Switchable chimeric antigen receptors

Abstract

The invention provides switchable chimeric antigen receptors (CARs) and their use for treatment.

Description

Attorney Docket No: 252457.000033SWITCHABLE CHIMERIC ANTIGEN RECEPTORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 705,954, filed October 10, 2024, the contents of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 8, 2025, is named 252457_000033_SL.xml and is 156,397 bytes in size.FIELD OF THE INVENTION

[0003] The invention relates to switchable chimeric antigen receptors (CARs) and their use for treatment.BACKGROUND

[0004] Chimeric antigen receptor (CAR) T-cells have emerged as a game-changer in the treatment of some advanced liquid cancers, with curative responses now documented (1). While promising clinical outcomes have been reported for their use against non-hematological solid tumors (2) it is widely held that combinatorial or / and co-engineering strategies will be needed to extend meaningful responses to more patients and cancer-types, and to prevent relapse (3). Examples include co-administration of low-dose irradiation or immune checkpoint inhibition (ICI) (4-6), and gene-modifications to enforce the secretion of immunomodulatory molecules (7) or to abrogate the expression of exhaustion-associated genes (8). CAR therapies have been associated with adverse events in some patients, which is already consequential for second generation (2G)- CAR T cells as a monotherapy (9). There is a need to develop CAR T-cell therapies with lower probability of adverse events.Attorney Docket No: 252457.000033SUMMARY OF THE INVENTION

[0005] As specified in the Background section above, there is a great need in the art for minimizing side effects of chimeric antigen receptor (CAR)-based therapies. The present invention addresses this and other needs by providing switchable CARs which allow for tight, user-controlled titration of effector function, and timing of activation for killing of target cells.

[0006] In one aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first polypeptide chain and the second polypeptide chain comprise a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR- mediated signaling.

[0007] In some embodiments, the first and second member of the dimerization pair are located extracellularly.

[0008] In some embodiments, the first member of the dimerization pair is located in the first polypeptide chain between the extracellular region and the first TM region; and the second member of the dimerization pair is located in the second polypeptide chain N-terminal to the second TM region.

[0009] In some embodiments, the first and second member of the dimerization pair are located intracellularly.

[0010] In some embodiments, the first member of the dimerization pair is located in the first polypeptide chain C-terminal to the first TM region, and the second member of the dimerizationAttorney Docket No: 252457.000033 pair is located in the second polypeptide chain between the second TM region and the intracellular signaling ED.

[0011] In some embodiments, the first polypeptide chain comprises a first co-stimulatory ED and the second polypeptide chain comprises a second co-stimulatory ED, and wherein the first member of the dimerization pair is located in the first polypeptide chain C-terminal to the first co- stimulatory ED, and the second member of the dimerization pair is located in the second polypeptide chain between the second co-stimulatory ED and the intracellular signaling ED.

[0012] In some embodiments, the first polypeptide chain comprises a first co-stimulatory ED, the second polypeptide chain comprises a second co-stimulatory ED, and wherein the first member of the dimerization pair is located in the first polypeptide chain C-terminal to the first TM region and N-terminal to the first co-stimulatory ED, and the second member of the dimerization pair is located in the second polypeptide chain between the second TM region and the second co- stimulatory ED.

[0013] In some embodiments, the second polypeptide chain comprises an extracellular region which does not comprise a target-binding capacity.

[0014] In one aspect provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first member of a dimerization pair; iii) a first linker region; iv) a first transmembrane (TM) region; and v) a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) a second member of the dimerization pair; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; and v) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, andAttorney Docket No: 252457.000033 wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

[0015] In one aspect provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first co-stimulatory endodomain (ED); and v) a first member of a dimerization pair, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) a second member of the dimerization pair; and vi) an intracellular signaling ED, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0016] In one aspect provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first member of a dimerization pair; and v) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region;Attorney Docket No: 252457.000033 iii) a second TM region; iv) a second member of the dimerization pair; v) a second co-stimulatory ED; and vi) an intracellular signaling ED, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0017] In some embodiments, the first and / or second member of the dimerization pair is derived from a Bcl-2 protein or a Bcl-xL protein.

[0018] In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking the transmembrane domain. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0019] In some embodiments, the first and / or second member of the dimerization pair comprises the amino acid sequence MAHPGRTGYDNREIVMKYIHYKLSQRGYEWDAGDDVEENRTEAPEGTESEVVHLTLR QAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGV MCVESVNREMSPEVDNIAEWMTEYENRHEHTWIQDNGGWDAFVEEYGP (SEQ ID NO: 8), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8.

[0020] In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xE protein. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xE protein lacking the transmembrane domain. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xE protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0021] In some embodiments, the first and / or second member of the dimerization pair comprises the amino acid sequenceMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFEL RYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEAttorney Docket No: 252457.000033MQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERED (SEQ ID NO:74), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 74.

[0022] In some embodiments, the activator is a small molecule or a polypeptide.

[0023] In some embodiments, the activator is selected from venetoclax, navitoclax and A- 1155463.

[0024] In some embodiments, the first member and the second member of the dimerization pair are derived from a Bcl-2 protein and the activator is navitoclax or venetoclax.

[0025] In some embodiments, the first member and the second member of the dimerization pair are derived from a Bcl-xL protein and the activator is A-l 155463.

[0026] In some embodiments, the first polypeptide chain and / or the second polypeptide chain further comprises a degron sequence that mediates proteasomal degradation of the CAR in the presence of a ligand.

[0027] In some embodiments, the degron sequence is located intracellularly within the first polypeptide chain and / or the second polypeptide chain.

[0028] In some embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and / or second polypeptide chain.

[0029] In some embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and / or between the co-stimulatory ED and the intracellular signaling ED in the second polypeptide chain.

[0030] In some embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and / or C-terminal to the intracellular signaling ED in the second polypeptide chain.

[0031] In some embodiments, the degron sequence is a Cereblon (CRBN) binding moiety and the ligand is lenalidomide.

[0032] In some embodiments, the degron sequence comprises LQCEICGFTCRQKGNLLRHIKLH (SEQ ID NO: 39), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 39.

[0033] In some embodiments, the first polypeptide chain comprises a first linker region interposed between the first member of a dimerization pair and the first TM region.

[0034] In some embodiments, the second polypeptide chain comprises a second linker region interposed between the second member of the dimerization pair and the second TM region.Attorney Docket No: 252457.000033

[0035] In some embodiments, the first polypeptide chain comprises a first linker region interposed between the extracellular region and the first TM region.

[0036] In some embodiments, the second polypeptide chain comprises a second linker region N- terminal to the second TM region.

[0037] In some embodiments, the first and / or second linker region is an immunoglobulin hinge region or a hinge region derived from CD8, CD8a, or CD28.

[0038] In some embodiments, the first and / or second linker region is a hinge region derived from CD8a.

[0039] In some embodiments, the first polypeptide chain comprises a target -binding capacity in the extracellular region.

[0040] In some embodiments, the target-binding capacity is provided by an antigen-binding polypeptide, a receptor, or a natural ligand for a target cell antigen or receptor.

[0041] In some embodiments, the target-binding capacity is provided by an antigen-binding polypeptide.

[0042] In some embodiments, the antigen-binding polypeptide is an antibody or an antibody fragment.

[0043] In some embodiments, the antigen-binding polypeptide is a single chain antibody.

[0044] In some embodiments, the antigen recognized by the antigen-binding polypeptide is selected from a tumor-associated antigen, an infection-associated antigen and an auto-antigen.

[0045] In some embodiments, the tumor-associated antigen is associated with a solid tumor.

[0046] In some embodiments, the antigen recognized by the antigen-binding polypeptide is selected from CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CA125, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, EpCAM, EPhA2, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL-2, IL-6, IL-8, insulinAttorney Docket No: 252457.000033 growth factor- 1 (IGF-I), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1 , MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleophosmin, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, NeuGcGM3, N-glycolyl GM3 ganglioside, Neu5Gc, GM3- Ganglioside, GD3, GM2, carbohydrate antigens, ganglioside antigens, Lewis Y, Lewis B, CD 123, and Kappa chain of immunoglobulin.

[0047] In some embodiments, the antigen recognized by the antigen-binding polypeptide is PSMA.

[0048] In some embodiments, the antigen recognized by the antigen-binding polypeptide is CD 19.

[0049] In some embodiments, the antigen recognized by the antigen-binding polypeptide is EpCAM.

[0050] In some embodiments, the antigen recognized by the antigen-binding polypeptide is EPhA2.

[0051] In some embodiments, the extracellular region of the first polypeptide chain comprises a natural ligand for a target cell antigen or receptor.

[0052] In some embodiments, the natural ligand for a target cell antigen or receptor is an NKG2D ectodomain.

[0053] In some embodiments, the extracellular region of the first polypeptide chain comprises a T-cell receptor (TCR) based recognition domain.

[0054] In some embodiments, the TCR based recognition domain is a single chain TCR.

[0055] In some embodiments, the first and / or second TM region is derived from CD8, CD8a, CD4, CD3-zeta, CD3-epsilon, CD28, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (OX-40), CD137, CD154, DAP10, or DAP12.

[0056] In some embodiments, the first and second TM regions are the same.

[0057] In some embodiments, the first and second TM regions are derived from CD8a.

[0058] In some embodiments, the extracellular region of the second polypeptide chain comprises a stabilizing domain.Attorney Docket No: 252457.000033

[0059] In some embodiments, the extracellular region of the second polypeptide chain comprises an extracellular region derived from DAP 10 or DAP 12.

[0060] In some embodiments, the first and / or second co-stimulatory ED is derived from 4- IBB (CD137), CD28, ICOS, CD134 (OX-40), BTLA, CD27, CD30, GITR, CD226, or HVEM.

[0061] In some embodiments, first and / or second co-stimulatory EDs are derived from 4- IBB (CD 137).

[0062] In some embodiments, the first and the second co-stimulatory EDs are different.

[0063] In some embodiments, the intracellular signaling ED is derived from DAP10, DAP12, Fc epsilon receptor I gamma chain (FCER1G), FcR beta CD3-delta, CD3-epsilon, CD3-gamma, CD3-zeta, CD226, CD66d, CD79A, or CD79B.

[0064] In some embodiments, the intracellular signaling ED is derived from CD3-zeta.

[0065] In some embodiments, the first and / or second polypeptide chain further comprises one or more additional polypeptide sequences.

[0066] In some embodiments, the one or more additional polypeptide sequences are selected from one or more additional co-stimulatory EDs, signal sequences, separation sequences, epitope tags, and polypeptides that produce a detectable signal.

[0067] In some embodiments, the signal sequence is derived from CD8a.

[0068] In some embodiments, the epitope tag is cMyc.

[0069] In some embodiments, the separation sequence is T2A.

[0070] In some embodiments, the extracellular region of the first polypeptide chain comprises the amino acid sequenceVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYN QKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGG GSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLD LKR (SEQ ID NO: 4), DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS (SEQ ID NO: 33),Attorney Docket No: 252457.000033QVKLQQSGAELVRPGASVKLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIYPSYIYT NYNQEFKDKVTLTVDESSSTAYMQLSSPTSEDSAVYYCTRSPYGYDEYGLDYWGQGTT VTVSSGGGGSGGGGSGGGGSDIELTQSPSSLTVTAGEKVTMNCKSSQSLLNSRNQKNYL TWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDY VYPETFGAGTKEEIKR (SEQ ID NO: 35) or QVQEEESGGGEVQPGGSERESCAASGFTFSSYTMSWVRQAPGQAEEWMGTISSGGTYT YYPDSVKGRFTISRDNAKNSEYEQMNSERAEDTAVYYCAREAIFTYWGRGTEVTVSSG GGGSGGGGSGGGGSDIQETQSPSSESASVGDRVTITCKASQDINNYESWYQQKPGQAPR EEIYRANREVDGVPDRFSGSGYGTDFTETINNIESEDAAYYFCEKYDVFPYTFGQGTKVE IK (SEQ ID NO: 37).

