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WO2024178128A1 - Lysis sensing receptors and uses thereof - Google Patents

Lysis sensing receptors and uses thereof Download PDF

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Publication number
WO2024178128A1
WO2024178128A1 PCT/US2024/016733 US2024016733W WO2024178128A1 WO 2024178128 A1 WO2024178128 A1 WO 2024178128A1 US 2024016733 W US2024016733 W US 2024016733W WO 2024178128 A1 WO2024178128 A1 WO 2024178128A1
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cells
lsr
cell
population
vector
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Paul R. Caron
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Elysion Therapeutics Inc
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Elysion Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Cancer immunotherapy including CAR-T cell therapy, is used to provoke immune responses attacking tumor cells while sparing normal tissues.
  • the present disclosure provides technologies related to compositions comprising a lysis sensing CAR Targeting an intracellular product of tumor cells and related methods.
  • Targeted immunotherapies rely on the use of immune cells or molecules that engage immune cells to treat a variety of diseases, including primarily cancer but also infectious diseases and autoimmune disorders. Recently, engineering T cells to express chimeric antigen receptors (CARs) that target tumor antigens has allowed the successful eradication of leukemic cells in humans.
  • CARs chimeric antigen receptors
  • CARs have two functional elements: an extracellular antigen recognition element, typically derived from an antibody, and an intracellular stimulatory element or set of elements. Upon recognition of an antigen on a tumor cell, CARs activate cytotoxic activity of T cells, which in turn results in tumor cell lysis.
  • the present disclosure identifies challenges with certain existing CAR-T cell therapies, including, for example that an antigen must be expressed on a tumor cell surface to be available for recognition.
  • the present disclosure identifies challenges with existing CAR-T cell therapies in that most tumor cell surface antigens that have been identified or utilized as targets of such CAR-T cell therapies are also expressed on healthy cells, reducing tolerance to CAR-T therapy and limiting effectiveness.
  • TCRs T cell receptors
  • the present disclosure provides an insight that connecting T cell activation to presence of lysed/lysing cells could dramatically improve engineered T cell therapy (e.g., CAR-T therapy).
  • CAR-T therapy e.g., CAR-T therapy.
  • the present disclosure provides CAR constructs that respond specifically to cell lysis, described herein as a Lysis Sensor Receptor (LSR).
  • LSR Lysis Sensor Receptor
  • the present disclosure describes LSR constructs comprising, for example, an extracellular lysis- antigen-binding moiety, a transmembrane element, and an intracellular T cell receptor co- stimulation element.
  • the present disclosure further provides engineered immune cell populations (e.g., engineered T cells) that express such LSRs, for example as a Co-CAR receptor.
  • engineered immune cell populations e.g., engineered T cells
  • the present disclosure further provides various insights and technologies relating to such LSR constructs and/or their expression by engineered cells including, for example, methods of making and/or using such constructs and/or cells that express them.
  • the present disclosure provides an insight that immune cells expressing such a LSR construct may be useful to enhance immune responses (e.g., by activating endogenous T cells), for example in the treatment of cancer.
  • LSRs provide a mechanism to dramatically expand the universe of antigens that can be used to stimulate an immune response in diseased tissues by bypassing the limitations of other immunotherapies imposed by the intracellular location of potential target antigens.
  • the present disclosure recognizes that LSRs have a further advantage over other activators of the immune response in that their activating antigens are only made available after cell lysis, thereby reducing the risk of toxicities that can result from antigens which may be expressed on the surface of both healthy and diseased cells.
  • LSRs are designed to selectively bind to antigens that are disease-specific, such as cancer- specific mutants or viral proteins, further reducing the potential for inadvertent immune cell activation by processes involved in healthy cell turnover.
  • Dependence on cell lysis serves as a positive feedback loop which increases the activity of immune cells in diseased tissues while minimizing their activation in other areas of the body.
  • populations of T cells e.g., a population of T cells isolated from a human
  • engineered T cell subpopulations will recognize diseased cells even if disease-specific antigens presented to TCR subclones change.
  • LSRs will amplify the response of any T cells in the vicinity of a diseased cell that are capable of recognizing a disease-specific antigen, thereby stimulating cytotoxicity and reinforcing clonal expansion of T cells with effective TCRs.
  • This approach provides a distinct advantage in the treatment of diseases with rapidly changing characteristics, such as tumor cells or viral infections, by limiting the need for artificial amplification of tumor antigen presentation or genetic engineering to customize LSRs for specific antigens.
  • LSRs will stimulate T cells encoding TCRs that recognize these new antigens without the need to develop a new therapy and thereby limiting the potential for resistance to develop.
  • use of LSRs as described herein can provide immune cell therapies, e.g., for solid tumors, that are (i) more effective, (ii) safer and (iii) more durable than existing cell therapies.
  • FIG.1 shows an image depicting a positive feedback loop between activated engineered T cells expressing a LSR reacting with and lysing tumor cells in their proximity causing lysis of more tumor cells, resulting in more activation of LSR-expressing T cells.
  • FIG.2 shows an image depicting certain exemplary LSR constructs comprising an extracellular single chain variable fragment (scFv) operably linked to a transmembrane element, and one or more T cell co-stimulation elements.
  • an exemplary LSR construct further comprises a CD3 ⁇ co-stimulation element.
  • FIG.3 shows an image depicting exemplary models for LSR activation by a lysis-associated-antigen (e.g., antigens that are made available to a LSR upon the lysis of a diseased cell (e.g., intracellular antigens/epitopes)).
  • a lysis-associated-antigen e.g., antigens that are made available to a LSR upon the lysis of a diseased cell (e.g., intracellular antigens/epitopes)
  • FIG.4 shows an image depicting exemplary anti-KRAS LSR constructs.
  • FIG.5 shows an image depicting activation and oligomerization of LSRs by KRAS binding.
  • FIG.6 shows a table depicting a number of oncogenes mutated in a high percentage of tumors; several of the proteins encoded by these oncogenes are intracellular Page 3 of 65 11825822v1 Docket No.: 2017422-0003 proteins.
  • the present disclosure provides LSR constructs (and associated technologies, as described herein) that target such intracellular oncogene-encoded proteins.
  • FIG.7 shows an image depicting an exemplary Anti-P53 LSR construct.
  • FIG.8 shows an image depicting exemplary steps of a LSR T therapy.
  • FIG.9 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Abecma (idecabtagene vicleucel).
  • FIG.10 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Breyanzi (lisocabtagene maraleucel).
  • FIG.11 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Kymriah (tisagenlecleucel).
  • FIG.12 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Tecartus (brexucabtagene autoleucel).
  • FIG.13 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Yescarta (axicabtagene ciloleucel).
  • FIG.14 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Carvykti (ciltacabtagene autoleucel).
  • FIG.15 depicts two exemplary LSR construct designs according to an embodiment of the present disclosure.
  • FIG.16 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct.
  • FIG.17 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct, Meso-CAR-T cells, and dual T cells expressing both a Meso-CAR and a KRAS-LSR (e.g., dual KRAS-LSR/Meso-CAR-T cell).
  • FIG.18 shows a graph depicting percent cell killing of A1847 cells by dual KRAS-LSR/Meso-CAR-T cells, Meso-CAR-T cells, and KRAS-LSR-T cells over time (0 to 21 hours).
  • FIG.19 shows a schematic depicting a proposed mechanism of action for increased T cell activation and increased cell killing by an exemplary KRAS LSR.
  • FIG.20 shows a graph depicting flow cytometry data depicting detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct, EpCAM-CAR-T cells, and dual T cells expressing both a EpCAM-CAR and a KRAS-LSR (e.g., dual KRAS- LSR/EpCAM-CAR-T cell).
  • FIG.21 shows a graph depicting percent cell killing of SW-1990 cells by dual KRAS-LSR/EpCAM-CAR-T cells, EpCAM-CAR-T cells, and KRAS-LSR-T cells over time (0 to 20 hours).
  • FIG.22 shows a graph depicting IFN-gamma levels in supernatant collected from A1847 cells (e.g., human ovarian cancer cells) cultured with Meso-CAR-T cells, Meso-CAR/KRAS-LSR T cells (e.g., T cells expressing both a KRAS-targeting LSR and a Meso-CAR, or non-transduced T cells.
  • Meso-CAR/KRAS-LSR T cells have a higher degree of T cell activation, as measured by IFN-gamma expression, than Meso-CAR-T cells alone.
  • FIG.23 shows a graph depicting fraction cell killing of A1847 cells by KRAS- LSR-T cells, Meso-CAR-T cells, a mixture of KRAS-LSR-T cells and Meso-CAR-T cells, and dual KRAS-LSR/ Meso-CAR-T cells over time (0 to 48 hours).
  • FIG.24 shows a graph depicting fraction cell killing of A1847 cells by KRAS- LSR-T cells with addition of purified KRAS protein or supernatant from A1847 cells previously killed by Meso-CAR-T cells.
  • FIG.25 shows a graph depicting fraction cell killing of A1847 cells by dual KRAS-LSR/Meso-CAR-T cells with and without addition of an anti-KRAS antibody. Addition of anti-KRAS antibody reduced rate of cell killing by dual KRAS LSR/Meso- CAR-T cells.
  • Administration typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment.
  • CDR refers to a complementarity determining region within an immunoglobulin (e.g., antibody, T cell receptor) variable region.
  • canonical antibodies include heavy and light chains, each of Page 5 of 65 11825822v1 Docket No.: 2017422-0003 which is comprised of a variable region and a constant region. There are three CDRs in each such variable region, designated CDR1, CDR2 and CDR3.
  • T cell receptors comprise a and B chains, or d and g chains, each of which includes 3 CDRs..
  • a “set of CDRs” or “CDR set” refers, as will be clear from context and understood by those skilled in the art, either to the group of three CDRs that occur in a particular chain, or to the set of six CDRs (i.e., three heavy chain and three light chain CDRs) that are found together in a particular immunoglobulin (and e.g., contribute to or determine its antigen-specificity). .
  • Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.); those skilled in the art appreciate the differences between and among these systems and are capable of understanding CDR boundaries to the extent required to understand and to practice the claimed invention.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • corresponding to refers to a relationship between two or more entities.
  • corresponding to may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition).
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • a monomeric residue in a polymer may be identified as “corresponding to” a residue in an appropriate Page 6 of 65 11825822v1 Docket No.: 2017422-0003 reference polymer.
  • residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190 th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids.
  • sequence alignment strategies including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
  • software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, Scala
  • Dosage form or unit dosage form Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent.
  • an active agent e.g., a therapeutic or diagnostic agent
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
  • individual doses are separated from one another by a time period of the same length; in Page 7 of 65 11825822v1 Docket No.: 2017422-0003
  • a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount.
  • different doses within a dosing regimen are of different amounts.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen). [0044] Effector function: as used herein refers a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand.
  • Effector functions include but are not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC).
  • an effector function is one that operates after the binding of an antigen, one that operates independent of antigen binding, or both.
  • Engineered In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence.
  • a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and/or if produced other than natural expression in a cell.
  • a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • the manipulation is or comprises a genetic manipulation, so that its genetic Page 8 of 65 11825822v1 Docket No.: 2017422-0003 information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a particular agent of interest e.g., a protein, a nucleic acid, and/or a particular form thereof
  • progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • Framework or "framework region: as used herein, refers to the sequences of an immunoglobulin (e.g., antibody) variable region minus the CDRs.
  • a framework sequence can be determined by different systems, likewise a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs divide the framework regions on the heavy and light chains into four sub-regions (FRl, FR2, FR3 and FR4) on each chain, in which CDRl is positioned between FRl and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions, FR1, for example, represents the first framework region closest to the amino terminal end of the variable region and 5' with respect to CDR1, and FRs represents two or more of the sub-regions constituting a framework region.
  • FR1 represents one of the four sub-regions, FR1, for example, represents the first framework region closest to the amino terminal end of the variable region and 5' with respect to CDR1, and FRs represents two or more of the sub-regions constituting a framework region.
  • homology refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic”amino acids, Page 9 of 65 11825822v1 Docket No.: 2017422-0003 and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.
  • Typical amino acid categorizations are summarized below: Page 10 of 65 11825822v1 Docket No.: 2017422-0003
  • a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences.
  • Calculation of the percent homology between two nucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position.
  • the percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Identity refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • Calculation of percent identity of two nucleic acid or polypeptide sequences can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared.
  • Percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • ALIGN program version 2.0
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • an appropriate reference measurement Page 12 of 65 11825822v1 Docket No.: 2017422-0003 may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (2) designed, produced, prepared, and/or manufactured by the hand of man.
  • a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated).
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
  • carriers or excipients e.g., buffer, solvent, water, etc.
  • a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature.
  • a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated” polypeptide.
  • a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
  • Operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control element "operably linked" to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element.
  • "operably linked" control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • Prevent or prevention refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. In some embodiments, prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
  • Reference As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
  • Specific binding refers to an ability to discriminate between possible binding partners in the environment in which binding is to Page 14 of 65 11825822v1 Docket No.: 2017422-0003 occur.
  • a binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts.
  • specific binding is assessed by detecting or determining degree and/or rate of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree and/or rate of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity.
  • Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
  • tumor refers to an abnormal growth of cells or tissue.
  • a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • a tumor is associated with, or is a manifestation of, a cancer.
  • a tumor may be a disperse tumor or a liquid tumor.
  • a tumor may be a solid tumor.
  • Tumor-specific refers to an agent that is associated with tumor cells and not with appropriate reference non-tumor cells.
  • neoantigens e.g., that arise in a tumor, for example by mutation that occurs in the tumor
  • Tumor-associated refers to an agent that is more likely to be present (or detectable) in and/or on tumor cells than appropriate reference non-tumor cells.
  • Variant As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function;
  • a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three- dimensional space.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • moieties e.g., carbohydrates, lipids, phosphate groups
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid.
  • a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
  • a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference.
  • a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference.
  • a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference.
  • a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference.
  • a reference polypeptide or nucleic acid is one found in nature.
  • a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector refers to a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non- episomal mammalian vectors
  • vectors are capable of directing the expression of genes to which they are Page 17 of 65 11825822v1 Docket No.: 2017422-0003 operatively linked. Such vectors are referred to herein as "expression vectors.”
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
  • Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.
  • Adoptive Cell Therapy (ACT) strategies are revolutionizing cancer care by using immune cells – typically cultured immune cells as therapeutic agents to attack patients’ tumors.
  • TIL tumor-infiltrating lymphocyte
  • CAR chimeric antigen receptor
  • TIL tumor infiltrating lymphocytes
  • targets of these TIL are not known, it is presumed that TIL isolated from a tumor are enriched in T cells directed against cancer cells.
  • TIL are typically expanded and activated ex vivo, and optionally selected or purified to enrich for cells demonstrated to target antigens expressed by tumors.
  • These tumor-associated or tumor-specific antigens can include antigens associated with tumor drivers, neoantigens (novel antigens arising from genetic mutations) or other antigens that are enriched on tumor cells compared to healthy cells).
  • An expanded population of TIL is then administered to a patient, who is usually the same person from whom the original TIL were isolated.
  • TIL therapy was first demonstrated to achieve regression in melanoma Page 18 of 65 11825822v1 Docket No.: 2017422-0003 patients in 1988 (see, Rosenberg et al., N. Engl. J. Med 319:1676, 1988, the contents of which is hereby incorporated by reference herein in its entirety), but initial responses were short-lived and administered T-cells were undetectable in the recipients within just a few days. It was subsequently determined that lymphoablation prior to administration of a TIL preparation could dramatically improve durability of response, and apparent successful engraftment of administered TIL (see, for example, Dudley et al., Science 298:850, 2002, the contents of which is hereby incorporated by reference herein in its entirety).
  • Lymphoablation can be utilized prior to any adoptive cell therapy. More recently, improvements in TIL therapy have been driven by use of certain factors (e.g., cytokines, certain antibodies, such as certain anti-PD-1 and/or anti-CTLA-4 antibodies, etc) to enhance survival and activation of TIL (see, Zhao and Cao, 2019, the contents of which is hereby incorporated by reference herein in its entirety). In some embodiments one or more such agents (e.g., in certain embodiments one or more cytokines) are added exogenously. In some embodiments, TIL are genetically engineered to express one or more such agents (e.g., one or more cytokines).
  • certain factors e.g., cytokines, certain antibodies, such as certain anti-PD-1 and/or anti-CTLA-4 antibodies, etc
  • TIL are genetically engineered to express one or more such agents (e.g., one or more cytokines).
  • TCR therapies which include both naturally occurring TILs and engineered cells involve administering T cells whose T-cell receptors (TCRs) target particular antigens of interest (e.g., tumor antigen, such as a tumor-associated antigen or a tumor-specific antigen).
  • T-cell receptors are transmembrane heterodimers of either ⁇ - and ⁇ - chains or ⁇ - and ⁇ - chains containing CDRs that together determine characteristics of the receptor’s antigen binding (e.g., its specificity and/or affinity, etc).
  • Cells in the body have a mechanism for displaying on their surface a sampling of every protein that is being made by a cell.
  • MHCs Major Histocompatibility Complexes
  • TCRs there are 10 10 (10 billion) distinct TCRs in an individual providing the opportunity for recognition of a broad range of potential antigens (see, J Theor Biol.2016 Jan 21; 389: 214–224, the contents of which is hereby incorporated by reference herein in its Page 19 of 65 11825822v1 Docket No.: 2017422-0003 entirety).
  • these TCRs are understood to recognize a specific HLA type.
  • T cells have been engineered with TCRs designed to target select tumor-specific antigens, their application is limited to individuals who also have a matching HLA type.
  • CAR-T therapy involves engineering T cells to express a chimeric antigen receptor (CAR) construct, to enable T cells to be activated independent of a T cell receptor.
  • CAR chimeric antigen receptor
  • Such a CAR construct links an antigen binding moiety, typically a single chain variable fragment (scFv) of an antibody known to bind the relevant antigen of interest, with TCR signaling domain(s) and/typically, one or more additional T cell co-stimulation elements (e.g., derived from receptors such as CD28, OX40, CD137, etc).
  • CAR constructs target surface antigens, and typically do so with very high avidity.
  • a limitation of CAR-T therapies is that CARs cannot penetrate cells and thereby cannot recognize intracellular antigens.
  • CAR-T therapy is also distinguished from TCR therapy through its ability to recognize antigens independent of HLA type.
  • CAR-T therapies are generated as autologous therapies to limit potential for mismatches in HLA type that can arise when generating CAR-T therapies from allogeneic donors. These mismatches can lead to the rejection of therapy.
  • the United States Food and Drug Administration has approved at least six adoptive cell therapies, all of which are CAR-T therapies, approved for treatment of hematological cancers.
  • CAR-T therapies have been approved for the treatment of large B-cell lymphoma , multiple myeloma, follicular lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia.
  • Certain approved CAR-T therapies have reported stunning success, including recurrence rates reduced by 80% or more.
  • cytokine release syndrome also known as “cytokine storm” has plagued certain CAR-T therapies, which seem to induce much more dramatic such reactions than have been observed with TCR (e.g., TIL and/or engineered TCR) therapies (see, for example, Kalos et al., Sci. Transl.
  • CAR-T therapies that have been approved by the FDA have exclusively targeted antigens that are exposed on proteins expressed on a cell surface. These therapies have been primarily focused on common surface proteins that are expressed on many hematologic malignancies. By contrast, solid tumors have a high degree of heterogeneity with few common surface-expressed antigens which limits the potential of generating broadly effective CAR-T therapies. In addition to the scarcity of tumor-specific targets, most surface-expressed proteins on tumor cells are also expressed on healthy cells, leading to toxicities due to effects of the highly cytotoxic CAR-T cells in non-tumor tissues. For example, HER2 is a surface-expressed protein that is also expressed in tissues such as the lung.
  • TIL therapy is that patients are treated with a population of cells that contain distinct TCRs, increasing the probability that the treatment contains cells that can target multiple tumor-specific antigens – antigens that exist at the time of treatment and antigens that may emerge as the tumor cells emerge that have altered antigens (see, Wang et al. BMC Medicine 2021).
  • Co-stimulation is a fundamental component of T cell biology and of adoptive cell therapy, specifically including CAR-T cell therapy.
  • Effective potency of a CAR-T cell is dependent on incorporation of one or more intracellular T cell receptor co-stimulation protein elements, which serve to increase CAR-T cell persistence and enhance cell cytotoxicity in response to antigen binding to an extracellular antigen binding moiety (see, Harrison et al, 2021, the contents of which is hereby incorporated by reference herein in its entirety).
  • First generation CARs lacked any co-stimulation elements and had limited clinical efficacy.
  • Second generation CARs have a single co-stimulation element as exemplified by FDA approved CAR-T products Abecma Page 21 of 65 11825822v1 Docket No.: 2017422-0003 (idecabtagene vicleucel), Breyanzi (lisocabtagene maraleucel), Kymriah (tisagenlecleucel) and Carvykti (ciltacabtagene autoleucel), which contain a 4-1BB co-stimulation element, and Tecartus (brexucabtagene autoleucel) and Yescarta (axicabtagene ciloleucel), which contain a CD28 co-stimulation element.
  • FDA approved CAR-T products Abecma Page 21 of 65 11825822v1 Docket No.: 2017422-0003 (idecabtagene vicleucel), Breyanzi (lisocabtagene maraleucel), Kymriah (tisagenle
  • LSR constructs comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements).
  • T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary elements.
  • an exemplary LSR construct comprises a CD28 co-stimulation element.
  • an exemplary LSR comprises a 4-1BB co-stimulation element.
  • an exemplary LSR comprises a OX40 co-stimulation element. In some embodiments, an exemplary LSR comprises a CD27 co-stimulation element. In some embodiments, an exemplary LSR comprises a GITR co-stimulation element. In some embodiments, an exemplary LSR comprises a ICOS co-stimulation element. [0073] In some embodiments, a LSR as described herein comprises one or more T cell receptor co-stimulation elements. In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element and a 4-1BB co-stimulation element.
  • a LSR as described herein comprises one or more T cell receptor co-stimulation elements as well as a CD3 ⁇ activation element.
  • an exemplary LSR comprises a CD28 co-stimulation element, a 4-1BB co- stimulation element, and a CD3 ⁇ activation element.
  • Lysis-Sensing Adaptive Cell Therapy [0075] The present disclosure provides an insight that linking T cell activation to cell lysis can provide a number of therapeutic benefits. For example, the present disclosure appreciates that certain tumor environments (including specifically certain solid tumor environments) are characterized by cell lysis (e.g., have a relatively elevated level of cell lysis products).
  • the present disclosure further provides an insight that linking of T cell activation to cell lysis can be achieved through use of a Lysis Sensor Receptor (LSR) as described and provided herein.
