Massively parallel mixed lymphocyte reactions

Abstract

Compositions and methods are provided for multiplexed screening of chimeric antigen receptor (CAR)-T cells. The subject methods allow engineered T cells from multiple donors to be pooled and tested simultaneously. Pooling of T cells from multiple subjects for screening decreases the cost of cell therapy development and makes pre-clinical development work for autologous and allogeneic engineered T cell therapies more efficient.

Description

MASSIVELY PARALLEL MIXED LYMPHOCYTE REACTIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63 / 548,724, filed February 1 , 2024, which application is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Testing engineered human cell-based therapeutics, in particular, engineered human immune cells such as chimeric antigen receptor (CAR)-T cells is costly and time consuming, which impedes research and development and diagnostic and therapeutic applications of potentially curative cellular medicines. There remains a need for more efficient, inexpensive methods of screening immune cells for use in cellular therapies, especially cells from multiple human donors and patients.SUMMARY OF THE I VENTION

[0003] Compositions and methods are provided for multiplexed screening of CAR-T cells. The subject methods allow engineered T cells from multiple donors to be pooled and tested simultaneously. Pooling of T cells from multiple subjects for screening decreases the cost of cell therapy development and makes pre-clinical development work for autologous and allogeneic engineered T cell therapies more efficient.

[0004] In one aspect, a method of multiplexed screening to identify CAR-T cells that are immunoreactive against a target cell is provided, the method comprising: providing a pooled population of CAR-T cells, wherein the CAR-T cells comprise T cells from a plurality of donors, wherein each T cell is engineered to express a chimeric antigen receptor that specifically binds to a target antigen on the target cell, and wherein expression of an endogenous T cell receptor (TCR) is eliminated in each T cell; contacting the pooled population of CAR-T cells with the target cell; and measuring an immune response of the population of CAR-T cells or a subset of the population of CAR- T cells to identify the CAR-T cells that are immunoreactive against the target cell.

[0005] In certain embodiments, measuring the immune response comprises measuring cytotoxicity against the target cell, cytokine secretion, expression of an activation marker, affinity of binding to the target antigen on the target cell, or proliferation of the CAR-T cells that are immunoreactive against the target cell, or any combination thereof. In someembodiments, measuring expression of an activation marker comprises detecting CD69, HLA-DR, IL2RA, or CD25, or any combination thereof. In some embodiments, measuring the cytotoxicity comprises detecting perforin, a proapoptotic serine protease, or a granzyme released from the CAR-T cell. In some embodiments, measuring cytotoxicity comprises detecting death of the target cell. In some embodiments, measuring cytokine secretion comprises detecting interferon (IFN)-y, tumor necrosis factor (TNF)-a, TNF-£, interleukin (IL)-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, or IL-25, or any combination thereof.

[0006] In certain embodiments, the CAR-T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell that has been genetically modified to express the chimeric antigen receptor, and wherein expression of the endogenous TCR is eliminated.

[0007] In certain embodiments, each T cell is further engineered to express a fluorescent protein, wherein T cells from the same donor are engineered to express the same fluorescent protein, and wherein T cells from different donors are engineered to express different fluorescent proteins.

[0008] In certain embodiments, the method further comprises genetically barcoding the population of CAR-T cells, wherein each CAR-T cell comprises a DNA barcode indicating which donor provided the T cell from which the CAR-T cell was generated.

[0009] In certain embodiments, measuring an immune response comprises measuring cytotoxicity against the target cell, cytokine secretion, expression of activation markers, affinity of binding to the target antigen on the target cell, or proliferation of the CAR-T cells that are immunoreactive against the target cell.

[0010] In certain embodiments, the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen on the target cell. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigenbinding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen. In some embodiments, the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain. In some embodiments, theintracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit [35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain. In certain embodiments, the chimeric antigen receptor further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.

[0011] In certain embodiments, the target cell is a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.

[0012] In certain embodiments, the antigen on the target cell is a tumor antigen or tumor- associated antigen.

[0013] In certain embodiments, the pathogen is a virus, a bacterium, a fungus, or a parasite. In some embodiments, the antigen on the target cell is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

[0014] In certain embodiments, the autoreactive immune cell is an autoreactive T cell or B cell. In some embodiments, the antigen on the target cell is an antigen on the autoreactive T cell or B cell.

[0015] In certain embodiments, the T cells are engineered using a gene editing system selected from the group consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, a zinc finger nuclease system, a transcription activatorlike effector nuclease (TALEN) system, and a meganuclease system.

[0016] In certain embodiments, the T cells are engineered to express the chimeric antigen receptor by transfecting the T cells with a recombinant polynucleotide encoding the chimeric antigen receptor. In some embodiments, the engineered T cells have their endogenous TCR removed. In some embodiments, T cells engineered to express the chimeric antigen receptor are substantially purified, wherein T cells still expressing an endogenous TCR are eliminated.

[0017] In certain embodiments, the method further comprises contacting the CAR-T cells with an antigen presenting cell or an artificial antigen presenting particle presenting the target antigen.

[0018] In another aspect, a CAR-T cell is provided, wherein the CAR-T is identified as being immunoreactive against a target cell using a method of multiplexed screening, described herein. In some embodiments, a composition comprising the CAR-T cell and a pharmaceutically acceptable excipient is provided. In certain embodiments, a methodof performing cellular therapy is provided, the method comprising administering a therapeutically effective amount of the composition to a subject. In some embodiments, the CAR-T cell is autologous or allogeneic.

[0019] In certain embodiments, the chimeric antigen receptor of the CAR-T cell specifically binds to a tumor antigen or tumor-associated antigen. In certain embodiments, a method of treating cancer in a subject is provided, the method comprising administering a therapeutically effective amount of the CAR-T cell comprising the chimeric antigen receptor that specifically binds to the tumor antigen or tumor- associated antigen to the subject.

[0020] In certain embodiments, the chimeric antigen receptor of the CAR-T cell specifically binds to a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen. In certain embodiments, a method of treating an infection in a subject is provided, the method comprising administering a therapeutically effective amount of the CAR-T cell comprising the chimeric antigen receptor that specifically binds to the viral antigen, bacterial antigen, fungal antigen or parasite antigen to the subject.

[0021] In certain embodiments, the chimeric antigen receptor of the CAR-T cell specifically binds to an antigen on an autoreactive T cell or B cell. In certain embodiments, a method of treating an autoimmune disease in a subject is provided, the method comprising administering a therapeutically effective amount of the CAR-T cell comprising the chimeric antigen receptor that specifically binds to the antigen on an autoreactive T cell or B cell to the subject.

[0022] In certain embodiments, the CAR-T cell further comprises a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the CAR-T cell. In some embodiments, the binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR). In some embodiments, the synthetic notch receptor comprises i) an extracellular ligand-binding domain that specifically binds to a second target antigen on the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the CAR-T cell.

[0023] In another aspect, a kit is provided, the kit comprising: a population of CAR-T cells, wherein the CAR-T cells comprise T cells from a plurality of donors, wherein each T cell is engineered to express a chimeric antigen receptor that specifically binds to an antigen on the target cell, and wherein expression of an endogenous T cell receptor (TCR) is eliminated in each T cell; and instructions for measuring an immune response against the target cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1. Donor Module. Knockout of the endogenous TCR genes in CAR-T cells derived from different donors allows pooling of CAR-T cells from different donors without cross-reaction. Normally, T cells from one donor may recognize T cells from another donor as foreign and become activated to kill T cells from another donor. TCR knockout enables pooled multiplexed screening to identify CAR-T cells with clinical potential.

[0025] FIG. 2. Mixed T cell reactions of pooled T cells from 5 donors. Activation of pooled T cells from multiple donors is dependent on TCR status. The percent activation of pooled T cells with and without endogenous TCRs was determined by detection of CD69, a marker of T cell activation.

[0026] FIG. 3. Pooled donor screening using massively parallel mixed lymphocyte reactions. Traditional testing of genetically modified T cells for therapeutic, diagnostic, or research purposes requires every unique donor’s T cells to be kept separate and tested individually. Massively parallel mixed lymphocyte reactions allow multiple donors T cells to be tested simultaneously in a large pool. By editing the T cells to integrate new genetic material at the site of a TCR component (e.g. TRAC locus), the majority of successfully edited T cells will lose expression of the TCR on their surface. T cells without a TCR on their surface can be selected for by depletion of TCR positive (e.g. CD3+) T cells, leaving only T cells without their endogenous TCR, the majority of which have had the new genetic material introduced (e.g. chimeric antigen receptor, among others). The genetic material introduced also includes a DNA barcode unique to each donor. These barcoded, edited TCR negative T cells from multiple donors can then be combined together to form a pool of barcoded T cells from multiple donors, which can now be tested together in experimental assays as TCR negative T cells do not show significant alloreactivity against other donor’s similarly TCR negative cells. At the beginning and end of the test assay, the donor barcodes can be read out by DNA sequencing to assess the function of each donor's T cells individually within the larger donor pool.DETAILED DESCRIPTION OF THE INVENTION

[0027] Compositions and methods are provided for multiplexed screening of chimeric antigen receptor (CAR)-T cells. The subject methods allow engineered T cells from multiple donors to be pooled and tested simultaneously. Pooling of T cells from multiple subjects for screening decreases the cost of cell therapy development and makes pre- clinical development work for autologous and allogeneic engineered T cell therapies more efficient.

[0028] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular devices, systems, software, and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0031] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an”, and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof, such as polynucleotides, known to those skilled in the art, and so forth.

[0033] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Definitions

[0034] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0035] “Biocompatible” or “cytocompatible” as used herein, refers to a property of a material that allows for prolonged contact with a cell or tissue without causing toxicity or significant damage.

[0036] The terms “engineered” or “recombinant” in reference to a T cell, gene, nucleic acid and / or protein as used herein, refer to a T cell, gene, nucleic acid and / or protein that has been altered through human intervention. Accordingly, the term “naturally occurring” as used herein in reference to a T cell, gene, nucleic acid and / or protein as used herein, refer to a T cell, gene, nucleic acid and / or protein existing in nature and without any human intervention. Exemplary human interventions comprise transfection with a heterologous polynucleotide, molecular cloning resulting in a deletion, insertion, modification and / or rearrangement with respect to a naturally occurring sequence such as a naturally occurring sequence in a T cell, gene, nucleic acid and / or protein herein described.

