the selection marker is selected from CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and/or CD45RO.

[00213] In some embodiments, the multimerization reagent comprises at least one Z-binding site, e.g., Z1, for reversible binding of the agent, e.g., first agent, and the agent, e.g., first agent, also comprises at least one C-binding partner, e.g., C1, wherein the C-binding partner, e.g., C1, is capable of reversibly binding to the Z-binding site, e.g., Z1, of the multimerization reagent. Thus, the agent, e.g., the first agent, when contacted or incubated with the multimerization agent, can be reversibly bound to the multimerization reagent via the reversible bond formed between the binding partner C, e.g., C1, and the binding site Z, e.g., Z1. Additionally, the one or more additional agents, e.g., the second agent, can comprise a binding partner C, e.g., C2, wherein the binding partner C2 is capable of being reversibly bound to a binding site Z, e.g., Z2, respectively, of the multimerization reagent. In some embodiments, the one or more additional agents, e.g., the second agent, when contacted or incubated with the multimerization agent, is reversibly bound to the multimerization reagent via the reversible bond formed between the binding partner C, e.g., C1, and the binding site Z, e.g., Z2. In some cases, C1 and C2 may be the same or substantially the same and/or comprise the same or substantially the same portion. In some cases, Z1 and Z2 may be the same or substantially the same and/or comprise the same or substantially the same portion.

[00214] In some embodiments, the use as binding partner C1 and C2, moieties that bind to the same binding site of the multimerization agent has the advantage that the same competition reagent (of the first binding partner C1 and also of the second binding partner C2) or its analogue can be used to interrupt and, in some cases, terminate the expansion of the target cell population (e.g., T cells) and release this target cell population (e.g., T cells) from the multimerization agent.

[00215] In some cases for producing binding agents (e.g., first, second, and/or additional receptor binding agents, e.g., stimulatory agents or selection agents) to comprise a binding partner C, the binding partner C, e.g., C1 or C2, can be provided by the respective expression vector used for recombinant production of the agent (e.g., antibody fragment), such that the binding partner C, e.g., C1 or C2, is part of a fusion peptide with the agent at the N-terminus or the C-terminus. In some embodiments, in the context of an agent that is an antibody or antigen-binding fragment, the binding partner C, e.g., C1 or C2, can be present at the C-terminus of the light chain or the heavy chain. Likewise, this methodology of cloning a recombinant protein, such as the variable domains of an antibody molecule, and recombinantly producing a respective protein, e.g., antibody fragment, is well known to the person skilled in the art, see, e.g., Skerra, A. (1994). In some embodiments, an antibody molecule can be generated from artificial binding molecules with antibody-like properties against a given target, such as CD3 or CD28 or other accessory or stimulatory agent molecules as described, such as by well-known evolutionary methods such as phage display (reviewed, e.g., in Kay, B.K. et al. (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1st Ed., Academic Press, New York NY; Lowman, H.B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, or Rodi, D.J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93), ribosome display (reviewed in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405) or mRNA display as reported in Wilson, D.S. et al. (2001) Proc. Natl. Acad. Sci. USA98, 3750-3755.

II. REVERSIBLE REAGENT SYSTEMS AND RELATED USES

[00216] In some embodiments, the methods employ reversible systems in which at least one agent (e.g., a receptor binding agent or selection agent) capable of binding to a molecule on the surface of a cell (cell surface molecule), is reversibly associated with a reagent. In some cases, the reagent contains a plurality of binding sites capable of reversibly binding to the agent (e.g., receptor binding agent or selection agent). In some cases, the reagent is a multimerization reagent. In some embodiments, the at least one agent (e.g., receptor binding agent or selection agent) contains at least one binding site B that can specifically bind an epitope or region of the molecule and also contains a binding partner C that specifically binds to the at least one binding site Z of the reagent. In some cases, the binding interaction between the binding partner C and the at least one binding site Z is a non-covalent interaction. In some embodiments, the binding interaction, such as non-covalent interaction, between the binding partner C and the at least one binding site Z is reversible.

[00217] In some embodiments, the reversible association can be mediated in the presence of a substance, such as a competing reagent (also called an eluent reagent), that is or contains a binding site that is also capable of binding to at least one Z-binding site. Generally, the substance (e.g., competing reagent) can act as a competitor due to a higher binding affinity for the Z-binding site present in the reagent and/or due to being present in higher concentrations than the C-binding partner, thereby separating and/or dissociating the C-binding partner from the reagent. In some embodiments, the affinity of the substance (e.g., competing reagent) for the at least one Z-binding site is greater than the affinity of the C-binding partner of the agent (e.g., receptor binding agent or selection agent) for the at least one Z-binding site. Thus, in some cases, the bond between the Z-binding site of the reagent and the C-binding partner of the agent (e.g., receptor binding agent or selection agent) can be disrupted by addition of the substance (e.g., competing reagent), thereby rendering the association of the agent (e.g., receptor binding agent or selection agent) and reagent reversible.

[00218] Reagents that can be used in such reversible systems are described and known in the art, see, e.g., U.S. Patent Nos. 5,168,049; 5,506,121; 6,103,493; 7,776,562; 7,981,632; 8,298,782; 8,735,540; 9,023,604; and International Published PCT Application Nos. WO2013/124474 and WO2014/076277. Non-limiting examples of reagents and binding partners capable of forming a reversible interaction, as well as substances (e.g., competing reagents) capable of reversing such binding, are described below.

A. Reagent

[00219] In some embodiments, the reagent contains one or a plurality of Z-binding sites that is capable of reversibly binding to C-binding partners comprised by the agent (e.g., receptor binding agent or selection agent). In some embodiments, the reagent contains a plurality of Z-binding sites, each of which is capable of specifically binding to C-binding partner that is included in the agent (e.g., receptor binding agent or selection agent), such that the reagent is capable of reversibly binding to a plurality of agents (e.g., receptor binding agent or selection agent), e.g., is a multimerization reagent. In some embodiments, the reagent is an oligomer or polymer of individual molecules (e.g., monomers) or complexes that make up an individual molecule (e.g., tetramer), each containing at least one Z-binding site. In some embodiments, the reagent contains at least two Z-binding sites, at least three Z-binding sites, at least four Z-binding sites, such as at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or more Z-binding sites. The binding sites may all be the same or the plurality of binding sites may contain one or more different binding sites. connection (e.g. Z1, Z2, Z3, etc.).

[00220] In some embodiments, two or more agents (e.g., receptor binding agents or selection agents) associate with, such as are reversibly bound to, the reagent, such as via one or a plurality of Z-binding sites present on the reagent. In some cases, this results in the agents (e.g., receptor binding agents or selection agents) being intimately disposed to each other such that an avidity effect can occur if a target cell having (at least two copies of) a cell surface molecule comes into contact with the agent (e.g., receptor binding agent or selection agent) that has one or more B-binding sites capable of binding to a particular molecule.

[00221] In some embodiments, two or more different agents (e.g., receptor binding agents or selection agents) that are the same, i.e., containing the same binding site B, can be reversibly linked to the reagent. In some embodiments, it is possible to use at least two different (types of) agents (e.g., receptor binding agents or selection agents), and in some cases, three or four different (types of) agents, e.g., two or more different receptor binding agents and/or selection agents. For example, in some embodiments, the reagent can be reversibly linked to a first agent (e.g., receptor binding agent or selection agent) containing a B1, B2, B3, or B4 binding site, etc. and a second agent (e.g., receptor binding agent or selection agent) containing another binding site, e.g., another of a B1, B2, B3, or B4 binding site. In some cases, the binding site of the first agent and the second agent can be the same. For example, in some aspects, each of at least two agents (e.g., receptor binding agent or selection agent) may bind to the same molecule. In some cases, the binding site of the first agent and the second agent may be different. In some aspects, each of at least two agents (e.g., receptor binding agent or selection agent) may bind to a different molecule, such as a first molecule, second molecule, and so on. In some cases, different molecules, such as cell surface molecules, may be present on the same target molecule. In other cases, different molecules, such as cell surface molecules, may be present on different target cells that are present in the same cell population. In some cases, a third, fourth agent, and so on (e.g., receptor binding agent or selection agent) may be associated with the same reagent, each containing a different binding site.

[00222] In some embodiments, the two or more different agents (e.g., receptor binding agents or selection agents) contain the same binding partner C. In some embodiments, the two or more different agents (e.g., receptor binding agents or selection agents) contain different binding partners. In some aspects, a first agent (e.g., receptor binding agent or selection agent) can have a binding partner C1 that can specifically bind to a binding site Z1 present on the reagent and a second agent (e.g., receptor binding agents or selection agent) can have a binding partner C2 that can specifically bind to the binding site Z1 or to a binding site Z2 present on the reagent. Thus, in some examples, the plurality of binding sites Z comprised by the reagent includes binding sites Z1 and Z2, wherein it is capable of reversibly binding to binding partners C1 and C2, respectively, comprised by the agent (e.g., receptor binding agent or selection agent). In some embodiments, C1 and C2 are the same, and/or Z1 and Z2 are the same. In other aspects, one or more of the plurality of Z binding sites may be different. In other examples, one or more of the plurality of C binding partners may be different. It is within the skill of a skilled artisan to choose any combination of different C binding partners that are compatible with a reagent containing the Z binding sites, so long as each of the C binding partners is capable of interacting, such as specifically binding, with one of the Z binding sites.

[00223] In some embodiments, the reagent is a streptavidin, a streptavidin mutein or analog, avidin, an avidin mutein or analog (such as neutravidin), or a mixture thereof, wherein such reagent contains one or more Z-binding sites for reversible association with a C-binding partner. In some embodiments, the C-binding partner can be a biotin, a biotin derivative or analog, or a streptavidin-binding peptide or other molecule that is capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin, or an avidin mutein or analog. In some embodiments, the reagent is or contains streptavidin, avidin, a streptavidin mutein or analog, or a mutein or analog, or avidin that reversibly binds biotin, a biotin analog, or a biologically active fragment thereof. In some embodiments, the reagent is or contains a streptavidin analog or mutein or an avidin mutein or analog that reversibly binds a streptavidin-binding peptide. In some embodiments, the substance (e.g., competitive reagent) can be a biotin, a biotin derivative or analog, or a streptavidin-binding peptide capable of competing for binding with the C-binding partner to the one or more Z-binding sites. In some embodiments, the C-binding partner and the substance (e.g., competitive reagent) are different, and the substance (e.g., competitive reagent) exhibits a higher binding affinity for the one or more Z-binding sites compared to the affinity of the binding partner.

[00224] In some embodiments, the streptavidin can be wild-type streptavidin, streptavidin muteins, or analogs, such as streptavidin-like polypeptides. Likewise, avidin, in some aspects, includes wild-type avidin or muteins or avidin analogs such as neutravidin, an avidin deglycosylated with modified arginines that typically exhibits a more neutral PI and is available as an alternative to native avidin. Generally, neutral, deglycosylated forms of avidin include those commercially available forms such as "Extravidin", available from Sigma Aldrich, or "NeutrAvidin" available from Thermo Scientific or Invitrogen, for example.

[00225] In some embodiments, the reagent is a streptavidin or a streptavidin mutein or analog. In some embodiments, wild-type streptavidin (streptavidin-wt) has the amino acid sequence disclosed by Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882 (SEQ ID NO: 1). In general, streptavidin naturally occurs as a tetramer of four identical subunits, i.e., it is a homo-tetramer, where each subunit contains a single binding site for biotin, a biotin derivative or analog, or a biotin mimic. An exemplary sequence of a streptavidin subunit is the amino acid sequence set forth in SEQ ID NO: 1, however, such a sequence may also include a sequence present in its homologues from other Streptomyces species. In particular, each streptavidin subunit can exhibit a strong binding affinity for biotin with an equilibrium dissociation constant (KD) on the order of about 10-14 M. In some cases, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463:1059-63)), a divalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. Biol., 426:199-214), or it can be present in either a monomeric or dimeric form (Wu et al. (2005) J. Biol. Chem., 280:23225-31; Lim et al. (2010) Biochemistry, 50:868291).

[00226] In some embodiments, the streptavidin can be in any form, such as wild-type or unmodified streptavidin, such as a streptavidin from a Streptomyces species or a functionally active fragment thereof that includes at least one functional subunit containing a binding site for biotin, a biotin derivative or analog, or a biotin mimetic, such as generally containing at least one functional subunit of a wild-type streptavidin from Streptomyces avidinii set forth in SEQ ID NO: 1 or a functionally active fragment thereof. For example, in some embodiments, the streptavidin can include a fragment of wild-type streptavidin, wherein it is shortened at the N- and/or C-terminus. Such minimal streptavidins include any that are N-terminally in the region of amino acid positions 10 to 16 of SEQ ID NO: 1 and C-terminally in the region of amino acid positions 133 to 142 of SEQ ID NO: 1. In some embodiments, a functionally active fragment of streptavidin contains the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, streptavidin as set forth in SEQ ID NO: 2 may also contain an N-terminal methionine at a position corresponding to Ala13 numbered in SEQ ID NO: 1. Reference to the position of residues in streptavidin or streptavidin muteins is with reference to the residue numbering in SEQ ID NO: 1.

[00227] In some aspects, streptavidin muteins include polypeptides that are distinguished from the sequence of an unmodified or wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but that include at least one functional subunit containing a binding site for biotin, a biotin derivative or analog, or a streptavidin-binding peptide. In some aspects, streptavidin-like polypeptides and streptavidin muteins can be polypeptides that are essentially immunologically equivalent to wild-type streptavidin and are in particular capable of binding biotin, biotin derivatives, or biotin analogs with the same or different affinity as wild-type streptavidin. In some cases, streptavidin-like polypeptides or streptavidin muteins can contain amino acids that are not part of wild-type streptavidin or can include only a portion of wild-type streptavidin. In some embodiments, streptavidin-like polypeptides are polypeptides that are not identical to wild-type streptavidin, since the host lacks the enzymes that are necessary to transform the host-produced polypeptide into the wild-type streptavidin structure. In some embodiments, streptavidin may also be present as streptavidin tetramers and streptavidin dimers, in particular streptavidin homotetramers, streptavidin homodimers, streptavidin heterotetramers, and streptavidin heterodimers. Generally, each subunit typically has a binding site for biotin or biotin analogs or for streptavidin-binding peptides. Examples of streptavidins or streptavidin muteins are mentioned, for example, in WO 86/02077, DE 19641876 Al, US 6,022,951, WO 98/40396 or WO 96/24606.

[00228] In some embodiments, a streptavidin mutein may contain amino acids that are not part of an unmodified or wild-type streptavidin or may include only a portion of an unmodified or wild-type streptavidin. In some embodiments, a streptavidin mutein contains at least one subunit that may have one or more additional amino acid substitutions (replacements) compared to a subunit of an unmodified or wild-type streptavidin, such as compared to the subunit of an unmodified or wild-type streptavidin set forth in SEQ. ID NO: 1 or a functionally active fragment thereof, e.g., set forth in SEQ ID NO: 2. In some embodiments, at least one subunit of a streptavidin mutein can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid differences compared to a wild-type or unmodified streptavidin and/or contains at least one subunit comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO: 1 or 2, wherein such streptavidin mutein exhibits functional activity to bind biotin, a biotin derivative or analog, or biotin mimetic. In some embodiments, the amino acid replacements (substitutions) are conservative or non-conservative mutations. Examples of streptavidin muteins are known in the art, see for example, U.S. Patent No. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; or 6,368,813; or International Published PCT Application No. WO2014/076277.

[00229] In some embodiments, streptavidin or a streptavidin mutein includes proteins containing one or more than one functional subunit containing one or more Z-binding sites for biotin, a biotin derivative or analog, or a streptavidin-binding peptide, such as two or more, three or more, four or more, and in some cases 5, 6, 7, 8, 9, 10, 11, 12 or more functional subunits. In some embodiments, streptavidin or a streptavidin mutein may include a monomer; a dimer, including a heterodimer or a homodimer; a tetramer, including a homotetramer, a heterotetramer, a monovalent tetramer, or a divalent tetramer; or may include higher ordered multimers or oligomers thereof.

[00230] In some embodiments, the binding affinity of streptavidin or a streptavidin mutein for a peptide ligand binding partner is less than 1 x 10-4 M, 5 x 10-4 M, 1 x 10-5 M, 5x 10-5 M, 1 x 10-6 M, 5 x 10-6 M, or 1 x 10-7 M, but generally greater than 1 x 10-13 M, 1 x 10-12 M, or 1 x 10-11 M. For example, peptide sequences (Streptavidin tags) such as those described in U.S. Patent No. 5,506,121 can act as biotin mimics and demonstrate a binding affinity for streptavidin, e.g., with a K of approximately between 10-4M and 10-5M. In some cases, binding affinity can also be improved by preparing a mutation within the streptavidin molecule, see, e.g., U.S. Patent No. 6,103,493 or International Published PCT Application No. WO2014/076277. In some embodiments, binding affinity can be determined by methods known in the art, such as any described below.

[00231] In some embodiments, the reagent, such as a streptavidin or streptavidin mutein, exhibits binding affinity for a peptide ligand binding partner, wherein the peptide ligand binding partner can be the binding partner C present in the agent (e.g., receptor binding agent or selection agent). In some embodiments, the peptide sequence contains a sequence with the general formula set forth in SEQ ID NO: 9, such as contains the sequence set forth in SEQ ID NO: 10. In some embodiments, the peptide sequence has the general formula set forth in SEQ ID NO: 11, such as set forth in SEQ ID NO: 12. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also referred to as Strep-tag®, set forth in SEQ ID NO:7). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II, set forth in SEQ ID NO:8). In some embodiments, the peptide linker contains a sequential arrangement of at least two streptavidin binding modules, wherein the distance between the two modules is at least 0 and no more than 50 amino acids, wherein one binding module is 3 to 8 amino acids long and contains at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine, or methionine, and wherein the other binding module has the same or a different streptavidin peptide linker as set forth in SEQ ID NO:11 (see, e.g., International Published PCT Application No. WO02/077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide linker contains a sequence having the formula set forth in any of SEQ ID NO: 13 or 14. In some embodiments, the peptide linker has the amino acid sequence set forth in any of SEQ ID NOS: 15-19.

[00232] In some embodiments, the reagent is or contains a streptavidin mutein. In some embodiments, the streptavidin muteins contain one or more mutations (e.g., amino acid substitutions) compared to the wild-type streptavidin set forth in SEQ ID NO: 1 or a biologically active portion thereof. For example, biologically active portions of streptavidin can include streptavidin variants that are shortened at the N- and/or C-terminus, which in some cases is referred to as a minimal streptavidin. In some embodiments, an N-terminally shortened minimal streptavidin, to which any of the mutations may be made, begins N-terminally in the region of amino acid positions 10 to 16 and ends C-terminally in the region of amino acid positions 133 to 142 as compared to the sequence set forth in SEQ ID NO: 1. In some embodiments, an N-terminally shortened streptavidin, to which any of the mutations may be made, contains the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the minimal streptavidin contains an amino acid sequence from position Ala13 to Ser139 and optionally has an N-terminal methionine residue instead of Ala13. For this purpose, the numbering of amino acid positions refers to the entire wild-type streptavidin numbering mentioned in SEQ ID NO: 1 (e.g., Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882, cf. also FIG. 3).

[00233] In some embodiments, the streptavidin mutein is a mutant as described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within the region of amino acid positions 44 to 53, based on the wild-type streptavidin amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains a mutation at one or more residues 44, 45, 46, and/or 47. In some embodiments, the streptavidin mutein contains a substitution of Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid, e.g., Val, Ala, Ile, or Leu, any amino acid at position 45, an aliphatic amino acid, such as a hydrophobic aliphatic amino acid at position 46, and/or a substitution of Val at position 47 with a basic amino acid, e.g., Arg or Lys, such as generally Arg. In some embodiments, Ala is at position 46 and/or Arg is at position 47 and/or Val or Ile is at position 44. In some embodiments, the streptavidin mutant contains residues Val44-Thr45-Ala46-Arg47, as set forth in exemplary streptavidin muteins containing the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 (also known as streptavidin mutant 1, SAM1). In some embodiments, the streptavidin mutein contains residues Ile44-Gly45-Ala46-Arg47, as set forth in exemplary streptavidin muteins containing the amino acid sequence set forth in SEQ ID NO: 5 or 6 (also known as SAM2). In some cases, such a streptavidin mutein is described, for example, in US Patent 6,103,493, and is commercially available under the tradename Strep-Tactin®.

[00234] In some embodiments, the streptavidin mutein is a mutant as described in International Published PCT Application Nos. WO 2014/076277. In some embodiments, the streptavidin mutein contains at least two cysteine residues in the region of amino acid positions 44 to 53 with reference to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, cysteine residues are present at positions 45 and 52 to create a disulfide bridge connecting these amino acids. In such an embodiment, amino acid 44 is typically glycine or alanine and amino acid 46 is typically alanine or glycine and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutein contains at least one mutation or amino acid difference in the region of amino acid residues 115 to 121 with reference to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains at least one mutation at amino acid position 117, 120, and 121 and/or a deletion of amino acids 118 and 119 and substitution of at least amino acid position 121.

[00235] In some embodiments, the streptavidin mutein contains a mutation at a position corresponding to position 117, wherein the mutation can be to a large hydrophobic residue like Trp, Tyr, or Phe, or a charged residue like Glu, Asp, or Arg, or a hydrophilic residue like Asn or Gin, or in some cases the hydrophobic residues Leu, Met, or Ala, or the polar residues Thr, Ser, or His. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120, wherein the mutation can be to a small residue like Ser or Ala or Gly, and a mutation at a position corresponding to position 121, wherein the mutation can be to a hydrophobic residue, such as a bulky hydrophobic residue like Trp, Tyr, or Phe. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 of the wild-type streptavidin set forth in SEQ ID NO: 1 or a biologically active fragment thereof, wherein the mutation may be a hydrophilic residue such as Leu, Ile, Met, or Val, or generally Tyr or Phe, and a mutation at a position corresponding to position 121 compared to the wild-type streptavidin positions set forth in SEQ ID NO: 1 or a biologically active fragment thereof, wherein the mutation may be to a small residue such as Gly, Ala, or Ser, or to Gln, or to a hydrophobic residue such as Leu, Val, Ile, Trp, Tyr, Phe, or Met. In some embodiments, these muteins may also contain residues Val44-Thr45-Ala46-Arg47 or residues Ile44-Gly45-Ala46-Arg47. In some embodiments, the streptavidin mutein contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121. In some embodiments, the mutein streptavidin contains the amino acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28, contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121, and exhibits functional activity to bind biotin, a biotin analog, or a streptavidin-binding peptide.

[00236] In some embodiments, a streptavidin mutein may contain any of the above mutations in any combination, and the resulting streptavidin mutein may exhibit a binding affinity that is less than 2.7 x 10-4 M for the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also called Strep-tag®, set forth in SEQ ID NO: 7) and/or less than 1.4 x 10-4 M for the peptide ligand (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also called Strep-tag® II, set forth in SEQ ID NO: 8) and/or is less than 1 x 10-4 M, 5 x 10-4 M, 1 x 10-5 M, 5x 10-5 M, 1 x 10-6 M, 5 x 10-6 M or 1 x 10-7 M, but generally greater than 1 x 10-13 M, 1 x 10-12 M or 1 x 10-11 M for any of the peptide ligands mentioned in any of SEQ ID NOS:7-19.

[00237] In some embodiments, the streptavidin mutein exhibits the amino acid sequence set forth in any of SEQ ID NOs: 3-6 27 or 28, or an amino acid sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in any of SEQ ID NO: 3-6, 27 or 28, and exhibits a binding affinity that is less than 2.7 x 10-4 M for the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also referred to as Strep-tag®, set forth in SEQ ID NO: 7) and/or less than 1.4 x 10-4 M for the peptide ligand (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also called Strep-tag® II, mentioned in SEQ ID NO: 8) and/or is less than 1 x 10-4 M, 5 x 104 M, 1 x 10-5 M, 5x 10-5 M, 1 x 10-6 M, 5 x 10-6 M or 1 x 10-7 M, but generally greater than 1 x 10-13 M, 1 x 10-12 M or 1 x 10-11 M for any of the peptide ligands mentioned in any of SEQ ID NOS:7-19.

[00238] In some embodiments, the streptavidin mutein also exhibits binding to other streptavidin ligands, such as but not limited to biotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA (hydroxyazobenzenebenzoic acid), and/or dimethyl-HABA. In some embodiments, the streptavidin mutein exhibits a binding affinity for another streptavidin ligand, such as biotin or desthiobiotin, that is greater than the binding affinity of the streptavidin mutein for a biotin mimetic peptide ligand as set forth in any of SEQ ID NOS: 7-19. Thus, in some embodiments, biotin or a biotin analog or derivative (e.g., desthiobiotin) can be employed as a competition reagent in the provided methods. For example, as an example, the interaction of a mutein streptavidin designated Strep-tactin® (e.g., containing the sequence set forth in SEQ ID NO: 4) with the peptide ligand designated Strep-tag® II (e.g., set forth in SEQ ID NO: 8) is characterized by a binding affinity with a KD of approximately 10-6 M compared to approximately 10-13 M for the biotin-streptavidin interaction. In some cases, biotin, which can bind with high affinity to Strep-tactin® with a KD of between or about 10-10 and 10-13 M, may compete with Strep-tag® II for the binding site.

[00239] In some cases, the reagent contains at least two K-chelating groups that may be capable of binding to a transition metal ion. In some embodiments, the reagent may be capable of binding to an oligohistidine affinity tag, a glutathione-S-transferase, calmodulin or an analog thereof, calmodulin-binding peptide (CBP), a FLAG peptide, an HA tag, maltose-binding protein (MBP), an HSV epitope, a myc epitope, and/or a biotinylated carrier protein.

[00240] In some embodiments, the reagent is an oligomer or polymer. In some embodiments, the oligomer or polymer can be generated by directly or indirectly binding individual molecules of the protein as it naturally exists, by directly or indirectly binding individual molecules of a monomer, or by a complex of subunits that make up an individual molecule (e.g., by directly or indirectly binding dimers, trimers, tetramers, etc. of a protein as it naturally exists). For example, a tetrameric homodimer or heterodimer of streptavidin or avidin can be referred to as an individual molecule or smaller building block of a respective oligomer or polymer. In some embodiments, the oligomer or polymer can contain linkage of at least 2 individual protein molecules (e.g., is a 2-mer), or can be at least one 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer, or 50-mer of individual protein molecules (e.g., monomers, tetramers).

[00241] The oligomers may be generated using any methods known in the art, such as any described in Published U.S. Patent Application No. US2004/0082012. In some embodiments, the oligomer or polymer contains two or more individual molecules that may be cross-linked, such as by a polysaccharide or a bifunctional linker.

[00242] In some embodiments, the oligomer or polymer is obtained by crosslinking individual molecules or a complex of subunits that constitute an individual molecule in the presence of a polysaccharide. In some embodiments, the oligomers or polymers can be prepared by introducing carboxyl residues into a polysaccharide, e.g., dextran. In some aspects, individual molecules of the reactant (e.g., monomers, tetramers) can be coupled via primary amino groups of internal lysine residues and/or the free N-terminus to the carboxyl groups on the dextran backbone using conventional carbodiimide chemistry. In some embodiments, the coupling reaction is carried out at a molar ratio of about 60 moles of individual molecules of the reactant (e.g., monomers, tetramers) per mole of dextran.

[00243] In some embodiments, the reagent is an oligomer or a polymer of one or more streptavidin or avidin or of any streptavidin analog or mutein or an avidin analog or mutein (e.g., neutravidin). In some embodiments, the Z-binding site is a natural biotin-binding site of avidin or streptavidin for which there may be up to four binding sites on an individual molecule (e.g., a tetramer contains four Z-binding sites), whereby a homo-tetramer may contain up to 4 binding sites that are the same, i.e., Z1, whereas a hetero-tetramer may contain up to 4 binding sites that may be different, e.g., containing Z1 and Z2. In some embodiments, the oligomer is generated or produced from a plurality of individual molecules (e.g., a plurality of homo-tetramers) of the same streptavidin, streptavidin mutein, avidin, or avidin mutein, in which case each Z-binding site, e.g., Z1, of the oligomer is the same. For example, in some cases, an oligomer may contain a plurality of Z1 binding sites, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more Z1 binding sites. In some embodiments, the oligomer is generated or produced from a plurality of individual molecules that may be heterotetrimers of a streptavidin, streptavidin mutein, avidin, or avidin mutein and/or from a plurality of two or more different individual molecules (e.g., different homo-tetramers) of streptavidin, streptavidin mutein, avidin, or avidin mutein that differ in their Z-binding sites, e.g., Z1 and Z2, in which case a plurality of different Z-binding sites, e.g., Z1 and Z2, may be present in the oligomer. For example, in some cases, an oligomer may contain a plurality of Z1 binding sites and a plurality of Z binding sites, which, in combination, may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more combined Z1 and Z2 binding sites.

[00244] In some cases, the respective oligomer or polymer may be cross-linked by a polysaccharide. In one embodiment, oligomers or polymers of streptavidin or avidin or streptavidin or avidin analogs (e.g., neutravidin) may be prepared by introducing carboxyl residues into a polysaccharide, e.g., dextran, essentially as described in Noguchi, A, et al., Bioconjugate Chemistry (1992) 3132-137 in a first step. In some such aspects, streptavidin or avidin or their analogs may then be linked via primary amino groups of internal lysine residues and/or the N-terminus to carboxyl groups on the dextran backbone using conventional carbodiimide chemistry in a second step. In some cases, cross-linked oligomers or polymers of streptavidin or avidin or any streptavidin or avidin analog can also be obtained by cross-linking bifunctional molecules serving as a ligand, such as glutaraldehyde, or by other methods described in the art.

[00245] In some embodiments, the oligomer or polymer is obtained by crosslinking individual molecules or a complex of subunits that constitute an individual molecule using a bifunctional ligand or other chemical linker, such as glutaraldehyde, or by other methods known in the art. In some aspects, crosslinked oligomers or polymers of streptavidin or avidin or any mutein or analog of streptavidin or avidin can be obtained by crosslinking individual streptavidin or avidin molecules through bifunctional molecules serving as a linker, such as glutaraldehyde, or by other methods described in the art. It is possible, for example, to generate oligomers of streptavidin muteins by introducing thiol groups into the streptavidin mutein (this can be done, for example, by reacting the streptavidin mutein with 2-iminothiolane (Trauts reagent) and activating, for example, in a separate reaction, amino groups available on the streptavidin mutein. In some embodiments, this activation of amino groups can be achieved by reacting the streptavidin mutein with a commercially available heterobifunctional crosslinker such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo SMCC) or Succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH). In some such embodiments, the two reaction products thus obtained are mixed, typically leading to the reaction of the thiol groups contained in a batch of modified streptavidin mutein with the activated amino acids (such as by maleimide functions) of the other batch of modified streptavidin mutein. In some cases, by this reaction, multimers/oligomers of streptavidin mutein are formed. These oligomers may have any suitable number of individual molecules, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more, and the degree of oligomerization may be varied according to the reaction condition.

[00246] In some embodiments, the oligomeric or polymeric reagent can be isolated by size exclusion chromatography and any desired fraction can be used as the reagent. For example, in some embodiments, after reacting the modified streptavidin mutein in the presence of 2-iminothiolane and a heterobifunctional crosslinker such as sulfo SMCC, the oligomeric or polymeric reagent can be isolated by size exclusion chromatography and any desired fraction can be used as the reagent. In some embodiments, the oligomers do not have (and need not have) a single molecular weight, but may instead observe a statistical weight distribution, such as a Gaussian distribution. In some cases, any oligomer with more than three streptavidin or mutein tetramers, e.g., homotetramers or heterotetramers, can be used as a soluble reagent, such as typically 3 to 50 tetramers, e.g., homotetramers or heterotetramers, 10 to 40 tetramers, e.g., homotetramers or heterotetramers, or 25 to 35 tetramers, e.g., homotetramers or heterotetramers. The oligomers can have, for example, 3 to 25 streptavidin mutein tetramers, e.g., homotetramers or heterotetramers. In some aspects, with a molecular weight of about 50 kDa for the streptavidin muteins, the soluble oligomers can have a molecular weight of about 150 kDa to about 2000 kDa, about 150 kDa to about 1500 kDa, about 150 kDa to about 1250 kDa, about 150 kDa to 1000 kDa, about 150 kDa to about 500 kDa, or about 150 kDa to about 300 kDa, about 300 kDa to about 2000 kDa, about 300 kDa to about 1500 kDa, about 300 kDa to about 1250 kDa, about 300 kDa to 1000 kDa, about 300 kDa to about 500 kDa, or about 150 kDa to about 300 kDa. kDa, about 500 kDa to about 2000 kDa, about 500 kDa to about 1500 kDa, about 500 kDa to about 1250 kDa, about 500 kDa to 1000 kDa, about 1000 kDa to about 2000 kDa, about 1000 kDa to about 1500 kDa, about 1000 kDa to about 1250 kDa, about 1250 kDa to about 2000 kDa or about 1500 kDa to about 2000 kDa. Generally, because each streptavidin/mutein molecule has four biotin binding sites, such a reagent can provide 12 to 160 Z-binding sites, such as 12 to 100 Z-binding sites.

B. Reagent Format 1. Support

[00247] In some embodiments, the reagent is comprised on a support, such as a solid support or surface, e.g., bead, or a stationary phase (chromatography matrix). In some such embodiments, the reagent is reversibly immobilized on the support. In some cases, the reagent is immobilized to the support via covalent bonds. In some aspects, the reagent is reversibly immobilized to the support non-covalently.

[00248] In some embodiments, the support is a solid support. Any solid support (surface) can be used for reversible immobilization of the reagent. Illustrative examples of solid supports on which the reagent can be immobilized include a magnetic bead, a polymeric bead, a cell culture plate, a microtiter plate, a membrane, or a hollow fiber. In some aspects, hollow fibers can be used as a bioreactor in the Quantum® Cell Expansion System, available from TerumoBCT Inc. (Lakewood, CO, USA). In some embodiments, the reagent is covalently attached to the solid support. In other embodiments, non-covalent interactions can likewise be used for immobilization, e.g., on plastic substrates. In some embodiments, the reagent can, for example, be a streptavidin or avidin mutein that reversibly binds to a streptavidin-binding peptide. Such streptavidin muteins can be covalently attached to any surface, e.g. resin (beads) used for chromatographic purification and are commercially available in such form from IBA GmbH, Gottingen, e.g. as Strep-Tactin® Sepharose, Strep-Tactin® Superflow®, Strep-Tactin® Superflow® High Capacity or Strep-Tactin® MacroPrep®. Other illustrative examples that are readily commercially available are immobilized metal affinity chromatography (IMAC) resins such as TALON® resins (Westburg, Leusden, Netherlands) that can be used for the reversible immobilization of oligohistidine-tagged (His-tagged) proteins, such as for the reversible binding of an agent (e.g., receptor binding agent or selection agent) that contains as a C-binding partner an oligohistidine tag, such as a penta- or hexahistidine tag. Other examples include calmodulin sepharose available from GE Life Sciences that can be used in conjunction with an agent (e.g., receptor binding agent or selection agent) that contains a calmodulin-binding peptide as a C-binding partner or sepharose to which glutathione is coupled. In some cases, the C-binding partner is glutathione-S-transferase.

[00249] In some embodiments, the support contains a stationary phase. Thus, in some embodiments, the reagent is comprised on a stationary phase (also called a chromatography matrix). In some such embodiments, the reagent is reversibly immobilized on the stationary phase. In some cases, the reagent is reversibly immobilized on the stationary phase via covalent bonds. In some aspects, the reagent is reversibly immobilized on the stationary phase non-covalently.

[00250] Any material may be employed as a chromatography matrix. In general, a suitable chromatography material is essentially innocuous, i.e., not detrimental to cell viability, such as when used in a chromatography column packed under desired conditions. In some embodiments, the stationary phase remains in a predefined location, such as a predefined position, while the location of the sample is being changed. Thus, in some embodiments, the stationary phase is the part of a chromatographic system through which the mobile phase flows (by flow or in a batch mode) and where the distribution of the components contained in the liquid phase (dissolved or dispersed) between the phases occurs.

[00251] In some embodiments, the chromatography matrix is in the form of a solid or semi-solid phase, while the sample containing the target cell to be isolated/separated is a fluid phase. The chromatography matrix can be a particulate material (of any suitable size and shape) or a monolithic chromatography material, including a paper substrate or membrane. Thus, in some aspects, the chromatography can be both column chromatography as well as planar chromatography. In some embodiments, in addition to standard chromatography columns, columns that allow for bidirectional flow such as PhyTip® columns available from PhyNexus, Inc. San Jose, CA, USA or pipette tips can be used for column-based/flow through mode-based methods. Thus, in some cases, pipette tips or columns that allow for bidirectional flow are also comprised of chromatography columns useful in the present methods. In some cases, such as where a particulate matrix material is used, the particulate matrix material may, for example, have an average particle size of from about 5 μm to about 200 μm, or from about 5 μm to about 400 μm, or 5 μm to about 600 μm. In some aspects, the chromatography matrix may, for example, be or include a polymeric resin or a metal oxide or a metalloid oxide. In some aspects, such as where planar chromatography is used, the matrix material may be any material suitable for planar chromatography, such as conventional cellulose or organic polymer-based membranes (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or silica-coated glass plates. In one embodiment, the chromatography matrix/stationary phase is a non-magnetic material or non-magnetizable material.

[00252] In some embodiments, non-magnetic or non-magnetizable chromatography stationary phases that are suitable in the present methods include derivatized silica or a cross-linked gel. In some aspects, a cross-linked gel may be based on a natural polymer, such as a naturally occurring class of polymers. For example, a natural polymer on which a chromatography stationary phase may be based is a polysaccharide. In some cases, a respective polysaccharide is generally cross-linked. An example of a polysaccharide matrix includes, but is not limited to, an agarose gel (e.g., Superflow™ agarose or a Sepharose® material such as Superflow™ Sepharose® which are commercially available in different bead and pore sizes) or a cross-linked Dextran(s) gel. Another illustrative example is a particulate cross-linked agarose matrix to which dextran is covalently attached, which is commercially available (in various bead sizes and with various pore sizes) as Sephadex® or Superdex®, both available from GE Healthcare. Another illustrative example of such a chromatography material is Sephacryl®, which is also available in different bead and pore sizes from GE Healthcare.

[00253] In some embodiments, a crosslinked gel may also be based on a synthetic polymer, such as a class of polymers that do not occur in nature. In some aspects, such a synthetic polymer, upon which a chromatography stationary phase is based, is a polymer that has polar monomer units and is therefore itself polar. Thus, in some cases, such a polar polymer is hydrophilic. Hydrophilic, also called lipophobic, molecules in some aspects contain moieties that can form dipole-dipole interactions with water molecules. In general, hydrophobic, also called lipophilic, molecules tend to separate from water.

[00254] Illustrative examples of suitable synthetic polymers are polyacrylamide(s), a styrene-divinylbenzene gel, and a copolymer of an acrylate and a diol or of an acrylamide and a diol. An illustrative example is a polymethacrylate gel, commercially available as a Fractogel®. Another example is a copolymer of ethylene glycol and methacrylate, commercially available as a Toyopearl®. In some embodiments, a chromatography stationary phase may also include natural and synthetic polymeric components, such as a composite matrix or a composite or a copolymer of a polysaccharide and agarose, e.g., a polyacrylamide/agarose composite, or of a polysaccharide and N,N'-methylenebisacrylamide. An illustrative example of a copolymer of a dextran and N,N'-methylenebisacrylamide is the aforementioned Sephacryl® series of materials. In some embodiments, a derivatized silica may include silica particles that are coupled to a synthetic polymer or a natural polymer. Examples of such embodiments include, but are not limited to, polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, and poly(N-isopropylacrylamide)-grafted silica.

[00255] In some embodiments, the chromatography matrix is a gel filtration matrix, for example when used in a removal cartridge as described herein. Generally, a gel filtration can be characterized by the property for which it is designed. Accordingly, a gel filtration matrix in some aspects allows for the separation of cells or other biological entities largely based on their size. In some such aspects, the respective chromatography matrix is typically a particulate porous material as mentioned above. The chromatography matrix may have a certain exclusion limit, which is typically defined in terms of a molecular weight above which molecules are entirely excluded from entering the pores. In some embodiments, the respective molecular weight defining the size exclusion limit may be selected to be below the weight corresponding to the weight of a target cell. In such an embodiment, the target cell is prevented from entering the pores of the size exclusion chromatography matrix. Similarly, a stationary phase may have pores that are smaller in size than the size of a chosen target cell. In illustrative embodiments, the chromatography matrix has an average pore size of 0 to about 500 nm.

[00256] In some embodiments, components present in a sample, such as agents (e.g., receptor binding agents or selection agents) or a competing reagent, may have a size that is below the exclusion limit of the pores and thus may enter the pores of the chromatography matrix. In some aspects, of such components that are able to partially or completely enter the pore volume, larger molecules, with less access to the pore volume, may elute first, while smaller molecules generally elute last. In some embodiments, the exclusion limit of the chromatography matrix is selected to be below the maximum width of the target cell. Thus, in some aspects, components that have access to the pore volume may remain longer in/on the chromatography matrix than on the target cell. Thus, in some cases, target cells may be collected in the eluate of a chromatography column separately from other materials/components of a sample. Therefore, in some aspects, components such as an agent (e.g., receptor binding agent or selection agent), or where applicable a competing reagent, may elute at a later point in time from a gel filtration matrix than the target cell. In some embodiments, this effect may be further enhanced, such as if the gel permeation matrix contains a reagent (such as covalently bound thereto) that contains Z-binding sites that are capable of binding agents (e.g., receptor binding agents or selection agents) and/or a competing reagent present in a sample. In some cases, the agent (e.g., receptor binding agent or selection agent) and/or the competing reagent may be bound by the Z-binding sites of the reagent and thereby immobilized on the matrix. In some aspects, this method is performed in a stripping cartridge.

[00257] In some embodiments, a chromatography matrix employed in the present methods may also include magnetically attractive matter, such as one or more magnetically attracted particles or a ferrofluid. A respective magnetically attractive particle may comprise a reagent with a binding site that is capable of binding to a target cell. In some cases, magnetically attractive particles may contain diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic material. In general, superparamagnetic material responds to a magnetic field with an induced magnetic field without a resulting permanent magnetization. Iron oxide-based magnetic particles are, for example, commercially available as Dynabeads from Dynal Biotech, as Magnetic MicroBeads from Miltenyi Biotec, as Magnetic Porous Glass Beads from CPG Inc., as well as from various other sources, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences or Novagen Inc., to name but a few. Magnetic nanoparticles based on superparamagnetic Co and FeCo, as well as ferromagnetic Co nanocrystals, have been described, for example, by Hutten, A. et al. (J. Biotech. (2004), 112, 47-63). In other embodiments, a chromatography matrix employed in the present methods is void of any magnetically attractive matter.

[00258] In some embodiments, provided is an apparatus that contains at least one arrangement of a first and a second stationary phase, such as a chromatography column for cell selection (a selection cartridge) and a second chromatography column (a stripping cartridge) for reagent removal. The apparatus may comprise a plurality of arrangements of first and second stationary phases (chromatography columns) being fluidly connected in series. The apparatus may comprise a sample inlet that is fluidly connected to the first stationary phase of the first arrangement of first and second stationary phases. In some embodiments, the apparatus may also comprise a sample outlet for cells, the sample outlet being fluidly connected to the second stationary phase of the last of the at least one arrangement of a first and second stationary phases for chromatography. In some aspects, the apparatus may also comprise a competition reagent container that is fluidly connected to at least one of the first stationary phases of the arrangements of first and second stationary phases.

2. Soluble

[00259] In some embodiments, the reagent is not bound to a solid support, i.e., is present in soluble form or is soluble. In principle, the same reagent can be used as in the case of a reagent that is immobilized on a support, such as a solid support or stationary phase. For example, any of the examples of reagents described above can be used without immobilizing or binding such reagent to a support, e.g., not binding the solid support or stationary phase. In some embodiments, the reagent contains a plurality of binding sites, Z, for reversibly binding to a binding agent through interaction with a binding partner, C. In some cases, the reagent is an oligomer or polymer of individual molecules or an oligomer or polymer of a complex of subunits that make up the individual molecule (e.g., oligomers or polymers of a dimeric, trimeric, or tetrameric protein). In some embodiments, the reagent can, for example, be a streptavidin mutein oligomer, a calmodulin oligomer, a compound (oligomer) that provides at least two K-chelating groups, wherein at least two chelating groups are capable of binding to a transition metal ion, thereby rendering the reagent capable of binding to an oligo-histidine affinity tag, multimeric glutathione-S-transferase, or a biotinylated carrier protein.

[00260] In some embodiments, the reagent is characterized by the absence of a solid support (surface) attached to the reagent. For example, in some embodiments, the reagent does not comprise or is not attached (directly or indirectly) to a particle, bead, nanoparticle, microsphere, or other solid support. In some embodiments, the reagent is not rigid, inflexible, or stiff or does not comprise or is not attached to a rigid, inflexible, or hard surface. In some embodiments, the reagent is flexible or substantially flexible. In some cases, the reagent is capable of conforming or adapting to the shape of the surface of cells. In some embodiments, the reagent does not include or does not comprise a shape that is spherical or substantially spherical.