  • LSR Lysis Sensor Receptor
  • Page 22 of 65 11825822v1 Docket No.: 2017422-0003 the present disclosure provides Lysis Sensor Receptors, which are activated by antigens that are made available to these receptors upon the lysis of a diseased cell (e.g., intracellular antigens/epitopes).
  • the present disclosure further comprises immune cells engineered to express such LSRs.
  • immune cells include T cells with an endogenous TCR; in some embodiments, such T cells include an engineered TCR.
  • such T cells include a CAR.
  • a LSR may be expressed in immune cells that have disease- fighting activity that are not dependent on a TCR or CAR including natural killer (or NK) cells.
  • introduction of LSRs into T cells can lead to activation of T cells that are in close proximity to a diseased tissue, such as a tumor. In some embodiments, such introduction achieves local activation of T cell cytotoxic activity in the vicinity of lysed cells.
  • Lysis Sensor Receptor Constructs [0078] Among other things, the present disclosure provides Lysis Sensor Receptor constructs (LSRs) that comprise a lysis-associated-antigen binding moiety, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements).
  • LSRs Lysis Sensor Receptor constructs
  • the present disclosure describes LSR constructs that, when expressed on a cell (e.g., an immune cell, e.g., a T cell), a lysis-associated-antigen binding moiety is located extracellularly, e.g., such that it is arranged and constructed to bind a target lysis-associated- antigen, and a signaling element is located in cytoplasm, e.g., such that it is arranged and constructed for signal transduction and/or cell activation.
  • Lysis-associated-antigens [0080] The present disclosure teaches that targeting antigens associated with cell lysis (lysis-associated-antigens) as described herein can provide valuable benefits (e.g., therapeutic benefits).
  • a lysis-associated-antigen is a protein, nucleic acid, carbohydrate, lipid or small molecule that becomes exposed at higher levels extracellularly upon lysis of a cell.
  • antigens are specific to diseased cells or are present at substantially higher levels in diseased cells compared to healthy cells.
  • such antigens include proteins encoded by tumor-specific mutations, proteins whose levels are increased due to altered signaling in tumor cells, nucleic acids expressed at higher levels in tumor cells and antigens derived from structural and metabolic changes associated with tumorigenesis.
  • the present disclosure describes lysis-associated-antigens such Page 23 of 65 11825822v1 Docket No.: 2017422-0003 as those driven by pathogens.
  • lysis-associated-antigens include genes encoded by a pathogen that are required for pathogen replication or survival within cells including components of replication and packaging machinery.
  • a lysis-associated-antigen is an intracellular antigen that is exposed to an extracellular environment by cell lysis.
  • established CAR-T technologies are required to target surface antigens; established TCR technologies are required to target antigens, often intracellular antigens presented to an extracellular cell surface in MHC complexes.
  • Lysis Sensor Receptor constructs provided by the present disclosure target intracellular antigens independent of MHC presentation.
  • a LSR described herein has a structure that resembles portions of a CAR with the exception that it targets intracellular antigens. As such, LSRs are activated only upon lysis of diseased cells and serve as a means to specifically stimulate an immune response in situations where lysis of diseased cells has occurred either as a result of disease progression, a patient’s immune response or action of another therapy.
  • a lysis-associated-antigen may be or comprise an oncogenic protein, a protein that drives cancer cells to grow and divide uncontrollably.
  • Exemplary tumor-specific lysis-associated-antigens include protein products of common oncogenes such as K-RAS, N-RAS, H-RAS, c-Myc, SRC family kinases and other intracellular signal transduction molecules, and intracellular elements of epidermal growth factor receptors and other receptor tyrosine kinases as well as protein products of mutated tumor suppressor proteins such as p53, BRCA1/2 and PTEN.
  • oncogene addiction describes the concept that cancer cells become highly dependent on oncogenes for survival and thus the oncogene is likely to persist throughout the tumor and maintained as the tumor progresses.
  • KRAS mutations are found in the majority of pancreatic ductal carcinomas.
  • a LSR targets KRAS variants with a mutation occurring at amino acid 12, optionally G12D, G12V or G12C.
  • KRAS G12D is found in roughly one third of pancreatic tumors and one sixth of colon cancers.
  • a LSR targets KRAS variants with a mutation at positions other than amino acid 12.
  • a lysis-associated-antigen may be a protein product of a gene that is mutated in one percent or more of all solid tumors optionally selected from BRAF, IDH1, PI3Kalpha, P53 or others.
  • a lysis-associated-antigen may be BRAF.
  • a lysis-associated-antigen may be IDH1.
  • a lysis-associated-antigen may be PI3Kalpha.
  • a lysis- associated antigen may be P53.
  • a lysis-associated-antigen is or comprises one or more neoepitopes.
  • Neoepitopes are characterized as altered protein sequences that arise from mutations in tumor cells. By definition, neoepitopes are not present in healthy cells and are tumor-specific. Whereas there is a potential for neoepitopes to be recognized by TCRs, neoepitopes that can be recognized in this manner are limited to those than can be presented by MHC class II receptors in an individual patient. It is an insight of the present disclosure that there is no such limitation on neoepitopes that could be targeted by LSRs, with the only requirement that they be present upon cell lysis.
  • Lysis-associated-antigen binding moiety [0085] Among other things, the present disclosure provides technologies (e.g., compositions, methods, producer cells, etc.) that are or comprise LSR constructs described herein. In some embodiments, technologies described herein comprise a LSR construct comprising a lysis-associated-antigen binding moiety. In some embodiments, a lysis- associated-antigen binding moiety binds specifically to a lysis-associated-antigen, as described herein. [0086] In some embodiments, a lysis-associated-antigen binding moiety is or comprises antigen binding elements of an immunoglobulin (e.g., an antibody, a T cell receptor, etc).
  • an immunoglobulin e.g., an antibody, a T cell receptor, etc.
  • a lysis-associated-antigen binding moiety is or comprises a set of CDRs.
  • a lysis-associated-antigen binding moiety is or comprises a single chain variable fragment (scFv).
  • lysis-associated-antigen binding moiety comprises a KRAS binding moiety as described by Singh et al., 2022, the contents of which is hereby incorporated by reference herein in its entirety.
  • a lysis-associated- antigen binding moiety comprises a KRAS binding moiety as described by Shin et al., 2017, the contents of which is hereby incorporated by reference herein in its entirety.
  • a lysis-associated antigen-binding moiety comprises a KRAS binding moiety as described by U.S. Pat. No.11,174,314 B2, the relevant disclosures of which are incorporated by reference herein.
  • a lysis-associated-antigen binding moiety comprises a STAT3 binding moiety as described by Singh et al., 2022.
  • an exemplary LSR construct comprises a KRAS mutant-specific scFV sequence according to SEQ ID NO: 1.
  • Exemplary KRAS mutant-specific scFV sequence (SEQ ID NO: 1) EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHD GSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQG TTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNY VSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAA WDDKFGVHWVFGGGTKLTVL
  • Critical to immune cell activation is the ability of binding of a ligand to an extracellular element to trigger a change in intracellular signaling.
  • LSR multimerization can be a result of antigens bound to intracellular cell structures such as membranes, protein complexes, structural elements, nucleic acids and glycoproteins. Alternatively or additionally, antigens released upon cell lysis may bind to extracellular elements resulting in multimer structures.
  • a LSR cell therapy may comprise immune cells Page 26 of 65 11825822v1 Docket No.: 2017422-0003 containing LSRs targeting different antigens or different antigenic sites on a single antigen (see, Han et al., 2019, the contents of which is hereby incorporated by reference herein in its entirety).
  • a Lysis Sensor Receptor construct for use in accordance with the present disclosure comprises a transmembrane element that, for example, may link a lysis-associated-antigen binding moiety as described herein to a signaling element as described herein.
  • a transmembrane element that, for example, may link a lysis-associated-antigen binding moiety as described herein to a signaling element as described herein.
  • a transmembrane element of a LSR construct regulates LSR expression and/or signaling activity (see, for example, Fujiwara et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety).
  • a transmembrane element may correspond to one that is found in a naturally occurring protein.
  • a transmembrane element may be designed or otherwise engineered, for example, to be or include a substantially hydrophobic segment that is thermodynamically stable in a cell membrane, whether or not its sequence is found in any known transmembrane protein (e.g., any known human transmembrane protein).
  • transmembrane domains examples include, for example, those described in U.S. Pat. No.7,052,906 Bl and PCT Publication No. WO 2000/032776 A2, the relevant disclosures of which are incorporated by reference herein.
  • a transmembrane element for use in accordance with the present disclosure corresponds to one found in a membrane protein selected from the group consisting of: CD8 ⁇ , CDS ⁇ , 4-1BB/CD137, CD28, CD34, CD4, FcERiy, CD16, OX40/CD134, CD3s, CD3E, CD3y, CD3o, TCRa, CD32, CD64, VEGFR2, FAS, and FGFR2B.
  • a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD8a.
  • a transmembrane element for use in accordance with the present disclosure corresponds to one found in 4- lBB/CD137.
  • a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD28 or CD34.
  • a Page 27 of 65 11825822v1 Docket No.: 2017422-0003 transmembrane element for use in accordance with the present disclosure corresponds to one found in CD8a.
  • Signaling element [0097] As described herein, effective CAR-T cell therapy requires co-stimulation to increase CAR-T cell persistence and enhance cell cytotoxicity in response to antigen binding.
  • a LSR utilized in accordance with the present disclosure comprises one or more signaling elements (e.g., co-stimulation element(s)) which leads to immune effector function as described herein.
  • Exemplary signaling elements that may be used in accordance with the present disclosure include those selected from the group consisting of MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R
  • MHC class I molecule TNF receptor proteins
  • a co-stimulation element for use in accordance with the present disclosure are described in U.S. Pat. No.11,084,880 B2, incorporated herein by reference.
  • a co-stimulation element is derived from a group consisting of CD28, OX40, 4I-BB, GITR, ICOS-1, CD27, AP10, and any combination thereof.
  • a co-stimulation element comprises a CD3 ⁇ Page 28 of 65 11825822v1 Docket No.: 2017422-0003 (CD3 ⁇ or CD3C) activating element.
  • a co-stimulation element does not comprise a CD3 ⁇ (CD3 ⁇ or CD3C) activating element.
  • a LSR construct may comprise more than one co- stimulation element (e.g., 2, 3 or more). In some embodiments, a LSR construct comprises two or more of the same co-stimulation elements. In some embodiments, a LSR construct comprises two or more co-stimulation elements from different co- stimulatory proteins, such as any two or more co-stimulatory proteins described herein. [0100] The present disclosure describes LSR constructs comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, an exemplary LSR construct comprises one or more T cell receptor co-stimulation elements.
  • T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary elements.
  • an exemplary LSR construct comprises a CD28 co-stimulation element.
  • An exemplary CD28 co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 2.
  • an exemplary LSR construct comprises a 4-1BB co-stimulation element.
  • An exemplary 4-1BB co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 3.
  • an exemplary LSR construct comprises a OX40 co-stimulation element.
  • an exemplary LSR construct comprises a CD27 co-stimulation element. In some embodiments, an exemplary LSR construct comprises a GITR co-stimulation element. In some embodiments, an exemplary LSR construct comprises a ICOS co-stimulation element. [0101] In some embodiments, an exemplary LSR construct comprises a CD28 co- stimulation element and a 4-1BB co-stimulation element. [0102] In some embodiments, an exemplary LSR construct comprises one or more T cell receptor co-stimulation elements as well as a CD3 ⁇ activation element. An exemplary CD3 ⁇ activation element sequence may be or comprise a sequence according to SEQ ID NO: 4.
  • an exemplary LSR construct does not comprise a CD3 ⁇ activation element.
  • an exemplary LSR comprises a CD28 co-stimulation element, a 4-1BB co-stimulation element, and a CD3 ⁇ activation element.
  • Exemplary CD28 co-stimulation element sequence SEQ ID NO: 2 ASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRLGPTRKHYQPYAPPRDFAAYRS [0105] Exemplary 4-1BB co-stimulation element sequence (SEQ ID NO: 3) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE [0106] Exemplary CD3 ⁇ activation element sequence (SEQ ID NO: 4) LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR Other element(
  • a LSR construct may comprise a masking element that is cleaved off or moved in a tumor microenvironment.
  • constructs of the present disclosure may comprise a masking element as described by U.S. Pat. No.9,120,853 B2, the contents of which are hereby incorporated by reference in its entirety.
  • a LSR construct as described herein comprises a signaling peptide sequence.
  • An exemplary signaling peptide sequence may be or comprise a sequence according to SEQ ID NO: 5.
  • a LSR construct as described herein further comprises, a polypeptide tag (e.g., a FLAG-tag, or FLAG octapeptide, or FLAG epitope).
  • a polypeptide tag e.g., a FLAG-tag, or FLAG octapeptide, or FLAG epitope
  • a polypeptide tag is used to assess expression of a LSR in T cells.
  • An Page 30 of 65 11825822v1 Docket No.: 2017422-0003 exemplary polypeptide tag sequence may be or comprise a sequence according to SEQ ID NO: 6.
  • Exemplary polypeptide tag sequence (SEQ ID NO: 6) DYKDDDDK
  • WO 2017/172952 the contents of which is hereby incorporated by reference herein in its entirety.
  • the present disclosure provides technologies (e.g., compositions, vectors, virions, producer cells).
  • technologies comprise a single construct.
  • such technologies comprise multiple constructs.
  • the present disclosure provides compositions or preparation of virions each comprised of a single construct as described herein.
  • a single construct may deliver a polynucleotide that encodes an exemplary LSR.
  • a construct is or comprises a LSR construct.
  • a single construct composition or system may comprise any or all of the exemplary construct components described herein.
  • an exemplary single construct is at least 85%, 90%, 95%, 98% or 99% identical to the sequences described herein.
  • constructs may undergo additional modifications including codon-optimization, introduction of novel but functionally equivalent (e.g., silent mutations), addition of reporter sequences, and/or other routine modification.
  • constructs, virions, populations of virions, or producer cells comprise a LSR polypeptide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with that of SEQ ID NO: 7.
  • constructs, virions, populations of virions, or producer cells comprise a LSR polynucleotide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with that of SEQ ID NO: 8.
  • Exemplary KRAS-LSR construct 1 polypeptide sequence Page 31 of 65 11825822v1 Docket No.: 2017422-0003 MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMH WVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDT AVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSA APGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGT SASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKDDDDKASAIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRLLHS
  • LSR constructs as described herein are polynucleotide constructs.
  • Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and constructs that incorporate a polynucleotide comprising a coding sequence operably linked to an expression control sequence, wherein the coding sequence encodes a LSR.
  • cosmids e.g., naked or contained in liposomes
  • constructs that incorporate a polynucleotide comprising a coding sequence operably linked to an expression control sequence wherein the coding sequence encodes a LSR.
  • Those of skill in the art will be capable of selecting suitable constructs, as well as cells, for making any of a nucleic acids described herein.
  • a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell).
  • a construct can be a cosmid (e.g., pWE or sCos series).
  • cosmid e.g., pWE or sCos series.
  • nucleic acids e.g., to cells as described herein.
  • methods for delivery of a polynucleotide construct e.g., a LSR construct
  • a polynucleotide construct e.g., a LSR construct
  • Hou et al., Page 33 of 65 11825822v1 Docket No.: 2017422-0003 2021, and U.S. Pat. No.11,084,880 B2 each of which is incorporated in its entirety herein by reference.
  • a viral vector comprising a polynucleotide construct encoding a LSR to facilitate introduction into immune cells.
  • a viral vector can be a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno- associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, a HSV vector among others.
  • HAV-1 human immunodeficiency virus 1
  • HMV-2 human immunodeficiency virus 2
  • VMV visna-maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • a viral vector lacking replicating ability so as not to self replicate in an infected cell is preferably used.
  • the present disclosure provides producer cells so that virions are produced in the producer cell that comprise the viral capsid comprising a polynucleotide construct as described herein. Methods of introducing and expressing genes in a cell are known in the art.
  • a polynucleotide construct encoding a LSR construct described herein is transferred into a producer cell.
  • the present disclosure describes a preparation of virions comprising a viral capsid and a LSR construct described herein to facilitate introduction into cells (e.g., immune cells).
  • a polynucleotide construct encoding a LSR construct described herein is operably linked to a promoter and incorporated into an expression vector.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • LSR Activation The present disclosure contemplates a variety of mechanisms that may be involved in LSR activation upon antigen (i.e., lysis-associated-antigen) binding. Page 34 of 65 11825822v1 Docket No.: 2017422-0003 [0129]
  • antigen binding may induce a conformational change, for example in an intracellular domain of the LSR, thereby achieving direct activation.
  • antigen binding may alter interaction(s) between a LSR and one or more other agents (e.g., other proteins), thereby achieving transduction of a relevant signal. Still further, alternatively or additionally, in some embodiments, antigen binding could trigger dimerization or higher order multimerization of a LSR (e.g., through binding of dimeric or oligomeric antigen ligands and/or through binding of multiple ligands that form multimeric structures, for example).
  • different LSRs may target different antigenic regions (e.g., epitopes) of a same target antigen (e.g., target protein, carbohydrate, complex, etc), such that antigen binding by two or more such LSRs co-localizes different LSRs, thereby achieving signal activation.
  • target antigen e.g., target protein, carbohydrate, complex, etc
  • a LSR construct comprises an oligomerization element for the purpose of coordinating protein oligomerization and subsequent signal transduction.
  • a LSR construct comprises a homodimerization, heterodimerization, trimerization, tetramerization, or oligomerization element, or combinations thereof, in order to achieve oligomerization.
  • Exemplary oligomerization elements that may be used in accordance with certain embodiments of the present disclosure are described in, for example, Engel & Kammerer, 2000, Thomas et al., 2013, and Mittl et al., 2000, the contents of which are hereby incorporated by reference in its entirety.
  • an oligomerization element is or comprises a short flexible peptide linker which joins the light and heavy chains of a scFV as described in, for example, Ahmad et al., 2012, the contents of which are hereby incorporated by reference in its entirety.
  • LSR-Expressing Cells [0131] Among other things, the present disclosure provides preparations of cells engineered to express a LSR construct as described herein. [0132] In some embodiments, provided cells (e.g., provided cell preparations) are immune cells. In some embodiments, provided cells (e.g., provided cell preparations) are T cells. In some embodiments, provided cells (e.g., provided cell preparations) are NK cells.
  • provided cells are macrophages.
  • provided cells are T cells that express a TCR.
  • TCR is a natural TCR; in some embodiments, such TCR is an engineered TCR.
  • provided cells are T cells that express a CAR.
  • provided cells are cells that have been cultured.
  • provided cells are cells that have been isolated from a subject and cultured (e.g., expanded) ex vivo.
  • provided cells are T cells, such as TIL cells.
  • provided cells include a plurality of different T cells (e.g., a plurality of T cells expressing different TCRs), each of which has been engineered to express the same LSR.
  • the present disclosure provides an insight that provided cell preparations will maintain their endogenous TCR heterogeneity.
  • provided cells e.g., a provided preparation of cells is expanded from a sample obtained from a subject.
  • such sample is or comprises blood or other bodily fluid, or a tissue sample.
  • a sample is obtained from a site that is in the vicinity of a tumor. In some embodiments, such site comprises part or all of a tumor.
  • a sample is or comprises peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • a sample is or comprises bone marrow.
  • a sample is or comprises a spleen, lymph node, or thymus sample.
  • a sample is or comprises tumor tissue (e.g., that may comprise TILs).
  • a population of cells (e.g., T cells) is obtained from a subject and engineered to express a LSR.
  • a population of cells (e.g., T cells) is isolated from a patient to whom the engineered population of cells is to be administered.
  • a population of T cells is obtained.
  • cells of an obtained population express T cell receptor(s).
  • cells of an obtained population of T cells are engineered to alter expression of their natural TCR (e.g., to replace it with an engineered TCR) and/or to express a CAR.
  • a Page 36 of 65 11825822v1 Docket No.: 2017422-0003 population of T cells is engineered according to methods described by, for example, Ellis et al., 2021, the contents of which are hereby incorporated by reference in its entirety.
  • a method for producing an engineered population of T cells is transduction with a viral vector or a polynucleotide construct comprising a coding sequence for a LSR.
  • examples of methods for preparation of cells for engineering and methods for engineering cells include, for example, those described in U.S. Pat. No.11,266,739 B2, the contents of which are hereby incorporated by reference in its entirety.
  • the present disclosure describes a method of generating a population of engineered cells (e.g., immune cells) comprising introducing an in vitro transcribed polynucleotide construct (e.g., RNA or synthetic RNA) into a cell, wherein the polynucleotide construct encodes a LSR construct as described herein (see, for example, U.S. Pat. No.11,453,719 B2, which is herein incorporated by reference in its entirety).
  • the present disclosure provides methods for preparing a population of cells engineered to express a LSR construct as described herein for administering to a subject.
  • an engineered population of T cells may be expanded and activated as described in U.S. Pat. No.11,084,880 B2, or references cited therein, each of which is herein incorporated by reference in its entirety.
  • Expression of a LSR construct in an engineered population of T cells may be evaluated by ways known in the art including but not limited to, Western Blot analysis using an antigen for a lysis- associated-antigen binding moiety, an antibody against a co-stimulation element(s), and any combination thereof, and analysis of in vitro expansion of engineered LSR-expressing T cells by flow cytometry.
  • the present disclosure describes assays for measuring activity of a population of T cells engineered to express a LSR construct as described herein, including but not limited to anti-cancer activity in in vitro and animal models.
  • Applications/Uses Methods of treatment [0143]
  • technologies of the present disclosure are used to treat a disease, disorder, or condition.
  • diseases may include tumors, cancers, or other proliferative diseases, and/or infectious agents or disease.
  • a method of treatment comprises administration of at least two successive doses of LSR-expressing cells (e.g., cells engineered to express an LSR as described herein).
  • a second dose is administered if (and optionally only if) an intervening assessment has determined that the subject continues to suffer from the relevant disease, disorder or condition and/or that the disease, disorder or condition progressed.
  • a subject who receives therapy with a preparation of engineered cells (i.e., LSR-expressing cells) as described herein is also receiving other therapy for the disease, disorder or condition.
  • engineered cells i.e., LSR-expressing cells
  • a subject who receives therapy with a preparation of engineered cells i.e., LSR-expressing cells
  • the disease disorder or condition is or comprises cancer
  • such subject may receive provided therapy in combination with, for example, one or more other immune-oncology therapies such as, for example other adaptive cell therapy, e.g., targeting one or more disease-associated antigens and/or checkpoint inhibitor therapy.
  • such subject may be receiving or have received cancer therapy that is or comprises chemotherapy, surgery and/ or radiation therapy.
  • Regulating administered cells e.g., the present disclosure describes technologies for regulating LSR-expressing cells (e.g., immunosuppressive agents, small Page 38 of 65 11825822v1 Docket No.: 2017422-0003 molecule drugs, etc) described herein, as described by, for example, Brandt et al.2020, Park et al., 2021, the contents of which is hereby incorporated by reference herein in its entirety.