[0071] In some embodiments, the intracellular signaling ED comprises the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 22).

[0072] In some embodiments, the first and / or second linker region comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 10).

[0073] In some embodiments, the first and / or second TM region comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12).

[0074] In some embodiments, the first and / or second co-stimulatory ED comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14).

[0075] In some embodiments, the first polypeptide chain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 51, SEQ ID NO: 55, or SEQ ID NO: 59.

[0076] In some embodiments, the second polypeptide chain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 26, SEQ ID NO: 43, or SEQ ID NO: 45.

[0077] In one aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising:Attorney Docket No: 252457.000033(i) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 26;(ii) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 43;(iii) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 45;(iv) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 51, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:26;(v) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 55, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:26; or(vi) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 59, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 26.

[0078] In one aspect, provided herein is a nucleic acid molecule encoding the switchable CAR of the present disclosure.

[0079] In some embodiments, the nucleic acid molecule comprises:(i) a) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 23; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25;(ii) a) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 23; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 42;(iii) a) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 23; and b) a nucleotideAttorney Docket No: 252457.000033 sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 44;(iv) a) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 50; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25;(v) a) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 54; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25; or(vi) a) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 58; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25.

[0080] In one aspect, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the switchable CAR of the present disclosure.

[0081] In some embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is SEQ ID NO: 23, SEQ ID NO: 50, SEQ ID NO: 54, or SEQ ID NO: 58.

[0082] In one aspect, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding the second polypeptide chain of the switchable CAR of the present disclosure.

[0083] In some embodiments, the nucleotide sequence encoding the second polypeptide chain of the CAR is SEQ ID NO: 25, SEQ ID NO: 42, or SEQ ID NO: 44.

[0084] In some embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter.

[0085] In some embodiments, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter.

[0086] In some embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter, and the first and second promoters are the same.Attorney Docket No: 252457.000033

[0087] In some embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter, and the first and second promoters are different.

[0088] In some embodiments, the nucleotide sequences encoding the first and second polypeptide chains of the CAR are operably linked to a single promoter.

[0089] In some embodiments, the first and / or second promoter is a T lymphocyte-specific promoter or an NK cell-specific promoter.

[0090] In some embodiments, the nucleic acid molecule is a DNA molecule.

[0091] In some embodiments, the nucleic acid molecule is an RNA molecule.

[0092] In one aspect, provided herein is an expression system comprising a first nucleotide sequence encoding the first polypeptide chain of the switchable CAR of the present disclosure and a second nucleotide sequence encoding the second polypeptide chain of the switchable CAR of the present disclosure.

[0093] In one aspect, provided herein is a recombinant vector comprising the nucleic acid molecule of the present disclosure.

[0094] In some embodiments, the vector is a viral vector.

[0095] In some embodiments, the vector is selected from a retroviral vector, a lenti viral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, and a vaccinia virus vector.

[0096] In some embodiments, the vector is a lentiviral vector.

[0097] In one aspect, provided herein is an isolated host cell comprising the switchable CAR of the present disclosure.

[0098] In one aspect, provided herein is an isolated host cell comprising the nucleic acid molecule of the present disclosure.

[0099] In one aspect, provided herein is an isolated host cell comprising the vector of any one of the present disclosure.

[0100] In some embodiments, the host cell is a mammalian cell.

[0101] In some embodiments, the host cell is selected from a cytotoxic cell, a T cell, a stem cell, a progenitor cell, and a cell derived from a stem cell or a progenitor cell.Attorney Docket No: 252457.000033

[0102] In some embodiments, the T cell is selected from T-helper cells, cytotoxic T-cells, T- regulatory cells (Treg), and gamma-delta T cells.

[0103] In some embodiments, the cytotoxic cell is a cytotoxic T cell or a NK cell.

[0104] In some embodiments, the host cell has been activated and / or expanded ex vivo.

[0105] In some embodiments, the host cell is an allogeneic cell.

[0106] In some embodiments, the host cell is an autologous cell.

[0107] In some embodiments, the host cell has been isolated from a subject having a disease.

[0108] In some embodiments, the subject is human.

[0109] In one aspect, provided herein is a pharmaceutical composition comprising the host cell of the present disclosure and a pharmaceutically acceptable carrier and / or excipient.

[0110] In one aspect, provided herein is a method for producing the host cell of the present disclosure comprising genetically modifying said cell with the nucleic acid molecule of the present disclosure, the expression system of the present disclosure, or the vector of the present disclosure.

[0111] In some embodiments of the method for producing the host cell of the present disclosure, the genetic modification is conducted ex vivo.

[0112] In some embodiments of the method for producing the host cell of the present disclosure, the method further comprises activation and / or expansion of the cell ex vivo.

[0113] In one aspect, provided herein is a method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of the present disclosure and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to said antigen and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0114] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is selected from a tumor- associated antigen, an infection-associated antigen and an auto-antigen.

[0115] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is a tumor-associated antigen.

[0116] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the tumor-associated antigen is associated with a solid tumor.Attorney Docket No: 252457.000033

[0117] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the tumor-associated antigen is PSMA.

[0118] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is an infection-associated antigen.

[0119] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is an auto-antigen.

[0120] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is CD 19.

[0121] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is EpCAM.

[0122] In some embodiments of the method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, the antigen is EPhA2.

[0123] In one aspect, provided herein is a method for stimulating elimination of a cell comprising PSMA in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of the present disclosure, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0124] In one aspect, provided herein is a method for stimulating elimination of a cell comprising CD 19 in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of the present disclosure, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0125] In one aspect, provided herein is a method for stimulating elimination of a cell comprising EpCAM in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of the present disclosure, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.Attorney Docket No: 252457.000033

[0126] In one aspect, provided herein is a method for stimulating elimination of a cell comprising EPhA2 in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of the present disclosure, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0127] In one aspect, provided herein is a method for treating a cancer in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of the present disclosure and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said cancer and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0128] In some embodiments of the method for treating a cancer in a subject in need thereof, the cancer is from a solid tumor.

[0129] In some embodiments of the method for treating a cancer in a subject in need thereof, the cancer is selected from carcinoma, melanoma, prostate cancer, sarcoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, and retinoblastoma.

[0130] In some embodiments of the method for treating a cancer in a subject in need thereof, the cancer is a leukemia or a lymphoma.

[0131] In one aspect, provided herein is a method for treating prostate cancer in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount cytotoxic T cells or NK cells comprising the switchable CAR of the present disclosure and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0132] In one aspect, provided herein is a method for treating an infection in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of the present disclosure and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigenAttorney Docket No: 252457.000033 associated with said infection and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0133] In one aspect, provided herein is a method for treating an inflammatory condition or an autoimmune disease in a subject in need thereof, wherein the method comprises administering to the subject (i) an effective amount of T-helper cells or Treg cells comprising the switchable CAR of the present disclosure and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said inflammatory condition or an autoimmune disease, and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR- mediated signaling.

[0134] In some embodiments of the method for treating an inflammatory condition or an autoimmune disease in a subject in need thereof, the method results in reducing an immune response to a transplanted organ or tissue.

[0135] In some embodiments of the methods of the present disclosure, the method comprises: a) isolating T cells or NK cells from the subject; b) genetically modifying said T cells or NK cells ex vivo with the nucleic acid molecule of the present disclosure, the expression system of the present disclosure, or the vector of the present disclosure; c) optionally, expanding and / or activating said T cells or NK cells before, after or during step (b); and d) introducing the genetically modified T cells or NK cells into the subject.

[0136] In some embodiments of the methods of the present disclosure, the subject is human.

[0137] In one aspect, provided herein is a method for activating the switchable CAR in the host cell of the present disclosure, comprising contacting the host cell with an activator, wherein the activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0138] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair are derived from a Bcl-2 protein or a Bcl-xL protein.Attorney Docket No: 252457.000033

[0139] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein. In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking the transmembrane domain. In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0140] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises the amino acid sequence MAHPGRTGYDNREIVMKYIHYKLSQRGYEWDAGDDVEENRTEAPEGTESEVVHLTLR QAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGV MCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGP (SEQ ID NO: 8), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8.

[0141] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein. In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking the transmembrane domain. In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0142] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first and / or second member of the dimerization pair comprises the amino acid sequence MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFEL RYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEAttorney Docket No: 252457.000033MQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERED (SEQ ID NO: 74), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 74.

[0143] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the activator is a small molecule or a polypeptide.

[0144] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the activator is selected from navitoclax, venetoclax and A-l 155463.

[0145] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first member and the second member of the dimerization pair are derived from a Bcl-2 protein and the activator is navitoclax or venetoclax.

[0146] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, the first member and the second member of the dimerization pair are derived from a Bcl-xL protein and the activator is A-l 155463.

[0147] In some embodiments, venetoclax is administered at about 1 - 50 mg / kg.

[0148] In some embodiments, venetoclax is administered at about 2.5 - 5 mg / kg.

[0149] In some embodiments, venetoclax is administered at a dose not to exceed about 100-600 mg / day.

[0150] In some embodiments, venetoclax is administered at a dose not to exceed about 400 mg / day.

[0151] In some embodiments, venetoclax is administered at sub-chemotherapeutic dose.

[0152] In some embodiments, venetoclax is administered orally, intravenously, subcutaneously, via intratumoral injection, or via peri tumoral injection.

[0153] In some embodiments of the method for activating the switchable CAR in the host cell of the present disclosure, wherein the first polypeptide chain and / or the second polypeptide chain of the CAR further comprises a degron sequence that mediates proteasomal degradation of the CAR in the presence of a ligand, the method further comprises contacting the host cell of the present disclosure with the ligand, resulting in downregulation of CAR-mediated signaling.

[0154] In some embodiments, the ligand is lenalidomide.

[0155] In some embodiments, lenalidomide is administered at about 1-25 mg / kg.

[0156] In some embodiments, lenalidomide is administered at about 2-5 mg / kg.

[0157] In some embodiments, lenalidomide is administered at a dose not to exceed about 50 mg / day- 100 mg / day.Attorney Docket No: 252457.000033

[0158] In some embodiments, lenalidomide is administered at sub-chemotherapeutic dose.

[0159] In one aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region comprising a binding means for specifically interacting with a target; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first polypeptide chain and the second polypeptide chain comprise a means for dimerizing the two polypeptides in the presence of an activator resulting in activation of CAR- mediated signaling.

[0160] In one aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, the improvement comprising: said first polypeptide chain and said second polypeptide chain comprises a first member and a second member of a dimerization pair, respectively, and the firstAttorney Docket No: 252457.000033 and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

[0161] In one aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising a first polypeptide chain and a second polypeptide chain, the improvement comprising: said first polypeptide chain does not comprise an intracellular signaling ED, said second polypeptide chain does not comprise a target-binding capacity, and said first polypeptide chain and said second polypeptide chain comprises a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

[0162] In some embodiments of the CAR, each of the first polypeptide chain and the second polypeptide chain further comprises a degron sequence that mediates proteasomal degradation of the CAR in the presence of a ligand.

[0163] These and other aspects described herein will be apparent to those of ordinary skill in the art in the following description, claims and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 shows schematic representations of exemplary activatable chimeric antigen receptor (ON-switch CAR) configurations according to some embodiments of the disclosure, and their assembly in the presence of a drug catalyst (activator molecule), e.g., venetoclax.

[0165] Figure 2 shows a schematic representation of the architecture of an exemplary ON- switch CAR. The recognition chain (R-chain) polypeptide chain may comprise: CD8 leader sequence - PSMA scFv - truncated mutant Bcl-2 protein - Serine-Glycine (Ser / Gly) linker - CD8 hinge - CD8 TM region - 4 IBB endodomain. The signaling chain (S-chain) polypeptide chain may comprise: CD8 leader sequence - c-Myc tag - Ser / Gly linker - truncated mutant Bcl-2 protein - CD8 hinge - CD8 TM region - 4 IBB endodomain - CD3^. The R-chain and the S-chain may be separated by a T2A self-cleaving peptide.