[00261] In some embodiments, substantially all, i.e., greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the reagent is composed of or contains organic material. For example, in some embodiments, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the reagent is composed of or contains lipids, carbohydrates, proteins, peptides, or mixtures thereof. In some embodiments, the reagent is composed of or contains an essential absence of inorganic material, an inorganic core, e.g., metal, e.g., iron, synthetic or inorganic polymers such as styrene polymers, e.g., polystyrene, latex, silica, or magnetic cores. For example, in some embodiments, the relative percentage of inorganic material in the reagent or comprised as part of the reagent is less than 20%, 15%, 10%, 5%, or less.

[00262] In some embodiments, the majority (i.e., greater than 50%), such as greater than 60%, 70%, 80%, 90%, 95%, 99%, or more of the total volume of the reagent in aqueous solution consists of the individual protein molecules that make up the reagent, such as oligomers or polymers of individual molecules, or a complex of subunits that make up an individual molecule (e.g., tetrameric molecule). In some embodiments, the total density of the soluble reagent is less than 1.2 g/cm3, 1.1 g/cm3, 1.0 g/cm3, or less.

[00263] In some embodiments, the soluble reagent, e.g., not being bound to a support or solid support (e.g., not being bound to a bead), has a relatively small size, such as generally less than or about less than 20 nM in size, such as less than or about less than 15 nM, less than or about less than 10 nM, less than or about less than 5 nM or smaller.

[00264] In some embodiments, the soluble reagent, e.g., not being bound to a support or solid support (e.g., not being bound to a bead), is biologically inert, i.e., is non-toxic to living cells. In some embodiments, the reagent may be biodegradable, e.g., may be degraded by enzymatic activity or eliminated by phagocytic cells.

[00265] In some embodiments, it is possible to react the reagent (e.g., a streptavidin or mutein, such as tetrameric streptavidin muteins) with a carrier, such as an organic carrier. In some aspects, in addition to a reaction with a polysaccharide, it is also possible to use physiologically or pharmaceutically acceptable proteins, such as serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)) as the carrier protein. In such an instance, the reagent, such as streptavidin or a streptavidin mutein (as an individual tetramer or also in the form of oligomers), can be coupled to the carrier protein via non-covalent interaction. In some such embodiments, biotinylated BSA (which is commercially available from various suppliers, such as ThermoFisher Scientific, Sigma Aldrich, or Vectorlabs, to name but a few) can be reacted with the reagent (e.g., streptavidin mutein). In some aspects, some of the reagent oligomers (e.g., streptavidin oligomers) can non-covalently bind via one or more Z-binding sites to the biotinylated carrier protein, leaving the majority of the oligomer's Z-binding sites available for binding to the agent (e.g., receptor binding agent or selection agent) and any other agent as described herein. Thus, by such an approach, a soluble reagent with a multiplicity of Z-binding sites can be prepared.

[00266] In other embodiments, a reagent, such as a streptavidin mutein (as an individual tetramer or also in the form of an oligomer), can be covalently coupled to a synthetic carrier, such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used for this purpose, for example, and the PEG molecule and the respective reagent can be soluble. Typically, PEG molecules up to a molecular weight of 1000 Da are soluble in water or culture media that can be used in the present methods. In some cases, such a PEG-based reagent can be prepared using commercially available activated PEG molecules (e.g., PEG-NHS derivatives available from NOF North America Corporation, Irvine, California, USA, or activated PEG derivatives available from Creative PEGWorks, Chapel Hills, North Carolina, USA) with streptavidin mutein amino groups.

C. Agents

[00267] In some embodiments, the agent (e.g., receptor binding agent or selection agent) has one or more binding sites, B, for binding to the molecule on the cell surface, e.g., cell surface molecule. Thus, in some cases, the agent (e.g., receptor binding agent or selection agent) contains a binding site B or a plurality of binding sites B, wherein the specific binding between the agent (receptor binding agent or selection agent) and the molecule on the surface of the target cells comprises interaction between B and the molecule. In some embodiments, the agent contains only a single binding site, i.e. monovalent. In some embodiments, the agent (e.g., receptor binding agent or selection agent) has at least two, such as a plurality of binding sites B including three, four, or five binding sites B capable of binding to the cell surface molecule. In some such aspects, the at least two or a plurality of binding sites B may be identical. In some embodiments, one or more of the at least two or a plurality of B binding sites may be different (e.g., B1 and B2).

[00268] In some embodiments, one or more different agents (e.g., one or more receptor binding agents, selection agent, or other different agent that binds to a molecule in a cell) are reversibly bound to the reagent. In some embodiments, at least 2, 3, 4, or more different agents are reversibly bound to the same reagent. In some embodiments, at least two different agents are reversibly bound to the same reagent, whereby each reagent comprises a binding site B or a plurality of binding sites B for specific binding between the agent and the molecule. In some embodiments, the at least two or more agents contain the same binding site B, e.g., for binding to the same or substantially the same molecule. In some embodiments, the at least two or more agents contain different binding sites B, e.g., for binding to different molecules. In some embodiments, a first agent (e.g., a first receptor binding agent or a first selection agent) contains a B1, B2, B3, B4, etc. binding site and a second agent (e.g., a second receptor binding agent or second agent) contains another of a B1, B2, B3, B4, etc. binding site. In some embodiments, a first agent (e.g., a first selection agent) contains a B1 binding site and a second agent (e.g., a second selection agent) contains a B3 binding site. In some embodiments, a first agent (e.g., a first receptor binding agent) contains a B2 binding site and a second agent (e.g., a second receptor binding agent) contains a B4 binding site. In any such embodiments, the first agent and the second agent can contain a binding partner, C1 or C2. In some embodiments, C1 and C2 can be the same. In some embodiments, C1 and C2 are different. In some embodiments, the first agent and the second agent contain the same binding partner, C1.

[00269] In some cases, the dissociation constant (K) of the binding between the reagent (e.g., via binding site B) and binding site Z of the reagent can have a value in the range of from about 10-2 M to about 10-13 M, or from about 10-3 M to about 10-12 M, or from about 10-4 M to about 10-11 M, or from about 10-5 M to about 10-10 M. In some embodiments, the dissociation constant (K) for the binding between the binding agent and the molecule is low affinity, e.g., in the range of a K of from about 10-3 to about 10-7 M. In some embodiments, the dissociation constant (K) for the binding between the binding agent and the molecule is high affinity, e.g., in the range of a K of from about 10-7 to about 10-12 M. 1 x io-10 mM.

[00270] In some embodiments, dissociation of the binding of the agent via binding site B and the molecule occurs sufficiently rapidly, for example, to allow the target cell to be only transiently labeled or associated with the agent after the reversible binding between the reagent and the agent has been broken. In some cases, when expressed in terms of the k off rate (also called the dissociation rate constant for the binding between the agent (via binding site B) and the molecule, the k off rate is about 0.5 x 10-4 sec-1 or greater, about 1 x 10-4 sec-1 or greater, about 2 x 10-4 sec-1 or greater, about 3 x 10-4 sec-1 or greater, about 4 x 10-4 sec-1 or greater, about 5 x 10-4 sec-1 or greater, about 1 x 10-3 sec-1 or greater, about 1.5 x 10-3 sec-1 or greater, about 2 x 10-3 sec-1 or greater, about 3 x 10-3 sec-1 or greater, about 4 x 10-3 sec-1, about 5 x 10-3 sec-1 or greater, about 1 x 10-2 sec-1 or greater, or about 5 x 10-1 sec-1 or greater. It is within the skill of the artisan to empirically determine the appropriate k off rate range for a particular agent and cellular molecule interaction (see, e.g., published US application US2014/0295458). For example, an agent with a very high k off rate of, e.g., greater than 4.0 x 10-4 sec-1 may be used such that, upon disruption of the binding complexes, most of the agent can be removed or dissociated within one hour. In other cases, an agent with a lower k off rate of, e.g., 1.0 x 10-4 sec-1, may be used such that upon disruption of the binding complexes, most of the agent can be removed or dissociated from the cell within about 3 1/2 hours.

[00271] In some embodiments, the K of this binding, as well as the rate K, KA, koff, and kon of the binding formed between the binding site B of the agent (e.g., receptor binding agent or selection agent) and the cell surface molecule can be determined by any suitable means, for example, by fluorescence titration, equilibrium dialysis, or surface plasmon resonance.

[00272] In some aspects, the cell surface molecule is a molecule against which an agent, such as a binding agent (e.g., receptor binding agent or selection agent) can be directed. In some embodiments, the cell surface molecule is a peptide or a protein, such as a receptor, e.g., a membrane receptor protein. In some embodiments, the receptor is a lipid, a polysaccharide, or a nucleic acid. In some embodiments, the cell surface molecule that is a protein can be a peripheral membrane protein or an integral membrane protein. The cell surface molecule can, in some embodiments, have one or more membrane-spanning domains. As some illustrative examples, a membrane protein with a transmembrane domain may be a G-protein-coupled receptor such as an olfactory receptor, a rhodopsin receptor, a rhodopsin pheromone receptor, a peptide hormone receptor, a taste receptor, a GABA receptor, an opiate receptor, a serotonin receptor, a Ca2+ receptor, melanopsin, a neurotransmitter receptor such as a ligand-activated, voltage-activated or mechanically activated receptor including the neuropeptide receptor for acetylcholine, nicotinic, adrenergic, norepinephrine, catecholamine, L-DOPA, dopamine and serotonin (biogenic amine, endorphin/enkephalin), a receptor kinase such as a serine/threonine kinase, a tyrosine kinase, a porin/channel such as a chloride channel, a potassium channel, a sodium channel, an OMP protein, a phospholipid transporter, and so on. ABC (ATP-Binding Cassette-Transporter) such as an amino acid transporter, the Na-glucose transporter, the Na/iodide transporter, an ion transporter such as the Light Harvesting complex, cytochrome c oxidase, Na/K, H/K, Ca ATPase, a cell adhesion receptor such as a metalloprotease, an integrin or a cadherin.

[00273] In some embodiments, the cell surface molecule can be an antigen that defines a desired population or subpopulation of cells, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, helper T cells, e.g., CD4+ helper T cells, B cells, or natural killer cells), monocytes, or stem cells, e.g., CD34-positive peripheral stem cells or stem cells expressing Nanog or Oct-4. Examples of T cells include cells such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Treg). An illustrative example of Treg is CD4CD25CD45RA Treg cells, and an illustrative example of memory T cells is CD62LCD8+-specific central memory T cells. The cell surface molecule can also be a marker for a tumor cell.

[00274] As described above, in some embodiments, the agent, such as a binding agent (e.g., receptor binding agent or selection agent) has, in addition to the binding site B that is capable of binding to the cell surface molecule, a binding partner C. In some aspects, this binding partner C is capable of binding to a binding site Z of the reagent, wherein the reagent has one or more binding sites for the binding partner C. In some embodiments, the non-covalent bond can be formed between the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) and the reagent binding site(s) Z of the reagent can be of any desired strength and affinity, and can be removable or reversible under the conditions under which the method is performed. The agent (e.g., receptor binding agent or selection agent) may include at least one, including two, three or more additional binding partners C and the reagent may include at least two, such as three, four, five, six, seven, eight or more binding sites Z for the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent). As described in U.S. Patent 7,776,562, U.S. Patent 8,298,782 or International Patent Application Publication No. WO 2002/054065, any combination of a binding partner C and a reagent having one or more corresponding binding sites Z may be chosen, for example, such that the binding partner C and the binding site Z are capable of binding reversibly in complex, such as to cause an avidity effect.

[00275] The binding partner C included in the agent (e.g., receptor binding agent or selecting agent) may, for example, be hydrocarbon-based (including polymeric) and include nitrogen, phosphorus, sulfur, carben, halogen, or pseudohalogen groups. In some aspects, it may be an alcohol, an organic acid, an inorganic acid, an amine, a phosphine, a thiol, a disulfide, an alkane, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a saccharide, an oligosaccharide, or a polysaccharide. As additional examples, it may also be a cation, an anion, a polycation, a polyanion, a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube, or carbon nanofoam. Generally, such a binding partner C has a higher affinity for the binding site of the reagent than for other matter. Examples of a respective binding partner C include, but are not limited to, a crown ether, an immunoglobulin, a fragment thereof, and a protein binding molecule with antibody-like functions.

[00276] In some embodiments, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) includes biotin and the reagent includes a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) includes a biotin analog that reversibly binds to streptavidin or avidin, and the reagent includes streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective biotin analog. In some embodiments, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) includes a streptavidin or avidin-binding peptide and the reagent includes streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective streptavidin- or avidin-binding peptide.

[00277] In some embodiments, the reagent is a streptavidin, such as a streptavidin mutein including any described above (e.g., set forth in SEQ ID NOS: 3-6), and the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) may include a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide may include a sequence having the general formula set forth in SEQ ID NO: 9, such as containing the sequence set forth in SEQ ID NO: 10. In some embodiments, the peptide sequence has the general formula set forth in SEQ ID NO: 11, such as set forth in SEQ ID NO: 12. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also referred to as Strep-tag®, set forth in SEQ ID NO: 7). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also referred to as Strep-tag® II, set forth in SEQ ID NO: 8). In some embodiments, the peptide linker contains a sequential arrangement of at least two streptavidin binding modules, wherein the distance between the two modules is at least 0 and no more than 50 amino acids, wherein one binding module is 3 to 8 amino acids long and contains at least the sequence His-Pro-Xaa (SEQ ID NO: 9), wherein Xaa is glutamine, asparagine, or methionine, and wherein the other binding module has the same or a different streptavidin peptide linker as set forth in SEQ ID NO: 11 (see, e.g., International Published PCT Application No. WO02/077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide linker contains a sequence having the formula set forth in any of SEQ ID NO: 13 or 14. In some embodiments, the peptide linker has the amino acid sequence set forth in any of SEQ ID NOS: 15-19. In most cases, all of these streptavidin-binding peptides bind to the same binding site, namely the biotin-binding site of streptavidin. If one or more of these streptavidin-binding peptides are used as binding partners C, e.g., C1 and C2, the multimerization reagent is typically a streptavidin mutein.

[00278] In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide may include the peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also referred to as Strep-tag® II, set forth in SEQ ID NO: 8) conjugated to a nickel-charged trisNTA (also referred to as His-STREPPER or His/Strep-tag®II Adapter).

[00279] In some embodiments, the binding partner C of the agent (e.g., receptor binding agent or selection agent) includes a moiety known to the skilled artisan as an affinity tag. In such an embodiment, the reagent can include a corresponding binding partner, e.g., an antibody or antibody fragment, known to bind to the affinity tag. As some illustrative examples of known affinity tags, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) can include dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG peptide, HA tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) (SEQ ID NO: 20), the VSV-G tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys) (SEQ ID NO: 21), the HSV tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro- Glu-Asp) (SEQ ID NO: 22), the T7 epitope (Ala-Ser-Met-Thr- Gly-Gly- Gln-Gln-Met-Gly) (SEQ ID NO: 22), maltose binding protein (MBP), the HSV epitope of the herpes simplex virus D glycoprotein sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 24), the c-myc transcription factor "myc" epitope of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 25), the V5 tag (sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) (SEQ ID NO: 26) or glutathione-S-transferase (GST). In such embodiments, the complex formed between the one or more Z-binding sites of the reagent, which can be an antibody or antibody fragment, and the antigen can be competitively disrupted by adding free antigen, i.e., free peptide (epitope tag) or free protein (such as MBP or CBP). In some embodiments, the affinity tag can also be an oligonucleotide tag. In some cases, such an oligonucleotide tag can, for example, be used to hybridize to an oligonucleotide having a complementary sequence, linked to or included in the reagent.

[00280] Other examples of a suitable C-binding partner include, but are not limited to, a lectin, protein A, protein G, a metal, a metal ion, nitrile triacetic acid (NT A) derivatives, RGD motifs, a dextran, polyethyleneimine (PEI), a redox polymer, a glycoprotein, an aptamer, a dye, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, poly U, or oligo-dT. Lectins such as Concavalin A are known to bind to glycosylated polysaccharides and proteins. An illustrative example of a dye is a triazine dye, such as Cibacron blue F3G-A (CB) or red HE-3B, which specifically bind to NADH-dependent enzymes. Typically, Green A binds to Co A proteins, human serum albumin, and dehydrogenases. In some cases, the dyes 7-aminoactinomycin D and 4´,6-diamidino-2-phenylindole bind to DNA. In general, metal cations such as Ni, Cd, Zn, Co, or Cu are typically used to bind affinity tags, such as an oligohistidine-containing sequence, including hexahistidine or the His-Asn-His-Arg-His-Lys-His tag. -Gly-Gly-Gly-Cys (MAT tag) (SEQ ID NO: 35) and N-methacryloyl-(L)-cysteine methyl ester.

[00281] In some embodiments, the binding between the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) and the one or more binding sites Z of the reagent occurs in the presence of a divalent, trivalent, or tetravalent cation. In this regard, in some embodiments, the reagent includes a divalent, trivalent, or tetravalent cation, typically retained, e.g., complexed, by means of a suitable chelator. In some embodiments, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) can include a moiety that includes, e.g., complexes, a divalent, trivalent, or tetravalent cation. Examples of a respective metal chelator include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimer-capto-1-propanol (dimercaprol), porphine, and heme. As an example, EDTA forms a complex with most monovalent, divalent, trivalent, and tetravalent metal ions, such as silver (Ag+), calcium (Ca2+), manganese (Mn2+), copper (Cu2+), iron (Fe2+), cobalt (Co+), and zirconium (Zr4+), while BAPTA is specific for Ca2+. As an illustrative example, a standard method used in the art is the formation of a complex between an oligohistidine tag and copper (Cu2+), nickel (Ni2+), cobalt (Co2+), or zinc (Zn2+) ions, which are presented by means of the chelator nitrilotriacetic acid (NTA).

[00282] In some embodiments, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) includes a calmodulin binding peptide and the reagent includes multimeric calmodulin as described in U.S. Patent 5,985,658 , for example. In some embodiments, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) includes a FLAG peptide and the reagent includes an antibody that binds to the FLAG peptide, for example, the FLAG peptide that binds to the monoclonal antibody 4E11 as described in U.S. Patent 4,851,341 . In one embodiment, the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) includes an oligohistidine tag and the reagent includes an antibody or a transition metal ion that binds the oligohistidine tag. In some cases, disruption of all of these binding complexes can be achieved by chelation of metal ions, e.g., chelation of calcium, e.g., by adding EDTA or EGTA. In some embodiments, calmodulin, antibodies such as 4E11, or chelated metal ions or free chelators can be multimerized by conventional means, e.g., by biotinylation and complexation with streptavidin or avidin or their oligomers or by introducing carboxyl residues into polysaccharide, e.g., dextran, essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137 in a first step and attaching calmodulin or antibodies or chelated metal ions or free chelators via primary amino groups to carboxyl groups on the polysaccharide, e.g., dextran, backbone using conventional carbodiimide chemistry in a second step. In some such embodiments, the bond between the binding partner C that is included in the agent (e.g., receptor binding agent or selection agent) and the one or more binding sites Z of the reagent can be disrupted by metal ion chelation. Metal chelation can, for example, be accomplished by addition of EGTA or EDTA.

[00283] In some embodiments, the agent (e.g., receptor binding agent or selection agent) that specifically binds to the cell surface molecule can, for example, be comprised of an antibody, a fragment thereof, or a proteinaceous binding molecule with antibody-like functions. In some embodiments, the binding site B of the agent is an antibody combining site, such as is or contains one or more complementarity determining regions (CDRs) of an antibody. Examples of (recombinant) antibody fragments include, but are not limited to, Fab fragments, Fv fragments, single chain Fv fragments (scFv), a divalent antibody fragment such as a F(ab')2F(ab')2 fragment, diabodies, triabodies (Iliades, P., et al., FEB S Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L. J., et al., Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, the agent (e.g., receptor binding agent or selection agent) may comprise an artificial bivalent proteinaceous binding molecule, such as a dimeric lipocalin mutein which is also known as "duocalin."

[00284] In some embodiments, the agent (e.g., receptor binding agent or selection agent) may have a single binding site B, i.e., may be monovalent. Examples of monovalent agents (e.g., receptor binding agents or selection agents) include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to, a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv), including a divalent single-chain Fv fragment.

[00285] In some embodiments, the agent is an antibody or an antigen-binding fragment thereof, such as Fab fragments, Fv fragments, single-chain Fv fragments (scFv), a divalent antibody fragment such as an F(ab')2 fragment. In some embodiments, the agent is or is derived from a parent antibody that is known to bind to a cell molecule of interest. Various antibody molecules or fragments thereof against cell surface molecules are well known in the art and any of a variety of these can be used as agents in the methods described herein. In some embodiments, the agent is an antibody or fragment thereof that contains one or more amino acid substitutions in the variable heavy chain of a parent or reference antibody, e.g., to generate an antibody with an altered affinity or that exhibits a sufficiently fast off rate as described above. For example, examples of such mutations are known in the context of mutants of the anti-CD4 antibody 13B8.2 (see, e.g., U.S. Patent No. 7,482,000, U.S. Patent Application Publication No. US2014/0295458, or International Patent Application Publication No. WO2013/124474), and any of these mutations may be generated in another parental or reference antibody.

[00286] In some aspects, the agent (e.g., receptor binding agent or selection agent) which may be monovalent, for example, comprises a monovalent antibody fragment or a monovalent artificial binding molecule (proteinaceous or otherwise) such as a lipocalin family polypeptide-based mutein (also known as "Anticalin®"), or a bivalent molecule such as an antibody or a fragment in which both binding sites are retained, such as an F(ab')2 fragment.

[00287] An example of a proteinaceous binding molecule with antibody-like functions includes a polypeptide-based mutein of the lipocalin family (see, e.g., WO 03/029462, Beste et al., Proc. Natl. Acad. Sci. USA (1999) 96, 1898-1903). Generally, lipocalins, such as bilin-binding protein, human neutrophil gelatinase-associated lipocalin, human Apo lipoprotein D, or human tear lipocalin, have natural ligand-binding sites that can be engineered to bind to a particular target. Other examples of a proteinaceous binding molecule with antibody-like binding properties that can be used as an agent (e.g., receptor binding agent or selecting agent) that specifically binds to the cell surface molecule include, but are not limited to, so-called glucobodies (see, e.g., International Patent Application Publication No. WO 96/23879), proteins based on ankyrin structure (Mosavi, L.K., et al., Protein Science (2004) 13, 6, 1435-1448) or crystal structure (e.g., International Patent Application Publication No. WO 01/04144), the proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, AdNectins, tetranectins, and avimers. Generally, avimers, including multivalent avimer proteins developed by exon shuffling of a family of human receptor domains, contain so-called A domains that occur as multidomain chains in several cell surface receptors (Silverman, J., et al., Nature Biotechnology (2005) 23, 1556-1561). Adnectins, usually derived from a human fibronectin domain, typically contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins, usually derived from the respective human homotrimeric protein, also typically contain the loop regions in a C-type lectin domain that can be engineered for desired binding. Peptoids, which can in some cases act as protein ligands, are typically oligo(N-alkyl)glycines that differ from peptides in that the side chain is attached to the amide nitrogen rather than the carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have much greater cellular permeability than peptides (see, e.g., Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).

[00288] Other examples of suitable proteinaceous binding molecules include, but are not limited to, an EGF-like domain, a Kringle domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gla domain, an SRCR domain, a Kunitz/Bovine Pancreatic Trypsin Inhibitor domain, Tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand C-type domain, an Amphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, an LDL receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an immunoglobulin domain or an immunoglobulin-like domain (e.g., camel heavy chain domain or antibodies), a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP-type four-disulfide core domain, a C-type F5/8 domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-like EGF domain, a C2 domain, "Kappabodies" (Ill et al. Protein Eng (1997) 10, 949-57, a so-called "minibody" (Martin et al., EMBO J (1994) 13, 5303-5309), a diabody (Holliger et al., PNAS USA (1993) 90, 6444-6448), a so-called "Janusis" (Traunecker et al., EMBO J (1991) 10, 3655-3659, or Traunecker et al., Int J Cancer (1992) Suppl 7, 51-52), a nanobody, a microbody, an affilin, an affibody, a knottin, ubiquitin, a zinc-binding protein, an autofluorescent protein, or a leucine-rich repeat protein. In some embodiments, a nucleic acid molecule with antibody-like functions can be an aptamer. Generally, an aptamer folds into a defined three-dimensional motif and shows high affinity for a given target structure.

1. Receptor Binding Agents

[00289] In some embodiments, the agent is a receptor binding agent. In some embodiments, the receptor binding agent binds to a molecule (e.g., receptor) on the surface of a cell, which binding between the agent and the molecule is capable of inducing or modulating a signal in the cells. In some cases, the cell surface molecule (e.g., receptor) is a signaling molecule. In some such cases, the receptor binding agent is capable of specifically binding to a signaling molecule expressed by one or more of the cells. In some cases, the receptor binding agent is a stimulatory agent, which can be any agent that is capable of inducing a signal in a cell (e.g., a T cell) by binding to a cell surface molecule, such as a receptor.

[00290] In some embodiments, the signal may be immunostimulatory, in which case the receptor binding agent or stimulatory agent is capable of inducing or modulating a signal that is engaged in or that stimulates an immune response by the cell (e.g., T cell), e.g., increasing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell. In some embodiments, the signal may be inhibitory, in which case the receptor binding agent or stimulatory agent is capable of inducing or modulating a signal in the cell (e.g., T cell) that is engaged in or that inhibits an immune response, e.g., inhibiting or decreasing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell. In some embodiments, the receptor binding agent, e.g., the stimulatory agent, is a receptor agonist, a receptor antagonist, or a mixed receptor agonist/antagonist.

[00291] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is a first receptor binding agent, e.g., first stimulatory agent. In some aspects, the first receptor binding agent, e.g., first stimulatory agent, binds to a first molecule, such as a receptor molecule, on the surface of the cells. Thus, in some instances, the first receptor binding agent, e.g., first stimulatory agent, induces or modulates a signal. In some aspects, the induction or modulation of a signal by the first receptor binding agent, e.g., first stimulatory agent, affects the activation, stimulation, and/or expansion (proliferation) of the cells. Thus, in some instances, the first receptor binding agent, e.g., first stimulatory agent, provides a primary activation signal to the cells, thereby activating the cells. In some embodiments, the first receptor binding agent, e.g., the first stimulatory agent, provides the T cell activation signal (e.g., signal via T cell receptor (TCR) engagement).

[00292] In some embodiments, the incubation or culturing of cells according to the methods provided is carried out or performed in the presence of one or more agents. In some embodiments, the incubation or culturing of cells is in the presence of the first receptor binding agent, e.g., as described above, and one or more second receptor binding agents, e.g., a second stimulatory agent.

[00293] In some aspects, the receptor binding agent, e.g., stimulatory agent, is a second receptor binding agent, e.g., second stimulatory agent. In some cases, the second receptor binding agent, e.g., second stimulatory agent, binds to a molecule on the surface of cells, such as a cell surface molecule, e.g., receptor molecule. In some embodiments, the second receptor binding agent, e.g., second stimulatory agent, induces or modulates a signal, e.g., a second or additional signal. In some embodiments, the second receptor binding agent, e.g., second stimulatory agent is capable of enhancing, decreasing, or modifying a signal emitted through the first molecule.

[00294] In some aspects, the second receptor binding agent, e.g., second stimulatory agent, can enhance or potentiate a signal induced by the first receptor binding agent, e.g., first stimulatory agent. In some embodiments, the second receptor binding agent, e.g., second stimulatory agent, binds to an accessory molecule and/or can stimulate or induce an accessory or secondary and/or additional signal in the cell. In some aspects, the second receptor binding agent, e.g., second stimulatory agent, binds to a costimulatory molecule and/or provides a costimulatory signal. In some embodiments, the second receptor binding agent, e.g., second stimulatory agent, provides T cell activation signal (e.g., costimulatory signal). In some embodiments, the second receptor binding agent, e.g., second stimulatory agent, provides T cell activation signal (e.g., cytokine signals, accessory signals, survival signals, and/or environmental signals).

[00295] In some embodiments, the receptor binding agent, e.g., the stimulatory agent, which may be the second receptor binding agent, e.g., second stimulatory agent, binds, e.g., specifically binds, to a molecule, e.g., a second molecule, which may be a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, an adhesion molecule, a member of the TNF family or the TNF receptor family, and/or a Wnt receptor or coreceptor, e.g., the Frizzled (Fz) family of receptors.

[00296] In some embodiments, the incubation or culturing of cells according to the provided methods is performed or carried out in the presence of three or more agents, such as three or more binding agents. In some embodiments, the incubation or culturing of cells is in the presence of one or more additional receptor binding agents, e.g., a third receptor binding agent, that modulates one or more additional signals. For example, in some embodiments, the incubation or culturing of cells according to the provided methods is performed or carried out in the presence of at least three different receptor binding agents, e.g., a first receptor binding agent, e.g., first stimulatory agent, providing a primary T cell activation signal, e.g., signal 1 (e.g., signal via T cell receptor (TCR) engagement); a second receptor binding agent, e.g., a second stimulatory agent, providing signal 2 (e.g., a costimulatory signal or an accessory signal); and an additional (e.g., third) receptor binding agent, e.g., an additional stimulatory agent, providing signal 3 (e.g., cytokine signals, survival signals, and/or environmental signals). Any number of receptor binding agents, e.g., stimulatory agents, may be selected in any combination for any of the methods described herein.

[00297] In some embodiments, the receptor binding agent, e.g., the first, second, and/or additional (e.g., third) receptor binding agent, e.g., stimulatory agent, can be a binding agent that specifically binds to a receptor, e.g., a receptor expressed on the cell surface. In some cases, the receptor binding agent, e.g., the stimulatory agent, is an antibody or an antigen-binding fragment thereof. In some cases, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., stimulatory agent, that specifically binds to the receptor can be selected from the group consisting of an anti-receptor antibody, a divalent antibody fragment of an anti-receptor antibody, a monovalent antibody fragment of an anti-receptor antibody, and a proteinaceous receptor binding molecule with antibody-like binding properties. The bivalent antibody fragment may be an F(ab')2 fragment, or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, a proteinaceous receptor-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a lipocalin family polypeptide, a glucobody, a protein based on the ankyrin structure, a protein based on the crystal structure, an adnectin, or an avimer. In some cases, the receptor-binding agent, e.g., the first, second, and/or additional receptor-binding agent, e.g., stimulatory agent, that specifically binds to the receptor is an antibody or an antigen-binding fragment, e.g., a Fab fragment.

[00298] In some embodiments, the receptor binding agent, e.g., the first, second, and/or additional (e.g., third) receptor binding agent, e.g., stimulatory agent, can be a ligand that binds, e.g., specifically to the receptor. In some embodiments, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, variant, or modified forms thereof. For example, in some embodiments, the receptor binding agent is a cytokine that binds to a cytokine receptor. In some embodiments, the receptor binding agent is a chemokine that binds to a chemokine receptor. In some embodiments, the receptor binding agent is an extracellular domain or a portion of a cytokine or chemokine or of an adhesion molecule that binds to a cell surface adhesion molecule. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an extracellular domain, or a portion thereof, of an endogenous ligand and/or a cognate ligand that is itself a transmembrane or cell surface protein.

[00299] In some embodiments, the receptor binding agent, e.g., first, second and/or additional (e.g., third) receptor binding agent, e.g., stimulatory agent, binds to a molecule on the surface of a cell which can be a lymphocyte including, but not limited to, a B cell, a T cell, or a natural killer cell. Illustrative examples of cells are B cells bearing CD40 or CD137 (both of which cell populations can be proliferated by binding only a first agent that provides an activation signal, e.g., 4-1BB ligand; or an αCD40 antibody molecule or an αCD137 antibody molecule (e.g., Zhang et al., 2010, J Immunol, 184: 787-795)). Other illustrative examples of agents (first or second) that can be used for B cell expansion are agents that bind to IgG, CD19, CD28, or CD14, e.g., αCD19, αIgG, αCD28, or αCD14 antibody molecules or antigen-binding portions thereof. It is also contemplated that the first or second agents for B cell expansion may comprise ligands for Toll-like receptors or interleukins, such as IL-21 (see, e.g., Dienz O, et al., 2009, J. Exp. Med. 206: 69). It is noted that lipopolysaccharide-dependent activation of B cells is also encompassed by the present invention, since a lipopolysaccharide can also be used as the first agent and can be equipped with a C1 binding partner as used herein.

[00300] Other illustrative examples of suitable cell populations include populations of T cells that expand after being activated by binding of a first agent to TCR/CD3 and binding of a second agent to an accessory molecule on the T cell, such as CD28. In this case, the first agent stimulates a signal associated with the TCR/CD3 complex on the T cells and the second agent provides a secondary stimulus by binding to CD28 as an accessory molecule. Agents that can be used for T cell expansion may also include interleukins, such as IL-2, IL-7, IL-15, or IL-21 (see, for example, Cornish et al. 2006, Blood. 108 (2)): 600-8, Bazdar and Sieg, 2007, Journal of Virology, 2007, 81 (22): 12670-12674, Battalia et al., 2013, Immunology, 139 (1): 109-120). Other illustrative examples for agents that can be used for T cell expansion are agents that bind to CD8, CD45, or CD90, such as antibodies to αCD8, αCD45, or αCD90. Illustrative examples of T cell population including antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells (an illustrative example of memory T cells are CD62L+CD8+ specific central memory T cells) or regulatory T cells (an illustrative example of Treg are CD4+CD25+CD45RA+ Treg cells).

[00301] Another illustrative example of a suitable cell population includes natural killer cells (NK cells), which can for example be expanded with agents that bind to CD16 or CD56, such as, for example, antibodies to αCD16 or αCD56. An illustrative example for such a CD16 antibody is the 3G8 antibody having a VH sequence set forth in SEQ ID NO: 48 and a VL sequence set forth in SEQ ID NO: 49 (see, for example, Hoshino et al., Blood. 1991 Dec 15;78(12):3232-40.). Another agent that can be used for NK cell expansion can be IL-15 (see, for example, Vitale et al. 2002. The Anatomical Record. 266:87-92). Yet another illustrative example of a suitable cell population includes monocytes, which can for example be expanded using an agent that binds to CD14, such as an αCD14 antibody molecule.

[00302] In some aspects, the receptor binding agent, e.g., the stimulatory agent, specifically targets a molecule expressed on the surface of target cells, wherein the molecule is a TCR receptor or chimeric antigen. For example, the molecule expressed on the surface of the target cell is selected from a T cell or B cell antigen receptor complex, a CD3 chain, a CD3 zeta, an antigen binding portion of a T cell receptor or a B cell receptor, or a chimeric antigen receptor. In some cases, the receptor binding agent targets peptide:MHC class I complexes.

[00303] In some embodiments, the stimulatory agent binds to a His-tagged extracellular domain of a molecule expressed on the surface of target cells. In some cases, the stimulatory agent contains the peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II, set forth in SEQ ID NO: 8) conjugated to a nickel-loaded trisNTA (also called His-STREPPER or His/Strep-tag®II adaptor). In some embodiments, the molecule expressed on the surface of His-tagged target cells is CD19.

[00304] In some aspects, the receptor binding agent, e.g., the stimulatory agent, specifically binds to the antibody portion of the recombinant receptor, e.g., CAR. In some cases, the antibody portion of the recombinant receptor includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is from a human IgG, such as IgG4 or IgG1. In some cases, the reagent is loaded with αIgG that recognizes the IgG4 spacer.

[00305] In some embodiments, two or more receptor binding agents are reversibly bound to the same reagent (e.g., multimerization reagent), such as the same oligomeric streptavidin mutein reagent. In some embodiments, two or more receptor binding agents, e.g., a first receptor binding agent, a second receptor binding agent, and/or one or more additional (e.g., third) receptor binding agents, are reversibly bound to the same reagent (e.g., multimerization reagent) via the plurality of binding sites capable of reversibly binding the two or more receptor binding agents.

[00306] In some embodiments, two or more receptor binding agents are reversibly linked to two or more different reagents (e.g., multimerization reagent), such as two or more different oligomeric streptavidin mutein reagents. In some such embodiments, at least one receptor binding agent (e.g., a first receptor binding agent) is reversibly linked to a first reagent (e.g., a first multimerization reagent) via the plurality of binding sites capable of reversibly binding to the receptor binding agent, and an additional receptor binding agent (e.g., a second receptor binding agent or third receptor binding agent) is reversibly linked to a second reagent (e.g., a second multimerization reagent) via the plurality of binding sites capable of reversibly binding to the receptor binding agent.

a. Receptor Binding Agents that Stimulate T Cell Activation

[00307] In some embodiments, the first receptor binding agent, e.g., first stimulatory agent, can stimulate a signal associated with the TCR/CD3 complex (e.g., signal 1 via TCR engagement) on cells, e.g., T cells. In some aspects, the first receptor binding agent, e.g., first stimulatory agent, can be a binding agent that specifically binds to CD3. In some cases, a first receptor binding agent, e.g., first stimulatory agent, that specifically binds to CD3 can be selected from the group consisting of an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. The divalent antibody fragment may be an F(ab')2 fragment, or a divalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, a proteinaceous CD3-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a lipocalin family polypeptide, a glucobody, a protein based on the ankyrin structure, a protein based on the crystal structure, an adnectin, or an avimer.

[00308] In some embodiments, an anti-CD3 Fab fragment can be derived from the CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™; see, also U.S. Patent No. 4,361,549). The heavy chain variable domain and light chain variable domain of the anti-CD3 antibody OKT3 are described in Arakawa et al., J. Biochem. 120, 657662 (1996) and comprise the amino acid sequences set forth in SEQ ID NOS: 31 and 32, respectively.

b. Receptor Binding Agent That Stimulates the Signal Costimulatory and/or Accessory

[00309] In some embodiments, the receptor binding agent, e.g., second receptor binding agent, stimulatory agent, binds to a molecule that provides a costimulatory or accessory signal, to the cell expressing the molecule, e.g., a T cell or a B cell. In some embodiments, the receptor binding agent, e.g., stimulatory agent, binds to a molecule that stimulates or activates a T cell activation signal, e.g., costimulatory signal, to the cell expressing the molecule, e.g., a T cell. In some embodiments, the receptor binding agent provides an accessory signal to the cell expressing the molecule, e.g., a T cell.

[00310] In any of the embodiments provided herein, the receptor binding agent, e.g., the second receptor binding agent, e.g., stimulatory agent, can be a binding agent that specifically binds to a receptor, e.g., a receptor expressed on the cell surface.

[00311] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is an antibody or an antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof that binds to a cell surface molecule that stimulates or activates the T2 cell activation signal, e.g., costimulatory signal, upon binding of the agent. In some cases, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., stimulatory agent, that specifically binds to the receptor can be selected from the group consisting of an anti-receptor antibody, a divalent antibody fragment of an anti-receptor antibody, a monovalent antibody fragment of an anti-receptor antibody, and a proteinaceous receptor binding molecule having antibody-like binding properties. The divalent antibody fragment may be an F(ab')2 fragment or a divalent single-chain Fv fragment while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, a proteinaceous receptor-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a lipocalin family polypeptide, a glucobody, a protein based on ankyrin structure, a protein based on a crystal structure, an adnectin, or an avimer. In some cases, the receptor-binding agent, e.g., the first, second, and/or additional receptor-binding agent, e.g., stimulatory agent, that specifically binds to the receptor is an antibody or an antigen-binding fragment, e.g., a Fab fragment.

[00312] In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be a ligand that binds, e.g., specifically to the receptor, e.g., a cell surface molecule that stimulates or activates an activating signal, e.g., costimulatory signal, upon binding of the agent. In some embodiments, the receptor binding agent, e.g., a first, second, and/or additional receptor binding agent, an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, a variant, or modified forms thereof. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an extracellular domain or a portion thereof, of an endogenous ligand, and/or a cognate ligand.

[00313] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is capable of binding to any one or more of CD28, CD5, CD4, CD8, MHC1, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L, and LIGHT. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an antibody, divalent antibody (e.g., an F(ab')2 fragment or a divalent single-chain Fv fragment), monovalent antibody (e.g., a Fab fragment, an Fv fragment, or a single-chain Fv fragment (scFv)), or binding to any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L, and LIGHT, wherein such agent is capable of inducing or modulating a signal in cells upon binding of the receptor binding agent, e.g., stimulatory agent, to the molecule.

[00314] In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be a ligand for any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L, and LIGHT, wherein such agent is capable of inducing or modulating a signal in cells upon binding of the receptor binding agent, e.g., stimulatory agent, to the molecule. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an extracellular domain, or a portion thereof, of an endogenous ligand and/or a cognate ligand. For example, in some embodiments, the receptor binding agent is or contains the extracellular domain, or a portion thereof, of B7-1 (CD80), B7-2 (CD86), ICOS-L, PD-L1, OX40L, CD27, 4-1BB (CD137), and/or CD30.

[00315] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is capable of specifically binding to a molecule on the surface of the target cells other than CD28, CD3, CD137, or CD40. In some cases, binding of the receptor binding agent, e.g., stimulatory agent, to the molecule on the surface of the target cells other than CD28, CD3, CD137, or CD40 induces or modulates a signal in the target cells and/or alters a function of the target cells, thereby generating cultured target cells.

[00316] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is capable of binding to any one or more than one member of the TNF family or TNF receptor family, e.g., a member of the TNF receptor superfamily and/or Wnt receptor or co-receptor, e.g., the Frizzled family of receptors (Fz).

[00317] In some embodiments, the molecule on the cell is a member of the TNF receptor superfamily, such as Tumor Necrosis Factor Receptor 1 (CD120a), Tumor Necrosis Factor Receptor 2 (CD120b), Lymphotoxin beta receptor (CD18), OX40 (CD134), CD40 (Bp50), Fas receptor (Apo-1, CD95), Decoy receptor 3 (TR6, M68), CD27 (S152, Tp55), CD30 (Ki-1), 4-1BB (CD137), Death receptor 4 (TRAILR1, Death receptor 5 (TRAILR2, CD262), Decoy receptor 1 (TRAILR3, LIT, TRID, CD263), Decoy receptor 2 (TRAILR4, TRUNDD, CD264), RANK (CD265), Osteoprotegerin (OCIF, TR1), TWEAK receptor (Fn14, CD266), TACI (IGAD2, CD267), BAFF receptor (CD268), Herpesvirus entry mediator (ATAR, TR2, CD270), nerve growth factor receptor (p75NTR, CD271), B cell maturation antigen (TNFRSF13A, CD269), glucocorticoid-induced TNFR-related (AITR, CD357), TROY (TAJ, TRADE), Death receptor 6 (CD358), Death receptor 3 (Apo-3, TRAMP, LARD, WS-1) or Ectodysplasin A2 receptor (XEDAR).

[00318] In some cases, the receptor binding agent that specifically binds to the TNF receptor superfamily protein is an antibody or an antigen-binding fragment thereof, e.g., a Fab fragment. In some embodiments, the receptor binding agent that specifically binds to the TNF receptor superfamily protein can be a ligand that specifically binds to the receptor, e.g., and/or an extracellular domain or portion thereof. In some embodiments, the ligand is or includes TNFa, Lymphotoxin beta (TNF-C), OX40L, CD154, FasL, FasL, LIGHT, TL1A, CD70, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, BAFF, LIGHT, NGF, BDNF, NT-3, NT-4, BAFF, GITR ligand, TL1A, or EDA-A2, or an extracellular domain or portion thereof of any of the transmembrane proteins.

[00319] In some embodiments, the molecule in the cell is a Wnt receptor or co-receptor, such as Frizzled (Fz) family receptor, lipoprotein receptor-related protein (LRP)-5/6, receptor tyrosine kinase (RTK), and receptor-related orphan receptor 2 (ROR2). In some embodiments, the molecule in the cell is, for example, Frizzled-1 (FZD1), Frizzled-2 (FZD2), Frizzled-3 (FZD3), Frizzled-4 (FZD4), Frizzled-5 (FZD5), Frizzled-6 (FZD6), Frizzled-7 (FZD7), Frizzled-8 (FZD8), Frizzled-9 (FZD9), or Frizzled-10 (FZD10).

[00320] In some cases, the receptor binding agent that specifically binds to the Wnt receptor or co-receptor is an antibody or an antigen-binding fragment thereof, e.g., a Fab fragment. In some embodiments, the receptor binding agent that specifically binds to the Wnt receptor or co-receptor can be a ligand that binds, e.g., specifically to the receptor. In some embodiments, the ligand is or includes, e.g., WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, or WNT16.

[00321] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is capable of binding to any one or more of CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an antibody, divalent antibody (e.g., an F(ab')2 fragment or a divalent single-chain Fv fragment), monovalent antibody (e.g., a Fab fragment, an Fv fragment, or a single-chain Fv fragment (scFv)), or ligand to any one or more of CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM, wherein such agent is capable of inducing or modulating a signal in cells upon binding of the receptor binding agent, e.g., stimulatory agent, to the molecule.

[00322] In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be a ligand for any one or more of CD28, 4-1BB (CD137), CD40, CD40L, Ligand for activation of T cells (LAT), CD27, OX40 (CD134), and Herpesvirus entry mediator (HVEM), wherein such agent is capable of inducing or shaping a signal in cells upon binding of the receptor binding agent, e.g., stimulatory agent, to the molecule. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an extracellular domain, or a portion thereof, of an endogenous ligand and/or a cognate ligand. For example, in some embodiments, the receptor binding agent is or contains the extracellular domain, or a portion thereof, of B7-1 (CD80), B7-2 (CD86), 4-1BBL, CD40, CD40L, CD27L (CD70), and/or OX40L.

[00323] In some embodiments, the molecule on the cell, e.g., T cell, can be CD28 and the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD28.