  • CRS cytokine release syndrome
  • a LSR construct may comprise a masking element that is cleaved off or moved in a tumor microenvironment.
  • Such a LSR construct comprises a masking element inhibiting activity of a lysis-associated-antigen binding moiety, with a linker sensitive to proteolytic cleavage.
  • a lysis-associated-antigen binding moiety is or comprises a conditional single chain variable fragment (scFv), wherein the scFv comprises a small molecule-based control element, so that LSR activity can be regulated by administration of a small molecule drug.
  • scFv conditional single chain variable fragment
  • affinity of a lysis- associated-antigen binding moiety towards a target protein is reduced by activity of a small molecule drug.
  • a LSR construct may comprise a virally derived NS3 protease and a degradation element such that the LSR is tagged for degradation. Moreover, activity of such a LSR is regulated by administration of a NS3 protease inhibitor.
  • Exemplification Example 1 Exemplary Lysis Sensor Receptor Constructs Targeting Lysis-Associated- Antigens [0151] The present Example provides exemplary compositions, preparations, constructs, virions, preparation of virions, and host cells for gene therapy and related methods that target an intracellular product of diseased cells.
  • the present disclosure provides an insight that connecting T cell activation to presence of lysed/lysing cells could dramatically improve engineered T cell therapy (e.g., CAR-T therapy).
  • CAR-T therapy e.g., CAR-T therapy
  • the present disclosure provides CAR constructs that Page 39 of 65 11825822v1 Docket No.: 2017422-0003 respond specifically to cell lysis, described herein as a Lysis Sensor Receptor (LSR).
  • LSR Lysis Sensor Receptor
  • a T cell e.g., immune effector cell
  • a lysis-associated-antigen(s) e.g., an intracellular disease antigen, e.g., tumor antigen
  • a LSR construct comprises an extracellular single chain variable fragment (scFv) of an antibody known to bind a relevant antigen of interest operably linked to a transmembrane element, and one or more T cell co-stimulation elements.
  • a LSR construct further comprises a CD3 ⁇ activation element.
  • the present Example describes certain exemplary models for LSR activation by a lysis-associated-antigen in accordance with the present disclosure.
  • the present disclosure describes that, in some embodiments, antigen binding leads to a direct conformational change in an intracellular domain of a LSR resulting in direct activation (as shown in FIG.3A). In some embodiments, antigen binding causes a change in interaction of a LSR with other endogenous proteins, resulting in signal activation (as shown in FIG.3B).
  • antigen binding causes dimerization or higher order oligomerization of a LSR through the binding of dimeric or oligomeric ligands (as shown in FIG.3C).
  • antigen binding causes dimerization through binding of multiple ligand molecules that form multimeric structures as a result of interactions with other macromolecules, such as nucleic acids, carbohydrates, lipids or proteins (as shown in FIG.3D).
  • an antigen brings LSRs that recognize different antigenic regions of a single protein or a protein complex together resulting in signal activation (as shown in FIG.3E).
  • Example 2 Exemplary Lysis Sensor Receptor Constructs Targeting KRAS
  • LSR Lysis Sensor Receptor
  • the present Page 40 of 65 11825822v1 Docket No.: 2017422-0003 disclosure harnesses this insight by providing LSR constructs, and engineered T cell populations that contain or express LSR constructs, targeting KRAS.
  • the present Example describes KRAS-targeting LSR construct(s) in which KRAS-biding-element(s) of a human anti-KRAS antibody (e.g., as may have arisen in a cancer patient) are linked to a transmembrane element and a T cell receptor co-stimulation element.
  • the present Example specifically exemplifies LSR construct(s) in which KRAS- binding-element(s) of a human anti-KRAS antibody (e.g., as described in Kim et al., 2018, the contents of which is hereby incorporated by reference herein in its entirety) are included in a single-chain antigen-binding domain (scFv) that is linked to a transmembrane element and one or more co-stimulation elements, as shown in FIG.4.
  • a LSR construct described herein comprises a KRAS-binding-element linked to a transmembrane element and CD28, OX40, and CD3 ⁇ co-stimulation elements.
  • LSR Constructs for use in accordance with the present disclosure.
  • Constructs comprising a coding sequence encoding a LSR described herein are generated and tested in producer cells according to standard protocols.
  • one or more LSR backbone constructs are designed for cloning into a lentiviral vector.
  • a preparation of virions comprising a LSR construct are produced by transfecting 293-T cells with a plasmid comprising a LSR construct described herein and three helper plasmids comprising a gag-pol gene, a rev gene, and a VSV-g envelope gene.
  • a LSR construct is cloned into an AAV vector.
  • 293 cells are transfected with a plasmid comprising a LSR construct, an AAV helper plasmid, and an adenoviral helper plasmid. Methods for production of an AAV vector in accordance with the present disclosure are described in, for example, Shin et al. 2013.
  • compositions, preparations, constructs, virions, population of virions, and host cells comprising a variant protoparvovirus VP1 capsid polypeptide can exhibit increased VP1 initiation relative to a reference VP1 capsid polypeptide.
  • Expression of LSR Constructs on T Cells Page 41 of 65 11825822v1 Docket No.: 2017422-0003 [0161]
  • a population of T cells is obtained from healthy human donors and transduced with a preparation of virions comprising a KRAS-targeting LSR described herein. Expression of a KRAS-targeting LSR may be determined by flow cytometry.
  • T cells A population of T cells is cultured in RPMI 1640 medium with 10% FBS and stimulated with anti-CD3/anti-CD28 Dynabeads (Invitrogen). T cells are transduced with a preparation of virions 24 hours after stimulation. Mock transduced cells are used as a negative control and T cells transduced with a CAR construct known in the art is used as a positive control. 4-6 days after transduction, expression of a LSR on the surface of an engineered T cell is evaluated by flow cytometry. [0162] The activation of a LSR, such as through KRAS binding, requires the ability of KRAS to oligomerize an LSR.
  • KRAS is post-translationally modified by myristoylation, the covalent addition of a 14-carbon unsaturated fatty acid. Myristoylation leads to the attachment of KRAS to the inner cell membrane where it is required to be localized for activity. Because multiple KRAS molecules are found localized to the inner cell membrane, binding to LSRs will lead to oligomerization of LSRs, as shown in FIG.5, resulting in activation of LSR signaling and stimulation of T cells. [0163] It is an insight of the present disclosure that altering the composition of the intracellular domains of LSRs can modulate their ability to stimulate T cell activity.
  • an exemplary LSR construct comprises a co-stimulation element comprising a CD28 and an OX40 co-stimulation element as described herein.
  • the present example recognizes that lack of a CD3 element in a LSR construct can reduce stimulatory activity, resulting in reduction of systemic toxicity.
  • a co-stimulation element comprising a CD3 element can be used in combination with a LSR that is highly specific to mutant forms of an intracellular product of diseased cells (e.g. KRAS) as systemic activation through binding of a LSR is expected to be low.
  • KRAS diseased cells
  • LSR constructs that target alternative markers (e.g., alternative intracellular markers).
  • alternative markers e.g., alternative intracellular markers.
  • FIG.6 a number of oncogenes are mutated in a high percentage of tumors; several of the proteins encoded by these oncogenes are intracellular proteins.
  • the present disclosure provides LSR constructs (and Page 42 of 65 11825822v1 Docket No.: 2017422-0003 associated technologies, as described herein) that target other intracellular oncogene- encoded proteins.
  • a LSR comprising a lysis-antigen-binding moiety for one or more P53 epitopes is within the scope of this disclosure exemplified in FIG.7.
  • one feature of the present disclosure is that certain embodiments do not require that the target of an LSR be a disease-specific (or disease-associated) target. That is, because the event of cell lysis may be disease-associated (or disease-specific), in some embodiments, LSR constructs may be directed at intracellular target(s) that are present in non-diseased cells (e.g., at a level or frequency comparable to or even higher than that at which they are present in diseased cells).
  • the present disclosure provides uniquely flexible targeting technologies.
  • Example 3 T cells engineered to express a LSR can be activated by exogenous mutant KRAS
  • the present Example teaches that T cells engineered to express a LSR can be activated by exogenous mutant KRAS.
  • CAR chimeric antigen receptor
  • the present disclosure notes that chimeric antigen receptor (CAR) activation is dependent on antigen density, CAR density, and co-stimulation elements (see, for example, Majzner et al., 2020).
  • CAR chimeric antigen receptor
  • the present Example demonstrates that engineered T cells expressing a KRAS-targeting LSR construct(s) show activation by exogenous mutant KRAS when stimulated by incubation with cell lysates from KRAS-mutant cell lines.
  • a KRAS-mutant cell line corresponds to a G12D KRAS mutant (e.g., AsPC-1, HPAF-II (pancreatic), GP2d, LS180 (colon), T3M-10 (lung).
  • a KRAS-mutant cell line corresponds to a G12V KRAS mutant (e.g., Capan-1, KP-3 (pancreatic), SW480 (colon), COLO 668 (lung).
  • a KRAS-mutant cell line corresponds to a G12C KRAS mutant (e.g., MIA PaCa-2 (pancreas), SW837 (colon), Calu-1, LU65 (lung).
  • a KRAS- mutant cell line corresponds to a KRAS variant with a mutation at a position other than amino acid 12.
  • KRAS-targeting LSR-expressing T cells or control T cells are expanded until the end of log-phase growth, they are artificially stimulated overnight and subsequently co- cultured for 16 hrs with either lysed AsPC-1-KRAS specific target cells, lysed non-KRAS target cells as positive control or no target cells as negative control at a 3 to 1 ratio of effector cells to target cells.
  • Culture supernatants are to be harvested to measure T cell Page 43 of 65 11825822v1 Docket No.: 2017422-0003 proliferation in response to co-culture conditions and cytokine production.
  • IFN-gamma and IL-2 concentration is measured by specific ELISA following manufacturer instructions (R&D).
  • T cells engineered to express a KRAS-targeting LSR will show increased cytokine production after co-culturing with KRAS mutant cells lines when compared to engineered T cells co-cultured with non-KRAS-mutant cells lines.
  • Cytotoxic activity of KRAS-targeting LSR-expressing-T cells is to be evaluated using a Cr release-assay.
  • methods of evaluating cytotoxic activity that may be used in accordance with the present disclosure are known in the art (e.g., flow cytometry, cell viability assays).
  • KRAS-targeting LSR-expressing-T cells are able to induce lysis of KRAS-expressing cell lines with little activity towards non-KRAS-mutant cell lines demonstrating that KRAS LSR T cells are specifically activated by lysed tumor cells only when these cells have KRAS mutations.
  • Example 4 T Cells Expressing a LSR Exert Antibody Dependent Cancer Cell Killing [0170] The present Example provides exemplary constructs, preparations, population of engineered T cells, and producer cells for CAR-T therapy and related methods that demonstrate enhanced killing of a KRAS mutation containing cell line as described herein.
  • anti-cancer activity of a KRAS-targeting LSR is evaluated in a preclinical animal model of pancreatic cancer using the cell line AsPC-1.
  • AsPC-1 cells are engineered to express Click-Beetle Green Luciferase (CB-G Luc + ) to track tumor progression by biolumiscent in vivo imaging (IVIS) and Living Image software (Perkin AElmer).
  • CB-G Luc + AsPC-1 cells are cultured for four weeks and later IV injected into NSG recipients.
  • Engineered T cells expressing the KRAS-targeting LSR are IV injected and tumor burden is evaluated by in vivo imaging.
  • a KRAS-mutant cell line (e.g, AsPC-1) is grown and expanded in vitro according to standard protocols and IV injected in NGS mice. Mice are to be administered 5xl0 6 T cells 7-8 days after tumor implantation. Cells are partially thawed in a 37 degree Celsius water bath and then completely thawed by addition of 1 ml of cold sterile PBS to the tube containing the cells. A population of thawed cells is transferred to a 15 ml falcon tube and adjusted to a final volume of 10 mLs with PBS. KRAS-targeting LSR T cells are washed twice at 1000 rpm for 10 minutes each time and then counted on a hemocytometer.
  • AsPC-1 AsPC-1
  • KRAS LSR T cells are normalized for transduction so that all mice are treated with the same percentage of cells, and resuspended at a concentration of 50x106 cells per mL of cold PBS and kept on ice until mice are dosed. Page 44 of 65 11825822v1 Docket No.: 2017422-0003 [0173] Five to seven mice per group are to be treated with either 100uL of KRAS- targeting LSR T cells, untransduced T cells (mock), or PBS alone. Mice are monitored according to standard protocols for health status, including body weight measurements, and tumor burden via in vivo imaging. Numbers of KRAS LSR T cells are also evaluated via peripheral blood FACS analysis.
  • mice treated with KRAS LSR T cells demonstrate expansion of KRAS LSR T cells compared to mock treated controls.
  • KRAS LSR T cell treated mice show significant reduction in tumor growth compared to controls. Lack of efficacy seen with untransduced T cells (non-lysis-associated-antigen targeting T cells) correlates with a reduced change in tumor growth compared to KRAS LSR T cell treated mice.
  • Example 5 Exemplary Dosing Regimen [0174] The present Example provides exemplary constructs, preparations, population of engineered T cells, and producer cells for LSR T therapy as described herein. Additionally, the present Example provides exemplary dosing regimens for LSR T therapy as described herein.
  • exemplary steps of administering a CAR therapy include, (1) collecting a patient’s peripheral blood, (1) isolating a population of T cells from a blood sample, (3) activation and amplification of such a population of T cells, (4) engineering the obtained population of T cells to express a designed CAR, (5) amplification and quality control measures of an engineered population of CAR-expressing T cells, and (6) transfusion of engineered CAR-T cells in the patient from whom the population of T cells was obtained.
  • a lymphodepleting regimen is cyclophosphamide and fludarabine. All of these dosing regimens involve not using a leukodepleting filter, Page 45 of 65 11825822v1 Docket No.: 2017422-0003 verifying a subject’s identity prior to infusion, premedication with acetaminophen and an H1 antihistamine, confirming availability of tocilizum prior to infusion, and dosing of a CAR-T therapy based on the number of CAR-positive viable T cells. All of these dosing regimens are for autologous use only. All of these dosing regimens are for intravenous use only.
  • Example 6 Production of Exemplary Lysis Sensor Receptor Constructs Targeting KRAS
  • the present Example documents expression of KRAS-targeting LSRs as described herein in T cells.
  • Two exemplary LSR constructs were designed and produced using a KRAS scFv (specifically, an scFv targeting mutant KRAS; the amino acid sequence of this scFv is provided as SEQ ID NO: 1, and is based on human KRAS antibody 1F4_4F10, as described in U.S. Patent No.11,174,314, which is herein incorporated by reference in its entirety), as depicted in FIG.15.
  • LSR Construct 1 includes CD28 and 4-1BB co-stimulation elements and a CD3 ⁇ activating element;
  • LSR Construct 2 includes only the CD28 and 4-1BB co-stimulation elements.
  • Each of these constructs further includes a Flag polypeptide tag used to assess expression in T cells.
  • the particular Flag tag aka FLAG octapeptide; FLAG epitope
  • LSR Constructs 1 and 2 were synthesized; their identities were confirmed by sequencing in both directions.
  • Lentivirus was generated using 293 FT cells, Lentivirus Packaging Mix and transfection agent (Alstem, Richmond, CA, USA) (e.g., as described in Berahovich et al., 2017, which is herein incorporated by reference in its entirety).
  • Virus titers were determined by quantitative RT-PCR using a Lenti-X qRT-PCR kit (Takara Bio, Mountain View, CA, USA) according to manufacturer’s protocol and a 7900HT thermal cycler (Thermo Fisher Scientific, South San Francisco, CA, USA).
  • PBMCs Human peripheral blood mononuclear cells
  • Ficoll-Paque solution GE Healthcare, Chicago, IL
  • PBMCs were suspended at 1 ⁇ 10 6 cells/mL in AIM V-AlbuMAX medium (Thermo Fisher) containing 10% FBS with 300 U/mL IL-2 (Thermo Fisher).
  • PBMC were activated with an equal number of CD3/CD28 Dynabeads (Thermo Fisher), and cultured in non-treated 24-well plates.
  • lentivirus was added to cultures at a multiplicity of infection (MOI) Page 46 of 65 11825822v1 Docket No.: 2017422-0003 of 5 with 1 ⁇ L of TransPlus transduction enhancer (AlStem).
  • MOI multiplicity of infection
  • the vector encoding the KRAS LSR also contained the polynucleotide encoding green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • FIG.16 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and LSR Construct 1.
  • the present Example documents expression of KRAS-LSRs as described herein in T cells.
  • Example 7 Expression of KRAS-LSR Increases T-Cell Killing
  • the present Example documents successful production of T cells expressing both a LSR and CAR in accordance with the present disclosure.
  • the present Example confirms that a KRAS-targeting LSR (e.g., KRAS-LSR) enhances cell killing activity of CAR-T cells.
  • KRAS-targeting LSR e.g., KRAS-LSR
  • the present Example confirms that a KRAS-LSR increased the rate and extent of cell killing by two different types of CAR-T cells: Meso-CAR-T cells and EpCAM-CAR-T cells.
  • T cells expressing a KRAS-targeting LSR i.e., KRAS-LSR-T cells
  • KRAS-LSR-T cells were created as described in Example 6, expressing LSR Construct 1.
  • Mesothelin-specific CAR- T cells e.g., Meso-CAR-T cells
  • T cells expressing both the KRAS-LSR and the Meso- CAR were created by simultaneously transfecting T cells with lentivirus comprising a polynucleotide encoding the KRAS-LSR construct and lentivirus comprising a polynucleotide encoding the Meso-CAR at a 4:1 ratio.
  • KRAS-LSR expression was measured using the FLAG-tag as described in Example 6.
  • A1847 target cells e.g., human ovarian cancer cells
  • adherent A1847 target cells were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 ⁇ 104 cells per well and monitored in culture overnight with impedance-based real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences).
  • RTCA real-time cell analysis
  • AIM V-AlbuMAX medium containing 10% FBS ⁇ 1 ⁇ 105 effector cells KRAS-targeting LSR-expressing T cells, Meso-CAR-T cells, or T cells that were transduced with both an LSR and CAR construct. Cells were monitored with a RTCA system, and impedance was plotted over time. CAR-T cell killing was calculated as (impedance of target cells with mock-transfected T cells—impedance of target cells with effector cells) ⁇ 100/impedance of target cells with mock transfected T cells.
  • T cells expressing both a KRAS-LSR and Meso-CAR showed higher percent cell killing of A1847 cells (e.g., human ovarian cancer cells) relative to Meso-CAR-T cells.
  • FIG.18 depicts that KRAS-LSR-T cells had no detected cell killing activity on their own, whereas Meso-CAR showed about 40% cell killing at 20 hours.
  • KRAS-LSR/Meso-CAR-T cells showed about 65% cell killing at 20 hours.
  • FIG.18 documents that co-expression of a LSR and CAR led to increased cell killing relative to Meso-CAR alone, thus documenting a synergistic effect of such co-expression.
  • FIG.19 shows a schematic depicting a proposed mechanism of action for increased T cell activation and increased cell killing by an exemplary KRAS LSR.
  • Dual CAR-T cells expressing a KRAS-LSR and an epithelial cell adhesion molecule-specific CAR e.g., EpCAM-CAR
  • EpCAM-CAR epithelial cell adhesion molecule-specific CAR
  • EpCAM-CAR expression was assessed using an anti- mouse FAB PE-conjugated antibody. As shown in FIG.20, 4% of transduced T cells showed expression of both a KRAS-LSR and an EpCAM-CAR. [0192] Those skilled in the art will appreciate that EpCAM expression has been described as a biomarker of cancer stem cells (CSCs) or circulating tumor cells (CTCs) and as associated with ovarian cancer, pancreatic cancer, and adenocarcinomas of various primary sites.
  • CSCs cancer stem cells
  • CTCs circulating tumor cells
  • the present Example documents effects of provided KRAS-targeting LSR with a EpCAM-targeting CAR-T in human pancreatic cancer cells. Specifically, T cells expressing both a KRAS-LSR and an EpCAM-CAR showed higher percent cell killing of SW-1990 cells (e.g., pancreatic cancer cells, e.g., pancreatic adenocarcinoma cells) relative to EpCAM-CAR-T cells alone, as depicted in FIG.21.
  • SW-1990 cells e.g., pancreatic cancer cells, e.g., pancreatic adenocarcinoma cells
  • the present Example confirms increased killing due to LSR construct expression in CAR-T targeting two different tumor antigens; these tumor antigens are associated with different types of tumors.
  • Those skilled in the art will be aware of a variety of tumor antigens against which CAR-T have been or can be developed, and will appreciate the usefulness of provided technologies in the context of such CAR-T.
  • several CD19-specific CAR-T and BCMA-specific CAR-T cell therapies have been FDA approved for treatment of Acute Lymphoblastic Leukemia, multiple myeloma, and different B cell malignancies (Mitra, Front Immunol.14:1188049, 2023, which is herein incorporated by reference in its entirety).
  • Example 8 Exemplary KRAS-LSR-T cells Showed Increased T Cell Activation [0195]
  • the present Example documents that co-expression of an exemplary LSR and a CAR in T cells, as described herein, results in a higher degree of T cell activation relative to expression of just the CAR in T cells.
  • A1847 cells e.g., human ovarian cancer cells
  • effector cells Meso-CAR-T cells
  • Meso-CAR/KRAS-LSR-T cells i.e., T cells expressing both a KRAS-targeting LSR and a Meso-CAR, or non-transduced T cells, at a effector to target ratio or E:T ratio of 5:1 or 10:1 in U-bottom 96-well plates with 200 ⁇ L of AIM V- AlbuMAX medium containing 10% FBS, in triplicate. After 24 hours a top 150 ⁇ L of medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells.
  • the present Example documents that T cells expressing both a LSR and a CAR have a higher degree of T cell activation relative to T cells expressing just the CAR (i.e., CAR-T cells).
  • CAR-T cells i.e., CAR-T cells
  • Meso- CAR/KRAS-LSR-T cells have a higher degree of T cell activation relative to Meso-CAR-T cells alone, and relative to a mixture of Meso-CAR-T cells and KRAS-LSR-T cells.
  • LSR and CAR constructs i.e., KRAS-targeting LSRs, Meso-CARs, and EpCAM-CARs
  • LSR and CAR constructs are exemplary and not limiting of the present disclosure; its findings can be expected to be applicable to LSRs targeting other antigens and/or to different CARs.
  • Example 9 Exemplary LSR Show Increased Tumor Specificity and Without Increased Non-specific Activity
  • T cells e.g., CAR-T cells
  • KRAS- LSR-T cells show tumor-specific efficacy (e.g., increased tumor-specificity relative to comparable CAR-T cells not expressing the KRAS-LSR-T cells) without increasing non-specific activity.