[0166] Figures 3A-3E demonstrate that Bcl-2 and venetoclax, as well as Bcl-xL and A- 1144563 constitute chemically-induced dimers. (Figure 3A) Schematic of mammalian two-hybrid system based on split chimeric transcription factors (TFs) that trigger reporter gene expression upon dimerization of the DNA binding domain (DBD) and the activation domain (AD). (Figure 3B) Gene expression of luciferase at 24 hours from a reporter plasmid encoding UAS-GAE4 responseAttorney Docket No: 252457.000033 elements for a two-hybrid system comprising GAL4 (galactose-responsive TF) and Rel65 (NF-kB p65) fused to Bcl-2 (left panel) or Bcl-xL (right panel) with + / - 100 nM venetoclax (Ven) or A- 1155463, respectively (or DMSO). (Figure 3C) Structure of Bcl-2 (PDB ID: 6o0k) with mutations Y108K and G145A that abrogate venetoclax binding (a small molecule compound shown in a skeletal form). (Figure 3D) Reporter gene expression (luciferase) at 24 hours from a reporter plasmid encoding UAS-GAL4 response elements for two-hybrid systems comprising GAL4 and Rel65 (NF-kB p65) fused to Bcl-2 (left panel) or Bcl-2 mutant (Y 108K, G145A) or Bcl-xL (right panel) or Bcl-xL mutant (Y101K, G138A) + / - increasing concentrations of venetoclax (Ven) or A-l 155463, respectively. (Figure 3E) Gene expression of luciferase at 24 hours from a reporter plasmid encoding UAS-GAL4 response elements for two-hybrid systems comprising GAL4 and Rel65 fused to Bcl-2 (left panel) or Bcl-xL (right panel) + / - increasing concentrations of DMSO, NVP-CGM097 (non-specific molecule), venetoclax (Ven) or A-l 155463. (ns = non-significant *= p<0.05 **= p<0.01. Ven = venetoclax; A-l l = small molecule A-l 155463; SEAP = secreted alkaline phosphatase.

[0167] Figures 4A-4E show that Bcl-2-based iON-CAR T cells targeting CD 19 were functional in vitro in the presence of venetoclax. (Figure 4A) Schematic of iON-CAR targeting CD 19. (Figure 4B) Evaluation of 2G- and iON-CAR expression by intracellular anti-Fab staining (n=6). (Figure 4C) Killing of CD19+BV173 tumor cells at 5 hours by CAR T cells + / - venetoclax; a one-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups; ****= p<0.0001, ns= non-significant. (Figure 4D) IncuCyte cytotoxicity assay of CD19+PC3- PIP tumor cells by the different CAR T cells + / - venetoclax (5 pM) over time (n=6 pooled HDs from two independent experiments). Target cell killing was reported as total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2). A two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing iON versus iON+Ven; ****= p<0.0001. (Figure 4E) Interferon-gamma (IFNy) production by CAR T cells in co-culture with CD19-PC3- PIP tumor cells + / - venetoclax (5 pM; effector to target ratio of 1: 1). A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups; ns= non-significant *= p<0.05 **= p<0.01. UTD = untransduced T cells; Ven = venetoclax; A-l l = small molecule A- 1155463; R-chain = receptor chain; S-chain = signaling chain; HD = T cells from healthy donors. (Figure 4F) CAR-engineered primary human T cells were specifically activated by PC3-PIP, as measured by IFNy cytokine production. A mixture CD4+ / CD8+ 1: 1 is shown. iON-CAR wasAttorney Docket No: 252457.000033 active only when venetoclax, at different doses, was added. (Data are represented as mean + / - s.e.m, n=3) A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups. ns=non-significant, *p<0.05, **** p<0.0001.

[0168] Figures 5A-5G show that Bcl-2-based iON-CAR T cells targeting PSMA were functional both in vitro and in vivo in the presence of venetoclax. (Figure 5A) Schematic of split iON-CAR targeting PSMA. (Figure 5B) Evaluation of 2G- and iON-CAR expression by intracellular anti-Fab staining (n=12). (Figure 5C) CD4+ (left) and CD8+ (right) T cell-surface staining in the presence of venetoclax (after 12 hours incubation) of 2G CAR by anti-Fab staining (n=3) and of iON CAR by anti-Fab (R-chain) and anti-cMyc staining (S-chain) (n=3). (Figure 5D) IncuCyte cytotoxicity assay of PSMA+ PC3-PIP tumor cells by the different CAR T cells + / - venetoclax over time (n=6 pooled from two independent experiments). Target cell killing is reported as total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2). A two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups; *= p<0.05. (Figure 5E) IFNy production by CAR T cells and UTD T cells upon coculture with PC3-PIP tumor cells + / - venetoclax (n=3), two independent replicates. A two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing iON vs 2G CAR; ****= p<0.0001: ns=non-significant. (Figure 5F) IFNy production by UTD and CAR T cells (n=3) upon co-culture with PC3-PIP tumor cells (effector: target ratio of 1:1) at various hours post venetoclax removal (after 12 hours pre-incubation or not). A two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups; *= p<0.05. **= p<0.005. (Figure 5G) Top: schematic of in vivo study. Bottom: PC3-PIP tumor control by UTD and CAR T cells + / - venetoclax (2.5 mg / kg) over days. Values are the mean ± s.e.m. of n = 7 mice per group. A two-way ANOVA with post hoc Tukey test was used for statistical analysis, statistical significance indicated considering all T-cell groups at day 23 (four independent replicates have been performed). UTD = untransduced; HD = T cells from healthy donors.

[0169] Figures 6A-6E show that all-in-one Bcl-2 and degron-based ON / OFF-switch CAR T cells targeting PSMA can be reversibly controlled with venetoclax and lenalidomide. (Figure 6A) Schematic of iON0-CAR targeting PSMA that was switched into active mode in the presence of venetoclax and can be downregulated in the presence of lenalidomide. (Figure 6B) Representative histogram plots for intracellular aFab CAR staining after overnight culture in venetoclax, lenalidomide, or venetoclax plus lenalidomide (n=3 HD), two independent experiments. (FigureAttorney Docket No: 252457.0000336C) IFNy production by CAR T cells upon co-culture with PC3-PIP cells + / - small molecules at concentrations as shown. Values are the mean + / - s.e.m. for n=3 HD. (Figure 6D) IncuCyte cytotoxicity assays of PC3-PIP cells and UTD or CAR T cells + / - venetoclax (5 pM) and + / - lenalidomide (1 pM) as shown. Target cell killing is reported as the total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2). Values are the mean ± s.e.m. of n=2 human T cell donors. Two independent replicates were performed (total n=4). Unpaired T test, parametric with Welch’s correction was used to determine statistical significance between UTD / CARs +Ven vs UTD / CARs +Ven+Len. (Figure 6E) Percentage of CAR-expressing T cells (intracellular staining) over time post-exposure to lenalidomide. Values are the mean ± s.e.m. for n=3 HD. UTD = untransduced; HD = T cells from healthy donors; Ven = venetoclax; Leni = lenalidomide; Receptor (R-)chain; Signaling (S-)chain.

[0170] Figures 7A-7I show additional reporter systems demonstrating homodimerization of Bcl-2 by venetoclax as well as Bcl-xL by A-l 155463. (Figures 7A-7B) Quantification of luciferase activity 24 hours after drug (100 nM) or vehicle (DMSO) treatment in HEK-293T cells transduced with luciferase reporter only (Neg = negative control) or with constitutive Gal4-Rel65 expression plasmid (pCMV-Gal4-Rel65-pA) (Pos = positive control). (Figure 7C) Reporter system comprising the fusion of Bcl-2 or Bcl-xL to TetR (repressor of the tetracycline resistance element) and VP 16 (Herpes simplex virus protein vmw65) which upon homodimerization in the presence of venetoclax (left) or A-l 155463 (right), respectively, drives SEAP expression from a reporter plasmid including Tet response elements. (Figure 7D) Schematic of Bcl-2 protein used to control transcription with the MS2-VPR associated with dead (d)Cas9:sgRNA RNP complex. The fusion protein MS2 (lightly speckled oval) and Bcl-2 (circle) dimerizes with BCL-2-VPR (dotted oval) in the presence of small molecule (yellow star; venetoclax or A-l 155463) that then binds to the stem-loop of sgRNA. The complex of RNP:MS2-VPR drives downstream SEAP reporter expression. (Figure 7E) Quantification of SEAP activity for the reporter system shown in Figure 7D, 24 hours after drug (100 nM; venetoclax (Ven) or A-l 155463 (A-l l)) or control (DMSO) treatment. (Figures 7F-7G) Quantification of SEAP activity 24 hours after drug (100 nM) or DMSO treatment of HEK-293T cells transduced with SEAP reporter only (Neg = negative control) or with constitutive TetR-VP16 expression plasmid (pCMV-TetR-VP16-pA) (Pos = positive control). (Figures 7H-7I) Quantification of SEAP activity 24 hours after drug (100 nM) or vehicle (DMSO) treatment in HEK-293T cells transduced with the SEAP reporter only (Neg =Attorney Docket No: 252457.000033 negative control) or with constitutive dCas9, insulin-sgRNA and MCP-VPR expression plasmid (Pos = positive control).

[0171] Figures 8A-8D show that in vitro function of anti-PSMA iON-CAR T against target tumor cells is regulated by venetoclax. (Figure 8A) Killing of PC3-PIP (PSMA+) tumor cells by UTD, 2G (top panel) and iON-CAR T (bottom panel) cells targeting PSMA in the presence and absence of 5 pM venetoclax. Values are the mean ± s.e.m. of n = 3 HD. Cytotoxicity is reported as total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2) A Two- way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups. (Figure 8B) Killing of PC3-PIP (PSMA+) tumor cells by UTD, 2G (top panel) and iON-CAR T (bottom panel) cells in the presence and absence of 5 pM, 2.5 pM, and 1.25 pM venetoclax. Values are the mean ± s.e.m. of n = 3 human donors. Cytotoxicity is reported as total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2) A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing 2G vs 2G + Ven and iON vs iON -i-Ven at all concentrations. ns=non-significant, *p<0.05, ** p<0.01. (Figure 8C) CAR-engineered primary human T cells were specifically activated by PC3-PIP, as measured by IFNy cytokine production. On the top left, IFNy production by CD4+ cell alone and on the top right IFNy production by CD8+ cell alone are reported. An IFNy production by mixture of CD4+ / CD8+ 1 : 1 is also shown (bottom panel). iON-CAR was active only when venetoclax, at different doses, was added. (Data are represented as mean + / - s.e.m, n=3) A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups. ns=non-significant, *p<0.05, **** p<0.0001 (Figure 8D) Killing of PC3 (PSMA-) tumor cells by UTD, 2G, and ON CAR-T cells in the presence and absence of 5 pM venetoclax. Values are the mean ± s.e.m. of n = 3 HD. Cytotoxicity is reported as total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2). A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups. ns=non-significant. (Figure 8E) CAR-engineered primary human T cells were not activated by PC3 (PSMA-), as measured by no cytokine production (IFN-y) in absence or presence of 5 pM venetoclax (Data are represented as mean + / - s.e.m, n=6 CD4+ and n=3 CD8+ T cells from HDs). A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups. ns=non-significant. UTD = untransduced; HD = T cells from healthy human donors; Ven = venetoclax.Attorney Docket No: 252457.000033

[0172] Figures 9A-9B show Bcl-2-based iON-CAR T cells are responsive to the BH3 mimetic Navitoclax. (Figure 9A) Killing of PC3-PIP (PSMA+) tumor cells by UTD, 2G (top panel) and iON-CAR T (bottom panel) cells in the presence and absence of 5 pM and 2.5 pM navitoclax (Navi). Values are the mean ± s.e.m. of n = 3 human donors. Cytotoxicity is reported as total area of dead cells, as measured by Cytotoxic Red reagent uptake (red area per pm2) A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing 2G vs 2G +Navi and iON vs iON +Navi at all tested concentrations. (Figure 9B) CAR-engineered primary human T cells were specifically activated by PC3-PiP, as measured by IFN-y cytokine production. CD4+ T cells are reported in presence and absence of navitoclax (Bcl-xL / Bcl-2 inhibitor) and A-l 155463 (Bcl-xL selective inhibitor) (top panel). CD8+ T cells are also shown in the same experimental conditions (bottom panel). In both the experiments, iON-CAR T cells specifically activated in presence of navitoclax, which targets Bcl-2 but not in presence of A-l 155463 which cannot bind Bcl-2. Data are represented as mean + / - s.e.m, n=3. A Two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups, ns = non-significant, *p<0.05, ** p<0.01. UTD = untransduced T cells; Navi = navitoclax.

