HK40026712A - Methods of administering chimeric antigen receptor immunotherapy - Google Patents

Description

Methods of administering chimeric antigen receptor immunotherapy

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 62/574,159, filed on 2017, month 10, 18, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates generally to T cell therapies, and more particularly to CD 19-directed genetically modified autologous T cell immunotherapies comprising Chimeric Antigen Receptors (CARs).

Background

Human cancers by their very nature comprise normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. At this point, the cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These aberrant tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.

A Chimeric Antigen Receptor (CAR) comprising a binding domain capable of interacting with a specific tumor antigen, allowing T cells to target and kill cancer cells expressing the specific tumor antigen.

Summary of The Invention

As described in detail below, the present disclosure is based, in part, on the surprising discovery that the administration methods disclosed herein recognize and manage the adverse side effects of CAR T cell immunotherapy.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

In one aspect, the invention provides a method of treating D L BC L caused by relapsed or refractory diffuse large B-cell lymphoma (D L BC L) non-finger type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma in a patient after two or more lines of systemic therapy, comprising treating the patient with about 1 × 10 per kg body weight of the patient⁶And about 2 × 10⁶Between doses of CAR-positive live T cells up to about 1 × 10⁸Administering by intravenous infusion to a patient in need thereof a suspension of axicabtagene ciloleucel, wherein axicabtagene ciloleucel isA CD 19-directed genetically modified autologous T cell immunotherapy comprising patient-own T cells harvested and genetically modified ex vivo by retroviral transduction to express a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single-chain variable fragment (scFv) linked to a CD28 and CD3-zeta costimulatory domain.

In another aspect, the invention provides methods of treating relapsed or refractory diffuse large B-cell lymphoma (D L BC L) and primary mediastinal large B-cell lymphoma (PMBC L) in a patient following two or more lines of systemic therapy comprising administering to the patient a therapeutically effective amount of about 0.4 × 10⁸And about 2 × 10⁸Between doses of CAR positive living T cells an axicabtagene ciloleucel suspension is administered by intravenous infusion to a patient in need thereof, wherein the axicabtagene ciloleucel is a CD 19-directed genetically modified autologous T cell immunotherapy comprising patient-own T cells harvested and genetically modified ex vivo by retroviral transduction to express a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single-chain variable fragment (scFv) linked to CD28 and CD3-zeta costimulatory domain.

In some embodiments, the intravenous infusion time is between 15 and 120 minutes. In some embodiments, the intravenous infusion time is up to 30 minutes.

In some embodiments, the infusion volume is between 50 and 100m L in some embodiments, the infusion volume is about 68m L.

In some embodiments, the immunotherapy is infused from an infusion bag. In some embodiments, the infusion bag is agitated during infusion.

In some embodiments, the immunotherapy is administered within 3 hours after thawing.

In some embodiments, the suspension further comprises albumin. In some embodiments, albumin is present in an amount of about 2-3% (v/v). In some embodiments, albumin is present in an amount of about 2.5% (v/v). In some embodiments, the albumin is human albumin.

In some embodiments, the suspension further comprises DMSO. In some embodiments, DMSO is present in an amount of about 4-6% (v/v). In some embodiments, DMSO is present in an amount of about 5% (v/v).

In one aspect, the invention provides a method of treating relapsed or refractory large B-cell lymphoma in a patient following two or more lines of systemic therapy, comprising: (a) administering to a patient in need thereof a CD 19-directed genetically modified autologous T cell immunotherapy; and (b) monitoring the patient for signs and symptoms of adverse reactions after infusion.

In some embodiments, the relapsed or refractory large B-cell lymphoma is D L BC L caused by diffuse large B-cell lymphoma (D L BC L), non-finger type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma.

In some embodiments, the adverse reaction is selected from the group consisting of: cytokine Release Syndrome (CRS), neurotoxicity, hypersensitivity, severe infection, cytopenia and hypogammaglobulinemia.

In some embodiments, the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphoblastic cell proliferation/macrophage activation syndrome (H L H/MAS), seizure, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia and anemia.

In some embodiments, the methods further comprise administering an I L-6 receptor inhibitor.

In some embodiments, the method further comprises administering an effective amount of toslizumab (tocilizumab) to treat a symptom of the adverse reaction.

In some embodiments, tositumumab is administered intravenously at a dose of about 8 mg/kg. In some embodiments, the toslizumab is administered intravenously over about 1 hour. In some embodiments, the tositulizumab is administered about every 8 hours. In some embodiments, tositulizumab is administered for no more than about 24 hours.

In some embodiments, the method further comprises administering a corticosteroid to treat a symptom of the adverse reaction.

In some embodiments, the corticosteroid is at least one of methylprednisone or dexamethasone.

In some embodiments, methylprednisolone is administered intravenously at a dose of about 1 mg/kg. In some embodiments, methylprednisolone is administered twice daily. In some embodiments, methylprednisolone is administered intravenously at a dose of about 1,000mg per day. In some embodiments, methylprednisolone is administered intravenously for about 3 days.

In some embodiments, dexamethasone is administered at a dose of about 10 mg. In some embodiments, dexamethasone is administered intravenously about every 6 hours.

In some embodiments, the adverse reaction is Cytokine Release Syndrome (CRS). In some embodiments, the signs and symptoms of Cytokine Release Syndrome (CRS) are monitored at least daily for about 7 days post-infusion. In some embodiments, the signs and symptoms of Cytokine Release Syndrome (CRS) are monitored at least daily following infusion for about 8 days, about 9 days, or about 10 days. In some embodiments, the signs and symptoms of Cytokine Release Syndrome (CRS) are monitored at least daily for about 10 days following infusion. In some embodiments, the signs and symptoms of Cytokine Release Syndrome (CRS) are monitored for about 4 weeks after infusion.

In some embodiments, the adverse reaction is neurotoxicity.

In some embodiments, signs and symptoms of neurotoxicity are monitored for up to 8 weeks after infusion.

In some embodiments, the method further comprises administering a non-sedating antiepileptic for seizure prevention.

In some embodiments, the non-sedating antiepileptic drug is levetiracetam (levetiracetam).

In some embodiments, the adverse reaction is cytopenia. In some embodiments, the cytopenia is thrombocytopenia, neutropenia, and/or anemia.

In some embodiments, the method further comprises administering at least one of erythropoietin, darbepotein α, platelet transfusion, Colony Stimulating Factor (CSF), granulocyte colony stimulating factor, filgrastim, pegfilgrastim, or granulocyte macrophage colony stimulating factor.

In some embodiments, the level of at least one of I L-6, I L-8, I L-10, I L-15, TNF- α, IFN- γ, and sI L2R α is measured.

In one aspect, the invention provides a container comprising a suspension of CD 19-directed genetically modified autologous T cells, about 5% dimethyl sulfoxide (DMSO), and about 2.5% human albumin (v/v). In another aspect, the container comprises about 0.4x10⁸-2x10⁸Between individuals, suspensions of CD 19-directed genetically modified autologous T cells (CAR positive live T cells).

In some embodiments, the volume of the bag is about 100m L, 250m L, 500m L, 750m L, 1000m L, 1500m L, 2000m L, or 3000m L.

In one aspect, the invention provides a method of treating a relapsed or refractory large B-cell lymphoma in a human following two or more lines of systemic therapy comprising administering to a human in need thereof CD 19-directed genetically modified autologous T cell immunotherapy comprising: (a) administering to the patient a composition comprising a CD 19-directed Chimeric Antigen Receptor (CAR) positive live T cell; (b) monitoring the patient for signs and symptoms of adverse reactions after administration; and (c) if Cytokine Release Syndrome (CRS) greater than grade 2 is observed in (b), intravenously administering tositumumab at a dose of about 8mg/kg over 1 hour, repeating tositumumab every 8 hours as needed if not responsive to intravenous fluid or increased supplemental oxygen; (d) if the CRS symptoms observed in (b) did not improve after 24 hours of (c), administering intravenously about 1mg/kg methylprednisolone twice daily or administering an equivalent dexamethasone dose and continuing corticosteroid use until the event is grade 1 or below, then gradually decreasing over 3 days; (e) administering intravenously tollizumab at a dose of 8mg/kg over 1 hour if CRS grade 3 is observed in (b), repeating tollizumab every 8 hours as needed if not responsive to intravenous fluid or increasing supplemental oxygen, and administering intravenously about 1mg/kg methylprednisolone twice daily or administering an equivalent dexamethasone dose and continuing corticosteroid use until the event is grade 1 or below, then gradually decreasing over 3 days; and (f) if CRS grade 4 is observed in (b), intravenously administering tositulizumab at a dose of about 8mg/kg over 1 hour, repeating tositulizumab every 8 hours as needed if not responsive to intravenous fluid or increased supplemental oxygen, and intravenously administering about 1,000mg methylprednisolone daily for 3 days.

In one aspect, the invention provides a method of treating a relapsed or refractory large B cell lymphoma in a patient following two or more lines of systemic therapy, comprising administering to a patient in need thereof a CD 19-directed, genetically modified autologous T cell immunotherapy comprising: (a) administering to the patient a composition comprising a CD 19-directed Chimeric Antigen Receptor (CAR) positive live T cell; (b) monitoring patients for signs and symptoms of adverse reactions after administration; and (c) if Cytokine Release Syndrome (CRS) and/or neurotoxicity is observed, managing Cytokine Release Syndrome (CRS) and/or neurotoxicity according to table 1 and/or table 2.

Other features and advantages of the disclosure will be apparent from the following detailed description, including the examples, and from the claims.