  • KRAS-LSR-T cells, Meso-CAR-T cells, and dual KRAS-LSR/Meso-CAR-T cells were prepared as described herein. Killing of A1847 cells was followed by RTCA. E:T ratio for all conditions was maintained at 10:1. For a mixed LSR + CAR sample, both the KRAS LSR and the meso CAR were added at a 5:1 E:T ratio. For all other samples the CAR or LSR cells were added at a 5:1 E:T ratio with an equivalent amount of mock transduced T cells used to bring an overall E:T ratio to 10:1.
  • dual KRAS-LSR/Meso-CAR-T cells increased activation as measured by IFN-gamma secretion relative to Meso-CAR-T cells.
  • Mixing the two populations of T cells, those with the LSR alone and those with the Meso-CAR alone, does not lead to increased T cell activation.
  • the increase in cell activation in the dual KRAS-LSR/Meso-CAR-T cells is believed to be due to the close proximity of the KRAS LSR to the killed A1847 cells, whereas the lack of increased activation from mixing of the two populations of T cells is believed to be due to the lower concentration of KRAS in the overall supernatant.
  • KRAS-LSR-T cells were further tested by addition of purified KRAS protein and A1847 cell lysates.1 ug/ml purified G12D KRAS (Acro Biosystems KRS-H51H4) was added to KRAS-LSR-T cells and cell killing of A1847 cells was followed using RTCA. Results were normalized to KRAS-LSR-T cells alone. Addition of purified KRAS did not increase cell killing in other controls (Data not shown). Supernatant from A1847 cells previously killed by Meso-CAR-T cells was collected and frozen.
  • AKRAS antibody raised against mutant KRAS was used to test specificity of KRAS-LSR-T cell activation.
  • Anti-KRAS mutant monoclonal antibody was used at a 1:1000 dilution (Thermofisher, cat#: MA5-36256). A1847 cell killing was followed by RTCA.
  • anti-KRAS mutant antibody reduced rate of cell killing by dual KRAS LSR/Meso-CAR-T cells. Increased cell killing that results from dual expression of the KRAS-LSR and the Meso-CAR in the same T cells is blocked by a monoclonal Page 51 of 65 11825822v1 Docket No.: 2017422-0003 antibody to mutant KRAS.
  • the present disclosure describes that LSR activation occurs through binding of KRAS released by cell lysis.
  • the present Example confirms that KRAS-LSR-T cells described herein show tumor-specific efficacy without an increase in non-specific activity.
  • the present Example confirms that purified KRAS protein increases activity of a KRAS-LSR-T cell.
  • the present Example also confirms that an anti-KRAS antibody inhibits activity of a KRAS-LSR-T cell.
  • Embodiment 1 Embodiment 1.
  • An engineered lysis sensor receptor comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element).
  • a lysis-associated-antigen binding moiety comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element).
  • a signaling element e.g., a T cell receptor co-stimulation element
  • the engineered LSR of embodiment 1, wherein the T cell receptor co-stimulation element is selected from a group consisting of CD28, OX40, 4I- BB, GITR, ICOS-1, CD27, AP10, and any combination thereof.
  • Embodiment 4 The engineered LSR of embodiment 3, wherein the co- stimulation element further comprises a CD3 zeta (CD3 Z or CD3C) activating element.
  • the engineered LSR of embodiment 3, wherein the co- stimulation element does not comprise a CD3 zeta (CD3 Z or CD3C) activating element.
  • a polynucleotide construct comprising a payload coding sequence operably linked to an expression control sequence, wherein the payload coding sequence encodes the LSR of embodiment 1.
  • Page 52 of 65 11825822v1 Docket No.: 2017422-0003 [0211]
  • Embodiment 7. A vector comprising the polynucleotide construct encoding the LSR of embodiment 1 to facilitate introduction into cells.
  • Embodiment 9 The vector of embodiment 8, wherein the viral vector lacks ability to replicate so as not to replicate in an infected cell.
  • HBV-1 human immunodeficiency virus 1
  • HMV-2 human immunodeficiency virus 2
  • VMV visna-maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • Embodiment 13 The preparation of virions of embodiment 11, wherein the viral capsid is an AAV capsid.
  • Embodiment 13 The preparation of virions of embodiment 11, wherein the polynucleotide construct comprises at least one inverted terminal repeat (ITR).
  • Embodiment 14 The preparation of virions of any one of the preceding embodiments, which virions are active to infect an immune effector cell.
  • Embodiment 15. The preparation of virions of embodiment 14, wherein the immune effector cell is a T cell. Page 53 of 65 11825822v1 Docket No.: 2017422-0003
  • Embodiment 16 The preparation of virions of embodiment 15, wherein the T cell is an autologous T cell.
  • Embodiment 17 The preparation of virions of embodiment 15, wherein the T cell is an allogenic T cell.
  • Embodiment 18 The preparation of virions of embodiment 14, wherein the immune effector cell is an NK cell.
  • Embodiment 19 The preparation of virions of embodiment 14, wherein the immune effector cell is an NK cell.
  • a construct comprising: (i) a first sequence that is or comprises a payload coding sequence operably linked to an expression control sequence, wherein the payload coding sequence encodes the LSR of embodiment 1 (ii) a second sequence that is or comprises a Rep protein-dependent origin of replication (ori); and (iii) at least one viral ITR; (b) the Rep protein that acts on the ori, (c
  • a gene editing system comprising: (a) a CRISPR/Cas gene editing system comprising: (i) a gRNA molecule having a nucleotide sequence comprising a targeting sequence specific to an integration site in the T cell genome, and (ii) a Cas9 protein or a nucleic acid encoding a Cas9 protein; and (b) a virion comprising a viral capsid and the polynucleotide construct of embodiment 6. [0225] Embodiment 21.
  • a population of T cells engineered to express a LSR comprising: Page 54 of 65 11825822v1 Docket No.: 2017422-0003 a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element).
  • Embodiment 22 The population of T cells of embodiment 21, wherein the population of T cells is isolated from a human.
  • Embodiment 23 The population of T cells of embodiment 22, wherein the human is a patient to whom the engineered population of T cells is to be administered.
  • Embodiment 24 Embodiment 24.
  • Embodiment 27 A method of producing a population of T cells according to any of the preceding embodiments comprising steps of: (a) obtaining a plurality of T cells; (b) introducing into the T cells: (i) the gene editing system of embodiment 20, so that a population of engineered T cells is produced. [0232] Embodiment 28.
  • Embodiment 29 A method of producing a population of engineered T cells according to any of the preceding embodiments comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the polynucleotide construct of embodiment 6.
  • Embodiment 29 A method of treating a disease, comprising a step of: Page 55 of 65 11825822v1 Docket No.: 2017422-0003 (a) administering to a subject suffering from a disease, disorder or condition, a population of T cells engineered to express a LSR that responds to/targets an intracellular product of diseased cell(s) in the subject.
  • Embodiment 30 A method of producing a population of engineered T cells according to any of the preceding embodiments comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the polynucleotide construct of embodiment 6.
  • Embodiment 31 The method of embodiment 29, wherein the diseased cells are tumor cells.
  • Embodiment 32 The method of embodiment 29, wherein the diseased cells are infected cells.
  • Embodiment 33 The method of embodiment 29, wherein the diseased cells are infected cells.
  • the step of administering comprises administering a population of T cells engineered by a process comprising steps of: (a) obtaining a plurality of T cells; (b) transducing the cells with a preparation of virions according to any one of embodiments 11-18, optionally further selecting or screening for the transduced cells; and (c) administering the transduced cells to a subject in need thereof.
  • Embodiment 34 A method of stimulating endogenous tumor infiltrating lymphocytes (TIL) in a subject, the method comprising steps of: (a) obtaining a population of TIL from a subject; and (b) engineering the population of T cells to express the LSR of embodiment 1, so that a population of engineered TIL is generated.
  • TIL tumor infiltrating lymphocytes
  • Embodiment 35 The method of embodiment 34, the method further comprising a step of: administering the population of engineered TIL. Page 56 of 65 11825822v1 Docket No.: 2017422-0003 [0240] Embodiment 36. The method of embodiment 34, wherein the obtained population of TIL comprises a plurality of TIL having TCRs directed to different antigens. [0241] Embodiment 37. The method of embodiment 35, wherein the administered population of engineered TIL comprises a plurality of engineered TIL having TCRs directed to different antigens. [0242] Embodiment 38. The method of treatment of embodiment 29, wherein the method of treatment comprises at least two successive doses. [0243] Embodiment 39.
  • Embodiment 40 The method of treatment of embodiment 29 wherein the treatment is administered in combination with another immune oncology therapy.
  • Embodiment 41 The method of treatment of embodiment 40 wherein the immune oncology therapy is a checkpoint inhibitor, a cytotoxic chemotherapy, or radiation therapy.
  • Embodiment 42 A method of characterizing a LSR according to any of embodiments 1-5, or a population of T cells according to embodiments 21-26.
  • a method of manufacturing a population of T cells comprising: (i) engineering the population of T cells to express the LSR of embodiments 1-5.
  • Embodiment 44 The method of manufacturing of embodiment 43, wherein the step of engineering comprises introducing the preparation of virions of embodiments 11-18 such that the payload encoding the LSR of embodiment 1 integrates at a site in the T cell genome. Page 57 of 65 11825822v1 Docket No.: 2017422-0003
  • Embodiment 45 The method of manufacturing a population of T cells according to embodiment 43 further comprising a step of isolating T cells from a human subject and administering to the human subject.

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Abstract

Disclosed herein are Lysis Sensor Receptors (LSR) comprising an extracellular domain with affinity and specific for an intracellular product of a diseased cell such as a cancer cell; a transmembrane element and at least one signaling element. Also provided herein are nucleic acids encoding such LSRs and cells (e.g., immune cells such as T cells) engineered to contain and/or express them. Also provided are various uses of such LSRs and engineered cells, including in the treatment of cancer, for example to enhance T cell activity (e.g., TIL and/or CAR-T cell activity) and/or to enhance antibody-based therapy, such as cancer immunotherapy.

Description

Docket No.: 2017422-0003 LYSIS SENSING RECEPTORS AND USES THEREOF Background [0001] Cancer immunotherapy, including CAR-T cell therapy, is used to provoke immune responses attacking tumor cells while sparing normal tissues. The present disclosure provides technologies related to compositions comprising a lysis sensing CAR Targeting an intracellular product of tumor cells and related methods. Summary [0002] Targeted immunotherapies rely on the use of immune cells or molecules that engage immune cells to treat a variety of diseases, including primarily cancer but also infectious diseases and autoimmune disorders. Recently, engineering T cells to express chimeric antigen receptors (CARs) that target tumor antigens has allowed the successful eradication of leukemic cells in humans. CARs have two functional elements: an extracellular antigen recognition element, typically derived from an antibody, and an intracellular stimulatory element or set of elements. Upon recognition of an antigen on a tumor cell, CARs activate cytotoxic activity of T cells, which in turn results in tumor cell lysis. [0003] Among other things, the present disclosure identifies challenges with certain existing CAR-T cell therapies, including, for example that an antigen must be expressed on a tumor cell surface to be available for recognition. Alternatively or additionally, the present disclosure identifies challenges with existing CAR-T cell therapies in that most tumor cell surface antigens that have been identified or utilized as targets of such CAR-T cell therapies are also expressed on healthy cells, reducing tolerance to CAR-T therapy and limiting effectiveness. [0004] The present disclosure appreciates that many epitopes preferentially or specifically associated with tumors (e.g., neoantigen epitopes, and many epitopes associated with tumor driver mutations) are primarily or wholly intracellular. Although intracellular proteins can be recognized by T cell receptors (TCRs) if presented to them, that presentation process (a) is not specific to tumor-associated (let alone tumor-specific) antigens; and (b) presents only small fragments of intracellular proteins, which may not include relevant epitopes (e.g., that are tumor-associated or tumor-specific). Thus, only small fragments of these proteins are presented to TCRs. This, in turn, limits the ability of the immune system Page 1 of 65 11825822v1 Docket No.: 2017422-0003 to detect intracellular mutations. Furthermore, as tumors accumulate mutations, the neoantigens that they present to T cells changes, rendering some previously responsive T cells ineffective. [0005] The present disclosure provides an insight that connecting T cell activation to presence of lysed/lysing cells could dramatically improve engineered T cell therapy (e.g., CAR-T therapy). Among other things, the present disclosure provides CAR constructs that respond specifically to cell lysis, described herein as a Lysis Sensor Receptor (LSR). The present disclosure describes LSR constructs comprising, for example, an extracellular lysis- antigen-binding moiety, a transmembrane element, and an intracellular T cell receptor co- stimulation element. The present disclosure further provides engineered immune cell populations (e.g., engineered T cells) that express such LSRs, for example as a Co-CAR receptor. The present disclosure further provides various insights and technologies relating to such LSR constructs and/or their expression by engineered cells including, for example, methods of making and/or using such constructs and/or cells that express them. [0006] Among other things, the present disclosure provides an insight that immune cells expressing such a LSR construct may be useful to enhance immune responses (e.g., by activating endogenous T cells), for example in the treatment of cancer. LSRs provide a mechanism to dramatically expand the universe of antigens that can be used to stimulate an immune response in diseased tissues by bypassing the limitations of other immunotherapies imposed by the intracellular location of potential target antigens. [0007] The present disclosure recognizes that LSRs have a further advantage over other activators of the immune response in that their activating antigens are only made available after cell lysis, thereby reducing the risk of toxicities that can result from antigens which may be expressed on the surface of both healthy and diseased cells. In some embodiments, LSRs are designed to selectively bind to antigens that are disease-specific, such as cancer- specific mutants or viral proteins, further reducing the potential for inadvertent immune cell activation by processes involved in healthy cell turnover. Dependence on cell lysis serves as a positive feedback loop which increases the activity of immune cells in diseased tissues while minimizing their activation in other areas of the body. [0008] The present disclosure describes that populations of T cells (e.g., a population of T cells isolated from a human) engineered to express a LSR maintain their endogenous TCR heterogeneity. Thus, in some embodiments, engineered T cell subpopulations will recognize diseased cells even if disease-specific antigens presented to TCR subclones change. Page 2 of 65 11825822v1 Docket No.: 2017422-0003 [0009] In some embodiments, LSRs will amplify the response of any T cells in the vicinity of a diseased cell that are capable of recognizing a disease-specific antigen, thereby stimulating cytotoxicity and reinforcing clonal expansion of T cells with effective TCRs. This approach provides a distinct advantage in the treatment of diseases with rapidly changing characteristics, such as tumor cells or viral infections, by limiting the need for artificial amplification of tumor antigen presentation or genetic engineering to customize LSRs for specific antigens. As a tumor (or other diseased tissue or cell, such as infected cells) evolves and presentation of antigens changes, LSRs will stimulate T cells encoding TCRs that recognize these new antigens without the need to develop a new therapy and thereby limiting the potential for resistance to develop. [0010] In some embodiments, use of LSRs as described herein can provide immune cell therapies, e.g., for solid tumors, that are (i) more effective, (ii) safer and (iii) more durable than existing cell therapies. Brief Description of the Drawing [0011] The drawing, comprising the below-described figures, forms part of the present specification and is included to further demonstrate and/or facilitate appreciation of certain aspects of the present disclosure. [0012] FIG.1 shows an image depicting a positive feedback loop between activated engineered T cells expressing a LSR reacting with and lysing tumor cells in their proximity causing lysis of more tumor cells, resulting in more activation of LSR-expressing T cells. [0013] FIG.2 shows an image depicting certain exemplary LSR constructs comprising an extracellular single chain variable fragment (scFv) operably linked to a transmembrane element, and one or more T cell co-stimulation elements. In some embodiments, an exemplary LSR construct further comprises a CD3ζ co-stimulation element. [0014] FIG.3 shows an image depicting exemplary models for LSR activation by a lysis-associated-antigen (e.g., antigens that are made available to a LSR upon the lysis of a diseased cell (e.g., intracellular antigens/epitopes)). [0015] FIG.4 shows an image depicting exemplary anti-KRAS LSR constructs. [0016] FIG.5 shows an image depicting activation and oligomerization of LSRs by KRAS binding. [0017] FIG.6 shows a table depicting a number of oncogenes mutated in a high percentage of tumors; several of the proteins encoded by these oncogenes are intracellular Page 3 of 65 11825822v1 Docket No.: 2017422-0003 proteins. The present disclosure provides LSR constructs (and associated technologies, as described herein) that target such intracellular oncogene-encoded proteins. [0018] FIG.7 shows an image depicting an exemplary Anti-P53 LSR construct. [0019] FIG.8 shows an image depicting exemplary steps of a LSR T therapy. [0020] FIG.9 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Abecma (idecabtagene vicleucel). [0021] FIG.10 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Breyanzi (lisocabtagene maraleucel). [0022] FIG.11 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Kymriah (tisagenlecleucel). [0023] FIG.12 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Tecartus (brexucabtagene autoleucel). [0024] FIG.13 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Yescarta (axicabtagene ciloleucel). [0025] FIG.14 shows an image depicting the dosage and administration regimen for the FDA approved CAR-T therapy Carvykti (ciltacabtagene autoleucel). [0026] FIG.15 depicts two exemplary LSR construct designs according to an embodiment of the present disclosure. [0027] FIG.16 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct. [0028] FIG.17 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct, Meso-CAR-T cells, and dual T cells expressing both a Meso-CAR and a KRAS-LSR (e.g., dual KRAS-LSR/Meso-CAR-T cell). [0029] FIG.18 shows a graph depicting percent cell killing of A1847 cells by dual KRAS-LSR/Meso-CAR-T cells, Meso-CAR-T cells, and KRAS-LSR-T cells over time (0 to 21 hours). [0030] FIG.19 shows a schematic depicting a proposed mechanism of action for increased T cell activation and increased cell killing by an exemplary KRAS LSR. [0031] FIG.20 shows a graph depicting flow cytometry data depicting detection of KRAS-LSR-T cells expressing GFP and a KRAS-LSR construct, EpCAM-CAR-T cells, and dual T cells expressing both a EpCAM-CAR and a KRAS-LSR (e.g., dual KRAS- LSR/EpCAM-CAR-T cell). Page 4 of 65 11825822v1 Docket No.: 2017422-0003 [0032] FIG.21 shows a graph depicting percent cell killing of SW-1990 cells by dual KRAS-LSR/EpCAM-CAR-T cells, EpCAM-CAR-T cells, and KRAS-LSR-T cells over time (0 to 20 hours). [0033] FIG.22 shows a graph depicting IFN-gamma levels in supernatant collected from A1847 cells (e.g., human ovarian cancer cells) cultured with Meso-CAR-T cells, Meso-CAR/KRAS-LSR T cells (e.g., T cells expressing both a KRAS-targeting LSR and a Meso-CAR, or non-transduced T cells. Meso-CAR/KRAS-LSR T cells have a higher degree of T cell activation, as measured by IFN-gamma expression, than Meso-CAR-T cells alone. [0034] FIG.23 shows a graph depicting fraction cell killing of A1847 cells by KRAS- LSR-T cells, Meso-CAR-T cells, a mixture of KRAS-LSR-T cells and Meso-CAR-T cells, and dual KRAS-LSR/ Meso-CAR-T cells over time (0 to 48 hours). [0035] FIG.24 shows a graph depicting fraction cell killing of A1847 cells by KRAS- LSR-T cells with addition of purified KRAS protein or supernatant from A1847 cells previously killed by Meso-CAR-T cells. Addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells, resulted in moderate cell killing of A1847 cells by KRAS-LSR-T cells. Addition of purified G12D KRAS resulted in increased killing of A1847 cells by KRAS-LSR-T cells relative to addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells. [0036] FIG.25 shows a graph depicting fraction cell killing of A1847 cells by dual KRAS-LSR/Meso-CAR-T cells with and without addition of an anti-KRAS antibody. Addition of anti-KRAS antibody reduced rate of cell killing by dual KRAS LSR/Meso- CAR-T cells. Definitions [0037] Administration: As used herein, the term “administration” typically refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., that is or comprises one or more cells, tissues, organisms, etc), for example to achieve delivery of an agent that is, is included in, or is otherwise delivered or generated by, such composition or treatment. [0038] CDR: as used herein, the term “CDR” refers to a complementarity determining region within an immunoglobulin (e.g., antibody, T cell receptor) variable region. Those skilled in the art are aware that canonical antibodies include heavy and light chains, each of Page 5 of 65 11825822v1 Docket No.: 2017422-0003 which is comprised of a variable region and a constant region. There are three CDRs in each such variable region, designated CDR1, CDR2 and CDR3. Analogously, T cell receptors comprise a and B chains, or d and g chains, each of which includes 3 CDRs.. A "set of CDRs" or "CDR set" refers, as will be clear from context and understood by those skilled in the art, either to the group of three CDRs that occur in a particular chain, or to the set of six CDRs (i.e., three heavy chain and three light chain CDRs) that are found together in a particular immunoglobulin (and e.g., contribute to or determine its antigen-specificity). . Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.); those skilled in the art appreciate the differences between and among these systems and are capable of understanding CDR boundaries to the extent required to understand and to practice the claimed invention. [0039] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied. [0040] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate Page 6 of 65 11825822v1 Docket No.: 2017422-0003 reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure. [0041] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents. [0042] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms. [0043] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in Page 7 of 65 11825822v1 Docket No.: 2017422-0003 some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen). [0044] Effector function: as used herein refers a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). In some embodiments, an effector function is one that operates after the binding of an antigen, one that operates independent of antigen binding, or both. [0045] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and/or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and/or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic Page 8 of 65 11825822v1 Docket No.: 2017422-0003 information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity. [0046] Framework" or "framework region: as used herein, refers to the sequences of an immunoglobulin (e.g., antibody) variable region minus the CDRs. Because a CDR sequence can be determined by different systems, likewise a framework sequence is subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four sub-regions (FRl, FR2, FR3 and FR4) on each chain, in which CDRl is positioned between FRl and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, FR1, for example, represents the first framework region closest to the amino terminal end of the variable region and 5' with respect to CDR1, and FRs represents two or more of the sub-regions constituting a framework region. [0047] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic”amino acids, Page 9 of 65 11825822v1 Docket No.: 2017422-0003 and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution. Typical amino acid categorizations are summarized below:
Figure imgf000011_0001
Page 10 of 65 11825822v1 Docket No.: 2017422-0003
Figure imgf000012_0001