[00324] In some embodiments, the molecule on the cell, e.g., T cell, can be CD28 and the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD28. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to CD28 can be selected from the group consisting of an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, and a proteinaceous CD28 binding molecule having antibody-like binding properties. The divalent antibody fragment may be an F(ab')2 fragment, or a divalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). A proteinaceous CD28-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a lipocalin family polypeptide, a glucobody, an ankyrin backbone-based protein, a crystal support-based protein, an adnectin, and an avimer.

[00325] In some embodiments, an anti-CD28 Fab fragment can be derived from the CD28.3 antibody (deposited as a synthetic single-chain Fv construct under GenBank Accession No. AF451974.1; see also Vanhove et al., BLOOD, 15 July 2003), Vol. 102, No. 2, pages 564-570) whose heavy and light chains comprise SEQ ID NO: 33 and 34, respectively.

[00326] In some embodiments, the molecule on the cell, e.g., T cell or B cell, can be CD137 and the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD137. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to CD137 can be selected from the group consisting of an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, and a proteinaceous CD137 binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, the anti-CD137 antibody may be LOB12, IgG2a or LOB12.3, IgG1 as described in Taraban et al. Eur J Immunol. 2002 Dec;32(12):3617-27. See also for example, US6569997, US6303121, Mittler et al. Immunol Res. 2004;29(1-3):197-208.

[00327] In some embodiments, the molecule on the cell, e.g., B cells, can be CD40 and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., a second stimulatory agent) specifically binds to CD40. In some aspects, the receptor binding agent (which can be a second receptor binding agent, e.g., a second stimulatory agent) that specifically binds to CD40 can be selected from the group consisting of an anti-CD40 antibody, a divalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, the anti-CD40 antibody may be the chimeric anti-human CD40 monoclonal antibody Teneliximab and anti-human CD40 (Affymetrix cat. no. 14-0409-80), or any described in, e.g., US Patent 2002/0142358, US Patent 2007/0077242, WO 2001/083755, Zhang et al., 2010, J Immunol,184:787-795.

[00328] In some embodiments, the molecule on the cell, e.g., T cell, can be CD40L (CD154) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD40L. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to CD40L can be selected from the group consisting of an anti-CD40L antibody, a divalent antibody fragment of an anti-CD40L antibody, a monovalent antibody fragment of an anti-CD40L antibody, and a proteinaceous CD40L binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, the anti-CD40L antibody may in some aspects be Hu5C8, as described in Blair et al. JEM vol. 191 no. 4 651-660. See also, e.g., WO1999061065 , US20010026932 , US7547438 , WO2001056603.

[00329] In some embodiments, the molecule on the cell, e.g., T cell, can be Inducible T-cell Costimulator (ICOS) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be the second receptor binding agent, e.g., second stimulatory agent) specifically binds to ICOS. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be the second receptor binding agent, e.g., second stimulatory agent) that specifically binds to ICOS can be selected from the group consisting of an anti-ICOS antibody, a divalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, and a proteinaceous ICOS binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. See, e.g., US20080279851 and Deng et al. Hybrids Hybridomics. 2004 Jun; 23 (3): 176-82.

[00330] In some embodiments, the molecule on the cell, e.g., T cell, can be Ligand for Activation of T cells (LAT) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be the second receptor binding agent, e.g., second stimulatory agent) specifically binds to LAT. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be the second receptor binding agent, e.g., second stimulatory agent) that specifically binds to LAT can be selected from the group consisting of an anti-LAT antibody, a divalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, and a proteinaceous LAT binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. See, e.g., Zhang et al., Cell. 198 Jan. 9; 92 (1): 83-92 and Facchetti et al., Am J Pathol. 1999 April; 154 (4): 1037-1046.

[00331] In some embodiments, the molecule on the cell, e.g., T cell, can be CD27 and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD27. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to CD27 can be selected from the group consisting of an anti-CD27 antibody, a divalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, and a proteinaceous CD27 binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. See, for example, WO2008051424, US9023999, US 8481029, US 9169325, US 9102737, US2016/0185870 and He et al., J Immunol. 2013 Oct 15; 191 (8): 4174-83.

[00332] In some embodiments, the molecule on the cell, e.g., T cell, can be OX40 (CD134) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to OX40. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to OX40 can be selected from the group consisting of an anti-OX40 antibody, a divalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40 antibody, and a proteinaceous binding molecule of OX40 having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. See, for example, US Patent 2010/0196359, US Patent 2015/0307617, WO 2015/153513, WO203038191 and Melero et al. Clin Cancer Res. 2013 Mar 1;19(5):1044-53.

[00333] In some embodiments, the molecule on the cell, e.g., T cell, can be herpesvirus entry mediator (HVEM) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be the second receptor binding agent, e.g., second stimulatory agent) specifically binds to HVEM. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be the second receptor binding agent, e.g., second stimulatory agent) that specifically binds to HVEM can be selected from the group consisting of an anti-HVEM antibody, a divalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, and a proteinaceous HVEM-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. See, for example, WO2006054961, WO2007001459,Parket et al. Cancer Immunol Immuno. 2012 Feb;61(2):203-14.

[00334] In some embodiments, the molecule on the cell, e.g., T cell, can be CD90 and the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD90. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to CD90 can be selected from the group consisting of an anti-CD90 antibody, a divalent antibody fragment of an anti-CD90 antibody, a monovalent antibody fragment of an anti-CD90 antibody, and a proteinaceous CD90 binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. See, for example, anti-human CD90 antibody clone 5E10 (Stemcell Technologies, Cat. No. 60045 or BD Biosciences Cat. No. 550402) and anti-CD90 antibody G7 (Biolegend, Cat. No. 105201).

[00335] In some embodiments, the molecule on the cell, e.g., T cell, can be CD95 and the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to CD95. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to CD95 can be selected from the group consisting of an anti-CD95 antibody, a divalent antibody fragment of an anti-CD95 antibody, a monovalent antibody fragment of an anti-CD95 antibody, and a proteinaceous CD95 binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the anti-CD95 antibody can be mouse monoclonal anti-human CD95 CH11 (Upstate Biotechnology, Lake Placid, NY) or can be anti-CD95 mAb 7C11 or anti-APO-1, as described in e.g., WO 2015/197874 , US 9309320 , US 7935339 , and Paulsen et al. Cell Death & Differentiation 18.4 (2011): 619-631.

c. Receiver Binding Agent Providing Additional Signal

[00336] In some embodiments, the molecule on the cell, e.g., T cell, to which the receptor binding agent, which may be a second or additional receptor binding agent, binds is a cell surface molecule that stimulates or activates a cytokine signal, chemokine signal, cell adhesion signal, T cell activation signal, or additional signals, e.g., environmental signals, upon binding of the agent. In some embodiments, the molecule on the cell, e.g., T cell, to which the receptor binding agent, which may be a second or additional receptor binding agent, specifically binds is a cytokine receptor or a chemokine receptor. In some cases, the receptor binding agent, e.g., additional receptor binding agent, is or contains an adhesion molecule, is a factor that induces cytokine production, chemokine production, expression of an adhesion molecule, and/or is involved in the stimulation and/or modulation of an accessory signal and/or an additional signal, e.g., an environmental signal

[00337] In any of the embodiments provided herein, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., stimulatory agent, can be a binding agent that specifically binds to a receptor, e.g., on the cell surface.

[00338] In some embodiments, the receptor binding agent, e.g., stimulatory agent, is an antibody or an antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof that binds to a cell surface molecule that stimulates or activates a cytokine signal, chemokine signal, cell adhesion signal, T-cell activation signal, or additional signals, e.g., environmental signals, upon binding of the agent. In some cases, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., stimulatory agent, that specifically binds to the receptor can be selected from the group consisting of an anti-receptor antibody, a divalent antibody fragment of an anti-receptor antibody, a monovalent antibody fragment of an anti-receptor antibody, and a proteinaceous receptor binding molecule with antibody-like binding properties. The bivalent antibody fragment may be an F(ab')2 fragment, or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, a proteinaceous receptor-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a lipocalin family polypeptide, a glucobody, a protein based on the ankyrin structure, a protein based on the crystal structure, an adnectin, or an avimer. In some cases, the receptor-binding agent, e.g., the first, second, and/or additional receptor-binding agent, e.g., stimulatory agent, that specifically binds to the receptor is an antibody or an antigen-binding fragment, e.g., a Fab fragment.

[00339] In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be a ligand that binds, e.g., specifically to the receptor, e.g., a cell surface molecule that stimulates or activates a cytokine signal, chemokine signal, cell adhesion signal, T cell activation signal, or additional signals, e.g., environmental signals, upon binding of the agent. In some embodiments, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, a variant, or modified forms thereof. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an extracellular domain, or a portion thereof, of an endogenous ligand, and/or a cognate ligand.

[00340] In some embodiments, the receptor binding agent, e.g., a second or an additional receptor binding agent, is or contains a ligand that specifically binds to a cytokine receptor. In some embodiments, the receptor binding agent, e.g., a second or an additional receptor binding agent, is or comprises a ligand of the cytokine receptor, e.g., a cytokine or a portion thereof.

[00341] Exemplary cytokine receptors include, but are not limited to, IL-2R, IL-7R, IL-21R, CD132 (IL receptor common gamma chain), IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-17R, TNFR1, and TNFR2. Exemplary ligands, e.g., cytokines, include, but are not limited to, IL-2, IL-7, IL-21, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, type I interferons (e.g., IFNα and/or IFNβ), IL-12, IL-17, IL-9, and TNF, and biologically active fragments thereof.

[00342] In some embodiments, the receptor binding agent, e.g., a second or an additional receptor binding agent, is an antibody or an antigen-binding fragment thereof, that specifically binds to a cytokine receptor. In some cases, the receptor binding agent, e.g., a second or an additional receptor binding agent, that specifically binds to the cytokine receptor can be selected from the group consisting of an anti-(cytokine receptor) antibody, a divalent antibody fragment of an anti-(cytokine receptor) antibody, a monovalent antibody fragment of an anti-(cytokine receptor) antibody, and a proteinaceous cytokine receptor binding molecule having antibody-like binding properties. The divalent antibody fragment can be an F(ab’)2 fragment, or a divalent single-chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

[00343] In some embodiments, the receptor binding agent, e.g., the additional receptor binding agent, is or contains a ligand that specifically binds to a chemokine receptor. In some embodiments, the receptor binding agent, e.g., a second or an additional receptor binding agent, is or comprises a ligand of the chemokine receptor, e.g., a chemokine or a portion thereof.

[00344] Exemplary chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. Exemplary ligands, e.g., chemokines, include, but are not limited to, CXCL9, CXCL10, CCL19, CCL21, and CCL25 or biologically active fragments thereof.

[00345] In some embodiments, the receptor binding agent, e.g., a second or an additional receptor binding agent, is an antibody or antigen-binding fragment thereof, that specifically binds to a chemokine receptor. In some cases, the receptor binding agent, e.g., a second or an additional receptor binding agent, that specifically binds to the chemokine receptor can be selected from the group consisting of an anti-(chemokine receptor) antibody, a divalent antibody fragment of an anti-(chemokine receptor) antibody, a monovalent antibody fragment of an anti-(chemokine receptor) antibody, and a proteinaceous chemokine receptor binding molecule with antibody-like binding properties. The divalent antibody fragment may be an F(ab’)2 fragment, or a divalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

[00346] In some cases, the receptor binding agent that specifically binds to the adhesion molecule or cytokine-inducing factors is an antibody or an antigen-binding fragment thereof, e.g., a Fab fragment. In some embodiments, the receptor binding agent that specifically binds to the adhesion molecule or cytokine-inducing factors can be a ligand that binds, e.g., specifically to the receptor. In some embodiments, the receptor binding agent is an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, variant, or modified forms thereof, of the adhesion molecule and/or the receptor. In some embodiments, the receptor binding agent, e.g., stimulatory agent, can be an extracellular domain, or a portion thereof, of an endogenous ligand and/or a cognate ligand that is itself a transmembrane or cell surface protein.

[00347] In some cases, the molecule on the cell, e.g., adhesion molecule, is CD44, CD31, CD18/CD11a (LFA-1; full-length alpha and beta chain sequence set forth in SEQ ID NOS: 71 and 72, respectively), CD29, CD54 (ICAM-1), CD62L (L-selectin; full-length sequence set forth in SEQ ID NO: 73), CD29/CD49d (VLA-4; full-length sequence set forth in SEQ ID NO: 75), CD106 (VCAM-1; full-length sequence set forth in SEQ ID NO: 74), or is a biologically active fragment thereof. In some embodiments, the receptor binding agent is an antibody or an antigen-binding fragment thereof that binds, e.g., specifically to an adhesion molecule, e.g., CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), CD29/CD49d (VLA-4), CD106 (VCAM-1), or a biologically active fragment thereof. In some embodiments, the receptor binding agent is a ligand or a portion thereof, that binds, e.g., specifically to an adhesion molecule, e.g., CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), CD29/CD49d (VLA-4), CD106 (VCAM-1). Such receptor binding agents include an agent that is or comprises an extracellular domain (ECD), or a portion thereof, of an endogenous ligand and/or a cognate ligand of an adhesion molecule. In some embodiments, the receptor binding agent is an extracellular domain, or a portion thereof, of an adhesion molecule, and can bind one or more adhesion molecules to the surface of a cell. Examples of such receptor binding agents include the extracellular domain of LFA-1α (ECD set forth in SEQ ID NO: 77); LFA-1β (ECD set forth in SEQ ID NO: 78); L-selectin (ECD set forth in SEQ ID NO: 79); VCAM-1 (ECD set forth in SEQ ID NO: 80); and VLA-4 (ECD set forth in SEQ ID NO: 81), or any portion thereof.

[00348] In some embodiments, the factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule is or contains a nuclear factor, such as retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[00349] In some embodiments, the receptor binding agent, e.g., a second or additional receptor binding agent, is or contains a ligand that specifically binds to a cytokine receptor.

[00350] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-2R and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds to IL-2R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds IL-2R can be selected from the group anti-IL-2R antibody, a divalent antibody fragment of an anti-IL-2R antibody, a monovalent antibody fragment of an anti-IL-2R antibody, and a proteinaceous IL-2R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment can be any of as described in, for example, EP 2425250, US Patent 2011/0053209, WO 2009/145831, US Patent 2010/0055098, and Volk et al., Clin Exp Immunol. 1989 Apr;76(1):121-125.

[00351] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-7R (CD127) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to IL-7R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to IL-7R can be selected from the group consisting of an anti-IL-7R antibody, a divalent antibody fragment of an anti-IL-7R antibody, a monovalent antibody fragment of an anti-IL-7R antibody, and a proteinaceous IL-7R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment can be any of those described in, e.g., WO 2016/059512, WO 2015/189302, Lee et al. Proc Natl Acad Sci USA. 2012 Jul 31; 109(31): 12674-9, and Chung et al., Blood (2007) 110(8): 2803-2810.

[00352] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-21R (CD360) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds IL-21R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds IL-21R can be selected from the group consisting of an anti-IL-21R antibody, a divalent antibody fragment of an anti-IL-21R antibody, a monovalent antibody fragment of an anti-IL-21R antibody, and a proteinaceous IL-21R binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment can be any of as described in, e.g., US Patent 2006/0159655, US Patent 2006/0039902, US Patent 2003/0108549, and Guo et al., J Transl Med. 2010;8:50.

[00353] In some embodiments, the molecule on the cell, e.g., T cell, can be the IL receptor common gamma chain (yc; or CD132) and the receptor binding agent, e.g., stimulatory agent (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds the common gamma chain. The common gamma chain (yc; or CD132), also known as the interleukin-2 receptor gamma subunit or IL-2RG, is a cytokine receptor subunit that is common to the receptor complexes for interleukin receptors: IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds the common gamma chain may be selected from the group consisting of an anti-γc or anti-CD132 antibody, a divalent antibody fragment of an anti-γc or anti-CD132 antibody, a monovalent antibody fragment of an anti-γc or anti-CD132 antibody, and a common proteinaceous gamma chain binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment can be any of those described in, e.g., anti-yc or anti-CD132 antibody TUGh4 (AB_2123585 (BioLegend Cat. No. 338607) or AB_2123584 (BioLegend Cat. No. 338608)), Itano et al. 1996. J. Exp. Med. 178:389, Kondo et al. 1993. Science 262:1874, Hodge et al. 2012. Blood. 120:3774, Matsubara et al. 2014. Sci Rep. 4:5043 and Massoud et al. 2015. PNAS. 112: 11030-11035, Pérez-Simón, Blood 2015 125: 424426.

[00354] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-1R (CD121), such as IL-1R1 or IL-1R2, and the receptor binding agent, e.g., stimulatory agent (e.g., being the second receptor binding agent, e.g., second stimulatory agent) specifically binds the IL-1R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., being the second receptor binding agent, e.g., second stimulatory agent) that specifically binds the IL-1R can be selected from the group consisting of an anti-IL-1R antibody, a divalent antibody fragment of an anti-IL-1R antibody, a monovalent antibody fragment of an anti-IL-1R antibody, and a proteinaceous IL-1R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment may be, for example, human IL-1R1 antibody (R&D Systems cat. no. AF269).

[00355] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-15R (CD215), and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) specifically binds to IL-15R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be a second receptor binding agent, e.g., second stimulatory agent) that specifically binds to IL-15R can be selected from the group consisting of an anti-IL-15R antibody, a divalent antibody fragment of an anti-IL-15R antibody, a monovalent antibody fragment of an anti-IL-15R antibody, and a proteinaceous IL-15R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment may be any one described in, for example, US Patent 2004/0185048, US Patent 6013480 or may be the human anti-IL-15R antibody eBioJM7A4 (Affymetrix cat. no. 12-7159-41).

[00356] In some embodiments, the molecule on the cell, e.g., T cell, can be interferon gamma receptor (IFNyR; CD119) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds IFNyR. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds IFNYR can be selected from the group consisting of an anti-IFNyR antibody, a divalent antibody fragment of an anti-IFNyR antibody, a monovalent antibody fragment of an anti-IFNyR antibody, and a proteinaceous IFNyR-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen binding fragment may be, for example, anti-human CD119 (IFN gamma receptor 1) antibody GIR 208 (eBioscience cat. no. 13-1199-80) and anti-human IFN gamma receptor antibody ab25448 (Abcam cat. no. ab25448).

[00357] In some embodiments, the molecule on the cell, e.g., T cell, can be tumor necrosis factor alpha receptor (TNFαR), e.g., including TNFR1 (CD120a) and TNFR2 (CD120b), and the receptor binding agent, e.g., stimulatory agent (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds TNFαR. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds TNFαR can be selected from the group consisting of an anti-TNFαR antibody, a divalent antibody fragment of an anti-TNFαR antibody, a monovalent antibody fragment of an anti-TNFαR antibody, and a proteinaceous TNFαR-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment may be any as described in WO 2012/087928, WO 2010/120374 and anti-human IFN gamma receptor antibody ab19139 (Abcam cat. no. ab19139) and ab74315 (Abcam cat. no. ab74315).

[00358] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-4R and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds IL-4R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds IL-4R can be selected from the group of an anti-IL-4R antibody, a divalent antibody fragment of an anti-IL-4R antibody, a monovalent antibody fragment of an anti-IL-4R antibody, and a proteinaceous IL-4R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen binding fragment can be any as described in WO2005047331.

[00359] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-10R and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds to IL-10R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds to IL-10R can be selected from the group consisting of an anti-IL-10R antibody, a divalent antibody fragment of an anti-IL-10R antibody, a monovalent antibody fragment of an anti-IL-10R antibody, and a proteinaceous IL-10R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen binding fragment can be any one as described in US Patent 7,553,932, US Patent 8,071,374, Castro et al., J Exp Med. 2000 Nov 20;192(10):1529-34 or anti-IL-10R antibody 9D7 (ThermoFisher cat. no. 06-1067).

[00360] In some embodiments, the molecule on the cell, e.g., T cell, can be the type I Interferon receptor, e.g., IFNα receptor (IFNAR), including IFNAR1 and IFNAR2, and the receptor binding agent, e.g., stimulatory agent (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds the IFNAR. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds the IFNAR can be selected from the group consisting of an anti-IFNAR antibody, a divalent antibody fragment of an anti-IFNAR antibody, a monovalent antibody fragment of an anti-IFNAR antibody, and a proteinaceous IFNAR-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment can be any one as described in, e.g., US Patent 2013/0254912, US Patent 7662381, US Patent 8460668, Baccala et al., J. Immunol. 2012, 189(12):5976-5984 and the anti-IFNAR2 antibody clone MMHAR-2 (PBL Sciences cat. No. 21385-1).

[00361] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-12R and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds IL-12R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds IL-12R can be selected from the group consisting of an anti-IL-12R antibody, a divalent antibody fragment of an anti-IL-12R antibody, a monovalent antibody fragment of an anti-IL-12R antibody, and a proteinaceous IL-12R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment may be any as described in EP 0638644 or US5853721.

[00362] In some embodiments, the molecule on the cell, e.g., T cell, can be IL-17R (CD217) and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds IL-17R. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds IL-17R can be selected from the group consisting of an anti-IL-17R antibody, a divalent antibody fragment of an anti-IL-17R antibody, a monovalent antibody fragment of an anti-IL-17R antibody, and a proteinaceous IL-17R binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some cases, the antibody or antigen-binding fragment can be any of the following: WO2008054603, EP2487188, Papp et al., J Invest Dermatol. 2012 Oct;132(10):2466-9, Moseley et al., 2003. Cytokine Growth Factor Rev. 14:155, Rong et al., 2009. Cell. Res. 19:208, Toy et al., 2006. J. Immunol. 177:36, or human anti-CD217 antibody clone W15177A (Biolegend).

[00363] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-2 ligand or a biologically active portion thereof. In some aspects, the IL-2 ligand has the amino acid sequence set forth in SEQ ID NO: 50 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 50, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00364] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-7 ligand or a biologically active portion thereof. In some aspects, the IL-7 ligand has the amino acid sequence set forth in SEQ ID NO: 51 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 51, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00365] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-21 ligand or a biologically active portion thereof. In some aspects, the IL-21 ligand has the amino acid sequence set forth in SEQ ID NO: 52 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 52, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00366] In some conditions, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-1 ligand or a biologically active portion thereof. In some aspects, the IL-1 ligand has the amino acid sequence set forth in SEQ ID NO: 53 and/or 54 (IL-1α or IL-1β, respectively) or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 53 and/or 54 (IL-1α or IL-1β, respectively), or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains receptor binding activity and/or a functional activity to modulate a receptor. or more signals to the receiver.

[00367] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-15 ligand or a biologically active portion thereof. In some aspects, the IL-15 ligand has the amino acid sequence set forth in SEQ ID NO: 55 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 55, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00368] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-9 ligand or a biologically active portion thereof. In some aspects, the IL-9 ligand has the amino acid sequence set forth in SEQ ID NO: 81 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 81, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00369] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IFNγ ligand or a biologically active portion thereof. In some aspects, the IFNγ ligand has the amino acid sequence set forth in SEQ ID NO: 56 or a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 56, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00370] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a TNFα ligand or a biologically active portion thereof. In some aspects, the TNFα ligand has the amino acid sequence set forth in SEQ ID NO: 57 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 57, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00371] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-4 ligand or a biologically active portion thereof. In some aspects, the IL-4 ligand has the amino acid sequence set forth in SEQ ID NO: 58 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 58, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00372] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-10 ligand or a biologically active portion thereof. In some aspects, the IL-10 ligand has the amino acid sequence set forth in SEQ ID NO: 59 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 59, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00373] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a Type I Interferon, e.g., IFNα, IFNβ, IFNK, IFNδ, IFNε, IFNT, IFNW, and IFNZ (also known as limitin) or a biologically active portion thereof. In some aspects, the Type I interferon is IFN-α or IFN-β that has the amino acid sequence set forth in SEQ ID NO: 60 or 61 (IFN-α2 or IFN-β1, respectively) or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 60 or 61, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains receptor binding activity and/or a functional activity to modulate one or more signals to the receiver.

[00374] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-12 ligand or a biologically active portion thereof. In some aspects, the IL-12 ligand has the amino acid sequence set forth in SEQ ID NO: 62 and/or 63 (IL-12α or IL-12β, respectively) or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 62 and/or 63 (IL-12α or IL-12β, respectively), or a biologically active portion thereof, wherein the variant or biologically active portion thereof retains the activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00375] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as an IL-17 ligand or a biologically active portion thereof. In some aspects, the IL-17 ligand has the amino acid sequence set forth in SEQ ID NO: 64 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 64, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00376] In some embodiments, the receptor binding agent, e.g., a second or additional receptor binding agent, is or contains a ligand that specifically binds to a chemokine receptor.

[00377] In some embodiments, the molecule on the cell, e.g., T cell, can be CXCR3 and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) is CXCR3. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds CXCR3 can be selected from the group consisting of an anti-CXCR3 antibody, a divalent antibody fragment of an anti-CXCR3 antibody, a monovalent antibody fragment of an anti-CXCR3 antibody, and a proteinaceous CXCR3-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen binding fragment can be any as described in WO2003024404.

[00378] In some embodiments, the molecule on the cell, e.g., T cell, can be CCR7 and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds CCR7. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds CCR7 can be selected from the group consisting of an anti-CCR7 antibody, a divalent antibody fragment of an anti-CCR7 antibody, a monovalent antibody fragment of an anti-CCR7 antibody, and a proteinaceous CCR7-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen-binding fragment can be any as described in US6153441.

[00379] In some embodiments, the molecule on the cell, e.g., T cell, can be CXCR1 and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds CXCR1. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds CXCR1 can be selected from the group consisting of an anti-CXCR1 antibody, a divalent antibody fragment of an anti-CXCR1 antibody, a monovalent antibody fragment of an anti-CXCR1 antibody, and a proteinaceous CXCR1-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen-binding fragment may be anti-CXCR1 antibody [42705.111] (Abcam; ab10400).

[00380] In some embodiments, the molecule on the cell, e.g., T cell, can be CXCR4 and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds CXCR4. In some aspects, the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds CXCR4 can be selected from the group consisting of an anti-CXCR4 antibody, a divalent antibody fragment of an anti-CXCR4 antibody, a monovalent antibody fragment of an anti-CXCR4 antibody, and a proteinaceous CXCR4-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen binding fragment can be any as described in WO2003024404.

[00381] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a ligand of CXCL9 or a biologically active portion thereof. In some aspects, the CXCL9 ligand has the amino acid sequence set forth in SEQ ID NO: 65 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 65, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00382] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a ligand of CXCL10 or a biologically active portion thereof. In some aspects, the CXCL10 ligand has the amino acid sequence set forth in SEQ ID NO: 66 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 66, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00383] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a ligand of CCL19 or a biologically active portion thereof. In some aspects, the CCL19 ligand has the amino acid sequence set forth in SEQ ID NO: 67 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 67, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00384] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a ligand of CCL21 or a biologically active portion thereof. In some aspects, the CCL21 ligand has the amino acid sequence set forth in SEQ ID NO: 68 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 68, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00385] In some embodiments, the receptor binding agent, e.g., stimulatory agent, (e.g., which may be an additional receptor binding agent, e.g., additional stimulatory agent) may include a ligand of a stimulatory receptor or another receptor capable of inducing a signal in the cell, such as a ligand of CCL25 or a biologically active portion thereof. In some aspects, the CCL25 ligand has the amino acid sequence set forth in SEQ ID NO: 69 or is a variant thereof that exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the sequence set forth in SEQ ID NO: 69, or is a biologically active portion thereof, wherein the variant or biologically active portion thereof retains activity to bind to the receptor and/or a functional activity to modulate one or more signals to the receptor.

[00386] In some cases, the receptor binding agent, e.g., additional receptor binding agent, is or contains an adhesion molecule or is a factor that induces cytokine production, chemokine production, expression of an adhesion molecule, and/or is involved in the stimulation and/or modulation of an accessory signal and/or an additional signal.

[00387] In some embodiments, the molecule on the cell, e.g., T cell, can be CD62L and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds to CD62L. In some aspects, the agent, e.g., receptor binding stimulatory agent (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds to CD62L can be selected from the group consisting of an anti-CD62L antibody, a divalent antibody fragment of an anti-CD62L antibody, a monovalent antibody fragment of an anti-CD62L antibody, and a proteinaceous CD62L binding molecule having antibody-like binding properties. The antibody or antigen binding fragment can be derived from any known in the art, e.g., DREG56 antibody (e.g., ATCC HB300); See, for example, Stemberger et al. 2012PLoS One. 2012;7(4):e35798.

[00388] In some embodiments, the molecule on the cell, e.g., T cells, can be RORyt and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds RORyt. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds RORyt can be selected from the group consisting of an anti-RORyt antibody, a divalent antibody fragment of an anti-RORyt antibody, a monovalent antibody fragment of an anti-RORyt antibody, and a proteinaceous RORyt-binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the antibody or antigen-binding fragment can be any of as described in CA2572334.

[00389] In some embodiments, the molecule on the cell, e.g., T cell, can be RORα and the receptor binding agent, e.g., stimulatory agent, (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) specifically binds RORα. In some aspects, the receptor binding agent, e.g., stimulatory agent (e.g., which can be an additional receptor binding agent, e.g., additional stimulatory agent) that specifically binds RORα can be selected from the group consisting of an anti-RORα antibody, a divalent antibody fragment of an anti-RORα antibody, a monovalent antibody fragment of an anti-RORα antibody, and a proteinaceous RORα binding molecule having antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, the anti-RORα antibody is RORα Antibody H-65 (Santa Cruz Biotechnology cat. no. sc-28612).

2. Selection Agents

[00390] In some embodiments, the agent is a selection agent. In some embodiments, the selection agent binds to a molecule on the surface of a cell, such as a cell surface molecule. In some cases, the cell surface molecule is a selection marker. In some such cases, the selection agent is capable of specifically binding to a selection marker expressed by one or more cells. In some embodiments, a selection agent or agents that are reversibly bound to a reagent can be used to facilitate selection or isolation of cells.

[00391] In any of the embodiments provided herein, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., stimulatory agent, can be a binding agent that specifically binds a receptor, e.g., a receptor expressed on the cell surface. In some cases, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., stimulatory agent, that specifically binds the receptor can be selected from the group consisting of an anti-receptor antibody, a divalent antibody fragment of an anti-receptor antibody, a monovalent antibody fragment of an anti-receptor antibody, and a proteinaceous receptor binding molecule having antibody-like binding properties. The bivalent antibody fragment can be an F(ab')2 fragment, or a bivalent single-chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, a proteinaceous receptor binding molecule with antibody-like binding properties can be an aptamer, a mutein based on a lipocalin family polypeptide, a glucobody, a protein based on the ankyrin structure, a protein based on the crystal structure, an adnectin, or an avimer. In some cases, the receptor binding agent, e.g., the first, second and/or additional receptor binding agent, e.g., stimulatory agent, that specifically binds the receptor is an antibody or an antigen-binding fragment thereof, e.g., a Fab fragment.

[00392] In some aspects, the cell surface molecule, e.g., selection marker, can be an antigen that defines a desired cell population or subpopulation, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, helper T cells, e.g., CD4+ helper T cells, B cells, or natural killer cells), monocytes, or stem cells, e.g., CD34-positive peripheral stem cells or stem cells expressing Nanog or Oct-4. In some embodiments, the selection marker can be a marker expressed on the surface of T cells or a subset of T cells, such as CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and/or CD45RO. Examples of T cells include cells such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Treg). An illustrative example of Treg includes CD4CD25CD45RA Treg cells and an illustrative example of memory T cells includes CD62LCD8+ specific central memory T cells. The cell surface molecule, e.g., selection marker, can also be a marker for a tumor cell.

[00393] In some embodiments, the selection marker can be CD4 and the selection agent specifically binds CD4. In some aspects, the selection agent that specifically binds CD4 can be selected from the group consisting of an anti-CD4 antibody, a divalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4 antibody, and a proteinaceous CD4 binding molecule with antibody-like binding properties. In some embodiments, an anti-CD4 antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g., CD4 Fab fragment) can be derived from the 13B8.2 antibody or a functionally active mutant of 13B8.2 that retains specific binding to CD4. For example, exemplary mutants of the 13B8.2 or m13B8.2 antibody are described in U.S. Pat. No. 7,482,000, U.S. Patent Application No. US2014/0295458 or International Patent Application Publication No. WO2013/124474; and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment designated "ml3B8.2" carries the variable domain of the murine CD4-binding antibody 13B8.2 and a constant domain containing human CH1 constant gamma-like domain for the heavy chain and human light chain constant kappa-like domain as described in U.S. Patent 7,482,000. In some embodiments, the anti-CD4 antibody, e.g., a mutant of the 13B8.2 antibody, contains the H91A amino acid substitution in the variable light chain, the Y92A amino acid substitution in the variable light chain, the H35A amino acid substitution in the variable heavy chain, and/or the R53A amino acid substitution in the variable heavy chain, each by Kabat numbering. In some aspects, compared to the variable domains of the 13B8.2 Fab fragment in ml3B8.2, the His residue at position 91 of the light chain (position 93 in SEQ ID NO: 30) is mutated to Ala and the Arg residue at position 53 of the heavy chain (position 55 in SEQ ID NO: 29) is mutated to Ala. In some embodiments, the reagent that is reversibly bound to anti-CD4 or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-206 or 68000-205 or 6-8002-100; IBA GmbH, Gottingen, Germany).

[00394] In some embodiments, the selection marker can be CD8 and the selection agent specifically binds CD8. In some aspects, the selection agent that specifically binds CD8 can be selected from the group consisting of an anti-CD8 antibody, a divalent antibody fragment of an anti-CD8 antibody, a monovalent antibody fragment of an anti-CD8 antibody, and a proteinaceous CD8 binding molecule with antibody-like binding properties. In some embodiments, an anti-CD8 antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g., CD8 Fab fragment) can be derived from the OKT8 antibody (e.g., ATCC CRL-8014) or a functionally active mutant thereof that retains specific binding to CD8. In some embodiments, the reagent that is reversibly bound to anti-CD8 or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8003 or 68000-201; IBA GmbH, Gottingen, Germany).

[00395] In some embodiments, the selection marker can be CD3 and the selection agent specifically binds CD3. In some aspects, the selection agent that specifically binds CD3 can be selected from the group consisting of an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, an anti-CD3 antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g., CD3 Fab fragment) can be derived from the OKT3 antibody (e.g., ATCC CRL-8001; see, e.g., Stemberger et al. PLoS One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding to CD3. In some embodiments, the reagent that is reversibly bound to anti-CD3 or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-201, 68001-100; IBA GmbH, Gottingen, Germany).

[00396] In some embodiments, the selection marker can be CD25 and the selection agent specifically binds CD25. In some aspects, the selection agent that specifically binds CD25 can be selected from the group consisting of an anti-CD25 antibody, a divalent antibody fragment of an anti-CD25 antibody, a monovalent antibody fragment of an anti-CD25 antibody, and a proteinaceous CD25 binding molecule with antibody-like binding properties. In some embodiments, an anti-CD25 antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g., CD25 Fab fragment) can be derived from the FRT5 antibody (see, e.g., Stemberger et al. 2012. PLoS One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding to CD25. In some embodiments, the reagent that is reversibly bound to anti-CD4 or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-205 or 6-8000-207 or 6-8004-050; IBA GmbH, Gottingen, Germany).

[00397] In some embodiments, the selection marker can be CD62L and the selection agent specifically binds CD62L. In some aspects, the selection agent that specifically binds CD62L can be selected from the group consisting of an anti-CD62L antibody, a divalent antibody fragment of an anti-CD62L antibody, a monovalent antibody fragment of an anti-CD62L antibody, and a proteinaceous CD62L binding molecule having antibody-like binding properties. In some embodiments, an anti-CD62L antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g., CD62L Fab fragment) can be derived from the DREG56 antibody (e.g., ATCC HB300; see, e.g., Stemberger et al. 2012, PLos One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding to CD62L. In some embodiments, the reagent that reversibly binds to anti-CD62L or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-204 or 6-8005-050; IBA GmbH, Gottingen, Germany).

[00398] In some embodiments, the selection marker can be CD45RA and the selection agent specifically binds CD45RA. In some aspects, the selection agent that specifically binds CD45RA can be selected from the group consisting of an anti-CD45RA antibody, a divalent antibody fragment of an anti-CD45RA antibody, a monovalent antibody fragment of an anti-CD45RA antibody, and a proteinaceous CD45RA binding molecule with antibody-like binding properties. In some embodiments, an anti-CD45RA antibody, such as a divalent antibody fragment or a monovalent antibody fragment (e.g., CD45RA Fab fragment) can be derived from the MEM56 antibody (e.g., Millipore 05-1413; See, e.g., Stemberger et al. PLoS One. 2012;7(4):e35798) or a functionally active mutant thereof that retains specific binding to CD45RA. In some embodiments, the reagent that is reversibly bound to anti-CD45RA or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-208 or 6-8007-050; IBA GmbH, Gottingen, Germany).

[00399] In some embodiments, the selection marker can be CD45RO and the selection agent specifically binds CD45RO. In some aspects, the selection agent that specifically binds CD45RO can be selected from the group consisting of an anti-CD45RO antibody, a divalent antibody fragment of an anti-CD45RO antibody, a monovalent antibody fragment of an anti-CD45RO antibody, and a proteinaceous CD45RO binding molecule with antibody-like binding properties. In some embodiments, the reagent that reversibly binds to the anti-CD45RO or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-209 or 6-8012-020; IBA GmbH, Gottingen, Germany).

[00400] In some embodiments, the selection marker can be CD154 and the selection agent specifically binds CD154. In some aspects, the selection agent that specifically binds CD154 can be selected from the group consisting of an anti-CD154 antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154 antibody, and a proteinaceous CD154-binding molecule with antibody-like binding properties. In some embodiments, the reagent that reversibly binds to anti-CD154 or a fragment thereof is commercially available or derived from a reagent that is commercially available (e.g., catalog No. 6-8000-202 or 6-5510-050; IBA GmbH, Gottingen, Germany).

[00401] In some embodiments, the selection marker can be CD16 and the selection agent specifically binds CD16. In some aspects, the selection agent that specifically binds CD16 can be selected from the group consisting of an anti-CD16 antibody, a divalent antibody fragment of an anti-CD16 antibody, a monovalent antibody fragment of an anti-CD16 antibody, and a proteinaceous CD16 binding molecule with antibody-like binding properties. In some embodiments, the reagent that reversibly binds to anti-CD16 or a fragment thereof is commercially available or derived from a reagent that is commercially available. In some aspects, the CD16 binding molecule comprises the heavy chain and/or light chain sequences set forth in SEQ ID NO: 36 and/or 37, respectively.

III. CELL CULTIVATION METHODS

[00402] Provided herein are methods of culturing cells that include incubating a composition containing target cells (e.g., T cells) in the presence of an agent (e.g., first, second, and/or additional), receptor binding agents, e.g., stimulatory agents, or selection agents) that are capable of binding to a molecule on the surface of the target cells (e.g., T cells) in the composition and that is reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the agent. In some embodiments, the incubation is carried out under conditions in which the agent binds, such as specifically binds, to the molecule on the cell. In some cases, for certain receptor binding agents (e.g., stimulatory agents), such binding can induce or modulate a signal in target cells (e.g., T cells) in the compositions, such as a primary signal or accessory signal as described. In some embodiments, binding of the agent to the molecule results in one or more stimulation, activation, expansion (proliferation), and/or differentiation of target cells in the composition.

[00403] In some embodiments, the provided method can be used to selectively induce ex vivo expansion of a population of cells, such as B cells, T cells, or natural killer cells. In some cases, stimulation can be in the absence of exogenous growth factors, such as lymphokines and accessory cells. In some embodiments, proliferation of these cells, such as B cells or T cells, can be induced without the need for antigen, thereby providing an expanded cell population, such as a T cell population that is polyclonal with respect to antigen reactivity. The methods described herein can provide for sustained proliferation of a selected population of T cells such as CD4+ or CD8+ T cells over an extended period of time to produce a multifold increase in the number of these cells relative to the original T cell population. In general, in the case of a (clonal) expansion of a lymphocyte population as described herein, all of the progeny can share the same antigen specificity as the cell population that was selected for expansion.

[00404] In some embodiments, the methods relate to expanding a population of antigen-specific T cells. In some embodiments, to produce a population of antigen-specific T cells, the T cells are contacted with an antigen in a form suitable for triggering a primary activation signal in the T cell, i.e., the antigen is presented to the T cell in such a manner that a signal is triggered in the T cell via the TCR/CD3 complex. For example, the antigen may be presented to the T cell by an antigen-presenting cell in conjunction with an MHC molecule. An antigen-presenting cell, such as a B cell, macrophage, monocyte, dendritic cell, Langerhans cell, or other cell that can present antigen to a T cell, can be incubated with the T cell in the presence of antigen (e.g., soluble antigen) such that the antigen-presenting cell presents the antigen to the T cell. Alternatively, a cell expressing an antigen of interest can be incubated with the T cell. For example, a tumor cell expressing tumor-associated antigens can be incubated with a T cell in conjunction to induce a tumor-specific response. Similarly, a cell infected with a pathogen, e.g., a virus, that presents antigens from the pathogen can be incubated with a T cell. Following antigen-specific activation of a population of T cells, the cells can be expanded according to the methods provided. For example, after antigen specificity has been established, T cells can be expanded by culture with an anti-CD3 antibody (used as the first agent) and an anti-CD28 antibody (used as the second agent) according to the methods described herein. In another embodiment, the first agent can be an MHC I:peptide complex that binds to a population of antigen-specific T cells. In such an embodiment, any antigen-specific peptide that is known and can be complexed with the respective MHC I molecule can be used. Alternatively, it is also possible to use as the first agent the natural ligand of a receptor that triggers cell expansion. For example, the extracellular domain of CD19 can be used to cause activation of intracellular signaling cascades of cells transduced to express the chimeric CD19-binding antigen receptor (CAR). Exemplary aspects of the foregoing are shown in the Examples.

[00405] In some embodiments, an in vitro method of culturing a population of cells is provided, comprising contacting a sample comprising a composition comprising a plurality of cells with a multimerization reagent. The multimerization reagent has been reversibly immobilized thereon (linked thereto) to an agent (first, second, and/or additional, receptor binding agent, e.g., stimulatory or selection agent), which can be used for selection, stimulation, expansion, and/or differentiation of cells. In some embodiments, an agent, e.g., first agent, that provides a primary activation signal to the cells, wherein the multimerization reagent comprises at least one Z1 binding site for reversible binding of the first agent. The first agent comprises at least one C1 binding partner, wherein the C1 binding partner is capable of reversibly binding to the Z1 binding site of the multimerization reagent, wherein the first agent is bound to the multimerization reagent via the reversible bond formed between the C1 binding partner and the Z1 binding site. The first agent binds to a receptor molecule on the surface of the cells, thereby providing a primary activation signal to the cells and thereby activating the cells.

[00406] In some embodiments, the multimerization reagent is immobilized on a support, such as a solid surface. In some embodiments, the multimerization reagent is not bound to a support, such as not bound to a solid surface or stationary phase.

[00407] For example, in some embodiments, an in vitro method of expanding a population of cells is provided, comprising contacting a sample comprising a population of cells with a multimerization reagent, wherein the multimerization reagent is not immobilized on a solid support, i.e., is in a soluble form, and has bound thereto an agent (first or second, receptor-binding agent, e.g., stimulatory, or selection agent), which can be used for the selection, stimulation, expansion, and/or differentiation of cells. In some embodiments, an agent, e.g., the first agent, that provides a primary activation signal to the cells is reversibly bound to the multimerization reagent. The multimerization reagent comprises at least one binding site, e.g., Z1 for binding of the first agent, wherein the first agent comprises at least one binding partner, e.g., C1, wherein the binding partner C1 is capable of binding to the Z1 binding site of the multimerization reagent. In some embodiments, the first agent is linked to the multimerization reagent via the bond formed between the C1 binding partner and the Z1 binding site, and the first agent binds to a receptor molecule on the surface of the cells, thereby providing a primary activation signal to the cells and thereby activating the cells. In some embodiments, when a soluble multimerization agent is used, the bond between the C binding moiety, e.g., C1, and the Z binding site, e.g., Z1, need not be reversible.

[00408] In some embodiments, the provided methods also include using a multimerization reagent having bound thereto one or more additional agents, e.g., a second agent or a third agent, such as targeting an accessory or costimulatory molecule or other signaling molecule in the cell, which stimulates one or more additional signals, e.g., a costimulatory or accessory signal. In some cases, the multimerization agent is immobilized on a support, e.g., a solid support or stationary phase. In some embodiments, the multimerization agent is not immobilized on a support, i.e., it is in soluble form.

[00409] In some embodiments, the one or more additional agents, e.g., second agent, comprises a binding partner C (e.g., C1, C2, C3, etc.) that is capable of being reversibly bound to a binding site Z (e.g., Z1, Z2, Z3, etc.). In some embodiments, the one or more additional agents, e.g., second agent, comprises a binding partner, e.g., C2, wherein the binding partner, e.g., C2, is capable of being reversibly bound to a binding site, e.g., Z2 of the multimerization reagent, wherein the second agent is bound to the multimerization reagent via the reversible bond formed between the binding partner C2 and the binding site Z2. In some embodiments, the bond formed between the binding partner C1 and the binding site Z1 may be reversible, and the bond formed between the binding partner C2 and the binding site Z2 may be reversible. In this case, the dissociation constant (KD) for the reversible binding between said binding site Z1 and said binding partner C1 and/or for the reversible binding between said binding site Z2 and said binding partner C2 may be in the range of 10-2 M to 10-13 M. In some aspects, such as when the multimerization reagent is not bound to a support (e.g., not bound to a solid support or stationary phase), the bond formed between the binding partner C1 and the binding site Z1 may be irreversible and/or also the bond formed between the binding partner C2 and the binding site Z2 may be irreversible.