[0173] Figures 10A-10C show Anti-PSMA iON-CAR T cells control tumors in the presence of venetoclax. (Figure 10A) On the top: cartoon of the experimental design for in vivo experiments. NSG mice were subcutaneously (s.c.) inoculated with PC3-PIP tumor cells, on day 5 received daily peri-tumoral (p.t.) injections of venetoclax 2.5mg / kg to assess the effect of the drug on tumor growth per se. On the bottom panel: tumor growth over time is shown, saline vs saline + venetoclax, and no significant difference was shown between the groups. Values are the mean ± s.e.m. of n = 6 mice per group. A two-way ANOVA with post hoc Tukey test was used for statistical analysis. (Figure 10B) On the top: cartoon of the experimental design for in vivo experiments. NSG mice were subcutaneously (s.c.) inoculated with PC3-PIP tumor cells, and on day 5 received CAR-T cells or UTD-T cells, followed by daily peri-tumoral (p.t.) injections of venetoclax 2.5mg / kg. On middle and bottom: tumor growth is shown upon UTD and CAR-T cell transfer, with and without daily injection of venetoclax. Values are the mean ± s.e.m. of n =7 mice per group. A two-way ANOVA with post hoc Tukey test was used for statistical analysis, comparing all groups. Statistical significance: considering all T-cell groups at day 28 and day 25. ***=p<0.001, **=p<0.01 iON vs iON+Ven. 2G CAR and iON CAR level of expression was not normalized to equal number. (Figure 10C) Tumor growth following UTD and CAR-T cellAttorney Docket No: 252457.000033 transfer, with and without daily injection of venetoclax. Values are the mean ± s.e.m. of n = 6 mice per group. A two-way ANOVA with post hoc Tukey test was used for statistical analysis. Statistical significance: considering all T-cell groups at day 22. 2G CAR and ON CAR level of expression were normalized to equal number. UTD = untransduced T cells; Ven = venetoclax.

[0174] Figures 11A-11C show in vitro function of iON and iON0 CAR T cells targeting PSMA+ PC3-PIP target cells. (Figure 11A) Transduction efficiency of UTD, iON and iON0 V2 (version 2) CAR T cells on CD4+ and CD8 T cells derived from healthy donor, after venetoclax o / n incubation (n=6). (Figure 11B) IFNy quantification by ELISA after 24 hours co-culture of UTD, iON, and iON0 V2 CAR T cells with PSMA+ tumor cells at 1: 1 Effector: Target (E:T) Ratio. Data are represented as mean + / - SEM, n=3. (Figure 11C) Incucyte assay 24 hours coculture of UTD (top), iON (middle), and iON0 V2 (bottom) CAR T cells with PSMA+ tumor cells at 2: 1 E:T Ratio. Data are represented by total red area (cell death) per pm2), and as mean + / - SEM, n=3. One-way ANOVA (panel b) and Two-way ANOVA (panel c) were used to determine statistical significance.

[0175] Figures 12A-12D show in vitro and in vivo function of anti-EpCAM iON CAR T cells. (Figure 12A) Transduction efficiency of UTD, EpCAM 2G and EpCAM iON CAR T cells on CD4+ and CD8 T cells derived from healthy donor, after venetoclax o / n incubation (n=3). (Figure 12B) IFNy quantification by ELISA after 24h co-culture of UTD, EpCAM 2G and EpCAM iON CAR T cells with EpCAM-i- tumor cells at 1:1 Effector: Target (E:T) Ratio, at different concentrations of venetoclax and vehicle. Data are represented as mean + / - SEM, n=3. (Figure 12C) Incucyte assay 24 hours co-culture of UTD (top), EpCAM 2G (middle) and EpCAM iON (bottom) CAR T cells with EpCAM-i- tumor cells at 2:1 E:T Ratio. Data are represented total red area (cell death) per pm2), and as mean + / - SEM, n=3. (Figure 12D) In vivo validation of EpCAM iON CAR T cells. Eight-week-old male NSG mice were injected with 5xl06Epcarn+ tumor cells subcutaneously. When tumor was palpable, 2xl06T cells were injected peritumorally, and drug or vehicle were administered every other day peritumorally. Data are represented as mean + / - SEM, n=6 mice per group. One-way ANOVA (panel b) and Two-way ANOVA (panel c and d) were used to determine statistical significance.Attorney Docket No: 252457.000033DETAILED DESCRIPTION

[0176] Chimeric antigen receptors (CARs) are hybrid molecules comprising a tumor antigentargeting moiety, typically an scFv, followed by a linker, a transmembrane (TM) domain, and various endodomains (EDs) involved in T-cell activation. First generation CARs include the ED of CD3-zeta (CD3Q only, required for “signal 1” of T cell activation, while second and third generation CARs also have one or more co-stimulatory EDs, respectively, such as CD28 and 4- 1BB, to provide “signal 2”.

[0177] The adoptive transfer of scFv-directed T lymphocytes, so-called CAR-T cells, has emerged as a potent treatment against various advanced cancers. For example, recent clinical trials with CD19-targeted CAR T cells have yielded up to 90% complete remission rates for patients suffering advanced acute lymphoblastic leukemia (ALL), a “liquid” tumor32-34. “Solid” tumors, however, remain a significant challenge to CAR therapy. This is in part due to the fact that there are few bona fide tumor antigens that are not found on healthy tissue, and as such, important “on- target / off-tumor” toxicities have occurred in CAR T-cell treated patients, in some instances even leading to death35. Early strategies to address such challenges included drug-inducible “suicide genes”36, 37, negative regulatory co-receptors (iCARs) that upon engagement with specific antigens will stop effector function38, and split-signaling CAR-Ts that require co-engagement of two ligands for full T-cell activation39. Though important elements for engineering therapeutic T cells, such strategies generally lack controlled timing and intensity of T cell activity. Designs incorporating suicide switches, for instance, permanently eliminate T cells, thereby irreversibly terminating a complex and costly treatment that may ultimately be too sluggish to prevent cross-reaction during initial cell transfer.

[0178] Chemically-induced dimerization (CID) systems, that is, protein-protein interfaces reliant upon the presence of a small chemical compound to bind one another, offer a promising approach for developing inducible ON-switch CARs in which the components necessary for antigen engagement and T-cell activation are split amongst two chains (10, 40). Ideally, CID-based ON-switches for CARs are non-immunogenic, will not interfere with cellular function, and the associated small molecule is well-tolerated, has favorable pharmacokinetic (PK) properties, and is clinically approved.Attorney Docket No: 252457.000033

[0179] T cells can be gene-modified with CARs to recognize and kill tumor cells. While CAR T-cell therapy has been approved to treat some B cell malignancies, for non-hematological solid tumors both toxicity and lack of efficacy remain important obstacles.

[0180] The ability to remotely control the activity of CARs with small molecules can improve both the safety and efficacy of gene-modified T cells. Split ON- and OFF-switch CAR designs involve the dissociation of tumor-antigen binding from the activation endodomain of CD3-zeta on two separate chains, the signaling (S-) and receptor (R-)chains, that either associate or are disrupted in the presence of a small molecule. The present disclosure is based, at least in part, on the development of an inducible iON-CAR comprising in the ectodomain of both the S- and R-chains a homodimerization domain derived from Bcl-2 or Bcl-xL protein which associate in the presence of a BH3 mimetic. As illustrated herein in the Examples, iON-CAR T cells comprising iON-CAR with a homodimerization domain derived from Bcl-2 responded to target tumor cells in vitro in the presence of BH3 mimetic venetoclax or navitoclax in a dose-dependent manner, while there was no impact of the drugs on the function of equivalent 2G-CAR T cells. Post-withdrawal of venetoclax, iON-CAR T cells lost their ability to respond to target tumor cells within 48 hours as evaluated by IFNy production. Importantly, iON-CAR T cells were also reliant upon the presence of venetoclax for in vivo activity. Lastly, by fusing degron sequences to the endodomain of the iON-CAR chains, an all-in-one ON / OFF-switch split CAR design, termed the “iON0-CAR” herein, was generated, that could be fully downregulated within 4 to 6 hours on-command upon administration of lenalidomide. These remotely-controlled CAR designs can offer elevated safety to patients along with improved tumor control because the engineered T cells can be periodically rested to alleviate exhaustion which can occur upon chronic antigen exposure.

[0181] The present disclosure provides ON-switch CARs (“ON-CARs” or “activatable CARs”) which effectively and selectively kill target cells (e.g., cancer cells) upon expression by engineered T cells and provide enhanced safety due to their ability to be activated by dimer- activating molecules. In one non-limiting embodiment, ON-CAR comprises two polypeptide chains, wherein an extracellular domain (e.g., a target-binding domain, e.g., an scFv) and intracellular signaling endodomain (ED) (e.g., CD3-zeta) are present on different polypeptide chains, and wherein the two chains dimerize via a first member of a dimerization pair and a second member of a dimerization pair, which form a dimer in the presence of an activator, thereby resulting in activation of CAR-mediated signaling. Specifically, the addition of an activator (e.g., a smallAttorney Docket No: 252457.000033 molecule drug) which interacts with the first and the second member of the dimerization pair with high affinity brings together the two polypeptide chains thereby activating CAR-mediated signaling. In a further embodiment, switchable CARs comprise one or more degron sequences, wherein the degron sequence(s) is fused to the co-stimulatory endodomain (ED) and / or intracellular signaling ED of the polypeptide chains of ON-CAR. Specifically, the degrons sequence(s) incorporated in the polypeptide chains of ON-CAR mediate CAR downregulation in the presence of a ligand (e.g., a small molecule drug). The CARs which can be further downregulated in the presence of a ligand due to presence of the degron sequence(s) on the CARs are referred herein as ON / OFF-switch CARs (“ON0-CAR”, “ON0 CAR”). Illustrated herein are an exemplary Bcl-2 based ON-switch CAR that can be activated in a dose-dependent manner in the presence of venetoclax, as well as an exemplary all-in-one ON / OFF-switch CAR that is reversibly controlled by venetoclax and lenalidomide.Definitions

[0182] The term “chimeric antigen receptor” or “CAR” as used herein is defined as a cell-surface receptor comprising an extracellular domain, a TM region, and a cytoplasmic domain, comprising an intracellular signaling ED and optionally at least one co-stimulatory signaling domain, all in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic domain are not naturally found together on a single receptor protein. The chimeric antigen receptors of the present disclosure may be for use with lymphocytes such as T cells and natural killer (NK) cells.