As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Page 11 of 65 11825822v1 Docket No.: 2017422-0003 [0048] Identity: As used herein, the term “identity” refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as a corresponding position in the second sequence, then the two molecules (i.e., first and second) are identical at that position. Percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. [0049] “Improve,” “increase”, “inhibit” or “reduce”: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement Page 12 of 65 11825822v1 Docket No.: 2017422-0003 may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. [0050] Isolated: as used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated). In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be "isolated" when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced. Page 13 of 65 11825822v1 Docket No.: 2017422-0003 [0051] Operably linked: as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, "operably linked" control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest. [0052] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. [0053] Prevent or prevention: as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. In some embodiments, prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. [0054] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control. [0055] Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to Page 14 of 65 11825822v1 Docket No.: 2017422-0003 occur. A binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree and/or rate of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree and/or rate of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations. [0056] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition. [0057] Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments, a tumor may be a disperse tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor. [0058] Tumor-specific: As used herein, the term “tumor-specific” refers to an agent that is associated with tumor cells and not with appropriate reference non-tumor cells. In some embodiments, neoantigens (e.g., that arise in a tumor, for example by mutation that occurs in the tumor) are tumor-specific antigens. Page 15 of 65 11825822v1 Docket No.: 2017422-0003 [0059] Tumor-associated: As used herein, the term “tumor-associated” refers to an agent that is more likely to be present (or detectable) in and/or on tumor cells than appropriate reference non-tumor cells. [0060] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three- dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In Page 16 of 65 11825822v1 Docket No.: 2017422-0003 some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature. In some embodiments, a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid. [0061] Vector: as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are Page 17 of 65 11825822v1 Docket No.: 2017422-0003 operatively linked. Such vectors are referred to herein as "expression vectors." Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Detailed Description of Certain Embodiments Adoptive Cell Therapy [0062] Adoptive Cell Therapy (ACT) strategies are revolutionizing cancer care by using immune cells – typically cultured immune cells as therapeutic agents to attack patients’ tumors. Most ACT therapies can be grouped into one of three types: (i) tumor-infiltrating lymphocyte (TIL) therapy, a T-cell receptor therapy designed to boost activity of naturally occurring immune cells found in a patient’s tumor, (ii) TCR cell therapy which relies on creating T cells enriched with specific T-cell receptors (TCR) to mediate tumor targeting, and (iii) chimeric antigen receptor (CAR) therapy, which instead utilizes an engineered CAR construct to target T cells (reviewed, for example, in Rosenberg & Restifo, Science 348:62, 2015; June et al., Science 359:1361, 2018; etc, the contents of which is hereby incorporated by reference herein in its entirety). [0063] Initial adoptive cell therapies utilized tumor infiltrating lymphocytes (TIL) that were isolated from patients’ tumors. Although targets of these TIL are not known, it is presumed that TIL isolated from a tumor are enriched in T cells directed against cancer cells. These TIL are typically expanded and activated ex vivo, and optionally selected or purified to enrich for cells demonstrated to target antigens expressed by tumors. These tumor-associated or tumor-specific antigens, can include antigens associated with tumor drivers, neoantigens (novel antigens arising from genetic mutations) or other antigens that are enriched on tumor cells compared to healthy cells). An expanded population of TIL is then administered to a patient, who is usually the same person from whom the original TIL were isolated. TIL therapy was first demonstrated to achieve regression in melanoma Page 18 of 65 11825822v1 Docket No.: 2017422-0003 patients in 1988 (see, Rosenberg et al., N. Engl. J. Med 319:1676, 1988, the contents of which is hereby incorporated by reference herein in its entirety), but initial responses were short-lived and administered T-cells were undetectable in the recipients within just a few days. It was subsequently determined that lymphoablation prior to administration of a TIL preparation could dramatically improve durability of response, and apparent successful engraftment of administered TIL (see, for example, Dudley et al., Science 298:850, 2002, the contents of which is hereby incorporated by reference herein in its entirety). Lymphoablation can be utilized prior to any adoptive cell therapy. More recently, improvements in TIL therapy have been driven by use of certain factors (e.g., cytokines, certain antibodies, such as certain anti-PD-1 and/or anti-CTLA-4 antibodies, etc) to enhance survival and activation of TIL (see, Zhao and Cao, 2019, the contents of which is hereby incorporated by reference herein in its entirety). In some embodiments one or more such agents (e.g., in certain embodiments one or more cytokines) are added exogenously. In some embodiments, TIL are genetically engineered to express one or more such agents (e.g., one or more cytokines). [0064] TCR therapies, which include both naturally occurring TILs and engineered cells involve administering T cells whose T-cell receptors (TCRs) target particular antigens of interest (e.g., tumor antigen, such as a tumor-associated antigen or a tumor-specific antigen). T-cell receptors are transmembrane heterodimers of either α- and β- chains or δ- and γ- chains containing CDRs that together determine characteristics of the receptor’s antigen binding (e.g., its specificity and/or affinity, etc). Cells in the body have a mechanism for displaying on their surface a sampling of every protein that is being made by a cell. This includes all normal proteins as well as aberrant proteins if a cell is cancerous or proteins from pathogens if a cell has been infected. These proteins are broken down into short fragments, or peptides, and these peptides are loaded into Major Histocompatibility Complexes, or MHCs, to be displayed on the outside of a cell. TCRs recognize their cognate antigens only when such antigens are presented by the MHC. [0065] The specific makeup of an individual’s MHC is determined by their Human Leukocyte Antigen, or HLA, type. For a TCR to recognize an antigen, it should match both the HLA type expressed by cells in that patient and a specific antigen peptide. It has been estimated that there are 1010 (10 billion) distinct TCRs in an individual providing the opportunity for recognition of a broad range of potential antigens (see, J Theor Biol.2016 Jan 21; 389: 214–224, the contents of which is hereby incorporated by reference herein in its Page 19 of 65 11825822v1 Docket No.: 2017422-0003 entirety). However, within a given individual these TCRs are understood to recognize a specific HLA type. In cases where T cells have been engineered with TCRs designed to target select tumor-specific antigens, their application is limited to individuals who also have a matching HLA type. [0066] CAR-T therapy involves engineering T cells to express a chimeric antigen receptor (CAR) construct, to enable T cells to be activated independent of a T cell receptor. Such a CAR construct links an antigen binding moiety, typically a single chain variable fragment (scFv) of an antibody known to bind the relevant antigen of interest, with TCR signaling domain(s) and/typically, one or more additional T cell co-stimulation elements (e.g., derived from receptors such as CD28, OX40, CD137, etc). CAR constructs target surface antigens, and typically do so with very high avidity. A limitation of CAR-T therapies is that CARs cannot penetrate cells and thereby cannot recognize intracellular antigens. CAR-T therapy is also distinguished from TCR therapy through its ability to recognize antigens independent of HLA type. However, in practice, most CAR-T therapies are generated as autologous therapies to limit potential for mismatches in HLA type that can arise when generating CAR-T therapies from allogeneic donors. These mismatches can lead to the rejection of therapy. [0067] The United States Food and Drug Administration has approved at least six adoptive cell therapies, all of which are CAR-T therapies, approved for treatment of hematological cancers. Specifically, as of November 2022, CAR-T therapies have been approved for the treatment of large B-cell lymphoma , multiple myeloma, follicular lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia. Certain approved CAR-T therapies have reported stunning success, including recurrence rates reduced by 80% or more. However, cytokine release syndrome (also known as “cytokine storm” has plagued certain CAR-T therapies, which seem to induce much more dramatic such reactions than have been observed with TCR (e.g., TIL and/or engineered TCR) therapies (see, for example, Kalos et al., Sci. Transl. Med.3:95ra73, 2011, the contents of which is hereby incorporated by reference herein in its entirety). [0068] Upon administration to patients, activation of potent CAR-T therapies often leads to the production of a large number of inflammatory cytokines, a condition known as cytokine release syndrome (CRS). CRS can be a life-threatening consequence of CAR-T therapy leading to modifications of treatment regimens to limit its severity and to the development of CAR-T therapies with safety switches which lead to the inactivation of the Page 20 of 65 11825822v1 Docket No.: 2017422-0003 therapy with the intent of halting the escalation of CRS (see, Zhao and Cao, 2019, the contents of which is hereby incorporated by reference herein in its entirety). [0069] CAR-T therapies that have been approved by the FDA have exclusively targeted antigens that are exposed on proteins expressed on a cell surface. These therapies have been primarily focused on common surface proteins that are expressed on many hematologic malignancies. By contrast, solid tumors have a high degree of heterogeneity with few common surface-expressed antigens which limits the potential of generating broadly effective CAR-T therapies. In addition to the scarcity of tumor-specific targets, most surface-expressed proteins on tumor cells are also expressed on healthy cells, leading to toxicities due to effects of the highly cytotoxic CAR-T cells in non-tumor tissues. For example, HER2 is a surface-expressed protein that is also expressed in tissues such as the lung. The first patient to be treated with a HER-2 CAR-T cell therapy developed by the National Cancer Institute fell into a coma soon after and died five days later (Zhao and Cao, 2019, the contents of which is hereby incorporated by reference herein in its entirety). [0070] Furthermore, expression of surface-expressed antigens can be lost, resulting in evasion of therapies that target them. An advantage of TIL therapy is that patients are treated with a population of cells that contain distinct TCRs, increasing the probability that the treatment contains cells that can target multiple tumor-specific antigens – antigens that exist at the time of treatment and antigens that may emerge as the tumor cells emerge that have altered antigens (see, Wang et al. BMC Medicine 2021). Due to the dependence of TIL therapy on TCR activation, it can detect protein changes that occur within cells where the majority of tumor-specific changes occur (see, Schietinger et al., 2008, the contents of which is hereby incorporated by reference herein in its entirety). Co-stimulation [0071] Among other things, the present disclosure recognizes that co-stimulation is a fundamental component of T cell biology and of adoptive cell therapy, specifically including CAR-T cell therapy. Effective potency of a CAR-T cell is dependent on incorporation of one or more intracellular T cell receptor co-stimulation protein elements, which serve to increase CAR-T cell persistence and enhance cell cytotoxicity in response to antigen binding to an extracellular antigen binding moiety (see, Harrison et al, 2021, the contents of which is hereby incorporated by reference herein in its entirety). First generation CARs lacked any co-stimulation elements and had limited clinical efficacy. Second generation CARs have a single co-stimulation element as exemplified by FDA approved CAR-T products Abecma Page 21 of 65 11825822v1 Docket No.: 2017422-0003 (idecabtagene vicleucel), Breyanzi (lisocabtagene maraleucel), Kymriah (tisagenlecleucel) and Carvykti (ciltacabtagene autoleucel), which contain a 4-1BB co-stimulation element, and Tecartus (brexucabtagene autoleucel) and Yescarta (axicabtagene ciloleucel), which contain a CD28 co-stimulation element. Third generation CARs containing at least two co- stimulation elements have been reported to have superior antitumor activity and longer persistence (see, Harrison et al, 2021, the contents of which is hereby incorporated by reference herein in its entirety). [0072] The present disclosure describes LSR constructs comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary elements. In some embodiments, an exemplary LSR construct comprises a CD28 co-stimulation element. In some embodiments, an exemplary LSR comprises a 4-1BB co-stimulation element. In some embodiments, an exemplary LSR comprises a OX40 co-stimulation element. In some embodiments, an exemplary LSR comprises a CD27 co-stimulation element. In some embodiments, an exemplary LSR comprises a GITR co-stimulation element. In some embodiments, an exemplary LSR comprises a ICOS co-stimulation element. [0073] In some embodiments, a LSR as described herein comprises one or more T cell receptor co-stimulation elements. In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element and a 4-1BB co-stimulation element. [0074] In some embodiments, a LSR as described herein comprises one or more T cell receptor co-stimulation elements as well as a CD3ζ activation element. In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element, a 4-1BB co- stimulation element, and a CD3ζ activation element. Lysis-Sensing Adaptive Cell Therapy [0075] The present disclosure provides an insight that linking T cell activation to cell lysis can provide a number of therapeutic benefits. For example, the present disclosure appreciates that certain tumor environments (including specifically certain solid tumor environments) are characterized by cell lysis (e.g., have a relatively elevated level of cell lysis products). The present disclosure further provides an insight that linking of T cell activation to cell lysis can be achieved through use of a Lysis Sensor Receptor (LSR) as described and provided herein. Page 22 of 65 11825822v1 Docket No.: 2017422-0003 [0076] Among other things, the present disclosure provides Lysis Sensor Receptors, which are activated by antigens that are made available to these receptors upon the lysis of a diseased cell (e.g., intracellular antigens/epitopes). The present disclosure further comprises immune cells engineered to express such LSRs. In some embodiments, such immune cells include T cells with an endogenous TCR; in some embodiments, such T cells include an engineered TCR. Alternatively or additionally, in some embodiments, such T cells include a CAR. In some embodiments, a LSR may be expressed in immune cells that have disease- fighting activity that are not dependent on a TCR or CAR including natural killer (or NK) cells. [0077] In some embodiments, introduction of LSRs into T cells can lead to activation of T cells that are in close proximity to a diseased tissue, such as a tumor. In some embodiments, such introduction achieves local activation of T cell cytotoxic activity in the vicinity of lysed cells. Lysis Sensor Receptor Constructs [0078] Among other things, the present disclosure provides Lysis Sensor Receptor constructs (LSRs) that comprise a lysis-associated-antigen binding moiety, a transmembrane element, and a signaling element (e.g., a T cell co-stimulation element or elements). [0079] The present disclosure describes LSR constructs that, when expressed on a cell (e.g., an immune cell, e.g., a T cell), a lysis-associated-antigen binding moiety is located extracellularly, e.g., such that it is arranged and constructed to bind a target lysis-associated- antigen, and a signaling element is located in cytoplasm, e.g., such that it is arranged and constructed for signal transduction and/or cell activation. Lysis-associated-antigens [0080] The present disclosure teaches that targeting antigens associated with cell lysis (lysis-associated-antigens) as described herein can provide valuable benefits (e.g., therapeutic benefits). A lysis-associated-antigen is a protein, nucleic acid, carbohydrate, lipid or small molecule that becomes exposed at higher levels extracellularly upon lysis of a cell. Preferably, such antigens are specific to diseased cells or are present at substantially higher levels in diseased cells compared to healthy cells. In the case of tumors, such antigens include proteins encoded by tumor-specific mutations, proteins whose levels are increased due to altered signaling in tumor cells, nucleic acids expressed at higher levels in tumor cells and antigens derived from structural and metabolic changes associated with tumorigenesis. Additionally, the present disclosure describes lysis-associated-antigens such Page 23 of 65 11825822v1 Docket No.: 2017422-0003 as those driven by pathogens. For example, in some embodiments, lysis-associated-antigens include genes encoded by a pathogen that are required for pathogen replication or survival within cells including components of replication and packaging machinery. [0081] In some embodiments, a lysis-associated-antigen is an intracellular antigen that is exposed to an extracellular environment by cell lysis. As described herein, established CAR-T technologies are required to target surface antigens; established TCR technologies are required to target antigens, often intracellular antigens presented to an extracellular cell surface in MHC complexes. Lysis Sensor Receptor constructs provided by the present disclosure target intracellular antigens independent of MHC presentation. In some embodiments, a LSR described herein has a structure that resembles portions of a CAR with the exception that it targets intracellular antigens. As such, LSRs are activated only upon lysis of diseased cells and serve as a means to specifically stimulate an immune response in situations where lysis of diseased cells has occurred either as a result of disease progression, a patient’s immune response or action of another therapy. [0082] In some embodiments, a lysis-associated-antigen may be or comprise an oncogenic protein, a protein that drives cancer cells to grow and divide uncontrollably. Exemplary tumor-specific lysis-associated-antigens include protein products of common oncogenes such as K-RAS, N-RAS, H-RAS, c-Myc, SRC family kinases and other intracellular signal transduction molecules, and intracellular elements of epidermal growth factor receptors and other receptor tyrosine kinases as well as protein products of mutated tumor suppressor proteins such as p53, BRCA1/2 and PTEN. The present disclosure recognizes that an important insight into tumorigenesis is the concept of oncogene addiction, which describes the concept that cancer cells become highly dependent on oncogenes for survival and thus the oncogene is likely to persist throughout the tumor and maintained as the tumor progresses. By contrast, most random mutations that occur in a tumor cell do not provide any selective advantage for tumor survival leading to tumor heterogeneity (see, Sharma and Settleman, 2007, the contents of which is hereby incorporated by reference herein in its entirety). [0083] It is an insight of the present disclosure that mutations in RAS family members are commonly found in many cancers. KRAS mutations are found in the majority of pancreatic ductal carcinomas. In some embodiments, a LSR targets KRAS variants with a mutation occurring at amino acid 12, optionally G12D, G12V or G12C. KRAS G12D is found in roughly one third of pancreatic tumors and one sixth of colon cancers. In some Page 24 of 65 11825822v1 Docket No.: 2017422-0003 embodiments a LSR targets KRAS variants with a mutation at positions other than amino acid 12. In some embodiments, a lysis-associated-antigen may be a protein product of a gene that is mutated in one percent or more of all solid tumors optionally selected from BRAF, IDH1, PI3Kalpha, P53 or others. In some embodiments, a lysis-associated-antigen may be BRAF. In some embodiments, a lysis-associated-antigen may be IDH1. In some embodiments, a lysis-associated-antigen may be PI3Kalpha. In some embodiments, a lysis- associated antigen may be P53. [0084] In some embodiments, a lysis-associated-antigen is or comprises one or more neoepitopes. Neoepitopes are characterized as altered protein sequences that arise from mutations in tumor cells. By definition, neoepitopes are not present in healthy cells and are tumor-specific. Whereas there is a potential for neoepitopes to be recognized by TCRs, neoepitopes that can be recognized in this manner are limited to those than can be presented by MHC class II receptors in an individual patient. It is an insight of the present disclosure that there is no such limitation on neoepitopes that could be targeted by LSRs, with the only requirement that they be present upon cell lysis. Lysis-associated-antigen binding moiety [0085] Among other things, the present disclosure provides technologies (e.g., compositions, methods, producer cells, etc.) that are or comprise LSR constructs described herein. In some embodiments, technologies described herein comprise a LSR construct comprising a lysis-associated-antigen binding moiety. In some embodiments, a lysis- associated-antigen binding moiety binds specifically to a lysis-associated-antigen, as described herein. [0086] In some embodiments, a lysis-associated-antigen binding moiety is or comprises antigen binding elements of an immunoglobulin (e.g., an antibody, a T cell receptor, etc). In some embodiments, a lysis-associated-antigen binding moiety is or comprises a set of CDRs. [0087] In some embodiments, a lysis-associated-antigen binding moiety is or comprises a single chain variable fragment (scFv). [0088] It is an insight of the present disclosure that antibodies and scFvs have been described for potential lysis-associated-antigens demonstrating that these antigens are readily recognizable by antibody-based proteins generated by various technologies. The findings of naturally occurring antibodies to these antigens in cancer patients is consistent with extracellular exposure of these antigens to components of the immune system (see, for Page 25 of 65 11825822v1 Docket No.: 2017422-0003 example, Collins & Pasca Di Magliano 2014, the contents of which is hereby incorporated by reference herein in its entirety). Thus, in some embodiments, lysis of tumor cells will make such antigens available for binding to LSRs. [0089] In some embodiments, a lysis-associated-antigen binding moiety comprises a KRAS binding moiety as described by Singh et al., 2022, the contents of which is hereby incorporated by reference herein in its entirety. In some embodiments, a lysis-associated- antigen binding moiety comprises a KRAS binding moiety as described by Shin et al., 2017, the contents of which is hereby incorporated by reference herein in its entirety. In some embodiments, a lysis-associated antigen-binding moiety comprises a KRAS binding moiety as described by U.S. Pat. No.11,174,314 B2, the relevant disclosures of which are incorporated by reference herein. In some embodiments, a lysis-associated-antigen binding moiety comprises a STAT3 binding moiety as described by Singh et al., 2022. [0090] Among other things, in some embodiments, an exemplary LSR construct comprises a KRAS mutant-specific scFV sequence according to SEQ ID NO: 1. [0091] Exemplary KRAS mutant-specific scFV sequence (SEQ ID NO: 1) EVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMHWVRQAPGKGLEWVAVISHD GSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDTAVYYCATSLYSSMDLWGQG TTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSAAPGQKVTISCSGSNSNIGKNY VSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGTSASLAISGLQSEDEADYYCAA WDDKFGVHWVFGGGTKLTVL [0092] Critical to immune cell activation is the ability of binding of a ligand to an extracellular element to trigger a change in intracellular signaling. For many immune cell receptors, activation of cell signaling is mediated by protein-protein interactions, driven by receptor multimerization. For traditional CARs, this multimerization can be a result of CAR binding to multiple copies of an antigen on a cell surface of a target cell. Among other things, the present disclosure describes that LSR multimerization can be a result of antigens bound to intracellular cell structures such as membranes, protein complexes, structural elements, nucleic acids and glycoproteins. Alternatively or additionally, antigens released upon cell lysis may bind to extracellular elements resulting in multimer structures. [0093] It is an insight of the present disclosure that a LSR could target more than one antigen as exemplified by a bispecific CAR that targets both CS1 and BCMA (see, for example, Zha et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety). In some embodiments, a LSR cell therapy may comprise immune cells Page 26 of 65 11825822v1 Docket No.: 2017422-0003 containing LSRs targeting different antigens or different antigenic sites on a single antigen (see, Han et al., 2019, the contents of which is hereby incorporated by reference herein in its entirety). For example, in some embodiments LSRs that target N-terminal domains and C- terminal domains of an antigen, such as