[00410] In some cases, the one or more additional agents, e.g., a second agent, binds to the accessory molecule on the surface of the cells, thereby stimulating the activated cells. In this embodiment, the first agent can stimulate a signal associated with the TCR/CD3 complex on the T cells and can be a binding agent that specifically binds CD3. In this embodiment, the accessory molecule on the T cell can be CD28 and the second agent that binds the accessory molecule is a binding reagent that specifically binds CD28. Alternatively, in some embodiments, it is found that targeting of other accessory molecules can also be employed, which can, in some cases, alter, such as enhance, one or more aspects, properties, or characteristics of the cultured cells. In some embodiments, the accessory molecule can be one or more of CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, or HVEM (e.g., an anti-CD90 antibody, an anti-CD95 antibody, an anti-CD137 antibody, and an anti-CD154 antibody, anti-ICOS antibody, anti-LAT antibody, anti-CD27 antibody, anti-OX40 antibody, or anti-HVEM antibody, respectively). Exemplary agents, such as receptor binding agents (e.g., stimulatory agents) are described below.

[00411] In some embodiments, the one or more additional agents, e.g., third agent, binds to an additional molecule that modulates survival, proliferation, and/or one or more other properties of the cells. Such targeting molecules include, but are not limited to, cytokine receptors, chemokine receptors, or adhesion molecules. In some aspects, stimulation by such additional molecules can result in one or other improved aspects of the stimulated or cultured cells, including, for example, increased persistence, survival, and/or less exhausted phenotype or activity. Exemplary agents, such as receptor binding agents (e.g., stimulatory agents), are described below.

[00412] In some embodiments, the provided method can be performed at any temperature at which the viability of the cell population is at least essentially non-compromised. In some embodiments, the condition at which the incubation or culture is performed includes any conditions that are at least essentially non-detrimental, non-damaging, or at least essentially non-compromising viability, e.g., under which the percentage of the cell population that is to be expanded to full viability is at least 70%, including at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5%. In some embodiments, the provided method is performed at a temperature of about 20°C or higher. Depending on the cell population to be expanded, a suitable temperature range may, for example, be from about 20°C to about 45°C, including from about 25°C to about 40°C, or from about 32°C to 37°C. In some embodiments, a method according to the invention is carried out at a constant temperature value, or at a temperature value selected from ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, 1°C or ± about 0.5°C. The person skilled in the art is able to empirically determine a suitable temperature, taking into account the nature of the cells and the expansion conditions. Typically, human cells are expanded at a temperature such as 37°C.

[00413] According to the present disclosure, there are also provided multimerized agents, or composition comprising multimerization reagents reversibly linked to one or more receptor binding agents, that are capable of stimulating, e.g., expanding or modulating a signal, in a population of cells. Such a multimerized agent that is capable of stimulating, e.g., expanding or modulating a signal, in a population of cells is a multimerization reagent that is not bound to a support (e.g., in soluble form) and comprises at least one binding site Z, e.g., Z1, for the reversible binding of a first agent that provides a primary activating signal to the cells, wherein the multimerization reagent has reversibly bound thereto, said first agent that provides a primary activating signal to the cells; wherein the first agent comprises at least one binding partner C, e.g., C1, wherein the binding partner C1 is capable of reversibly binding to at least one binding site Z1 of the multimerization reagent, wherein the first agent is bound to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1. It should be noted herein that such a multimerization agent may have immobilized thereon any of the first agents that are described herein.

[00414] In some embodiments, a multimerized agent provided herein can further comprise at least one binding site, e.g., Z2, for reversibly binding a second agent that stimulates an accessory molecule on the surface of cells, wherein the multimerization reagent has reversibly bound thereto the second agent that stimulates an accessory molecule on the surface of cells, wherein the second agent comprises a binding partner, e.g., C2, wherein the C2 binding partner is capable of binding to the at least one Z2 binding site of the multimerization reagent. In this embodiment, the second agent is bound to the multimerization reagent via the bond formed between the C2 binding partner and the Z2 binding site. In some embodiments, the second agent is any that can bind to CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, or HVEM (e.g., an anti-CD90 antibody, an anti-CD95 antibody, an anti-CD137 antibody, and an anti-CD154 antibody, anti-ICOS antibody, anti-LAT antibody, anti-CD27 antibody, anti-OX40 antibody, or anti-HVEM antibody, respectively).

[00415] In some embodiments, a multimerized agent provided herein can also comprise at least one binding site, e.g., Z that is the same or different from Z1 or Z2, for reversibly binding an additional agent that binds to an additional molecule that modulates survival, proliferation, and/or one or more other properties of cells, wherein the multimerization reagent reversibly binds to the additional agent that stimulates an accessory molecule on the surface of cells, wherein the additional agent comprises a binding partner, e.g., C that is the same or different from C1 or C2, wherein the binding partner C is capable of binding to the at least one Z binding site of the multimerization reagent.

[00416] In some embodiments, culturing the composition containing target cells (e.g., T cells) with the multimerized agent (e.g., anti-CD3/anti-CD28 mutein streptavidin or oligomer thereof) can be performed in a bioreactor such as a hollow fiber bioreactor (e.g., Quantum® Cell Expansion System hollow fiber bioreactor) or a plastic bag bioreactor (e.g., Cellbag® used in GE Healthcare's Xuri Cell Expansion System W25).

[00417] In some embodiments, the method further includes contacting the cultured target cells (e.g., T cells) in the reaction mixture (e.g., containing the target cells, e.g., T cells, bound to the multimerization reagent via, e.g., the first agent and the second agent) with (i) a competition reagent (e.g., free first binding partner C, e.g., C1) or an analogue thereof capable of disrupting the bond between the first binding partner, e.g., C1, and the binding site, e.g., Z1, and/or (as needed) (ii) a second competition reagent, e.g., free second binding partner, e.g., C2, or an analogue thereof, capable of disrupting the bond between the second binding partner C2 and the binding site Z2. In doing so, the reversible binding between said C1 binding partner of the first agent and said Z1 binding sites, as well as the reversible binding between said C2 binding partner of the second agent and said Z2 binding site of said multimerization reagent is disrupted, thereby releasing into an eluate the T cells bound to the multimerization reagent via the first agent and the second agent and disrupting the stimulation and/or expansion of the T cells.

[00418] In some embodiments, the competition reagent (e.g., the first and/or second competition reagent) is added within 5 days of starting the incubation, such as within 4 days, 3 days, 2 days, or 1 day of starting the incubation. Thus, by controlling the time at which stimulation is disrupted, one or more particular characteristics of the cultured T cells eluted from the multimerized agent can be altered as described herein.

[00419] In some embodiments, the method further includes separating or removing one or more of the components remaining after reversible dissociation of the components. In some embodiments, any unbound or residual biotin in the cultured target cells (e.g., T cells) can be separated or removed. In some embodiments, the multimerization reagent is removed or separated from the cells in the cultured target cell composition. For example, in some embodiments, the separation/removal can be accomplished using a second stationary phase. To this end, a mixture comprising the target cells (e.g., T cells) and the soluble multimerization reagent are exposed, before or after being applied to the first stationary phase described above, to chromatography on a suitable second stationary phase. This secondary stationary phase can be a gel filtration matrix and/or affinity chromatography matrix, wherein the gel filtration and/or affinity chromatography matrix comprises an affinity reagent. The affinity reagent comprised on the chromatography resin includes a binding partner D that (specifically) binds to the Z1 binding site and/or Z2 binding site, if present, of the multimerization reagent, thereby immobilizing the multimerization reagent on the stationary phase. If a streptavidin-based multimerization reagent is used and both the first and second agents have a streptavidin-binding peptide as a C1 or C2 binding partner, the binding partner D that is included in the affinity reagent of this second stationary phase may be biotin. The soluble oligomer of streptavidin or a mutein of streptavidin that is used as the multimerization reagent then binds to biotin that is usually covalently coupled to a chromatography matrix, such as biotin-sepharose, which is commercially available. In some such embodiments, cultured cells (e.g., cultured T cells) can be recovered from the multimerization reagent.

A. Cells

[00420] In some embodiments, the sample of the cell population may be from any suitable source, typically a whole sample of a body tissue or a body fluid such as blood. In the latter case, the sample may be, for example, a population of peripheral blood mononuclear cells (PBMC) that may be obtained by standard isolation methods, such as a Ficoll gradient of blood cells. The cell population to be stimulated or expanded may, however, also be in purified form and may have been isolated using a reversible cell labeling/isolation technology as described in U.S. Pat. No. 7,776,562 , U.S. Pat. No. 8,298,782 , International Patent Pub. No. WO02/054065 or International Patent Pub. No. WO2013/011011. Alternatively, the cell population may also be obtained by cell separation by negative magnetic immunoadhesion, as described in US patent 6,352,694 B1 or European patent EP 0 700 430 B1. If an isolation method described herein is used in basic research, the sample may be cells from in vitro cell culture experiments. The sample will typically be prepared in the form of a fluid, such as a solution or dispersion.

[00421] The cells contained in the composition containing target cells are generally eukaryotic cells, such as mammalian cells, and are typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, or are cells of the immune system, such as cells of innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from an individual and/or isolated from an individual and frozen.

[00422] In some embodiments, the reversibly linked agents, such as multimerized agents, provided herein are capable of expanding a lymphocyte population or a subpopulation within the lymphocyte population. The lymphocyte population to be expanded may be any suitable population, e.g., a B cell population, a T cell population, or a natural killer cell population. The T cell population may be an antigen-specific T cell population, a helper T cell population, a cytotoxic T cell, a memory T cell, a regulatory T cell, or a natural killer T cell population. Accordingly, in such embodiments of the multimerized agent, the first agent is capable of stimulating a signal associated with the TCR/CD3 complex on T cells. The first agent present in the multimerized agent may thus be a binding reagent that specifically binds to CD3, while the second agent that binds to the accessory molecule, such as may be a binding agent that specifically binds to CD28, CD137 CD90, CD95, CD137, CD154, ICOS LAT, CD27, OX40 or HVEM.

[00423] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD3+, CD4+, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation capabilities, expansion, recirculation, localization and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Methods include off-the-shelf methods. In some aspects, such as off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them as described herein, and reintroducing them into the same patient, either before or after cryopreservation.

[00424] In some embodiments, the T cells, such as CD3+, CD4+, or CD8+ cells, are no longer enriched for another marker. In some embodiments, the T cells are no longer enriched for CD62L+ cells.

[00425] Among the subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells are naïve T cells (TN), effector T cells (TEFF), memory T cells and their subtypes such as stem cell memory T cells (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T cells (MAIT), naturally occurring and adaptive regulatory T cells (Treg), helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[00426] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

Cell Preparation

[00427] In some embodiments, preparing the cells includes one or more culturing and/or preparation steps. The cells may be isolated from a sample, such as a biological sample, e.g., one obtained or derived from an individual. In some embodiments, the individual from which the cells are isolated is one who has the disease or condition or is in need of a cell therapy or to whom the cell therapy will be administered. The individual in some embodiments is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are being isolated, processed, and/or modified.

[00428] Thus, the cells in some embodiments are primary cells, e.g., primary human cells. Samples include tissue, fluid, and other samples taken directly from the individual, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and organ samples, including processed samples derived therefrom.

[00429] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organ and/or cells derived therefrom. Samples include, in the context of cellular therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[00430] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, e.g., from mouse, rat, non-human primate, or pig.

[00431] In some embodiments, isolating cells includes one or more steps of separating cells based on preparation and/or non-affinity. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, e.g., to remove undesirable components, enrich for desired components, lyse, or remove cells sensitive to specific reagents. In some examples, cells are separated based on one or more properties, such as density, adherent properties, size, sensitivity, and/or resistance to particular components.

[00432] In some examples, cells from the circulating blood of an individual are obtained, for example, by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects, contain cells other than red blood cells and platelets.

[00433] In some embodiments, blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in a buffer or medium appropriate for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is devoid of calcium and/or magnesium and/or most or all divalent cations. In some aspects, a wash step is performed in a semi-automated “full flow” centrifuge (e.g., the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a wash step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, e.g., Ca++/Mg++-free PBS. In certain embodiments, components of a blood cell sample are removed, and the cells are resuspended directly in culture media.

[00434] In some embodiments, the methods include density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysing the red blood cells and centrifuging through a Percoll or Ficoll gradient.

[00435] In some embodiments, isolation methods include separating different cell types based on the expression or presence on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. Separation methods can include any of those disclosed herein, including methods utilizing reversible reagent systems, e.g., agents (such as receptor binding agents or selection agents) and reagents as described herein.

[00436] In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, isolation in some aspects includes separating cells and cell populations based on the level of expression or expression by the cells of one or more markers, typically cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such markers, generally followed by washing steps and separation of cells that bound the antibody or binding partner from cells that did not bind the antibody or binding partner.

[00437] Such separation steps may be based on positive selection, in which cells that bind the reagent are retained for later use, and/or negative selection, in which cells that have not bound the antibody or binding partner are retained. In some examples, both fractions are retained for later use. In some aspects, negative selection may be particularly useful when there is no antibody available that specifically identifies a cell type in a heterogeneous population, such that separation is best performed based on markers expressed by cells other than the desired population.

[00438] Separation need not result in 100% enrichment or removal of a particular population of cells or cells expressing a particular marker. For example, positive selection or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[00439] In some examples, multiple cycles of separation steps are performed, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can be simultaneously positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[00440] For example, in some aspects, specific subpopulations of T cells, such as cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.

[00441] For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28-conjugated magnetic beads (e.g., DYNABEADS M-450 CD3/CD28 T Cell Expander).

[00442] In some embodiments, isolation is accomplished by enrichment for a particular population of cells by positive selection, or depletion of a particular population of cells by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (+ marker) at a relatively higher level (high marker) on the positively or negatively selected cells, respectively.

[00443] In some embodiments, T cells are separated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. These CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more subpopulations of naive, memory, and/or effector T cells.

[00444] In some embodiments, CD8+ cells are enriched or depleted of stem, central memory, effector memory, and/or naïve central memory cells, such as positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T cells (MCT) is performed to enhance efficacy so as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, the combination of MCT-enriched CD8+ T cells and CD4+ T cells further improves efficacy.

[00445] In embodiments, memory T cells are present in CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched or depleted of CD62L-CD8+ and/or CD62L+ CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[00446] In some embodiments, enrichment for central memory T cells (TCM) is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is accomplished by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T cells (TCM) is accomplished by starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on CD14 and CD45RA expression, and a positive selection based on CD62L. Such selections in some aspects are performed simultaneously and in other aspects are performed sequentially, in any order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more additional positive or negative selection steps.

[00447] CD4+ T helper cells are classified into effector, central memory, and effector cells by identifying cell populations bearing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naïve CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L and CD45RO-.

[00448] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is attached to a solid support or matrix, such as a magnetic bead or a paramagnetic bead, to allow for the separation of cells for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or magnetic affinity) separation techniques (reviewed in Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[00449] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, is generally directly or indirectly linked to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that is desired to separate, e.g., i.e., desired to negatively or positively select.

[00450] In some embodiments, the magnetic particle or bead contains a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452,342 B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in Owen, U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[00451] Incubation is generally performed under conditions in which antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, that specifically bind to such antibodies or binding partners, that are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[00452] In some aspects, the sample is placed in a magnetic field, and cells that have magnetically responsive or magnetizable particles attached to them will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where both positive and negative fractions are retained and further processed or subjected to further separation steps.

[00453] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are bound to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, the streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[00454] In some embodiments, the magnetically responsive particles are left attached to cells that are to be subsequently incubated, cultured, and/or modified; in some aspects, the particles are left attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of unlabeled competing antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[00455] In some embodiments, the affinity-based selection is via magnetic activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high purity selection of cells having magnetized particles bound thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are eluted sequentially after the application of the external magnetic field. That is, the cells bound to magnetized particles are held in place while the unbound species are eluted. Then, after this first elution step is complete, the species that were trapped in the magnetic field and were prevented from being eluted are released in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labeled and depleted from the heterogeneous population of cells.

[00456] In certain embodiments, the isolation or separation is accomplished using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culturing, and/or formulation steps of the methods. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize errors, user handling, and/or contamination. In one example, the system is a system as described in International Patent Publication No. WO2009/072003, or US 20110003380 A1.

[00457] In some embodiments, the system or apparatus performs one or more, e.g., all of the isolation, processing, engineering, and formulation steps in an integrated or independent system, and/or in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows the user to program, control, evaluate the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[00458] In some aspects, the separation and/or other steps are performed using the CliniMACS system (Miltenyi Biotec), e.g., for automated cell separation at clinical scale level in a closed, sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and multiple squeeze valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a support for the selection column. The peristaltic pump controls the flow rate throughout the tubing assembly and, together with the squeeze valves, ensures controlled flow of buffer through the system and continuous suspension of the cells.

[00459] The CliniMACS system in some aspects uses magnetizable particles coupled to antibodies that are provided in a sterile, non-pyrogenic solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. A cell preparation bag is then attached to the tube set, which in turn is attached to a bag containing buffer and a cell collection bag. The tube set consists of pre-assembled sterile tubes, including a pre-column and a separation column, and are for single use only. After the separation program is initiated, the system automatically applies the cell sample to the separation column. Labeled cells are retained within the column, while unlabeled cells are removed by a series of wash steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained on the column. In some embodiments, cell populations for use with the methods described herein are labeled and are retained on the column. In some embodiments, cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field and are collected within the cell collection bag.

[00460] In certain embodiments, the separation and/or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system, in some aspects, is equipped with a cell processing unit that allows for automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include integrated camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the product from the cell of origin. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber that performs cell culture protocols, such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Inlet ports may allow for sterile removal and replenishment of media, and cells may be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9):651–660, Terakura et al. (2012)Blood. 1:72–82, and Wang et al. (2012) J Immunother. 35(9):689–701.

[00461] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells labeled for multiple cell surface markers are transported in a fluid stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale sorting (FACS). In certain embodiments, a cell population described herein is collected and enriched (or depleted) by utilizing microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. 2010) Lab Chip 10, 1567-1573 and Godin et al. (2008) J Biophoton, 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing the isolation of well-defined T cell subsets with high purity.

[00462] In some embodiments, the antibodies or binding partners are labeled with one or more detectable markers, to facilitate separation for positive and/or negative selection. For example, separation can be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on binding of antibodies or other binding partners specific to one or more cell surface markers is carried out in a fluid stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[00463] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, prior to or after isolation, incubation, and/or handling. In some embodiments, freezing and subsequent thawing removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of freezing solutions and parameters known in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with the media, such that the final concentration of DMSO and HSA is 10% and 4%, respectively. The cells are then frozen at -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[00464] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culturing, growing, stimulating, activating, and/or propagating. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[00465] The conditions may include one or more particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[00466] In some embodiments, the stimulating conditions or agents include one or more agents, e.g., ligand, that is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent activates or initiates the TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, e.g., bound to a solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method can further comprise the step of adding anti-CD3 and/or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/mL). In some embodiments, the stimulating agents include IL-2 and/or IL-15, e.g., an IL-2 concentration of at least about 10 units/mL.

[00467] In some aspects, the incubation is carried out according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):651660, Terakura et al. (2012) Blood. 1:72-82 and/or Wang et al. (2012) J Immunother. 35(9):689-701.

[00468] In some embodiments, the T cells are expanded by adding to the composition feeder cells, such as undivided peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the undivided feeder cells may comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the T cell populations.

[00469] In some embodiments, the stimulation conditions include temperature suitable for growth of human T lymphocytes, e.g., at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation can further include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects are provided in any suitable amount, such as a ratio of LCL feeder cells to starting T lymphocytes of at least about 10:1.

[00470] In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating native or antigen-specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated for cytomegalovirus antigens by isolating T cells from infected individuals and stimulating the cells in vitro with the same antigen.

B. Apparatus and Manufacturing Articles

[00471] In some embodiments, an apparatus or article of manufacture is also provided. In some embodiments, an arrangement of a bioreactor and a first stationary phase for chromatography is provided. The bioreactor is suitable for expanding cells, and the stationary phase is suitable for cell separation and reagent removal. The first stationary phase is a gel filtration and/or affinity chromatography matrix, wherein the gel filtration and/or affinity chromatography matrix comprises an affinity reagent, such as a multimerization reagent as described, wherein the reagent comprises a Z-binding site (e.g., Z1) specifically binding to a C-binding partner (e.g., C1) comprised in a first agent and/or the affinity reagent, such as a multimerization reagent as described, comprises a Z-binding site (e.g., Z2) specifically binding to a C-binding partner (e.g., C2) comprised in a second agent. In some embodiments, the gel chromatography and/or affinity chromatography comprises an affinity reagent, such as a multimerization reagent as described, comprising the binding site Z (e.g., the same or different from the first or second agent) specifically binding to a binding partner C comprised in the additional agent (e.g., the same or different from that comprised in the first or second agent). The first stationary phase is thus suitable for immobilizing the first agent, the second agent and/or the additional agent, the binding partner C, such as the first binding partner C1 and/or the second free binding partner C2. Furthermore, the bioreactor and the stationary phase are fluidly connected. This arrangement can be used in serial expansion, as explained above, and can be integrated into known cell expansion systems, such as the Quantum® Cell Expansion System) or the Xuri W25 Cell Expansion System.

[00472] In this arrangement, the first stationary phase is comprised of a chromatography column or is a planar stationary phase. The arrangement may further comprise a second stationary phase that is fluidly bound to the first stationary phase. The secondary stationary phase may be a gel filtration matrix and/or affinity chromatography matrix, wherein the gel filtration and/or affinity chromatography matrix comprises an affinity reagent. This affinity reagent may comprise a binding partner D that (specifically) binds to the Z1 binding site of the multimerization reagent, thereby being suitable for immobilizing the multimerization reagent on the stationary phase.

[00473] Also provided is an apparatus for purification (e.g., selection) and culture, such as stimulation or expansion, of a composition of cells, the apparatus comprising at least one arrangement of a bioreactor and a first stationary phase or a second stationary phase for chromatography as defined above.

[00474] The apparatus may further comprise a plurality of arrangements of a bioreactor and a stationary phase being fluidly connected in series.

[00475] The apparatus may comprise a sample inlet being fluidly connected to the bioreactor of a bioreactor arrangement and the stationary phase for chromatography. The apparatus may also comprise a sample outlet for purified and expanded target cells, the sample outlet being fluidly connected to the stationary phase of the latter of at least one arrangement of a bioreactor and the stationary phase for chromatography.

[00476] In some embodiments, the apparatus may be designed as a functionally closed system.

C. Exemplary Characteristics of Cultured Cells

[00477] In some embodiments, the cultured target cells, (e.g., cultured T cells), which may include cultured cells generated or produced in accordance with the methods provided herein, exhibit one or more specified phenotypic and/or functional characteristics based on or related to their proliferation capacity, expression of surface markers, differentiation state, activation state, and/or metabolic profile. In some embodiments, culturing the target cells (e.g., T cell culture) in accordance with any of the methods provided results in a change in a parameter associated with the function (e.g., increase or decrease in a functional activity) or phenotype (e.g., greater or lesser expression of a marker or markers) of cells as compared to the corresponding or respective function or phenotype of cells in the composition prior to incubation in accordance with the methods provided herein. In some embodiments, the cultured T cells exhibit a change in a parameter between expansion and/or proliferation capacity, CD4+/CD8+ T cell distribution or ratio, surface marker expression, functional activity, or metabolic profile.

[00478] In some embodiments, the change in the parameter, as measured in the cultured T cells, is compared or referenced to the same or similar parameter, as measured in a reference T cell composition or preparation. Typically, the T cells in the reference T cell composition or preparation include or are derived from the same or substantially the same T cell composition prior to incubation with the reversibly bound agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin), unless such cells were not subjected to incubation or were subjected to a different incubation. In some embodiments, the reference T cell preparation is subjected to incubation using substantially the same protocol or conditions (e.g., type of stimulatory agent or agents, format of stimulatory agent or agents, substantially the same starting cell numbers, washes, presence or absence of additional reagents, incubation time, incubation temperature), except at least one aspect, and in some cases only one aspect, of such incubation in a different reference T cell preparation than the incubation that produces the cultured T cells. In some embodiments, the reference T cell preparation is one in which the incubation is not performed using a multimerization reagent, but instead in which the receptor binding agents, e.g., stimulatory agents, are presented in an alternative formation, such as directly and/or non-reversely linked or conjugated to a support (e.g., solid support), e.g., a bead, particle, magnetic particle or bead, nanoparticle, or microsphere.

[00479] In some embodiments, the reference T cell composition or preparation is the composition containing T cells prior to incubation with a reversibly bound agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin).

[00480] In some embodiments, cultured T cells are generated by incubation with a reversibly bound agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin) for less than 5 days and/or where the association of such agent with one or more molecules on the cell is disrupted (e.g., in the presence of a competing reagent, e.g., biotin or a biotin analog), such as disrupted within 5 days of initiating incubation with such agent. For example, in some aspects, cultured T cells are generated or produced following incubation with a reversibly bound agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin) as described herein, wherein the incubation is terminated and/or disrupted within 5 days after initiation of such incubation (such as within or about 4, 3, 2, or 1 day, or less), and/or when a competing agent (e.g., biotin) that dissociates the reversibly bound agent from the cells is added to the incubated cells within 5 days after initiation of such incubation (such as within or about 4, 3, 2, or 1 day, or less). In some embodiments, the reference T cell preparation is generated or produced after incubation with the same or substantially the same reversibly linked agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), but where the incubation is carried out for more than 5 days, it is not terminated and/or disrupted to diminish or terminate the induced or modulated signal in the cell, and/or where the T cell preparation is produced without the addition of a competing agent (e.g., biotin or biotin analog) that dissociates the reagent from the cells.

[00481] In some embodiments, cultured T cells are generated by incubation with a reversibly bound agent (e.g., multimerizing agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin) wherein the receptor-binding agent (e.g., stimulatory agent) is one that does not bind to CD28 and/or induce signaling, i.e., is not an anti-CD28 antibody or fragment thereof. For example, in some embodiments, cultured T cells are produced or generated after incubation with a reversibly linked reagent, wherein one or more stimulatory agents are reversibly linked to a mutein streptavidin, wherein at least one stimulatory agent is specific for CD3 (e.g., anti-CD3 antibody or a fragment thereof) and a second stimulatory agent can be specific for one or more of CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, or HVEM (e.g., an anti-CD90 antibody, an anti-CD95 antibody, an anti-CD137 antibody, and an anti-CD154 antibody, anti-ICOS antibody, anti-LAT antibody, anti-CD27 antibody, anti-OX40 antibody, or anti-HVEM antibody, respectively, or antigen-binding fragments thereof). In some embodiments, the reference T cell preparation is a culture of T cells generated or produced after incubation with a reversibly bound agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin), but wherein the reagent comprises an agent that specifically binds to CD28 and/or induces or modulates CD28 signaling. For example, in some embodiments, the reference T cell preparation generated or produced after incubation of a T cell composition with anti-CD3/anti-CD28 Dynabeads beads, anti-CD3/anti-CD28 ExPact, or another anti-CD3/anti-CD28 stimulatory agent. In some embodiments, such another anti-CD3/anti-CD28 stimulatory agent is one wherein the antibody reagents are bound to a support (e.g., solid support), for example, a bead, particle, magnetic particle or bead, nanoparticle, or microsphere. In some embodiments, cultured T cells are prepared by incubation with a reversibly bound agent (e.g., multimerizing agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric streptavidin) that is soluble, i.e., not bound to a support (e.g., solid support).

[00482] For example, in some embodiments, cultured T cells are provided, such as prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation with a reversibly linked agent as described herein (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), wherein the cultured T cells are characterized by an increased capacity for expansion and/or proliferation compared to a reference T cell composition or preparation. In some embodiments, the increased expansion and/or proliferation capacity comprises an increase in the number or percentage of CD3+ T cells, CD4+ T cells, and/or CD8+ T cells in the cultured T cells by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of CD3+ T cells, CD4+ T cells, and/or CD8+ T cells, respectively, in the reference T cell composition or preparation.

[00483] In some embodiments, cultured T cells are provided, such as prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation with a reversibly linked agent as described herein (e.g., multimerizing agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), wherein the cultured T cells are characterized by an increased capacity for CD3+ T cell expansion and/or proliferation compared to a reference T cell culture. In some embodiments, the increased capacity for expansion and/or proliferation comprises an increase in the number or percentage of CD3+ T cells in the cultured T cells by at least about 2-fold (such as at least about any of 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of CD3+ T cells in the reference T cell composition or preparation.

[00484] In some embodiments, cultured T cells are provided, such as prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation with a reversibly linked agent as described herein (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), wherein the cultured T cells are characterized by an increased expansion and/or proliferation capacity of CD4+ T cells compared to a reference T cell composition or preparation. In some embodiments, the increased capacity for expansion and/or proliferation comprises an increase in the number or percentage of CD4+ T cells in the cultured T cells by at least about 2-fold (such as at least about any of 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of CD4+ T cells in the reference T cell composition or preparation.

[00485] In some embodiments, cultured T cells are provided, such as prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation with a reversibly linked agent as described herein (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), wherein the cultured T cells are characterized by an increased capacity for CD8+ T cell expansion and/or proliferation compared to a reference T cell composition or preparation. In some embodiments, the increased capacity for expansion and/or proliferation comprises an increase in the number or percentage of CD8+ T cells in the cultured T cells by at least about 2-fold (such as at least about any of 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of CD8+ T cells in the reference T cell composition or preparation.

[00486] In some embodiments, cultured T cells are provided, such as prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation with a reversibly linked agent as described herein (e.g., multimerizing agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), wherein the cultured T cells are characterized by an altered CD8+/CD4+ T cell distribution or normalized T cell distribution, such as an altered CD8+/CD4+ ratio or normalized CD8+/CD4+ T cell ratio, compared to a reference T cell composition or preparation. The CD8+/CD4+ ratio or normalized ratio may be increased or decreased. In some embodiments, the altered CD8+/CD4+ T cell ratio results from an increase in the number or percentage or normalized number or percentage of CD8+ T cells in the cultured T cells relative to or compared to the number or percentage or normalized number or percentage in a reference composition or preparation. In some embodiments, the number of CD8+ T cells in the cultured T cells is increased by at least about 2-fold (such as at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of CD8+ T cells or to the normalized number or percentage of CD8+ T cells in the reference T cell composition or preparation. In some embodiments, the CD8+/CD4+ T cell ratio or the normalized CD8+/CD4+ ratio is increased by at least about 2-fold (such as at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the CD8+/CD4+ T cell ratio or the normalized CD8+/CD4+ ratio in the reference T cell composition or preparation. In some embodiments, the number, percentage, or ratio in the cultured T cells or in a composition or preparation is normalized to the number, percentage, or ratio in the starting composition containing the T cells prior to incubation.

[00487] In some embodiments, provided are cultured T cells prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation with a reversibly linked agent as described herein (e.g., multimerizing agent, such as incubation with a stimulatory agent reversibly linked to an oligomeric mutein streptavidin), and wherein the cultured T cells are characterized by an altered surface marker expression profile compared to a reference T cell composition or preparation. In some embodiments, the altered surface marker expression profile is due to a change in the number or percentage of one or more T cell subsets that are positive, negative, high, or low for one or more surface markers selected from CD45RA, CD45RO, CD62L CD69, CCR7, CD27, CD28, CD122, T-bet, IL-7Ra, CD95, IL-2Rβ, CXCR3, LFA-1, KLRG1. In some embodiments, the number or percentage of the T cell subset in the cultured T cells is increased by at least about 2-fold (such as at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference composition or preparation.

[00488] In some embodiments, the T cell subset in the cultured T cells (e.g., a T cell subset that is increased in the cultured T cells compared to the reference composition or preparation) exhibits a reduced or decreased differentiation or activation state compared to the reference T cell composition or preparation. In some embodiments, the T cell subset is not or does not include an effector T cell (ET) or effector memory T cell (EMT) phenotype. In some embodiments, the T cell subset contains a surface phenotype that is one or more of CD62L+, CCR7+, CD27+, CD28+, or KLRG1low/-. In some embodiments, such a T cell subset in the cultured T cells is increased by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference T cell composition or culture.

[00489] In some embodiments, the T cell subset in the cultured T cells (e.g., a T cell subset that is increased in the cultured T cells compared to the reference composition or preparation) is positive for CD62L and/or IL-7Rα (CD127) and/or negative or low for t-bet. In some embodiments, the T cell subset is positive for CD45RA and/or negative or low for CD45RO. In some embodiments, the T cell subset is positive for one or more of CCR7, CD45RA, CD62L, CD27, CD28, IL-7Rα (CD127), CD95, IL-2Rβ, CXCR3, and LFA-1 and/or negative for CD45RO. In some embodiments, such a T cell subset in the cultured T cells is increased by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference T cell composition or culture.

[00490] In some embodiments, the T cell subset in the cultured T cells (e.g., a T cell subset that is increased in the cultured T cells compared to the reference composition or preparation) is or includes cells that are positive for CD62L (CD62L+). In some embodiments, such a T cell subset in the cultured T cells is increased by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference T cell composition or culture.

[00491] In some embodiments, the T cell subset in the cultured T cells (e.g., a T cell subset that is increased in the cultured T cells compared to the reference composition or preparation) is or includes CD62L+ cells and a) any one or more of CD45RA low/+, CD45RO low/+, CCR7+, and CD27+ and b) any one or more of t-betlow, IL-7Ra+ (CD127+), CD95+, IL-2Rp+, CXCR3+, and LFA-1+. In some embodiments, the T cell subset can also be CD3+, CD4+, or CD8+. In some embodiments, such a T cell subset in the cultured T cells is increased by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference T cell composition or culture.

[00492] In some embodiments, the T cell subset, such as a CD62L+ T cell subset, in the cultured T cells is or includes or shares phenotypic characteristics with memory T cells or particular subsets thereof, such as long-lived memory T cells. In some embodiments, such memory T cells are central memory T cells (TCM) or stem memory T cells (TSCM). In some embodiments, the memory T cells are TSCM cells. TSCM cells can be described as having one or more phenotypic differences or functional characteristics compared to other memory T cell subsets or compared to naive T cells, such as less differentiated or more naive (see, e.g., Ahlers and Belyakov, 2010) Blood, 115:1678); Cieri et al. (2015) Blood, 125:2865; Flynn et al. (2014) Clinical & Translational Immunology, 3, e20; Gattinoni et al. (2012) Nat. Med. 17:1290–1297; Gattinoni et al. (2012) Nat. Reviews, 12:671; Li et al. (2013) PLOS ONE, 8:e67401; and published PCT application No. WO2014/039044). In some cases, TSCM cells are considered the only memory T cells capable of generating effector T cells and all three memory T cell subsets (TSCM, TCM, and TEM). In some respects, TSCM cells have the greatest survival and proliferation response to antigenic or homeostatic stimuli of all memory T cell subsets and the least attrition absent cognate antigen. In some embodiments, less differentiated TSCM cells may exhibit greater expansion, long-term viability, and target cell killing following adoptive transfer than other memory T cells, and thus may be able to mediate more effective treatment with fewer transferred cells than would be possible for TCM or TEM cells.

[00493] In some aspects, examples of phenotypic or functional characteristics that have been reported or are known for TSCM cells include, for example, that such cells a) are CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, IL-7Rα.+, CD95+, IL-2Rβ+, CXCR3+, and LFA-1+; b) are CD45RA+, CCR7+, CD62L+, and CD95+; c) are CD45RA+, CD45RO+, CCR7+, CD62L+, CD27+, CD28+, CD95+, and IL-2Rβ+; d) are CD45RO-, CD45RA+, CCR7+, CD62L+, CD27+, CD28+, CD127+, and CD95+; e) are CD45RA+, CD44+/-, CD62L+, CD127+, IL-2R <0045>+, CD28+, CD43-, KLRG1-, Peforin-, and GranzymeB-; f) express high levels of CCR7 CD62L, CD27, and CD28, intermediate levels of CD95 and IL-2Rβ, low levels of CD45RA, and do not express CD45RO or KLRG-1; or g) express high levels of CD62L, low levels of CD44 and t-bet, and are Sca-1+; and/or have intermediate IL-2 production capacity, low IFNY production capacity, low cytotoxicity, and high self-renewal capacity.

[00494] In some embodiments, the T cell subset in the cultured T cells (e.g., a T cell subset that is increased in the cultured T cells compared to the reference composition or preparation) is or includes memory T cells, such as memory T cells. In some embodiments, the memory T cells are central memory (CMT) T cells. In some embodiments, the T cell subset has a CD45RA-, CD45ROlow/+, CCR7+, CD62L+, CD27+, CD28+, CD95+ CD122+, and/or KLGR1low phenotypic characteristic. In some embodiments, such a T cell subset in the cultured T cells is increased by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference T cell composition or culture.

[00495] In some embodiments, the memory T cells are stem central memory (TSCM) T cells. In some embodiments, the T cell subset has a CD45RA+, CD45RO+, CCR7+, CD62L+, CD27+, CD28+, CD95+, CD122+, and/or KLGR1- phenotypic characteristic. In some embodiments, the T cell subset has a CD45RA+, CD45RO-, CCR7+, CD62L+, CD27+, CD28+, CD95+, CD122+, and/or KLGR1- phenotypic characteristic. In some embodiments, the T cell subset has a CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+, and/or LFA-1+ phenotypic characteristic. In some embodiments, the T cell subset has a CD45RA+, CCR7+, CD62L+, and/or CD95+ phenotypic characteristic. In some embodiments, the T cell subset has a CD45RA+, CD45RO+, CCR7+, CD62L+, CD27+, CD28+, CD95+, and/or IL-2Rβ+ phenotypic characteristic. In some embodiments, the T cell subset has a CD45RO-, CD45RA+, CCR7+, CD62L+, CD27+, CD28+, CD127+, and/or CD95+ phenotypic characteristic. In some embodiments, the T cell subset has a CD45RA+, CD44+/-, CD62L+, CD127+, IL-2Rβ+, CD28+, CD43-, KLRG1, Pephorin-, and/or Granzyme B- phenotypic characteristic. In some embodiments, the T cell subset expresses high levels of CCR7, CD62L, CD27, and/or CD28, intermediate levels of CD95 and/or IL-2Rβ, low levels of CD45RA, and/or does not express CD45RO and/or KLRG-1. In some embodiments, the T cell subset expresses high levels of CD62L, low levels of CD44 and t-bet, and/or is Sca-1+. In some embodiments, the T cell subset has an intermediate phenotypic characteristic of IL-2 production capacity, low IFNY production capacity, low cytotoxicity, and/or high self-renewal capacity. In some embodiments, such a T cell subset in the cultured T cells is increased by at least about 2-fold (such as by at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) compared to the number or percentage of the T cell subset in the reference T cell composition or culture.

[00496] In some embodiments, the T cell subset, such as any T cell subset described above, is present at a greater percentage of the total T cells in the cultured T cells or at a greater number of total T cells in the cultured T cells compared to the T cells in a reference T cell composition or preparation. In some embodiments, the percentage of the T cell subset in the cultured T cells as a percentage of the total T cells or total cells in the culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the percentage of the T cell subset in the cultured cells, such as any T cell subset described above, is greater, e.g., at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or greater, than the corresponding percentage of the cell subset in a T cell in a T cell composition isolated or enriched directly from a human subject based on the surface expression of one or more markers comprising the phenotype, but without incubation or culture. In some embodiments, the total number, relative number, or normalized number of the T cell subset in the cultured cells, such as any T cell subset described above, is greater, e.g., at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or greater, than the number, relative number, or normalized number of the T cell subset in a reference T cell composition or preparation, such as any reference T cell composition or preparation described above, per the T cell composition prior to incubation with the reversibly bound agent (e.g., multimerized agent, such as incubation with a stimulatory agent reversibly bound to an oligomeric mutein streptavidin) in accordance with any of the modes provided herein. In some embodiments, the number of T cells corresponding to the subset of T cells present in the T cell culture is at least or at least about 1 x 106 cells, 2 x 106 cells, 3 x 106 cells, 4 x 106 cells, 5 x 106 cells, or more.

[00497] In some embodiments, the T cell subset is CD62L+ and/or IL-7Rα+ (CD127+) and the percentage of the CD62L+ and/or IL-7Rα+ (CD127+) subset in the cultured T cells as a percentage of the total T cells or the total cells in the culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the T cell subset is CD45RA-, CD45ROlow/+, and/or KLRG1low and the percentage of the CD45RA-, CD45ROlow/+, and/or KLRG1low subset in the cultured T cells as a percentage of the total T cells or the total cells in the culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the T cell subset is CD45RA low/+, CD45RO low/+, and/or KLRG1-, and the percentage of the CD45RA low/+, CD45RO low/+, and/or KLRG1- subset in the cultured T cells as a percentage of total T cells or total cells in the culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95% or more.

[00498] In some embodiments, the T cell subset is or includes TCM cells. In some embodiments, the percentage of the TCM subset in the cultured T cells as a percentage of the total T cells or total cells in the culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[00499] In some embodiments, the T cell subset is or includes TSCM cells. In some embodiments, the percentage of the TSCM subset in the cultured T cells as a percentage of the total T cells or total cells in the culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[00500] In some embodiments, the T cell subset, such as CD62L+ T cells, has or exhibits a) a low level of TCR rearrangement excision circles (TREC); and/or b) expresses a proliferation marker (e.g., Ki-67); and/or c) exhibits the ability to proliferate in the presence of a stimulatory agent; and/or d) exhibits an ability to produce a cytokine selected from IFN-gamma, TNF, and IL-2 in the presence of a stimulatory agent; and/or e) is refractory to attrition in the absence of a stimulatory agent; and/or f) is capable of generating TSCM, TCM, TEM, and TEFF cells; and/or g) has low cytotoxicity; and/or h) can produce the same or greater response following adoptive transfer of fewer cells than with TCM or TEM cells. In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine (e.g., IL-15 or IL-17), or is an agent that is capable of initiating a signal associated with the TCR/CD3 complex on T cells. In some embodiments, the ability to produce a cytokine comprises a low ability to produce IFNγ and/or an intermediate ability to produce IL-2.

[00501] In some embodiments, cultured T cells are provided, such as prepared according to any of the methods provided herein, wherein the cultured T cells are generated or produced following incubation as described herein, and wherein the cultured T cells are characterized by a modified functional activity profile compared to a reference T cell composition or preparation. In some embodiments, the cultured T cells or a specific subset of T cells present in the culture exhibit an altered functional activity profile compared to a reference composition or preparation or compared to the subset of T cells in the reference composition or preparation, such as altered (e.g., increased or decreased) functional activity by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold. In some embodiments, the functional activity is selected from one or more of a) a low level of TCR rearrangement excision circles (TREC); and/or b) expression of a proliferation marker (e.g., Ki-67); and/or c) ability to proliferate in the presence of a stimulatory agent; and/or d) ability to produce a cytokine selected from IFN-gamma, TNF, and IL-2 in the presence of a stimulatory agent; and/or e) are refractory to attrition in the absence of a stimulatory agent; and/or f) are capable of generating TSCM, TCM, TEM, and TEFF cells; and/or g) have low cytotoxicity. In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine (e.g., IL-15 or IL-17), or is an agent that is capable of initiating a signal associated with the TCR/CD3 complex on T cells. In some embodiments, the ability to produce a cytokine comprises a low ability to produce IFNγ and/or an intermediate ability to produce IL-2. In some embodiments, the T cell subset comprises memory T cells, such as long-lived memory T cells, in the cultured T cells. In some embodiments, the memory T cells are TSCM cells.

[00502] In some embodiments, the cultured T cells or a specific subset of T cells present in the culture can produce the same or greater response after adoptive transfer of fewer cells than can be achieved by a reference composition or preparation or by the subset of T cells from the reference composition or preparation. In some embodiments, such a response is achieved with at least about 2-fold (such as at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) fewer cells. In some embodiments, the response is increased or is greater by at least about 2-fold (such as at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more)

[00503] In some embodiments, the percentage of the T cell subset in the cultured cells, such as any T cell subset described above, is greater, e.g., at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or greater, than the corresponding subset of cells in a preparation of T cells that were incubated in the presence of a GSK-P inhibitor. In some embodiments, the cultured T cell composition does not contain a GSK-P inhibitor.

[00504] In some embodiments, the percentage of the T cell subset in the cultured cells, such as any T cell subset described above, is greater, e.g., at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or greater, than the corresponding subset of cells that were incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15. In some embodiments, the cultured T cell composition does not contain a recombinant (e.g., exogenous) IL-7 cytokine or a recombinant (e.g., exogenous) IL-15 cytokine.