[0183] The terms “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTE; CD4+ T cell), a cytotoxic T cell (CTE; CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), CD4+CD8+ T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. Also included are “NKT cells”, which refer to a specialized population of T cells that express a semi-invariant aP T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8- cells. The TCR on NKT cells is unique in thatAttorney Docket No: 252457.000033 it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Also included are “gamma-delta T cells (y8 T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated a- and P-TCR chains, the TCR in y8 T cells is made up of a y-chain and a 8-chain. y8 T cells can play a role in immunosurveillance and immunoregulation, and were found to be an important source of IL- 17 and to induce robust CD8+ cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs”, which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs are typically transcription factor Foxp3 -positive CD4+T cells and can also include transcription factor Foxp3- negative regulatory T cells that are IL-10-producing CD4+T cells.

[0184] As used herein, the term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) or molecule capable of being bound by a T-cell receptor. An antigen is also able to provoke an immune response. An example of an immune response may involve, without limitation, antibody production, or the activation of specific immunologically competent cells, or both. A skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins / antigens, killed or inactivated whole cells or lysates.

[0185] The term “tumor-targeting moiety” refers to a target-specific binding element that may be any ligand that binds to the antigen of interest or a polypeptide or fragment thereof, wherein the ligand is either naturally derived or synthetic. Examples of tumor-targeting moieties include, but are not limited to, antibodies; polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab’, F(ab’)2, and Fv fragments; polypeptides derived from T Cell receptors, such as, for example, TCR variable domains; secreted factors (e.g., cytokines, growth factors) that can be artificially fused to signaling domains (e.g., “zytokines”); and any ligand or receptor fragment (e.g., CD27, NKG2D) that binds to the antigen of interest.Attorney Docket No: 252457.000033Combinatorial libraries could also be used to identify peptides binding with high affinity to the therapeutic target.

[0186] Host cells of the present disclosure may include T cells and natural killer cells that contain the DNA or RNA sequences encoding the CAR and express the CAR on the cell surface. Host cells may be used for enhancing T cell activity, natural killer cell activity, treatment of cancer, and treatment of autoimmune disease.

[0187] The terms “activation” or “stimulation” means to induce a change in their biologic state by which the cells (e.g., T cells and NK cells) express activation markers, produce cytokines, proliferate and / or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals. Co-stimulatory signals can amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity. A “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0188] The term “proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells. The term “expansion” refers to the outcome of cell division and cell death.

[0189] The term “linker”, “linker region”, “hinge”, or “linker domain” as used herein generally means any oligo- or polypeptide that functions to link the antigen-binding moiety to the TM region.

[0190] The term “differentiation” refers to a method of decreasing the potency or proliferation of a cell or moving the cell to a more developmentally restricted state.

[0191] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become produced, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. , the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or transmembrane.

[0192] The term “transfection” means the introduction of a “foreign” (i.e., extrinsic or extracellular) nucleic acid into a cell using recombinant DNA technology. The term “genetic modification” means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA orAttorney Docket No: 252457.000033RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences operably linked to polynucleotide encoding the chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “genetically engineered.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0193] The term “transduction” means the introduction of a foreign nucleic acid into a cell using a viral vector.

[0194] The terms “genetically modified” or “genetically engineered” refers to the addition of extra genetic material in the form of DNA or RNA into a cell.

[0195] As used herein, the term “derivative” in the context of proteins or polypeptides (e.g., CAR constructs or domains thereof) refers to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide it is a derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (z.e., additions, deletions and / or substitutions) relative to the polypeptide it is a derivative of; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a derivative of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a derivative of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a derivative of.Attorney Docket No: 252457.000033

[0196] Percent sequence identity can be determined using any method known to one of skill in the art. In a specific embodiment, the percent identity is determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, moderate, and typical stringency conditions) have been described, see, e.g., U.S. Patent Application Publication No. US 2005 / 0048549 (e.g., paragraphs 72-73).

[0197] The terms “vector”, “cloning vector,” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc. In certain embodiments, the vector is a viral vector such as, but not limited to, viral vector is an adenoviral, adeno-associated, alphaviral, herpes, lentiviral, retroviral, or vaccinia vector.

[0198] The terms “treat” or “treatment” of a state, disorder, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e., arresting, reducing, or delaying the development of the state, disorder, condition, or disease, or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder, or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0199] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severityAttorney Docket No: 252457.000033 of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0200] The phrase “pharmaceutically acceptable”, as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0201] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human, and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0202] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.

[0203] By “enhance” or “promote,” or “increase”, or “expand” or “improve” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in T cell expansion, activation, effector function, persistence, and / or an increase in cancer cell death killing ability, among others apparent from the understanding in the art and the description herein. In certain embodiments, an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal pointsAttorney Docket No: 252457.000033 in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.

[0204] By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refers generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (z.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. In certain embodiments, a “decrease” or “reduced” amount can be a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage.

[0205] The term “protein” is used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0206] The terms “nucleic acid”, “nucleotide”, and “polynucleotide” encompass both DNA and RNA unless specified otherwise. By a “nucleic acid sequence” or “nucleotide sequence” is meant the nucleic acid sequence encoding an amino acid, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by linkers

[0207] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0208] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.Attorney Docket No: 252457.000033

[0209] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ.

[0210] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein.

[0211] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.Chimeric Antigen Receptors

[0212] In one aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second costimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first polypeptide chain and the second polypeptide chain comprise a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.Attorney Docket No: 252457.000033

[0213] In various embodiments, the first and the second member of the dimerization pair may be located extracellularly. In various embodiments, the first and the second member of the dimerization pair may be located intracellularly. In various embodiments, the first member of the dimerization pair may be located extracellularly and the second member of the dimerization pair may be located intracellularly. In various embodiments, the first member of the dimerization pair may be located intracellularly and the second member of the dimerization pair may be located extracellularly.

[0214] In various embodiments, the first member of the dimerization pair may be located in the first polypeptide chain between the extracellular region and the first TM region, and the second member of the dimerization pair may be located in the second polypeptide chain N-terminal to the second TM region.

[0215] In certain embodiments, the first member of the dimerization pair may be located in the first polypeptide chain C-terminal to the first TM region, and the second member of the dimerization pair may be located in the second polypeptide chain between the second TM region and the intracellular signaling ED.

[0216] In some embodiments of any of the switchable CARs of the disclosure, the CAR molecule further comprises one or more degron sequences that mediates proteasomal degradation of the CAR in the presence of a ligand. In certain embodiments, the degron sequence is located intracellularly. In certain embodiments, the degron sequence is present on either of the first polypeptide chain and second polypeptide chain intracellularly. In certain embodiments, the degron sequence is present on each of the first polypeptide chain and second polypeptide chain intracellularly. In certain embodiments, the degron sequence is located C-terminal to the costimulatory ED in the first polypeptide chain and second polypeptide chain. In certain embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and between the co-stimulatory ED and the intracellular signaling ED in the second polypeptide chain. In certain embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and C-terminal to the intracellular signaling ED in the second polypeptide chain.

[0217] In various embodiments, the first polypeptide chain may optionally comprise a first co- stimulatory ED and the second polypeptide chain may comprise a second co-stimulatory ED. In such embodiments, the first member of the dimerization pair may be located in the first polypeptideAttorney Docket No: 252457.000033 chain C terminal to the first co-stimulatory endodomain (ED), and the second member of the dimerization pair may be located in the second polypeptide chain between the second costimulatory ED and the intracellular signaling ED.

[0218] In certain embodiments, the first polypeptide chain may optionally comprise a first costimulatory ED, the second polypeptide chain may comprise a second co-stimulatory ED. In such embodiments, the first member of the dimerization pair may be located in the first polypeptide chain C terminal to the first TM region and N terminal to the optional first co-stimulatory ED, and the second member of the dimerization pair may be located in the second polypeptide chain between the second TM region and the second co-stimulatory ED.

[0219] In some embodiments of any of the CARs of the disclosure, the first polypeptide chain may comprise one or more of a first co-stimulatory ED. In some embodiments, the co-stimulatory ED may be absent from the first polypeptide such that the first polypeptide does not comprise a first co-stimulatory ED.

[0220] In some embodiments of any of the CARs of the disclosure, the second polypeptide chain may comprise one or more of a second co-stimulatory ED. In some embodiments, the co- stimulatory ED may be absent from the second polypeptide such that the second polypeptide does not comprise a second co-stimulatory ED.

[0221] In some embodiments, the first polypeptide chain comprises a first co-stimulatory ED and the second polypeptide chain comprises a second co-stimulatory ED.

[0222] In some embodiments of any of the CARs disclosed herein, the first and / or second co- stimulatory ED may be derived from, for example, without limitation, 4-1BB (CD137), CD28, ICOS, CD134 (OX-40), BTLA, CD27, CD30, GITR, CD226, or HVEM.

[0223] In some embodiments, the first co-stimulatory ED and the second co-stimulatory ED may be derived from the same co-stimulatory ED. As a non-limiting example, when the first co- stimulatory ED and the second co-stimulatory ED are derived from the same co-stimulatory ED, the co-stimulatory domain may be derived from 4- IBB (CD 137). In some embodiments, the first co-stimulatory ED and the second co-stimulatory ED may be derived from different co-stimulatory domains. As a non-limiting example, when the first co-stimulatory ED and the second co- stimulatory ED are derived from different co-stimulatory EDs, the first co-stimulatory domain may be derived from 4- IBB (CD 137) and the second co-stimulatory ED may be derived from CD28. In some embodiments, the first and second co-stimulatory EDs of the first and second polypeptideAttorney Docket No: 252457.000033 may be the same. In some embodiments, the first and second co-stimulatory EDs of the first and second polypeptide may be different.

[0224] In various embodiments of any of the CARs disclosed herein, the second polypeptide chain may comprise an extracellular region which does not comprise a target-binding capacity. In some embodiments, the extracellular region of the second polypeptide domain may comprise a stabilizing domain. By way of a non-limiting example, the stabilizing domain may be derived from DAP 10 or DAP 12. The extracellular region of the second polypeptide chain may stabilize the CAR protein in the membrane and optionally can be used for the detection purpose. In some embodiments, the extracellular region of the second polypeptide chain may comprise a second target-binding moiety.

[0225] In another aspect provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first member of a dimerization pair; iii) a first linker region; iv) a first transmembrane (TM) region; and v) a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) a second member of the dimerization pair; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; and v) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR- mediated signaling.

[0226] In yet another aspect is provided a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first co-stimulatory endodomain (ED); and v) a first member of a dimerization pair, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) a second member of the dimerization pair; and vi) an intracellular signaling ED, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR- mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.Attorney Docket No: 252457.000033

[0227] In still yet another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first member of a dimerization pair; and v) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second transmembrane (TM) region; iv) a second member of the dimerization pair; v) a second co-stimulatory ED; and vi) an intracellular signaling ED, wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0228] In another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region comprising a binding means for specifically interacting with a target; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first polypeptide chain and the second polypeptide chain comprise a means for dimerizing the two polypeptides in the presence of an activator resulting in activation of CAR-mediated signaling.

[0229] In another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co- stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, the improvement comprising: said first polypeptide chain and said second polypeptide chain comprises a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.Attorney Docket No: 252457.000033

[0230] In another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising a first polypeptide chain and a second polypeptide chain, the improvement comprising: said first polypeptide chain does not comprise an intracellular signaling ED, said second polypeptide chain does not comprise a target-binding capacity, and said first polypeptide chain and said second polypeptide chain comprises a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

[0231] In various embodiments of any of the CARs of the disclosure, the first member and the second member of the dimerization pair may be derived from proteins that do not interact in vivo. In some embodiments, the dimerization of the dimer formed by the first and second member of the dimerization pair may be promoted by an activator molecule. The promotion may result in activation of CAR-mediated signaling. In certain embodiments, the activator molecule may be a small molecule. In certain embodiments, the activator molecule may be a polypeptide. Nonlimiting examples of activator molecules that may be useful in accordance with the present disclosure including, without limitation, navitoclax, A-1331852, A-l 155463, venetoclax, ABT- 199 (GDC-0199), obatoclax mesylate (GX15-070), HA14-1, ABT-737, TW-37, AT101, sabutoclax, gambogic acid, ARRY 520 trifluoroacetate, iMAC2, maritoclax, methylprednisolone, MIMI, ML 311, glossypol, BH3I-1, and 2-methoxy-antimycin A3.