P53 are introduced into an immune cell. Thus, in some embodiments, multimerization of distinct LSRs by an antigen, even in a monomeric form, will trigger immune cell activation. Transmembrane element [0094] In many embodiments, a Lysis Sensor Receptor construct for use in accordance with the present disclosure comprises a transmembrane element that, for example, may link a lysis-associated-antigen binding moiety as described herein to a signaling element as described herein. Those skilled in the art will be familiar with a variety of transmembrane elements that can be used in the practice of the present disclosure. Alternatively or additionally, in some embodiments, a transmembrane element of a LSR construct regulates LSR expression and/or signaling activity (see, for example, Fujiwara et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety). [0095] In some embodiments, a transmembrane element may correspond to one that is found in a naturally occurring protein. Alternatively or additionally, in some embodiments, a transmembrane element may be designed or otherwise engineered, for example, to be or include a substantially hydrophobic segment that is thermodynamically stable in a cell membrane, whether or not its sequence is found in any known transmembrane protein (e.g., any known human transmembrane protein). Examples of synthetic transmembrane domains that are known in the art include, for example, those described in U.S. Pat. No.7,052,906 Bl and PCT Publication No. WO 2000/032776 A2, the relevant disclosures of which are incorporated by reference herein. [0096] In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in a membrane protein selected from the group consisting of: CD8α, CDS~, 4-1BB/CD137, CD28, CD34, CD4, FcERiy, CD16, OX40/CD134, CD3s, CD3E, CD3y, CD3o, TCRa, CD32, CD64, VEGFR2, FAS, and FGFR2B. In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD8a. In some examples, a transmembrane element for use in accordance with the present disclosure corresponds to one found in 4- lBB/CD137. In some embodiments, a transmembrane element for use in accordance with the present disclosure corresponds to one found in CD28 or CD34. In some embodiments, a Page 27 of 65 11825822v1 Docket No.: 2017422-0003 transmembrane element for use in accordance with the present disclosure corresponds to one found in CD8a. Signaling element [0097] As described herein, effective CAR-T cell therapy requires co-stimulation to increase CAR-T cell persistence and enhance cell cytotoxicity in response to antigen binding. In many embodiments, a LSR utilized in accordance with the present disclosure comprises one or more signaling elements (e.g., co-stimulation element(s)) which leads to immune effector function as described herein. Those skilled in the art, reading the present disclosure, would be aware of appropriate source(s) of co-stimulation elements for particular applications as described herein. [0098] Reports have described successful Co-CAR constructs that utilize, for example, co-stimulation elements from CD28 and OX40 proteins (see, Omer et al., 2022). Exemplary signaling elements (e.g., co-stimulation element(s)) that may be used in accordance with the present disclosure include those selected from the group consisting of MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83. Exemplary co-stimulation elements for use in accordance with the present disclosure are described in U.S. Pat. No.11,084,880 B2, incorporated herein by reference. In some embodiments, a co-stimulation element is derived from a group consisting of CD28, OX40, 4I-BB, GITR, ICOS-1, CD27, AP10, and any combination thereof. In some embodiments, a co-stimulation element comprises a CD3ζ Page 28 of 65 11825822v1 Docket No.: 2017422-0003 (CD3ζ or CD3C) activating element. In some embodiments, a co-stimulation element does not comprise a CD3ζ (CD3ζ or CD3C) activating element. [0099] In some embodiments, a LSR construct may comprise more than one co- stimulation element (e.g., 2, 3 or more). In some embodiments, a LSR construct comprises two or more of the same co-stimulation elements. In some embodiments, a LSR construct comprises two or more co-stimulation elements from different co- stimulatory proteins, such as any two or more co-stimulatory proteins described herein. [0100] The present disclosure describes LSR constructs comprising one or more signaling elements (e.g., T cell receptor co-stimulation elements). In some embodiments, an exemplary LSR construct comprises one or more T cell receptor co-stimulation elements. In some embodiments, T cell receptor co-stimulation elements are derived from CD28, 4-1BB, OX40, CD27, GITR, ICOS as well as proprietary elements. In some embodiments, an exemplary LSR construct comprises a CD28 co-stimulation element. An exemplary CD28 co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 2. In some embodiments, an exemplary LSR construct comprises a 4-1BB co-stimulation element. An exemplary 4-1BB co-stimulation element sequence may be or comprise a sequence according to SEQ ID NO: 3. In some embodiments, an exemplary LSR construct comprises a OX40 co-stimulation element. In some embodiments, an exemplary LSR construct comprises a CD27 co-stimulation element. In some embodiments, an exemplary LSR construct comprises a GITR co-stimulation element. In some embodiments, an exemplary LSR construct comprises a ICOS co-stimulation element. [0101] In some embodiments, an exemplary LSR construct comprises a CD28 co- stimulation element and a 4-1BB co-stimulation element. [0102] In some embodiments, an exemplary LSR construct comprises one or more T cell receptor co-stimulation elements as well as a CD3ζ activation element. An exemplary CD3ζ activation element sequence may be or comprise a sequence according to SEQ ID NO: 4. In some embodiments, an exemplary LSR construct does not comprise a CD3ζ activation element. [0103] In some embodiments, an exemplary LSR comprises a CD28 co-stimulation element, a 4-1BB co-stimulation element, and a CD3ζ activation element. Page 29 of 65 11825822v1 Docket No.: 2017422-0003 [0104] Exemplary CD28 co-stimulation element sequence (SEQ ID NO: 2) ASAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRLGPTRKHYQPYAPPRDFAAYRS [0105] Exemplary 4-1BB co-stimulation element sequence (SEQ ID NO: 3) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE [0106] Exemplary CD3ζ activation element sequence (SEQ ID NO: 4) LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR Other element(s) [0107] Among other things, the present disclosure describes a LSR construct comprising one or more elements that regulate activity of a LSR. For example, in some embodiments, a LSR construct may comprise a masking element that is cleaved off or moved in a tumor microenvironment. In some embodiments, constructs of the present disclosure may comprise a masking element as described by U.S. Pat. No.9,120,853 B2, the contents of which are hereby incorporated by reference in its entirety. [0108] The potency and survival of LSR cell therapies is dependent in part to the strength of a co-stimulation signal transmitted by a LSR receptor. It is an insight of the present disclosure that four factors may be optimized to generate an effective therapy: (1) the strength and number of co-stimulation elements, (2) the expression density of a LSR on engineered immune cells, (3) the affinity of a lysis-antigen-binding moiety for a target antigen, and (4) the density and availability of an antigen after cell lysis (see, Rodriguez- Marquez et al., 2022, Majzner et al., 2020, the contents of which is hereby incorporated by reference herein in its entirety). [0109] Among other things, in some embodiments, a LSR construct as described herein comprises a signaling peptide sequence. An exemplary signaling peptide sequence may be or comprise a sequence according to SEQ ID NO: 5. [0110] Exemplary signaling peptide sequence (SEQ ID NO: 5) MLLLVTSLLLCELPHPAFLLIP [0111] Among other things, in some embodiments, a LSR construct as described herein further comprises, a polypeptide tag (e.g., a FLAG-tag, or FLAG octapeptide, or FLAG epitope). In some embodiments, a polypeptide tag (e.g., a FLAG-tag, or FLAG octapeptide, or FLAG epitope) is used to assess expression of a LSR in T cells. An Page 30 of 65 11825822v1 Docket No.: 2017422-0003 exemplary polypeptide tag sequence may be or comprise a sequence according to SEQ ID NO: 6. [0112] Exemplary polypeptide tag sequence (SEQ ID NO: 6) DYKDDDDK [0113] Those of skill in the art, reading the present disclosure, would be aware of polypeptide tags and methods of adding a polypeptide tag using recombinant DNA technology (see, e.g., WO 2017/172952, the contents of which is hereby incorporated by reference herein in its entirety. Exemplary Lysis Sensor Receptor Construct Sequences [0114] Among other things, in some embodiments, the present disclosure provides technologies (e.g., compositions, vectors, virions, producer cells). In some embodiments, such technologies comprise a single construct. In some embodiments, such technologies comprise multiple constructs. In some embodiments, the present disclosure provides compositions or preparation of virions each comprised of a single construct as described herein. In some embodiments, a single construct may deliver a polynucleotide that encodes an exemplary LSR. In some embodiments, a construct is or comprises a LSR construct. [0115] In some embodiments, a single construct composition or system may comprise any or all of the exemplary construct components described herein. In some embodiments, an exemplary single construct is at least 85%, 90%, 95%, 98% or 99% identical to the sequences described herein. One skilled in the art would recognize that constructs may undergo additional modifications including codon-optimization, introduction of novel but functionally equivalent (e.g., silent mutations), addition of reporter sequences, and/or other routine modification. [0116] In some embodiments, constructs, virions, populations of virions, or producer cells comprise a LSR polypeptide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with that of SEQ ID NO: 7. In some embodiments, constructs, virions, populations of virions, or producer cells comprise a LSR polynucleotide sequence that shows at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% overall sequence identity with that of SEQ ID NO: 8. [0117] Exemplary KRAS-LSR construct 1 polypeptide sequence (SEQ ID NO: 7) Page 31 of 65 11825822v1 Docket No.: 2017422-0003 MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGVVQPGRSLRLSCAASGFTSRHPGMH WVRQAPGKGLEWVAVISHDGSKKYYADSVKGRFTISRDNSKNTLFVQLSSLRPEDT AVYYCATSLYSSMDLWGQGTTVTVSSGSTSGSGKPGSGEGSTKGQSVVTQPPSVSA APGQKVTISCSGSNSNIGKNYVSWFQQVPGTAPKLLIFEDNQRPSGIPDRFSASKSGT SASLAISGLQSEDEADYYCAAWDDKFGVHWVFGGGTKLTVLDYKDDDDKASAIEV MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRLLHSDYMNMTPRRLGPTRKHYQPYAPPRDFAAYRSKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [0118] Exemplary KRAS-LSR construct 1 polynucleotide sequence (SEQ ID NO: 8) ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC TCCTGATCCCAGAAGTGCAACTTGTACAGTCTGGAGGTGGCGTTGTACAGCCTG GGCGGAGCCTCCGACTCAGCTGCGCCGCATCAGGATTTACAAGCCGACATCCGG GAATGCACTGGGTGCGCCAAGCACCAGGGAAAGGCTTGGAGTGGGTGGCCGTT ATTAGCCACGACGGTTCTAAAAAGTACTATGCAGACTCCGTGAAGGGGCGGTTT ACCATCTCCAGGGATAACTCTAAGAACACGCTGTTTGTCCAACTGTCTTCCTTGC GGCCGGAAGATACTGCGGTCTACTACTGCGCTACTTCTCTTTACAGCAGCATGG ACCTGTGGGGCCAAGGAACCACCGTGACTGTGAGTAGCGGCTCCACCTCTGGAT CCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCCAGTCCGTAGTTACCC AGCCTCCTAGTGTAAGCGCGGCCCCGGGTCAAAAAGTGACGATCAGCTGTTCTG GCTCTAACAGTAACATTGGGAAGAATTATGTAAGTTGGTTTCAACAGGTGCCGG GCACGGCACCCAAGCTCCTTATTTTTGAGGATAACCAACGACCATCAGGTATCC CCGACAGATTTTCTGCGTCTAAATCTGGTACGTCCGCGAGCCTTGCTATAAGTGG TCTTCAGTCAGAGGATGAAGCTGACTACTATTGTGCTGCCTGGGATGATAAATT TGGCGTCCATTGGGTCTTTGGTGGGGGGACAAAGCTCACTGTATTGGACTACAA AGACGATGACGACAAGGCTAGCGCAATTGAAGTTATGTATCCTCCTCCTTACCT AGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTT GTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGT TGGGGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC TGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGAC TCCAAGACGCCTAGGTCCCACTCGCAAGCATTACCAGCCCTATGCCCCACCACG Page 32 of 65 11825822v1 Docket No.: 2017422-0003 CGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTA GCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTC AGCAGGAGCGCAGACGCTCCAGCTTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTG GGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC CCTCGCTAA Encoding Nucleic Acids [0119] In some embodiments, the present disclosure provides nucleic acids encoding LSRs as described herein, and compositions that comprise and/or deliver them (e.g., to cells as described herein). [0120] Among other things, the present disclosure provides that some LSR constructs as described herein are polynucleotide constructs. Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and constructs that incorporate a polynucleotide comprising a coding sequence operably linked to an expression control sequence, wherein the coding sequence encodes a LSR. Those of skill in the art will be capable of selecting suitable constructs, as well as cells, for making any of a nucleic acids described herein. In some embodiments, a construct is a plasmid (i.e., a circular DNA molecule that can autonomously replicate inside a cell). In some embodiments, a construct can be a cosmid (e.g., pWE or sCos series). [0121] Those of skill in the art, reading the present disclosure, would be aware of methods of generating a polynucleotide construct in accordance with the present disclosure. Examples of methods of generating a polynucleotide construct as described herein (e.g., in vitro transcription, enzymatic ligation, genetic engineering techniques), as described by, for example, Wood, 1982, incorporated in its entirety herein by reference. [0122] Those of skill in the art, reading the present disclosure, would be aware of methods for delivery of nucleic acids (e.g., to cells as described herein). Examples of methods for delivery of a polynucleotide construct (e.g., a LSR construct) that are known in the art include, for example, those described in U.S. Pat. No.10,898,574 B2, Hou et al., Page 33 of 65 11825822v1 Docket No.: 2017422-0003 2021, and U.S. Pat. No.11,084,880 B2, each of which is incorporated in its entirety herein by reference. [0123] Among other things, the present disclosure describes a vector comprising a polynucleotide construct encoding a LSR to facilitate introduction into immune cells. In some embodiments, a viral vector can be a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudo type vector), an adenovirus vector, an adeno- associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, a HSV vector among others. Examples of lentiviral vectors for use in accordance with the present disclosure include, for example, human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). Examples of viral vectors that may be used in accordance with the present disclosure are described in U.S. Pat. No.11,382,965 B2, the contents of which are incorporated in its entirety herein by reference. [0124] In some embodiments, a viral vector lacking replicating ability so as not to self replicate in an infected cell is preferably used. [0125] Among other things, in some embodiments, the present disclosure provides producer cells so that virions are produced in the producer cell that comprise the viral capsid comprising a polynucleotide construct as described herein. Methods of introducing and expressing genes in a cell are known in the art. In some embodiments, a polynucleotide construct encoding a LSR construct described herein is transferred into a producer cell. [0126] Moreover, the present disclosure describes a preparation of virions comprising a viral capsid and a LSR construct described herein to facilitate introduction into cells (e.g., immune cells). [0127] Among other things, in some embodiments, a polynucleotide construct encoding a LSR construct described herein is operably linked to a promoter and incorporated into an expression vector. In some embodiments, the expression vector may be provided to a cell in the form of a viral vector. LSR Activation [0128] The present disclosure contemplates a variety of mechanisms that may be involved in LSR activation upon antigen (i.e., lysis-associated-antigen) binding. Page 34 of 65 11825822v1 Docket No.: 2017422-0003 [0129] For example, in some embodiments, antigen binding may induce a conformational change, for example in an intracellular domain of the LSR, thereby achieving direct activation. Alternatively or additionally, in some embodiments, antigen binding may alter interaction(s) between a LSR and one or more other agents (e.g., other proteins), thereby achieving transduction of a relevant signal. Still further, alternatively or additionally, in some embodiments, antigen binding could trigger dimerization or higher order multimerization of a LSR (e.g., through binding of dimeric or oligomeric antigen ligands and/or through binding of multiple ligands that form multimeric structures, for example). Yet further alternatively or additionally, in some embodiments, different LSRs may target different antigenic regions (e.g., epitopes) of a same target antigen (e.g., target protein, carbohydrate, complex, etc), such that antigen binding by two or more such LSRs co-localizes different LSRs, thereby achieving signal activation. [0130] Those skilled in the art will be aware of different elements, or combinations thereof that may be included in LSRs provided herein to accomplish signal transduction by one or more such mechanisms (see, for example, Schlessinger, 1998, and Wu, 2013, each of which is which is incorporated in its entirety herein by reference). For example, in some embodiments, a LSR construct comprises an oligomerization element for the purpose of coordinating protein oligomerization and subsequent signal transduction. In some embodiments, a LSR construct comprises a homodimerization, heterodimerization, trimerization, tetramerization, or oligomerization element, or combinations thereof, in order to achieve oligomerization. Exemplary oligomerization elements that may be used in accordance with certain embodiments of the present disclosure are described in, for example, Engel & Kammerer, 2000, Thomas et al., 2013, and Mittl et al., 2000, the contents of which are hereby incorporated by reference in its entirety. In some embodiments, an oligomerization element is or comprises a short flexible peptide linker which joins the light and heavy chains of a scFV as described in, for example, Ahmad et al., 2012, the contents of which are hereby incorporated by reference in its entirety. LSR-Expressing Cells [0131] Among other things, the present disclosure provides preparations of cells engineered to express a LSR construct as described herein. [0132] In some embodiments, provided cells (e.g., provided cell preparations) are immune cells. In some embodiments, provided cells (e.g., provided cell preparations) are T cells. In some embodiments, provided cells (e.g., provided cell preparations) are NK cells. Page 35 of 65 11825822v1 Docket No.: 2017422-0003 In some embodiments, provided cells (e.g., provided cell preparations) are macrophages. In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express a TCR. In some embodiments, such TCR is a natural TCR; in some embodiments, such TCR is an engineered TCR. In some embodiments, provided cells (e.g., provided cell preparations) are T cells that express a CAR. [0133] In some embodiments, provided cells (e.g., provided cell preparations) are cells that have been cultured. In some embodiments, provided cells (e.g., provided cell preparations) are cells that have been isolated from a subject and cultured (e.g., expanded) ex vivo. In some such embodiments, provided cells are T cells, such as TIL cells. [0134] In some embodiments, provided cells (e.g., provided cell preparations) include a plurality of different T cells (e.g., a plurality of T cells expressing different TCRs), each of which has been engineered to express the same LSR. Among other things, the present disclosure provides an insight that provided cell preparations will maintain their endogenous TCR heterogeneity. [0135] In some embodiments, provided cells (e.g., a provided preparation of cells) is expanded from a sample obtained from a subject. In some embodiments, such sample is or comprises blood or other bodily fluid, or a tissue sample. In some embodiments, a sample is obtained from a site that is in the vicinity of a tumor. In some embodiments, such site comprises part or all of a tumor. In some embodiments, a sample is or comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, a sample is or comprises bone marrow. In some embodiments, a sample is or comprises a spleen, lymph node, or thymus sample. In some embodiments, a sample is or comprises tumor tissue (e.g., that may comprise TILs). [0136] Those skilled in the art, reading the present disclosure, would be aware of appropriate source(s) of sample(s) for particular applications as described herein. [0137] In some embodiments, a population of cells (e.g., T cells) is obtained from a subject and engineered to express a LSR. In some embodiments, a population of cells (e.g., T cells) is isolated from a patient to whom the engineered population of cells is to be administered. [0138] In some embodiments, a population of T cells is obtained. In some embodiments, cells of an obtained population express T cell receptor(s). In some embodiments, cells of an obtained population of T cells are engineered to alter expression of their natural TCR (e.g., to replace it with an engineered TCR) and/or to express a CAR. In some embodiments, a Page 36 of 65 11825822v1 Docket No.: 2017422-0003 population of T cells is engineered according to methods described by, for example, Ellis et al., 2021, the contents of which are hereby incorporated by reference in its entirety. [0139] In some embodiments, a method for producing an engineered population of T cells is transduction with a viral vector or a polynucleotide construct comprising a coding sequence for a LSR. Those skilled in the art will be familiar with various technologies for engineering cells (e.g., to express LSRs and/or engineered CARs and/or TCRs, etc) in accordance with the present disclosure. For example, technologies such as electroporation, particle (e.g., gold particle, lipid nanoparticle, etc) delivery, transfection (e.g., chemical transfection), vesicle (e.g., exosome or other lipid vesicle) delivery, viral delivery, etc. are available (see, for example, Alzubi et al., 2021, Paunovska et al., 2022). Additionally or alternatively, examples of methods for preparation of cells for engineering and methods for engineering cells (e.g., to express LSRs and/or engineered CARs and/or TCRs, etc) in accordance with the present disclosure that are known in the art include, for example, those described in U.S. Pat. No.11,266,739 B2, the contents of which are hereby incorporated by reference in its entirety. [0140] Alternatively or additionally, the present disclosure describes a method of generating a population of engineered cells (e.g., immune cells) comprising introducing an in vitro transcribed polynucleotide construct (e.g., RNA or synthetic RNA) into a cell, wherein the polynucleotide construct encodes a LSR construct as described herein (see, for example, U.S. Pat. No.11,453,719 B2, which is herein incorporated by reference in its entirety). [0141] Among other things, the present disclosure provides methods for preparing a population of cells engineered to express a LSR construct as described herein for administering to a subject. In some embodiments, an engineered population of T cells may be expanded and activated as described in U.S. Pat. No.11,084,880 B2, or references cited therein, each of which is herein incorporated by reference in its entirety. Expression of a LSR construct in an engineered population of T cells may be evaluated by ways known in the art including but not limited to, Western Blot analysis using an antigen for a lysis- associated-antigen binding moiety, an antibody against a co-stimulation element(s), and any combination thereof, and analysis of in vitro expansion of engineered LSR-expressing T cells by flow cytometry. Page 37 of 65 11825822v1 Docket No.: 2017422-0003 [0142] Among other things, the present disclosure describes assays for measuring activity of a population of T cells engineered to express a LSR construct as described herein, including but not limited to anti-cancer activity in in vitro and animal models. Applications/Uses Methods of treatment [0143] Among other things, in some embodiments, technologies of the present disclosure are used to treat a disease, disorder, or condition. In some embodiments, such diseases may include tumors, cancers, or other proliferative diseases, and/or infectious agents or disease. In some embodiments, provided herein are methods treating a disease comprising administering a population of cells engineered to express a LSR construct as described herein that targets a lysis-associated antigen (e.g., an intracellular lysis-associated antigen) produced by diseased cell(s) in the subject. [0144] In some embodiments, a method of treatment comprises administration of at least two successive doses of LSR-expressing cells (e.g., cells engineered to express an LSR as described herein). In some embodiments, a second dose is administered if (and optionally only if) an intervening assessment has determined that the subject continues to suffer from the relevant disease, disorder or condition and/or that the disease, disorder or condition progressed. Exemplary dosing regimens that may be useful in accordance with certain embodiments of the present disclosure are described in, for example, U.S. Pat. No. 11,266,739 B2, incorporated in its entirety herein by reference. [0145] In some embodiments, a subject who receives therapy with a preparation of engineered cells (i.e., LSR-expressing cells) as described herein is also receiving other therapy for the disease, disorder or condition. For example, where the disease disorder or condition is or comprises cancer, such subject may receive provided therapy in combination with, for example, one or more other immune-oncology therapies such