[00505] In some embodiments, the cultured T cell composition was produced or generated according to any of the modes provided herein wherein a substance, such as a competing agent, was added to the T cells to disrupt, such as to decrease and/or terminate, signaling from the stimulatory agent or agents. In some embodiments, the cultured T cell composition contains the presence of a substance, such as a competing agent, e.g., biotin or a biotin analog, e.g., D-Biotin. In some embodiments, the substance, such as a competing agent, e.g., biotin or a biotin analog, e.g., D-Biotin, is present in an amount that is at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 1000-fold, or greater, than the amount of the substance in a reference composition or preparation of cultured T cells, in which the substance was not added exogenously during incubation. In some embodiments, the amount of the substance, such as a competing agent, e.g., biotin or a biotin analog, e.g., D-Biotin, in the cultured T cell composition is at or about 10 μM to 100 μM, 100 μM to 1 mM, 100 μM to 500 μM, or 10 μM to 100 μM.

IV. METHODS OF GENETICALLY MODIFIED CULTURED CELLS, ANTIGEN RECEPTORS AND GENETICALLY MODIFIED CELLS

[00506] In some embodiments, the cultured cells contain or are engineered to contain a modified receptor, e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR). Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells of a certain type, such as T cells or CD8+ or CD4+ cells, are enriched or selected. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[00507] Thus, in some embodiments, the cultured cells include one or more genetically engineered nucleic acids and thereby express recombinant or genetically modified products of such nucleic acids. In some embodiments, the gene transfer is accomplished by first stimulating the cultured cells, such as by combining them with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells and expansion in culture to numbers sufficient for clinical applications.

[00508] In some contexts, overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) may be toxic to an individual. Thus, in some contexts, the engineered cells include gene segments that render the cells susceptible to negative selection in vivo, such as following administration in adoptive immunotherapy. For example, in some aspects, the cultured cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, e.g., a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 2:223, 1977) that confers sensitivity to ganciclovir; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)). In some aspects, the cultured cells are further modified to promote the expression of cytokines or other factors.

A. Nucleic acids encoding antigen receptors, e.g., chimeric antigen receptors

[00509] Methods, nucleic acids, compositions, and kits for producing genetically modified cells are provided. Genetic engineering generally involves introducing a nucleic acid encoding the recombinant or modified component into a composition containing cultured cells, such as by retroviral transduction, transfection, or transformation.

[00510] In some embodiments, the nucleic acids are heterologous, that is, they are not normally present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not normally found in the cell being modified and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are non-naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

1. Chimeric Antigen Receptors (CARs)

[00511] The cells often express recombinant receptors, such as antigen receptors, including functional non-TCR antigen receptors, e.g., chimeric antigen receptors (CARs), and other antigen-binding receptors, such as transgenic T cell receptors (TCRs). Also among the receptors are other chimeric receptors.

[00512] Exemplary antigen receptors, including CARs, and methods for modifying and introducing such receptors into cells, include those described, for example, in Ped. Pub. International Patent Applications WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061, US patent application publication numbers US2002131960, US2013287748, US20130149337, US Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 1,282,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, Nos. 7,354,762, 1,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 Apr; 3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 Oct; 24(5):633-39; Wu et al., Cancer, 2012 Mar 18(2):160-75. In some aspects, the antigen receptors include a CAR as described in US Patent No. 7,446,190, and those described in Ped Pub. International Patent No. WO/2014055668 A1. Examples of CARs include CARs as disclosed in any of the above-mentioned publications such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent No. 7,446,190, and US Patent No.: 8,389,282. Chemical receptors, such as CARs, generally include an extracellular antigen-binding domain, such as a portion of an antibody molecule, usually a variable heavy chain (VH) region and/or light chain (VL) region of the antibody, e.g., an scFv antibody fragment.

[00513] In some embodiments, the target antigen of the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on the cells of the disease or condition, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.

[00514] Antigens targeted by the receptors in some embodiments include the orphan receptor tyrosine kinase ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2 and MAGE A3, CE7, Wilms tumor 1 (WT-1), a cyclin such as cyclin A1 (CCNA1) and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens.

[00515] In some embodiments, the CAR binds to a pathogen-specific antigen. In some embodiments, the CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

[00516] In some embodiments, the antibody portion of the recombinant receptor, e.g., CAR, includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is from a human IgG, such as IgG4 or IgG1. In some aspects, the constant region portion serves as a spacer region between the antigen recognition component, e.g., scFv, and the transmembrane domain. The spacer can have a length that provides increased responsiveness of the cell upon antigen binding, compared to the absence of the spacer. Exemplary spacers, e.g., hinge regions, include those described in International Patent Publication No. WO2014031687. In some examples, the spacer is either about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those that are at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include the IgG4 hinge alone, the IgG4 hinge linked to the CH2 and CH3 domains, or the IgG4 hinge linked to the CH3 domain.

[00517] This antigen recognition domain is generally linked to one or more intracellular signaling components, such as signaling components that mimic activation via an antigen receptor complex, such as a TCR complex in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the antigen binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the receptor, e.g., CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to prevent binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[00518] The transmembrane domain is in some embodiments derived from a natural source or a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprise at least the transmembrane region(s) of) the T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and/or CD154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, linkage is by linkers, spacers, and/or transmembrane domain(s).

[00519] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[00520] The receptor, e.g., CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen binding portion is linked to one or more cellular signaling modules. In some embodiments, the cellular signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-Z) or Fc receptor Y and CD8, CD4, CD25, or CD16.

[00521] In some embodiments, upon binding of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cells engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of coreceptors that in the natural context act in conjunction with such receptors to initiate signal transduction after antigen receptor engagement.

[00522] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating a secondary or costimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed on the same cell and provides the component for generating the secondary or costimulatory signal.

[00523] T cell activation is, in some aspects, described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate primary antigen-dependent activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of these signaling components.

[00524] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR contain a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3zeta.

[00525] In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both activator and costimulatory components.

[00526] In some embodiments, the activation domain is included within a CAR, while the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CARs and/or a costimulatory CAR. In some embodiments, the cells additionally include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013), such as a CAR that recognizes an antigen other than that associated with and/or specific to the disease or condition whereby an activation signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[00527] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 intracellular domain (e.g., CD3-zeta). In some embodiments, the intracellular signaling domain comprises CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domains to a CD3 zeta intracellular domain.

[00528] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

[00529] In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, that can be used to confirm transduction or modification of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, an NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687.

[00530] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.

[00531] In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[00532] In some embodiments, the marker has no therapeutic function and/or has no effect other than being used as a marker for genetic engineering, e.g., to select for successfully modified cells. In other embodiments, the marker can be a therapeutic molecule or molecules that otherwise exert some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or decrease the responses of cells upon adoptive transfer and ligand encounter.

[00533] In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that provides only a CD3 chain-induced signal upon antigen binding; in some aspects, a second generation CAR is one that provides both such a signal and a costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains from different costimulatory receptors.

[00534] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes a scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta chain (CD3Z). In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T-cell costimulatory molecule. In some aspects, the T-cell costimulatory molecule is CD28 or 41BB.

[00535] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including receptors and other provided polypeptides, e.g., ligands or peptides, may include amino acid residues including natural and/or unnatural amino acid residues. The terms also include post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, polypeptides may contain modifications relative to a native or natural sequence, so long as the protein retains the desired activity. Such modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of the host producing the proteins or errors due to PCR amplification.

[00536] In some embodiments, the receptor, e.g., CAR, expressed by the cells in the consecutive dose contains at least one immunoreactive epitope like the receptor, e.g., CAR, expressed by the cells from the first dose. In some aspects, the receptor, e.g., CAR, expressed by the cells administered in the consecutive dose is identical to the receptor, e.g., CAR, expressed by the first dose or is substantially identical to the receptor, e.g., CAR, expressed by the cells administered in the first dose.

[00537] Recombinant receptors, such as CARs, expressed by the cells administered to the subject at the various doses generally recognize or specifically bind to a molecule that is expressed by, associated with, and/or specific to, the disease or condition or its cells to be treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, to the cell, thereby promoting an immune response directed toward the disease or condition. For example, in some embodiments, the cells in the first dose express a CAR that specifically binds to an antigen expressed by a cell or tissue of or associated with the disease or condition.

2. TCRs

[00538] In some embodiments, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells. In some embodiments, a high affinity T cell clone for a target antigen (e.g., a cancer antigen) is identified, isolated from a patient, and introduced into the cells. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with genes from the human immune system (e.g., the human leukocyte antigen, or HLA, system). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808. In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354.

[00539] In some embodiments, after obtaining the T cell clone, the TCR alpha and beta chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosomal skipping peptide such that both chains are co-expressive. In some embodiments, the TCR gene transfer is accomplished via retroviral or lentiviral vectors, or via transposons (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy 13:1050-1063 Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy 18:1748-1757 Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy 18:674-683.

3. Multi-direction

[00540] In some embodiments, the cells and methods include multi-targeting strategies, such as expressing two or more genetically engineered receptors on the cell, each recognizing the same or a different antigen and typically including a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Pub. No. WO 2014055668 A1 (describing combinations of activating and costimulatory CARs, e.g., targeting two different antigens individually present on off-target, e.g., normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013) (describing cells that express both an activating and an inhibitory CAR, such as those in which the activating CAR binds to an antigen expressed on normal or nondiseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on normal cells or cells not desired to be treated).

[00541] For example, in some embodiments, the cells include a receptor that expresses a first genetically modified antigen receptor (e.g., CAR or TCR) that is capable of inducing an activation signal to the cell, generally upon specific binding to the antigen recognized by the first receptor, e.g., the first antigen. In some embodiments, the cell further includes a second genetically modified antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, that is capable of inducing a costimulatory signal to the immune cell, generally upon specific binding to a second antigen recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

[00542] In some embodiments, the first and/or second genetically engineered antigen receptor (e.g., CAR or TCR) is capable of inducing an activation signal to the cell. In some embodiments, the receptor includes an intracellular signaling component containing ITAM or ITAM-like motifs. In some embodiments, the activation induced by the first receptor involves a signal transduction or change in protein expression in the cell resulting in the initiation of an immune response, such as phosphorylation of ITAM and/or initiation of the ITAM-mediated signal transduction cascade, formation of an immunological synapse and/or clustering of molecules near the bound receptor (e.g., CD4 or CD8, etc.), activation of one or more transcription factors, such as NF-κB and/or AP-1, and/or induction of gene expression of factors such as cytokines, proliferation, and/or survival.

[00543] In some embodiments, the first and/or second receptor includes intracellular signaling domains of costimulatory receptors, such as CD28, CD137 (4-1 BB), OX40, and/or ICOS. In some embodiments, the first and second receptors include an intracellular signaling domain of a costimulatory receptor that are different. In one embodiment, the first receptor contains a costimulatory signaling region of CD28 and the second receptor contains a costimulatory signaling region of 4-1BB, or vice versa.

[00544] In some embodiments, the first and/or second receptor includes an intracellular signaling domain containing ITAM or ITAM-like motifs and an intracellular signaling domain of a costimulatory receptor.

[00545] In some embodiments, the first receptor contains an intracellular signaling domain containing ITAM or ITAM-like motifs and the second receptor contains an intracellular signaling domain of a costimulatory receptor. The costimulatory signal in combination with the activating signal induced in the same cell is one that results in an immune response, such as a robust and sustained immune response such as increased gene expression, secretion of cytokines and other factors, and T cell-mediated effector functions such as cell death.

[00546] In some embodiments, neither binding of the first receptor alone nor binding of the second receptor alone induces a robust immune response. In some aspects, if only one receptor is bound, the cell becomes tolerated or unresponsive to the antigen, or inhibited, and/or is not induced to proliferate or secrete factors or perform effector functions. In some such embodiments, however, when the plurality of receptors are bound, such as upon encounter with a cell expressing both the first and second antigens, a desired response is achieved, such as full immune activation or stimulation, e.g., as indicated by secretion of one or more cytokines, proliferation, persistence, and/or performance of an immune effector function, such as cytotoxic killing of a target cell.

[00547] In some embodiments, the two receptors induce, respectively, an activating and inhibitory signal from the cell, such that binding of one of the receptors to its antigen activates the cell or induces a response, but binding by the second inhibitory receptor to its antigen induces a signal that suppresses or diminishes that response. Examples are combinations of activating CARs and inhibitory CARs or iCARs. Such a strategy can be used, for example, in which the activating CAR binds to an antigen expressed in a disease or condition but that is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen that is expressed on normal cells but not cells of the disease or condition.

[00548] In some embodiments, the multi-target strategy is employed in a case where an antigen associated with a particular disease or condition is expressed on a non-diseased cell and/or is expressed on the modified cell itself, either transiently (e.g., following stimulation in association with genetic engineering) or permanently. In such cases, by requiring the binding of two separate and individually specific antigen receptors, specificity, selectivity, and/or efficacy may be improved.

[00549] In some embodiments, the plurality of antigens, e.g., the first and second antigens, are expressed on the target cell, tissue, or disease or condition, such as a cancer cell. In some aspects, the cell, tissue, disease, or condition is multiple myeloma or a multiple myeloma cell. In some embodiments, one or more of the plurality of antigens are generally also expressed on a cell that is not desired to be targeted with the cell therapy, such as a normal or non-diseased cell or tissue, and/or the engineered cells themselves. In such embodiments, by requiring the binding of multiple receptors to elicit a response from the cell, specificity and/or efficacy is achieved.

B. Vectors and methods for genetic engineering

[00550] Various methods for introducing genetically modified components, e.g., antigen receptors, e.g., CARs or TCRs, are well known and can be used with the provided methods and compositions. Exemplary methods include those for transferring nucleic acids encoding the receptors, including via viral transduction, e.g., retroviral or lentiviral, transposition, and electroporation.

[00551] In some embodiments, recombinant nucleic acids are transferred to cells in culture using recombinant infectious virus particles, such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi:10.1038/gt. 2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11):550-557.

[00552] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, e.g., a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focal formation virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, they are capable of infecting host cells of various species, including humans. In one embodiment, the gene to be expressed replaces retroviral gag, pol, and/or env sequences. Several illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, A.D. (1990) Human Gene Therapy 1: 5-14 Scarpa et al., Virology 180: 849-852, 1991; Burns et al., (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet, Develop, 3: 102-109.

[00553] Methods of lentiviral transduction are known, and exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2):497-505.

[00554] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells by transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; microparticle bombardment facilitated by tungsten particles (Johnston, Nature, 346: 776–777 (1990)); and coprecipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031–2034 (1987)).

[00555] Other approaches and vectors for transferring the nucleic acids encoding the recombinant products are those described, for example, in International Patent Pub. No. WO2014055668 and U.S. Patent No. 7,446,190.

[00556] In some embodiments, the cells, e.g., T cells, can be transfected during or after expansion, e.g., with a T cell receptor (TCR) or a chemical antigen receptor (CAR). This transfection for the introduction of the desired receptor gene can be performed with any suitable retroviral vector, e.g., . The genetically modified cell population can then be released from the initial stimulus (the CD3/CD28 stimulus, e.g.) and subsequently stimulated with a second type of stimulus, e.g., via a newly introduced receptor). This second type of stimulus can include an antigenic stimulus in the form of a peptide/MHC molecule, the cognate (cross-linked) ligand of the genetically introduced receptor (e.g., natural ligand of a CAR), or any ligand (such as an antibody) that binds directly within the structure of the new receptor (e.g., recognizing constant regions within the receptor). See, e.g., Cheadle et al., "Chimeric antigen receptors for T-cell based therapy." Methods Mol Biol. 2012; 907: 64566 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[00557] Additional nucleic acids, for example, include genes for introduction that enhance the efficacy of the therapy, such as by promoting the viability and/or function of the transferred cells; genes that provide a genetic marker for selection and/or evaluation of the cells, such as to assess survival or localization in vivo; genes that enhance safety, e.g., by rendering the cell susceptible to negative selection in vivo, as described by Lupton S. D. et al., Mol. and Cell Biol. 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also Lupton et al. publications PCT/US91/08442 and PCT/US94/05601 describing the use of selectable bifunctional fusion genes derived from the fusion of a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, at columns 1417.

[00558] In some cases, a vector may be used that does not require the cells, e.g., T cells, to be activated. In some cases, the cells may be selected and/or transduced prior to activation. Thus, the cells may be modified prior to or after culturing the cells as described herein, and in some cases, at the same time or during at least a portion of the culturing. In some embodiments, the cells that are to be modified are the cultured cells, or in some cases, the cells may be transduced prior to culturing as described herein.

V. COMPOSITIONS, FORMULATIONS AND METHODS OF ADMINISTRATION

[00559] Also provided are compositions containing the modified receptor (e.g., modified antigen receptor), such as CAR or TCR, and compositions containing the modified cells, including pharmaceutical compositions and formulations. Also provided are compositions containing a plurality of cultured T cells or target cells produced by any of the methods provided herein and, optionally, a pharmaceutically acceptable excipient. Also provided are methods of use and uses of the compositions, such as in the treatment of diseases, conditions, and disorders in which the antigen is expressed, or in methods of detection, diagnosis, and prognosis.

A. Compositions/Formulations

[00560] The term "pharmaceutical formulation" refers to a preparation that is of such a form that it allows the biological activity of a contained active ingredient to be effective, and that does not contain additional components that would be unacceptably toxic to an individual to whom the formulation would be administered.

[00561] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to an individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[00562] In some aspects, the choice of carrier is determined in part by the particular cell and/or the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, by Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[00563] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005)

[00564] The formulation or composition may also contain more than one active ingredient useful for the indication, disease or condition to be treated with the cells, preferably those with complementary activities of the cell, wherein the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic and arylsulfonic acids, for example p-toluenesulfonic acid.

[00565] The active ingredients can be entrapped in microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as a cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target host cells (e.g., T cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and U.S. Patents 4,235,871 , 4,501,728 , 4,737,028 , and 5,019,369 .

[00566] The pharmaceutical composition in some aspects may employ timed release, delayed release, and extended release delivery systems such that release of the composition occurs prior to, and with sufficient time to cause sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems may avoid repeated administrations of the composition, thereby increasing convenience for the individual and the physician.

[00567] The pharmaceutical composition in some embodiments contains cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic evaluation of treated subjects. For repeated administrations over several days or more, depending on the condition, treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and may be determined. The desired dosage may be administered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by administration by continuous infusion of the composition.

[00568] The cells may be administered using standard administration techniques, formulations and/or devices. Provided that they are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells may be autologous or heterologous. For example, immunosorbent or progenitor cells may be obtained from an individual and administered to the same individual or to a different compatible individual. Peripheral blood-derived immunoresponsive cells or their progeny may be administered (e.g., in vivo, ex vivo or in vitro) by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[00569] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral” when used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject utilizing peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[00570] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions may comprise carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[00571] Sterile injectable solutions may be prepared by incorporating the cells in a solvent, such as mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methyl cellulose), pH buffering agents, gelling or viscosity-increasing additives, preservatives, flavoring agents, colors and the like, depending on the route of administration and desired preparation. Standard texts may, in some aspects, be consulted for preparing suitable preparations.

[00572] Various additives that increase the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers, may be added. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. Prolonged absorption of the injectable pharmaceutical form may be achieved by the use of agents that delay absorption, for example, aluminum monostearate and gelatin.

[00573] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules.

[00574] Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

B. Methods of Administration

[00575] Provided are methods of administering the cells, populations, and compositions, and uses of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancers. In some embodiments, the cells, populations, and compositions are administered to a subject or patient having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T-cell therapy. In some embodiments, the cells and compositions prepared by the provided methods, such as modified compositions and final production compositions after incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods treat, e.g., ameliorate one or more symptoms of the disease or condition, such as by decreasing tumor burden in a cancer expressing an antigen recognized by a modified T cell.

[00576] Methods for administering cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, methods of adoptive T cell therapy are described, e.g., in U.S. Patent Application Publication 2003/0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[00577] As used herein, a "subject" is a mammal, such as a human or other animal, and is typically human. In some embodiments, the subject, e.g., patient, to whom the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or a howler monkey. The subject may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[00578] When used herein, "treatment" (and its grammatical variations such as "treat" or "treating") refers to the complete or partial amelioration or reduction of a disease or condition or disorder, or an associated symptom, adverse effect or outcome, or phenotype. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastases, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. The terms do not imply complete cure of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[00579] As used herein, "delaying the development of a disease" means to delay, prevent, slow, retard, stabilize, suppress, and/or postpone the development of the disease (such as cancer). This delay may be of varying length, depending on the history of the disease and/or the individual being treated. As is apparent to one of skill in the art, a sufficient or significant delay may, in effect, encompass prevention, in that the individual does not develop the disease. For example, advanced stage cancer, such as the development of metastasis, may be delayed.

[00580] "Prevention" when used herein includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual who may be predisposed to the disease but who has not yet been diagnosed with the disease. In some embodiments, the cells and compositions provided are used to delay the development of a disease or slow the progression of a disease.

[00581] As used herein, to "suppress" a function or activity is to reduce the function or activity when compared to the same conditions except for a condition or parameter of interest, or alternatively, when compared to another condition. For example, cells that suppress tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of the cells.

[00582] An "effective amount" of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/quantities and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic outcome.

[00583] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder and/or the pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the cell populations administered. In some embodiments, the provided methods involve administering the cells and/or compositions in effective amounts, e.g., therapeutically effective amounts.

[00584] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in individuals prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[00585] The disease or condition that is treated may be any in which the expression of an antigen is associated with and/or involved in the etiology of a disease state or disorder, e.g., causes, exacerbates, or is otherwise involved in such a disease, condition, or disorder. Exemplary diseases and conditions may include diseases or conditions associated with malignancy or transformation of cells (e.g., cancer), autoimmune or inflammatory disease, or an infectious disease, e.g., caused by a bacterial, viral, or other pathogen. Exemplary antigens, which include antigens associated with various diseases and conditions that may be treated, are described above. In particular embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease or condition.

[00586] Thus, the methods and uses provided include methods and uses for adoptive cell therapy. In some embodiments, the methods include administering the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition, or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or pathology to be treated, for example, through adoptive cell therapy, such as adoptive T-cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition, to ameliorate one or more symptoms of the disease or condition.

[00587] In some embodiments, the cell therapy, e.g., adoptive T-cell therapy, is performed by autologous transfer, in which cells are isolated and/or otherwise prepared from the individual receiving the cell therapy or a sample derived from such an individual. Thus, in some aspects, the cells are derived from an individual, e.g., patient, in need of a treatment and the cells, after isolation and processing, are administered to the same individual.

[00588] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is accomplished by allogeneic transfer, in which the cells are isolated and/or prepared other than by a subject receiving or receiving the cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. The cells may be administered by any suitable means. Dosage and administration may depend in part on whether administration is brief or chronic. Various dosing regimens include, but are not limited to, single or multiple administrations over multiple time points, bolus administration, and pulse infusion.

[00589] In certain embodiments, the cells, or individual populations of cell subtypes, are administered to the subject in a range of from about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as for example, 1 million to 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the preceding values) and in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, 45 billion cells) or any value in between and/or per kilogram of body weight. Again, dosages may vary depending on the specific attributes of the disease or disorder and/or the patient and/or other treatments. In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously or sequentially with, in any order, another therapeutic intervention, such as an antibody or modified cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in conjunction with another therapeutic intervention, either simultaneously or sequentially, in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to increase persistence. In some embodiments, the methods comprise administering a chemotherapeutic agent.

[00590] Following administration of the cells, the biological activity of the modified cell populations in some embodiments is measured, for example, by any of a number of known methods. Parameters to be assessed include the specific binding of a genetically modified or natural T cell or other immune cell to antigen, in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the modified cells to kill target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described, for example, in Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009) and Herman et al., J. Immunological Methods, 285 (1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and/or secretion of one or more cytokines, such as CD107a, IFNY, IL-2, and TNF. In some aspects, the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[00591] In certain embodiments, the modified cells are further modified in any number of ways such that their therapeutic or prophylactic efficacy is increased. For example, the modified CAR or TCR expressed by the population can be conjugated directly or indirectly via a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, e.g., Wadwa et al., J. Drug Targeting 3:111 (1995), and U.S. Patent 5,087,616.

VI. DEFINITIONS

[00592] Unless otherwise defined, all terms of the art, notations and other technical and scientific terms or terminology used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[00593] When used herein, the singular forms "a", "an", and "the" include the plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is understood that the aspects and variations described herein include aspects and variations "consisting of" and/or "consisting essentially of".

[00594] Throughout this description, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be deemed to have specifically described all possible sub-ranges, as well as individual numerical values within that range. For example, where a range of values is given, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value within that stated range, is encompassed by the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed by the claimed subject matter, subject to any limit specifically excluded in the stated range. Where the stated range includes one or both of these limits, ranges that exclude one or both of the included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[00595] The term "about" when used herein refers to the usual error range for the respective value as readily known to the person skilled in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, the description referring to "about X" includes the description of "X".

[00596] As used herein, a composition refers to any mixture of two or more products, substances or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[00597] The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid construct as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Among vectors are viral vectors, such as retroviral vectors, e.g., gamma-retroviral and lentiviral vectors.

[00598] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom without regard to passage number. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as assessed or selected in the originally transformed cell are included herein.

[00599] As used herein, "enrichment" when referring to one or more cell types or cell populations, refers to increasing the number or percentage of the cell type or population, e.g., compared to the total number of cells or volume of the composition, or relative to other cell types, such as by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present in the cell population or cell to be depleted. The term does not require the complete removal of other cells, cell types or populations from the composition and does not require that the cells so enriched be present at or even near 100% in the enriched composition.

[00600] As used herein, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence in or on the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, e.g., by labeling with an antibody that specifically binds to the marker and detecting said antibody, wherein the label is detectable by flow cytometry at a level substantially above the label detected by performing the same procedure with an isotype-matched control under identical reference conditions and/or at a level substantially similar to that of the cell known to be positive for the marker and/or at a level substantially higher than that of a cell known to be negative for the marker.

[00601] When used herein, a statement that a cell or population of cells is "negative" for a particular marker refers to the absence of substantial detectable presence in or on the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by labeling with an antibody that specifically binds to the marker and detecting said antibody, wherein the label is not detected by flow cytometry at a level substantially above the label detected by performing the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that of the cell known to be positive for the marker and/or at a substantially similar level when compared to that of a cell known to be negative for the marker.

[00602] The term "expression" when used herein refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[00603] As used herein, a control refers to a sample that is substantially identical to the test sample except that it is not treated with a test parameter or, if it is a plasma sample, may be from a normal volunteer unaffected with the condition of interest. A control may also be an internal control.

VII. EXEMPLARY TYPES

[00604] Among the modalities provided are:

[00605] 1. A method for culturing T cells, the method comprising:

[00606] (a) incubating a composition comprising T cells in the presence of a receptor binding agent wherein i) is reversibly bound to a reagent comprising a plurality of binding sites capable of reversibly binding to the receptor binding agent and ii) is capable of specifically binding to a molecule on the surface of the T cells in a manner that induces or modulates a signal in T cells in the composition; and

[00607] (b) within 5 days after the start of said incubation, disrupting the reversible binding between the receptor binding agent and the reagent, thereby generating cultured T cells.

[00608] 2. The method of embodiment 1, wherein the incubation is carried out under conditions where the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in one or more T cells in the composition.

[00609] 3. The method of mode 1 or mode 2, in which said rupture is carried out more than 30 minutes after the start of said incubation.

[00610] 4. The method of any of embodiments 1-3, wherein:

[00611] said rupture is carried out between 1 hour and 4 days after the start of said incubation, between 6 hours and 3 days after the start of said incubation, between 12 hours and 2 days after the start of said incubation or between 1 day and 3 days after the start of said incubation; or

[00612] said rupture is effected between about 1 hour and about 4 days after the start of said incubation, between about 6 hours and about 3 days after the start of said incubation, between about 12 hours and about 2 days after the start of said incubation, or between about 1 day and about 3 days after the start of said incubation; or

[00613] said rupture is effected greater than or equal to about 1 hour after the start of said incubation and within 1 day, 2 days, 3 days or 4 days after the start of said incubation.

[00614] 5. The method of any of embodiments 1-4, wherein:

[00615] the receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells; and/or

[00616] the receptor binding agent specifically binds to an element of a TCR/CD3 complex; and/or

[00617] the receptor binding agent specifically binds to CD3.

[00618] 6. The method of any of embodiments 1-5, wherein the molecule is a component of the TCR/CD3 complex or is CD3.

[00619] 7. The method of any of embodiments 1-6, wherein the molecule is a first molecule and the receptor binding agent is also capable of specifically binding to a second molecule on the surface of one or more of the T cells, which second molecule is optionally capable of inducing or enhancing, decreasing or modifying a signal delivered through the first molecule on the T cells.

[00620] 8. The method of any of embodiments 1-7, wherein:

[00621] the receptor binding agent comprises a C1 binding partner; and

[00622] the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent and the reagent.

[00623] 9. The method of any of embodiments 1-8, wherein said disruption comprises introducing to the cells a composition comprising a substance capable of reversing the binding between the receptor binding agent and the reagent.

[00624] 10. The method of any of embodiments 1-9, wherein the receptor binding agent is a first receptor binding agent and the incubation is also carried out in the presence of a second receptor binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells, which second molecule is optionally capable of enhancing, diminishing, or modifying a signal delivered through the first molecule to the T cells.

[00625] 11. The method of embodiment 10, wherein the reagent comprises a plurality of binding sites capable of reversibly binding to the second receptor binding agent, whereby the second receptor binding agent is reversibly bound to the reagent.

[00626] 12. The method of embodiment 11, wherein the plurality of binding sites capable of reversibly binding to the first receptor binding agent and the plurality of binding sites capable of reversibly binding to the second receptor binding agent may be the same or different.

[00627] 13. The method of embodiment 11 or embodiment 12, wherein:

[00628] the second receptor binding agent comprises a C1 or C2 binding partner, which is capable of reversibly binding to two or more Z1 binding sites, whereby the first and second receptor binding agents are reversibly bound to the reagent via the two or more Z1 binding sites; and/or

[00629] the second receptor binding agent comprises a C2 binding partner and the reagent also comprises a plurality of Z2 binding sites, which is capable of binding to the C2 binding partner to form the reversible bond between the second receptor binding agent and the reagent.

[00630] 14. The method of embodiment 13, wherein:

[00631] C2 and C1 are the same or substantially the same, or contain the same or substantially the same moiety; and/or

[00632] Z1 and Z2 are the same or substantially the same or contain the same or substantially the same moiety.

[00633] 15. The method of any of embodiments 1-14, wherein the reagent is a first reagent, and the incubation is carried out in the presence of at least one second reagent that is reversibly bound to the second receptor binding agent.

[00634] 16. The method of any of embodiments 10-15, wherein the incubation is carried out under conditions wherein the second receptor binding agent specifically binds to the second molecule, thereby inducing or modulating a signal that enhances, diminishes, or modifies a signal delivered through the first molecule to the T cells.

[00635] 17. The method of any of embodiments 7-16, wherein said disruption comprises introducing to the cells a composition comprising a substance capable of reversing the binding between the first receptor binding agent and the reagent and/or the second receptor binding agent and the reagent.

[00636] 18. The method of any of embodiments 7-17, wherein said disruption terminates or reduces the signal induced or modulated by the first receptor binding agent and terminates or reduces the signal induced or modulated by the second receptor binding agent.

[00637] 19. A method for culturing T cells, the method comprising:

[00638] incubating a composition comprising T cells in the presence of:

[00639] a first receptor binding agent that is capable of specifically binding to a first molecule expressed on the surface of T cells in a manner that induces or modulates a signal associated with the TCR/CD3 complex on the T cell; and

[00640] a second receptor binding agent wherein i) is reversibly linked to a reagent comprising a plurality of binding sites capable of reversibly binding to the second receptor binding agent and ii) is capable of specifically binding to a second molecule on the surface of T cells in a manner that induces or modulates a second signal on the T cells to enhance, diminish or modify a signal delivered through the first molecule; and

[00641] within 5 days after the start of said incubation, disrupting the reversible binding between the second receptor binding agent and the reagent, thereby generating cultured T cells.

[00642] 20. The method of embodiment 19, wherein the incubation is carried out under conditions wherein the first receptor binding agent specifically binds to the first molecule and/or the second receptor binding agent specifically binds to the second molecule, thereby inducing or modulating one or more signals in the T cells.

[00643] 21. The method of embodiment 19 or embodiment 20, wherein:

[00644] the second receptor binding agent comprises a C1 binding partner; and

[00645] the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the second receptor binding agent and the reagent.

[00646] 22. The method of any of embodiments 7-21, wherein:

[00647] the second signal is a signal other than a signal associated with the TCR/CD3 complex;

[00648] the second signal is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex; or

[00649] the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family.

[00650] 23. The method of any of embodiments 7-22, wherein the second molecule is selected from CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM.

[00651] 24. The method of any of embodiments 7-23, wherein the second molecule is CD28.

[00652] 25. The method of any of embodiments 7-24, wherein said disruption comprises introducing to the cells a composition comprising a substance capable of reversing the binding between the second receptor binding agent and the reagent, which may be the second reagent.

[00653] 26. The method of any of embodiments 7-25, wherein:

[00654] said disruption terminates or reduces the signal induced or modulated by the second receptor binding agent; or

[00655] the additional molecule is CD28 and disruption terminates or reduces the CD28 costimulatory signal on T cells.

[00656] 27. The method of any of embodiments 1-26, comprising, after said disruption, also incubating the composition comprising the T cells.

[00657] 28. The method of embodiment 27, wherein:

[00658] the incubation and another incubation are carried out in the same vessel; and/or

[00659] the other incubation is carried out in the presence of the substance; and/or

[00660] the method does not comprise removing the substance, receptor binding agent, second receptor binding agent and/or reagent from the cell composition prior to further incubation.

[00661] 29. The method of any of embodiments 1-28, wherein:

[00662] the incubation and/or other incubation is carried out at or about 37°C ± 2°C; and/or

[00663] the incubation and/or other incubation is carried out in the presence of another agent that is capable of delivering a signal to the T cells.

[00664] 30. The method of embodiment 29, wherein the other agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells, and/or CD8+ T cells.

[00665] 31. The method of embodiment 30, wherein the other agent is a cytokine selected from IL-2, IL-15, and IL-7.

[00666] 32. The method of any of embodiments 27-32, wherein the further incubation is carried out for a time that is no more than 14 days, no more than 12 days, no more than 10 days, no more than 8 days, or no more than 6 days.

[00667] 33. A method for culturing T cells, comprising:

[00668] incubating a composition comprising T cells in the presence of a receptor binding agent that specifically binds to a CD28 molecule on the surface of T cells under conditions to effect signaling through CD28 on the cells; and

[00669] within 5 days after the start of said incubation, eliminate or reduce the binding of the receptor binding agent and the CD28 molecule, whereby CD28 signaling is terminated or reduced in the cells, thereby generating cultured T cells.

[00670] 34. The method of embodiment 33, in which the elimination or reduction is carried out within 4 days, within 3 days, within 2 days, or within 1 day after the start of incubation.

[00671] 35. The method of embodiment 33 or embodiment 34, in which the elimination or reduction comprises:

[00672] washing the cells, whereby any receptor binding agent that is not specifically bound to CD28 is removed or reduced from the composition; or

[00673] reversing the binding interaction between the receptor binding agent and the CD28 molecule, optionally also comprising washing the cells to remove or reduce the receptor binding agent from the composition.

[00674] 36. The method of any of embodiments 33-35, wherein during at least a portion of the incubation and/or subsequent to the incubation, the T cells are incubated in the composition in the presence of an agent that specifically binds to a molecule of the TCR/CD3 complex, whereby a signal associated with the TCR/CD3 complex is induced or modulated in the cells.

[00675] 37. A method for culturing T cells, comprising incubating a composition comprising T cells in the presence of a receptor binding agent which is i) reversibly bound to a reagent comprising a plurality of binding sites capable of reversibly binding to the receptor binding agent; and ii) is capable of specifically binding to a molecule on the surface of the T cells other than CD28 or CD3 in a manner that induces or modulates a signal in T cells, thereby generating cultured T cells.

[00676] 38. The method of embodiment 37, wherein the incubation is carried out under conditions where the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in the T cells.

[00677] 39. The method of embodiment 37 or embodiment 38, in which the signal is not a signal associated with the TCR/CD3 complex.

[00678] 40. The method of any of embodiments 37-39, wherein the receptor binding agent comprises a binding partner C1, and the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent and the reagent.

[00679] 41. The method of any of embodiments 37-40, wherein the receptor binding agent is a second receptor binding agent and the molecule is a second molecule, and the incubation is also carried out in the presence of a first receptor binding agent that is capable of specifically binding to a first molecule on the surface of one or more of the T cells, wherein the first molecule is optionally capable of inducing or modulating a first signal in one or more T cells in the composition.

[00680] 42. The method of embodiment 41, wherein:

[00681] the first receptor binding agent is reversibly bound to the reagent, said reagent comprising a plurality of binding sites for the first receptor binding agent and the second receptor binding agent; or

[00682] The first receptor binding agent is reversibly linked to a second reagent comprising a plurality of binding sites capable of reversibly binding to the first receptor binding agent.

[00683] 43. The method of embodiment 41 or embodiment 42, wherein:

[00684] the first receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells; and/or

[00685] the first receptor binding agent specifically binds to an element of a TCR/CD3 complex; and/or

[00686] the first receptor binding agent specifically binds to CD3.

[00687] 44. The method of any of embodiments 41-43, wherein:

[00688] the specific binding of the second receptor binding agent to the second molecule is capable of enhancing, diminishing, or modifying a signal delivered through the first molecule; or

[00689] the specific binding of the second receptor binding agent to the second molecule is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex.

[00690] 45. A method for culturing T cells, comprising incubating a composition comprising T cells in the presence of:

[00691] a first receptor binding agent wherein i) is reversibly linked to a reagent comprising a plurality of binding sites capable of reversibly binding to the first receptor binding agent and ii) is capable of specifically binding to a first molecule on the surface of T cells in a manner that induces or modulates a signal associated with the TCR/CD3 complex on T cells in the composition; and

[00692] a second receptor binding agent wherein i) is reversibly bound to the reagent also comprising a plurality of binding sites for the second receptor binding agent or to a second reagent comprising a plurality of binding sites capable of reversibly binding to the second receptor binding agent and ii) is capable of specifically binding to a second molecule on the surface of T cells in a manner that induces or modulates a second signal on T cells in the composition, said second molecule being different from CD28, and

[00693] wherein the incubation is carried out under conditions in which the signal and/or the second signal is/are induced or modulated in T cells in the composition, thereby generating cultured T cells.

[00694] 46. The method of embodiment 44, wherein the first receptor binding agent specifically binds to an element of a TCR/CD3 complex and/or the first receptor binding agent specifically binds to CD3.

[00695] 47. The method of embodiment 45 or embodiment 46, wherein:

[00696] the specific binding of the second receptor binding agent to the second molecule is capable of inducing or modulating a signal other than a signal associated with the TCR/CD3 complex; and/or

[00697] the specific binding of the second receptor binding agent to the second molecule is capable of enhancing, diminishing, or modifying a signal delivered through the first molecule; or

[00698] the specific binding of the second receptor binding agent to the second molecule is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex.

[00699] 48. The method of any of embodiments 37-47, wherein:

[00700] the molecule, which may be the second molecule, is selected from CD90 (Thy-1), CD95 (Apo-/Fas), CD137, (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 and HVEM; and/or

[00701] the receptor binding agent, which may be the second receptor binding agent, specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

[00702] 49. The method of any of embodiments 37-48, wherein the molecule, which may be a second molecule, is not CD137 and/or the receptor binding agent, which may be a second receptor binding agent, non-specifically binds to CD137.

[00703] 50. The method of any of embodiments 42-49, wherein:

[00704] the first receptor binding agent and the second receptor binding agent reversibly bind to the reagent; and

[00705] then

[00706] i) the first receptor binding agent and the second receptor binding agent each individually comprise a binding partner C1, and the plurality of binding sites comprise two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the first and second receptor binding agents and the reagent; or

[00707] ii) the first receptor binding agent comprises a C1 binding partner, the second receptor binding agent comprises a C2 binding partner, and the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner and the C2 binding partner to form the reversible bond between the first and second receptor binding agents and the reagent; or

[00708] iii) the first receptor binding agent comprises a C1 binding partner, the second receptor binding agent comprises a C2 binding partner, and the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner to form the reversible bond between the first receptor binding agent and the reagent, and two or more binding sites, Z2, each of which is capable of binding to the C2 binding partner to form the reversible bond between the second receptor binding agent and the reagent.

[00709] 51. A method for culturing target cells, comprising incubating a composition comprising target cells in the presence of a receptor binding agent that is i) reversibly bound to a reagent that is a streptavidin or mutein analog comprising a plurality of binding sites capable of reversibly binding to the receptor binding agent; and ii) is capable of specifically binding to a molecule on the surface of the target cells in a manner that induces or modulates a signal in target cells in the composition, wherein the streptavidin or mutein comprises a net negative charge or exhibits an isoelectric point lower than the isoelectric point of the streptavidin mutein set forth in SEQ ID NO:4 or SEQ ID NO:6, thereby generating cultured target cells.

[00710] 52. A method for culturing target cells, comprising incubating a composition comprising target cells in the presence of a receptor binding agent that is i) reversibly bound to a reagent that is a streptavidin analog or mutein comprising a plurality of binding sites capable of reversibly binding to the receptor binding agent; and ii) is capable of specifically binding to a molecule on the surface of target cells in a manner that induces or modulates a signal in target cells in the composition, in which the streptavidin analog or mutein exhibits a higher affinity for a streptavidin-binding peptide comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than a streptavidin or mutein comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-6, thereby generating cultured target cells.

[00711] 53. The method of embodiment 51 or embodiment 52, in which the incubation is carried out under conditions in which the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in one or more target cells in the composition.

[00712] 54. The method of any of embodiments 51-53, wherein:

[00713] the plurality of binding sites comprises two or more binding sites, Z1; and

[00714] The receptor binding agent comprises a binding partner C1, which is capable of reversibly binding to the binding site Z1, wherein the reversible binding between C1 and Z1 effects the reversible binding between the receptor binding agent and the reagent.

[00715] 55. The method of embodiment 54, wherein the streptavidin analog or mutein comprises a plurality of Z1 binding sites, in which a plurality of receptor binding agents are reversibly bound to the reagent.

[00716] 56. The method of any of embodiments 51-56, wherein:

[00717] target cells comprise blood cells;

[00718] target cells comprise leukocytes;

[00719] target cells comprise lymphocytes;

[00720] target cells comprise B cells;

[00721] the target cells comprise a B cell population;

[00722] target cells comprise T cells;

[00723] the target cells comprise a T cell population; and/or

[00724] Target cells comprise natural killer (NK) cells.

[00725] 57. The method of any of embodiments 51-56, wherein the target cells comprise antigen-specific T cells or a population thereof, a helper T cell or population thereof, a cytotoxic T cell or population thereof, a memory T cell or population thereof, a regulatory T cell or population thereof, an NK cell or population thereof, antigen-specific B cells or a population thereof, a memory B cell or population thereof, or a regulatory B cell or population thereof.

[00726] 58. The method of any of embodiments 51-57, wherein the target cells are T cells.

[00727] 59. The method of any of embodiments 51-58, wherein the molecule is present on the surface of T cells, wherein the receptor binding agent is capable of inducing or modulating a signal in T cells in the composition.

[00728] 60. The method of any of embodiments 51-59, wherein:

[00729] the receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells; and/or

[00730] the receptor binding agent specifically binds to an element of a TCR/CD3 complex; and/or

[00731] the receptor binding agent specifically binds to CD3.

[00732] 61. The method of any of embodiments 51-60, wherein the molecule is a first molecule and the receptor binding agent is capable of specifically binding to the first molecule and a second molecule on the surface of one or more of the target cells, wherein binding to the second molecule is optionally capable of enhancing, decreasing, or modifying a signal delivered through the first molecule.

[00733] 62. The method of any of embodiments 51-61, wherein the receptor binding agent is a first receptor binding agent and the incubation is also carried out in the presence of a second receptor binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the target cells, wherein binding to the second molecule is optionally capable of inducing or modulating a signal to enhance, diminish, or modify a signal delivered through the first molecule.

[00734] 63. The method of embodiment 62, wherein the incubation is carried out under conditions wherein the second receptor binding agent specifically binds to the second molecule, thereby inducing or modulating a signal in target cells in the composition to enhance, decrease, or modify a signal delivered through the first molecule.

[00735] 64. The method of embodiment 62 or embodiment 63, wherein the streptavidin mutein or analog comprises a plurality of binding sites capable of reversibly binding to the second receptor binding agent, whereby the second receptor binding agent is reversibly bound to the streptavidin mutein or analog.

[00736] 65. The method of embodiment 64, wherein the second receptor binding agent comprises a C1 or C2 binding partner, which is capable of reversibly binding to two or more Z2 binding sites present on the streptavidin analog or mutein.

[00737] 66. The method of any of embodiments 61-65, wherein:

[00738] the additional molecule is selected from CD28, CD90 (Thy- 1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 and HVEM; and/or

[00739] the second receptor binding agent specifically binds to CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

[00740] 67. The method of any of embodiments 61-66, wherein:

[00741] the additional molecule is selected from CD40 and CD137; and/or

[00742] the second receptor binding agent specifically binds to CD40 or CD137.

[00743] 68. The method of any of embodiments 11-67, wherein the second receptor binding agent comprises a plurality of different receptor binding agents, each of which is capable of individually binding to the same or a different second molecule on the surface of T cells in the composition to collectively induce or modulate one or more signals in the cells.

[00744] 69. The method of any of embodiments 37-68, further comprising breaking the reversible bond between the first and/or second receptor binding agent and the reagent.