Detailed Description

The present disclosure relates to engineered cells (e.g., T cells) comprising a CD19CAR genetic modified autologous T cell immunotherapy designated for the treatment of adult patients with relapsed or refractory large B cell lymphoma following two-or more-line system therapy, including diffuse large B cell lymphoma (D L BC L) non-finger type, primary mediastinal large B cell lymphoma, higher grade B cell lymphoma, and D L BC L caused by follicular lymphoma.

To prepare a CD 19-directed genetically modified autologous T cell immunotherapy, patient-own T cells may be harvested and genetically modified ex vivo by retroviral transduction to express a Chimeric Antigen Receptor (CAR) comprising a murine anti-CD 19 single-chain variable fragment (scFv) linked to CD28 and CD3-zeta costimulatory domains. In some embodiments, the CAR comprises a murine anti-CD 19 single chain variable fragment (scFv) linked to a 4-1BB and CD3-zeta costimulatory domain. anti-CD 19CAR T cells can be expanded and infused back into the patient, where they can recognize and eliminate target cells expressing CD 19.(Axi-cel^TM(ii) a axicabtagene ciloleucel) is an example of such CD 19-directed genetically modified autologous T cell immunotherapy. See Kochenderfer, et al, (J Immunother 2009; 32: 689702). Additional CD 19-directed CAR therapies include JCAR017, JCAR015, JCAR014, kymeriah (tisagenlecucel). See, Sadelain et al Nature Rev. cancer Vol.3(2003), Ruella et al, Curr HematolMalig Rep., Springer, NY (2016), and Sadelain et al cancer Discovery (Apr 2013).

CD 19-directed, genetically modified, autologous T cell immunotherapy can be prepared from patients' peripheral blood mononuclear cells, which are typically obtained via standard leukapheresis (leukapheresis) procedures the monocytes can be enriched for T cells and activated with anti-CD 3 antibodies in the presence of I L-2 and then transduced with replication incompetent retroviral vectors containing an anti-CD 19CAR transgene.

In addition to T cells, CD 19-directed genetically modified autologous T cell immunotherapy may contain NK and NK-T cells. In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy formulation contains about 5% dimethyl sulfoxide (DMSO) and about 2.5% albumin (human) (v/v).

CD 19-directed genetically modified autologous T cells bind to cancer cells expressing CD19 and normal B cells. Studies have shown that upon engagement of anti-CD 19CAR T cells with target cells expressing CD19, CD28 and CD3-zeta costimulatory domains activate downstream signaling cascades that lead to T cell activation, proliferation, acquisition of effector functions, and secretion of inflammatory cytokines and chemokines. This series of events results in killing of cells expressing CD 19.

In one aspect, the invention provides a method of treating D L BC L caused by relapsed or refractory diffuse large B-cell lymphoma (D L BC L) non-finger type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma in a patient after two or more lines of systemic therapy, comprising treating the patient with about 1 × 10 per kg body weight of the patient⁶And about 2 × 10⁶Between doses of CAR-positive live T cells up to about 1 × 10⁸Maximum dose of individual CAR positive live T cells CD 19-directed genetically modified autologous T cell suspension was administered by intravenous infusion to patients in need thereof.

Definition of

In order that the invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" should be understood to be inclusive and encompass both "or" and "unless the context clearly dictates otherwise.

The term "and/or" as used herein is to be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

As used herein, the terms "such as" and "i.e.," are used by way of example only and are not intended to be limiting, and should not be construed as referring only to those items explicitly recited in the specification.

The term "or more", "at least", "over", etc., e.g., "at least one" should be understood to include, but not be limited to, at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 104, 107, 100, 102, 110, 109, 110, 109, 100, 110, 109, 45, 46, 47, 48, 111. 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated values. But also any larger number or fraction therebetween.

Conversely, the term "not more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2,1 and 0 nucleotides. But also any smaller number or fraction therebetween.

The terms "plurality", "at least two", "two or more", "at least a second", etc. should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 101, 104, 105, 106, 107, 109, 108, 106, 109, 108, 105, 106, 109, 103, 45, 47, 48, 110. 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. But also any larger number or fraction therebetween.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It should be understood that the language "comprising" is used herein to describe aspects and also to provide other similar aspects described as "consisting of and/or" consisting essentially of.

Unless specifically stated or otherwise apparent from the context, the term "about," as used herein, refers to a value or composition within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within 1 or over 1 standard deviation as practiced in the art. "about" or "approximately" can mean a range of up to 10% (i.e., + -10%). Thus, "about" may be understood as being greater than or less than the stated value within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001%. For example, about 5mg may include any amount between 4.5mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean values up to an order of magnitude or up to 5-fold. When a particular value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition.

As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range is to be understood as encompassing the value of any integer within the recited range, and where appropriate, including fractions thereof (e.g., tenths and hundredths of integers).

The units, prefixes, and symbols used herein are provided in their international system of units (SI) accepted form. Numerical ranges include the numbers defining the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Juo, "The circumcise Dictionary of biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press; "The dictionary Cell & Molecular Biology", 5th ed., (2013), Academic Press; and "The Oxford dictionary Of Biochemistry And Molecular Biology", Cammacack et al.

By "administering" is meant physically introducing the agent into the subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection and including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, such as orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods.

In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds, each H chain comprising a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region comprising three constant domains, CH1, CH2, and CH3. each light chain comprises a light chain variable region (abbreviated herein as V L) and a light chain constant region comprising one constant domain, the C L VH and V L regions may be further subdivided into regions of hypervariability, referred to as Complementarity Determining Regions (CDRs), interspersed with regions of more conservation, referred to as Framework Regions (FRs). Each VH and V L comprises three CDRs and four FRs, arranged from amino-terminal to carboxy-terminal in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. the variable regions of the heavy and light chains comprise constant domains that interact with antigen, and the constant regions of the tissue-mediated binding system of the host cell, e.g., the first complement system mediated by the classical complement factor (C6851).

Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain fvs (scfvs), camelized antibodies, affibodies, Fab fragments, F (ab') 2 fragments, disulfide linked fvs (sdfvs), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies), Minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, an antibody described herein refers to a polyclonal antibody population.

An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that comprises an antigen-binding portion (e.g., a CDR) from an antibody from which the molecule is derived. The antigen binding molecule may include antigen Complementarity Determining Regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab ', F (ab') 2, and Fv fragments, dabs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide binding domain) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or binds to a viral or bacterial antigen. In some embodiments, the antigen binding molecule binds CD 19. In a further embodiment, the antigen binding molecule is an antibody fragment that specifically binds an antigen, including one or more Complementarity Determining Regions (CDRs) thereof. In a further embodiment, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of a high affinity multimer (avimer).

"antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production or activation of specific immunocompetent cells or both. One skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The antigen may be expressed endogenously, i.e. from genomic DNA, or may be expressed recombinantly. The antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly. In addition, larger molecule fragments may serve an antigenic role. In some embodiments, the antigen is a tumor antigen.

"CD 19-directed genetically modified autologous T cell immunotherapy" refers to a suspension of Chimeric Antigen Receptor (CAR) positive T cells. An example of such immunotherapy is axicabtagene ciloleucel (also known as Axi-cel)^TM，) Developed by Kite Pharmaceuticals, Inc.

The term "neutralizing" refers to an antigen binding molecule, scFv, antibody or fragment thereof that binds to a ligand and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof directly blocks a binding site on the ligand or alters the binding capacity of the ligand by an indirect means, such as a structural or energetic change in the ligand. In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof prevents the protein to which it binds from performing a biological function.

The term "autologous" refers to any substance derived from the same individual into which it is later reintroduced. For example, engineered autologous cell therapy (eACT) as described herein^TM) The methods of (a) involve collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, and then administering back to the same patient.

The term "allogeneic" refers to any material that is derived from one individual and then introduced into another individual of the same species, such as allogeneic T cell transplantation.

The terms "transduction" and "transduced" refer to a process by which foreign DNA is introduced into cells via a viral vector (see Jones et al, "Genetics: printles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculovirus vector, an EB virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

In some embodiments, the methods disclosed herein may be used to reduce the size of tumors derived from, for example, cancers of the bone, pancreas, skin, head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, multiple myeloma, hodgkin's disease, non-hodgkin's lymphoma (NH L), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (D4656), multiple myeloma, hodgkin's lymphoma, non-hodgkin's lymphoma (L), primary splenic large B cell lymphoma (PMBC), refractory splenic lymphoma, neuroblastoma (e), refractory splenic lymphoma, neuroblastoma, melanoma, neuroblastoma, melanoma, prostate.

As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. An anti-tumor effect may also refer to the prevention of tumorigenesis, e.g. a vaccine.

As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell "cytokine" as used herein means a protein released by one cell population that acts on another cell as an intercellular mediator cytokine may be endogenously expressed by cells or administered to a subject. cytokines may be released by immune cells (including macrophages, B cells, T cells and mast cells) to propagate an immune response. cytokines may induce various responses in recipient cells. cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors and acute phase proteins. for example, homeostatic cytokines (including interleukin (I L) 7 and I45-15) promote survival and proliferation of immune cells, while pro-inflammatory cytokines may promote inflammatory responses. examples of homeostatic cytokines include but are not limited to I L-2, I961-4, I962-5, Fas3-7, I L-10, I5-5, TNF-5, soluble cytokines including but not limited to VEGF-2-11, TNF-5, VEGF-2, VEGF-11, VEGF-2, VEGF-3, VEGF-2, VEGF-3, VEGF-2, VEGF-3, VEGF-2, VEGF-3, VEGF-2, VEGF-3, VEGF-2, VEGF-3.