as, for example other adaptive cell therapy, e.g., targeting one or more disease-associated antigens and/or checkpoint inhibitor therapy. Alternatively or additionally, such subject may be receiving or have received cancer therapy that is or comprises chemotherapy, surgery and/ or radiation therapy. Regulating administered cells [0146] Among other things, in some embodiments, the present disclosure describes technologies for regulating LSR-expressing cells (e.g., immunosuppressive agents, small Page 38 of 65 11825822v1 Docket No.: 2017422-0003 molecule drugs, etc) described herein, as described by, for example, Brandt et al.2020, Park et al., 2021, the contents of which is hereby incorporated by reference herein in its entirety. [0147] As described herein, cytokine release syndrome (CRS) is a well-known consequence of CAR-T therapy. It is an insight of the present disclosure that methods developed to regulate CAR T activity may be used to regulate activity of LSR-expressing cells described herein. In some embodiments, activity of a LSR-expressing cell (e.g., engineered T cell) is inhibited by administering immunosuppressive agents (e.g., corticosteroids). [0148] As described herein, in some embodiments, a LSR construct may comprise a masking element that is cleaved off or moved in a tumor microenvironment. Such a LSR construct comprises a masking element inhibiting activity of a lysis-associated-antigen binding moiety, with a linker sensitive to proteolytic cleavage. In such embodiments, activity of LSR-expressing cells is regulated by tumor-associated proteases removing a masking peptide in the tumor microenvironment. [0149] In some embodiments, a lysis-associated-antigen binding moiety is or comprises a conditional single chain variable fragment (scFv), wherein the scFv comprises a small molecule-based control element, so that LSR activity can be regulated by administration of a small molecule drug. In such embodiments, affinity of a lysis- associated-antigen binding moiety towards a target protein is reduced by activity of a small molecule drug. [0150] In some embodiments, a LSR construct may comprise a virally derived NS3 protease and a degradation element such that the LSR is tagged for degradation. Moreover, activity of such a LSR is regulated by administration of a NS3 protease inhibitor. Exemplification Example 1: Exemplary Lysis Sensor Receptor Constructs Targeting Lysis-Associated- Antigens [0151] The present Example provides exemplary compositions, preparations, constructs, virions, preparation of virions, and host cells for gene therapy and related methods that target an intracellular product of diseased cells. [0152] The present disclosure provides an insight that connecting T cell activation to presence of lysed/lysing cells could dramatically improve engineered T cell therapy (e.g., CAR-T therapy). Among other things, the present disclosure provides CAR constructs that Page 39 of 65 11825822v1 Docket No.: 2017422-0003 respond specifically to cell lysis, described herein as a Lysis Sensor Receptor (LSR). As shown in FIG.1, a T cell (e.g., immune effector cell) comprising a LSR as described herein is activated by a lysis-associated-antigen(s) (e.g., an intracellular disease antigen, e.g., tumor antigen) released upon lysis of a target cell, resulting in T cell cytotoxic activity, which results in more T cell effector activation in a positive feedback loop. Exemplary LSR construct structures are depicted in FIG.2. In some embodiments, a LSR construct comprises an extracellular single chain variable fragment (scFv) of an antibody known to bind a relevant antigen of interest operably linked to a transmembrane element, and one or more T cell co-stimulation elements. In some embodiments, a LSR construct further comprises a CD3ζ activation element. [0153] Moreover, the present Example describes certain exemplary models for LSR activation by a lysis-associated-antigen in accordance with the present disclosure. [0154] Among other things, the present disclosure describes that, in some embodiments, antigen binding leads to a direct conformational change in an intracellular domain of a LSR resulting in direct activation (as shown in FIG.3A). In some embodiments, antigen binding causes a change in interaction of a LSR with other endogenous proteins, resulting in signal activation (as shown in FIG.3B). In some embodiments, antigen binding causes dimerization or higher order oligomerization of a LSR through the binding of dimeric or oligomeric ligands (as shown in FIG.3C). In some embodiments, antigen binding causes dimerization through binding of multiple ligand molecules that form multimeric structures as a result of interactions with other macromolecules, such as nucleic acids, carbohydrates, lipids or proteins (as shown in FIG.3D). In some embodiments, an antigen brings LSRs that recognize different antigenic regions of a single protein or a protein complex together resulting in signal activation (as shown in FIG.3E). Example 2: Exemplary Lysis Sensor Receptor Constructs Targeting KRAS [0155] The present Example describes certain exemplary Lysis Sensor Receptor (LSR) constructs in accordance with the present disclosure. [0156] Anti-KRAS antibodies have been reported in certain cancer patients (see, for example, Collins & Pasca Di Magliano 2014, the contents of which is hereby incorporated by reference herein in its entirety). The present disclosure provides an insight that, as KRAS is predominantly an intracellular protein, the observation of anti-KRAS antibodies in cancer patients may indicate that sufficient KRAS can be released from lysed cells in such patients that their immune systems can mount some level of successful response. The present Page 40 of 65 11825822v1 Docket No.: 2017422-0003 disclosure harnesses this insight by providing LSR constructs, and engineered T cell populations that contain or express LSR constructs, targeting KRAS. [0157] For example, the present Example describes KRAS-targeting LSR construct(s) in which KRAS-biding-element(s) of a human anti-KRAS antibody (e.g., as may have arisen in a cancer patient) are linked to a transmembrane element and a T cell receptor co-stimulation element. The present Example specifically exemplifies LSR construct(s) in which KRAS- binding-element(s) of a human anti-KRAS antibody (e.g., as described in Kim et al., 2018, the contents of which is hereby incorporated by reference herein in its entirety) are included in a single-chain antigen-binding domain (scFv) that is linked to a transmembrane element and one or more co-stimulation elements, as shown in FIG.4. In some embodiments, a LSR construct described herein comprises a KRAS-binding-element linked to a transmembrane element and CD28, OX40, and CD3ζ co-stimulation elements. Generation of LSR Constructs [0158] The present Example describes generation of LSR constructs for use in accordance with the present disclosure. Constructs comprising a coding sequence encoding a LSR described herein are generated and tested in producer cells according to standard protocols. In some embodiments, one or more LSR backbone constructs are designed for cloning into a lentiviral vector. In some embodiments, a preparation of virions comprising a LSR construct are produced by transfecting 293-T cells with a plasmid comprising a LSR construct described herein and three helper plasmids comprising a gag-pol gene, a rev gene, and a VSV-g envelope gene. Methods for production of a lentiviral vector that may be utilized in accordance with some embodiments of the present disclosure are described in, for example, Merten et al., 2016. [0159] In some embodiments, a LSR construct is cloned into an AAV vector. In some embodiments, 293 cells are transfected with a plasmid comprising a LSR construct, an AAV helper plasmid, and an adenoviral helper plasmid. Methods for production of an AAV vector in accordance with the present disclosure are described in, for example, Shin et al. 2013. [0160] Accordingly, in some embodiments, the present disclosure describes compositions, preparations, constructs, virions, population of virions, and host cells comprising a variant protoparvovirus VP1 capsid polypeptide can exhibit increased VP1 initiation relative to a reference VP1 capsid polypeptide. Expression of LSR Constructs on T Cells Page 41 of 65 11825822v1 Docket No.: 2017422-0003 [0161] In some embodiments, a population of T cells is obtained from healthy human donors and transduced with a preparation of virions comprising a KRAS-targeting LSR described herein. Expression of a KRAS-targeting LSR may be determined by flow cytometry. A population of T cells is cultured in RPMI 1640 medium with 10% FBS and stimulated with anti-CD3/anti-CD28 Dynabeads (Invitrogen). T cells are transduced with a preparation of virions 24 hours after stimulation. Mock transduced cells are used as a negative control and T cells transduced with a CAR construct known in the art is used as a positive control. 4-6 days after transduction, expression of a LSR on the surface of an engineered T cell is evaluated by flow cytometry. [0162] The activation of a LSR, such as through KRAS binding, requires the ability of KRAS to oligomerize an LSR. KRAS is post-translationally modified by myristoylation, the covalent addition of a 14-carbon unsaturated fatty acid. Myristoylation leads to the attachment of KRAS to the inner cell membrane where it is required to be localized for activity. Because multiple KRAS molecules are found localized to the inner cell membrane, binding to LSRs will lead to oligomerization of LSRs, as shown in FIG.5, resulting in activation of LSR signaling and stimulation of T cells. [0163] It is an insight of the present disclosure that altering the composition of the intracellular domains of LSRs can modulate their ability to stimulate T cell activity. In some embodiments, an exemplary LSR construct comprises a co-stimulation element comprising a CD28 and an OX40 co-stimulation element as described herein. Among other things, the present example recognizes that lack of a CD3 element in a LSR construct can reduce stimulatory activity, resulting in reduction of systemic toxicity. In some embodiments, a co-stimulation element comprising a CD3 element can be used in combination with a LSR that is highly specific to mutant forms of an intracellular product of diseased cells (e.g. KRAS) as systemic activation through binding of a LSR is expected to be low. [0164] The present Example specifically describes LSR constructs that target KRAS. Those skilled in the art, reading this Example and particularly in the context of the present disclosure will appreciate that analogous strategies can be implemented to provide LSR constructs that target alternative markers (e.g., alternative intracellular markers). [0165] For example, as shown in FIG.6, a number of oncogenes are mutated in a high percentage of tumors; several of the proteins encoded by these oncogenes are intracellular proteins. In some embodiments, the present disclosure provides LSR constructs (and Page 42 of 65 11825822v1 Docket No.: 2017422-0003 associated technologies, as described herein) that target other intracellular oncogene- encoded proteins. To give but one example, a LSR comprising a lysis-antigen-binding moiety for one or more P53 epitopes is within the scope of this disclosure exemplified in FIG.7. [0166] Furthermore, one feature of the present disclosure is that certain embodiments do not require that the target of an LSR be a disease-specific (or disease-associated) target. That is, because the event of cell lysis may be disease-associated (or disease-specific), in some embodiments, LSR constructs may be directed at intracellular target(s) that are present in non-diseased cells (e.g., at a level or frequency comparable to or even higher than that at which they are present in diseased cells). Thus, the present disclosure provides uniquely flexible targeting technologies. Example 3: T cells engineered to express a LSR can be activated by exogenous mutant KRAS [0167] The present Example teaches that T cells engineered to express a LSR can be activated by exogenous mutant KRAS. [0168] Without wishing to be bound to any theory, the present disclosure notes that chimeric antigen receptor (CAR) activation is dependent on antigen density, CAR density, and co-stimulation elements (see, for example, Majzner et al., 2020). The present Example demonstrates that engineered T cells expressing a KRAS-targeting LSR construct(s) show activation by exogenous mutant KRAS when stimulated by incubation with cell lysates from KRAS-mutant cell lines. In some embodiments a KRAS-mutant cell line corresponds to a G12D KRAS mutant (e.g., AsPC-1, HPAF-II (pancreatic), GP2d, LS180 (colon), T3M-10 (lung). In some embodiments, a KRAS-mutant cell line corresponds to a G12V KRAS mutant (e.g., Capan-1, KP-3 (pancreatic), SW480 (colon), COLO 668 (lung). In some embodiments a KRAS-mutant cell line corresponds to a G12C KRAS mutant (e.g., MIA PaCa-2 (pancreas), SW837 (colon), Calu-1, LU65 (lung). In some embodiments, a KRAS- mutant cell line corresponds to a KRAS variant with a mutation at a position other than amino acid 12. [0169] KRAS-targeting LSR-expressing T cells or control T cells are expanded until the end of log-phase growth, they are artificially stimulated overnight and subsequently co- cultured for 16 hrs with either lysed AsPC-1-KRAS specific target cells, lysed non-KRAS target cells as positive control or no target cells as negative control at a 3 to 1 ratio of effector cells to target cells. Culture supernatants are to be harvested to measure T cell Page 43 of 65 11825822v1 Docket No.: 2017422-0003 proliferation in response to co-culture conditions and cytokine production. IFN-gamma and IL-2 concentration is measured by specific ELISA following manufacturer instructions (R&D). T cells engineered to express a KRAS-targeting LSR will show increased cytokine production after co-culturing with KRAS mutant cells lines when compared to engineered T cells co-cultured with non-KRAS-mutant cells lines. Cytotoxic activity of KRAS-targeting LSR-expressing-T cells is to be evaluated using a Cr release-assay. Alternatively or additionally, the present disclosure recognizes that methods of evaluating cytotoxic activity that may be used in accordance with the present disclosure are known in the art (e.g., flow cytometry, cell viability assays). KRAS-targeting LSR-expressing-T cells are able to induce lysis of KRAS-expressing cell lines with little activity towards non-KRAS-mutant cell lines demonstrating that KRAS LSR T cells are specifically activated by lysed tumor cells only when these cells have KRAS mutations. Example 4: T Cells Expressing a LSR Exert Antibody Dependent Cancer Cell Killing [0170] The present Example provides exemplary constructs, preparations, population of engineered T cells, and producer cells for CAR-T therapy and related methods that demonstrate enhanced killing of a KRAS mutation containing cell line as described herein. [0171] In one exemplary embodiment, anti-cancer activity of a KRAS-targeting LSR is evaluated in a preclinical animal model of pancreatic cancer using the cell line AsPC-1. AsPC-1 cells are engineered to express Click-Beetle Green Luciferase (CB-G Luc+) to track tumor progression by biolumiscent in vivo imaging (IVIS) and Living Image software (Perkin AElmer). CB-G Luc+ AsPC-1 cells are cultured for four weeks and later IV injected into NSG recipients. Engineered T cells expressing the KRAS-targeting LSR are IV injected and tumor burden is evaluated by in vivo imaging. [0172] In the present Example, a KRAS-mutant cell line (e.g, AsPC-1) is grown and expanded in vitro according to standard protocols and IV injected in NGS mice. Mice are to be administered 5xl06 T cells 7-8 days after tumor implantation. Cells are partially thawed in a 37 degree Celsius water bath and then completely thawed by addition of 1 ml of cold sterile PBS to the tube containing the cells. A population of thawed cells is transferred to a 15 ml falcon tube and adjusted to a final volume of 10 mLs with PBS. KRAS-targeting LSR T cells are washed twice at 1000 rpm for 10 minutes each time and then counted on a hemocytometer. KRAS LSR T cells are normalized for transduction so that all mice are treated with the same percentage of cells, and resuspended at a concentration of 50x106 cells per mL of cold PBS and kept on ice until mice are dosed. Page 44 of 65 11825822v1 Docket No.: 2017422-0003 [0173] Five to seven mice per group are to be treated with either 100uL of KRAS- targeting LSR T cells, untransduced T cells (mock), or PBS alone. Mice are monitored according to standard protocols for health status, including body weight measurements, and tumor burden via in vivo imaging. Numbers of KRAS LSR T cells are also evaluated via peripheral blood FACS analysis. Mice treated with KRAS LSR T cells demonstrate expansion of KRAS LSR T cells compared to mock treated controls. In addition, KRAS LSR T cell treated mice show significant reduction in tumor growth compared to controls. Lack of efficacy seen with untransduced T cells (non-lysis-associated-antigen targeting T cells) correlates with a reduced change in tumor growth compared to KRAS LSR T cell treated mice. Example 5: Exemplary Dosing Regimen [0174] The present Example provides exemplary constructs, preparations, population of engineered T cells, and producer cells for LSR T therapy as described herein. Additionally, the present Example provides exemplary dosing regimens for LSR T therapy as described herein. [0175] Among other things, the present disclosure describes steps of a method of administering a LSR T cell therapy. Methods for administering CAR-T cell therapy known in the art may be used in accordance with the present disclosure. As shown in FIG.8, exemplary steps of administering a CAR therapy (e.g., a LSR therapy as described herein) include, (1) collecting a patient’s peripheral blood, (1) isolating a population of T cells from a blood sample, (3) activation and amplification of such a population of T cells, (4) engineering the obtained population of T cells to express a designed CAR, (5) amplification and quality control measures of an engineered population of CAR-expressing T cells, and (6) transfusion of engineered CAR-T cells in the patient from whom the population of T cells was obtained. [0176] As of 2017, six CAR-T therapies have been approved: Abecma (idecabtagene vicleucel), Breyanzi (lisocabtagene maraleucel), Kymriah (tisagenlecleucel), Tecartus (brexucabtagene autoleucel), Yescarta (axicabtagene ciloleucel), Carvykti (ciltacabtagene autoleucel). The dosing regimens for these approved therapies are attached as figures herein. [0177] As can be seen in FIGs.9-14, all of these dosing regimens involve a step of administering a lymphodepleting regimen before infusion of a population of engineered T cells. In some embodiments, a lymphodepleting regimen is cyclophosphamide and fludarabine. All of these dosing regimens involve not using a leukodepleting filter, Page 45 of 65 11825822v1 Docket No.: 2017422-0003 verifying a subject’s identity prior to infusion, premedication with acetaminophen and an H1 antihistamine, confirming availability of tocilizum prior to infusion, and dosing of a CAR-T therapy based on the number of CAR-positive viable T cells. All of these dosing regimens are for autologous use only. All of these dosing regimens are for intravenous use only. Example 6: Production of Exemplary Lysis Sensor Receptor Constructs Targeting KRAS [0178] The present Example documents expression of KRAS-targeting LSRs as described herein in T cells. [0179] Two exemplary LSR constructs were designed and produced using a KRAS scFv (specifically, an scFv targeting mutant KRAS; the amino acid sequence of this scFv is provided as SEQ ID NO: 1, and is based on human KRAS antibody 1F4_4F10, as described in U.S. Patent No.11,174,314, which is herein incorporated by reference in its entirety), as depicted in FIG.15. Specifically, as indicated in FIG.15, LSR Construct 1 includes CD28 and 4-1BB co-stimulation elements and a CD3ζ activating element; LSR Construct 2 includes only the CD28 and 4-1BB co-stimulation elements. Each of these constructs further includes a Flag polypeptide tag used to assess expression in T cells. Those skilled in the art will be familiar with such tags; the particular Flag tag (aka FLAG octapeptide; FLAG epitope) used in exemplified LSR Constructs 1 and 2 is set forth in SEQ ID NO: 6. [0180] LSR Constructs 1 and 2 were synthesized; their identities were confirmed by sequencing in both directions. They were each cloned into a pCD510 lentiviral vector (Systems Bioscience, Palo Alto, CA, USA). Lentivirus was generated using 293 FT cells, Lentivirus Packaging Mix and transfection agent (Alstem, Richmond, CA, USA) (e.g., as described in Berahovich et al., 2017, which is herein incorporated by reference in its entirety). Virus titers were determined by quantitative RT-PCR using a Lenti-X qRT-PCR kit (Takara Bio, Mountain View, CA, USA) according to manufacturer’s protocol and a 7900HT thermal cycler (Thermo Fisher Scientific, South San Francisco, CA, USA). Lentiviral titers were expressed in pfu/mL and ranged 1–10 × 108 pfu/mL. [0181] Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque solution (GE Healthcare, Chicago, IL). PBMCs were suspended at 1 × 106 cells/mL in AIM V-AlbuMAX medium (Thermo Fisher) containing 10% FBS with 300 U/mL IL-2 (Thermo Fisher). PBMC were activated with an equal number of CD3/CD28 Dynabeads (Thermo Fisher), and cultured in non-treated 24-well plates. At 24 and 48 h, lentivirus was added to cultures at a multiplicity of infection (MOI) Page 46 of 65 11825822v1 Docket No.: 2017422-0003 of 5 with 1 μL of TransPlus transduction enhancer (AlStem). As a control for transduction efficiency, the vector encoding the KRAS LSR also contained the polynucleotide encoding green fluorescent protein (GFP). KRAS-targeting LSR-expressing T cells were counted every two to three days and fresh medium with 300 U/mL IL-2 was added to cultures to maintain a cell density at 1 × 106 cells/mL. [0182] To measure KRAS-LSR expression, 5 × 105 cells were suspended in 100 μL of buffer (1× PBS with 0.5% BSA+ 0.5 mM EDTA + 0.1% NaN3) and incubated on ice with 1 μL of human serum (Jackson Immunoresearch, West Grove, PA, USA) for 10 min. Anti-FLAG APC (Biolegend; 637308) was used to assess expression of FLAG-Tagged KRAS LSR. Cells were rinsed with buffer and acquired on an Agilent NovoCyte 300 Flow cytometer. Cells were analyzed first for light scatter versus GFP staining, then GFP−live gated cells were plotted for APC staining. FIG.16 depicts flow cytometry data indicating detection of KRAS-LSR-T cells expressing GFP and LSR Construct 1. [0183] Accordingly, the present Example documents expression of KRAS-LSRs as described herein in T cells. Example 7: Expression of KRAS-LSR Increases T-Cell Killing [0184] The present Example documents successful production of T cells expressing both a LSR and CAR in accordance with the present disclosure. Moreover, the present Example confirms that a KRAS-targeting LSR (e.g., KRAS-LSR) enhances cell killing activity of CAR-T cells. In fact, the present Example confirms that a KRAS-LSR increased the rate and extent of cell killing by two different types of CAR-T cells: Meso-CAR-T cells and EpCAM-CAR-T cells. [0185] T cells expressing a KRAS-targeting LSR (i.e., KRAS-LSR-T cells) were created as described in Example 6, expressing LSR Construct 1. Mesothelin-specific CAR- T cells (e.g., Meso-CAR-T cells) were created using a similar method by transducing T cells with lentivirus comprising a polynucleotide construct encoding a mesothelin-specific CAR (PMC958 ProMab Biotechnologies). T cells expressing both the KRAS-LSR and the Meso- CAR were created by simultaneously transfecting T cells with lentivirus comprising a polynucleotide encoding the KRAS-LSR construct and lentivirus comprising a polynucleotide encoding the Meso-CAR at a 4:1 ratio. [0186] KRAS-LSR expression was measured using the FLAG-tag as described in Example 6. To measure Meso-CAR expression, cells were suspended in 100 µL of buffer 1× PBS with 0.5% BSA+ 0.5 mM EDTA + 0.1% NaN3 and incubated on ice with 1 µL of Page 47 of 65 11825822v1 Docket No.: 2017422-0003 human serum for 10 min. Biotin-human Mesothelin protein (Acro Biosystems; MSN- H82E9) was added to the cells and incubated for 30 min at 4 ◦C, and after washing, PE- conjugated streptavidin (Biolegend; 405204) was added at 1:100 dilution for 30 min incubation at 4 ◦C. Cells were rinsed with 3 mL of washing buffer, then stained for 10 min with 7-AAD (Biolegend;420404), suspended in buffer, and acquired on an Agilent NovoCyte 300 Flow cytometer. As depicted in FIG.17, 7% of transduced T cells showed expression of both a KRAS-LSR and a Meso-CAR. [0187] Those skilled in the art will appreciate that mesothelin expression has been described as associated with a variety of different solid tumors, including, for example, mesotheliomas, epithelial ovarian cancers, and pancreatic adenocarcinomas, and also in lung and uterine malignancies as well as cholangiocarcinoma. See, for example, Pende, Cancers 14:1550, 2022, and references cited therein. The present Example documents effects of provided KRAS-targeting LSR with a Mesothelin-targeting CAR-T in human ovarian cancer cells. [0188] Specifically, adherent A1847 target cells (e.g., human ovarian cancer cells) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 × 104 cells per well and monitored in culture overnight with impedance-based real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences). After 24 hours, medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 × 105 effector cells: KRAS-targeting LSR-expressing T cells, Meso-CAR-T cells, or T cells that were transduced with both an LSR and CAR construct. Cells were monitored with a RTCA system, and impedance was plotted over time. CAR-T cell killing was calculated as (impedance of target cells with mock-transfected T cells—impedance of target cells with effector cells) × 100/impedance of target cells with mock transfected T cells. [0189] T cells expressing both a KRAS-LSR and Meso-CAR showed higher percent cell killing of A1847 cells (e.g., human ovarian cancer cells) relative to Meso-CAR-T cells. FIG.18 depicts that KRAS-LSR-T