[0037] The term “biological sample” encompasses a clinical sample, including, but not limited to, a bodily fluid, tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirate, lymph node aspirate, cystic aspirate, a paracentesis sample, a thoracentesis sample, and the like.

[0038] The terms “obtained” or “obtaining” as used herein can also include the physical extraction or isolation of a biological sample (e.g., comprising immune cells) from a subject. Accordingly, a biological sample comprising immune cells can be isolated from a subject (and thus “obtained”) by the same person or same entity that subsequently isolates immune cells from the sample. When a biological sample is “extracted” or “isolated” from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample was “obtained” by the first party (and also “isolated” by the first party), and then subsequently “obtained” (but not “isolated”) by the second party. Accordingly, in some embodiments, the step of obtaining does not comprise the step of isolating a biological sample.

[0039] In some embodiments, the step of obtaining comprises the step of isolating a biological sample. Methods and protocols for isolating various biological samples (e.g., a blood sample, a biopsy sample, an aspirate, etc.) will be known to one of ordinary skill in the art and any convenient method may be used to isolate a biological sample.

[0040] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and / or in which the entity of interest is partially or substantially purified.

[0041] "Substantially" or "essentially" means nearly totally or completely, for instance, 95% or greater of some given quantity.

[0042] "Substantially purified" generally refers to isolation of a component of a sample (e.g., cell or substance), such that the component comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises at least 70%, preferably at least 80%-85%, more preferably at least 90-99% of the sample.

[0043] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention. Subjects include, but are not limited to, mammals, including human and non-human mammals such as non-human primates, including chimpanzeesand other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals. In the context of the disclosure, the term "subject" generally refers to an individual who will be administered or who has been administered one or more compositions described herein (e.g., cellular therapy with cells screened according to the methods described herein).

[0044] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms for use in the present invention depend on the particular compound employed and the effect to be achieved, the pharmacodynamics associated with each compound in the host, and the like.

[0045] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted as well as those in which prevention is desired, including those with a genetic predisposition or increased susceptibility to developing a disease.

[0046] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In someembodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0047] A "therapeutically effective amount" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose or amount can be administered in one or more administrations.

[0048] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0049] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0050] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0051] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0052] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences,respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in AppL Math. 2:482 489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0053] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0054] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed bydigestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra', DNA Cloning, supra', Nucleic Acid Hybridization, supra.

[0055] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0056] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0057] "Recombinant host cells," "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0058] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0059] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0060] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0061] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0062] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0063] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cellmembrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0064] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0065] The term “hybridization” refers to the specific binding of a nucleic acid to a complementary nucleic acid via Watson-Crick base pairing.

[0066] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non- viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[0067] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about I Q- 12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0068] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0069] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[0070] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0071] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0072] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[0073] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be allele-specific (e.g., a major or minor allele).

[0074] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0075] "Administering" a nucleic acid, such as a viral vector or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Cas12a, Cas12d, Cas13, or Cpf1 )) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.

[0076] A "barcode" refers to one or more nucleotide sequences that are used to identify a nucleic acid or cell with which the barcode is associated. Barcodes can be 3-1000 or more nucleotides in length, preferably 10-250 nucleotides in length, and more preferably 10-30 nucleotides in length, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. Barcodes may be used, for example, to identify a single cell, subpopulation of cells, colony, or sample from which a nucleic acid originated. Barcodes may also be used to identify the position (i.e., positional barcode) of a cell, colony, or sample from which a nucleic acid originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, or the position of a tube, flask, or other container in a rack. In particular, a barcode may be used to identify a genetically modified cell from which a nucleic acid originated. In some embodiments, a barcode is used to identify a donor T cell from which a CAR-T cell originated. Alternatively, a unique barcode may be used to identify each guide-RNA and donor polynucleotide used in multiplexed or multi-stepgenome editing. Furthermore, multiple barcodes can be used in combination to identify different features of a nucleic acid or cell. For example, positional barcoding (e.g., to identify the position of a cell, colony, culture, or sample in an array, multi-well plate, or rack) can be combined with barcodes identifying a T cell donor and / or barcodes identifying guide-RNAs or donor polynucleotides used in genome editing.

[0077] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and / or imino molecules. No particular size is implied by use of the terms “polypeptide,” “peptide,” and “protein” and these terms are used interchangeably. The “terms include post-expression modifications of the polypeptide, peptide, or protein such as glycosylation, acetylation, phosphorylation, and the like. Further, polypeptides, peptides, or proteins, as described herein may include additional molecules such as labels (e.g., fluorescent, bioluminescent, or radioactive), tags (e.g., histidine tag, epitope tag), or other chemical moieties.

[0078] The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, monoclonal antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, single-domain antibodies, nanobodies, bispecific antibodies, tri-specific antibodies, and other multi-specific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0079] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0080] “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between theVn and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0081] The terms “specific binding,” “specifically binds,” “selectively binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10-5M or less (e.g., 10-6M or less, 10-7M or less, 10‘8M or less, 10-9M or less, 10'1° M or less, 10’11M or less, 10-12M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g. as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.

[0082] The term “antigen-binding fragment” as used herein refers to any antibody fragment that specifically binds to a target antigen including, but not limited to, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody including one or more complementarity determining regions (CDRs).

[0083] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the [3-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation ofthe antibody in antibody-dependent cellular toxicity. VL and VH sequences can be reformatted as fragments, as single chain binding domains, linked to chimeric antigen receptors, and the like.

[0084] The term “antigen binding domain (ABD)” refers to a domain that specifically binds to a target antigen. The antigen binding domain region of an antibody may comprise a heavy-chain variable domain (VH) and a light-chain variable domain (VL) in non-covalent association as a single polypeptide or as a dimer. The three complementaritydetermining regions of the heavy chain variable domain (CDR H1 , H2, H3) and three complementarity-determining regions of the light chain variable domain (CDR L1 , L2, L3) interact to define an antigen-binding site on the surface of an antibody. Collectively, the six CDRs of the light chain and heavy chain variable domains confer antigen-binding specificity to an antibody. An antigen binding domain region of a CAR may comprise all six CDRs of an antibody or a single variable domain or half of an Fv fragment comprising only three CDRs specific for an antigen, which still retains the ability to recognize and bind the target antigen. In some embodiments, the antigen-binding domain binds to one or more target antigens expressed on the surface of a target cell (e.g., cell surface markers).

[0085] The term "T cell" includes all types of immune cells expressing CD3 including T- helper cells (CD4+cells), cytotoxic T-cells (CD8+cells), natural killer T cells, T-regulatory cells (Treg) and gamma-delta T cells. The term "T cell" also includes genetically modified T cells, including T cells engineered to express a chimeric antigen receptor (CAR) and T cells from which the gene encoding the endogenous T cell receptor has been inactivated or deleted (i.e. , TCR gene knockout).

[0086] The terms “T cell receptor” and “TCR” are used interchangeably and generally refer to a receptor found on the surface of T cells or T lymphocytes that is responsible for recognizing antigenic peptides bound to major histocompatibility complex (MHC) molecules. The TCR is a membrane-anchored heterodimeric protein comprising two different protein chains. In the majority of human T cells, the TCR consists of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB genes, respectively). In about 5% of human T cells, the TCR consists of gamma and delta (y / 8) chains (encoded by TRG and TRD genes, respectively). T cells expressing a TCR comprising alpha and beta chains are referred to as a(3 T cells, and T cells expressing a TCR comprising gamma and delta chains are referred to as yS T cells The ratio of cc|3 T cells to y8 T cells differsbetween species and may be altered by disease (such as leukemia). The variable domains of the TCR a-chain and p-chain each have three hypervariable or complementarity-determining regions (CDRs). CDR 1 and CDR3 bind to the antigenic peptide. CDR2 recognizes the MHC. The constants domains of the TCR a-chain and p- chain each have a cysteine that forms a disulfide bond that links the two chains. The TCR receptor a and chains associate with six additional adaptor proteins, including a delta chain, a gamma chain, two epsilon chains, and two zeta chains to form an octameric complex. The adaptor proteins comprise signaling motifs involved in TCR signaling.

[0087] Chimeric antigen receptor (CAR). A CAR may have any suitable architecture, as known in the art, comprising an antigen binding domain, usually provided in an scFv format, linked to T cell receptor effector functions. The term refers to artificial multimodule molecules capable of triggering or inhibiting the activation of an immune cell. A CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR will include one or more co-stimulatory domains and / or one or more co-inhibitory domains.

[0088] The antigen-binding domain of the CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest. In some embodiments, the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof. The antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof. Thus, in some embodiments, the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof. An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0089] In some embodiments, the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen. In some embodiments, the antigen binding domain is provided by a single chain variable fragment (scFv). A scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and / or amino acid composition of the linker peptide joining the VH and VL sequences. In some embodiments, any arbitrary single-chain peptide including about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[0090] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some embodiments, the transmembrane domain comprises at least the stalk and / or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit f35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and / or CD86. In some embodiments, the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3 CD4, CD8, or CD28. In other embodiments, the transmembrane domain is synthetic, in which case it will include predominantly hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.

[0091] In some embodiments, the CAR further comprises one or more linkers / spacers. For example, an extracellular spacer region may link the antigen binding domain to the transmembrane domain and / or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain. A spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[0092] Various types of linkers may be used in the CARs described herein. In some embodiments, the linker includes a peptide linker / spacer sequence. In some embodiments, the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.

[0093] In principle, there are no particular limitations to the length and / or amino acid composition of a linker peptide sequence. In some embodiments, a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR. In some embodiments the linker comprises the amino acid sequence (G4S)n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.

[0094] A cytoplasmic signaling domain, such as those derived from the T cell receptor - chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.

[0095] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which are incorporated herein by reference in their entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0096] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protein. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .

[0097] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FcsRIy, or the CD3 chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.

[0098] Second-generation CARs include a co-stimulatory signal in addition to the CD3 signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatorydomain will usually be membrane proximal relative to the CD3^ domain. Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR-T cells, CAR-T cells are further genetically modified to express or block molecules and / or receptors to enhance immune activity.