Examples of chemokines include, but are not limited to, I L-8, I L-16, eotaxin-3, macrophage-derived chemokine (MDC or CC L22), monocyte chemotactic protein 1(MCP-1 or CC L2), MCP-4, macrophage inflammatory protein 1 α (MIP-1 α, MIP-1a), MIP-1 β (MIP-1b), gamma-induced protein 10(IP-10), and thymus-and activation-regulated chemokine (TARC or CC L17).

A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., an engineered CAR T cell) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by decreased severity of disease symptoms, increased frequency and duration of asymptomatic phase of the disease, or prevention of injury or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to skilled practitioners, for example in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

As used herein, the term "lymphocyte" includes Natural Killer (NK) cells, T cells or B cells NK cells are a class of cytotoxic (cytotoxic) lymphocytes that represent the major components of the innate immune system, NK cells reject tumors and virally infected cells, which act through the process of apoptosis or programmed cell death since they do not require activation to kill cells, so called "natural killer". T cells play a major role in cell-mediated immunity (without antibody involvement). its T Cell Receptor (TCR) distinguishes itself from other lymphocyte types. thymus (a specialized organ of the immune system) is primarily responsible for the maturation of T cells. there are six types of T cells, namely: helper T cells (e.g. CD4+ cells), cytotoxic T cells (also called TC, cytotoxic T lymphocytes, CT L, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((I) like memory cells such as T cells) (e.g. CD4+ cells), T cells (also called TC), cytotoxic T lymphocytes), CT L, T cells like memory cells (CD 5842 + T + CD 465 + T cells) and CD 3655 + T cells which are also expressed as CD-T cells or CD-T cells + CD 465 + CD-T cells and CD-T cells which are not expressing CD-9 + CD-9 + CD 465 + CD-CD 465 + CD9 + CD2 + CD9 + CD2 + CD9 + cells which, and which, CD9, which are involved in cell line cells which, and which are involved in cell line cells which, and which, are involved in cell line cells which, also in.

The term "genetically engineered" or "engineered" refers to methods of modifying the genome of a cell, including, but not limited to, deletions of coding or non-coding regions or portions thereof, or insertions of coding regions or portions thereof. In some embodiments, the modified cell is a lymphocyte (e.g., a T cell), which can be obtained from a patient or donor. The cells may be modified to express exogenous constructs, such as Chimeric Antigen Receptors (CARs) or T Cell Receptors (TCRs) that integrate into the genome of the cells.

By "immune response" is meant the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including abs, cytokines, and complements) that result in the selective targeting, binding, damaging, destroying, and/or eliminating invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells in vertebrates, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or recurrence of a disease by a method that includes inducing, enhancing, suppressing or otherwise modifying an immune response^TM) And allogeneic T cell transplantation. However, one skilled in the art will appreciate that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. patent publication nos. 2014/0154228 and 2002/0006409, U.S. patent No. 7,741,465, U.S. patent No. 6,319,494, U.S. patent No. 5,728,388, and international publication No. WO 2008/081035.

T cells can be obtained from, e.g., Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors^TMIsolation and/or apheresis) is obtained from a unit of blood collected from a subject. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.

The term "engineered autologous cell therapy" (which may be abbreviated as "eACT^TM", also known as adoptive cell transfer) is the collection of patient's own T cells, which are then inheritedT cells can be engineered to express, for example, a Chimeric Antigen Receptor (CAR). CAR positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain the CAR scFv can be designed to target, for example, CD19, a transmembrane protein expressed by cells of the B cell lineage (including all normal B cell and B cell malignancies, including but not limited to diffuse large B cell lymphoma (D L BC L) non-finger type, primary mediastinal large B cell lymphoma, higher B cell lymphoma, and follicular lymphoma, D L BC 5, NH L, C LL, and non-T cell a LL.example T cell therapies and CARs are described in U.S. patent publication No. 2013/0287748, 2014/0050708, and these are incorporated by reference in their entirety.

As used herein, "patient" includes any human having cancer (e.g., lymphoma or leukemia). Herein, the terms "subject" and "patient" are used interchangeably.

As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, the in vitro cells may comprise T cells.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. There is no limit to the maximum number of amino acids that a protein or peptide contains at least two amino acids and may comprise the sequence of the protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which are of many types). "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signaling event. A "stimulatory molecule" is a molecule on a T cell, such as the T Cell Receptor (TCR)/CD3 complex that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on the T cell, thereby mediating a primary response (including but not limited to activation, initiation of an immune response, proliferation, etc.) of the T cell. Stimulatory ligands include, but are not limited to, anti-CD 3 antibodies, peptide-loaded mhc class i molecules, hyperactivating anti-CD 2 antibodies, and hyperactivating anti-CD 28 antibodies.

As used herein, "co-stimulatory signal" refers to a signal that, when combined with a primary signal (e.g., TCR/CD3 linkage), results in a T cell response (e.g., without limitation, up-or down-regulation of proliferation and/or key molecules).

As used herein, "co-stimulatory ligand" includes molecules on antigen presenting cells that specifically bind to cognate co-stimulatory molecules on T cells. binding of the co-stimulatory ligand provides a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc. the co-stimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule (e.g., provided by the binding of the T Cell Receptor (TCR)/CD complex to the peptide-loaded Major Histocompatibility Complex (MHC) molecule.) the co-stimulatory ligand may include, but is not limited to, 3/TR, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptor, B-1 (CD), B-2 (CD), CD ligand, CD, herpes virus invasion mediator (HVEM), human leukocyte antigen G (H A-G), I T, immunoglobulin-like transcript (I T)3, inducible co-stimulatory ligand (OS-), intracellular adhesion molecule (ICAM), ligand that specifically binds to B-H, lymphotoxin receptor, beta receptor, MICI-related protein A chain, MHC class I-T3, inducible co-stimulatory ligand (ICOS-), CD-ligand (CD) ligand (CD-H), CD-H) and TNF-2 (TNF-H) receptor-TNF-2, or TNF-H) receptor family proteins present on natural cell specific, or TNF-2 (TNF-H) cell death, or TNF-2, or TNF-4, or TNF-4, or TNF-family of cells.

A "co-stimulatory molecule" is a homologous binding partner that specifically binds to a co-stimulatory ligand on a T cell, thereby mediating a co-stimulatory response (e.g., but not limited to proliferation) of the T cell. the co-stimulatory molecule includes, but is not limited to, 4-1BB/CD137, B7-H3, BAFFR, B L AME (S L AMF8), BT 80 8, CD45, CD100(SEMA4 8), CD103, CD134, CD137, CD154, CD8, CD160(BY 8), CD8, CD19 8, CD8 (alpha; beta; epsilon; gamma; zeta; CD8, CD 8-7, CD 8-7, CD8, CD-7, CD8, CD-7-T8, CD-T8, CD-T8.

Herein, the terms "reduce" and "reducing" are used interchangeably and mean any change less than the original. "reduction" and "decrease" are relative terms and require comparison between before and after measurement. "reduce" and "reduce" include complete consumption.

By "treating" or "treatment" of a subject is meant any type of intervention or procedure performed on the subject, or administration of an active agent to the subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, severity, or recurrence of a symptom, complication, or condition, or biochemical indicator associated with the disease. In some embodiments, "treating" or "treatment" includes partial remission. In another embodiment, "treating" or "treatment" includes complete remission.

Various aspects of the disclosure are described in further detail in the following subsections.

Chimeric antigen receptors

Chimeric antigen receptors (CARs or CAR-T) are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells, including T cells, according to techniques known in the art. Using CARs, a single receptor can be programmed to recognize a particular antigen and, when bound to that antigen, activate immune cells to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells.

Engineered T cells and uses

CD 19-directed genetically modified autologous T cell immunotherapy indicated for the treatment of patients with relapsed or refractory large B cell lymphoma after two-or more-line systemic therapy, including diffuse large B cell lymphoma (D L BC L) non-finger type, primary mediastinal large B cell lymphoma, higher B cell lymphoma, and D L BC L caused by follicular lymphoma^TM，)。

The cells of the present disclosure may be useful for treatingT cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors^TMIsolation and/or apheresis) is obtained from a unit of blood collected from a subject. In some embodiments, cells collected by apheresis are washed to remove plasma fractions and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As should be appreciated, the washing step can be used, for example, by using a semi-automatic non-countercurrent centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMateTM, etc.). In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other salt solutions with or without buffers. In some embodiments, undesired components of the apheresis sample are removed. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.

In some embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes (e.g., by using centrifugation via PERCO LL TM gradients.) in some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA +, and CD45RO + T cells, may be further isolated by positive or negative selection techniques known in the art.

In some embodiments, PBMCs are used directly for genetic modification of immune cells (e.g., CARs) using methods as described herein. In some embodiments, after PBMC isolation, T lymphocytes may be further isolated and both cytotoxic and helper T lymphocytes sorted into naive, memory and effector T cell subsets, either before or after genetic modification and/or expansion.

In some embodiments, CD8+ cells are further sorted into naive, central memory and effector cells by identifying cell surface antigens associated with each of naive, central and effector cell types of CD8+ cells in some embodiments, expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD 62L and CD127 and is granzyme B negative.