[0232]

[0233] In some embodiments, the first member and the second member of the dimerization pair may be derived from a Bcl-2 protein. In some embodiments, the first member and the second member of the dimerization pair may be a truncated and / or mutant Bcl-2 protein. As a non-limiting example, the first member and the second member of the dimerization pair may be a truncated and / or mutant Bcl-2 protein lacking the transmembrane domain. As another non-limiting example, the first member and the second member of the dimerization pair may be a truncated and / or mutant Bcl-2 protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0234] A non-limiting example of an amino acid sequence of a truncated and / or mutant Bcl-2 protein is as follows (positions corresponding to the full-length Bcl-2 Y108 and G145 involved in venetoclax binding are bolded and underlined):MAHPGRTGYDNREIVMKYIHYKLSQRGYEWDAGDDVEENRTEAPEGTESEVVHLTLRAttorney Docket No: 252457.000033QAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGV MCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGP (SEQ ID NO: 8).

[0235] A non-limiting example of an amino acid sequence of a full-length Bcl-2 protein, identified by UniProt accession number Pl 0415, is as follows (positions Y108 and G145 involved in venetoclax binding are bolded and underlined):

[0236] MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSS QPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSP LVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALV GACITLGAYLGHK (SEQ ID NO: 103).

[0237] A non-limiting example of an amino acid sequence of a truncated Bcl-2 protein is as follows (positions corresponding to the full-length Bcl-2 Y 108 and G145 involved in venetoclax binding are bolded and underlined):MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPH PAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSS QLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIAL WMTEYLNRHLHTWIQDNGGWDAFVELYGPSMR (SEQ ID NO: 31).

[0238] A non-limiting example of an amino acid sequence of a truncated Bcl-2 protein is as follows (positions corresponding to the full-length Bcl-2 Y108 and G145 involved in venetoclax binding are bolded and underlined):PLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGR FATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHT WIQDNGGWDAFVELYGPSMR (SEQ ID NO: 106).

[0239] In some embodiments, a truncated Bcl-2 protein, or a mutant thereof, described herein may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 8, 31 or 106, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 8, 31 or 106.

[0240] In some embodiments, the first member and the second member of the dimerization pair may be a truncated and / or mutant Bcl-xL protein. As a non-limiting example, the first member andAttorney Docket No: 252457.000033 the second member of the dimerization pair may be a truncated and / or mutant Bcl-xL protein lacking the transmembrane domain. As another non-limiting example, the first member and the second member of the dimerization pair may be a truncated and / or mutant Bcl-xL protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0241] A non-limiting example of an amino acid sequence of a truncated and / or mutant Bcl- xL protein is as follows (positions corresponding to the full-length Bcl-xL Y101 and G138 are bolded and underlined):MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFEL RYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKE MQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERED (SEQ ID NO:74).

[0242] Another non-limiting example of an amino acid sequence of a truncated Bcl-xL protein is as follows (positions corresponding to the full-length Bcl-xL Y101 and G138 are bolded and underlined):HSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNEL FRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGG WDTFVELYGNNAAAESRKGQER (SEQ ID NO: 101).

[0243] A non-limiting example of an amino acid sequence of a full-length Bcl-xL protein (also known as Bcl-2-like protein 1), identified by UniProt accession number Q07817, is as follows (positions Y101 and G138 are bolded and underlined):MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHL ADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGT AYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLN DHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSR K (SEQ ID NO: 98).

[0244] Another non-limiting example of an amino acid sequence of a full-length Bcl-xL protein is as follows (positions Y101 and G138 are bolded and underlined):MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHL ADSPAVNGATAHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGT AYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNAttorney Docket No: 252457.000033DHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSR K (SEQ ID NO: 76).

[0245] In some embodiments, a truncated Bcl-xL protein, or a mutant thereof, described herein may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 101, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 74 or 101.Extracellular Region

[0246] The extracellular region of the first polypeptide may comprise an antigen-binding polypeptide, a receptor (e.g., TCR-based recognition domain), or a natural ligand for a target cell antigen or receptor. The extracellular region of the first polypeptide may comprise an antigenbinding polypeptide. Exemplary antigen-binding polypeptides include, but are not limited to, antibodies and antibody fragments. For example, the antigen-binding polypeptide may be a murine antibody, a rabbit antibody, a human antibody, a humanized antibody, a single chain variable fragment (scFv), a camelid antibody variable domain, a humanized version of a camelid antibody variable domain, a shark antibody variable domain (VNAR), a humanized version of a shark antibody variable domain, a single domain antibody variable domain, a nanobody (VHHs), and a camelized antibody variable domain.

[0247] The antigen recognized by the antigen-binding polypeptide may be a tumor cell associated antigen, an infection-associated antigen, or an auto-antigen. The tumor cell associated antigen may be associated with a solid tumor. In certain embodiments, the tumor associated antigen is PSMA. In certain embodiments, the tumor associated antigen is CD 19. In certain embodiments, the tumor associated antigen is EpCAM. In certain embodiments, the tumor associated antigen is EPhA2. In certain embodiments, the antigen recognized by the antigenbinding polypeptide may be tumor antigen N-glycolil (NGc) GM3 (NGcGM3).

[0248] In some embodiments, the antigen recognized by the antigen-binding polypeptide is selected from CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), PSA, CD44 surface adhesion molecule, mesothelin (anti-mesothelin antibodies as disclosed in Klampatsa, A. et al, Expert Opinion on Biological Therapy, 21(4), 473- 486; which is incorporated herein by reference in its entirety), carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), EGFRvIII, vascularAttorney Docket No: 252457.000033 endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2 (14g2a and the like ScFv antibodies as disclosed in Majzner, R.G. et al., Nature 603, 934-941 (2022) ; which is incorporated herein by reference in its entirety), carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CA125, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colonspecific antigen-p (CSAp), CSAp, EGP-I, EGP-2, EpCAM, EPhA2, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF- I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF-I), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1 , MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleophosmin, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, NeuGcGM3, N-glycolyl GM3 ganglioside, Neu5Gc, GM3 -Ganglioside, GD3, GM2, carbohydrate antigens, ganglioside antigens, Lewis Y, Lewis B, CD 123, and Kappa chain of immunoglobulin. In some embodiments, the mesothelin targeting polypeptide is selected from SSI, a murine-derived anti-mesotheling antibody or its variant thereof; LMB-100 / RG7787, a humanized antibody; amatuximab (MORAb-009); anetumab; HPN536; and the like as disclosed in Klampatsa, A. et al, Expert Opinion on Biological Therapy, 21(4), 473-486; which is incorporated herein by reference in its entirety.

[0249] In certain embodiments, the antigen recognized by the antigen-binding polypeptide may be PSMA. In some embodiments, the antigen-binding polypeptide may comprise a single chain variable fragment (scFv), wherein the scFv comprises an anti-PSMA scFv. Without wishing to be bound by theory, PSMA is a type II membrane protein originally characterized by the murine monoclonal antibody (mAb) 7E11-C5.3 and is expressed in all forms of prostate tissue, including carcinoma. PSMA helps fuel the development of prostate cancer cells. Indeed, prostate cancer cells have high levels of PSMA. In certain embodiments, the antigen recognized by the antigen-binding polypeptide is PSMA.

[0250] In certain embodiments, the antigen recognized by the antigen-binding polypeptide may be CD 19. In some embodiments, the antigen-binding polypeptide may comprise a single chainAttorney Docket No: 252457.000033 variable fragment (scFv), wherein the scFv comprises an anti-CD19 scFv. Without wishing to be bound by theory, the human CD 19 antigen is a 95 kD transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD 19 is classified as a type I transmembrane protein, with a single TM region, a cytoplasmic C-terminus, and extracellular N-terminus. CD 19 is a biomarker for normal and neoplastic B cells, as well as follicular dendritic cells. CD 19 is involved in establishing intrinsic B cell signaling thresholds through modulating both B cell receptordependent and independent signaling. CD 19 can function as a dominant signaling component of a multimolecular complex on the surface of mature B cells, alongside complement receptor CD21, and the tetraspanin membrane protein CD81 (TAPA-1), as well as CD225. Without wishing to be bound by theory, through study of CD 19 transgenic and knockout mouse models, CD 19 can play a role in maintaining the balance between humoral, antigen-induced response and tolerance induction.

[0251] Since CD 19 is a marker of B cells, CD 19 has been used to diagnose cancers that arise from B cells, notably B cell lymphomas, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Leukemia & Lymphoma, 1995, 18(5-6):385-397. The majority of B cell malignancies express normal to high levels of CD 19. The most current experimental antiCD 19 immunotoxins in development work by exploiting the widespread presence of CD 19 on B cells, with expression highly conserved in most neoplastic B cells, to direct treatment specifically towards B-cell cancers. Arthritis Res. & Ther., 2012, 14 Suppl. 5 (5):S1 and Nature Reviews Rheumatology, 2011, 7(3): 170-178. However, it is now emerging that the protein plays an active role in driving the growth of these cancers, most intriguingly by stabilizing the concentrations of the MYC oncoprotein. This suggests that CD19 and its downstream signaling may be a more attractive therapeutic target than initially suspected. Journ. Clin. Invest., 2012, 122(6):2257-66 and J. Immunol., 2012, 189(5):2318-25. The targeting of CD19, a cell surface molecule expressed in the vast majority of leukemias and lymphomas, has been successfully translated in the clinic. (Mol. Ther. 2017 May 3;25(5): 1117-1124. doi: 10.1016 / j.ymthe.2017.03.034. Epub 2017 Apr 26. Chimeric Antigen Receptors: A Cell and Gene Therapy Perspective. Riviere II, Sadelain M2.).

[0252] CD19-targeted therapies based on T cells that express CD19-specific chimeric antigen receptors (CARs) have been utilized for their antitumor abilities in patients with CD 19+ lymphoma and leukemia, first against Non-Hodgkin’s Lymphoma (NHL), then against CLL in 2011, and then against ALL in 2013. Leukemia & Lymphoma, 1995, 18(5-6):385-397; New England J. Med.,Attorney Docket No: 252457.0000332011, 365(8):725-33; Cell, 2017, 171(7): 1471; and Clinical Trial Number NCT01493453 at clinicaltrials.gov. Two CD-19-CAR T therapies have been approved: Gilead Sciences’ Yescarta (axicabtagene ciloleucel, KTE-C19) for third line or later (3L+) large B-cell lymphoma and Novartis’ Kymriah (tisagenlecleucel, CTL019) for acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). CAR-19 T cells are genetically modified T cells that express a targeting moiety on their surface that confers T cell receptor (TCR) specificity towards CD 19+ cells. CD 19 activates the TCR signaling cascade that leads to proliferation, cytokine production, and ultimately lysis of the target cells, which in this case are CD 19+ B cells. CAR- 19 T cells are more effective than anti-CD19 immunotoxins because they can proliferate and remain in the body for a longer period of time.