[0099] CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Trans! Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388- 98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0100] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains

[0101] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through -chain or costimulatory signals, e.g., through CD28 allows a more selective activation of the reprogrammed T cells by restricting Tandem CAR’s activity to cancer cell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thus ineffective in absence of the engagement of costimulatory receptor. The combinatorialantigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[0102] Inhibitory CARs (iCARs) are designed to regulate CAR-T cells activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.

[0103] An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182-197; Stamova et al. Antibodies 2012, 1 (2), 172-198; Farhadfar et al. Leak Res. (2016) 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1 ):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[0104] In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.

[0105] In some instances, a chimeric bispecific binding member may be a CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have two binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directedto a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc. (2015) 137(8):2832-5; Ma et al. Proc Natl Acad Sci U S A. (2016) 1 13(4):E450-8 and Cao et al. Angew Chem Int Ed Engl. (2016) 55(26) :7520-4; the disclosures of which are incorporated herein by reference in their entirety.

[0106] Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell. In some embodiments, a T cell is engineered to express a CAR. The term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and / or a T cell antigen receptor.

[0107] In some embodiments, the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[0108] In other embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor, and a cellular product suitable for all patients independent of HLA type provides an alternative.

[0109] Allogeneic T cells may be administered in combination with intensification of lymphodepletion to allow CAR-T cells to expand and clear malignant cells prior to host immune recovery, e.g., by administration of Alemtuzumab (monoclonal anti-CD52), purine analogs, etc. The allogeneic T cells may be modified for resistance to Alemtuzumab. Gene editing can be used to prevent expression of HLA class I molecules on CAR-T cells, e.g., by deletion of [32-microglobulin.

[0110] In addition to modifying T cells, induced pluripotent stem (iPS) cell-derived CAR- T cells can be used. For example, donor T cells can be transduced with reprogramming factors to restore pluripotency, and then re-differentiated into T effector cells.[oom] T cells for engineering as described above collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, efc.

[0112] Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, efc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.

[0113] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, efc., frequently supplemented with fetal calf serum (FCS).

[0114] The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0115] Engineered CAR-T cells may be infused into a subject in any physiologically acceptable medium by any convenient route of administration, normally intravascularly, though CAR-T cells may also be introduced by other routes, where the cells may find anappropriate site for growth. Usually, at least 1 x106cells / kg will be administered, at least 1x107cells / kg, at least 1 x108cells / kg, at least 1 x109cells / kg, at least 1 x1 O10cells / kg, or more, usually being limited by the number of T cells that are obtained during collection.

[0116] By “genetically engineered” or “genetically modified”, it is intended to mean that the genome of a cell has been altered. In some cases, the genome of the cell has been manipulated to express an expression product that is not normally naturally expressed by the cell. Examples of cells that have been genetically engineered include chimeric antigen receptor (CAR)-T cells that are T-cells that have been genetically engineered to express a CAR. A coding sequence encoding a CAR may be introduced on an expression vector into a cell to be engineered. For example, a CAR coding sequence may be introduced into the genome at the site of an endogenous T cell receptor gene. In some cases, cells are further engineered to delete an endogenous T cell receptor (i.e., TCR knockout). In some cases, a CRISPR / Cas9 system is used to genetically modify a T cell. A CRISPR / Cas9 system can be introduced into cells by transfection with a mRNA or plasmid that encodes Cas9 and a gRNA or by viral delivery of CRISPR components, e.g., using lentiviral, retroviral vectors, or non-integrating viruses, such as adenovirus and adeno-associated virus (AAV).

[0117] By “binding-triggered transcriptional switch” or “BTSS”, it is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain. Upon binding of the first member of the specific binding pair to the BITS the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal.

[0118] Examples of BTSS include the synNotch system, the modular extracellular sensor architecture (MESA) system, the TANGO system, the A2 Notch system, and the synthetic intramembrane proteolysis receptor (SNIPR) system, etc. The synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below. The MESA system may be as described in WO 2018 / 081039 A1 and comprises a self-containing sensing and signal transduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene. In the MESA system, binding of the ligand to the receptor induces dimerization that results inproteolytic trans-cleavage of the system to release a transcriptional activator previously sequestered at the plasma membrane. The TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.). Activation of the receptor fusion results in the recruitment of a signaling protein fused to a protease that then cleaves and releases the transcription factor to activate genes in the cell. The A2 Notch system may be as described in WO 2019099689 A1. Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell. The SNIPR system may be described as in Zhu et al. (2022) Cell 185(8) :1431 -1443. e16; herein incorporated by reference. Briefly, the SNIPR system uses a synthetic RiP receptor comprising an ectodomain comprising an extracellular regulatory element that specifically binds a ligand, a transmembrane domain, a juxtamembrane domain, and a transcription factor that can be cleaved from the SNIPR by a protease in response to binding of a ligand to the extracellular regulatory element.

[0119] In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS. In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing the BTTS and a CAR under control of the BTTS.

[0120] In certain cases, the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 . For example, the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds. In certain cases, the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.

[0121] In certain embodiments, the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally present in a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, anS2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain; and a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR). In certain cases, the cell may be a T-cell, such as, those described in U.S. Patent No. 9,670,281 , which is herein incorporated by reference.TCR Knockout

[0122] For multiplexed screening, the endogenous T cell receptor (TCR) is knocked out of the genome of the CAR-T cells to ensure selective cytotoxicity against target cells and prevent CAR-T cells derived from different donors from being immunoreactive against one another. TCRs are membrane-anchored heterodimeric proteins that require both protein chains of the TCR to be present in order to function. Accordingly, TCR receptors can be knocked out by deleting or disrupting expression of one or both of the protein chains that form the heterodimer. For TCR knockout in ap T cells, wherein the TCR consists of an alpha (a) chain (TRAC), encoded by a TRA gene, and a TCR beta (0) chain (TRBC), encoded by a TRB gene, expression of TRAC and / or TRBC can be disrupted. For TCR knockout in y6 T cells, wherein the TCR consists of a gamma chain (TRGC), encoded by a TRG gene, and a delta chain (TRDC), encoded by a TRD gene, expression of TRGC and / or TRDC can be disrupted.

[0123] Various gene editing approaches can be used for this purpose, including, without limitation, the use of genome editing systems comprising clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) nucleases, meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., CRISPR Gene Editing: Methods and Protocols (edited by Luo, Humana, 2019), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery (edited by Appasani and Church, Cambridge University Press, 2018); herein incorporated by reference in their entireties. These gene editing techniques involve creating a double-strand break (DSB) in the DNA at a target site ofthe intended gene edit. TCR knockout may be achieved through either the non- homologous end joining (NHEJ) or the microhomology-mediated end-joining (MMEJ) DNA repair pathways, which generate small nucleotide insertions or deletions (indels) at the site of the DSB. Gene knockout results if an indel shifts the reading frame or introduces a premature stop codon in a TCR coding sequence. In some embodiments, a CRISPR system is used with a guide RNA (gRNA) to direct a Cas9 RNA-guided nuclease to create a DSB at a target site in an exon of a TCR gene. For example, a TCRap dimer can be inactivated by using a CRISPR system with a gRNA designed to guide a Cas9 nuclease to a target site to create a site-specific DNA break in an exon encoding either an alpha or a beta protein chain, wherein introduction of an indel in the exon results in knockout of the TCR gene. Alternatively, CRISPR can be performed with a donor DNA template using homologous recombination (HR) to replace a portion of the genomic sequence with a modified sequence. For a description of methods of using a CRISPR system for TCR gene knockout, see, e.g., Eyquem et al. (2017) Nature 543:113-1 17, Georgiadis et al. (2018) Mol. Ther. 26:1215-1227, Kamali et al. (2021 ) BMC Biotechnology 21 : 9 (2021 ), Seki et al. (2018) J Exp Med 215(3):985-997, Qasim et al. (2017) Sci Transl Med 9(374):eaaj2013, and Stenger et al. (2020) Blood. 136(12):1407- 1418; herein incorporated by reference. For a description of methods of engineering a T cell to both express a CAR and knockout an endogenous TCR gene, see also co-owned Provisional Patent Application, entitled "Touchless Selection of Gene Modified Cell Therapies Through TRAC Intron Knockins," and co-owned Provisional Patent Application, entitled ""Simultaneous Gene Knockin and Knockout in Engineered Cell Therapies with mRNA expressed Guide RNAs,” filed even date herewith, the disclosures of which are hereby incorporated by reference herein in their entireties.CAR-T Cells

[0124] CAR-T cells are T cells that have been engineered to express a chimeric antigen receptor (CAR) that specifically binds to a target antigen. The CAR localizes T cells to sites where target cells are present that express the target antigen. Binding of a CAR-T cell to a target antigen on the surface of a cell activates the T cell resulting in secretion of cytokines, which regulate other immune cells, and killing of target cells. For example, CAR-T cells may be engineered to target an antigen that is expressed on the surface of tumors but not on healthy cells to selectively kill tumor cells. In another example, CAR-T cells may also be engineered to target an antigen that is expressed on the surface ofactivated fibroblasts or fibrotic tissue, which may be used to selectively eliminate fibrotic tissue. In another example, CAR-T cells may also be engineered to target an antigen that is expressed on the surface of a pathogen (e.g., bacterium, virus, fungus, or parasite) to eradicate a pathogen. In a further example, CAR-T cells may be engineered to target an antigen that is expressed on the surface of an autoreactive immune cell (e.g., autoreactive T cell or B cell) to eliminate autoreactive immune cells. Thus, CAR-T cells may be used for the treatment of various diseases, including cancer, fibrosis, infections such as bacterial infections (e.g., multidrug resistant bacteria), viral infections, fungal infections, and parasitic infections, and autoimmune diseases.

[0125] The T cell, from which the CAR-T cell is derived, may be autologous or allogeneic. In some embodiments, the CAR-T cell is an effector T cell (e.g., a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell) or a regulatory T cell (Treg) that has been genetically modified to express a CAR.

[0126] A CAR may have any suitable architecture, known in the art, wherein the CAR comprises an antigen binding domain linked to T cell receptor effector functions. The term “CAR” refers to an artificial multi-module molecule capable of triggering or inhibiting the activation of an immune cell. A CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR includes one or more co-stimulatory domains and / or one or more co- inhibitory domains.