In some embodiments, immune cells, such as T cells, are genetically modified after isolation using known methods, or immune cells are activated and expanded (or, in the case of progenitor cells, differentiated) in vitro before being genetically modified. In another embodiment, an immune cell, e.g., a T cell, is genetically modified (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) with a chimeric antigen receptor described herein and then activated and/or amplified in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. patent nos. 6,905,874; 6,867,041, respectively; and 6,797,514; and PCT publication No. WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety. Generally, such methodsComprising contacting PBMC or isolated T cells with a stimulating and co-stimulating agent (e.g., anti-CD 3 and anti-CD 28 antibodies, typically adhered to beads or other surfaces) in a medium with an appropriate cytokine (e.g., I L-2.) the anti-CD 3 and anti-CD 28 antibodies adhered to the same beads act as "surrogate" Antigen Presenting Cells (APCs)A system, a CD3/CD28 activator/stimulator system for physiologically activating human T cells. In other embodiments, T cells are activated and stimulated for proliferation with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. patent nos. 6,040,177 and 5,827,642 and PCT publication No. WO 2012/129514 (the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient having a cancer or tumor. In some embodiments, the donor subject is a human patient that does not have a cancer or tumor.

In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and/or adjuvant. In some embodiments, the composition comprises an excipient.

In some embodiments, the composition is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as oral administration. It is within the ability of those skilled in the art to prepare such pharmaceutically acceptable compositions. In some embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically in the pH range of about 5 to about 8. In some embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the composition described herein with or without an additional therapeutic agent in a pharmaceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the compositions described herein (with or without at least one additional therapeutic agent) are formulated as sterile isotonic solutions that are suitably preserved. In some embodiments, the preparation involves a formulation of the desired molecule with a polymeric compound (e.g., polylactic acid or polyglycolic acid), beads, or liposomes that provides controlled or sustained release of the product, which is then delivered via depot injection (depot injection). In some embodiments, the desired molecule is introduced using an implantable drug delivery device.

In some embodiments, the method of treating cancer in a subject in need thereof comprises T cell therapy. In some embodiments, the T cell therapy disclosed herein is an engineered autologous cell therapy (eACT)^TM). According to this embodiment, the method may include collecting blood cells from the patient. The isolated blood cells (e.g., T cells) can then be engineered to express the CARs or TCRs disclosed herein. In particular embodiments, the CAR T cells or TCR T cells are administered to a patient. In some embodiments, the CART cells or TCR T cells treat a tumor or cancer in the patient. In some embodiments, the CAR T cells or TCR T cells reduce the size of the tumor or cancer.

In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject other than a patient. The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10⁴At least about 10 cells⁵At least about 10 cells⁶At least about 10 cells⁷At least about 10 cells⁸At least about 10 cells⁹Or at least about 10¹⁰And (4) respectively. In another embodiment, the therapeutically effective amount of T cells is about 10⁴One cell, about 10⁵One cell, about 10⁶One cell, about 10⁷One cell or about 10⁸In some embodiments, the therapeutically effective amount of CAR T cells is about 2 × 10⁶Individual cell/kg, about 3 × 10⁶Individual cell/kg, about 4 × 10⁶Individual cell/kg, about 5 × 10⁶Individual cell/kg, about 6 × 10⁶Individual cell/kg, about 7 × 10⁶Individual cell/kg, about 8 × 10⁶Individual cell/kg, about 9 × 10⁶Individual cell/kg, about 1 × 10⁷Individual cell/kg, about 2 × 10⁷Individual cell/kg, about 3 × 10⁷Individual cell/kg, about 4 × 10⁷Individual cell/kg, about 5 × 10⁷Individual cell/kg, about 6 × 10⁷Individual cell/kg, about 7 × 10⁷Individual cell/kg, about 8 × 10⁷Individual cell/kg, or about 9 × 10⁷Cells/kg. in some embodiments, a therapeutically effective amount of CAR-positive living T cells is about 1 × 10 per kg body weight⁶And about 2 × 10⁶Between CAR-positive viable T cells up to about 1 × 10⁸Maximum dose of individual CAR-positive live T cells.

In some embodiments, the therapeutically effective amount of CAR-positive living T cells is about 0.4 × 10⁸And about 2 × 10⁸CAR-positive live T cells between individuals. In some embodiments, the therapeutically effective amount of CAR-positive living T cells is about 0.4x10⁸0.5x10⁸0.6x10⁸0.7x10⁸0.8x10⁸0.9x10⁸1, about 1.0x10⁸1, about 1.1x10⁸1, about 1.2x10⁸1, about 1.3x10⁸1, about 1.4x10⁸1, about 1.5x10⁸1, about 1.6x10⁸1, about 1.7x10⁸1, about 1.8x10⁸1, about 1.9x10⁸Or about 2.0x10⁸Individual CAR-positive live T cells.

Method of treatment

The methods disclosed herein can be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.

Cancers that may be treated include non-vascularized, substantially non-vascularized or vascularized tumors, cancers may also include solid or non-solid tumors in some embodiments the cancer is a hematological cancer in some embodiments the cancer is a cancer of leukocytes in other embodiments the cancer is a cancer of plasma cells in some embodiments the cancer is a leukemia, lymphoma or myeloma in some embodiments the cancer is an acute lymphoblastic leukemia (A LL) including non-T cell A LL, acute lymphoid leukemia (A LL LL) and hemophagocytic lymphohistiocytic cell cytostasis (H LL H), B cell prolymphocytic leukemia, B cell acute lymphoid leukemia ("BA LL LL) 634, blastic plasmacytoid cell neoplasms, Burkitt lymphoma, chronic lymphocytic leukemia (C LL), chronic myelogenous leukemia (CM L), chronic lymphocytic leukemia (CM 8655), chronic myeloblastic lymphoblastic leukemia (CMM), lymphoblastic myeloblastic lymphoblastic myelomatosis (" CMM "), lymphoblastic leukemia (CMM), lymphoblastic leukemia (CMM (CMOSm (CMM), lymphoblastic lymphoblasti.

In some embodiments, the cancer is myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute myeloid leukemia.

In some embodiments, the method further comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent of choice is a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens and related beneficial biomarkers are described in U.S. provisional patent applications 62/262,143 and 62/167,750, which are hereby incorporated by reference in their entirety. These describe, for example, methods of conditioning a patient in need of a T cell therapy comprising administering to the patient a specified beneficial dose of cyclophosphamide (200 mg/m)²Day-2000 mg/m²Day) and the indicated dose of fludarabine (20 mg/m)²Day-900 mg/m²Day). One such dosage regimen involves treating the patient, including administering to the patient about 500mg/m per day²Cyclophosphamide per day and about 60mg/m²Fludarabine/day for 3 days, and then administering a therapeutically effective amount of the engineered T cells to the patient.

In some embodiments, the antigen binding molecule, transduced (or otherwise engineered) cells (e.g., CAR), and chemotherapeutic agent are each administered in an amount effective to treat the disease or condition in the subject.

In some embodiments, a composition comprising a CAR-expressing immune effector cell disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and Cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa) and uretepa (uredepa); ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetylomelamine; nitrogen mustards (nitrogen mustards), such as chlorambucil (chlorambucil), and naphazel (naphazel)Chlornaphazazine, cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechloroethamine), mechlorethamine hydrochloride (mechlorethamine), melphalan (melphalalan), neomustard (novembichin), benzene mustard (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracilmustine), nitrosoureas (nitroureas), carmustine (carmustine), chlorzotocin (chlorzotocin), carmustine (streptomycin), streptomycin (gentamycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin, streptomycin (streptomycin, streptomycin), streptomycin), or streptomycin), streptomycin (streptomycin), streptomycin), streptomycin (streptomycin ), or streptomycin, streptomycin), or streptomycin, streptomycin), streptomycin (streptomycin), or streptomycin (streptomycin ), streptomycin (streptomycin ), or streptomycin (streptomycin), streptomycin (streptomycin), streptomycin (streptomycin), or streptomycin (Glycosides (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, such as folinic acid (folinic acid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); amsacrine (amsacrine); tabularil (bestrabucil); bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etiolate (ellitiniumacetate); etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); podophyllinic acid (podophyllic acid); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);razoxan (razoxane), Sisofhenan (sizoiran), germanium spirans (spirogemanium), Alternaria tenuissima (tenuazonic acid), triaminequinone (triaziquone), 2 '-trichlorotriethylamine, urethane (urethan), vindesine (vindesine), dacarbazine (dacarbazine), mannitol mustard (manomicine), mitobronitol (mitobronitol), dibromodulcitol (olamitolactol), pipobromane (pipobroman), Garcinia (gacytosine), cytarabine (Ara-C'), cyclophosphamide, thiotepa (thiotepa), taxoids (taxoids), such as littrowel (paclitaxel) (TACLATACLA L, Stobbe-L, and Squalex (Myxol)Rhone-Poulencrorer, chlorambucil (chlorembeucil), gemcitabine, 6-thioguanine (thioguanine), mercaptopurine (mercaptoprine), methotrexate (methotrexate), platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin), vinblastine (vinblastine), platinum, etoposide (VP-16), ifosfamide (ifosfamide), mitomycin C, mitoxantrone (mitoxantrone), vincristine, vinorelbine, navelbine (navelbine), neuroblastoma (novaluron), teniposide (teniposide), daunomycin (daunomycin), aminopterin (aminopterin), ibandronate (ibandronate), CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornidine (DMeomycin), trexatilin (amitriptorelin), a (ketoprofen), a salt of an (ketoprofen), a derivative of an antibiotic, a prodrug, a (ketoprofen), a prodrug, a prodrug, a prodrug, a prodrugDoxorubicin (hydroxydoxorubicin), vincristineAnd prednisone.

In some embodiments, the chemotherapeutic agent is administered simultaneously with or within a week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

A variety of additional therapeutic agents may be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumabPembrolizumab (pembrolizumab)Pembrolizumab, abaclizumab (pidilizumab) (CureTech), and alezumab (Atezolizumab) (Roche).

Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein include, but are not limited to, ibrutinib (ibrutinib)Oxamumumab (ofatumumab)Rituximab (rituximab)Bevacizumab (bevacizumab)Trastuzumab (trastuzumab)trastuzumabemtansineImatinib (imatinib)Cetuximab (cetuximab)Panitumumab (panitumumab)Cartuzumab (Catitumoxomab), ibritumomab (ibritumomab), Aframumab, tositumomab (tositumomab), benitumomab (brentuximab), alemtuzumab (alemtuzumab), gemtuzumab (gemtuzumab), erlotinib (erlotinib), gefitinib (gefitinib), vandetanib (vandetanib), afatinib (lapatinib), neratinib (neratinib), axitinib (axitinib), masitinib (masitinib), pazopaniib (pazotinib), sunitinib (sunitinib), sorafenib (soratinib), toceranib (netentiib), sunitinib (sunitinib), sunitinib (soratinib), toseranib (sunitinib), sunitinib (sunitinib), sunitinib (sorafenib), sunitinib (sunitinib), sunitinib (, Cabozantinib (cabozantinib), imatinib (imatinib), dasatinib (dasatinib), nilotinib (nilotinib), panatinib (ponatinib), radotinib (raditinib), bosutinib (bosutinib), lestatinib (lestauauritinib), ruxolitinib (ruxolitinib), palitinib (pacitinib), cobitinib (cobimetinib), semtinib (selumetinib), trametinib (trametinib), bismertinib (binetinib), aratinib (aletinib), ceritinib (ceritinib), crizotinib (critinib), everitinib (critinib), aflibericicepacitin (afliberitinib), oxytetratinib (adotinib), interleukin (mTOR, e inhibitors such as ovirolimus (des), and Everolimus (elsinos), such as Everolimus (elsinomycin), and temolimus (trestinib), such as Everolimus (elsinomycin (e), and (e-g)ib) and vemuraglib (vismodegib), CDK inhibitors such as CDK inhibitors (palbociclib).

In some embodiments, the composition comprising the CAR immune cell is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, triamcinolone, nonsteroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include acetaminophen (acetaminophen), oxycodone (oxycodone), tramadol, or propoxyphene hydrochloride (tramadol of propofol hydrochloride). Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists, (e.g., etanercept)Adalimumab (adalimumab)And infliximab (infliximab)Chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecule. Exemplary DMARDs include azathioprine (azathioprine), cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, and beta-cyclodextrin,Hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline (minocycline).

In some embodiments, the compositions described herein are administered in combination with cytokines, examples of which are lymphokines, monokines, and traditional polypeptide hormones, including growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and luteinizing hormone (L H), liver growth factor (HGF), Fibroblast Growth Factor (FGF), prolactin, placental lactogen, mullerian tube inhibitory substance (mullerian-inhibiting substance), mouse gonadotropin-related peptides, inhibin, activin, vascular endothelial growth factor, integrin, Thrombopoietin (TPO), Nerve Growth Factor (NGF) such as NGF-beta, platelet growth factor (TGF), transforming growth factor (alpha) such as TGF-and erythropoietin-beta, insulin-like growth factor-I and-II,) Osteoinductive factors, interferons such as interferon- α, β and- γ, Colony Stimulating Factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (I β 0) such as I β 1-1, I β 2-1alpha, I β 3-2, I β 4-3, I β 5-4, I β 6-5, I β 7-6, I β 8-7, I L-8, I L-9, I L-10, I L-11, I L-12, I L-15, tumor necrosis factors such as TNF-alpha or beta-TNF a, and other IF polypeptide factors including L and kit ligand (K L). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell cultures, and biological equivalents of natural sequence cytokines.

Administration of CD 19-directed genetically modified autologous T cell immunotherapy

Indications and methods of use

In some embodiments, CD 19-directed genetically modified autologous T cell immunotherapy is indicated for treating adult patients with relapsed or refractory large B cell lymphoma following two-line or more-line system therapy, including diffuse large B cell lymphoma (D L BC L) non-finger type, primary mediastinal large B cell lymphoma, higher-grade B cell lymphoma, and D L BC L from follicular lymphoma.

Dosage and administration

In some embodiments, an infusion bag of CD 19-directed genetically modified autologous T cell immunotherapy comprises a suspension of Chimeric Antigen Receptor (CAR) positive T cells in about 68m L a target dose may be about 1 × 10 per kg body weight⁶And about 2 × 10⁶Between CAR-positive live T cells, up to 2 × 10⁸Individual CAR-positive live T cells. In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy is Axi-cel^TM(axicabtagene ciloleucel)。

CD 19-directed genetically modified autologous T cell immunotherapy is used autologous. The identity of the patient must match the patient identifier on the CD 19-directed genetically modified autologous T cell immunotherapy kit and infusion bag. If the information on the patient-specific label does not match the expected patient, CD 19-directed genetically modified autologous T cell immunotherapy cannot be administered.

In some embodiments, the availability of CD 19-directed genetically modified autologous T cell immunotherapy must be confirmed prior to initiating a lymphocyte depletion (lymphopleating) protocol.

In some embodiments, the patient is pre-treated with lymphodepleting chemotherapy prior to CD 19-directed infusion of genetically modified autologous T cell immunotherapy. In some embodiments, cyclophosphamide is administered intravenously at 500mg/m on the fifth, fourth, and third days prior to infusion of the CD 19-directed genetically modified autologous T cell immunotherapy²And intravenous administration of fludarabine 30mg/m²The lymphocyte depleting chemotherapy regimen of (a).

In some embodiments, the patient is pre-drug treated with acetaminophen prior to the CD 19-directed infusion of genetically modified autologous T cell immunotherapy by oral administration at a dose of between about 500-1000mg, about 600-1000mg, about 700-1000mg, about 800-1000mg, about 900-1000mg, about 500-900mg, about 500-800mg, about 500-700mg, about 500-600mg, about 600-900mg, about 600-800mg, about 600-700mg, about 700-900mg, about 700-800mg or about 800-900 mg. In some embodiments, the patient is pre-drug treated with acetaminophen by oral administration at a dose of about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, about 850mg, about 875mg, about 900mg, about 925mg, about 950mg, about 975mg, or about 1000mg prior to infusion of the CD 19-directed genetically modified autologous T cell immunotherapy.

In some embodiments, the patient is pre-drug treated by oral administration of acetaminophen 650mg and intravenous administration of diphenhydramine 12.5mg prior to infusion of the CD 19-directed genetically modified autologous T cell immunotherapy or oral administration about 1 hour prior to CD 19-directed genetically modified autologous T cell immunotherapy.

In some embodiments, prophylactic use of systemic steroids is avoided as it may interfere with the activity of CD 19-directed genetically modified autologous T cell immunotherapy.

Preparation of CD 19-directed genetically modified autologous T cell immunotherapy for infusion

The timing of thawing and infusion of CD 19-directed genetically modified autologous T cell immunotherapy is coordinated. In some embodiments, the infusion time is predetermined and the start time for thawing of the CD 19-directed genetically modified autologous T cell immunotherapy is adjusted so that it is available for infusion when the patient is ready.

In some embodiments, patient identity is confirmed prior to thawing of the CD 19-directed genetically modified autologous T cell immunotherapy. Prior to preparation of the CD 19-directed genetically modified autologous T cell immunotherapy, the identity of the patient is matched to the patient identifier on the CD 19-directed genetically modified autologous T cell immunotherapy cassette. In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy product bag is not removed from the cassette if the information on the patient-specific label does not match the expected patient.

In some embodiments, once patient identity is confirmed, the CD 19-directed genetically modified autologous T cell immunotherapy product bag is removed from the cassette and patient information on the cassette label is confirmed to match the bag label.

In some embodiments, the method includes checking the integrity of the product bag container for any gaps, such as cracks or fissures, prior to thawing. In some embodiments, the infusion bag is placed in a second sterile bag according to local guidelines.

In some embodiments, the method comprises thawing the CD 19-directed genetically modified autologous T cell immunotherapy at about 37 ℃ using a water bath or dry-melt process until there is no visible ice in the infusion bag. In some embodiments, the method comprises mixing or agitating the contents of the bag to disperse the clumped cellular material. In some embodiments, the contents of the bag are gently mixed or agitated. In some embodiments, the method comprises checking the bag for the presence of visible residual cell clumps and continuing mixing or stirring. The small pieces of cell material should be dispersed with gentle manual mixing. In some embodiments, the methods do not include washing, rapid centrifugation, and/or resuspending CD 19-directed genetically modified autologous T cell immunotherapy in fresh media prior to infusion.

In some embodiments, once thawed, CD 19-directed genetically modified autologous T cell immunotherapy can be stored at room temperature (20 ℃ to 25 ℃) for up to 3 hours.

Administration of

In some embodiments, the presently disclosed methods of administering CD 19-directed genetically modified autologous T cell immunotherapy include one or more of the following as a step or consideration:

ensure availability of tositulizumab and emergency equipment before infusion and during recovery period.

Do not use leukocyte depletion filters.

Central venous access was recommended for infusion of CD 19-directed genetically modified autologous T cell immunotherapy.

Confirm that the patient's identity matches the patient identifier on the CD 19-directed genetically modified autologous T cell immunotherapy product bag.

Fill the tubing with saline prior to infusion.

Infusion of the entire contents of the CD 19-directed genetically modified autologous T cell immunotherapy bag by gravity or peristaltic pump over 30 minutes. After thawing, the CD 19-directed genetically modified autologous T cell immunotherapy was stable at room temperature for up to 3 hours.

Gently agitate the product bag to prevent cell clumping during CD 19-directed genetically modified autologous T cell immunotherapy infusion.