[0253] In certain embodiments, the antigen recognized by the antigen-binding polypeptide may be tumor antigen N-glycolyl (NGc) GM3 (NGcGM3). In some embodiments, the antigenbinding polypeptide may comprise a single chain variable fragment (scFv), wherein the scFv comprises an 14F7-derived scFv that targets NGcGM3. Additional information on 14F7 can be found in Bjerregaard- Andersen, K., Sci. Rep. 2018, 10836, incorporated by reference herein in its entirety. Exemplary scFv include, but are not limited to, those comprising: (i) a VH domain fused to a second VH domain, (ii) a VH domain fused to a linker, wherein the linker is fused to a second VH, (iii) a VH domain fused to a 7 AH domain, (iv) a VH domain fused to a linker, wherein the linker is fused to a 7AH domain, (v) a VH domain fused to a 7BH domain, (vi) a VH domain fused to a linker, wherein the linker is fused to a 7BH domain, (vii) a VH domain fused to an 8BH domain, (viii) a VH domain fused to a linker, wherein the linker is fused to an 8BH domain, (ix) a VH domain fused to a 2Am domain, (x) a VH domain fused to a linker, wherein the linker is fused to a 2Am domain, (xi) a VH domain fused to a 3Fm domain, and (xii) a VH domain fused to a linker, wherein the linker is fused to a 3Fm domain. The VH domain may be a murine domain. 2Am and 3Fm are murine domains; 7AH, 7BH, and 8BH are human domains. Exemplary 14F7- derived scFvs that target NGcGM3 are listed in SEQ ID NOs: 66, 68, or 70 (Int. Pat. Pub. No. WO 2021 / 123908 and U.S. Pat. App. Pub. No, US20230141511A1; which are incorporated herein by reference in their entireties).

[0254] In certain embodiments, the antigen recognized by the antigen-binding polypeptide may be EpCAM. In some embodiments, the antigen-binding polypeptide may comprise a single chain variable fragment (scFv), wherein the scFv comprises an anti-EpCAM scFv. WithoutAttorney Docket No: 252457.000033 wishing to be bound by theory, EpCAM is expressed on most normal epithelial cells and gastrointestinal carcinomas and functions as a homotypic calcium-independent cell adhesion molecule. EpCAM is being used as a target for immunotherapy treatment of human carcinomas. Mutations in the gene encoding EpCAM result in congenital tufting enteropathy. In certain embodiments, the antigen recognized by the antigen-binding polypeptide is EpCAM.

[0255] In certain embodiments, the antigen recognized by the antigen-binding polypeptide may be EPhA2. In some embodiments, the antigen-binding polypeptide may comprise a single chain variable fragment (scFv), wherein the scFv comprises an anti- EPhA2 scFv. Without wishing to be bound by theory, EPhA2 belongs to the ephrin receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-related receptors have been implicated in mediating developmental events, particularly in the nervous system. In certain embodiments, the antigen recognized by the antigen-binding polypeptide is EPhA2. In some embodiments, the EPhA2 recognizing polypeptide can be any EPhA2 binding molecule such as anti-EPhA2 scFv as disclosed in Lin Q et al, Front. Oncol. 11 : 694941 ; which is incorporated herein by reference in its entirety.

[0256] Various non-limiting exemplary antigen recognized by the antigen-binding polypeptide are also displayed in Tables 1-3.

[0257] In some embodiments, the antigen-binding polypeptide may comprise a VH sequence, a VL sequence, and / or CDRs thereof, such as those described in the cited publications, the contents of each publication are incorporated herein by reference in their entirety for all purposes (Table 1).Table 1. Exemplary antigen-binding polypeptides comprising a VH sequence, a VL sequence, and / or CDRs thereofAttorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033

[0258] In some embodiments, the antigen-binding polypeptide may comprise an scFv derived from an antibody or antibody fragment that binds to an antigen target such as those described in the cited publications, the contents of each publication are incorporated herein by reference in their entirety for all purposes (Table 2).Table 2. Exemplary antigen-binding polypeptides comprising an scFv derived from an antibody or antibody fragment that binds to an antigen targetAttorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033

[0259] In some embodiments, the antigen-binding polypeptide may comprise an antigen-binding polypeptide derived from a CAR that binds to an antigen target, such as those described in the cited publications, the contents of each publication are incorporated herein by reference in their entirety for all purposes (Table 3).Table 3. Exemplary the antigen-binding polypeptides comprising an antigen-binding polypeptide derived from a CAR that binds to an antigen targetAttorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033Attorney Docket No: 252457.000033

[0260] In some embodiments, the extracellular region may be a natural ligand for a target cell antigen or receptor. As a non-limiting example, the natural ligand for a target cell antigen or receptor may be an NKG2D ectodomain. In some embodiments, the NKG2D constructs are as disclosed in Sentman et al., Cancer J. 2014 ; 20(2): 156-159; which is incorporated herein by reference in its entirety.

[0261] In various embodiments, the extracellular region may be a T-cell receptor (TCR) based recognition domain. In some embodiments, the TCR based recognition domain may be a single chain TCR.

[0262] In certain embodiments, the extracellular region of the first polypeptide sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.

[0263] In certain embodiments, the extracellular region of the first polypeptide sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33.Attorney Docket No: 252457.000033

[0264] In certain embodiments, the extracellular region of the first polypeptide sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35.

[0265] In certain embodiments, the extracellular region of the first polypeptide sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: SEQ ID NO: 37.

[0266] In various embodiments of any of the CARs of the present disclosure, the second polypeptide chain may not comprise a target-binding capacity. In some embodiments, the extracellular region which does not comprise the target-binding capacity may comprise a stabilizing domain. In some embodiments, the extracellular region which does not comprise the target-binding capacity is derived from DAP 10. Examples of extracellular regions derived from DAP10 include, but are not limited to, the DAP10 ectodomain, and the TM region. The DAP10 extracellular region derived from the DAP 12 ectodomain may comprise the sequence QTTPGERSSLPAFYPGTSGSCSGCGSLSLP (SEQ ID NO: 29). In some embodiments, the extracellular region which does not comprise the target-binding capacity is derived from DAP12. Examples of extracellular regions derived from DAP12 include, but are not limited to, the DAP12 ectodomain, and the TM region. The DAP12 extracellular region derived from the DAP12 ectodomain may comprise the sequence GVLAGIVMGDLVLTVLIALAV (SEQ ID NO: 71). The DAP 12 extracellular region derived from the DAP 12 TM region may comprise the amino acid sequence of LRPVQAQAQSDCSCSTVSP (SEQ ID NO: 72).

[0267] In certain embodiments, the extracellular region which does not comprise the targetbinding capacity further comprises a tag sequence selected from an Myc (cMyc) tag, a His tag, a FLAG tag, a Biotin tag, an Fc-tag, a hemagglutinin (HA) tag, and / or a combination thereof.

[0268] In some embodiments, the extracellular region of the first polypeptide chain of the CARs disclosed herein comprises an antigen-binding polypeptide comprising a single chain variable fragment (scFv). By way of a non-limiting example, the scFv may comprise any of various scFv sequences, or variants or derivatives thereof, including, e.g., those described in Int. Pat. Pub. Nos. WO2018231759 and WO2020223279; U.S. Pat. App. Pub. Nos. US20200101142A1,Attorney Docket No: 252457.000033US20230074330A1, and US20230075314A1, the disclosure of each of which is incorporated herein by reference in its entirety for all purposes.

[0269] In various embodiments, the scFv comprises an anti-PSMA scFv. An exemplary anti- PSMA scFv sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In various embodiments, the nucleic acid molecule encoding the anti-PSMA scFv sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,or 100% identical to SEQ ID NO: 3.

[0270] In various embodiments, the scFv comprises an anti-CD19 scFv. An exemplary antiCD 19 scFv sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33. In various embodiments, the nucleic acid molecule encoding the anti-CD19 scFv sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32 or SEQ ID NO: 64.

[0271] In various embodiments, the scFv comprises an anti-EpCAM scFv. An exemplary anti- EpCAM scFv sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35. In various embodiments, the nucleic acid molecule encoding the anti-EpCAM scFv sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34.

[0272] In various embodiments, the scFv comprises an anti-EPhA2 scFv. An exemplary anti- EPhA2 scFv sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37. In various embodiments, the nucleic acid molecule encoding the anti-EPhA2 scFv sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36.Attorney Docket No: 252457.000033Dimerization Pair Sequences

[0273] The present disclosure provides CAR molecules comprising a first and a second member of a dimerization pair, wherein said first and second member of the dimerization pair is derived from anti-apoptotic proteins such as, e.g., Bcl-2 family proteins and mutants thereof. Nonlimiting examples of Bcl-2 family proteins that can be used as the first and / or second member of the dimerization pair for constructing a CAR molecule of the present disclosure are BCL2 (Bcl- 2), BCL-XL (Bcl-xL; BCL2L1), BCL-W (Bcl-W; BCL2L2), BCL-B (BCL2L10), MCL-1, and mutants thereof. Such first and second member of the dimerization pair present on the first and second polypeptide chains of the CAR molecule of the present disclosure bind to an activator of the present disclosure such as, e.g., a BH3 mimetic drug (e.g., venetoclax), to achieve dimerization.

[0274] In some embodiments, the first and / or second member of the dimerization pair comprises a sequence(s) derived from a Bcl-2 family protein(s), the sequence(s) comprising a hydrophilic region of a Bcl-2 family protein(s). Said sequence(s) can comprise a hydrophilic region of a Bcl-2 family protein(s) which is capable of binding to the BH3 mimetics as described herein for activating CAR-mediated signalling.

[0275] In some embodiments, the first and second member of the dimerization pair are located extracellularly. When the dimerization pairs are located extracellularly, the first member of the dimerization pair may be located C-terminal to the target-binding region in the extracellular region.

[0276] In some embodiments, the first and second member of the dimerization pair are located intracellularly. When the dimerization pairs are located intracellularly, the first and second member of the dimerization pair may be located N-terminal to each of the co-stimulatory endodomains (EDs) in respective polypeptide chains. When the dimerization pairs are located intracellularly, the first and second member of the dimerization pair may be located C-terminal to each of the co-stimulatory endodomains (EDs) in respective polypeptide chains.

[0277] In some embodiments, the first and / or second member of the dimerization pair are as described in U.S. Pat. App. Pub. No. US 20210113615; incorporated herein by reference in its entirety.

[0278] In some embodiments, the first and / or second member of the dimerization pair of the CAR may comprise an amino acid sequence of a truncated and / or mutant Bcl-2 protein. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking the transmembrane domain. In some embodiments, the first and / orAttorney Docket No: 252457.000033 second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices. In some embodiments, the first and / or second member of the dimerization pair of the CAR may comprise a truncated and / or mutant Bcl-2 sequence comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence SEQ ID NO: 8. In embodiments, the truncated and / or mutant Bcl-2 sequence comprises SEQ ID NO: 8 or a sequence having an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8. In some embodiments, the nucleic acid molecule encoding the truncated and / or mutant Bcl-2 sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence SEQ ID NO: 7. In some embodiments, the first and / or second member of the dimerization pair of the CAR may comprise a truncated and / or mutant Bcl-2 sequence comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 106.

[0279] In some embodiments, to form a truncated Bcl-2 protein, or a mutant thereof, disclosed herein, one or more portions of the amino acid sequence of the truncated Bcl-2 protein, or a mutant thereof, is eliminated (e.g., removed) as compared to the amino acid sequence of the full-length Bcl-2 protein. For example, to form a truncated Bcl-2 protein, or a mutant thereof, disclosed herein, one or more of the following amino acid sequences may be removed from the amino acid sequence of the full-length Bcl-2 protein: (i) a signal peptide sequence, or a portion thereof; (ii) a loop domain such as a flexible loop domain (FLD), or a portion thereof; (ii) a domain comprising, consisting of, or consisting essentially of a domain which can extend from the transmembrane domain to the outer mitochondrial membrane, or a portion thereof.

[0280] A non-limiting example of an amino acid sequence of a signal peptide of a full-length Bcl-2 protein is the sequence MAHAGRTGYDNREIVMKYIHYKLSQRGYEW (SEQ ID NO: 104). In some embodiments, a signal peptide described herein may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 104, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 104. In some embodiments, the signal peptideAttorney Docket No: 252457.000033 comprises the sequence of SEQ ID NO: 104, or an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 104.