[0127] The antigen-binding domain of a CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest. In some embodiments, the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof. The antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof. Thus, in some embodiments, the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof. An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof. In someembodiments, the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0128] In some embodiments, the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen. In some embodiments, the antigen binding domain is provided by a single chain variable fragment (scFv). A scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and / or amino acid composition of the linker peptide joining the VH and VL sequences. In some embodiments, any arbitrary single-chain peptide including about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[0129] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some embodiments, the transmembrane domain comprises at least the stalk and / or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit P5, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and / or CD86. In some embodiments, the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3 , CD4, CD8, or CD28. In other embodiments, the transmembrane domain is synthetic, in which case it will include predominantlyhydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.

[0130] In some embodiments, the CAR further comprises one or more linkers / spacers. For example, an extracellular spacer region may link the antigen binding domain to the transmembrane domain and / or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain. A spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[0131] Various types of linkers may be used in the CARs described herein. In some embodiments, the linker includes a peptide linker / spacer sequence. In some embodiments, the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.

[0132] In principle, there are no particular limitations to the length and / or amino acid composition of a linker peptide sequence. In some embodiments, a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR. In some embodiments the linkercomprises the amino acid sequence (G4S)n where n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.

[0133] A cytoplasmic signaling domain, such as those derived from the T cell receptorchain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.

[0134] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies, Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which is incorporated herein by reference in its entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0135] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protein. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .

[0136] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FccRIy, or the CD3( chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.

[0137] Second-generation CARs include a co-stimulatory signal in addition to the CD3^ signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokinesecretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain will usually be membrane proximal relative to the CD3 domain. Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR-T cells, CAR-T cells are further genetically modified to express or block molecules and / or receptors to enhance immune activity.

[0138] CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Trans! Med (2013) 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91 -106; Barrett et al. Anna Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388- 98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; herein incorporated by reference in their entireties.

[0139] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains.

[0140] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through -chain or costimulatory signals, e.g., through CD28 allows a more selective activation of the reprogrammed T cells by restricting Tandem CAR’s activity to cancer cell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thusineffective in absence of the engagement of costimulatory receptor. The combinatorial antigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[0141] Inhibitory CARs (iCARs) are designed to regulate CAR-T cell activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.

[0142] An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BITE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182-197; Stamova et al. Antibodies 2012, 1 (2), 172-198; Farhadfar et al. Leak Res. (2016) 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1 ):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[0143] In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BITE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.

[0144] In some instances, a chimeric bispecific binding member may be a CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have two binding regions, onespecific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc. (2015) 137(8):2832-5; Ma et al. Proc Natl Acad Sci U S A. (2016) 1 13(4):E450-8 and Cao et al. Angew Chem Int Ed Engl. (2016) 55(26)7520-4; the disclosures of which are incorporated herein by reference in their entirety.

[0145] Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell. In some embodiments, a patient's own T cells or T cells from a donor are engineered to express a CAR. In some embodiments, the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, e.g., Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor- Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”. In other embodiments, stem cells, differentiated into T cells, are engineered to express a CAR. In some embodiments, induced pluripotent stem cell (iPSC)-derived T cells are engineered to express a CAR. See, e.g., Zhou et al. (2022) Cancers (Basel) 14(9):2266, Nezhad et al. (2021 ) Pharm Res 38(6):931 -945; herein incorporated by reference in their entireties.

[0146] A biological sample comprising T cells, from which CAR-T cells are generated, may be collected from a subject or a donor. The biological sample may include, without limitation, blood, lymphoid tissue (e.g., bone marrow, spleen, tonsils, lymph nodes), mucosal tissue (e.g., lungs, small intestine, and large intestine), skin, or a tissue where T cells have infiltrated. The T cells may be separated from a mixture of cells prior to engineering the T cells to generate CAR-T cells. Alternatively, T cells may be engineered and cultured without separation from other cells.

[0147] T cells may be separated from other cells using any suitable cell separation technique such as, but not limited to, centrifugation-based cell separation, positive or negative selection against surface markers on cells (e.g., with antibody-coated beads), affinity chromatography, panning and immunopanning techniques, fluorescence activated cell sorting (FACS), or magnetic-activated cell sorting (MACS). Affinity reagentsmay be employed comprising specific receptors or ligands specific for cell surface molecules. The T cells may be separated from dead cells by employing viability dyes (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the T cells.

[0148] The cells may be collected in any appropriate medium that maintains the viability of the cells. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., which may be supplemented with fetal calf serum (FCS). The collected cells may be used immediately or frozen (e.g., at liquid nitrogen temperatures) prior to use.

[0149] In some embodiments, CAR-T cells are expanded in culture prior to screening, as described further below, or use in therapy. The CAR-T cells require activation for expansion in vitro or ex vivo, which can be accomplished by co-incubating T cells with natural antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles that present antigen and / or activating signals to the CAR-T cells. See, e.g., Rhodes et al. (2018) Mol Immunol. 98:13-18, Couture et al. (2019) Front Immunol. 10:1081 , Turtle (2010) Cancer J. 16(4):374-81 , Wang et al. (2017) Theranostics 7(14):3504-3516, Est-Witte et al. (2021 ) Semin Immunol. 56:101541 , Perica et al. (2014) Nanomedicine. 10 (1 ): 1 19-129, Latouche et al. (2000) Nature Biotechnology. 18 (4): 405-409; herein incorporated by reference.

[0150] In some embodiments, a CAR-T cell is further engineered to comprise a binding- triggered transcriptional switch (BTSS) that regulates expression of the chimeric antigen receptor or activity of the CAR-T cell. By BTSS, is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain. Upon binding of the first member of the specific binding pair to the BTTS the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal. In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS.

[0151] Examples of binding-triggered transcriptional switches include the synNotch system, the modular extracellular sensor architecture (MESA) system, the TANGO system, the A2 Notch system, and the synthetic intramembrane proteolysis receptor(SNIPR) system, etc. The synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below. The MESA system may be as described in WO 2018 / 081039 A1 and comprises a self-containing sensing and signal transduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene. In the MESA system, binding of the ligand to the receptor induces dimerization that results in proteolytic trans-cleavage of the system to release a transcriptional activator previously sequestered at the plasma membrane. The TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.). Activation of the receptor fusion results in the recruitment of a signaling protein fused to a protease that then cleaves and releases the transcription factor to activate genes in the cell. The A2 Notch system may be as described in WO 2019099689 A1. Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell. The SNIPR system may be described as in Zhu et al. (2022) Cell 185(8):1431 -1443. e16; herein incorporated by reference. Briefly, the SNIPR system uses a synthetic RIP receptor comprising an ectodomain comprising an extracellular regulatory element that specifically binds a ligand, a transmembrane domain, a juxtamembrane domain, and a transcription factor that can be cleaved from the SNIPR by a protease in response to binding of a ligand to the extracellular regulatory element.

[0152] In certain cases, the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 . For example, the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds. In certain cases, the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.

[0153] In certain embodiments, the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally presentin a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolytic cleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain; and a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR). In certain cases, the cell may be a T-cell, such as, those described in U.S. Patent No. 9,670,281 , which is herein incorporated by reference.Engineering a T cell to Express a CAR

[0154] A recombinant polynucleotide comprising a coding sequence encoding a CAR may be introduced into a T cell to be engineered by any suitable method known in the art. In some embodiments, a donor polynucleotide comprising a coding sequence encoding the CAR is integrated into the genome of a T cell at the site of an endogenous gene encoding a T cell receptor by homology-directed repair (HDR) using clustered regularly interspaced short palindromic repeats (CRISPR) technology. The coding sequence encoding the CAR may be integrated into an exon or an intron of an endogenous TCR gene (e.g., TRA, TRB, TRG, or TRD). For a description of methods of integrating a coding sequence encoding a CAR into an exon of the TCR alpha chain locus (TRAC) gene using a CRISPR / Cas9 system, see, e.g., Roth et al. (2018) Nature 559(7714):405-409, Freen-van Heeren et al. (2020) Cytokine X 3(1 ):100049, Albers et al. (2019) Life Sci Alliance 2(2):e201900367, Eyquem et al. (2017) Nature 543:113-1 17; Ren et al. (2017) Protein Cell 8(9):634-643; Ren et al. (2017) Oncotarget 8(10):17002- 1701 1 ). For a description of methods of integrating a coding sequence encoding a CAR into an intron of the TRAC gene, see, e.g., co-owned Provisional Patent Application entitled "Touchless Selection of Gene Modified Cell Therapies Through TRAC Intron Knockins," filed even date herewith, the disclosure of which is hereby incorporated by reference herein in its entirety. A CRISPR / Cas9 system can be introduced into human cells with a viral vector that encodes Cas9 and a guide RNA (gRNA). Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, andadeno-associated virus (AAV) vectors. For a description of methods of introducing a CRISPR system into cells with various viral vectors, see, e.g., Shalem et al. (2014) Science 343:84-87, Williams et al. (2016) Sci Rep. 6:2561 1 , Ran et al. (2015) Nature 520:186-191 , Swiech et al. (2015) Nat BiotechnoL 33:102-106; herein incorporated by reference.

[0155] Alternatively, a viral vector can be used to introduce a coding sequence encoding a CAR into a T cell. A recombinant polynucleotide comprising a coding sequence encoding a CAR can be inserted into an expression vector to create an expression cassette capable of producing the CAR in a host T cell. Expression cassettes typically include control elements operably linked to the coding sequence, which allow for the expression of the gene in vivo in the subject species. For example, typical promoters for mammalian cell expression include the SV40 early promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus promoter, the elongation factor 1 -a (EF1 -a) promoter, the beta-2-microglobulin (P2m) promoter, the human phosphoglycerate kinase (hPGK) promoter, and the RPBSA promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression. In some embodiments, the promoter is a T cell-specific promoter that drives or regulates expression in T cells. T cell-specific promoters include, but are not limited to, the distal Lek (dLck) promoter and the CD38-promoter. In some embodiments, the promoter is a conditional promoter, optionally wherein the conditional promoter is selected from TET (tetracycline-response elements, TET-ON / TET-OFF), Lac, dCas-transactivator, Zinc- finger-TF, TALENs-ZF Gal4-uas, synNotch and inducible promoters based on endogenous signals TNF-alpha, and cFOS promoter.

[0156] Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator / polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0157] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et aL, EMPO J. (1985) 4:761 , the enhancer / promoter derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41 :521 , such as elements included in the CMV intron A sequence.