After infusion of the entire contents of the product bag, the tube is flushed with saline at the same infusion rate to ensure delivery of all the product.

CD 19-directed genetically modified autologous T cell immunotherapy comprising human blood cells genetically modified with a replication-incompetent retroviral vector. Treatment and disposition are performed following general precautions and local biosafety guidelines to avoid potential transmission of infectious diseases.

Monitoring

In some embodiments, administration of the CD 19-directed genetically modified autologous T cell immunotherapy is performed at a certified medical facility.

In some embodiments, the methods disclosed herein comprise monitoring the patient CRS and signs and symptoms of neurotoxicity at a certified medical facility for 7 days at least daily after infusion. In some embodiments, the methods disclosed herein comprise monitoring patients CRS and signs and symptoms of neurotoxicity at certified medical institutions for at least 10 days following infusion.

In some embodiments, the patient is instructed to remain in proximity to the certified medical facility for at least 4 weeks after the infusion.

Management of severe adverse reactions

In some embodiments, the method comprises administration of an adverse reaction. In some embodiments, the adverse reaction is selected from the group consisting of: cytokine Release Syndrome (CRS), neurotoxicity, hypersensitivity, severe infection, cytopenia and hypogammaglobulinemia.

In some embodiments, the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphoblastic cell proliferation/macrophage activation syndrome (H L H/MAS), seizure, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia and anemia.

Cytokine release syndrome

In some embodiments, the method comprises identifying the CRS based on clinical presentation. In some embodiments, the methods comprise assessing and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, management is performed according to the recommendations in Table 1. Patients experiencing CRS levels ≧ 2 (e.g., hypotension, hypoxia that does not respond to fluid or requires supplemental oxygenation) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, echocardiography is considered for evaluating cardiac function for patients experiencing severe CRS. For severe or life-threatening CRS, intensive care support therapy may be considered. In some embodiments, a biological analog or equivalent of tositulizumab may be used in place of tositulizumab in the methods disclosed herein.

CRS grading and management guidelines

(a)Lee DW et al.,(2014).Current concepts in the diagnosis andmanagement of cytokine release syndrome.Blood.2014 Jul 10；124(2):188–195。

(b) For the management of neurotoxicity see table 2.

(c) For detailed information see(toslizumab) prescription information, https:// www.gene.com/download/pdf/actumra _ descriptive. pdf (last visit 2017, 10 months and 18 days). The time of first approval in the united states is shown as 2010.

Neurotoxicity

In some embodiments, the methods comprise monitoring patients for signs and symptoms of neurotoxicity (table 2). In some embodiments, the method comprises excluding other causes of the neurological condition. Patients experiencing grade 2 or greater neurotoxicity should be monitored by continuous cardiac telemetry and pulse oximetry. Intensive care support therapy is provided for severe or life-threatening neurotoxicity. For any grade 2 neurotoxicity, non-sedative antiepileptic drugs (e.g., levetiracetam) are contemplated for use in preventing seizures.

TABLE 2 neurotoxicity grading and management guidelines

Dosage forms and advantages

In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy may be obtained as a cell suspension for infusion.

In some embodiments, a single dose of CD 19-directed genetically modified autologous T cell immunotherapy comprises about 1 × 10 per kg body weight in about 68m L suspension in an infusion bag⁶And about 2 × 10⁶Target doses of CAR-positive live T cells between individuals (or for 100kg and above patients,maximum 2 × 10⁸Individual CAR-positive live T cells). In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy is axicabtagene ciloleucel

In some embodiments, a single dose of CD 19-directed genetically modified autologous T cell immunotherapy is present in a container such a container may be sterile in some embodiments, the container is an infusion bag in some embodiments, the infusion bag has a volume of about 100m L, 150m L, 200m L0, 250m L, 300m L, 500m L, 750m L, 1,000m L, 1,500m L, 2,000m L, or 3,000m L.

Risk assessment and mitigation strategies (REMS)

Due to the risks of CRS and neurotoxicity, in some embodiments, CD 19-directed genetically modified autologous T cell immunotherapy can be obtained by limited procedures under risk assessment and mitigation strategies (REMS). Typical components of REMS are:

medical institutions that distribute and administer CD 19-directed genetically modified autologous T cell immunotherapy must register and comply with REMS requirements.

The certified medical institution must be immediately available to use tositumumab on site and ensure that a minimum of two doses of tositumumab per patient are available for infusion within 2 hours after infusion of CD 19-directed genetically modified autologous T cell immunotherapy if treatment with CRS is required.

Certified medical institutions must ensure that healthcare providers prescribing, distributing, or administering CD 19-directed genetically modified autologous T cell immunotherapy receive training on CRS and neurotoxicity management.

Cytokine Release Syndrome (CRS)

In some embodiments, the medical facility ensures that two doses of tositumumab are available prior to infusion of the CD 19-directed genetically modified autologous T cell immunotherapy. In some embodiments, the medical facility ensures that four doses of tositumumab are available prior to infusion of the CD 19-directed genetically modified autologous T cell immunotherapy. In some embodiments, the method comprises monitoring the patient CRS at a certified medical facility for signs and symptoms for 7 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient CRS at a certified medical facility for signs and symptoms for 7-10 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient CRS at a certified medical institution for signs and symptoms for 8 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient CRS at a certified medical institution for signs and symptoms for 9 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient CRS at a certified medical facility for signs and symptoms for 10 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient for signs or symptoms of CRS for 4 weeks following infusion. In some embodiments, the method comprises instructing the patient to seek medical attention immediately at any time if signs or symptoms of CRS occur. In some embodiments, the method comprises starting treatment with supportive care, tositumumab, or tositumumab and a corticosteroid, as indicated by the first body characterization of CRS.

Neurotoxicity

In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity at a certified medical facility for 7 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient CRS at a certified medical facility for signs and symptoms for 7-10 days at least daily after infusion. In some embodiments, the method comprises monitoring the patient CRS at a certified medical facility for signs and symptoms for 10 days at least daily after infusion. In some embodiments, the methods comprise monitoring the patient for signs or symptoms of neurotoxicity 4 weeks after infusion and treatment in time.

Hypersensitivity reactions

Allergic reactions may occur with infusion of CD 19-directed genetically modified autologous T cell immunotherapy. In some embodiments, severe hypersensitivity, including anaphylaxis, may be due to dimethyl sulfoxide (DMSO) or residual gentamicin in CD 19-directed genetically modified autologous T cell immunotherapy.

Virus reactivation

In some embodiments, Hepatitis B Virus (HBV) reactivation (in some cases leading to fulminant hepatitis, liver failure, and death) may occur in patients treated with B cell directed drugs. In some embodiments, the methods comprise screening for HBV, HCV, and HIV according to clinical guidelines prior to collecting cells for manufacture.

Long term cytopenia

In some embodiments, the patient may exhibit a reduction in bleeding cells for several weeks following lymphodepleting chemotherapy and CD 19-directed infusion of genetically modified autologous T cell immunotherapy. In some embodiments, the method comprises monitoring blood cell counts after CD 19-directed infusion of genetically modified autologous T cell immunotherapy.

Globulinemia with low gamma globulin level

In some embodiments, B cell aplasia and hypogammaglobulinemia may occur in a patient treated with autologous T cell immunotherapy with CD 19-directed genetic modification. In some embodiments, the methods comprise monitoring immunoglobulin levels following treatment with CD 19-directed genetically modified autologous T cell immunotherapy and management with infection prevention measures, antibiotic prevention, and immunoglobulin replacement.

In some embodiments, vaccination with a live virus vaccine is not advised at least 6 weeks prior to initiation of lymphodepleting chemotherapy, during treatment with CD 19-directed genetically modified autologous T cell immunotherapy, and until immune recovery following treatment with CD 19-directed genetically modified autologous T cell immunotherapy.

Secondary malignant tumor

In some embodiments, a patient treated with CD 19-directed genetically modified autologous T cell immunotherapy is likely to develop a secondary malignancy. In some embodiments, the method comprises monitoring for life secondary malignancy.

Tumor lysis syndrome: (TLS)

To minimize the risk of T L S, in some embodiments, the method comprises assessing the patient for elevated uric acid or high tumor burden and administering allopurinol or alternative prophylaxis prior to axicam ciloleucel infusion.Influence on ability to drive and use machine

Due to the potential for neurological events, including mental state changes or seizures, patients receiving CD 19-directed genetically modified autologous T cell immunotherapy had a risk of conscious or coordinated changes or reductions within 8 weeks after infusion of CD 19-directed genetically modified autologous T cell immunotherapy. In some embodiments, the method includes advising the patient to avoid driving and engaging in dangerous occupations or activities during this initial period, such as operating heavy or potentially dangerous machinery.

Storage and handling

In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy supplied in an infusion bag contains a frozen suspension of genetically modified autologous T cells of about 68m L in 5% DMSO and 2.5% albumin (human) in some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy supplied in an infusion bag contains a frozen suspension of genetically modified autologous T cells of about 68m L in 5% DMSO and 2.5% albumin (human) (NDC71287-119-01) in some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy comprises Cryostor cs10 in some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy comprises greater than 300mg sodium per infusion, in some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy supplied in an infusion bag contains greater than 300mg sodium in some embodiments, the CD 19-directed autologous T cell immunotherapy supplied in an infusion bag contains greater than 5% DMSO and 2.5% modified (DMSO) in less than L, more than 3660, more than 365-75% DMSO, more than L in 365% DMSO, more than 365-100% DMSO, more than L, more than 365-3660, more than L in 365% DMSO, more than 365-5% DMSO, more than L, more than 365-75, more than 365-5-75, more than L, more than 365-3, more than 0, more than 365-5-75, more than 1, more than L, more than 0, more than 365, more than 3, more than 1, more than 3, more than 1, more than 3, more than 1, more than 3.