cells had no detected cell killing activity on their own, whereas Meso-CAR showed about 40% cell killing at 20 hours. KRAS-LSR/Meso-CAR-T cells showed about 65% cell killing at 20 hours. FIG.18 documents that co-expression of a LSR and CAR led to increased cell killing relative to Meso-CAR alone, thus documenting a synergistic effect of such co-expression. Moreover, as also shown in FIG.18, LSR-boosted cell killing activity was delayed by about 5 hours, consistent with LSR activation following initial cell killing and release of intracellular KRAS. Page 48 of 65 11825822v1 Docket No.: 2017422-0003 [0190] Without wishing to be bound to any theory, FIG.19 shows a schematic depicting a proposed mechanism of action for increased T cell activation and increased cell killing by an exemplary KRAS LSR. [0191] Dual CAR-T cells expressing a KRAS-LSR and an epithelial cell adhesion molecule-specific CAR (e.g., EpCAM-CAR) (ProMab Biotechnologies, PMC1057) were also produced as described herein. EpCAM-CAR expression was assessed using an anti- mouse FAB PE-conjugated antibody. As shown in FIG.20, 4% of transduced T cells showed expression of both a KRAS-LSR and an EpCAM-CAR. [0192] Those skilled in the art will appreciate that EpCAM expression has been described as a biomarker of cancer stem cells (CSCs) or circulating tumor cells (CTCs) and as associated with ovarian cancer, pancreatic cancer, and adenocarcinomas of various primary sites. See, for example, Spizzo, Journal of Clinical Pathology.64(5): 415–420, 2011, and Liu, Exp Hematol Onco.l 11, 97, 2022, and references cited therein, each of which is herein incorporated by reference in their entirety. [0193] The present Example documents effects of provided KRAS-targeting LSR with a EpCAM-targeting CAR-T in human pancreatic cancer cells. Specifically, T cells expressing both a KRAS-LSR and an EpCAM-CAR showed higher percent cell killing of SW-1990 cells (e.g., pancreatic cancer cells, e.g., pancreatic adenocarcinoma cells) relative to EpCAM-CAR-T cells alone, as depicted in FIG.21. [0194] Accordingly, the present Example confirms increased killing due to LSR construct expression in CAR-T targeting two different tumor antigens; these tumor antigens are associated with different types of tumors. Those skilled in the art will be aware of a variety of tumor antigens against which CAR-T have been or can be developed, and will appreciate the usefulness of provided technologies in the context of such CAR-T. For example, several CD19-specific CAR-T and BCMA-specific CAR-T cell therapies have been FDA approved for treatment of Acute Lymphoblastic Leukemia, multiple myeloma, and different B cell malignancies (Mitra, Front Immunol.14:1188049, 2023, which is herein incorporated by reference in its entirety). Page 49 of 65 11825822v1 Docket No.: 2017422-0003 Example 8: Exemplary KRAS-LSR-T cells Showed Increased T Cell Activation [0195] The present Example documents that co-expression of an exemplary LSR and a CAR in T cells, as described herein, results in a higher degree of T cell activation relative to expression of just the CAR in T cells. [0196] A1847 cells (e.g., human ovarian cancer cells) were cultured with effector cells, Meso-CAR-T cells, Meso-CAR/KRAS-LSR-T cells (i.e., T cells expressing both a KRAS-targeting LSR and a Meso-CAR, or non-transduced T cells, at a effector to target ratio or E:T ratio of 5:1 or 10:1 in U-bottom 96-well plates with 200 μL of AIM V- AlbuMAX medium containing 10% FBS, in triplicate. After 24 hours a top 150 μL of medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells. Supernatant was transferred to a new 96-well plate and analyzed by ELISA for IFN-gamma levels using a kit from Thermo Fisher (South San Francisco, CA, USA) according to manufacturer’s protocol. As depicted in FIG.22, Meso-CAR/KRAS- LSR-T cells have a higher degree of T cell activation, as measured by IFN-gamma expression, relative to Meso-CAR-T cells alone, and relative to a mixture of Meso-CAR-T cells and KRAS-LSR-T cells. [0197] Accordingly, the present Example documents that T cells expressing both a LSR and a CAR have a higher degree of T cell activation relative to T cells expressing just the CAR (i.e., CAR-T cells). Specifically, the present Example documents that Meso- CAR/KRAS-LSR-T cells have a higher degree of T cell activation relative to Meso-CAR-T cells alone, and relative to a mixture of Meso-CAR-T cells and KRAS-LSR-T cells. [0198] Those skilled in the art, reading the present Example, will appreciate that the particular LSR and CAR constructs utilized (i.e., KRAS-targeting LSRs, Meso-CARs, and EpCAM-CARs) are exemplary and not limiting of the present disclosure; its findings can be expected to be applicable to LSRs targeting other antigens and/or to different CARs. Example 9: Exemplary LSR Show Increased Tumor Specificity and Without Increased Non-specific Activity [0199] The present Example documents that T cells (e.g., CAR-T cells) expressing a KRAS-targeting LSR as exemplified herein (referred to in the present Example as “KRAS- LSR-T cells”) show tumor-specific efficacy (e.g., increased tumor-specificity relative to comparable CAR-T cells not expressing the KRAS-LSR-T cells) without increasing non- specific activity. Page 50 of 65 11825822v1 Docket No.: 2017422-0003 [0200] KRAS-LSR-T cells, Meso-CAR-T cells, and dual KRAS-LSR/Meso-CAR-T cells were prepared as described herein. Killing of A1847 cells was followed by RTCA. E:T ratio for all conditions was maintained at 10:1. For a mixed LSR + CAR sample, both the KRAS LSR and the meso CAR were added at a 5:1 E:T ratio. For all other samples the CAR or LSR cells were added at a 5:1 E:T ratio with an equivalent amount of mock transduced T cells used to bring an overall E:T ratio to 10:1. [0201] As indicated in FIG.22, dual KRAS-LSR/Meso-CAR-T cells increased activation as measured by IFN-gamma secretion relative to Meso-CAR-T cells. Mixing the two populations of T cells, those with the LSR alone and those with the Meso-CAR alone, does not lead to increased T cell activation. Without wishing to be bound to any theory, the increase in cell activation in the dual KRAS-LSR/Meso-CAR-T cells is believed to be due to the close proximity of the KRAS LSR to the killed A1847 cells, whereas the lack of increased activation from mixing of the two populations of T cells is believed to be due to the lower concentration of KRAS in the overall supernatant. [0202] Activation of KRAS-LSR-T cells by KRAS was further tested by addition of purified KRAS protein and A1847 cell lysates.1 ug/ml purified G12D KRAS (Acro Biosystems KRS-H51H4) was added to KRAS-LSR-T cells and cell killing of A1847 cells was followed using RTCA. Results were normalized to KRAS-LSR-T cells alone. Addition of purified KRAS did not increase cell killing in other controls (Data not shown). Supernatant from A1847 cells previously killed by Meso-CAR-T cells was collected and frozen. After thawing, supernatant was added to KRAS LSR-T cells at a 1:5 ratio and cell killing of A1847 cells was followed by RTCA. No cell killing was observed by addition of supernatant alone in other controls (Data not shown). Addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells resulted in moderate cell killing of A1847 cells by KRAS-LSR-T cells, as depicted in FIG.23. Addition of purified G12D KRAS resulted in increased killing of A1847 cells by KRAS-LSR-T cells relative to addition of supernatant from A1847 cells previously killed by Meso-CAR-T cells, as depicted in FIG.23. [0203] AKRAS antibody raised against mutant KRAS was used to test specificity of KRAS-LSR-T cell activation. Anti-KRAS mutant monoclonal antibody was used at a 1:1000 dilution (Thermofisher, cat#: MA5-36256). A1847 cell killing was followed by RTCA. As shown in FIG.24, anti-KRAS mutant antibody reduced rate of cell killing by dual KRAS LSR/Meso-CAR-T cells. Increased cell killing that results from dual expression of the KRAS-LSR and the Meso-CAR in the same T cells is blocked by a monoclonal Page 51 of 65 11825822v1 Docket No.: 2017422-0003 antibody to mutant KRAS. Without wishing to be bound to any theory, the present disclosure describes that LSR activation occurs through binding of KRAS released by cell lysis. [0204] Accordingly, the present Example confirms that KRAS-LSR-T cells described herein show tumor-specific efficacy without an increase in non-specific activity. In particular, the present Example confirms that purified KRAS protein increases activity of a KRAS-LSR-T cell. The present Example also confirms that an anti-KRAS antibody inhibits activity of a KRAS-LSR-T cell. Exemplary Embodiments [0205] Embodiment 1. An engineered lysis sensor receptor (LSR) comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element). [0206] Embodiment 2. The engineered LSR of embodiment 1, wherein the lysis-associated-antigen binding moiety responds to/targets an intracellular product of diseased cell(s) in a subject, optionally an intracellular tumor antigen. [0207] Embodiment 3. The engineered LSR of embodiment 1, wherein the T cell receptor co-stimulation element is selected from a group consisting of CD28, OX40, 4I- BB, GITR, ICOS-1, CD27, AP10, and any combination thereof. [0208] Embodiment 4. The engineered LSR of embodiment 3, wherein the co- stimulation element further comprises a CD3 zeta (CD3 Z or CD3C) activating element. [0209] Embodiment 5. The engineered LSR of embodiment 3, wherein the co- stimulation element does not comprise a CD3 zeta (CD3 Z or CD3C) activating element. [0210] Embodiment 6. A polynucleotide construct comprising a payload coding sequence operably linked to an expression control sequence, wherein the payload coding sequence encodes the LSR of embodiment 1. Page 52 of 65 11825822v1 Docket No.: 2017422-0003 [0211] Embodiment 7. A vector comprising the polynucleotide construct encoding the LSR of embodiment 1 to facilitate introduction into cells. [0212] Embodiment 8. The vector of embodiment 7, wherein the vector is an expression vector, an episomal vector, a viral vector, a retroviral vector (e.g., an oncoretrovirus vector, a lentivirus vector, or a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a sendai virus vector, an Epstein-Barr virus (EBV) vector, or a HSV vector, among others. [0213] Embodiment 9. The vector of embodiment 8, wherein the viral vector lacks ability to replicate so as not to replicate in an infected cell. [0214] Embodiment 10. The vector of embodiment 8, wherein the vector is a lentiviral vector selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV). [0215] Embodiment 11. A preparation of virions comprising a viral capsid and the polynucleotide construct of embodiment 6. [0216] Embodiment 12. The preparation of virions of embodiment 11, wherein the viral capsid is an AAV capsid. [0217] Embodiment 13. The preparation of virions of embodiment 11, wherein the polynucleotide construct comprises at least one inverted terminal repeat (ITR). [0218] Embodiment 14. The preparation of virions of any one of the preceding embodiments, which virions are active to infect an immune effector cell. [0219] Embodiment 15. The preparation of virions of embodiment 14, wherein the immune effector cell is a T cell. Page 53 of 65 11825822v1 Docket No.: 2017422-0003 [0220] Embodiment 16. The preparation of virions of embodiment 15, wherein the T cell is an autologous T cell. [0221] Embodiment 17. The preparation of virions of embodiment 15, wherein the T cell is an allogenic T cell. [0222] Embodiment 18. The preparation of virions of embodiment 14, wherein the immune effector cell is an NK cell. [0223] Embodiment 19. A producer cell engineered to contain one or more of: (a) a construct comprising: (i) a first sequence that is or comprises a payload coding sequence operably linked to an expression control sequence, wherein the payload coding sequence encodes the LSR of embodiment 1 (ii) a second sequence that is or comprises a Rep protein-dependent origin of replication (ori); and (iii) at least one viral ITR; (b) the Rep protein that acts on the ori, (c) a viral capsid protein, so that virions are produced in the producer cell that comprise the viral capsid protein encapsidating the construct. [0224] Embodiment 20. A gene editing system comprising: (a) a CRISPR/Cas gene editing system comprising: (i) a gRNA molecule having a nucleotide sequence comprising a targeting sequence specific to an integration site in the T cell genome, and (ii) a Cas9 protein or a nucleic acid encoding a Cas9 protein; and (b) a virion comprising a viral capsid and the polynucleotide construct of embodiment 6. [0225] Embodiment 21. A population of T cells engineered to express a LSR comprising: Page 54 of 65 11825822v1 Docket No.: 2017422-0003 a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element). [0226] Embodiment 22. The population of T cells of embodiment 21, wherein the population of T cells is isolated from a human. [0227] Embodiment 23. The population of T cells of embodiment 22, wherein the human is a patient to whom the engineered population of T cells is to be administered. [0228] Embodiment 24. The population of T cells of embodiment 21, wherein the T cells are obtained from a site that is in the vicinity of a tumor. [0229] Embodiment 25. The population of T cells of embodiment 24, wherein the site comprises part or all of the tumor. [0230] Embodiment 26. The population of T cells of embodiment 21, wherein the population of T cells also express a T cell receptor (TCR). [0231] Embodiment 27. A method of producing a population of T cells according to any of the preceding embodiments comprising steps of: (a) obtaining a plurality of T cells; (b) introducing into the T cells: (i) the gene editing system of embodiment 20, so that a population of engineered T cells is produced. [0232] Embodiment 28. A method of producing a population of engineered T cells according to any of the preceding embodiments comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the polynucleotide construct of embodiment 6. [0233] Embodiment 29. A method of treating a disease, comprising a step of: Page 55 of 65 11825822v1 Docket No.: 2017422-0003 (a) administering to a subject suffering from a disease, disorder or condition, a population of T cells engineered to express a LSR that responds to/targets an intracellular product of diseased cell(s) in the subject. [0234] Embodiment 30. The method of treatment of embodiment 29, wherein the subject has a disease associated with expression of a tumor or disease-related antigen (e.g., a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the disease-related antigen). [0235] Embodiment 31. The method of embodiment 29, wherein the diseased cells are tumor cells. [0236] Embodiment 32. The method of embodiment 29, wherein the diseased cells are infected cells. [0237] Embodiment 33. The method of embodiment 29, wherein the step of administering comprises administering a population of T cells engineered by a process comprising steps of: (a) obtaining a plurality of T cells; (b) transducing the cells with a preparation of virions according to any one of embodiments 11-18, optionally further selecting or screening for the transduced cells; and (c) administering the transduced cells to a subject in need thereof. [0238] Embodiment 34. A method of stimulating endogenous tumor infiltrating lymphocytes (TIL) in a subject, the method comprising steps of: (a) obtaining a population of TIL from a subject; and (b) engineering the population of T cells to express the LSR of embodiment 1, so that a population of engineered TIL is generated. [0239] Embodiment 35. The method of embodiment 34, the method further comprising a step of: administering the population of engineered TIL. Page 56 of 65 11825822v1 Docket No.: 2017422-0003 [0240] Embodiment 36. The method of embodiment 34, wherein the obtained population of TIL comprises a plurality of TIL having TCRs directed to different antigens. [0241] Embodiment 37. The method of embodiment 35, wherein the administered population of engineered TIL comprises a plurality of engineered TIL having TCRs directed to different antigens. [0242] Embodiment 38. The method of treatment of embodiment 29, wherein the method of treatment comprises at least two successive doses. [0243] Embodiment 39. The method of treatment of embodiment 38, wherein the second dose further comprises an indication that a disease, disorder or condition in a subject has progressed. [0244] Embodiment 40. The method of treatment of embodiment 29 wherein the treatment is administered in combination with another immune oncology therapy. [0245] Embodiment 41. The method of treatment of embodiment 40 wherein the immune oncology therapy is a checkpoint inhibitor, a cytotoxic chemotherapy, or radiation therapy. [0246] Embodiment 42. A method of characterizing a LSR according to any of embodiments 1-5, or a population of T cells according to embodiments 21-26. [0247] Embodiment 43. A method of manufacturing a population of T cells comprising: (i) engineering the population of T cells to express the LSR of embodiments 1-5. [0248] Embodiment 44. The method of manufacturing of embodiment 43, wherein the step of engineering comprises introducing the preparation of virions of embodiments 11-18 such that the payload encoding the LSR of embodiment 1 integrates at a site in the T cell genome. Page 57 of 65 11825822v1 Docket No.: 2017422-0003 [0249] Embodiment 45. The method of manufacturing a population of T cells according to embodiment 43 further comprising a step of isolating T cells from a human subject and administering to the human subject. Equivalents [0250] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: Page 58 of 65 11825822v1

Claims

Docket No.: 2017422-0003 Claims We claim: 1. An engineered lysis sensor receptor (LSR) comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element). 2. The engineered LSR of claim 1, wherein the lysis-associated-antigen binding moiety responds to/targets an intracellular product of diseased cell(s) in a subject, optionally an intracellular tumor antigen. 3. The engineered LSR of claim 1, wherein the T cell receptor co-stimulation element is selected from a group consisting of CD28, OX40, 4I-BB, GITR, ICOS-1, CD27, AP10, and any combination thereof. 4. The engineered LSR of claim 3, wherein the co-stimulation element further comprises a CD3 zeta (CD3 Z or CD3C) activating element. 5. The engineered LSR of claim 3, wherein the co-stimulation element does not comprise a CD3 zeta (CD3 Z or CD3C) activating element. 6. A polynucleotide construct comprising a payload coding sequence operably linked to an expression control sequence, wherein the payload coding sequence encodes the LSR of claim 1. 7. A vector comprising the polynucleotide construct encoding the LSR of claim 1 to facilitate introduction into cells. 8. The vector of claim 7, wherein the vector is an expression vector, an episomal vector, a viral vector, a retroviral vector (e.g., an oncoretrovirus vector, a lentivirus vector, or a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a sendai virus vector, an Epstein-Barr virus (EBV) vector, or a HSV vector, among others. Page 59 of 65 11825822v1 Docket No.: 2017422-0003 9. The vector of claim 8, wherein the viral vector lacks ability to replicate so as not to replicate in an infected cell. 10. The vector of claim 8, wherein the vector is a lentiviral vector selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV). 11. A preparation of virions comprising a viral capsid and the polynucleotide construct of claim 6. 12. The preparation of virions of claim 11, wherein the viral capsid is an AAV capsid. 13. The preparation of virions of claim 11, wherein the polynucleotide construct comprises at least one inverted terminal repeat (ITR). 14. The preparation of virions of any one of the preceding claims, which virions are active to infect an immune effector cell. 15. The preparation of virions of claim 14, wherein the immune effector cell is a T cell. 16. The preparation of virions of claim 15, wherein the T cell is an autologous T cell. 17. The preparation of virions of claim 15, wherein the T cell is an allogenic T cell. 18. The preparation of virions of claim 14, wherein the immune effector cell is an NK cell. 19. A producer cell engineered to contain one or more of: (a) a construct comprising: Page 60 of 65 11825822v1 Docket No.: 2017422-0003 (i) a first sequence that is or comprises a payload coding sequence operably linked to an expression control sequence, wherein the payload coding sequence encodes the LSR of claim 1 (ii) a second sequence that is or comprises a Rep protein-dependent origin of replication (ori); and (iii) at least one viral ITR; (b) the Rep protein that acts on the ori, (c) a viral capsid protein, so that virions are produced in the producer cell that comprise the viral capsid protein encapsidating the construct. 20. A gene editing system comprising: (a) a CRISPR/Cas gene editing system comprising: (i) a gRNA molecule having a nucleotide sequence comprising a targeting sequence specific to an integration site in the T cell genome, and (ii) a Cas9 protein or a nucleic acid encoding a Cas9 protein; and (b) a virion comprising a viral capsid and the polynucleotide construct of claim 6. 21. A population of T cells engineered to express a LSR comprising: a) a lysis-associated-antigen binding moiety; b) a transmembrane element; and c) a signaling element (e.g., a T cell receptor co-stimulation element). 22. The population of T cells of claim 21, wherein the population of T cells is isolated from a human. 23. The population of T cells of claim 22, wherein the human is a patient to whom the engineered population of T cells is to be administered. 24. The population of T cells of claim 21, wherein the T cells are obtained from a site that is in the vicinity of a tumor. Page 61 of 65 11825822v1 Docket No.: 2017422-0003 25. The population of T cells of claim 24, wherein the site comprises part or all of the tumor. 26. The population of T cells of claim 21, wherein the population of T cells also express a T cell receptor (TCR). 27. A method of producing a population of T cells according to any of the preceding claims comprising steps of: (a) obtaining a plurality of T cells; (b) introducing into the T cells: (i) the gene editing system of claim 20, so that a population of engineered T cells is produced. 28. A method of producing a population of engineered T cells according to any of the preceding claims comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the polynucleotide construct of claim 6. 29. A method of treating a disease, comprising a step of: (a) administering to a subject suffering from a disease, disorder or condition, a population of T cells engineered to express a LSR that responds to/targets an intracellular product of diseased cell(s) in the subject. 30. The method of treatment of claim 29, wherein the subject has a disease associated with expression of a tumor or disease-related antigen (e.g., a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the disease-related antigen). 31. The method of claim 29, wherein the diseased cells are tumor cells. 32. The method of claim 29, wherein the diseased cells are infected cells. 33. The method of claim 29, wherein the step of administering comprises administering a population of T cells engineered by a process comprising steps of: Page 62 of 65 11825822v1 Docket No.: 2017422-0003 (a) obtaining a plurality of T cells; (b) transducing the cells with a preparation of virions according to any one of claims 11-18, optionally further selecting or screening for the transduced cells; and (c) administering the transduced cells to a subject in need thereof. 34. A method of stimulating endogenous tumor infiltrating lymphocytes (TIL) in a subject, the method comprising steps of: (a) obtaining a population of TIL from a subject; and (b) engineering the population of T cells to express the LSR of claim 1, so that a population of engineered TIL is generated. 35. The method of claim 34, the method further comprising a step of: administering the population of engineered TIL. 36. The method of claim 34, wherein the obtained population of TIL comprises a plurality of TIL having TCRs directed to different antigens. 37. The method of claim 35, wherein the administered population of engineered TIL comprises a plurality of engineered TIL having TCRs directed to different antigens. 38. The method of treatment of claim 29, wherein the method of treatment comprises at least two successive doses. 39. The method of treatment of claim 38, wherein the second dose further comprises an indication that a disease, disorder or condition in a subject has progressed. 40. The method of treatment of claim 29 wherein the treatment is administered in combination with another immune oncology therapy. 41. The method of treatment of claim 40 wherein the immune oncology therapy is a checkpoint inhibitor, a cytotoxic chemotherapy, or radiation therapy. Page 63 of 65 11825822v1 Docket No.: 2017422-0003 42. A method of characterizing a LSR according to any of claims 1-5, or a population of T cells according to claims 21-26. 43. A method of manufacturing a population of T cells comprising: (i) engineering the population of T cells to express the LSR of claims 1-5. 44. The method of manufacturing of claim 43, wherein the step of engineering comprises introducing the preparation of virions of claims 11-18 such that the payload encoding the LSR of claim 1 integrates at a site in the T cell genome. 45. The method of manufacturing a population of T cells according to claim 43 further comprising a step of isolating T cells from a human subject and administering to the human subject. Page 64 of 65 11825822v1
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180258391A1 (en) * 2010-12-09 2018-09-13 The Trustees Of The University Of Pennsylvania Compositions and Methods for Treatment of Cancer
US20190248879A1 (en) * 2016-10-17 2019-08-15 Agency For Science, Technology And Research Anti-p53 antibodies

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180258391A1 (en) * 2010-12-09 2018-09-13 The Trustees Of The University Of Pennsylvania Compositions and Methods for Treatment of Cancer
US20190248879A1 (en) * 2016-10-17 2019-08-15 Agency For Science, Technology And Research Anti-p53 antibodies

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