[00745] 70. The method of embodiment 69, wherein the disruption is performed within 14 days after the start of incubation, within 12 days after the start of incubation, within 10 days after the start of incubation, within 8 days after the start of incubation, or within 6 days after the start of incubation.

[00746] 71. The method of any of embodiments 37-70, wherein at least a portion of the incubation is carried out in the presence of another agent that is capable of delivering a signal to the T cells.

[00747] 72. The method embodiment 71, in which the other agent is capable of enhancing or inducing the proliferation of T cells, CD4+ T cells, and/or CD8+ T cells.

[00748] 73. The method of embodiment 71 or embodiment 72, in which the other agent is a cytokine selected from IL-2, IL-15, and IL-7.

[00749] 74. The method of any of embodiments 71-73, wherein the other agent nonspecifically binds to CD28 and/or induces CD28 signaling.

[00750] 75. The method of any of embodiments 1-74, in which the T cells or target cells are primary cells from an individual

[00751] 76. The method of any of embodiments 1-75, in which the T cells or target cells are directly isolated from a subject.

[00752] 77. The method of any of embodiments 1-50 and 5776, wherein the T cells are unfractionated T cells, are enriched or isolated CD3+ T cells, are enriched or isolated CD4+ T cells, or are enriched or isolated CD8+ T cells.

[00753] 78. The method of any of embodiments 1-50 and 57-77, wherein prior to incubation, the T cells are not enriched for CD62L+ cells and/or are not enriched for naïve T cells.

[00754] 79. The method of any of embodiments 1-78, wherein the T cells or target cells are human cells.

[00755] 80. The method of any of embodiments 1-80, wherein:

[00756] the reagent has a size that is smaller than 20 nm, smaller than 10 nm, smaller than 5 nm, or smaller than 1 nm; or

[00757] the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3.

[00758] 81. The method of any of embodiments 1-80, wherein the reagent is not bound to a support or a solid support during said incubation.

[00759] 82. The method of any of embodiments 1-80, wherein the reagent is bound to a support during at least a portion of the incubation, whereby a plurality of the T cells or target cells are reversibly immobilized on the support during at least a portion of the incubation.

[00760] 83. The method of embodiment 82, in which the support is a solid support or a stationary phase.

[00761] 84. The method of any of embodiments 1-83, wherein:

[00762] the receptor binding agent, which may be the first receptor binding agent, comprises only one of said binding sites, B2; and/or

[00763] the receptor binding agent, which may be the first receptor binding agent, specifically binds to the molecule in a monovalent manner.

[00764] 85. The method of any of embodiments 1-84, wherein:

[00765] the second receptor binding agent comprises only one of said binding sites, B4; and/or

[00766] the second receptor binding agent specifically binds to the molecule in a monovalent manner.

[00767] 86. The method of embodiment 84 or embodiment 85, in which the binding site, B2 and/or B4, comprises an antibody combining site.

[00768] 87. The method of any of embodiments 1-86, wherein the receptor binding agent, which may be a first receptor binding agent, and/or the second receptor binding agent, is each individually selected from an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, and an MHC molecule and binding fragments thereof.

[00769] 88. The method of any of embodiments 1-87, wherein:

[00770] the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent comprises an antibody fragment;

[00771] the receptor binding agent, which may be a first receptor binding agent, and/or the second receptor binding agent comprises a Fab fragment;

[00772] the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent is a divalent antibody fragment selected from an F(ab’)2 fragment and a divalent single chain Fv (scFv) fragment;

[00773] the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment and an scFv fragment; and/or

[00774] The receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent is a proteinaceous binding molecule with antibody-like binding properties selected from aptamers, muteins based on a polypeptide of the lipocalin family, glucobodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins and avimers.

[00775] 89. The method of any of embodiments 1-88, wherein:

[00776] the receptor binding agent, which may be the first receptor binding agent, comprises an agent that specifically binds to CD3, optionally selected from the group consisting of an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3 binding molecule having antibody-like binding properties, and/or

[00777] the second receptor binding agent comprises an agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and/or HVEM, optionally selected from the group consisting of an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, an antibody fragment of an anti-CD28 antibody, a CD28 proteinaceous binding molecule with antibody-like binding properties, an anti-CD90 antibody, a divalent antibody fragment of an anti-CD90 antibody, an antibody fragment of an anti-CD90 antibody, a proteinaceous CD90 binding molecule with antibody-like binding properties, an anti-CD95 antibody, a divalent antibody fragment of an anti-CD95 antibody, an antibody fragment of an anti-CD95 antibody, a proteinaceous CD95 binding molecule with antibody-like binding properties antibody, an anti-CD154 antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154 antibody, a CD154 binding proteinaceous molecule with antibody-like binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a CD137 binding proteinaceous molecule with antibody-like binding properties, an anti-ICOS antibody, a divalent antibody fragment of an anti-ICOS antibody, an antibody fragment of an anti-ICOS antibody, a proteinaceous ICOS binding molecule with antibody-like binding properties, an anti-LAT antibody, a divalent antibody fragment of an anti-LAT antibody, an antibody fragment of an anti-LAT antibody, a proteinaceous LAT binding molecule with antibody-like binding properties, an anti-CD27 antibody, a divalent antibody fragment of an anti-CD27 antibody, an antibody fragment of an anti-CD27 antibody, a proteinaceous CD27 binding molecule with antibody-like binding properties, an anti-OX40 antibody, a divalent antibody fragment of an anti-OX40 antibody, an antibody fragment of an anti-OX40 antibody, a proteinaceous OX40 binding molecule with antibody-like binding properties, an anti-HVEM antibody, a divalent antibody fragment of an anti-HVEM antibody, an antibody fragment of an anti-HVEM antibody, a proteinaceous HVEM binding molecule with antibody-like binding properties, and 4-1BB ligand, and any mixture thereof.

[00778] 90. The method of any of embodiments 1-50 and 6889, wherein the reagent is or comprises streptavidin, avidin, a streptavidin analog or mutein that reversibly binds biotin, a biotin analog or a biologically active fragment thereof; an avidin or streptavidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent comprising at least two K-chelating groups, in which the at least two chelating groups are capable of binding a transition metal ion; an agent capable of binding an oligohistidine affinity tag; an agent capable of binding a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding calmodulin-binding peptide (CBP); an agent capable of binding a FLAG peptide; an agent capable of binding to an HA tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[00779] 91. The method of any of embodiments 1-94, wherein:

[00780] the reagent is or comprises a streptavidin or mutein analogue or an avidin or mutein analogue that reversibly binds biotin or a biologically active fragment;

[00781] the reagent is or comprises a streptavidin analogue or mutein or an avidin analogue or mutein that reversibly binds to a biotin analogue or biologically active fragment; and/or

[00782] the reagent is or comprises a streptavidin or mutein analogue or an avidin or mutein analogue that reversibly binds to a streptavidin-binding peptide.

[00783] 92. The method of any of embodiments 1-91, wherein the reagent is an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein that reversibly binds biotin or a biologically active fragment; a streptavidin or avidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent comprising at least two K-chelating groups, in which the at least two chelating groups are capable of binding a transition metal ion; an agent capable of binding an oligohistidine affinity tag; an agent capable of binding a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding calmodulin-binding peptide (CBP); an agent capable of binding a FLAG peptide; an agent capable of binding an HA tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[00784] 93. The method of any of embodiments 1-92, in which the reagent comprises an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein, or and an avidin analog or mutein.

[00785] 94. The method of embodiment 92 or embodiment 93, in which individual molecules of the oligomer or polymer are cross-linked by a polysaccharide or a bifunctional linker.

[00786] 95. The method of any of embodiments 1-94, in which the plurality of binding sites comprises at least 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 72 or more binding sites.

[00787] 96. The method of any of embodiments 90-95, in which the streptavidin-binding peptide is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

[00788] 97. The method of any of embodiments 1-96, in which:

[00789] the reagent comprises a streptavidin analogue or mutein comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1; or

[00790] the streptavidin analogue or mutein comprises the amino acid sequence Va144-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1.

[00791] 98. The method of any of embodiments 1-51 and 6897, wherein the streptavidin analog or mutein comprises:

[00792] a) the amino acid sequence set forth in any of SEQ ID NOS: 3-6, 27 and 28;

[00793] b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS:3-6, 27 and 28 and contains the amino acid sequence corresponding to Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 and that reversibly binds biotin or a biologically active form thereof, a biotin analog or mutein or a biologically active fragment thereof or a streptavidin-binding peptide; or

[00794] c) a functional fragment of a) or b) that reversibly binds to biotin or a biologically active form thereof, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide.

[00795] 99. The method of embodiment 97 or embodiment 98, in which the streptavidin analog or mutein further comprises an amino acid substitution or substitutions at a position corresponding to 117, 120, and/or 121 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1.

[00796] 100. The method of embodiment 99, in which:

[00797] the amino acid substitution(s) are selected from Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121, or Phe121; or

[00798] the amino acid substitution or substitutions are selected from one or more of Glu117, Gly120, or Tyr121; or

[00799] The amino acid substitutions are selected from Glu117, Gly120 or Tyr121.

[00800] 101. The method of any of embodiments 51-100, wherein the streptavidin analog or mutein comprises:

[00801] a) the amino acid sequence set forth in SEQ ID NO: 27 or 28;

[00802] b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identities to SEQ ID NOS:28 and contains the amino acid sequence corresponding to Va144, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121 and reversibly binds biotin or a biologically active fragment, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide; or

[00803] c) a functional fragment of a) or b) that reversibly binds biotin or a biologically active fragment thereof, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide.

[00804] 102. The method of any of embodiments 1-101, in which the binding partner C1 and/or the binding partner C2, independently, comprises a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19);

[00805] 103. The method of any of embodiments 1-36 and 69-102, wherein said disruption comprises introducing to the cells a composition comprising a substance capable of reversing the binding between the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent, and the reagent.

[00806] 104. The method of embodiment 103, in which the substance is a free binding partner and/or is a competing agent.

[00807] 105. The method of embodiment 103 or embodiment 104, wherein the substance in the composition is not harmful to the T cells or target cells and/or wherein the addition of said substance does not reduce the percentage of surviving T cells or target cells to less than 90%, 80%, 70%, 60%, or 50%, as compared to incubating the T cells or target cells, respectively, under comparable or the same conditions without the substance.

[00808] 106. The method of any of embodiments 1-36 and 69-105, wherein said disruption terminates or reduces the signal induced or modulated by one or both of the receptor binding agent or second receptor binding agent on the T cells or target cells.

[00809] 107. The method of any of embodiments 10-18, 22-32, and 69-107, wherein:

[00810] the reagent is or comprises a streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein or biologically active fragments thereof; and

[00811] the substance comprises a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analogue or biologically active fragment.

[00812] 108. The method of embodiment 107, wherein:

[00813] the substance is a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys- (GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19); and/or

[00814] the substance is C1 or an analogue thereof or is C2 or an analogue thereof.

[00815] 109. The method of any of embodiments 1-108, wherein:

[00816] the dissociation constant (KD) for the reversible binding between said Z1 binding site and said C1 binding partner and/or for the reversible binding between said Z2 binding site and said C2 binding partner is in the range of 10-2 M to 10-13 M.

[00817] 110. The method of any of embodiments 1-109, wherein:

[00818] prior to incubation, contacting the cells with a selection agent that specifically binds to a marker comprised of T cells or target cells of the composition, thereby generating or obtaining the composition comprising the T cells or target cells; or

[00819] at least a portion of the incubation is also carried out in the presence of a selection agent that specifically binds to a marker comprised of T cells or target cells of the composition, wherein the cultured T cells are enriched for T cells or target cells comprising the marker.

[00820] 111. The method of embodiment 110, wherein:

[00821] the selection agent is reversibly bound to the reagent, said reagent also comprising a plurality of binding sites capable of specifically binding to the selection agent; or

[00822] the selection agent is reversibly linked to a second reagent comprising a plurality of binding sites capable of specifically binding to the selection agent.

[00823] 112. The method of any of embodiments 1-110, wherein the signal induction or modulation effects an increase in expansion (proliferation) and/or activation of the cultured T cells as compared to incubating the T cells in the absence of the signal induction or modulation.

[00824] 113. The method of embodiment 112, in which the increase is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

[00825] 114. The method of any of embodiments 7-113, wherein the induction or modulation of the additional or second signal increases the expansion (proliferation) and/or activation of the T cells compared to incubating the T cells in the absence of the induction or modulation of the additional or second signal.

[00826] 115. The method of embodiment 114, wherein the increase is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

[00827] 116. The method of any of embodiments 1-115, wherein the method increases the expansion and/or proliferation of T cells in the composition, alters the metabolic profile of T cells in the composition, alters the subset of CD8+ T cells in the composition; and/or increases the percentage of long-lived memory T cells in the composition, each compared to T cells in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling.

[00828] 117. The method of any of embodiments 1-116, wherein the methods result in cultured T cells wherein:

[00829] the number or percentage of CD3+ T cells, CD4+ T cells, or CD8+ cells is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the number or percentage of CD3+ T cells, CD4+ T cells, or CD8+ T cells, respectively, in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to an analogous incubation but performed in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling;

[00830] the ratio of CD8+ T cells or the relative or normalized ratio of CD8+ T cells in the composition is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the ratio or the relative or normalized ratio of CD8+ T cells in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to an analogous incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling;

[00831] the number or percentage of CD62L+, optionally long-lived memory T cells or memory stem cells (TSCM), in the composition is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the number or percentage of the corresponding population of cells, CD62L+, long-lived memory T cells, or TSCM, in the composition prior to incubation, subsequent to incubation but in the absence of disruption, or subsequent to an analogous incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling.

[00832] 118. The method of any of embodiments 1-117, wherein the cultured T cells comprise greater than 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% of a T cell subset comprising a phenotype that is surface positive for CD62L (CD62L+) as a percentage of the total T cells in the composition or of the total cells in the composition.

[00833] 119. The method of embodiment 118, wherein the T cell subset further comprises a phenotype comprising:

[00834] a) CD127+; and/or

[00835] b) any one or more of CD45RA+, CD45RO-, CCR7+ and CD27+ and any one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+.

[00836] 120. The method of embodiment 118 or embodiment 119, in which the T cell subset:

[00837] a) comprises a low level of TCR recombination excision circles (TRECs); and/or

[00838] b) expresses a proliferation marker, optionally Ki-67; and/or

[00839] c) exhibits the ability to proliferate in the presence of a stimulating agent; and/or

[00840] d) exhibits the ability to produce a cytokine selected from IFN-gamma, TNF and IL-2 in the presence of a stimulating agent.

[00841] 121. The method of embodiment 120, wherein the stimulatory agent is an antigen, a homeostatic cytokine, optionally IL-15 and/or IL-17, or is an agent that is capable of initiating a signal associated with the TCR/CD3 complex on T cells.

[00842] 122. The method of any of embodiments 118-121, wherein the T cell subset is or comprises long-lived memory T cells.

[00843] 123. The method of any of embodiments 118-122, wherein the T cell subset is or comprises T stem memory cells (TSCM).

[00844] 124. The method of any of embodiments 1-123, further comprising introducing a recombinant nucleic acid molecule into T cells or target cells of the population, wherein the nucleic acid molecule encodes a recombinant protein, whereby the cells express the recombinant protein.

[00845] 125. The method of embodiment 124, wherein the recombinant receptor is a chimeric antigen receptor or transgenic T cell receptor (TCR).

[00846] 126. The method of any of embodiments 1-125, in which the method is performed in vitro or ex vivo.

[00847] 127. The method of any of embodiments 1-126, further comprising administering the cultured cells to a subject having a disease or condition.

[00848] 128. The composition, comprising a plurality of cultured T cells or target cells produced by the method of any of embodiments 1-127, and optionally a pharmaceutically acceptable excipient.

[00849] 129. The composition of embodiment 128, in which after addition of the substance, the cells were not incubated in vitro or ex vivo at a temperature greater than 30 °C for more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours.

[00850] 130. An article of manufacture, comprising:

[00851] a) a reagent comprising a plurality of binding sites capable of binding to a receptor binding agent; and

[00852] b) the receptor binding agent wherein i) is reversibly bound to the reagent and ii) is capable of specifically binding to a molecule on the surface of T cells in a manner that induces or modulates a signal in T cells, in which the molecule is not CD28 or CD3.

[00853] 131. The article of manufacture of embodiment 130, in which:

[00854] binding to the molecule induces or modulates a signal in a T cell other than a signal associated with the TCR/CD3 complex; and/or

[00855] binding to the molecule enhances or potentiates a signal associated with the TCR/CD3 complex.

[00856] 132. The article of manufacture of embodiment 130 or embodiment 131, wherein the molecule is selected from CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM.

[00857] 133. The article of manufacture of any of embodiments 130-132, in which the molecule is not CD137.

[00858] 134. An article of manufacture, comprising:

[00859] a) a reagent that is a streptavidin or mutein analogue comprising a plurality of binding sites capable of binding an agent, wherein the streptavidin or mutein analogue comprises a net negative charge or exhibits a lower isoelectric point than a streptavidin mutein comprising the amino acid sequence set forth in SEQ ID NO:4 or 6 and/or exhibits a higher affinity for a streptavidin-binding peptide comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:8) than a streptavidin or mutein comprising the amino acid sequence set forth in SEQ ID NO:4 or 6; and

[00860] b) the agent in which i) is reversibly bound to the reagent and ii) is capable of specifically binding to a molecule on the surface of a cell.

[00861] 135. The article of manufacture of any of embodiments 130-134, in which:

[00862] the receptor binding agent or agent comprises a C1 binding partner; and

[00863] the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent or agent and the reagent.

[00864] 136. The article of manufacture of any of embodiments 130-133, wherein the receptor binding agent is a second receptor binding agent and the molecule is a second molecule, and the reagent further comprises: c) a plurality of binding sites capable of reversibly binding to a first receptor binding agent, and d) the first receptor binding agent wherein i) is reversibly bound to the reagent and ii) is capable of specifically binding to a first molecule on the surface of a T cell.

[00865] 137. The article of manufacture of embodiment 134 or embodiment 136, wherein the agent or the first receptor binding agent specifically binds to an element of a TCR/CD3 complex and/or the first receptor binding agent specifically binds to CD3.

[00866] 138. The article of manufacture of embodiment 136 or embodiment 137, in which:

[00867] i) the first receptor binding agent and the second receptor binding agent each individually comprise a binding partner C1, and the plurality of binding sites comprise two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the first and second receptor binding agents and the reagent; or

[00868] ii) the first receptor binding agent comprises a C1 binding partner, the second receptor binding agent comprises a C2 binding partner, and the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner and the C2 binding partner to form the reversible binding between the first and second receptor binding agents and the reagent; or

[00869] iii) the first receptor binding agent comprises a C1 binding partner, the second receptor binding agent comprises a C2 binding partner, and the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the C1 binding partner to form the reversible bond between the first receptor binding agent and the reagent, and two or more binding sites, Z2, each of which is capable of binding to the C2 binding partner to form the reversible bond between the second receptor binding agent and the reagent.

[00870] 139. The article of manufacture of any of embodiments 130-139, in which:

[00871] the reagent has a size that is smaller than 20 nm, smaller than 10 nm, smaller than 5 nm, or smaller than 1 nm; or

[00872] the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3.

[00873] 140. The article of manufacture of any of embodiments 130-139, in which the reagent is not attached to a support or solid support.

[00874] 141. The article of manufacture of any of embodiments 130-140, in which the reagent is bound or immobilized to a support.

[00875] 142. The article of manufacture of embodiment 141, in which the support is a solid support or a stationary phase.

[00876] 143. The article of manufacture of embodiment 141 or embodiment 142, wherein the support comprises a bead, a particle, a nanoparticle, or a microsphere.

[00877] 144. The article of manufacture of any of embodiments 130-143, in which the agent, the receptor binding agent, which may be a first receptor binding agent, and/or the second receptor binding agent, is each individually selected from an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule having antibody-like binding properties, an Ig domain-containing molecule, and binding fragments thereof.

[00878] 145. The article of manufacture of any of embodiments 130-144, in which:

[00879] the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent comprises an antibody fragment;

[00880] the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent comprises a Fab fragment;

[00881] the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent is a divalent antibody fragment selected from an F(ab’)2 fragment and a divalent single-chain Fv (scFv) fragment; or

[00882] the agent, the receptor binding agent, which may be the second receptor binding agent, and/or the first receptor binding agent is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment and an scFv fragment.

[00883] 146. The article of manufacture of any of embodiments 130-145, in which:

[00884] the receptor binding agent or first agent comprises an agent that specifically binds to CD3, optionally selected from the group consisting of an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3 binding molecule having antibody-like binding properties, and/or

[00885] the receptor binding agent or agent, which may be the second receptor binding agent, comprises an agent that specifically binds to CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, or HVEM, optionally selected from the group consisting of an anti-CD90 antibody, a divalent antibody fragment of an anti-CD90 antibody, an antibody fragment of an anti-CD90 antibody, a proteinaceous CD90 binding molecule having antibody-like binding properties, an anti-CD95 antibody, a divalent antibody fragment of an anti-CD95 antibody, an antibody fragment of an anti-CD95 antibody, a proteinaceous CD95 binding molecule having antibody-like binding properties, an anti-CD154 antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154 ... CD154 binding proteinaceous molecule with antibody-like binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a CD137 binding proteinaceous molecule with antibody-like binding properties, an anti-ICOS antibody, a divalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, an ICOS binding proteinaceous molecule with antibody-like binding properties, an anti-LAT antibody, a divalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, a LAT binding proteinaceous molecule with antibody-like binding properties, an anti-CD27 antibody, a divalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, a CD27 binding proteinaceous molecule with antibody-like binding properties, an antibody anti-OX40, a divalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40 antibody, a proteinaceous OX40-binding molecule with antibody-like binding properties, an anti-HVEM antibody, a divalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, a proteinaceous HVEM-binding molecule with antibody-like binding properties, and any mixture thereof.

[00886] 147. The article of manufacture of any of embodiments 130-147, in which:

[00887] the reagent is or comprises streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein; or

[00888] the reagent comprises an oligomer or polymer of streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein.

[00889] 148. The kit, comprising the article of manufacture of any of embodiments 130-147 and optionally instructions for use.

[00890] 149. The kit of embodiment 148, also comprising a substance capable of reversing the binding between the receptor binding agent and the reagent

[00891] 150. The kit, comprising:

[00892] a) a reversible reagent comprising i) a reagent comprising a plurality of binding sites capable of reversibly binding to a receptor binding agent; and ii) the receptor binding agent that is reversibly bound to the reagent and is capable of specifically binding to a molecule expressed on the surface of target cells, and optionally wherein binding to the molecule induces or modulates a signal in the target cells; and

[00893] b) a substance capable of reversing the binding between the receptor binding agent and the reagent.

[00894] 151. The kit of embodiment 150, in which the target cells are T cells.

[00895] 151. The kit of mode 150 or mode 151, in which:

[00896] the reagent is or comprises streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein; or

[00897] the reagent comprises an oligomer or polymer of streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein.

[00898] 152. The kit of any of embodiments 149-151, in which the substance comprises a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analog or biologically active fragment.

[00899] 153. The kit of any of the modalities 150-152, in which:

[00900] the reagent has a size that is smaller than 20 nm, smaller than 10 nm, smaller than 5 nm, or smaller than 1 nm; or

[00901] the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3.

[00902] 154. The kit of any of embodiments 150-153, in which the reagent is not bound to a support or solid support.

[00903] 155. The composition, comprising a plurality of T cells genetically engineered to express a recombinant receptor that specifically binds to a target antigen, wherein:

[00904] greater than 35%, 40%, 50%, 60%, 70%, 80% or 90% of the cells comprise a T cell subset comprising a surface phenotype that is CD3+, CD4+ or CD8+ and CD62L+ and one or more of CD127+, CD45RA+, CD45RO-, CCR7+ and CD27+ and one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+ as a percentage of the total T cells in the composition or of the total cells in the composition; and each or both:

[00905] a) prior to or during genetic construction, the plurality of T cells comprising the T cell subset:

[00906] i) was not incubated in the presence of a GSK-P inhibitor;

[00907] ii) was not incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15; or

[00908] iii) was not enriched for CD62L+ cells; or

[00909] b) the composition does not comprise a GSK-P inhibitor or a recombinant homeostatic cytokine, optionally IL-7 or IL-15.

[00910] 156. The composition of embodiment 155, wherein the T cell subset comprises at least 5 X 106, at least 1 X 106, or at least 2 X 106 cells.

[00911] 157. The composition, comprising a plurality of T cells genetically engineered to express a recombinant receptor that specifically binds to a target antigen, wherein:

[00912] the genetically engineered T cells are derived from transduction of a T cell population comprising a T cell subset comprising a surface phenotype that is CD3+, CD4+ or CD8+ and CD62L+ and one or more of CD127+, CD45RA+, CD45RO-, CCR7+ and CD27+ and one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+, in which the T cell subset is present in a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to:

[00913] a) a population comprising primary T cells that have been isolated or enriched from a human subject based on the surface expression of one or more markers comprising the phenotype; or

[00914] b) a population of T cells that has been incubated in the presence of a GSK-P inhibitor;

[00915] c) a population of T cells that has been incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15; or

[00916] d) a population of T cells that were stimulated by anti-CD3 and anti-CD8, but in which the stimulation or activation was for more than 1 day, 2 days, 3 days, 4 days or 5 days and/or the stimulation was not disrupted in the presence of biotin or a biotin analogue.

[00917] 158. The composition of mode 156, in which:

[00918] the T cell subset is present in the population at or about greater than 35%, 40%, 50%, 60%, 70%, 80%, or 90% as a percentage of the total T cells in the population; or

[00919] the T cell subset comprises at least 5 x 106 cells, 1 x106 cells, 2 x106 cells or more.

[00920] 159. The composition of any of embodiments 155158, which is a pharmaceutical composition.

[00921] 160. A method of treatment, comprising administering to a subject having a disease or condition, the composition of embodiment 128, embodiment 129, and any of embodiments 154-158.

[00922] 161. The treatment method of embodiment 160, wherein the cells comprise a recombinant receptor, chimeric antigen receptor, or TCR and the recombinant receptor, chimeric antigen receptor, or transgenic TCR specifically binds to an antigen associated with the disease or condition.

[00923] 162. The treatment method of embodiment 160 or embodiment 161, in which the disease or condition is a cancer, an autoimmune disease or disorder, or an infectious disease.

[00924] 163. A method for culturing target cells, comprising incubating a composition comprising target cells in the presence of a receptor binding agent which is i) reversibly bound to a reagent comprising a plurality of binding sites capable of reversibly binding to the receptor binding agent; and ii) is capable of specifically binding to a molecule on the surface of the target cells other than CD28, CD3, CD137, or CD40 in a manner that induces or modulates a signal in the target cells and/or alters a function of the target cells, thereby generating cultured target cells.

[00925] 164. The method of embodiment 163, wherein the receptor deactivating agent specifically binds to a cytokine receptor, specifically binds to a chemokine receptor, is or comprises an adhesion molecule, or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule.

[00926] 165. The method of embodiment 163 or embodiment 164, in which the receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, TNFR2, IL-7R, IL-21R, and CD132 (IL receptor common gamma chain).

[00927] 166. The method of any of embodiments 163-165, in which:

[00928] the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R , Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1, TNFR2, IL-7R, IL-21R and CD132(IL receptor common gamma chain); and/or

[00929] the receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, TNF, IL-7, IL-21 and IL-9, or is a biologically active fragment thereof.

[00930] 167. The method of any of embodiments 163-165, in which:

[00931] the receptor binding agent specifically binds to a cytokine selected from IL-12R, IFN-gammaR, IL-4R and IL-17R; or

[00932] the receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15 and IL-17 or is a biologically active fragment thereof.

[00933] 168. The method of embodiment 163 or embodiment 164, in which the receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4.

[00934] 169. The method of embodiment 163, embodiment 164, or embodiment 168, in which:

[00935] the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or

[00936] the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[00937] 170. The method of any of embodiment 163, embodiment 164, embodiment 168, or embodiment 169, in which:

[00938] the receptor binding agent specifically binds to a cytokine selected from CXCR3, CCR7, CXCR1, and CXCR4; or

[00939] the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[00940] 171. The method of embodiment 163 or embodiment 164, in which the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof.

[00941] 172. The method of embodiment 171, wherein the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1, and VLA-4 or is a biologically active fragment thereof.

[00942] 173. The method of embodiment 163 or embodiment 164, in which the factor is a nuclear factor.

[00943] 174. The method of embodiment 163, embodiment 164 or embodiment 173, wherein the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[00944] 175. The method of any of embodiments 163-174, in which:

[00945] target cells comprise blood cells;

[00946] target cells comprise leukocytes;

[00947] target cells comprise lymphocytes;

[00948] target cells comprise B cells;

[00949] the target cells comprise a B cell population;

[00950] target cells comprise T cells;

[00951] the target cells comprise a T cell population; and/or

[00952] Target cells comprise natural killer (NK) cells.

[00953] 176. The method of any of embodiments 163-175, wherein the target cells comprise immune cells.

[00954] 177. The method of any of embodiments 163-176, wherein the target cells comprise antigen-specific T cells or a population thereof, a helper T cell or population thereof, a cytotoxic T cell or population thereof, a memory T cell or population thereof, a regulatory T cell or population thereof, an NK cell or population thereof, antigen-specific B cells or a population thereof, a memory B cell or population thereof, or a regulatory B cell or population thereof.

[00955] 178. The method of any of embodiments 163-177, in which the target cells are T cells.

[00956] 179. The method of any of embodiments 163-178, in which the target cells are primary cells from an individual.

[00957] 180. The method of any of embodiments 163-179, wherein the incubation is carried out under conditions in which the receptor binding agent specifically binds to the molecule, thereby inducing or modulating the signal in the target cells and/or altering a function in the target cells.

[00958] 181. The method of any of embodiments 163-180, wherein the receptor binding agent comprises a binding partner C1, and the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent and the reagent.

[00959] 182. The method of any of embodiments 163-181, wherein the receptor binding agent is an additional receptor binding agent, and the molecule is an additional molecule, and the incubation is also carried out in the presence of a first receptor binding agent, which is capable of specifically binding to a first molecule on the surface of one or more of the T cells, wherein the first molecule is optionally capable of inducing or modulating a first signal on one or more T cells in the composition.

[00960] 183. The method of embodiment 182, in which:

[00961] the first receptor binding agent is reversibly bound to the reagent, said reagent comprising a plurality of binding sites for the first receptor binding agent and the additional receptor binding agent; or

[00962] The first receptor binding agent is reversibly linked to a second reagent comprising a plurality of binding sites capable of reversibly binding to the first receptor binding agent.

[00963] 184. The method of embodiment 182 or embodiment 183, in which:

[00964] the first receptor binding agent is capable of initiating a signal associated with the TCR/CD3 complex on T cells; and/or

[00965] the first receptor binding agent specifically binds to an element of a TCR/CD3 complex; and/or

[00966] the first receptor binding agent specifically binds to CD3.

[00967] 185. The method of any of embodiments 182-184, wherein the first receptor binding agent comprises a C2 binding partner, and the plurality of binding sites comprises two or more binding sites, Z2, each of which is capable of binding to the C2 binding partner to form the reversible bond between the first receptor binding agent and the reagent.

[00968] 186. The method of any of embodiments 182-185, wherein the incubation is also carried out in the presence of a second receptor binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells, which second molecule is optionally capable of inducing or modulating a signal in target cells in the composition to enhance, diminish, or modify a signal delivered through the first molecule.

[00969] 187. The method of embodiment 186, in which:

[00970] the second signal is a signal other than a signal associated with the TCR/CD3 complex;

[00971] the second signal is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex; or

[00972] the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family.

[00973] 188. The method of any of embodiments 186 or embodiment 187, wherein the second molecule is selected from CD28 and CD137.

[00974] 189. The method of any of embodiments 186-188, in which the second molecule is CD28.

[00975] 190. The method of any of embodiments 186-188, wherein the second receptor binding agent comprises a C3 binding partner, and the plurality of binding sites comprises two or more binding sites, Z3, each of which is capable of binding to the C3 binding partner to form the reversible bond between the first receptor binding agent and the reagent.

[00976] 191. The method of any of embodiments 163-190, in which:

[00977] C1 and C2, C1 and C3, C2 and C3 or C1, C2 and C3 are the same or substantially the same, or contain the same or substantially the same moiety;

[00978] Z1 and Z2, Z1 and Z3, Z2 and Z3 or Z1, Z2 and Z3 are the same or substantially the same or contain the same or substantially the same moiety.

[00979] 192. The method of any of embodiments 163-191, wherein the reagent is immobilized, or is capable of being immobilized, on a support for at least a portion of the incubation, whereby the (additional, first and/or second) receptor binding agent is immobilized, or is capable of being immobilized, on the support.

[00980] 193. The method of embodiment 192, in which:

[00981] the support is or comprises a stationary phase; and/or

[00982] the support is or comprises a solid support.

[00983] 194. The method of any of embodiments 163-191, in which:

[00984] the reagent is not, and is not bound to or associated with, a solid support, stationary phase, a bead, a microparticle, a magnetic particle, and/or a matrix during said incubation; and/or

[00985] the reagent is flexible, does not contain a metal or magnetic core, is comprised entirely or primarily of organic multimer, is not spherical, is not substantially spherical or uniform in shape, and/or is not rigid;

[00986] the reagent has a size that is smaller than 20 nm, smaller than 10 nm, smaller than 5 nm, or smaller than 1 nm; or

[00987] the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3.

[00988] 195. The method of any of embodiments 163-194, in which:

[00989] the receptor binding agent, which may be the additional receptor binding agent, comprises a binding site, B1; and/or

[00990] the receptor binding agent, which may be the additional receptor binding agent, comprises only one of said binding sites, B1; and/or

[00991] the receptor binding agent, which may be the additional receptor binding agent, specifically binds to the molecule in a monovalent manner.

[00992] 196. The method of any of embodiments 163-195, in which:

[00993] the first receptor binding agent comprises a binding site, B2; and/or

[00994] the first receptor binding agent comprises only one of said binding sites, B2; and/or

[00995] the first receptor binding agent specifically binds to the molecule in a monovalent manner.

[00996] 197. The method of any of embodiments 163-196, in which:

[00997] the second receptor binding agent comprises a binding site, B4; and/or

[00998] the second receptor binding agent comprises only one of said binding sites, B4; and/or

[00999] the second receptor binding agent specifically binds to the molecule in a monovalent manner.

[001000] 198. The method of any of embodiments 195-197, in which the binding site, B2 and/or B4, comprises an antibody combining site.

[001001] 199. The method of any of embodiments 163-198, wherein the receptor binding agent, which may be an additional receptor binding agent, is selected from a cytokine, a chemokine, or an adhesion molecule.

[001002] 200. The method of any of embodiments 163-199, wherein the receptor binding agent (additional, first, and/or additional) is each individually selected from an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an Ig domain-containing molecule, an aptamer, and an MHC molecule and binding fragments thereof.

[001003] 201. The method of any of embodiments 163-200, in which:

[001004] the receptor binding agent (additional, first and/or additional) comprises an antibody fragment;

[001005] the receptor binding agent (additional, first and/or additional) comprises a Fab fragment;

[001006] the receptor binding agent (additional, first and/or additional) is a divalent antibody fragment selected from an F(ab’)2 fragment and a divalent single chain Fv (scFv) fragment;

[001007] the receptor binding agent (additional, first and/or additional) is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment and an scFv fragment; and/or

[001008] the receptor binding agent (additional, first and/or additional) is a proteinaceous binding molecule with antibody-like binding properties selected from aptamers, muteins based on a polypeptide of the lipocalin family, glucobodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins and avimers.

[001009] 202. The method of any of embodiments 163-201, wherein the reagent is or comprises streptavidin, avidin, a streptavidin analog or mutein that reversibly binds biotin, a biotin analog or a biologically active fragment thereof; an avidin or streptavidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent comprising at least two K chelating groups, in which the at least two chelating groups are capable of binding a transition metal ion; an agent capable of binding an oligohistidine affinity tag; an agent capable of binding a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding calmodulin-binding peptide (CBP); an agent capable of binding a FLAG peptide; an agent capable of binding to an HA tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[001010] 203. The method of any of embodiments 163-202, in which:

[001011] the reagent is or comprises a streptavidin or mutein analogue or an avidin or mutein analogue that reversibly binds biotin or a biologically active fragment;

[001012] the reagent is or comprises a streptavidin analogue or mutein or an avidin analogue or mutein that reversibly binds to a biotin analogue or biologically active fragment; and/or

[001013] the reagent is or comprises a streptavidin or mutein analogue or an avidin or mutein analogue that reversibly binds to a streptavidin-binding peptide.

[001014] 204. The method of any of embodiments 163-203, wherein the reagent is an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein that reversibly binds biotin, or a biologically active fragment; a streptavidin or avidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent comprising at least two K-chelating groups, in which the at least two chelating groups are capable of binding a transition metal ion; an agent capable of binding an oligohistidine affinity tag; an agent capable of binding a glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding calmodulin-binding peptide (CBP); an agent capable of binding a FLAG peptide; an agent capable of binding to an HA tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[001015] 205. The method of any of embodiments 163-204, wherein the reagent comprises an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein, or and an avidin analog or mutein.

[001016] 206. The method of embodiment 204 or embodiment 205, in which individual molecules of the oligomer or polymer are cross-linked by a polysaccharide or a bifunctional linker.

[001017] 207. The method of any of embodiments 163-206, wherein the plurality of binding sites comprises at least 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 72 or more binding sites.

[001018] 208. The method of any of embodiments 202-207, in which the streptavidin-binding peptide is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

[001019] 209. The method of any of embodiments 163-208, in which:

[001020] the reagent comprises a streptavidin analogue or mutein comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1; or

[001021] the streptavidin analogue or mutein comprises the amino acid sequence Va144-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1.

[001022] 210. The method of any of embodiments 163-209, wherein the streptavidin analog or mutein comprises:

[001023] a) the amino acid sequence set forth in any of SEQ ID NOS: 3-6, 27 or 28;

[001024] b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS:3-6, 27 or 28 and contains the amino acid sequence corresponding to Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 and that reversibly binds biotin or a biologically active form thereof, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide; or

[001025] c) a functional fragment of a) or b) that reversibly binds to biotin or a biologically active form thereof, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide.

[001026] 211. The method of embodiment 209 or embodiment 210, in which the streptavidin analog or mutein further comprises an amino acid substitution or substitutions at a position corresponding to 117, 120, and/or 121 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1.

[001027] 212. The method of embodiment 211, in which:

[001028] the amino acid substitution(s) are selected from Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121, or Phe121; or

[001029] the amino acid substitution or substitutions are selected from one or more of Glu117, Gly120 or Tyr121; or

[001030] amino acid substitutions are selected from Glu117, Gly120 or Tyr121.

[001031] 213. The method of any of embodiments 163-212, wherein the streptavidin analog or mutein comprises:

[001032] a) the amino acid sequence set out in SEQ ID NO: 27 or 28;

[001033] b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identities to SEQ ID NOS:28 and contains the amino acid sequence corresponding to Va144, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121 and reversibly binds biotin or a biologically active fragment, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide; or

[001034] c) a functional fragment of a) or b) that reversibly binds biotin or a biologically active fragment thereof, a biotin analogue or mutein or a biologically active fragment thereof or a streptavidin-binding peptide.

[001035] 214. The method of any of embodiments 163-213, in which the C1 binding partner and/or the C2 binding partner, independently, comprises a streptavidin-binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

[001036] 215. The method of any of embodiments 163-214, further comprising breaking the reversible bond between the first and/or second receptor binding agent and the reagent.

[001037] 216. The method of embodiment 215, wherein said disruption comprises introducing to the cells a composition comprising a substance capable of reversing the binding between the receptor binding agent, which may be the first receptor binding agent, and/or the second receptor binding agent, and the reagent.

[001038] 217. The method of embodiment 216, in which the substance is a free binding partner and/or is a competing agent.

[001039] 218. The method of embodiment 216 or embodiment 217, wherein the substance in the composition is not harmful to the T cells or target cells and/or wherein the addition of said substance does not reduce the percentage of surviving T cells or target cells to less than 90%, 80%, 70%, 60%, or 50%, as compared to incubating the T cells or target cells, respectively, under comparable or the same conditions without the substance.

[001040] 219. The method of any of embodiments 215-218, wherein said disruption terminates or reduces the signal induced or modulated by one or both of the receptor binding agent or second receptor binding agent on the T cells or target cells.

[001041] 220. The method of any of embodiments 215-219, in which:

[001042] the reagent is or comprises a streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein or biologically active fragments thereof; and

[001043] the substance comprises a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analogue or biologically active fragment.

[001044] 221. The method of embodiment 220, in which:

[001045] the substance is a streptavidin-binding peptide, is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys- (GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19); and/or

[001046] the substance is C1, C2 or C3 or an analogue thereof.

[001047] 222. The method of any of embodiments 163-221, in which:

[001048] the induction or modulation of the signal results in an altered activity selected from chemotaxis, adhesion, cytokine production, proliferation (expansion), cytotoxic activity, metabolic activity compared to target cells, optionally T cells, in the composition in the absence of the induction or modulation of the signal; and/or

[001049] the altered functional activity is selected from chemotaxis, adhesion, cytokine production, proliferation (expansion), cytotoxic activity, metabolic activity as compared to the functional activity of target cells, optionally T cells, in the composition prior to incubation; and/or

[001050] the cultured target cells exhibit an altered activity selected from chemotaxis, adhesion, cytokine production, proliferation (expansion), cytotoxic activity, metabolic activity compared to the functional activity of target cells, optionally T cells, in the composition prior to incubation.

[001051] 223. The method of embodiment 222, in which the activity is changed at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more.

[001052] 224. The method of any of embodiments 163-223, further comprising introducing a recombinant nucleic acid molecule into T cells or target cells of the population, wherein the nucleic acid molecule encodes a recombinant protein, whereby the cells express the recombinant protein.

[001053] 225. The method of embodiment 224, wherein the recombinant receptor is a chimeric antigen receptor or transgenic T cell receptor (TCR).

[001054] 226. The method of any of embodiments 163-225, in which the method is performed in vitro or ex vivo.

[001055] 227. The method of any of embodiments 163-226, further comprising administering the cultured cells to a subject having a disease or condition.

[001056] 228. The composition, comprising a plurality of cultured T cells or target cells produced by the method of any of embodiments 163-227, and optionally a pharmaceutically acceptable excipient.

[001057] 229. The composition of embodiment 228, in which after addition of the substance, the cells were not incubated in vitro or ex vivo at a temperature greater than 30 °C for more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours.

[001058] 230. An article of manufacture, comprising:

[001059] a) a reagent comprising a plurality of binding sites capable of binding to a receptor binding agent; and

[001060] b) the receptor binding agent wherein i) is reversibly bound to the reagent and ii) is capable of specifically binding to a molecule on the surface of target cells in a manner that induces or modulates a signal in the cells or alters a functional activity in the cells, in which the molecule is not CD28, CD3, CD137 or CD40.

[001061] 231. The article of manufacture of embodiment 230, in which the receptor binding agent specifically binds to a cytokine receptor, specifically binds to a chemokine receptor, is or comprises an adhesion molecule, or is a factor that induces cytokine production, chemokine production, and/or expression of an adhesion molecule.

[001062] 232. The article of manufacture of embodiment 230 or embodiment 231, in which the receptor binding agent specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR Type I, IL-12R, IL-15R, IL-17R, TNFR1, TNFR2, IL-7R, IL-21R, and CD132 (IL receptor common gamma chain).

[001063] 233. The article of manufacture of any of embodiments 230-232, in which:

[001064] the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R , Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1, TNFR2, IL-7R, IL-21R and CD132(IL receptor common gamma chain); and/or

[001065] the receptor binding agent is a ligand selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17, TNF, IL-7, IL-21 and IL-9, or is a biologically active fragment thereof.

[001066] 234. The article of manufacture of any of embodiments 230-232, in which:

[001067] the receptor binding agent specifically binds to a cytokine selected from IL-12R, IFN-gammaR, IL-4R, and IL-17R; or

[001068] the receptor binding agent is a ligand selected from IL-2, IL-4, IL-7, IL-10, IL-15 and IL-17 or is a biologically active fragment thereof.

[001069] 235. The article of manufacture of embodiment 68 or embodiment 69, wherein the receptor binding agent specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4.

[001070] 236. The article of manufacture of embodiment 230, embodiment 231, or embodiment 235, in which:

[001071] the receptor binding agent is a ligand that specifically binds to a cytokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; or

[001072] the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[001073] 237. The article of manufacture of any of mode 68, mode 69, mode 235, or mode 236, in which:

[001074] the receptor binding agent specifically binds to a cytokine selected from CXCR3, CCR7, CXCR1, and CXCR4; or

[001075] the receptor binding agent is a ligand selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[001076] 238. The article of manufacture of embodiment 230 or embodiment 231, wherein the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof.

[001077] 239. The article of manufacture of embodiment 238, in which the adhesion molecule is selected from LFA-1, L-selectin, VCAM-1, and VLA-4 or is a biologically active fragment thereof.

[001078] 240. The article of manufacture of embodiment 230 or embodiment 231, in which the factor is a nuclear factor.