In some embodiments, a CD 19-directed, genetically modified autologous T cell immunotherapy infusion bag containing a cell dispersion of about 68m L was supplied in an ethylene-vinyl acetate cryopreservation bag with a sealed addition tube and two available lancet ports.

In some embodiments, the CD 19-directed infusion bag of genetically modified autologous T cell immunotherapy is packaged individually in a metal box. In some embodiments, CD 19-directed infusion bags for genetically modified autologous T cell immunotherapy are packaged individually in a metal cassette (NDC 71287-119-02). In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy infusion bag is stored in the vapor phase of liquid nitrogen. In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy infusion bag is supplied in a liquid nitrogen dry tote.

In some embodiments, the method includes matching the identity of the patient with patient identifiers on the cassette and the infusion bag upon receipt. In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy is cryopreserved in the vapor phase of liquid nitrogen (less than or equal to minus 150 ℃). In some embodiments, the CD 19-directed genetically modified autologous T cell immunotherapy is thawed prior to use.

Examples

Example 1: clinical study of relapsed or refractory large B-cell lymphoma

A single-arm, open-label, multi-center test evaluated Axi-cel^TMEligible patients have a disease refractory to recent therapy or relapse within 1 year after autologous Hematopoietic Stem Cell Transplantation (HSCT) studies exclude patients with prior allogeneic HSCT, any history of central nervous system lymphoma, ECOG activity status of 2 or higher, absolute lymphocyte count of less than 100/μ L, creatinine clearance of less than 60m L/min, hepatic transaminase exceeding 2.5 times the upper normal limit, cardiac ejection fraction of less than 50%, or active severe infection.

After lymphodepleting chemotherapy, 2 × 10⁶Target dose of individual CAR-positive viable T cells/kg (maximum permissible dose: 2 × 10)⁸Individual cells) was administered Axi-cel by a single intravenous infusion^TM. The lymphocyte eliminating method is performed by cyclophosphamide 500mg/m²Vein and fludarabine 30mg/m²The venous components, both of which are Axi-cel^TMThe fifth, fourth and third day before administration. Bridging chemotherapy between leukopheresis and lymphodepleting chemotherapy is not allowed. All patients received Axi-cel in hospitalization^TMInfused and hospitalized for a minimum of 7 days thereafter.

Of 111 patients who underwent leukapheresis, 101 received Axi-cel^TMOf the treated patients, the median age was 58 years (range: 23 to 76 years), 67% male, and 89% caucasian, most (76%) had D L BC L, 16% had transformed follicular lymphoma, and 8% had primary mediastinal large B cell lymphoma, the median of the previous therapies was 3 (range: 1 to 10), 77% had disease refractory to second or more lines of therapy, and 21% had relapsed within 1 year of autologous HSCT.

1 of 111 patients did not receive the product due to manufacturing failure. The other nine patients were untreated, mainly due to progressive disease or severe adverse reactions after leukapheresis. The median time from leukapheresis to product delivery was 17 days (range: 14 to 51 days) and the median time from leukapheresis to infusion was 24 days (range: 16 to 73 days)) Median dose of 2.0 × 10⁶One CAR-positive live T cell/kg (range: 1.1 to 2.2 × 10)⁶Individual cells/kg).

Efficacy was determined by the independent review board and was established based on Complete Remission (CR) rate and duration of response (DOR) (tables 3 and 4). The median reaction time was 0.9 months (range: 0.8 to 6.2 months). Patients who achieve CR had longer duration of response compared to patients with Partial Remission (PR) optimal response (table 4). Of the 52 patients who reached CR, 14 had initially stable disease (7 patients) or PR (7 patients), with a median time to improvement of 2.1 months (range: 1.6 to 5.3 months).

TABLE 3 reaction rates

CI, confidence interval.

^aAccording to international working group standards revised in 2007, as assessed by the independent review board.

TABLE 4 duration of the reaction

CR, complete remission; DOR, duration of reaction; NE, not estimable; PR, partial remission.

^aIn all the reactors. DOR is measured from the date of the first objective response to the date of progression or death due to relapse or toxicity.

^bKaplan-Meier estimation.

^cThe a + sign represents the truncated value.

Example 2: axi-cel^TMPharmacodynamics and pharmacokinetics after infusion

At Axi-cel^TMPharmacodynamic responses are assessed at 4 week intervals after infusion by measuring transient elevations in blood of cytokines, chemokines and other molecules, to cytokines and chemokines such as I L-6, I L-8, I L-10, I L-15, TNF- α, IFN-. gamma.andThe levels of sI L2R α were analyzed.A peak increase was observed within the first 14 days post infusion and typically returned to baseline within 28 days due to Axi-cel^TMAt the target effect of (a), a period of B cell hypoplasia is expected.

Infusion Axi-cel^TMLater, anti-CD 19CAR T cells showed an initial rapid expansion followed by a 3 month drop to near baseline levels. The peak level of anti-CD 19CAR T cells occurred at Axi-cel^TMWithin the first 7-14 days after infusion. Age (23-76 years) and gender Pair Axi-cel^TMAUC of (1)_(0-28d)And Cmax had no significant effect.

The median anti-CD 19CAR T cell Cmax level in responders (n-73) was 205% higher (43.6 cells/μ L versus 21.2 cells/μ L) compared to the corresponding level in non-responders (n-23). the median AUC of responsive patients (n-73) was 251% (557.1 days × cells/μ L versus 222.0 days × cells/μ L) of the corresponding level in non-responders (n-23).

Some patients require toslizumab and corticosteroids for the management of CRS and neurotoxicity. Such as respectively by AUC_(0-28d)And Cmax, patients treated with tositulizumab (n-44) had 262% and 232% higher anti-CD 19CAR T cells compared to patients not receiving tositulizumab (n-57). Similarly, patients receiving corticosteroid (n-26) had 217% and 155% higher AUC compared to patients not receiving corticosteroid (n-75)_(0-28d)And Cmax.

Example 3: management of adverse effects following CD 19-directed genetically modified autologous T cell immunotherapy

Because clinical trials are conducted under a wide variety of conditions, the adverse reaction rate observed in a clinical trial of one drug cannot be directly compared with that observed in a clinical trial of another drug, and may not reflect the adverse reaction rate observed in practice.

The safety data described in this section reflect clinical trialsPair Axi-cel in (study 1)^TMOf 108 patients with relapsed/refractory B-cell NH L received CAR-positive T-cells based on the recommended dose (based on body weight.) patients with CNS disorders (such as seizures or cerebral vascular ischemia) or a history of autoimmune disease requiring systemic immunosuppression were ineligible.median follow-up duration of 8.7 months. median age of the study population was 58 years (range: 23 to 76 years), 68% were male baseline ECOG activity status of 43% with ECOG 0 and 57% with ECOG 1.

The most common adverse reactions (incidence > 20%) include CRS, fever, hypotension, encephalopathy, tachycardia, fatigue, headache, loss of appetite, chills, diarrhea, febrile neutropenia, infection-unspecified pathogens, nausea, hypoxia, tremor, cough, vomiting, dizziness, constipation and arrhythmia. Serious adverse reactions occurred in 52% of patients. The most common serious adverse reactions (> 2%) include encephalopathy, fever, lung infection, febrile neutropenia, arrhythmia, heart failure, urinary tract infection, renal insufficiency, aphasia, cardiac arrest, clostridium difficile infection, delirium, hypotension and hypoxia.

The most common (≧ 10%) grade 3 or higher responses include febrile neutropenia, fever, CRS, encephalopathy, infection-unspecified pathogen, hypotension, hypoxia and pulmonary infection.

Forty-five percent (49/108) of patients were in Axi-cel^TMTositulizumab was received after infusion.

Table 5 summarizes the use of Axi-cel at least 10%^TMAdverse reactions occurred in treated patients, and table 6 describes grade 3 or 4 laboratory abnormalities that occurred in at least 10% of patients.

TABLE 5 use of Axi-cel in at least 10% of study 1^TMOverview of adverse reactions observed in treated patients

Using Axi-cel in less than 10%^TMOther clinically important adverse reactions that occur in treated patients include the following:

disorders of the blood and lymphatic system: coagulopathy (2%)

Cardiac disorders: heart failure (6%) and sudden cardiac arrest (4%)

Immune system disorders hemophagocytic lymphohistiocytosis/macrophage activation syndrome (H L H/MAS) (1%), hypersensitivity reactions (1%)

Infection and infestation disorders: fungal infection (5%)

Neurological disorders: ataxia (6%), seizures (4%), computational difficulty (2%) and myoclonus (2%)

Respiratory, thoracic and mediastinal disorders: pulmonary edema (9%)

Disorders of the skin and subcutaneous tissues: rash (9%)

Vascular disorders: capillary leak syndrome (3%)

Laboratory abnormalities:

TABLE 6 grade 3 or 4 laboratory abnormalities in study 1 after treatment with CTCAE-based Axi-cel in > 10% of patients (N ═ 108)

Cytokine release syndrome

In use of Axi-cel^TMCRS was developed following treatment, including a fatal or life-threatening response. In study 1, 94% (101/108) of accepted Axi-cel^TMHas developed CRS in patients (D) of (D), and developed levels of 3 or more in patients including 13% (14/108) (L ee rating system)¹) CRS. After receiving Axi-cel^TMOf the patients who died later, 4 had progress at the time of deathMedian onset time is 2 days (range: 1 to 12 days), and median CRS duration is 7 days (range: 2 to 58 days). major manifestations of CRS include fever (78%), hypotension (41%), tachycardia (28%), hypoxia (22%), and chills (20%). serious events that may be associated with CRS include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, and hemophagic lymphoblastic hyperplasia/macrophage activation syndrome (H L H/MAS).