[0158] Additionally, 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same. Such sequences may include UTRs comprising an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a vector to allow, for example, coexpression of the CAR and another protein (e.g. fluorescent protein) from the same vector. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991 ) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298: Rees et al., BioTechniques (1996) 20:102-1 10; Kobayashi et al., BioTechniques (1996) 2 :399-402; and Mosser et al., BioTechniques (1997) 22 150-161. A multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picornavi ruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651 -1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et aL, Proc. Natl. Acad. Sci. (2003) 100(251:15125-15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697- 2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like. A variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al. , Mol. Cell Endocrinol. (2003) 212:51 -61 ), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):31 12- 31 19, Bert et al. (2006) RNA 12(6):1074-1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1 ):37-44). These elements are readily commercially available in plasmids sold, e.g., by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA) and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). An IRES sequence may be included in a vector, for example, to express multiple protein products in combination.

[0159] Alternatively, a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products from a single vector. 2A linker peptides are inserted between the coding sequences in the multicistronic construct. The 2A peptide, which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels. 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1 . See, e.g., Kim et al. (201 1 ) PLoS One 6(4):e18556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625-629, Furler et al. (2001 ) Gene Then 8(11 ):864-873; herein incorporated by reference in their entireties.

[0160] In certain embodiments, cells containing the construct are identified in vitro or in vivo by including a selection marker expression cassette in the construct. Selection markers confer an identifiable change to the cell permitting positive selection of cells having the construct. For example, fluorescent or bioluminescent markers (e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Dronpa, mCherry, mOrange, mPlum, Venus, YPet, phycoerythrin, or luciferase), cell surface markers, expression of a reporter gene (e.g., GFP, dsRed, GUS, lacZ, CAT), drug selection markers such as genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol may be used to identify cells. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Any selectable marker may be used as long as it is capable of being expressed in the cell to allow identification of genetically modified cells containing the construct. Further examples of selectable markers are well known to one of skill in the art.

[0161] In certain embodiments, the selection marker expression cassette encodes two or more selection markers. Selection markers may be used in combination; for example, a cell surface marker may be used with a fluorescent marker, or a drug resistance gene may be used with a suicide gene. In certain embodiments, the selection marker expression cassette is multicistronic to allow expression of multiple selection markers in combination. The multicistronic vector may include an IRES or viral 2A peptide to allow expression of more than one selection marker from a single vector.

[0162] In certain embodiments, a suicide gene is introduced into the T cells, for example, to improve their safety by allowing their destruction at will. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a nontoxic drug to a toxiccompound in the T cells. Examples include suicide genes encoding caspases, thymidine kinases, cytosine deaminases, intracellular antibodies, telomerases, and DNases. See, e.g., Jones et al. (2014) Front. Pharmacol. 5:254, Mitsui et al. (2017) Mol. Ther. Methods Clin. Dev. 5:51 -58, Greco et al. (2015) Front. Pharmacol. 6:95; herein incorporated by reference. In some cases, the suicide gene is expressed from an inducible promoter to provide a “safety switch” (i.e., kill cells by inducing the suicide gene). For example, an inducible caspase-9 suicide gene system can be incorporated into CAR-T cells as a "safety switch" (see, e.g., Straathof et al. (2005) Blood 105(1 1 ):4247-4254; Thomis et al. (2001 ) Blood 97(5):1249-1257; Tey et al. (2007) Biol. Blood Marrow Transplant. 13(8) :913-24; herein incorporated by reference). In some embodiments, a suicide gene is selected that expresses a human protein to minimize immune reactions in human patients treated with the CAR-T cells.

[0163] In some embodiments, CAR-T cells are further engineered to express a fluorescent protein to allow CAR-T cells derived from different donors to be distinguished in multiplexed screening. In some embodiments, T cells from the same donor are engineered to express the same fluorescent protein, and T cells from different donors are engineered to express different fluorescent proteins. Exemplary fluorescent proteins include, without limitation, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), superfolder GFP, emerald, Azami Green, mWasabi, TagGFP, TurboGFP, red fluorescent protein, blue fluorescent protein (BFP), EBFP, EBFP2, mTagBFP, Azurite, cyan fluorescent protein (CFP), mECFP, Cerulean, mCerulean, mTurquoise, CyPet, AmCyanl , Midori-lshi Cyan, yellow fluorescent protein (YFP), EYFP, Topaz, Venus, YPet, mCitrine, mBanana, orange fluorescent protein (OFP), Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, TagRFP, DsRed, DsRed2, mTangerine, red fluorescent protein (RFP), mRuby, mRuby2, mApple, mStrawberry, mCherry, mRaspberry, AsRed2, mRFP1 , JRed, dKeima-Tandem, Dronpa, mPlum, E2-Crimson, and aequorin.

[0164] In some embodiments, the CAR-T cell are barcoded. For example, each CAR-T cell can be barcoded with a distinct DNA sequence indicating which donor provided the T cell from which the CAR-T cell was generated.

[0165] Once complete, the constructs encoding the CAR, and optionally a fluorescent protein and / or barcode can be delivered to T cells. A number of viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (y-retroviruses and lentiviruses), poxviruses, adeno-associated viruses,baculoviruses, and herpes simplex viruses (see e.g., Warnock et al. (201 1 ) Methods Mol. Biol. 737:1 -25; Walther et al. (2000) Drugs 60(2):249-271 ; and Lundstrom (2003) Trends BiotechnoL 21 (3):117-122; herein incorporated by reference).

[0166] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al. (1991 ) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033- 8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (201 1 ) Curr Pharm Des. 17(24):2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (201 1 ) Viruses 3(2) :132-159; herein incorporated by reference).

[0167] A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911 -5921 ; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et aL, Gene Therapy (1994) 1 :51 -58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et aL, Human Gene Therapy (1993) 4:461 -476). Additionally, various adeno- associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92 / 01070 (published 23 January 1992) and WO 93 / 03769 (published 4 March 1993); Lebkowski et aL, Molec. Cell. BioL (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533- 539; Muzyczka, N. Current Topics in Microbiol, and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801 ; Shelling and Smith, Gene Therapy (1994) 1 :165-169; and Zhou et aL, J. Exp. Med. (1994) 179:1867-1875.

[0168] Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the CAR include those derived from the pox family of viruses, including vacciniavirus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the CAR can be constructed as follows. The DNA encoding the particular CAR coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0169] Alternatively, avi poxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with, respect to the production of vaccinia viruses. See, e.g., WO 91 / 12882; WO 89 / 03429; and WO 92 / 03545.

[0170] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0171] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present invention. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95 / 07995, WO 96 / 17072; as well as Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1 , 1998, and Dubensky, Jr., T. W., U.S. Patent No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02 / 099035, WO 02 / 080982, WO 01 / 81609, and WO 00 / 61772; herein incorporated by reference in their entireties.

[0172] A vaccinia-based infection / transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a CAR expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0173] As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94 / 26911 ; Studier and Moffatt, J. Mol. Biol. (1986) 189:1 13-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201 - 1206; Gao and Huang, Nuc. Acids Res. (1993) 21 :2867-2872; Chen et aL, Nuc. Acids Res. (1994) 22:21 14-2120; and U.S. Pat. No. 5,135,855.

[0174] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to cells. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1 :1 (mgDNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991.) 1097:1 -17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101 , pp. 512-527.

[0175] Liposomal preparations for use in the subject methods include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081 ); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.

[0176] Cationic liposomes are readily available. For example, N[1 -2 ,3-d ioley loxy)propyl]- N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No. WO 90 / 11092 for a description of the synthesis of DOTAP (1 ,2-bis(oleoyloxy)-3- (trimethylammonio)propane) liposomes.

[0177] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0178] The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in Methods of Immunology (1983), Vol. 101 , pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad.Sci. USA (1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431 ; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[0179] The DNA and / or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871 ,488.

[0180] The expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J. P., et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D. T., et al., Vaccine 11 (2):149-54, 1993.

[0181] Furthermore, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No. 5,831 ,005, issued Nov. 3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present invention.

[0182] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes encoding a CAR. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371 ,015; and 5,478,744. Also, needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0183] Alternatively, T cells may be engineered to express a CAR by transfecting a T cell with a messenger RNA encoding the CAR. Messenger RNA transfection of T cells canbe performed using electroporation, cationic-lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(1 ):533-542, Tchou et al. (2017) Cancer Immunol Res. 5(12):1152-1 161 , Ye et al. (2022) ACS Biomater Sci Eng. 8(2):722-733, Guevara et al. (2020) Front. Chem. 8:589959; herein incorporated by reference.Multiplexed Screening

[0184] CAR-T cells from multiple donors, wherein the CAR-T cells have knockouts of their endogenous TCRs, as described herein, can be pooled and tested simultaneously in multiplexed assays. Activation of CAR-T cells can be determined by measuring cell proliferation, expression of activation markers (e.g., CD69, HLA-DR, IL2RA, and / or CD25), and production of effector cytokines (e.g., IFN-y, TNF-a, TNF-|3, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, and / or IL-25). Multiplexed screening of CAR-T cell cytotoxic activity can be performed in vitro to validate activity against target cells before further testing individual CAR-T cell in vivo in animal models and human clinical trials.

[0185] Cytotoxicity of CD8+ CAR-T cells involves exocytosis of granules containing the pore-forming toxin, perforin, proapoptotic serine proteases, and granzymes that lyse target cells. Cytotoxicity of CD4+ CAR-T cells involves secretion of cytokines and apoptotic factors such as TNF-a, INF-y, and TRAIL that induce apoptosis of target cells or activate macrophages to engulf tumor cells. Perforin, proapoptotic serine proteases, granzymes, cytokines, and apoptotic factors can be measured, for example, using a multiplexed enzyme-linked immunosorbent assay (ELISA). Cytolysis can be assayed in vitro based on the release of compounds containing radioactive isotopes such as51Cr from radiolabeled target cells. Alternatively, a membrane-permeable live-cell labeling dye such as calcein acetoxymethyl ester of calcein (Calcein / AM) can be used to distinguish live cells from dead cells. In the Calcein / AM assay, intracellular esterases cleave the acetoxymethyl (AM) ester group to produce a membrane-impermeable calcein fluorescent dye that is retained in live cells. Apoptotic and dead cells without intact cell membranes do not retain the calcein fluorescent dye. A lactate dehydrogenase (LDH) assay can also be used to evaluate cytotoxicity. LDH is a cytoplasmic enzyme, which is released into the extracellular space when the plasma membrane is damaged. Cytotoxicity is monitored by detecting LDH release from cells. See, e.g., Lieberman (2003) Nat Rev Immunol 3(5):361 -370, Neri et al. (2001 ) Clin Diagn Lab Immunol8(6) :1 131 -1135, Smith et al. (2011 ) PLoS One 6(1 1 ):e26908, Chan et al. (2013) Methods Mol Biol 979:65-70; herein incorporated by reference in their entireties.