[001079] 241. The article of manufacture of embodiment 230, embodiment 231, or embodiment 240, wherein the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[001080] 242. The article of manufacture of any of embodiments 230-241, in which:

[001081] the receptor binding agent or agent comprises a C1 binding partner; and

[001082] the plurality of binding sites comprises two or more binding sites, Z1, each of which is capable of binding to the binding partner C1 to form the reversible bond between the receptor binding agent or agent and the reagent.

[001083] 243. The article of manufacture of any of embodiments 230-241, wherein the receptor binding agent is an additional receptor binding agent, and the molecule is an additional molecule, and the reagent further comprises: c) a plurality of binding sites capable of reversibly binding to a first receptor binding agent, and d) the first receptor binding agent wherein i) is reversibly bound to the reagent, and ii) is capable of specifically binding to a first molecule on the surface of a T cell.

[001084] 244. The article of manufacture of embodiment 243, wherein the first receptor binding agent specifically binds to an element of a TCR/CD3 complex and/or the first receptor binding agent specifically binds to CD3.

[001085] 245. The article of manufacture of embodiment 243 or embodiment 244, wherein the incubation is also carried out in the presence of a second receptor binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells, which second molecule is optionally capable of inducing or modulating a signal in target cells in the composition to enhance, diminish, or modify a signal delivered through the first molecule.

[001086] 246. The article of manufacture of embodiment 245, in which:

[001087] the second signal is a signal other than a signal associated with the TCR/CD3 complex;

[001088] the second signal is capable of enhancing or potentiating a signal associated with the TCR/CD3 complex; or

[001089] the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule, or is a member of the TNF family or the TNF receptor family.

[001090] 247. The article of manufacture of any of embodiment 245 or embodiment 246, in which the second molecule is selected from CD28 or CD137.

[001091] 248. The article of manufacture of any of embodiments 245-247, in which the second molecule is CD28.

[001092] 249. The article of manufacture of any of embodiments 230-248, in which the reagent is immobilized, or is capable of being immobilized, on a support for at least a portion of the incubation whereby the receptor binding agent (additional, first and/or second) is immobilized, or is capable of being immobilized, on the support.

[001093] 250. The article of manufacture of embodiment 249, in which:

[001094] the support is or comprises a stationary phase; and/or

[001095] the support is or comprises a solid support.

[001096] 251. The article of manufacture of any of embodiments 230-248, in which:

[001097] the reagent is not, and is not bound to or associated with, a solid support, stationary phase, a bead, a microparticle, a magnetic particle, and/or a matrix during said incubation; and/or

[001098] the reagent is flexible, does not contain a metal or magnetic core, is comprised entirely or primarily of organic multimer, is not spherical, is not substantially spherical or uniform in shape, and/or is not rigid;

[001099] the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm; or

[001100] the reagent has a density less than 1.2 g/cm3 or less than 1.0 g/cm3.

[001101] 252. The article of manufacture of any of embodiments 230-251, in which the receptor binding agent, which may be the additional receptor binding agent, the first receptor binding agent, and/or the second receptor binding agent, is each individually selected from an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule having antibody-like binding properties, an Ig domain-containing molecule, and binding fragments thereof.

[001102] 253. The article of manufacture of any of embodiments 230-252, in which:

[001103] the receptor binding agent, which may be the additional receptor binding agent, the first receptor binding agent and/or the second receptor binding agent comprises an antibody fragment;

[001104] the receptor binding agent, which may be the additional receptor binding agent, the first receptor binding agent and/or the second receptor binding agent comprises a Fab fragment;

[001105] the receptor binding agent, which may be the additional receptor binding agent, the first receptor binding agent and/or the second receptor binding agent, is a divalent antibody fragment selected from an F(ab’)2 fragment and a divalent single-chain Fv (scFv) fragment; or

[001106] the receptor binding agent, which may be the additional receptor binding agent, the first receptor binding agent and/or the second receptor binding agent is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment and an scFv fragment.

[001107] 254. The article of manufacture of any of embodiments 230-253, in which:

[001108] the reagent is or comprises streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein; or

[001109] the reagent comprises an oligomer or polymer of streptavidin, avidin, a streptavidin analogue or mutein or and an avidin analogue or mutein.

[001110] 255. The kit, comprising the article of manufacture of any of embodiments 230-254 and optionally instructions for use.

[001111] 256. The kit of embodiment 255, further comprising a substance capable of reversing binding between the receptor binding agent and the reagent

[001112] 257. The kit of embodiment 256, wherein the substance comprises a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analogue or biologically active fragment.

VIII. EXAMPLES

[001113] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1: Generation of a soluble stimulatory reagent containing reversibly multimerized anti-CD3 and anti-CD28 Fab fragments linked to an oligomeric streptavidin mutein reagent

[001114] Stimulating agents (anti-CD3 and anti-CD28 Fab fragments) were multimerized by reversible binding to a multimerization reagent, which was an oligomeric streptavidin mutein. The reagent contained multiple binding sites for peptide tags, which were present on the Fab fragments. Oligomeric streptavidin mutein was prepared by polymerizing the streptavidin mutein designated Strep-tactin® m1 (a streptavidin homotetramer containing the mutein amino acid sequence set forth in SEQ ID NO: 6, see for example US Patent No. 6,103,493 and Voss and Skerra (1997) Protein Eng., 1:975-982) with sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, product #22122 Thermo Scientific) and iminothiolane (product #26101 Thermo Scientific) according to the manufacturer's instructions (Thermo Scientific). Oligomeric streptavidin mutein molecules were separated from monomeric (unreacted) and dimeric streptavidin mutein by size exclusion chromatography.

[001115] Anti-CD3 and anti-CD28 Fab fragments were reversibly linked to oligomeric streptavidin mutein via a streptavidin peptide binding partner fused to each Fab fragment. The anti-CD3 Fab fragment was derived from the CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™; see similarly U.S. Patent No. 4,361,549), and contained the heavy chain variable domain and light chain variable domain of the anti-CD3 antibody OKT3 described in Arakawa et al. J. Biochem. 120, 657-662 (1996). These sequences are set forth in SEQ ID NOs:31 and 32, respectively. The anti-CD28 Fab fragment was derived from antibody CD28.3 (deposited as a synthetic single-chain Fv construct under GenBank Accession No. AF451974.1; see similarly Vanhove et al., BLOOD, July 15, 2003, Vol. 102, No. 2, pages 564-570) and contained the heavy and light chain variable domains of the anti-CD28 antibody CD28.3 set forth in SEQ ID NOS: 33 and 34, respectively. The Fab fragments were individually fused to the carboxy terminus of their heavy chain to a streptavidin peptide-binding sequence containing a sequential array of two streptavidin-binding modules having the amino acid sequence SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 16). Peptide-tagged Fab fragments have been recombinantly produced (see, International Patent Pub. Nos. WO 2013/011011 and WO 2013/124474).

[001116] To effect reversible binding, the anti-CD3 and anti-CD28 peptide-tagged Fab fragments were mixed with the multimerization reagent at approximately room temperature, thereby reversibly binding them to the reagent via interaction between dual streptavidin labels on the Fab fragments, which were binding partners capable of reversibly binding to the binding sites on the reagent. Specifically, in this study, approximately 0.5 μg of the anti-CD3 peptide-tagged Fab fragment and approximately 0.5 μg of the anti-CD28 peptide-tagged Fab fragment were added to approximately 3 μg of soluble oligomeric Streptatactin® at room temperature. In some cases, the peptide-labeled Fab fragments were premixed prior to immobilization on the soluble oligomeric mutein streptavidin backbone, which in some instances may result in a more even distribution of the different Fab molecules. The resulting soluble anti-CD3/anti-CD28 multimerized agent was used to stimulate T cells. In some cases, the resulting soluble anti-CD3/anti-CD28 multimerized agent was stored on ice prior to cell stimulation.

EXAMPLE 2: Assessment of T cell expansion after transiently controlled stimulation of T cells by incubation with a reversible agent

[001117] T cells were incubated with the reagent described in Example 1, containing multimerized reversibly bound agents (anti-CD3/anti-CD28 Fab fragments). The interaction between the multimerized anti-CD3 and anti-CD28 Fabs and the cells was disrupted at various time points after initiation by addition of D-biotin. D-biotin competes with the streptavidin label on the agents for binding to the binding partner on the streptavidin mutein, thereby disrupting binding.

[001118] In more detail, T cells were selected from a sample of fresh peripheral blood mononuclear cells (PBMCs) obtained from a Ficoll gradient, based on the surface expression of CD3, CD4, or CD8. Approximately 500,000 T cells (CD3+, CD4+, or CD8+) were seeded in 48-well plates in 1 mL of complete RPMI medium, supplemented with 50 U/mL IL-2. The reagent (containing reversibly linked anti-CD3/anti-CD28 Fabs) described in Example 1 was added on the day of initiation of cell culture (day 0), typically at least 20 minutes prior to stimulation. For the other sample, cells were spiked with anti-CD3/anti-CD28 beads (which were magnetic beads coated with anti-CD3- and anti-CD28- mAb, to which binding was not disrupted by the addition of D-Biotin, thereby providing a control to confirm that any effects observed with D-biotin addition were due to specific binding and disruption of the multimerized agents). Untreated (unstimulated) cells served as a negative control.

[001119] Cells were incubated at 37 °C for a total of eight days. As shown in FIG. 5A, D-Biotin was added at various time points to cell compositions (final concentration of 1 mM) that were incubated with the multimerized anti-CD3/anti-CD28 stimulatory agents in order to disrupt the reversible binding of the multimeric stimulatory agent complex to the cells, to reduce or attenuate signaling delivered to the cells through the multimerized anti-CD3 and anti-CD28 Fabs. Specifically, D-Biotin was added to culture wells (at one (1) hour, one day, two days, three days, four days, or five days after the start of incubation, or at harvest (day 8)) at a final concentration of 1 mM, without resuspension of cells. Cells were then incubated for 30 minutes at room temperature, followed by continued incubation at 37°C, where applicable, until day 8. Culture media were changed on days three and five. Individual test wells were resuspended to harvest cells, individually transferred to microcentrifuge tubes, and analyzed for cell counts to assess cell expansion and/or by flow cytometry for surface expression of CD4 or CD8. Exemplary results are depicted in FIGS. 5B and 5C. Data are representative of three independent experiments involving duplicates for each condition tested and controls.

[001120] As shown in FIG. 5B, incubation of T cells with the multimerized anti-CD3/anti-CD28 agents, followed by disruption by addition of D-Biotin at various time points, resulted in an increase in T cell numbers, consistent with T cell expansion and/or persistence to a degree at least comparable to, and in some cases improved over, incubation for eight days without disruption of binding. The results showed that addition of D-Biotin at early time points to dissociate the Fabs from the multimerization reagent (such as generally within one day for CD8+ cells or within 4 days for CD3+ cells) resulted in harvest at day 8 after initiation of stimulation, higher total CD3+ and CD8+ cell numbers compared to stimulations in which the signal was not disrupted. In particular, when CD8+ T cells were stimulated for 1 day with the soluble anti-CD3/anti-CD28 multimerized reagent before the addition of D-Biotin to disrupt the interaction and diminish the stimulatory signal, T cell numbers at harvest were nearly 3-fold greater compared to T cell expansion in the presence of anti-CD3/anti-CD28 reagents without disruption of binding. For CD4+ cells, cell numbers at harvest were generally comparable to those observed after stimulation in the presence of anti-CD3/anti-CD28 without disruption, particularly at time points when the anti-CD3/anti-CD28 signaling complex was transiently controlled by disruption of the multimerized complex on days 1 and 4 after stimulation with the anti-CD3/anti-CD28 multimerized reagent.

[001121] To assess the relative effects on CD4+ and CD8+ subsets under the various conditions, the proportion of CD4+ or CD8+ T cells in the stimulated CD3+ cell population was determined by flow cytometry. As shown in FIG. 5C, incubation of CD3+ cell cultures with the multimerized anti-CD3/CD28 agents led to a relatively higher proportion of CD8+ cells compared to CD4+ cells at harvest when disruption was performed at a relatively early time point, such as within one day after initiation of stimulation, and compared to other conditions.

EXAMPLE 3: Assessment of T cell expansion and phenotype after transiently controlled activation of CD3+ T cells by incubation with a reversible agent

[001122] The impact of temporal control of T cell stimulation using the reagent described in Examples 1 and 2, via disruption with D-Biotin, was evaluated. In this study, bulk CD3+ T cells were selected as described in Examples 1 and 2 and incubated with the multimerized CD3/CD28 agent or one of several control reagents. As shown in FIG. 6A, D-Biotin was added to certain samples, and not others, as described in Example 2, and the cells were cultured through day 8 (with media change on day 3) after initiation of stimulation. Untreated (unstimulated) cells served as a negative control. The cells were incubated at 37°C with media change on day three. After seven days, individual test wells were resuspended to harvest cells, individually transferred to microcentrifuge tubes, and analyzed by cell count to assess persistence and/or expansion of cells, and/or by flow cytometric analysis to assess surface expression of various markers (e.g., CD4, CD8, CD62L, and CD127) and/or nuclear expression of Ki-67 and T-bet. Exemplary results are depicted in FIGS. 6B–6F. Data are representative of three independent experiments involving duplicates for each condition tested and controls.

[001123] As shown in FIG. 6B, the results showed a greater number of CD3+ T cells in the harvest after stimulation of cells with the soluble anti-CD3/anti-CD28 multimerized reagent, temporally controlled by disruption on day 1 after initiation, compared to cells cultured under similar conditions with such reagents, without temporal control of the signal (i.e., on addition of D-Biotin). Approximately 3-fold greater numbers of CD3+ T cells were observed in the harvest after temporally controlled stimulation by addition of D-Biotin one day after initiating incubation with the soluble anti-CD3/anti-CD28 multimerized reagent, compared to cells stimulated in the presence of anti-CD3/anti-CD28 beads or the soluble reagent, without reversible disruption of binding. Additionally, the addition of D-Biotin had no substantial effect on the number of cells at harvest in the case of cultures stimulated with anti-CD3/anti-CD28 beads, indicating that the effects observed with the addition of D-Biotin to the soluble reagent were due to its ability to specifically bind and disrupt the binding of the components of this reagent.

[001124] Within the expanded CD3+ population, a higher proportion of CD8+ cells was observed at harvest after culture with the soluble reagent and temporal control using D-Biotin Disruption on day 1, as compared to other conditions tested (FIG. 6C). As shown in FIG. 6D, normalizing the CD4+ and CD8+ subsets within the CD3+ T cell population to the ratio of CD4+ and CD8+ T cells in the unstimulated controls, the results showed that when soluble reagent binding was disrupted on day 1 using D-Biotin, the relative degree of expansion (and/or survival) of CD8+ T cells compared to CD4+ T cells was greater as compared to other conditions tested. The results demonstrate that in a mixed cell culture (e.g., bulk T cells), temporal control of the signal, for example, by disruption of multimerized agent binding using a competing substance, can be used to tailor or adjust the relative expansion and/or persistence of particular subpopulations compared to others (e.g., CD4+ vs CD8+).

[001125] The results of this study also showed that the ability to temporarily control the CD3/CD28 signal using this reversible reagent can also be used to impact the relative representation, in the harvest composition, of various cell populations with different activation, differentiation, and phenotypic profiles. For example, as shown in FIG. 6E, stimulation of CD3+ T cells with soluble anti-CD3/anti-CD28 multimerized reagent for eight days, with the addition of D-Biotin on the first day, resulted in a final composition in which a higher percentage of CD4+ cells and a higher percentage of CD8+ cells showed high surface expression of CD62L and CD127, compared to other conditions, in which the cells were cultured for the same period without temporal control by disruption. Similarly, as shown in FIG. 6F, compared to conditions in which the signal was not disrupted, after stimulation with the disrupted multimerized reagent on day 1, a higher percentage of both CD4+ and CD8+ cells in the harvest were positive for the Ki-67 marker (at slightly lower levels than high-staining populations), indicative of proliferation, and exhibited low levels of T-bet staining. These observations are consistent with a conclusion that the ability to transiently control the signal using the reagent modalities provided may result in an increase in long-lived, less differentiated T cell populations, such as long-lived memory T cells. Without being limited by theory, increased expression of CD62L and CD127 is generally observed in less differentiated and/or relatively longer-lived T cell populations, such as naïve and long-lived memory populations, such as central memory T cells (CMT) and differentiated memory populations, such as so-called stem memory T cells (SMT). In contrast, these markers are generally lower in effector cells or terminally differentiated effector memory T cells. Ki-67 positivity indicates cell proliferation, suggesting that at least some cells within the augmented population may represent less differentiated, long-lived memory cells, as opposed to naïve T cells. T-bet staining generally increases after activation and is reduced in less differentiated cells, and has been reported to be lower in long-lived central memory T cells, and even lower in TSCM cells. Thus, the results indicate that in this study, temporal control of the signal by disruption of the multimerization reagent on day 1 resulted in an increase (compared to similar stimulation without disruption) in the persistence, expansion, and/or emergence of cells with a phenotypic marker profile similar to that present in less differentiated, long-lived memory cells. Thus, an increased percentage of cells harvested after temporally controlled stimulation using the reversible reagent may exhibit characteristics of long-lived memory cells, including stem T memory cells (TSCM).

EXAMPLE 4: Assessment of T cell expansion and phenotype after temporally controlled activation of CD8+ T cells by incubation with a reversible agent

[001126] To assess the impact on CD8+ cells without substantial CD4+ cell presence, a study was performed essentially as in Example 3, but in which the cultured cells were enriched for CD8+ cells, as opposed to stimulating bulk CD3+ cells. Exemplary results are depicted in FIGS. 7A-7C. Data are representative of three independent experiments encompassing duplicates for each condition tested and controls.

[001127] As shown in FIG. 7A, as was the case for CD3+ bulk cultures, cell numbers indicated increased survival and/or expansion of cells following signal disruption on day 1, compared to corresponding conditions when no D-Biotin was added or signal was not disrupted. The increases in cell number and relative expansion or persistence were similar to those observed for bulk CD3+ cells, and again, the addition of D-Biotin to the Dynabead® control sample indicated specificity of effect due to disruption of binding of the multimerized reagent components.

[001128] As shown in FIGS. 7B and 7C, even in the absence of substantial CD4+ cells, the temporally controlled conditions in this study resulted in increased T cell representation in the harvest, exhibiting phenotypic properties similar to long-lived memory and/or less differentiated cells.

EXAMPLE 5: Assessment of T cell stimulation following T cell stimulation with a soluble multimerized reagent functionalized to target CD3 and various costimulatory molecules

[001129] Additional studies were performed evaluating cells after incubation with alternative stimulatory agents and combinations thereof, illustrating that for some embodiments of the provided reagents, modular properties of the reagents can be used to design or tune output compositions to promote desired outcomes or phenotypes. Various multimerized agents containing anti-CD3 Fabs and one or two additional Fabs were generated as described in Example 1, except that in addition to the anti-CD3 and anti-CD28 Fabs described, additional Fabs that were capable of delivering, modulating, or enhancing a signal in T cells were used (anti-CD90, anti-CD95, anti-CD137, and anti-CD154). For different conditions, the Fabs were reversibly bound to the reagent in various double and triple combinations.

[001130] Functionalization of the reagent with the various Fabs was performed essentially as described in Example 1, with room temperature mixing of the Fab and the oligomeric reagent. To multimerize two different Fabs (double-Fab combinations), approximately 3 μg of oligomeric Strep-tactin® was mixed with approximately 0.5 μg of each Fab; to multimerize three different Fabs in the same reagent (triple-Fab combinations), approximately 4.5 μg of soluble oligomeric Strep-tactin® was mixed with approximately 0.5 μg of each Fab for the generation of multimerized Triple-Fab agents, containing anti-CD3 Fab, CD28 Fab and one of the additional Fabs (targeting CD90, CD95, CD137 or CD154). The resulting soluble multimerized reagents were used directly to stimulate T cells and optionally stored on ice prior to cell stimulation.

[001131] CD3+ T cells were isolated as described in Example 1, and 500,000 CD3+ T cells were seeded in 48-well plates in 1 mL of complete cell culture medium supplemented with 50 U/mL IL-2. Cells were stimulated with either one of several soluble Fab multimerizing agents or with anti-CD3/anti-CD28 beads (beads coated with anti-CD3 and anti-CD28 mAb). Untreated (unstimulated) cells served as a negative control. Cells were incubated at 37°C with medium change on day two post-priming. After six days, individual test wells were resuspended to harvest cells, transferred individually to microcentrifuge tubes, and analyzed for cell counts to assess cell expansion and/or by flow cytometric analysis for surface expression of CD4, CD8, CD45RO, CD45RA, CD69, or CD62L. Exemplary results are depicted in FIGS. 8A-8F. Data are representative of three independent experiments encompassing duplicates for each condition and controls tested.

[001132] As shown in FIG. 8A, stimulation of CD3+ T cells with each of the Fab multimerized agents resulted in a cell count at harvest (indicative of expansion and/or persistence) that was at least comparable to stimulation with anti-CD3/anti-CD28 beads, and in some cases higher. Stimulation with Fab multimerized agents that do not contain anti-CD28 Fab shifted the T cell composition of the expanded culture to a higher proportion of CD8+ cells compared to stimulation of cells with a reagent that includes an anti-CD28 Fab, such as by stimulation with anti-CD3/anti-CD28 beads or soluble anti-CD3/anti-CD28 multimerized agent. For example, as shown in FIG. 8B, normalization of the CD4+ and CD8+ subsets within the CD3+ T cell population to the proportion of CD4+ and CD8+ T cells in unstimulated controls, the results showed that the relative increase in the number of CD8+ T cells was greater than the relative increase in CD4+ T cells for cultures stimulated with multimeric reagents containing multimerized anti-CD3 Fab and multimerized anti-CD90, anti-CD95, anti-CD137, or anti-CD154 Fabs, but not those stimulated with such reagents containing multimerized anti-CD3 and anti-CD28 Fabs or those with anti-CD3/anti-CD28 beads or those containing three multimerized Fabs in which one of the multimerized Fabs was anti-CD28 Fab) did not lead to a higher proportion of CD8+ cells in the expanded T cell composition.

[001133] As shown in FIGS. 8C and 8D, under the conditions in this study, the use of multimerized Fab combinations other than those including anti-CD28 Fabs appeared to result in a relatively higher percentage of cells that were CD62L+, compared to similar stimulating conditions and agents including anti-CD28 Fab. This effect was observed in both the CD4++ (FIG. 8C) and CD8+ (FIG. 8D) cell populations at harvest, after normalizing for the respective subsets within the CD3+ T cell population. FIGS. 8E (CD4+ T cells) and 8F (CD8+ T cells), show expression of CD62L, CD69, CD45RA, and CD45RO under the various conditions. These results demonstrate that, in the embodiments provided herein, the modular nature of the reagents allows for tailoring of the reagent to desired outcomes, cell populations, and phenotypes, including through the use of different combinations of stimulatory agents (including those targeting various second or accessory signaling molecules, including those other than CD28 (e.g., CD90, CD95, CD137, or CD154). In some aspects, such tailoring can be used to shift cultures to produce a desired phenotype, such as to promote cells with a less differentiated surface phenotype, such as long-lived memory T cells.

EXAMPLE 6: Effect of the streptavidin mutein backbone of a soluble anti-CD3/anti-CD28 multimerized reagent on T cell stimulation

[001134] An alternative streptavidin mutein, which is a streptavidin homo-tetramer containing the mutein amino acid sequence set forth in SEQ ID NO: 27, see, e.g., International Published PCT Application No. WO 2014/076277), was used to generate a soluble oligomeric reagent using methods that were substantially the same as those described in Example 1. Anti-CD3 and anti-CD28 Fab fragments were immobilized on the soluble oligomeric streptavidin mutein (ST-MMB, via a streptavidin peptide binding partner fused to each Fab fragment, substantially as described in Example 1. Stimulation of T cells with the resulting anti-CD3/CD28 Fab multimerized reagent (ST-MMB) was compared to stimulation of T cells with the anti-CD3/CD28 Fab multimerized reagent generated in Example 1. (designated ST-MMA for comparison here).

[001135] CD3+ T cells were selected from a sample of fresh peripheral blood mononuclear cells (PBMCs) obtained from a Ficoll gradient. Approximately 500,000 CD3+ T cells were seeded in 48-well plates in 1 mL of complete cell culture medium supplemented with 50 U/mL IL-2. Cells were stimulated with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) generated as described in Example 1, soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB), or with anti-CD3/anti-CD28 beads (beads coated with anti-CD3 and anti-CD28 mAb; positive control). Untreated (unstimulated) cells served as a negative control. Cells were incubated at 37 °C without medium change. After five days, individual test wells were resuspended to harvest cells, transferred individually to microcentrifuge tubes, and analyzed for cell counts to assess cell expansion and/or by flow cytometric analysis for surface expression of CD4, CD8, CD69, or CD62L. Exemplary results are depicted in FIGS. 9A-9D. Data are representative of three independent experiments encompassing duplicates for each condition and controls tested.

[001136] As shown in FIG. 9A, the results showed that stimulation of CD3+ T cells with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) or with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB) resulted in comparable expansion of cells, which was superior to that achieved with stimulation with anti-CD3/anti-CD28 beads. The results in FIG. 9B showed that stimulation of cells with the soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB) led to a small increase in the proportion of CD8+ cells in the T cell composition of the expanded culture compared to stimulation of cells with the soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA). As shown in FIG. 9C , normalizing the CD4+ and CD8+ subsets within the CD3+ T cell population to the ratio of CD4+ and CD8+ T cells in the unstimulated controls, the results showed that the expansion of CD8+ T cells was substantially greater than the expansion of CD4+ T cells in cells that were stimulated with the soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB) compared to either the anti-CD3/anti-CD28 multimerized reagent (ST-MMA) or with anti-CD3/anti-CD28 beads. The results in FIG. 9D also showed that cells stimulated with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) shifted the T cell composition of the expanded culture to a higher proportion of CD62L+ cells compared to cells stimulated with either soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) or anti-CD3/anti-CD28 beads.

[001137] To assess the effects of the ST-MMB reagent specifically on CD8+ cells, similar experiments were performed as above, except that stimulation was allowed to proceed until day 6 and a medium change was included. In particular, approximately 500,000 CD8+ T cells were seeded in 48-well plates in 1 mL of complete cell culture medium supplemented with 50 U/mL IL-2. Cells were stimulated with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) generated as described in Example 1, soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB) or with anti-CD3/anti-CD28 beads (beads coated with anti-CD3 and anti-CD28-mAb; positive control). Untreated (unstimulated) cells served as a negative control. Cells were incubated at 37°C with medium change on day 4. After six days, individual test wells were resuspended to harvest cells, transferred individually to microcentrifuge tubes, and analyzed for cell counts to assess cell expansion and/or by flow cytometric analysis for surface expression of CD8, CD69, or CD62L. Exemplary results are depicted in FIGS. 10A and 10B. Data are representative of three independent experiments comprising duplicates for each condition tested and controls.

[001138] As shown in FIG. 10A, the results showed that stimulation of CD8+ T cells with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB) resulted in a greater than 2-fold increase in cell counts compared to CD8+ T cells stimulated with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) or with anti-CD3/anti-CD28 beads. The results in FIG. 10B show surface phenotypes of the cells, including the degree of surface expression of the CD62L+ and CD69+ T cell composition on T cells expanded with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMB) versus T cells expanded with soluble anti-CD3/anti-CD28 multimerized reagent (ST-MMA) or with anti-CD3/anti-CD28 beads.

Example 7: Stimulation/expansion of CD3+ T responder cells with αCD3/αCD28 Fab fragments that were reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®.

[001139] 300,000 CD3+ CD62L responder T cells (Tresp, isolated by serial magnetic enrichment of an unmobilized donor apheresis product) were labeled with 3 μM CFSE and stimulated with 5 μL of a 15 μL preparation of Streptactin® beads (10 mg magnetic particles/mL, loaded with 35 μg Streptactin®/mg beads) loaded with 0.5 μg αCD3 Fab fragment alone, 0.5 μg αCD28 Fab fragment alone, or a mixture of 0.5 μg αCD3 Fab fragment and 0.5 μg αCD28 Fab.

[001140] The αCD3 Fab fragment used was derived from the CD3-binding monoclonal antibody produced by the OKT3 hybridoma cell line. The OKT3 hybridoma cell line and the OKT3 antibody are described in US Patent 4,361,549, the cell line has been deposited under ATCC® accession number CRL-8001™). The FabαCD28 used was derived from the monoclonal anti-human CD28 antibody CD28.3 (Vanhove et al., BLOOD, July 15, 2003, Vol. 102, No. 2, pages 564-570). The nucleotide sequence of the variable domains of this CD28.3 antibody has been deposited in GenBank as a synthetic anti-human CD28 single-chain FFC28 antibody scFv28.3 under GenBank accession number AF451974.1 (shown in SEQ ID NOS: 33 and 34).

[001141] Both Fab fragments were recombinantly produced in E. coli as described in International Patent Pub. Nos. WO2013/011011 and WO 2013/124474 carrying as constant domains (CH1 and Ckappa) an IgG1 consensus sequence. The heavy chain of both Fab fragments was fused at the carboxyl terminus to a sequential arrangement of two streptavidin-binding modules (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK) (SEQ ID NO: 16), which is commercially available as "Twin-Strep-tag®" from IBA GmbH, Gottingen, Germany). The αCD3 Fab fragment was used as the first agent with the streptavidin-binding peptide serving as the C1 binding partner and the αCD28 Fab fragment was used as the second agent with the streptavidin-binding peptide serving as the C2 binding partner. The streptavidin mutein (tetrameric) "Strep-tactin®" serves as the reagent in which both Fab fragments were reversibly immobilized.

[001142] In the expansion experiment, Tresp cells stimulated with blank beads (without Fab) served as a negative control. Tresp cells were seeded in triplets in 48-well plates together with 300,000 CD3 cells, autologous feeder cells (irradiated with 30Gy) in 3 mL of complete cell culture medium (RPMI (Gibco) supplemented with 10% (v/v) fetal calf serum, L-glutamine, b-mercaptoethanol, HEPES, penicillin, streptomycin, and gentamicin) supplemented with 10 U/mL interleukin 2 (IL-2). Cells were incubated at 37 °C without medium change and analyzed after 4 days by FACS analysis. FACS labeling and analysis were performed after 10 min of incubation with 100 μM D-biotin. A representative graph for each condition is shown in FIG. 11A. The schematics show live CD3+ cells that were labeled with propidium iodide (PI) for live/dead discrimination. FIG. 11A is a histogram showing the size distribution (forward scatter) of stimulated cells. Figure 11A shows that a specific cell population of Tresp cells was stimulated and expanded (increased in size/number compared to the unstimulated “beads only” control) when incubated in the presence of beads on which a mixture of 0.5 μg of αCD3 Fab fragment and 0.5 μg of αCD28 Fab was immobilized, after being stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®. Figure 11B shows histograms of CFSE proliferation dye dilution representing the degree of proliferation according to the number of cells per cell division (indicated at the top of FIG. 11B, 0 represents undivided cells; 5 represents cells that have undergone at least 5 divisions). It can be seen from FIG. 11B that the T cell population stimulated with the beads on which a mixture of 0.5 μg αCD3 Fab fragment and 0.5 μg αCD28 Fab was immobilized, for the most part, underwent three cell divisions and represents a more uniform proliferation pattern than with a single stimulus alone (small number of cells within the undivided "0" peak). The increased absolute amount of proliferation (more cells proliferated uniformly after 4 d stimulation with αCD3- and αCD28-functionalized beads) is also represented by a more intense consumption of media as represented by an indicator color change to yellow (represented as clearer liquid in wells in FIG. 11C).

EXAMPLE 8: Stimulation/expansion of CD3+ T responder cells with αCD3/αCD28 Fab fragments that were reversibly immobilized on soluble Strep-tactin

[001143] In this example, CD3+ T responder cells (isolated by magnetic selection from a sample of fresh PBMCs obtained from a Ficoll gradient) were expanded following in vitro stimulation with αCD3/αCD28 Fab fragments that were reversibly immobilized on soluble oligomeric Strep-tactin, acting as a soluble reagent. Oligomeric streptavidin mutein was obtained by polymerizing Strep-tactin® with sulfoSMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, product # 22122 Thermo Scientific) and iminothiolane (product # 26101 Thermo Scientific) according to the manufacturer's protocol (Thermo Scientific). Oligomeric streptavidin muteins were separated from monomeric (unreacted) and dimeric streptavidin muteins by size exclusion chromatography and the thus obtained fraction of oligomeric streptavidin mutein (n > 3) was used as soluble reagent. For in vitro expansion, 300,000 CD3+ responder T cells (Tresp) were labeled with 2 μM Carboxyfluorescein succinimidyl ester (CFSE) and stimulated with varying amounts of a soluble oligomeric streptavidin mutein preparation on which a combination of the OKT3 αCD3 Fab fragment and the αCD28 Fab fragment of antibody 28.3 (both containing the above-mentioned Twin-Strep-tag® as streptavidin-binding peptide in the heavy chain) were immobilized. (“1x” corresponds to 3 μg of oligomeric streptavidin mutein functionalized with 0.5 μg of monomeric αCD3 Fab fragment and 0.5 μg of monomeric αCD28 Fab fragment, the numbers “0.5x”, “2x” and “5x” indicate the respective n-fold amount of “1x”). Tresp cells left unstimulated or stimulated with blank oligomeric streptavidin mutein (no Fab) served as negative controls. Tresp cells were seeded in duplicate in 48-well plates together with 300,000 autologous CD3-negative feeder cells (irradiated with 30 Gy) in 1 ml of cell culture medium supplemented with 20 U/ml IL-2. The cells were incubated at 37 °C without media exchange, and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis. Figure 12A shows the increase in the size distribution of proliferating cells after 5 days in culture compared with negative controls. FIG. 12B shows that CD3+resp T cells were adequately stimulated and proliferated vigorously when incubated with soluble oligomeric streptavidin mutein (compared with solid Streptactin magnetic particles in Example 7 and FIG. 11(A-C)) on which a mixture of αCD3 Fab and αCD28 Fab fragments was immobilized. The results in FIGS. 12A and 12B indicate that under these in vitro conditions the majority of CD3+ T responder cells divided (2 to 5 cell divisions) after engagement of the CD28 and TCR/CD3 surface complex with αCD3 and αCD28 Fab fragments that were reversibly immobilized on soluble oligomeric streptavidin mutein. After in vitro expansion, the soluble multimerized agents were dissociated and removed after treatment with D-biotin. The dissociation and removal of the monomeric Fab fragments was analyzed by flow cytometry by relabeling the cells with the phycoerythrin marker Strep-Tactin® (ST-PE). A representative histogram (dark gray histogram) is shown in comparison with the appropriate ST-PE-only negative control (light gray histogram). It can be seen from FIG. 12C that both Fab fragments were completely dissociated and were completely removed from the expanded cells. Figure 12D shows the absolute number of live (trypan blue-negative) cells after 5 days. The number was counted using a Neubauer counting chamber and plotted against the respective stimulation condition. Median cell numbers are shown in FIG. 12D; error bars indicate standard deviation (SD). Figure 12D shows that all mixtures of αCD3 Fab fragments and αCD28 Fab fragments that were immobilized in a soluble oligomeric streptavidin mutein reagent were equally effective in expanding CD3+ cells and resulted in an approximately 4-fold increase in absolute cell numbers.

EXAMPLE 9: Proliferation kinetics of purified CD4+ and CD8+ T responder cells stimulated in vitro with reversible αCD3/αCD28 Fab Streptamer multimers without medium exchange

[001144] In this example, the proliferation expansion kinetics of purified CD4+ and CD8+ T responder cells (Tresp) that were stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized soluble oligomeric streptavidin muteins were examined.

[001145] For this purpose, soluble oligomeric Strep-tactin® mutein of two different sizes served as the soluble reagent. The first type of oligomeric Streptokinin® was the oligomeric streptavidin mutein fraction (n > 3) obtained in Example 8 (also referred to herein as "conventional oligomeric streptavidin mutein backbone", illustrated by the triangle symbol with the point at the top in FIG.13(AB)). The second type of this oligomeric streptavidin mutein used as the soluble reagent was an oligomeric streptavidin mutein (n > 3) that reacted with biotinylated human serum albumin (also referred to herein as "large oligomeric streptavidin mutein backbone").

[001146] In this example, 500,000 purified CD4+ or CD8+ responder T cells (Tresp) were stimulated separately with these two different Streptamer multimers as explained above, i.e., with the oligomeric streptavidin mutein backbone of Example 8 (using a solution with a concentration of 1 mg oligomeric streptavidin mutein/mL) or with the large oligomeric streptavidin mutein backbones (0.1 mg/mL). 3 μL of the two different backbones were loaded with a combination of 0.5 μg of the αCD3 Fab and 0.5 μg of αCD28 Fab used in the previous Examples which contain the streptavidin-binding peptide SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 16) at the C-terminus of the heavy chain of the Fab fragment. In addition, 4.5 μL of the conventional oligomeric streptavidin mutein backbone were loaded with 0.5 μg of αCD3 Fab fragment, 0.5 μg of αCD8 Fab fragment (IBA GmbH, Gottingen, which also carries at the C-terminus of the Fab fragment the streptavidin-binding peptide SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 16) and 0.5 μg of αCD28 Fab fragment. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with commercially available anti-CD3/anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) as a positive control. Tresp cells were seeded in duplicates in 48-well plates in 1 ml of cell culture medium (RPMI 1640 (Gibco) supplemented with 10% (v/v) fetal calf serum, 0.025% (w/v) L-glutamine, 0.025% (w/v) L-arginine, 0.1% (w/v) HEPES, 0.001% (w/v) gentamicin, 0.002% (w/v) streptomycin, 0.002% (w/v) penicillin 0.002% (w/v) supplemented with 30U/ml IL-2. Cells were incubated at 37°C without media change, and cell counts were analyzed after 1, 3, and 6 days. In the experiments in FIG. 13(A-B), expansion was performed without media change. The results for CD4+ T responder cells are shown in FIG. 13A, the results for CD8+ T responder cells are shown in FIG. 13B, with the graphs representing the degree of proliferation according to the number of cells harvested per time point for CD4+ Tresp (FIG. 13A) and for CD8+ Tresp in FIG. 13B.

[001147] As can be seen in FIG. 13A, the "smaller" soluble reagent in which the αCD3 and αCD28 Fab fragments were reversibly immobilized provided the same amount of CD4+ T cell expansion as the anti-CD3/anti-CD28 beads (which are, to date, the standard reagent for T cell expansion), while the "larger" soluble multimerized agent provided even better expansion compared to the Dynabead. This improvement may be caused by the "larger" soluble multimerized being able to bind to more T cells at the same time than the "smaller" multimerized agent, thus being able to stimulate more CD4+ T cells than the "smaller" multimerized agent.

[001148] As is evident from FIG. 13B, using the soluble multimerized agents disclosed herein, CD8+ T cells could be expanded within the first 3 days at least as efficiently as with anti-CD3/anti-CD28 beads. Notably, in this time period, the expansion experiment that used a soluble reagent that in addition to the αCD3 and αCD28 Fab fragments (as first and second agent) carried reversibly immobilized on the αCD8 Fab fragment, showed the best degree of expansion under these culture conditions. This indicates that it is possible to use a stimulus that is specific for a particular subpopulation of cells (here the αCD8 Fab fragment) to increase or modulate the selectivity of the expansion, thus being able to obtain larger quantities of a desired cell (sub)population.

[001149] Thus, summarizing Example 9 above, it is shown that the functionality of the soluble multimerized agent in triggering T cell expansion is comparable to the current standard methodology of using anti-CD3/anti-CD28 beads for this purpose. However, since stimulation can be controlled (and terminated, if desired) by the addition of a competitor such as biotin in the case of a reversible streptavidin-based interaction between the first and second agents and the reagent, the compositions and methods described herein provide a significant advantage over anti-CD3/anti-CD28 bead technology since expansion conditions can be optimized (it would, for example, be possible to stop stimulation in the experiment of FIG. 13B after 3 days). Furthermore, since the soluble reagent can be easily removed from the reaction (e.g., by immobilizing the reagent on a biotinylated column after the expansion reaction), the expansion methods disclosed herein can be performed and automated in closed systems that are, for example, required for the production of GMP cells for therapeutic purposes, without having to deal with the removal of beads such as anti-CD3/anti-CD28 beads.

EXAMPLE 10: Proliferation kinetics of purified CD4+ and CD8+ T responder cells stimulated in vitro with reversible aCD3/aCD28 Fab Streptamer multimers with medium exchange

[001150] In this example, the proliferation expansion kinetics of purified CD4+ and CD8+ T responder cells (Tresp) that were stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on soluble oligomeric streptavidin muteins, were examined. For this purpose, soluble oligomeric Strep-tactin® mutein of two different sizes served as soluble reagent. The first type of oligomeric Streptokinin® was the oligomeric streptavidin mutein fraction (n 3) obtained in Example 8 (also referred to herein as "conventional oligomeric streptavidin mutein backbone", illustrated by the downward-pointing triangle symbol in FIG. 14(AB)). The second type of this oligomeric streptavidin mutein used as a soluble reagent was obtained by reacting the oligomeric Streptocatin (n > 3) obtained in Example 8 with biotinylated human serum albumin. This soluble oligomeric reagent is also referred to herein as "large oligomeric streptavidin mutein structure".

[001151] In this example, 400,000 purified CD4+ or CD8+ responder T cells (Tresp) were stimulated separately with these two different oligomeric streptavidin muteins as explained above, i.e., either with the oligomeric streptavidin mutein backbone of Example 8 (1.0 mg/mL) or with the large oligomeric streptavidin mutein backbones (0.1 mg/mL). 3 μL of both different backbones were loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab fragments described above. Additionally, 4.5 μL of the oligomeric streptavidin mutein construct from Example 8 was loaded with 0.5 μg of αCD3, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab fragment as described above. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3/anti-CD28 beads (on which the αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) as a positive control. Tresp cells were seeded in duplicate in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/mL IL-2. Cells were incubated at 37 °C with medium change on day 3, and cell counts were analyzed after 1, 3, and 6 days. The results for CD4+ T responder cells are shown in FIG. 14A, the results for CD8+ T responder cells are shown in FIG. 14B, with the graphs representing the degree of proliferation according to the number of cells harvested per time point for Tresp CD4+ (FIG. 14A) and for Tr8 CD8+ (FIG. 14B).

[001152] As can be seen in FIG. 14A, the soluble reagents in which the αCD3 Fab and αCD28 Fab fragments were reversibly immobilized (the multimerized agents) provided better expansion of CD4+ T cells than the anti-CD3/anti-CD28 beads.

[001153] As evident from FIG. 14B, using the multimerized agents, CD8+ T cells could be expanded in the first 6 days at least as efficiently as with anti-CD3/anti-CD28 beads. Notably, in this time period, the expansion experiment using the larger soluble reagent containing αCD3 and αCD28 Fab fragments (as first and second agents) showed the best degree of expansion under these culture conditions. This may again be caused by the "larger" soluble multimerized agent being able to bind to more T cells at the same time than the "smaller" multimerized agent, being able to stimulate more CD4+ T cells than the "smaller" multimerized agent.

EXAMPLE 11: Expansion kinetics of purified CD4+ and CD8+ T cell cultures with or without medium exchange

[001154] In this Example, the combined data from Examples 9 and 10 were normalized to the number of input cells for the "smaller" multimerized agent and positive and negative controls. No normalization data were obtained on the "larger" multimerized agent. As explained in Examples 9 and 10, 400,000 to 500,000 CD4+ or CD8+ T responder (Tresp) cells were stimulated with 3 μL of a multimerized agent preparation (1mg/ml; in which 0.5 μg of αCD3 Fab fragment and 0.5 μg of αCD28 Fab fragment were immobilized). Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3/anti-CD28 beads as a positive control. Tresp cells were seeded in duplicate in 48-well plates in 1 ml of cell culture medium supplemented with 30 U/ml IL-2. The cells were incubated at 37°C with media change (straight lines in FIG. 15(AB)) or without media change (dashed line in FIG. 15(AB)) on day 3, and cell counts were analyzed after 1, 3, and 6 days. As is evident from the normalized data in FIG. 15A, the “smaller” soluble reagent in which the αCD3 Fab and αCD28 Fab fragments were reversibly immobilized produced an approximately 2.5-fold expansion of CD4+ T cells, whereas expansion using anti-CD3/anti-CD28 beads produced an approximately 1.8-fold expansion rate. Thus, the use of a multimerized agent does provide an improvement in CD4+ T cell expansion over anti-CD3/anti-CD28 beads. Similarly, FIG. 15B confirms that CD8+ T cells can be expanded using the multimerized agents in the first 3 days at least as efficiently as with the anti-CD3/anti-CD28 beads.

EXAMPLE 12: Expansion kinetics and phenotype of activated/expanded polyclonal bulk CD3+ central memory T cells (TCM)

[001155] In this Example, 500,000 CD3+ CD62L+ CD45RA TCM cells (Tresp) were stimulated with 3 μL of a soluble oligomeric streptavidin mutein preparation of Example 8 (1 mg/ml) that was loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. Additionally, 4.5 μL of an oligomeric streptavidin mutein preparation loaded with 0.5 μg of αCD3, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab was used as an additional stimulation condition. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3/anti-CD28 beads (on which the αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) as a positive control. Tresp cells were seeded in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/mL IL-2 alone or 30 U/mL IL-2 and 5 ng/mL IL-15. The cells were incubated at 37 °C with medium change every 3 days, and cell counts were analyzed after 7 and 14 days. The graphs represent the degree of proliferation according to the number of cells harvested per time point, in FIG. 16A in medium supplemented with IL-2 alone and in medium supplemented with both IL-2 and IL-15 in FIG. 16B. As can be seen from FIG. 16A and FIG. 16B , the soluble reagent that reversibly bound to the αCD3 Fab fragment and the αCD28 Fab fragment produces better cell expansion than the anti-CD3/anti-CD28 beads. As further demonstrated by the flow cytometric analysis of CD62L and CD127 surface expression after 14 days of culture in variable cytokine media of FIG. 16C , the experimental approaches using multimerized agents retain, under both conditions chosen herein, a higher content of long-lived T cells expressing CD127 than expansion with anti-CD3/anti-CD28 beads. This illustrates another advantage of the methods of the present compositions and methods described herein.