Neurotoxicity

In use of Axi-cel^TMAfter treatment, life threatening or fatal neurotoxicity occurs. Neurotoxicity occurred in 87% of patients. Ninety-eight percent of all neurotoxicity occurs in Axi-cel^TMWithin the first 8 weeks of infusion, the median time to onset was 4 days (range: 1 to 43 days). Median neurotoxicity duration was 17 days. Grade 3 or higher neurotoxicity occurred in 31% of patients.

The most common neurological toxicities include encephalopathy (57%), headache (44%), tremor (31%), dizziness (21%), aphasia (18%), delirium (17%), insomnia (9%) and anxiety (9%). Prolonged encephalopathy lasting for up to 173 days was noted. Accompanied by Axi-cel^TMSevere events including leukoencephalopathy and seizures occurred. With Axi-cel^TMFatal and severe cases of cerebral edema occurred in the treated patients.

Severe infection with hepatitis B

At Axi-cel^TMSerious or life-threatening infections occur in patients after infusion. In study 1, infection occurred in 38% of patients (all grades). Grade 3 or higher infections occurred in 23% of patients. Infection with unspecified pathogens of grade 3 or higher occurs in 16% of patients, with bacterial infection in 9% and viral infection in 4%. Axi-cel should not be used^TMAdministered to a clinically significant patient with active systemic infection. At Axi-cel^TMPatients were monitored for signs and symptoms of infection before and after infusion and were treated appropriately. Prophylactic antimicrobial drugs were administered according to local guidelines.

At Axi-cel^TMAfter infusion, febrile neutropenia was observed in 36% of patients and possibly coincident with CRS. In the case of febrile neutropenia, infections were assessed and managed as indicated by medicine using broad spectrum antibiotics, fluids and other supportive care.

Immunogenicity

Axi-cel^TMAxi-cel has been evaluated using enzyme-linked immunosorbent assay (E L ISA)^TMTo detect binding antibodies against FMC63, FMC63 is the original antibody of anti-CD 19 CAR. Three patients tested positive for pre-dose anti-FMC 63 antibody at baseline and at 1, 3, or 6 months in study 1. There is no evidence of Axi-cel^TMInitial amplification and persistence kinetics of, or Axi-cel^TMThe safety or effectiveness of (a) has changed in these patients.

In study 1, (28%) Axi-cel occurred in patients^TMGrade 3 or higher cytopenias, including thrombocytopenia (18%), neutropenia (15%) and anemia (3%), which remain unresolved at day 30 post-infusion. Infusion Axi-cel^TMBlood counts were monitored afterwards.

In study 1, hypogammaglobulinemia occurred in 15% of patients.

All publications, patents, patent applications, and references, including prescription information, mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. To the extent that any definition or term provided in a reference, which is incorporated by reference, differs from the term and discussion provided herein, the term and definition shall govern.

Claims (30)

1. A method of treating D L BC L caused by relapsed or refractory diffuse large B-cell lymphoma (D L BC L) non-finger type, primary mediastinal large B-cell lymphoma, higher-grade B-cell lymphoma, or follicular lymphoma in a patient following two or more lines of systemic therapy, comprising:

at about 1 × 10 per kg body weight⁶And about 2 × 10⁶Between doses of CAR-positive live T cells up to about 1 × 10⁸(ii) the maximum dose of individual CAR positive viable T cells is administered by intravenous infusion to said patient in need thereof an axicabtagengecilileucel suspension,

wherein axicabtagene ciloleucel is a CD 19-directed genetically modified autologous T cell immunotherapy comprising said patient's own T cells harvested and genetically modified ex vivo by retroviral transduction to express a Chimeric Antigen Receptor (CAR) comprising an anti-CD 19 single-chain variable fragment (scFv) linked to a CD28 and CD3-zeta costimulatory domain.

2. The method of claim 1, wherein the intravenous infusion time is between 15 and 120 minutes.

3. The method of claim 1, wherein the intravenous infusion time is up to 30 minutes.

4. The method of any one of claims 1-3, wherein the infusion volume is between 50 and 100m L.

5. The method of any one of claims 1-4, wherein the infusion volume is about 68m L.

6. The method of any one of claims 1-5, wherein the immunotherapy is infused from an infusion bag.

7. The method of claim 6, wherein the infusion bag is agitated during the infusion.

8. The method of any one of claims 1-7, wherein the immunotherapy is administered within 3 hours after thawing.

9. The method of any one of claims 1 to 8, wherein the suspension further comprises albumin.

10. The method of claim 9, wherein albumin is present in an amount of about 2-3% (v/v).

11. The method of claim 10, wherein albumin is present in an amount of about 2.5% (v/v).

12. The method of any one of claims 9-11, wherein the albumin is human albumin.

13. The method of any one of claims 1-12, wherein the suspension further comprises DMSO.

14. A method of treating relapsed or refractory large B-cell lymphoma in a patient following two or more lines of systemic therapy comprising:

(a) administering to the patient in need thereof a CD 19-directed genetically modified autologous T cell immunotherapy; and

(b) the patient was monitored for signs and symptoms of adverse reactions after infusion.

15. The method of claim 14, wherein the relapsed or refractory large B-cell lymphoma is D L BC L caused by diffuse large B-cell lymphoma (D L BC L) non-finger type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma.

16. The method of claim 14, wherein the adverse reaction is selected from the group consisting of: cytokine Release Syndrome (CRS), neurotoxicity, hypersensitivity, severe infection, cytopenia and hypogammaglobulinemia.

17. The method of claim 14, wherein the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphoblastic cell proliferation/macrophage activation syndrome (H L H/MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia and anemia.

18. The method of claim 14, wherein the method further comprises administering an effective amount of toslizumab (tocilizumab) to treat symptoms of the adverse reaction.

19. The method of claim 18, further comprising administering a corticosteroid to treat a symptom of the adverse reaction.

20. The method of claim 14, wherein the adverse reaction is Cytokine Release Syndrome (CRS).

21. The method of claim 20, wherein the method comprises monitoring signs and symptoms of Cytokine Release Syndrome (CRS) for at least daily for about 7 days following infusion.

22. The method of claim 14, wherein the adverse reaction is neurotoxicity.

23. The method of claim 22, wherein said method further comprises administering a non-sedating antiepileptic drug for seizure prevention.

24. The method of claim 14, wherein the adverse reaction is cytopenia.

25. The method of claim 24, wherein the method further comprises administering at least one of erythropoietin, darbepotein α, a platelet transfusion, Colony Stimulating Factor (CSF), granulocyte colony stimulating factor, filgrastim (filgrastim), pegfilgrastim (pegfilgrastim), or granulocyte macrophage colony stimulating factor.

26. The method of claim 14, further comprising measuring cytokine and chemokine levels.

27. The method of claim 26, wherein the level of at least one of I L-6, I L-8, I L-10, I L-15, TNF- α, IFN- γ, and sI L2R α is measured.

28. A container comprising a suspension of CD 19-directed genetically modified autologous T cells, about 5% dimethyl sulfoxide (DMSO), and about 2.5% human albumin (v/v).

29. A method of treating relapsed or refractory large B cell lymphoma in a human following two or more lines of systemic therapy comprising administering to said human in need thereof CD 19-directed genetically modified autologous T cell immunotherapy comprising:

(a) administering to the patient a composition comprising a CD 19-directed Chimeric Antigen Receptor (CAR) positive live T cell;

(b) monitoring the patient for signs and symptoms of adverse reactions after administration; and

(c) administering tositulizumab IV at a dose of about 8mg/kg over 1 hour if Cytokine Release Syndrome (CRS) greater than grade 2 is observed in (b), repeating tositulizumab every 8 hours if necessary if not responsive to IV fluids or increased supplemental oxygen;

(d) if the CRS symptoms observed in (b) did not improve after 24 hours of (c), IV administering about 1mg/kg methylprednisolone twice daily or administering an equivalent dexamethasone dose and continuing corticosteroid use until the event is grade 1 or below, then gradually decreasing over 3 days;

(e) administering tositumumab IV at a dose of 8mg/kg over 1 hour if CRS grade 3 is observed in (b), repeating tositumumab every 8 hours as needed if not responsive to IV fluids or increased supplemental oxygen, and IV administering about 1mg/kg methylprednisolone twice daily or administering an equivalent dexamethasone dose and continuing corticosteroid use until the event is grade 1 or below, then gradually decreasing over 3 days; and

(f) administering tositumumab IV at a dose of about 8mg/kg over 1 hour if CRS grade 4 is observed in (b), repeating tositumumab every 8 hours as needed if not responsive to IV fluids or increased supplemental oxygen, and IV administering about 1,000mg methylprednisolone daily for 3 days.

30. A method of treating relapsed or refractory large B cell lymphoma in a patient following two or more lines of systemic therapy comprising administering to the patient in need thereof CD 19-directed genetically modified autologous T cell immunotherapy comprising:

(a) administering to the patient a composition comprising a CD 19-directed Chimeric Antigen Receptor (CAR) positive live T cell;

(b) monitoring said patient for signs and symptoms of adverse reactions after administration; and

(c) managing Cytokine Release Syndrome (CRS) and/or neurotoxicity according to table 1 and/or table 2 if Cytokine Release Syndrome (CRS) and/or neurotoxicity is observed.