[0186] Flow cytometry can also be used to assess cell proliferation, activation, and cytotoxicity. The percentage of target cells that are live, apoptotic, or dead can be determined by staining target cells with viability dyes such that the live and dead cell populations can be distinguished based on differences in fluorescence. For example, Annexin V-FITC can be used to label target cells that are at an early stage of apoptosis. Propidium iodide can be used to label target cells that are at a late stage of apoptosis or dead. Lipophilic dyes, such as PKH67 and PKH26 can be used to label the cell membranes of target cells for measuring proliferation of CAR-T cells by flow cytometry. In addition, T cell activation can also be detected by immunofluorescent labeling of activation markers such as CD69, HLA-DR, IL2RA, and CD25. See, e.g., Zaritskaya et al. (2010) Expert Rev Vaccines 9(6):601 -616, Fischer et al. (2002) J Immunol Methods 259(1 -2):159-169, Aubry et al. (1999) Cytometry 37(3):197-204, and Tario et al. (201 1 ) Methods Mol Biol 699:1 19-164; herein incorporated by reference in their entireties.

[0187] Cell proliferation can also be detected and quantified, for example, using a cell counter or staining of CAR-T cells with a fluorescent tracking dye, such as carboxyfluorescein succinimidyl ester (CFSE).

[0188] The CAR-T cells may be further tested for efficacy in treating a disease in vivo, e.g., in an animal. For example, CAR-T cells can be tested for cytotoxicity against cancerous cells in an animal with solid tumors. In some embodiments, human xenograft tumors are implanted in animals, followed by administration of CAR-T cells, and evaluation of antitumor responses. An exemplary animal model of cancer is a NOD Scid Gamma (NSG) mouse transplanted with human tumors. NSG mice are completely deficient in adaptive immunity and severely deficient in innate immunity, which avoids transplant rejection of CAR-T cells and patient-derived xenografts.

[0189] Antitumor responses can be evaluated by various methods known in the art. The volume of a subcutaneous tumor can be measured by using a digital caliper. Internal tumors can be measured by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or singlephoton emission computed tomography (SPECT). In some cases, the CAR-T cells are further modified to express a bioluminescent protein such as luciferase to allow monitoring of tumors by bioluminescence imaging or a fluorescent protein such as green fluorescent protein to allow monitoring of tumors by fluorescence imaging.

[0190] In addition, tumors can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of tumor specimens can be used to detect T cell infiltration into tumors and quantitate target antigen expression. Cytokine profiling of tumors treated with CAR-T cells can also be performed.

[0191] In another example, CAR-T cells can be tested for cytotoxicity against activated fibroblasts or fibrotic tissue in an animal with fibrosis. The extent of fibrosis can be monitored in an animal in vivo, for example, by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission computed tomography (SPECT). In addition, fibrotic tissue can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of fibrotic tissue specimens can be used to detect T cell infiltration into fibrotic tissue and quantitate target antigen expression. Cytokine profiling of fibrotic tissue treated with CAR-T cells can also be performed.

[0192] An animal model can be used not only to determine efficacy but also the toxicity or side effects of treatment with a CAR-T cell. Furthermore, this disclosure pertains to uses of CAR-T cells, identified by the above-described screening assays for treatment of a disease such as, but not limited to, cancer, fibrosis, an infection, or an autoimmune disease. A CAR-T cell, identified by the above-described screening assays for treatment of a disease, may be expanded in culture in the presence of a natural antigen-presenting cell (e.g., dendritic cell) or an artificial antigen-presenting cell or particle under selective conditions prior to formulation into a pharmaceutical composition and administration.Pharmaceutical Compositions

[0193] Pharmaceutical compositions comprising CAR-T cells, identified by the abovedescribed screening assays for treatment of a disease, can be prepared by formulating the CAR-T cells into dosage forms by known pharmaceutical methods. For example, a pharmaceutical composition comprising CAR-T cells can be formulated for parenteral administration, as capsules, liquids, film-coated preparations, suspensions, emulsions, and injections (such as venous injections, drip injections, and the like).

[0194] In formulation into these dosage forms, the CAR-T cells can be combined as appropriate, with pharmaceutically acceptable carriers or media, in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents,soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives.

[0195] The CAR-T cells may also be used in combination with other therapeutic agents for treating a disease. For example, for treatment of cancer, CAR-T cells may be used in combination with anti-cancer agents such as, but not limited to: chemotherapeutic agents such as cyclophosphamide, doxorubicin, vincristine, methotrexate, cytarabine, ifosfamide, etoposide, adriamycin, bleomycin, vinblastine, dacarbazine, chlormethine, oncovin, and procarbazine; immunotherapeutic agents such as antibodies (e.g., rituximab), cytokines (e.g., interferons, including type I (IFNa and IFN|3), type II (IFNy) and type III (IFN ) and interleukins, including interleukin-2 (IL-2)), adjuvant immunochemotherapy agents (e.g., polysaccharide-K), adoptive T-cell therapy agents, and immune checkpoint blockade therapy agents; steroids such as prednisolone, biologic therapeutic agents such as tyrosine-kinase inhibitors, such as Imatinib mesylate (Gleevec, also known as STI-571 ), Gefitinib (Iressa, also known as ZD1839), Erlotinib (marketed as Tarceva), Sorafenib (Nexavar), Sunitinib (Sutent), Dasatinib (Sprycel), Lapatinib (Tykerb), Nilotinib (Tasigna), and Bortezomib (Velcade); Janus kinase inhibitors, such as tofacitinib; ALK inhibitors, such as crizotinib; Bcl-2 inhibitors, such as obatoclax and gossypol; PARP inhibitors, such as Iniparib and Olaparib; PI3K inhibitors, such as perifosine; VEGF receptor 2 inhibitors, such as Apatinib; AN-152 (AEZS-108) doxorubicin linked to [D-Lys(6)]-LHRH; Braf inhibitors, such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors, such as trametinib; CDK inhibitors, such as PD-0332991 and LEE011 ; Hsp90 inhibitors, such as salinomycin; small molecule drug conjugates, such as Vintafolide; serine / threonine kinase inhibitors, such as Temsirolimus (Torisel), Everolimus (Afinitor), Vemurafenib (Zelboraf), Trametinib (Mekinist), and Dabrafenib (Tafinlar); pro-apoptotic agents such as oblimersen sodium, sodium butyrate, depsipetide, fenretinide, flavipirodol, gossypol, ABT-737, ABT-263 (Navitoclax), GX15- 070 and HA14-1 ; angiogenesis inhibitors such as bevacizumab, ramucirumab, ranibizumab, sorafenib, sunitinib, itraconazole, and carboxyamidotriazole; photoactive agents such as porfimer sodium, chlorins, bacteriochlorins, phthalocyanines, and aminolevulinic acid prodrugs; radiosensitizing agents such as cisplatin, fluoropyrimidines, gemcitabine, misonidazole, metronidazole, and taxanes; radioisotopes such as iodine- 131 , holmium-166, lutetium-177, radium-223, samarium-153, strontium-89, and yttrium- 90; or other therapeutic agents.

[0196] In some embodiments, the pharmaceutical composition comprising the CAR-T cells is a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for delivery of the CAR-T cells over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[0197] Usually, but not always, the subject who receives the CAR-T cells (i.e., the recipient) is also the subject from whom the original T cells (i.e., before genetic modification to express a CAR specific for a target cell) are harvested or obtained, which provides the advantage that the cells are autologous. However, T cells can be obtained from another subject (i.e., donor), a culture of cells from a donor, or from established cell culture lines and genetically modified, as described herein. T cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a biological sample comprising T cells from a close relative or matched donor, genetically modified to express a CAR, and administered to a subject in need of treatment. The patients or subjects who donate or receive the T cells are typically mammalian, and usually human. However, this need not always be the case, as veterinary applications are also contemplated. In certain embodiments, the CAR-T cells administered to a subject are autologous or allogeneic.Cellular Therapy with CAR-T Cells

[0198] CAR-T cells are administered to a subject in a therapeutically effective amount. The phrase “therapeutically effective amount” refers to the administration of the CAR-T cells to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. For example, in the case of cancer, a therapeutically effective amount of the CAR-T cells provides an anti-tumor effect, as defined herein. Therefore, for example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1 ) reduction in tumor size; (2) reduction in the number of cancer cells; (3) inhibition (i.e.,slowing to some extent, preferably halting) of tumor growth; (4) inhibition (i.e. , slowing to some extent, preferably halting) of cancer cell infiltration into peripheral organs; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor metastasis; and (6) some extent of relief from one or more symptoms associated with the cancer. Such therapeutic responses may be further characterized as to degree of improvement. Thus, for example, an improvement may be characterized as a complete response. By “complete response” is documentation of the disappearance of all symptoms and signs of all measurable or evaluable disease confirmed by physical examination, laboratory, nuclear and radiographic studies (i.e., CT (computer tomography) and / or MRI (magnetic resonance imaging)), and other non-invasive procedures repeated for all initial abnormalities or sites positive at the time of entry into the study. Alternatively, an improvement in the disease may be categorized as being a partial response. By “partial response” is intended a reduction of greater than 50% in the sum of the products of the perpendicular diameters of all measurable lesions when compared with pretreatment measurements.

[0199] In certain embodiments, antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles are used to stimulate proliferation and expansion of CAR-T cells in vitro or ex vivo prior to administration. In certain embodiments, the ex vivo method comprises contacting a population of T cells comprising a CAR-T cell with the antigen-presenting cells or artificial antigen-presenting cells or particles, wherein the population of T cells have been obtained from the subject to be treated, then genetically modified to express a CAR with an endogenous TCR knockout, as described herein. After one or more rounds of antigen-stimulation with the antigen-presenting cells or artificial antigen-presenting cells or particles and expansion of the CAR-T cells in culture, the autologous CAR-T cells are subsequently administered to the subject.