EXAMPLE 13: Yield and phenotype of expanded CD8+ T cells - varying the size of the soluble reagent and adding αCD8 Fab for stimulation

[001156] In this example, the expansion of CD8+ T responder cells stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized soluble oligomeric streptavidin muteins was examined. Additionally, the effect of adding αCD8 Fab to the reagent to increase the specificity of expansion for CD8+ T cells was examined.

[001157] To this end, 300,000 purified CD8+ responder T cells (Tresp) were stimulated separately with two different Streptactin-based reagents, namely the small multimerized agent of Example 8 (1 mg/mL) or the larger multimerized agent described above (0.1 mg/mL). 3 μL of both oligomeric streptavidin mutein reagent backbones were loaded with a combination of the 0.5 μg of αCD3 Fab and 0.5 μg of the αCD28 Fab fragments described above to form the multimerized agents. Additionally, 4.5 μL of the smaller oligomeric streptavidin mutein backbone was loaded with 0.5 μg of αCD3, 0.5 μL of αCD8 Fab, and 0.5 μL of αCD28 Fab fragments described above. In addition, 3 μL of the “smaller” oligomeric streptavidin mutein backbone only functionalized with 0.5 μL of αCD3 Fab fragment alone or 0.5 μL of αCD28 Fab fragment alone was used. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3/anti-CD28 beads served as a positive control. Tresp cells were seeded in duplicate in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/mL IL-2. Cells were incubated at 37°C with medium change after 3 days and analyzed after 6 days. FIG. 17A depicts the degree of proliferation according to the number of cells harvested on day 6 compared with negative controls and normalized to the positive control. FIG. 17A shows that expansion of CD8+ T cells using the multimerized agents results in higher yields of CD8+ T cells than expansion using anti-CD3/anti-CD28 beads. FACS analysis of CD8 surface expression and CD45RO surface expression (FIG. 17B) after cell culture shows that the same phenotype of CD8+ T cells was expanded by either multimerized agents or anti-CD3/anti-CD28 beads (the various stimulation conditions were compared using one-way ANOVA and no significant differences (n.s.) were detected). The improved yield of CD8+ cells using the expansion methods described here compared to anti-CD3/anti-CD28 beads may be due to the fact that soluble multimerized agents can access their target cell surface receptors better than antibodies that are immobilized on anti-CD3/anti-CD28 beads. This improved yield may be very advantageous when expanding a rare population of cells from an initial sample.

[001158] Furthermore, comparing the expansion yield achieved with the reagent in which both 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab fragments were immobilized together (second column from the left in FIG. 17B) to the yield using two reagents that were functionalized with only the αCD3 Fab fragment alone or the αCD28 Fab fragment alone (third column from the left in FIG. 17B), it can be seen that both experiments had the same expansion efficiency. Thus, these experiments show that using a reagent in which the first agent and the second agent are immobilized together is functionally equivalent to using for expansion two separate reagents that are loaded with only the first agent and the second agent, respectively.

EXAMPLE 14: Yield & phenotype of expanded CD8+ T cells - titration of separate soluble reagents with different proportions of the αCD3- and αCD28 Fab fragment immobilized on the same

[001159] In this Example, the yield and phenotype of expanded CD8+ T responder (Tresp) cells that were stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized in different amounts on soluble oligomeric streptavidin muteins were examined.

[001160] For this purpose, 300,000 CD8+ responder T cells (Tresp) were stimulated with varying amounts of a mixture of preparations of the "small" oligomeric streptavidin muteins (1 mg/ml) functionalized with αCD3 Fab alone and αCD28 Fab alone ("1x" corresponds to 1.5 μg of oligomeric streptavidin mutein functionalized with 0.5 μg of αCD3 alone and 1.5 μg of oligomeric streptavidin mutein functionalized with 0.5 μg of the αCD28 Fab fragment alone) or 3 μL of a preparation of the oligomeric streptavidin mutein loaded with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab or 4.5 μL of an oligomeric streptavidin mutein preparation loaded with 0.5 μg of αCD3, 0.5 μg of strep-labeled αCD8, and 0.5 μL of αCD28 Fab. Untreated Tresp cells served as a negative control and Tresp cells stimulated with anti-CD3/anti-CD28 beads as a positive control. Tresp cells were seeded in 48-well plates in 1 mL of cell culture medium with 30 U/mL IL-2. Cells were incubated at 37 °C without medium change and analyzed after 5 days. FIG. 18A represents the degree of proliferation according to the number of cells harvested on day 5 compared with the negative controls and normalized to the positive control. FIG. 18A shows that expansion of CD8+ T cells using the various multimerized agents results in higher CD8+ T cell yields than expansion using anti-CD3/anti-CD28 microbeads (especially the total cumulative reagent amount of the 5x condition resulted in optimal cell expansion, especially over time/increase in total cells initiating cell division). FACS analysis of CD8 surface expression and CD45RO surface expression (FIG. 18B) after cell culture shows that the same CD8+ T cell phenotype was expanded by both the multimerized agents and the commercially available anti-CD3/anti-CD28 beads.

EXAMPLE 15: Yield and subset compositions of CD3+ T cells expanded with the addition of αCD8 Fab for stimulation

[001161] The experiment shows the expansion of purified CD3+ T responder cells stimulated in vitro with αCD3/αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric streptavidin muteins of Example 8 that served as soluble reagent. In one experiment, in addition to the αCD3/αCD28 Fab fragments, a commercially available αCD8 Fab fragment from IBA GmbH, Gtingen, Germany (reference number 6-8000-203) was immobilized on the soluble oligomeric streptavidin mutein, to test whether it is possible to preferentially stimulate a specific T cell subpopulation in vitro with the reversible αCD3/αCD28 multimerized agents. In more detail, 500,000 purified CD3+ responder T cells (Tresp) were stimulated with 3 μL of an oligomeric streptavidin mutein preparation (1 mg/mL) loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. As an alternative approach, 4.5 μL of the oligomeric streptavidin muteins were loaded with 0.5 μg of αCD3, 0.5 μg of strep-labeled αCD8 Fab, and 0.5 μg of strep-labeled αCD28 Fab. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3/anti-CD28 beads (beads on which the αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) served as a positive control. As can be seen in FIG. 19A, the reagent that is reversibly loaded with the αCD3 Fab fragment, the αCD28 Fab fragment, and also the αCD8 Fab fragment provided the highest number of expanded CD3+ T cells. At approximately 1×106 the number of expanded cells, the yield was about 30% higher than that of expanding these T cells using commercially available anti-CD3/anti-CD28 beads. Furthermore and most importantly, as shown in FIG. 19B with this reagent containing the αCD3 Fab fragment, the αCD28 Fab fragment and the αCD8 Fab fragment, the amount of CD8+ T cells was the highest, compared to expansion with anti-CD3/anti-CD28 beads or a soluble reagent containing only the αCD3 Fab fragment and the αCD28 Fab fragment as the first and second agent as described herein. Thus, this experiment also shows the advantage of the compositions and methods described herein that, in addition to a first agent that provides a primary activation signal for the desired cell population and optionally a second agent that provides a costimulatory signal, another agent that is specific for the activation of the desired cell population can be immobilized in the reagent. Thus, by doing so, the compositions and methods described herein provide the possibility of preferentially expanding or selectively enriching any desired cell population or subpopulation from a sample that, for example, comprises a variety of different subpopulations.

EXAMPLE 16: Analysis of differential intracellular calcium mobilization in Jurkat cells

[001162] Real-time low-level cytometry analysis of differential intracellular calcium mobilization induced in Jurkat cells that are either labeled with the αCD3 antibody clone OKT3 or with OKT3 Fab fragments to be multimerized with Strep-tactin® was examined here. For this purpose, Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM and calcium release was triggered by injection of the αCD3 monoclonal antibody OKT3 (produced by the OKT3 hybridoma cell line, see above, black squares) or αCD3 Fab fragments (derived from the OKT3 parental cell line) that were multimerized through reversible binding of their streptavidin-binding peptide to fluorescently soluble Strep-Tactin conjugated with phycoerythrin. In the case of intact multimeric OKT3 Fab-Strep-Tactin complexes, calcium release was triggered over a time course identical to that of the parental antibody clone (dark gray triangles). Cell activation could be completely prevented by injection of predissociated D-biotin-treated Fab-Strep-Tactin complexes (light gray circles) identical to the injection of the PBS negative control (inverted white triangles). Application of ionomycin served as a positive control for calcium influx. Time-resolved changes in intracellular Ca 2+ concentration were monitored by flow cytometry based on the change in the FL6/FL7 ratio. It can be seen from FIG. 20A that both the parental OKT3 antibody and the multimerized monovalent OKT3 Fab fragment effected calcium release, meaning that the multimerized monovalent OKT3 Fab fragment is essentially as functional as the parental antibody. Notably, the multimeric OKT3 Fab fragment was unable to trigger calcium release if biotin was added to Streptactin, on which the OKT3 Fab fragment was immobilized prior to the addition of the Estreptactin-OKT3 Fab fragment. In this case, biotin disrupted the reversible bond formed between Streptactin as a multimerizing agent and the OKT3 Fab fragment. The monovalent Fab fragment was therefore displaced from the multimerizing agent and upon dissociation was unable to trigger calcium release by binding to CD3 of Jurkat cells.

[001163] In the experiments shown in FIG. 20B, indo-1-AM-labeled Jurkat cells were activated by OKT3-derived αCD3 Fab-Strep-Tactin complexes as described in FIG. 20A. Injection of intact (top graph) or pre-dissociated (bottom graph) complexes served as positive or negative controls, respectively. Furthermore, stimulation of cells with intact Fab-Strep-Tactin complexes followed by subsequent injection of D-biotin (near peak activation at = 140 s) resulted in abrupt disruption of αCD3 Fab multimer signaling (middle graph). Injection of ionomycin into the pre-dissociated Fab complex group served as a positive control. Data are representative of three different experiments. Importantly, FIG. 20B shows that the addition of D-biotin to the sample rapidly displaces the Fab fragment from the Streptactin multimerizing agent, effectively terminating calcium release even under continued calcium stimulation and demonstrating that the dissociated OKT3 Fab fragment is no longer biologically active. Similarly, the multimeric OKT3 Fab fragment was also unable to trigger calcium release when biotin was added to the Streptactin-OKT3 Fab fragment multimer prior to the addition of the Streptactin-OKT3 Fab sample to the Jurkat cells.

EXAMPLE 17: Reversible labeling of cells by αCD3 Fab multimers

[001164] This example examines the reversible labeling of cells by αCD3 Fab multimers. Freshly isolated PBMCs were labeled with the αCD3 monoclonal antibody clone OKT3 (left dot plot, parental clone for the Fab multimers) or cognate phycoerythrin (PE)-labeled OKT3 Fab multimers and analyzed before (second dot plot from left) or after D-biotin treatment (center dot plot). Remaining Fab monomers were then detected after subsequent wash steps using fresh PE-labeled Strep-Tactin® (second dot plot from right). Secondary Fab multimer labeling of reversibly labeled cells served as a control (right dot plot). Only live CD3 cells that are negative upon propidium iodide (PI) labeling for live/dead discrimination are shown in FIG. The numbers in the dot plots indicate the percentage of cells within the gates. This experiment shows that labeling of CD3+ PBMC with a multimerized anti-CD3 Fab fragment with Streptactin as a multimerization reagent is fully reversible by the addition of D-biotin and that the monovalent Fab fragment alone does not bind to the CD3 molecule present on PBMCs.

EXAMPLE 18: Reversible cell isolation by αCD28 Fab multimers

[001165] This example shows the isolation of cells by reversible binding of multimerized anti-CD28 Fab fragments to Strep-Tactin® magnetic particles (magnetic particles are available from IBA GmbH Gottingen, Germany). Fab fragments derived from the CD28.3 antibody described in Example 7 above were used for this purpose. CD28+ cells were selected/isolated by Fab multimer magnetic cell sorting from freshly isolated PMBC as essentially described in International Patent Application Pub. No. WO2013/011011. The results are shown in Figure. 22. Prior to sorting, cells were labeled with the cognate fluorescent αCD28 multimers (left dot plot) or with an antibody directed against the immunoglobulin kappa light chain (second dot plot from the left, α-Ig kappa mAb) as a labeling control. After selection, CD28+ cells were treated with D-biotin and subsequently washed to remove magnetic beads and Fab monomers. Released CD28+ cells were subsequently (re)labeled with αCD28 Fab multimers (second dot plot on the right) or with the α-Ig kappa mAb (right dot plot) to detect potentially remaining Fab monomers. Only live CD3+ cells (PI-negative) are shown. The numbers in the dot plots indicate the percentage of cells within the gates. Fig. 22 shows that CD28+ cells can be isolated from PMBC using such a multimerized anti-CD28 Fab fragment and that all isolation reagents including anti-CD28 Fab monomers can be removed after selection.

EXAMPLE 19: Early cluster formation following activation of purified CD4+ and CD8+ T responder cells stimulated in vitro with reversible aCD3/aCD28 Fab-streptamer multimers

[001166] In this example, 400,000 CD4+ or CD8+ T responder (Tresp) cells were stimulated with 3 μL of an oligomeric Streptactin multimerization reagent preparation (1 mg/ml) loaded with a combination of 0.5 μg αCD3 and 0.5 μg αCD28 Fab. Untreated (unstimulated) Tresp cells served as a negative control and Tresp cells stimulated with anti-CD3/anti-CD28 beads as a positive control. Tresp cells were seeded in duplicate in 48-well plates in 1 ml of cell culture medium supplemented with 30 U/ml IL-2. Cells were incubated at 37 °C and analyzed microscopically after 1 and 2 days. Stimulation of CD4+ Tresp (FIG. 23A) and CD8+ Tresp (FIG. 23B) is shown for anti-CD3/anti-CD28 beads (middle row) and multimerization agent (bottom row), respectively. The photographs represent the degree of cluster formation: For better visibility, exemplary clusters are indicated by circles for stimulation with soluble streptavidin mutein oligomers in FIG. 23A and FIG. 23B. Clusters within Dynabead stimulation are readily visible by the accumulation of dark stimulatory particles. As is evident, for both CD4+ and CD8+ T cells, early clusters formed when using the expansion method of the invention employing a soluble oligomeric multimerization reagent.

EXAMPLE 20: Antigen-specific selective expansion of TCM responder cells from central memory CD3+ T cells (kinetics and phenotype)

[001167] In this example, the kinetics and phenotype of antigen-specific (Ag-specific) expansion out of purified CD3+ CD62L+CD45RA-TCM responder cells were examined.

[001168] In more detail, CD3+ CD62L+CD45RA-TCM responder cells were stimulated in vitro with a peptide:MHC molecule complex (which acts as a first agent that provides a primary activation signal to the cells) and an αCD28 Fab fragment (which acts as a second reagent that stimulates an accessory molecule on the surface of the cells). Both the antigen-specific peptide complex with the MHC molecule and the αCD28 Fab fragment were reversibly immobilized on the soluble oligomeric streptavidin mutein (with n > 3) described in Example 8. The peptide that was used for antigen-specific expansion was the peptide CRVLCCYVL (SEQ ID NO: 38), amino acids 309-317 of immediate early protein 1 (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, 5, e1003383) representing a cytomegalovirus (CMV)-specific HLA-C7/IE-1 epitope. The peptide-presenting MHC I molecule carries at the C-terminus of the α-chain (heavy chain) the streptavidin-binding peptide (SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK, (SEQ ID NO: 16) which is commercially available as "Twin-Estrep-tag®" from IBA GmbH, Gottingen, Germany).

[001169] For this purpose, 500,000 CD3+ CD62L+ CD45RA (Tresp) responder TCM cells were specifically stimulated for Ag using 3 μL of a soluble oligomeric Streptactin multimerization reagent preparation functionalized with 0.5 μg of the peptide:MHC class I complexes equipped with the streptavidin-binding peptide and with 0.5 μg of the αCD28 Fab described above. Alternatively, 4.5 μL of a Streptactin multimerization reagent preparation was loaded with 0.5 μg of the following peptide:MHC class I complexes, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab. For comparison, polyclonal stimulation was performed using 3 μL of a streptactin multimerization reagent preparation (1 mg/mL) or loaded with a combination of 0.5 μg αCD3 Fab and 0.5 μg αCD28 Fab. Again as an alternative stimulation condition described above, 4.5 μL of a Streptactin multimerization reagent preparation reversibly loaded with 0.5 μg αCD3 Fab, 0.5 μg αCD8 Fab, and 0.5 μg αCD28 Fab was used. Untreated (unstimulated) Tresp cells served as a negative control and polyclonal Tresp cells stimulated with anti-CD3/anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) as a positive control. Tresp cells were seeded in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/mL IL-2 and 5 ng/mL IL-15. The cells were incubated at 37°C with medium changes every 3 days, and cell counts were analyzed after 7 and 14 days. Exemplary results are shown in FIG. 24(A–F). Exemplary flow cytometric analysis of the fraction of Ag-specific cells that were stimulated/expanded by the soluble Streptocotactin oligomer on which the peptide:MHC-I complex for an HLA-C7/IE-1 epitope (for CMV) was immobilized (FIG. 24A) shows that these antigen-specific T cells were specifically expanded. The graphs in FIG. 24B through FIG. 24E (which depict the degree of expansion of distinct Ag specificities according to the number of peptide multimer:MHCI positive cells harvested per time point in analogy to the expansion experiment shown in FIG. 24A) show that the multimerization reagent using the respective Ag-specific peptide complex and the MHC1 molecule provided the greatest number of expanded cells (ranging from a twenty-fold increase in the number of cells for Ag-specific cells recognizing the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF, (SEQ ID NO: 39)) restricted by HLA-A2402) (see FIG. 24B) to a 98-fold increase in the number of Ag-specific cells recognizing the HLA-B7/IE-1309-317 epitope (CRVLCCYVL (SEQ ID NO: 38)) of CMV (see FIG. 24E ), thus showing that the expansion method of the present invention is fully applicable to the expansion of Ag-specific cells. Finally, the exemplary flow cytometric analysis of the surface expression of CD62L and CD127 after 14 days of culture for the HLA-B7/Hexon5 epitope (for adenovirus) shown in FIG. 24F further confirms that experimental approaches using the soluble multimerization reagents of the present invention retain a higher content of long-lived memory T cells expressing CD127 under polyclonal and Ag-specific stimulating conditions.

EXAMPLE 21: Ag-specific expansion kinetics and phenotype of central memory T cells in bulk

[001170] This example examines the selective Ag-specific expansion kinetics of purified CD3+CD62L+CD45RA-TCM responder cells that were stimulated in vitro with a) antigen-specific peptide MHC I complexes and b) αCD28 Fab fragments that were reversibly immobilized as first and second agents on soluble oligomeric streptavidin muteins.

[001171] For this purpose, 500,000 CD3+CD62L+CD45RA responder TCM cells (Tresp) were specifically stimulated for Ag using 3 μL of a Streptactin multimerization reagent preparation functionalized with 0.5 μg of peptide:MHC class I complexes equipped with a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 (CPYSGTAYNSL, SEQ ID NO: 41) of the adenovirus Hexon 5 protein) restricted by HLA-B07) and 0.5 μg of αCD28 Fab. Alternatively, 4.5 μL of a streptactin multimerization reagent preparation loaded with 0.5 μg of this peptide:MHC class I complex, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab was used. For comparison, polyclonal stimulation was performed using 3 μL of a streptactin multimerization reagent preparation (1 mg/mL) or loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. Again as an alternative stimulation condition described above, 4.5 μL of a Streptactin multimer preparation loaded with 0.5 μg of αCD3 Fab, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab was used. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells were polyclonal stimulated with anti-CD3/anti-CD28 beads as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U/ml IL-2 and 5 ng/ml IL-15. The cells were incubated at 37°C with medium changes every 3 days, and cell counts were analyzed after 7 and 14 days. The images shown in FIG. 25 represent the degree of cluster formation on day 5; exemplary Ag-specific stimulation is illustrated for the adenovirus HLA-B7/Hexon 5 epitope. As can be seen in FIG. 25, such adenovirus antigen-specific cells can be specifically expanded from the original CD3+ CD45L+ CD45RA-TCM responder population.

EXAMPLE 22: Activation of intracellular signaling cascades following streptamer multimer stimulation of CD19-CAR-transduced Jurkat cells

[001172] In this example, the activation of intracellular signaling cascades of transduced Jurkat cells that were modified to express a tumor-specific chimeric antigen receptor (CAR), i.e., CD19, and that were stimulated using the oligomeric Streptocatin® of Example 8 as the soluble multimerization reagent was examined.

[001173] For this purpose, 300,000 Jurkat responder (Jresp) cells were stimulated with (A) varying amounts of a mixture of Streptactin multimerization reagent preparations (1 mg/ml) functionalized with αCD3 and αCD28 Fab fragments described herein (“x1” corresponds to 3 μg of Streptactin multimerization reagent functionalized with 0.5 μg of αCD3 and 0.5 μg of αCD28 Fab provides a “Strepactin-based polyclonal multimerization reagent”) or (B) 3 μL of a Streptactin multimerization reagent preparation functionalized with 0.5 μg (x1) or 1 μg) of the extracellular domain (ECD) of CD19 (the natural ligand for the αCD19-CAR - this provides a "CAR-specific Streptactin-based multimerization reagent") or 3 μL of a Streptactin multimerization reagent preparation loaded with 0.5 μg (x1) or 1 μg (x2) αIgG recognizing the IgG4 spacer within αCD19-CAR - this also provides a "CAR-specific Streptavidin mutein-based multimerization reagent". The CD19 ECD equipped with a hexa-histidine tag was obtained from Sino Biological/Life Technologies (SEQ ID NO: 47) and was functionalized by binding to the streptavidin-based demultimerization reagent by mixing the CD19 ECD with the His-STREPPER adapter molecule (IBA GmbH, Germany, order number 2-0920-005) in a 1:1 molecular ratio and incubating for 15 min at room temperature. The His-STREPPER adapter molecule contains a chelating moiety that binds to the hexahistidine tag and a streptavidin-binding peptide, transiently providing the target molecule, here the CD19 ECD with a streptavidin-binding peptide that can reversibly bind to a streptavidin mutein-based multimerization reagent. Jresp stimulated with anti-CD3/anti-CD28 beads (beads having these aCD3 and αCD28 monoclonal antibodies irreversibly immobilized) or PMA and Ionomycin served as positive controls. Jresp cells were seeded in 1.5 mL Eppendorf tubes in 200 μL of cell culture medium supplemented with 30 U/mL IL-2. Cells were incubated at 37°C and placed on ice and lysed after 0 min to 20 min of stimulation. Detection of phosphorylated ERK indicates active MAPK signaling, staining for housekeeping β-Actin indicates loading of equal amounts of total protein per condition and time point. As can be seen from the comparison of FIG. 26A showing activation of Jurkat cells by the “polyclonal Streptactin multimerization reagent” and FIG. 26B showing activation of Jurkat cells by the two “CAR-Specific Streptactin-based multimerization reagents,” Jurkat cells can be activated/expanded by binding of the extracellular domain of CD19 to the CD19-specific chimeric antigen receptor. Since downstream genetic processing of T cells is almost exclusively performed on preselected cell populations, a generic activation via cross-linking of CARs introduced via the IgG4 spacer domain (this is conserved in multiple CARs with different specificities) broadens the applicability for reversible cell stimulation/expansion in these in vitro cell processing situations.

[001174] Thus, this experiment shows that, in principle, any cell population that is activated by the binding of an agent (ligand) that provides a primary activation signal to the cell population can be expanded using a first agent reversibly immobilized in a multimerization reagent as described herein.

EXAMPLE 23: Parallel Antigen-Specific Expansion of TCM Responder Cells out of a Single Pool

[001175] In this Example, the parallel Antigen-specific (Ag-specific) expansion kinetics of a single pool of T responder cells stimulated in vitro with multiple reversible peptide:MHC/aCH28 Streptamer Fab multimers is examined.

[001176] 500,000 CD3+ CD62L+CD45RA(Tresp) responder TCM cells are stimulated simultaneously for multiple Ag specificities using, for each specificity, 3 μL of Streptactin multimers functionalized with 0.5 μg of the respective peptide:MHC class I complexes carrying a streptavidin-binding peptide and 0.5 μg of αCD28 Fab also carrying a streptavidin-binding peptide. As an alternative approach, 4.5 μL of Streptactin-based multimerization reagent functionalized with 0.5 μg of peptide:MHC class I complexes carrying a streptavidin-binding peptide, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab as described herein are used for each specificity. For comparison, polyclonal stimulation is performed using 3 μL of a Streptactin-based multimerization reagent preparation (1 mg/mL) reversibly loaded with a combination of 0.5 μL of αCD3 Fab and 0.5 μL of CDCD Fab. Again as an alternative stimulation condition described above, 4.5 μL of a Streptactin-based multimerization reagent preparation reversibly loaded with 0.5 μg αCD3 Fab, 0.5 μg αCD8 Fab, and 0.5 μg αCD28 Fab (each containing a streptavidin-binding peptide) can be used. Untreated (unstimulated) Tresp cells serve as a negative control, and polyclonal Tresp cells stimulated with anti-CD3/anti-CD28 beads (αCD3- and α28CDmAb-coated beads) as a positive control. Tresp cells are seeded in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/mL IL-2 and 5 ng/mL IL-15. Cells are incubated at 37 °C with media changes every 3 days and cell counts are analyzed after 7 and 14 days.

EXAMPLE 24: Preferential proliferation of CD8+ T cells among CD3+ T responder cells stimulated in vitro with streptavidin-based multimerization reagents reversibly functionalized with αCD3/αCD8/αCD28 Fab fragments

[001177] 300,000 CD3+ responder T cells (Tresp) are stimulated with 3 μL of a streptactin multimerization preparation (1 mg/mL) or a preparation of a multimerization reagent utilizing the large streptactin backbone (0.1 mg/mL) loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab or 4.5 μL of a Streptactin-based multimerization reagent preparation loaded with 0.5 μg of αCD3, 0.5 μg of αCD8 Fab and 0.5 μg of αCD28 Fab or 3 μL of a mixture of Streptactin-based multimerization reagent preparations with 0.5 μg of αCD3 Fab. αCD3 alone and 0.5 μg of αCD28 Fab alone (each Fab fragment again carries a streptavidin-binding peptide). Untreated Tresp cells serve as a negative control and Tresp is stimulated with anti-CD3/anti-CD28 beads (beads coated with αCD3 and αCD28-mAb) as a positive control. Tresp cells are seeded in duplicates in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/ml IL-2. Cells are incubated at 37 °C with medium change after 3 days and analyzed after 6 days.

EXAMPLE 25: Preferential proliferation of CD8+ T cells among CD3+ responder cells stimulated in vitro with streptavidin-based multimerization reagents reversibly functionalized with αCD3 Fab and αCD28 Fab fragments

[001178] 300,000 CD3+ responder T cells (Tresp) are stimulated with varying amounts of a mixture of preparations of a Streptactin-based multimerization reagent (1 mg/mL) functionalized with the αCD3 Fab fragment alone and the isolated αCD28 Fab fragment (1.5 μg of Streptactin-based multimerization reagent functionalized with 0.5 μg of an isolated αCD3 Fab fragment and 1.5 μg of Streptactin-based multimerization reagent functionalized with 0.5 μg of an isolated αCD28 Fab fragment), or varying amounts of a mixture of preparations of Streptactin-based multimerization reagent functionalized with the αCD3 Fab fragment and αCD28 Fab fragment with or without the αCD8 Fab fragment (each fragment Fab again contains a streptavidin-binding peptide) (3 μg of Streptactin-based multimerization reagent functionalized with 0.5 μg of αCD3 and 0.5 μg of αCD28 Fab fragment - without the αCD8 Fab fragment, or 4.5 μL of a Streptactin multimerization reagent preparation loaded with 0.5 μg of αCD3 Fab fragment, 0.5 μg of αCD8 Fab fragment and 0.5 μg of αCD28 Fab fragment, wherein the Fab fragment again carries a streptavidin-binding peptide). Untreated Tresp cells serve as a negative control, and Tresp stimulated with anti-CD3/anti-CD28 beads (beads coated with αCD3 and αCD28-mAb) as a positive control. Tresp cells are seeded in 48-well plates in 1 mL of cell culture medium supplemented with 30 U/mL IL-2. Cells are incubated at 37 °C with media change after 3 days and analyzed after 6 days.

EXAMPLE 26: Biotin-Mediated Disruption of Stimulation by Intracellular Calcium Mobilization Analysis in Primary T Cells

[001179] The impact of disruption of cellular stimulation via a reversible multimerization reagent (by addition of D-Biotin) on stimulation of primary human T cells was monitored by assessing Ca2+ flux. Purified CD3 T cells from healthy donor peripheral blood mononuclear cells (PBMC) were loaded with a calcium sensor dye, incubated with a reagent containing multimerized anti-CD3/anti-CD28 Fab fragments reversibly linked to an oligomeric streptavidin mutein reagent, and assessed for Ca2+ flux by flow cytometric analysis. The multimerized anti-CD3/anti-CD28 agent was added for 60 s, and the culture was either left intact or treated with addition of D-Biotin for 200 s. To confirm that induction of T cell calcium flux was still possible after disrupting the signal by dissociation of the Fab fragments from the multimerization reagent, ionomycin was added for 400 s.

[001180] As shown in FIG. 27, dissociation by addition of D-Biotin was able to disrupt the signal delivered into the cell via the anti-CD3/anti-CD28 multimerized agent as shown by a decrease in intracellular Ca2+ levels following the reagent-induced increase in Ca2+ levels. Addition of ionomycin following disruption of D-Biotin-mediated signaling restored intracellular Ca2+ to levels observed prior to D-Biotin addition, indicating that the cells remained responsive and healthy following dissociation from reagent dissociation and signaling disruption and were able to efflux calcium and in response to ionomycin exhibited intracellular levels that were similar levels when compared to those observed following initial stimulation.

EXAMPLE 27: Assessment of T cell expansion after temporally controlled activation of T cells by incubation with a reversible agent

[001181] After incubation with and dissociation of the anti-CD3/anti-CD28 signal, the expansion of T cells isolated from PBMCs of healthy donors was assessed. T cells were incubated for 7 days after addition of a reagent containing multimerized anti-CD3/anti-CD28 Fab fragments reversibly linked to an oligomeric streptavidin mutein reagent. On day 1 of culture, D-biotin was added to dissociate the multimerized agent or the cells were left intact (no D-Biotin added on day 1). As a control, cells were incubated with anti-CD3/anti-CD28 magnetic beads for 7 days. Total cell count and the fraction of annexin V-positive cells were assessed on day 0, 3, or 7 of culture. As shown in FIGS. 28A and 28B, CD3+ T cells cultured with the dissociated anti-CD3/anti-CD28 multimerized agent on day 1 (biotin added on day 1) showed increased expansion and reduced apoptosis (as indicated by decreased annexin V labeling) compared to the culture in which the cells were incubated for a full seven days with the multimerized agent (no D-Biotin added) and compared to the cultures in which the cells were incubated with the anti-CD3/anti-CD28 control beads.

[001182] In another assay, isolated CD3+ T cells, CD4+ T cells, or CD8+ T cells were labeled with CFSE dye and proliferation was assessed following stimulation as described above, except with the addition of biotin on day 2. Proliferation was determined by flow cytometry over the extent of CFSE dye dilution. As shown in FIG. 29, CD3+ cells stimulated with the multimerized anti-CD3/anti-CD28 agent with dissociation on d 1 (biotin added) showed increased proliferation, particularly on day 3, compared to cells stimulated with the multimerized agent but without signal disruption (no biotin).

EXAMPLE 28: Assessment of metabolic activity in T cells after temporally controlled activation by incubation with a reversible agent

[001183] The metabolic activity of cells as an indicator of cell proliferation was assessed by colorimetric monitoring of the cleavage of the stable tetrazolium salt WST-1 to a soluble formazan dye complex. Since cleavage of this agent generally depends on glycolytic production of NAD(P)H in viable cells, the extent of formazan dye uptake can often be directly correlated to the number of metabolically active cells in a culture. In this study, purified CD4 or CD8 T cells were incubated with the multimerized anti-CD3/anti-CD28 Fab fragments reversibly bound to an oligomeric streptavidin mutein reagent (with or without the addition of D-Biotin on day 2) or a control anti-CD3/anti-CD28 magnetic bead reagent. After culturing the cells on day 1, day 2, or day 3, the cells were incubated with the WST-1 reagent, and the metabolic activity levels were assessed by measuring the absorbance at 450 nm as a readout. The results were normalized to the number of cells in the culture to be analyzed and represented as the ratio of WST-1 per cell number.

[001184] As shown in FIG. 30, the results indicated that controlled stimulation of T cells with the anti-CD3/anti-CD28 multimeric reagent resulted in increased levels of a signal indicative of metabolic activity and cell expansion, during incubation in this study, even when biotin was added on d. 2 to dissociate the reagent and disrupt signaling.

EXAMPLE 29: Impact of Disruption of Signaling via a Multimeric Anti-CD3/anti-CD28 Agent on T Cell Expansion Capacity After Restimulation

[001185] CFSE dye-labeled CD3+ cells were cultured in the presence of a reversible reagent containing multimerized anti-CD3/anti-CD28 Fab fragments reversibly linked to an oligomeric form of a streptavidin mutein. On day 1, D-Biotin was added to some samples as indicated in Figure 31. Under some conditions, as indicated, cells were sorted on day 3 for CFSElow (unsplit) cells. Sorted cells were either cultured without further addition of stimulus (purified type (see FIG. 31)) or were additionally mixed with additional multimerized anti-CD3/anti-CD28 agent (purified type, restimulation (see FIG. 31)). Unsorted cells (cells not sorted based on CFSE levels on day 3) were also evaluated in parallel. Cell expansion, normalized to unstimulated cells, was monitored until day 15.

[001186] As shown in FIG. 31, it was observed that cells incubated with the multimeric reagent continued to expand throughout the duration of the study, up to day 15, for all conditions tested. Even in samples in which signaling induced by the multimeric agent was disrupted on day 1, and those in which the cells were purified in kind without additional addition of the reagent, they continued to proliferate throughout this time period. This result indicated that incubation with the stimulatory oligomeric reagent, even for only 1 day, was capable of inducing prolonged expansion of T cells over a 15-day period. This effect was observed even in samples in which the cells were purified in kind on day 3, to isolate cells that had not yet divided and in which there was no additional stimulation by re-addition of this or any stimulatory reagent. The result was consistent with the conclusion that the reversible oligomeric stimulatory agent was capable of inducing prolonged T cell expansion, for several days after disruption of reagent binding and/or after purification of nondividing cells, without additional reagent induction, an effect not observed in this assay under conditions in which cells were stimulated with beads.

EXAMPLE 30: Evaluation of T cell surface markers after temporally controlled activation by incubation with a reversible agent

[001187] Expression of various T cell markers was assessed following stimulation of T cells subjected to various conditions, including various types of retroviral-mediated transduction, by incubating them with the reversibly binding anti-CD3/anti-CD28 multimerized agent, with or without dissociation on day 1, induced by the addition of D-Biotin). CD3-purified T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC), transduced with one of several retroviral vector particles, each encoding a different chemical antigen receptor, were expanded in culture, frozen, and thawed. The cells were incubated for 7 days after addition of the reagent described in Example 1 containing multimerized anti-CD3/anti-CD28 Fab fragments, which were multimerized by reversible binding to an oligomeric streptavidin mutein. On day 1 of culture, D-biotin was added to dissociate the multimerized agent from the cells in some samples, whereas in others, the cells and reagent were left intact (no D-biotin added on day 1). In other samples, the cells were incubated with anti-CD3/anti-CD28 magnetic beads for 7 days. The percentage of cells positive for surface and/or intracellular expression of each T-cell activation number and/or other phenotypic markers was assessed at various time points.

[001188] For example, on day 7, cells were analyzed for expression of Ki-67 and T-bet markers by flow cytometry as described. As in other studies described herein, following stimulation with the reversible oligomeric reagent, when the reagent was dissociated to disrupt signaling on day 1, cells were observed to retain Ki67 positivity but had reduced expression of T-bet. This result indicated that, under various conditions, temporal control by reagent dissociation could be used to generate an output composition of cells with a less differentiated or stem-like phenotype, longer memory phenotype, and/or central memory and/or stem central memory phenotype.

[001189] Other markers evaluated included CD25 and CD69 and the markers CD45RA, CD45RO, CD27, CD127 and CXCR3, were evaluated by flow cytometry on day 3 and day 7. The results are consistent with the observation that disruption of reversible stimulatory reagent binding on day 1 of the culture period resulted in a higher percentage of T cells in the resulting production pool having characteristics of a phenotypic profile generally seen in less differentiated and/or relatively longer-lived and/or stem-like T cell populations, such as central memory T cells (CMT) and less differentiated memory, such as so-called stem memory T cells (SMT). These studies were further consistent with the observation that temporally controlled stimulation of oligomeric reagent utilization by the addition of D-Biotin at early time points (within 4 days) resulted in cellular compositions with an increase in cells with a phenotypic profile consistent with or similar to that present in a less differentiated, long-lived T cell population, such as long-lived memory T cells.

[001190] The present invention is not intended to be limited to the scope of the particular embodiments described, which are provided, by way of example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings presented herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present description.SEQUENCES

Claims (22)

1. A method for culturing T cells, comprising: (a) incubating a composition comprising T cells in the presence of: (1) an anti-CD3 antibody or antigen-binding fragment thereof, wherein the anti-CD3 antibody or antigen-binding fragment thereof is (i) linked to a streptavidin-binding peptide that is reversibly linked to a streptavidin mutein reagent and (ii) specifically binds to a component of the TCR/CD3 complex or CD3 on the surface of T cells in a manner that induces a TCR/CD3 complex-associated signal in T cells in the composition, and (2) a second antibody or antigen-binding fragment thereof that binds to an accessory molecule selected from CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), and CD154 (CD40L), wherein binding to the accessory molecule stimulates or induces an additional signal on the surface of the T cells. cell; wherein the induction of the signals affects the activation, stimulation and/or expansion of the T cells; and(b) within 5 days after the start of said incubation, disrupt the reversible binding between the streptavidin-binding peptide and the streptavidin mutein reagent, thereby generating cultured T cells. 2. The method of claim 1, wherein the streptavidin mutein reagent comprises a plurality of binding sites capable of reversibly binding to a streptavidin-binding peptide and the second antibody or antigen-binding fragment thereof is bound to a streptavidin-binding peptide that is reversibly bound to the streptavidin mutein reagent. 3. The method of claim 1, wherein the streptavidin mutein reagent is a first reagent, and the incubation is carried out in the presence of at least a second streptavidin mutein reagent, and wherein the second antibody or antigen-binding fragment is linked to a streptavidin-binding peptide that is reversibly linked to the second streptavidin mutein reagent. 4. The method of claim 1 or 2, wherein: (a) the second antibody or antigen-binding fragment thereof is an anti-CD28 Fab, wherein the anti-CD28 Fab is i) linked to a streptavidin-binding peptide that is reversibly linked to the streptavidin mutein reagent and ii) specifically binds to a CD28 molecule on the surface of T cells in a manner that induces additional signaling through CD28 on the T cells; (b) the anti-CD3 antibody or antigen-binding fragment thereof is an anti-CD3 Fab; (c) the anti-CD3 Fab and the anti-CD28 Fab are reversibly linked to the same streptavidin mutein reagent; and(d) within 5 days after the start of said incubation, disrupting the reversible binding between the streptavidin mutein reagent and both anti-CD28 Fab and anti-CD3 Fab, thereby generating cultured T cells. 5. The method of any one of claims 1 to 4, wherein: said rupture is performed more than 30 minutes after the start of said incubation; or said rupture is performed between 1 hour and 4 days after the start of said incubation, between 6 hours and 3 days after the start of said incubation, between 12 hours and 2 days after the start of said incubation, or between 1 day and 3 days after the start of said incubation; or said rupture is performed greater than or equal to 1 hour after the start of said incubation and within 1 day, 2 days, 3 days, or 4 days after the start of said incubation. 6. The method of any one of claims 1 to 3 and 5, wherein the second antibody or antigen-binding fragment thereof binds to CD28. 7. The method of claim 2, wherein: the second antibody or antigen-binding fragment thereof is an anti-CD28 antibody or antigen-binding fragment thereof; and within 5 days after the start of said incubation, disrupting the reversible binding between the streptavidin-binding peptide and the streptavidin mutein reagent, whereby CD28 signaling is terminated or reduced in the cells, thereby generating cultured T cells. 8. The method of any one of claims 1 to 7, wherein: (a) the streptavidin mutein comprises a net negative charge or exhibits a lower isoelectric point than the streptavidin mutein set forth in SEQ ID NO: 4 or 6; or (b) the streptavidin mutein exhibits a higher affinity for a streptavidin-binding peptide comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than a streptavidin or mutein comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-6. 9. The method of any one of claims 1 to 8, wherein: the T cells are primary cells from an individual; or the T cells are unfractionated T cells, are enriched or isolated CD3+ T cells, are enriched or isolated CD4+ T cells, or are enriched or isolated CD8+ T cells. 10. The method of any one of claims 1 to 9, wherein: each antibody or antigen-binding fragment thereof comprises an antibody fragment; each antibody or antigen-binding fragment thereof individually comprises a Fab fragment; each antibody or antigen-binding fragment thereof is individually a divalent antibody fragment selected from an F(ab')2 fragment and a divalent single-chain Fv (scFv) fragment; and/or each antibody or antigen-binding fragment thereof is individually a monovalent antibody fragment selected from a Fab fragment, an Fv fragment and a scFv fragment. 11. The method of any one of claims 1 to 10, wherein the streptavidin mutein reagent is not, and is not bound to or associated with, a solid support, a stationary phase, a bead, a microparticle, a magnetic particle and/or a matrix during said incubation. 12. The method of any one of claims 1 to 11, wherein: the streptavidin mutein reagent is or comprises a streptavidin mutein that reversibly binds to biotin or a biologically active fragment; the streptavidin mutein reagent is or comprises a streptavidin mutein that reversibly binds to a biotin analogue or a biologically active fragment; and/or the streptavidin mutein reagent is or comprises a streptavidin mutein that reversibly binds to a streptavidin-binding peptide. 13. The method of any one of claims 1 to 12, wherein the streptavidin mutein reagent comprises a streptavidin mutein oligomer or polymer, optionally wherein individual molecules of the oligomer or polymer are crosslinked with a polysaccharide or a bifunctional ligand. 14. The method of claim 13, wherein the oligomer polymer comprises three or more streptavidin mutein tetramers. 15. Method according to any one of claims 1 to 7 and 9 to 14, characterized in that: the streptavidin mutein reagent comprises the amino acid sequence Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to the positions in streptavidin in the amino acid sequence set forth in SEQ ID NO:1. 16. Method according to any one of claims 1 to 7 and 9 to 15, characterized in that the streptavidin mutein reagent comprises: a) the amino acid sequence set forth in any of SEQ ID NOS: 3-6, 27 and 28; or b) a functional fragment of a) that reversibly binds to biotin or a biologically active form thereof, a biotin analogue or a biologically active fragment thereof, or a streptavidin-binding peptide. 17. The method of any one of claims 1 to 16, wherein: the streptavidin-binding peptide that is bound to the anti-CD3 antibody or antigen-binding fragment thereof is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18). and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7), SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO: 15) and SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK (SEQ ID NO: 16). 18. The method of any one of claims 2 to 17, wherein the streptavidin-binding peptide that is linked to the second antibody or antigen-binding fragment thereof is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp- Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), Trp-Arg-His-Pro-Gln- Phe-Gly-Gly (SEQ ID NO: 7), SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO: 15) and SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK (SEQ ID NO: 16). 19. The method of any one of claims 1 to 18, wherein said disruption comprises introducing to the cells a composition comprising biotin or a biologically active fragment thereof, or a biotin analogue or a biologically active fragment thereof capable of reversing the binding between the streptavidin-binding peptide and the streptavidin mutein reagent, wherein the substance comprises biotin or a biologically active fragment thereof, or a biotin analogue or a biologically active fragment thereof. 20. An article of manufacture for carrying out the method as defined in any one of claims 1 to 19, comprising the streptavidin mutein reagent, which is reversibly linked to: a) the anti-CD3 antibody or antigen-binding fragment thereof; and b) the second antibody or antigen-binding fragment thereof, which are linked to the streptavidin mutein reagent via streptavidin-binding peptides. 21. Kit, characterized by the fact that it comprises the article of manufacture, as defined in claim 20, and optionally instructions for use. 22. The kit of claim 21, further comprising biotin, a biotin analogue or a biologically active fragment thereof to disrupt the reversible bond between the streptavidin mutein reagent and the streptavidin-binding peptide.