[0200] In certain embodiments, stimulation of proliferation and expansion of CAR-T cells with antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles are carried out in vitro. In certain embodiments, the in vitro method comprises contacting a population of T cells comprising a CAR-T cell with the antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles, wherein the T cells have been obtained from a donor, a culture of cells from a donor, or from established cell culture lines, then genetically modified to express a CAR with an endogenous TCR knockout, as described herein. The T cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and morepreferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a blood sample comprising T cells from a close relative or matched donor. After one or more rounds of antigen-stimulation with the antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles and expansion of the CAR-T cells in culture, the CAR-T cells may be subsequently administered to a subject.

[0201] In certain embodiments, proliferation and expansion of CAR-T cells occurs in vivo either by stimulation with an endogenous antigen-presenting cell or by coadministration of antigen-presenting cells or artificial antigen-presenting cells or particles with the CAR- T cells to the subject.

[0202] In the in vitro, ex vivo, or in vivo methods described herein, the subject may have cancer, wherein the CAR-T cells comprise a CAR that specifically binds to an antigen expressed on a cancerous cell. In some embodiments, the antigen is a tumor-specific antigen or a tumor-associated antigen expressed on a cancerous cell, wherein the antigen is used to activate a CAR-T cell designed for therapeutic use against a cancerous cell. Exemplary tumor-specific antigens and tumor-associated antigens include, without limitation, oncogene protein products, mutated or dysregulated tumor suppressor proteins, oncovirus proteins, oncofetal antigens, mutated or dysregulated differentiation antigens, overexpressed or aberrantly expressed cellular proteins (e.g., mutated or aberrantly expressed growth factors, mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, serine / threonine kinases and their regulatory subunits, G proteins, and transcription factors), and altered cell surface glycolipids and glycoproteins on cancerous cells. For example, tumor-specific antigens and tumor-associated antigens may include without limitation, dysregulated or mutated RAS, WNT, MYC, ERK, TRK, CTAG1 B, MAGEA1 , Bcr-Abl, p53, c-Sis, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), HER2 / neu, Src-family, Syk-ZAP-70 family proteins, and BTK family of tyrosine kinases, Abl, Raf kinase, cyclin-dependent kinases, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1 , epithelial tumor antigen (ETA), tyrosinase, melanoma- associated antigen (MAGE), and other abnormal or dysregulated proteins expressed on cancerous cells. In certain embodiments, the subject has leukemia, lymphoma, myeloma, prostate cancer, breast cancer, lung cancer, kidney cancer, lung cancer, ovarian cancer, intestine cancer, or glioblastoma. In other embodiments, the subject has fibrosis, wherein the CAR-T cells comprise a CAR that specifically binds to a fibrosis antigen expressed on activated fibroblasts or fibrotic tissue such as fibroblast activation protein (FAP). Incertain embodiments, the subject is undergoing or has previously undergone CAR-T cell immunotherapy.

[0203] The present disclosure contemplates the administration of the CAR-T cells, and compositions thereof, in any appropriate manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebroventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual, inhalation, local, e.g., injection directly into a target organ or tissue such as a tumor or fibrotic tissue.

[0204] In some embodiments, the CAR-T cells may comprise a binding-triggered transcriptional switch. In some embodiments, the method may further include activating a T cell such as a T cell expressing a chimeric Notch polypeptide, as described herein. In certain embodiments, the method of the present disclosure may be used for inducing T-cell proliferation without significantly increasing cytokine production by the T cell. For example, the method may include administering a T cell expressing a chimeric Notch polypeptide and CAPP having a protein displayed on the surface, where the protein binds to the Notch polypeptide resulting in expression of a cancer associated CAR on the cell surface. The CAPP further includes an antigen that binds the cancer associated CAR, where binding of the antigen on the particle to the cancer associated CAR results in activation of the T cell in absence of significant expression of cytokines. In certain embodiments, the level of cytokines produced by the T cells in the absence of cancer cells expressing the CAR antigen is substantially lower than the level of the cytokines produced by the T cells in the presence of cancer cells expressing the CAR antigen. Thus, use of particles functionalized with both a protein that binds to the chimeric Notch polypeptide and an antigen that binds to the CAR expressed in response to the binding of the protein to the chimeric Notch polypeptide provides for proliferation of the T-cells while having a substantially lower production of cytokines by the activated T cell.

[0205] In certain aspects, contacting a CAR-T cell expressing a BTTS, e.g., a chimeric Notch receptor polypeptide, as described herein with the CAPP of the present disclosure may modulate an activity of the CAR-T cell. In some cases, release of the intracellular domain modulates proliferation of the cell or of cells surrounding the cell. In some cases, release of the intracellular domain modulates apoptosis in the cell or in cells surrounding the cell. In some cases, release of the intracellular domain induces cell death by a mechanism other than apoptosis. In some cases, release of the intracellular domainmodulates gene expression in the cell through transcriptional regulation, chromatin regulation, translation, trafficking or post-translational processing. In some cases, release of the intracellular domain modulates differentiation of the cell. In some cases, release of the intracellular domain modulates migration of the cell or of cells surrounding the cell. In some cases, release of the intracellular domain modulates the expression and secretion of a molecule from the cell. In some cases, release of the intracellular domain modulates adhesion of the cell to a second cell or to an extracellular matrix. In some cases, release of the intracellular domain induces de novo expression a gene product in the cell. In some cases, where release of the intracellular domain induces de novo expression a gene product in the cell, the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody.Kits

[0206] Kit are provided to perform the subject methods for multiplexed screening of CAR- T cells. In some embodiments, the kit comprises CAR-T cells from different donors, wherein the CAR-T cells have their endogenous TCRs knocked out to allow the CAR-T cells to be pooled for multiplexed screening, as described herein. In some embodiments, the kit comprises a CRISPR system and / or a vector to genetically modify T cells for knockout of an endogenous TCR gene and / or expression of a chimeric antigen receptor. A kit may further comprise media suitable for culturing CAR-T cells. Additionally, the kit may include transfection agents, buffers, tissue culture plates, flasks, test tubes, vials, and the like, and optionally one or more other factors, such as cytokines (e.g., IL-2, IL-3, IL-6, IL-7, IL-15, TNFa, IFN-y, and GM-CSF), growth factors, antibiotics, or other media supplements, and the like.

[0207] Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0208] The kit may also provide a delivery device for administration of CAR-T cells to a patient. For example, kits may comprise a container having a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.

[0209] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.Examples of Non-Limiting Aspects of the Disclosure

[0210] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1 -43 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below.1. A method of multiplexed screening to identify chimeric antigen receptor (CAR)-T cells that are immunoreactive against a target cell, the method comprising: providing a pooled population of CAR-T cells, wherein the CAR-T cells comprise T cells from a plurality of donors, wherein each T cell is engineered to express a chimeric antigen receptor that specifically binds to a target antigen on the target cell, and wherein expression of an endogenous T cell receptor (TCR) is eliminated in each T cell; contacting the pooled population of CAR-T cells with the target cell; andmeasuring an immune response of the population of CAR-T cells or a subset of the population of CAR-T cells to identify the CAR-T cells that are immunoreactive against the target cell.2. The method of aspect 1 , wherein said measuring comprises measuring cytotoxicity against the target cell, cytokine secretion, expression of an activation marker, affinity of binding to the target antigen on the target cell, or proliferation of the CAR-T cells that are immunoreactive against the target cell.3. The method of aspect 2, wherein the activation marker is CD69, HLA-DR, IL2RA, or CD25, or any combination thereof.4. The method of aspect 2, wherein said measuring the cytotoxicity comprises detecting perforin, a proapoptotic serine protease, or a granzyme released from the CAR- T cell.5. The method of aspect 2, wherein said measuring cytotoxicity comprises detecting death of the target cell.6. The method of aspect 2, wherein said measuring cytokine secretion comprises detecting interferon (IFN)-y, tumor necrosis factor (TNF)-a, TNF-p, interleukin (IL)-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, or IL-25, or any combination thereof.7. The method of any one of aspects 1 -6, wherein the CAR-T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell that has been genetically modified to express the chimeric antigen receptor, and wherein expression of the endogenous TCR is eliminated.8. The method of any one of aspects 1 -7, wherein the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen on the target cell.9. The method of aspect 8, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.10. The method of aspect 8 or 9, wherein the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.11 . The method of aspect 8 or 9, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).12. The method of any one of aspects 8-1 1 , wherein the chimeric antigen receptor further comprises a costimulatory domain.13. The method of aspect 12, wherein the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.14. The method of any one of aspects 8-13, wherein the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.15. The method of any one of aspects 1 -14, wherein the CAR-T cell further comprises a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the CAR-T cell.16. The method of aspect 15, wherein the binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR).17. The method of aspect 16, wherein the synthetic notch receptor comprises i) an extracellular ligand-binding domain that specifically binds to a second target antigenon the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the CAR-T cell.18. The method of any one of aspects 1 -17, wherein the target cell is a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.19. The method of aspect 18, wherein the antigen on the target cell is a tumor antigen or a tumor-associated antigen.20. The method of aspect 18, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.21 . The method of aspect 20, wherein the antigen on the target cell is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.22. The method of aspect 18, wherein the autoreactive immune cell is an autoreactive T cell or B cell.23. The method of aspect 22, wherein the antigen on the target cell is an antigen on the autoreactive T cell or B cell.24. The method of any one of aspects 1 -23, wherein the T cells are engineered using a gene editing system selected from the group consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, a zinc finger nuclease system, a transcription activator-like effector nuclease (TALEN) system, and a meganuclease system.25. The method of any one of aspects 1 -24, wherein the T cells are engineered to express the chimeric antigen receptor by transfecting the T cells with a recombinant polynucleotide encoding the chimeric antigen receptor.26. The method of any one of aspects 1 -25, further comprising contacting the CAR-T cells with an antigen presenting cell or an artificial antigen presenting particle presenting the target antigen.27. The method of any one of aspects 1 -26, wherein each T cell is further engineered to express a fluorescent protein, wherein T cells from the same donor are engineered to express the same fluorescent protein, and wherein T cells from different donors are engineered to express different fluorescent proteins.28. The method of any one of aspects 1 -27, further comprising genetically barcoding the population of CAR-T cells, wherein each CAR-T cell comprises a DNA barcode indicating which donor provided the T cell from which the CAR-T cell was generated.29. The method of any one of aspects 1 -28, further comprising substantially purifying the T cells engineered to express the chimeric antigen receptor, wherein T cells still expressing an endogenous TCR are eliminated.30. A CAR-T cell identified as being immunoreactive against the target cell using the method of any one of aspects 1 -29.31 . The CAR-T cell of aspect 30, wherein the chimeric antigen receptor specifically binds to a tumor antigen or tumor-associated antigen.32. The CAR-T cell of aspect 30, wherein the chimeric antigen receptor specifically binds to a fibrosis antigen.33. The CAR-T cell of aspect 32, wherein the fibrosis antigen is fibroblast activation protein (FAP).34. The CAR-T cell of aspect 30, wherein the chimeric antigen receptor specifically binds to a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.35. The CAR-T cell of aspect 30, wherein the chimeric antigen receptor specifically binds to an antigen on an autoreactive T cell or B cell.36. A composition comprising the CAR-T cell of any one of aspects 30-35 and a pharmaceutically acceptable excipient.37. A method of performing cellular therapy, the method comprising administering a therapeutically effective amount of the composition of aspect 36 to a subject.38. The method of aspect 37, wherein the CAR-T cell is autologous or allogeneic.39. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of aspect31 to the subject.40. A method of treating fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of aspect32 or 33 to the subject.41 . A method of treating an infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of aspect 34 to the subject.42. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of aspect 35 to the subject.43. A kit comprising: a population of CAR-T cells, wherein the CAR-T cells comprise T cells from a plurality of donors, wherein each T cell is engineered to express a chimeric antigenreceptor that specifically binds to an antigen on the target cell, and wherein expression of an endogenous T cell receptor (TCR) is eliminated in each T cell; and instructions for measuring an immune response against the target cell.

[0211] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.EXPERIMENTAL

[0212] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0213] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0214] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.Example 1Massively Parallel Mixed Lymphocyte Reactions

[0215] Testing engineered human cell-based therapeutics, in particular engineered human immune cells such as CAR-T cells, is costly and time consuming, limiting research, development, diagnostic, and therapeutic applications of these potentially curative cellular medicines. We have developed a cell therapy development and testing technology that allows the engineered T cells from multiple human donors or patients to be tested simultaneously in the same reaction. This pooling of many donors increases the scale and decreases the cost of cell therapy development and will enable future diagnostic and pre-clinical development work in autologous and allogeneic engineered T cell therapies.

[0216] Pooled screening technologies have drastically increased the pace and scale of cell therapy development and are now widely used in the cell therapy industry. However we have never been able to pool together T cells from multiple individual donors due to the fact that these cells would recognize other donor’s T cells as foreign and thereby become activated and kill the other donor’s cells, while at the same time being targeted by the other donor’s cells themselves. This recognition as “foreign” of other donor’s T cells is mediated through the T Cell Receptor (TCR) complex. We and others have previously developed gene editing systems in human T cells that allows for removal of the TCR. Here, we go further and show that engineered T cells with their own TCR’s removed can now be safely pooled together in the same assay / flask / well without causing activation / kil ling of other donor’s “foreign” cells. In our preliminary studies we have shown that individual donor’s T cells behave similarly when tested in isolation or as part of large multi-donor pools. We have shown that as few as two donors can be pooled together simultaneously, or as many as dozens of donors together. Overall we term this technology “Massively Parallel Mixed Lymphocyte Reactions” and plan to use this in academic and future industry cell therapy development and diagnostic efforts.

[0217] The subject methods will be useful in cell therapy preclinical development work. MP-MLRs enable many more donors to be tested, thereby making it possible to find ideal modifications for the specific types of patients that their trials will be conducted in, as well as identify constructs that will be effective across many patients. Future T cell therapy products can be personalized to individual patients. By enabling many patients’ cells tobe tested simultaneously, MP-MLRs allow for personalized diagnostic testing of engineered patient T cells at reasonable cost and scale compared to testing individual donors one by one (analogous to how clinical DNA sequencing involves sample barcoding and pooling of specimens from many different patients all onto a single nextgeneration sequencing run).

[0218] Currently if you want to test an engineered cell therapy using cells from two different donors / patients, you have to set everything up twice - twice the cost, time, effort, etc. Scale that by dozens or hundreds of patients, and its clear why there is no comparable technology to lower the cost of testing many different human donors besides just brute force, resource intensive, one-by-one testing.

Claims

What is claimed is:

1. A method of multiplexed screening to identify chimeric antigen receptor (CAR)-T cells that are immunoreactive against a target cell, the method comprising: providing a pooled population of CAR-T cells, wherein the CAR-T cells comprise T cells from a plurality of donors, wherein each T cell is engineered to express a chimeric antigen receptor that specifically binds to a target antigen on the target cell, and wherein expression of an endogenous T cell receptor (TCR) is eliminated in each T cell; contacting the pooled population of CAR-T cells with the target cell; and measuring an immune response of the population of CAR-T cells or a subset of the population of CAR-T cells to identify the CAR-T cells that are immunoreactive against the target cell.

2. The method of claim 1 , wherein said measuring comprises measuring cytotoxicity against the target cell, cytokine secretion, expression of an activation marker, affinity of binding to the target antigen on the target cell, or proliferation of the CAR-T cells that are immunoreactive against the target cell.

3. The method of claim 2, wherein the activation marker is CD69, HLA-DR, IL2RA, or CD25, or any combination thereof.

4. The method of claim 2, wherein said measuring the cytotoxicity comprises detecting perforin, a proapoptotic serine protease, or a granzyme released from the CAR- T cell.

5. The method of claim 2, wherein said measuring cytotoxicity comprises detecting death of the target cell.

6. The method of claim 2, wherein said measuring cytokine secretion comprises detecting interferon (IFN)-y, tumor necrosis factor (TNF)-a, TNF-p, interleukin (IL)-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, or IL-25, or any combination thereof.

7. The method of any one of claims 1 -6, wherein the CAR-T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell thathas been genetically modified to express the chimeric antigen receptor, and wherein expression of the endogenous TCR is eliminated.

8. The method of any one of claims 1 -7, wherein the chimeric antigen receptor comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen on the target cell.

9. The method of claim 8, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.

10. The method of claim 8 or 9, wherein the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.

11. The method of claim 8 or 9, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

12. The method of any one of claims 8-1 1 , wherein the chimeric antigen receptor further comprises a costimulatory domain.

13. The method of claim 12, wherein the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.

14. The method of any one of claims 8-13, wherein the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.

15. The method of any one of claims 1 -14, wherein the CAR-T cell further comprises a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the CAR-T cell.

16. The method of claim 15, wherein the binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR).

17. The method of claim 16, wherein the synthetic notch receptor comprises i) an extracellular ligand-binding domain that specifically binds to a second target antigen on the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the CAR-T cell.

18. The method of any one of claims 1 -17, wherein the target cell is a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.

19. The method of claim 18, wherein the antigen on the target cell is a tumor antigen or a tumor-associated antigen.

20. The method of claim 18, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.21 . The method of claim 20, wherein the antigen on the target cell is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

22. The method of claim 18, wherein the autoreactive immune cell is an autoreactive T cell or B cell.

23. The method of claim 22, wherein the antigen on the target cell is an antigen on the autoreactive T cell or B cell.

24. The method of any one of claims 1 -23, wherein the T cells are engineered using a gene editing system selected from the group consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, a zinc finger nuclease system, a transcription activator-like effector nuclease (TALEN) system, and a meganuclease system.

25. The method of any one of claims 1 -24, wherein the T cells are engineered to express the chimeric antigen receptor by transfecting the T cells with a recombinant polynucleotide encoding the chimeric antigen receptor.

26. The method of any one of claims 1 -25, further comprising contacting the CAR-T cells with an antigen presenting cell or an artificial antigen presenting particle presenting the target antigen.

27. The method of any one of claims 1 -26, wherein each T cell is further engineered to express a fluorescent protein, wherein T cells from the same donor are engineered to express the same fluorescent protein, and wherein T cells from different donors are engineered to express different fluorescent proteins.

28. The method of any one of claims 1 -27, further comprising genetically barcoding the population of CAR-T cells, wherein each CAR-T cell comprises a DNA barcode indicating which donor provided the T cell from which the CAR-T cell was generated.

29. The method of any one of claims 1 -28, further comprising substantially purifying the T cells engineered to express the chimeric antigen receptor, wherein T cells still expressing an endogenous TCR are eliminated.

30. A CAR-T cell identified as being immunoreactive against the target cell using the method of any one of claims 1 -29.

31. The CAR-T cell of claim 30, wherein the chimeric antigen receptor specifically binds to a tumor antigen or tumor-associated antigen.

32. The CAR-T cell of claim 30, wherein the chimeric antigen receptor specifically binds to a fibrosis antigen.

33. The CAR-T cell of claim 32, wherein the fibrosis antigen is fibroblast activation protein (FAP).

34. The CAR-T cell of claim 30, wherein the chimeric antigen receptor specifically binds to a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

35. The CAR-T cell of claim 30, wherein the chimeric antigen receptor specifically binds to an antigen on an autoreactive T cell or B cell.

36. A composition comprising the CAR-T cell of any one of claims 30-35 and a pharmaceutically acceptable excipient.

37. A method of performing cellular therapy, the method comprising administering a therapeutically effective amount of the composition of claim 36 to a subject.

38. The method of claim 37, wherein the CAR-T cell is autologous or allogeneic.

39. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of claim 31 to the subject.

40. A method of treating fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of claim 32 or 33 to the subject.41 . A method of treating an infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of claim 34 to the subject.

42. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the CAR-T cell of claim 35 to the subject.

43. A kit comprising: a population of CAR-T cells, wherein the CAR-T cells comprise T cells from a plurality of donors, wherein each T cell is engineered to express a chimeric antigen receptor that specifically binds to an antigen on the target cell, and wherein expression of an endogenous T cell receptor (TCR) is eliminated in each T cell; and instructions for measuring an immune response against the target cell.

Images