[0281] A non-limiting example of an amino acid sequence of a loop domain such as a flexible loop domain (FLD) of a full-length Bcl-2 protein is the sequence DAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTS (SEQ ID NO: 105). In some embodiments, a loop domain described herein may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 105, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 105. In some embodiments, the loop domain comprises the sequence of SEQ ID NO: 105, or an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 105.

[0282] A non-limiting example of an amino acid sequence of a domain which can extend from the transmembrane domain to the outer mitochondrial membrane of a full-length Bcl-2 protein is the sequence PLFDFSWLSLKTLLSLALVGACITLGAYLGHK (SEQ ID NO: 107). In some embodiments, a domain which can extend from the transmembrane domain to the outer mitochondrial membrane may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 107, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 107. In some embodiments, the domain which can extend from the transmembrane domain to the outer mitochondrial membrane comprises the sequence of SEQ ID NO: 107, or an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 107.

[0283] In some embodiments, elimination of the transmembrane portion in the mitochondrial membrane of the truncated Bcl-2 sequence improves formation of a dimer between the first and the second member of the dimerization pair, for example, in the presence of any activator disclosed herein.

[0284] In some embodiments, the first and the second member of the dimerization pair of the CAR may be derived from the same a Bcl-2 protein. In some embodiments, the first and the second member of the dimerization pair of the CAR may be derived from a different Bcl-2 protein.

[0285] In some embodiments, the first and / or the second member of the dimerization pair comprises a Bcl-2 sequence derived from PDB ID 41vt (SEQ ID NO: 88).Attorney Docket No: 252457.000033

[0286] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31.

[0287] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 103 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 103.

[0288] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.

[0289] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 106 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 106.

[0290] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 or 103 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 106.Attorney Docket No: 252457.000033

[0291] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 106 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 or 103.

[0292] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88.

[0293] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 106 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88.

[0294] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 or 103 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88.

[0295] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or 106.Attorney Docket No: 252457.000033

[0296] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 or 103.

[0297] In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking the transmembrane domain. In some embodiments, the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

[0298] In some embodiments, to form a truncated Bcl-xL protein, or a mutant thereof, disclosed herein, one or more portions of the amino acid sequence of the truncated Bcl-xL protein, or a mutant thereof, is eliminated (e.g., removed) as compared to the amino acid sequence of the full-length Bcl-xL protein. For example, to form a truncated Bcl-xL protein, or a mutant thereof, disclosed herein, one or more of the following amino acid sequences may be removed from the amino acid sequence of the full-length Bcl-xL protein: (i) a signal peptide sequence, or a portion thereof; (ii) a loop domain such as a flexible loop domain (FLD), or a portion thereof; (ii) a domain comprising, consisting of, or consisting essentially of a domain which can extend from the transmembrane domain to the outer mitochondrial membrane, or a portion thereof. Any combination of such amino acid sequences may be eliminated (e.g., removed) from the full-length Bcl-xL protein sequence to form a suitable truncated Bcl-xL protein, or a mutant thereof, for use in the present disclosure. In certain embodiments, each of the signal peptide sequence, or portion thereof, the loop domain, or portion thereof, and the domain comprising, consisting of, or consisting essentially of the domain which can extend from the transmembrane domain to the outer mitochondrial membrane, or a portion thereof, have been eliminated (e.g., removed) from the full- length Bcl-xL protein to form a truncated Bcl-xL protein, or a mutant thereof, disclosed herein.

[0299] A non-limiting example of an amino acid sequence of a signal peptide of a full-length Bcl-xL protein is the sequence MSQSNRELVVDFLSYKLSQKGYSWSQFSDV (SEQ ID NO: 99). In some embodiments, a signal peptide described herein may comprise, consist of, or consistAttorney Docket No: 252457.000033 essentially of the sequence of SEQ ID NO: 99, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 99. In some embodiments, the signal peptide comprises the sequence of SEQ ID NO: 99, or an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 99.

[0300] A non-limiting example of an amino acid sequence of a loop domain such as a flexible loop domain (FLD) of a full-length Bcl-xL protein is the sequence EENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATG (SEQ ID NO: 100). In some embodiments, a loop domain described herein may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 100, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 100. In some embodiments, the loop domain comprises the sequence of SEQ ID NO: 100, or an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 100.

[0301] A non-limiting example of an amino acid sequence of a domain which can extend from the transmembrane domain to the outer mitochondrial membrane of a full-length Bcl-xL protein is the sequence FNRWFLTGMTVAGVVLLGSLFSRK (SEQ ID NO: 102). In some embodiments, a domain which can extend from the transmembrane domain to the outer mitochondrial membrane may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 102, or an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 102. In some embodiments, the domain which can extend from the transmembrane domain to the outer mitochondrial membrane comprises the sequence of SEQ ID NO: 102, or an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 102.

[0302] In some embodiments, elimination of the transmembrane portion in the mitochondrial membrane of the truncated Bcl-xL sequence improves formation of a dimer between the first and the second member of the dimerization pair, for example, in the presence of any activator disclosed herein.

[0303] In some embodiments, the first and the second member of the dimerization pair of the CAR may be derived from the same a Bcl-xL protein. In some embodiments, the first and theAttorney Docket No: 252457.000033 second member of the dimerization pair of the CAR may be derived from a different Bcl-xL protein.

[0304] In some embodiments, the first and / or the second member of the dimerization pair comprise a Bcl-xL sequence derived from PDB ID 4qvf (SEQ ID NO: 89).

[0305] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76.

[0306] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 98 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 98.

[0307] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74.

[0308] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101.

[0309] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76 or 98 and the secondAttorney Docket No: 252457.000033 member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74 or 101.

[0310] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74 or 101 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76 or 98.

[0311] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76 or 98 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89.

[0312] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74 or 101 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89.

[0313] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74 or 101.

[0314] In some embodiments, the first member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89 and the second member of the dimerization pair of the CAR comprises the sequence at least 81%, 82%, 83%, 84%, 85%,Attorney Docket No: 252457.00003386%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76 or 98.Linker Region

[0315] In various aspects of the CARs of the present disclosure, the first polypeptide chain may comprise a first linker region that may be interposed, for example, between the first member of a dimerization pair and the first TM region. In various aspects of the CARs of the present disclosure, the second polypeptide chain may comprise a second linker region that may be interposed, for example, between the second member of the dimerization pair and the second TM region. In some embodiments, the first polypeptide chain may comprise a first linker region interposed between the extracellular region and the first TM region. In some embodiments, the second polypeptide chain may comprise a second linker region N-terminal to the second TM region

[0316] Without wishing to be bound by theory, a linker region may be used to provide more flexibility and accessibility for the antigen-binding moiety. A linker region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. A linker region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively, the linker region may be a synthetic sequence that corresponds to a naturally occurring linker region sequence or may be an entirely synthetic linker domain sequence. Nonlimiting examples of linker region which may be used in accordance with the disclosure include a part of human CD8a (CD8a) chain, partial extracellular domain of CD28, FcyRIIIa receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof. In certain embodiments, additional linking amino acids are added to the linker region to ensure that the antigen-binding moiety is an optimal distance from the TM region. In certain embodiments, when the linker is derived from an Ig, the linker may be mutated to prevent Fc receptor binding.

[0317] In various embodiments, the linker region may comprise a Serine / Glycine (Ser / Gly) Linker comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6.Attorney Docket No: 252457.000033

[0318] In various embodiments, the linker region may comprise a Furin + GS Linker comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16.

[0319] In some embodiments, any of the CARs of the present disclosure may comprise a first and / or a second linker which may be a hinge derived from, for example, CD8, CD8a, or CD28. In some embodiments, the first and / or second linker may be a hinge region derived from CD 8. In some embodiments, the first and / or second linker may be a hinge region derived from CD 8 a. In some embodiments, the first and / or second linker may be a hinge region derived from CD28.

[0320] In some embodiments, the linker region may comprise a hinge derived from CD8-alpha (CD8a). Cluster of differentiation 8-alpha (CD8-alpha) is a co-receptor for the T-cell receptor (TCR). In T cells, CD8-alpha primarily functions as a co-receptor for MHC class I molecule peptide complex. CD8-alpha can interact with TCR and the MHC class I proteins to modulate signaling mechanisms that enable cytotoxic T-lymphocytes.

[0321] In various embodiments, the linker region may comprise a CD8a hinge comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

[0322] In certain embodiments, the linker region comprises an immunoglobulin IgG hinge or functional fragment thereof. In certain embodiments, the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the linker region comprises the CHI, CH2, CH3 and / or hinge region of the immunoglobulin. In certain embodiments, the linker region comprises the core hinge region of the immunoglobulin. The term “core hinge” can be used interchangeably with the term “short hinge” (also known as “SH”). Nonlimiting examples of suitable linker region are the core immunoglobulin hinge regions listed in Table 4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes). In certain embodiments, the linker region is a fragment of the immunoglobulin hinge. Non-limiting examples of exemplary peptide sequences of core hinge regions of IgG immunoglobulins are displayed in Table 4.Attorney Docket No: 252457.000033Table 4. Peptide sequences of Core Hinge Regions of IgG Immunoglobulins

[0323] In certain embodiments, the linker region comprises an IgGl hinge, or a variant thereof. In certain embodiments, the linker region comprises the core hinge structure of IgGl or a variant thereof. In certain embodiments, the linker region comprises an IgG2 hinge, or a variant thereof. In certain embodiments, the linker region comprises the core hinge structure of IgG2 or a variant thereof.Transmembrane Region

[0324] In certain embodiments, the transmembrane (TM) region is fused in frame between the extracellular region and the cytoplasmic domain. The TM region may be derived from the protein contributing to the extracellular region, the protein contributing the signaling or co-signaling domain, or by a totally different protein. In some instances, the TM region can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In some instances, the TM region can be selected or modified by amino acid substitution, deletions, or insertions to avoid-binding of proteins naturally associated with the TM region. In certain embodiments, the TM region includes additional amino acids to allow for flexibility and / or optimal distance between the domains connected to the TM region.

[0325] The TM region may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of TM regions of particular use in this disclosure may be derived from (z.e. comprise at least the TM region(s) of) the alpha (a), beta (P), or zeta (Q chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD 134, CD 137, CD 154. Alternatively, the TM region may be synthetic, in which case it may comprise predominantly hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan and / or valine can be found at each end of a synthetic TM region.

[0326] In certain embodiments, it is desirable to utilize the TM region of the C, eta (q), or FcsRly chains which contain a cysteine residue capable of disulfide bonding, so that the resultingAttorney Docket No: 252457.000033 chimeric protein able to form disulfide linked dimers with itself, or with unmodified versions of the ' , q, or FcsRly chains or related proteins. In some instances, the TM region is selected or modified by amino acid substitution to avoid-binding of such domains to the TM regions of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it is desirable to employ the TM region of C,, p or FcsRly and - P, MB1 (Iga.), B29 or CD3- y, ' , or ], in order to retain physical association with other members of the receptor complex.

[0327] Without limitation, the first and / or second transmembrane (TM) region may be derived from, for example, CD8, CD8a, CD4, CD3-zeta, CD3-epsilon, CD28, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (OX-40), CD137, CD154, DAP10, or DAP12. The first and second transmembrane (TM) regions of the first and second polypeptide may be the same. The first and second transmembrane (TM) regions of the first and second polypeptide may be different. In some embodiments, the first and second transmembrane (TM) regions are derived from CD8a. In some embodiments, the first and second transmembrane (TM) regions are derived from CD28.

[0328] In various embodiments, the TM region may comprise a CD8a-derived TM region comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 12.Cytoplasmic Domain

[0329] In certain embodiments, the cytoplasmic domain may comprise one or more of a costimulatory endodomain (ED). In certain embodiments, the cytoplasmic domain may comprise one or more of an intracellular signaling endodomain (ED). In certain embodiments, any number of various co-stimulatory EDs may be present. In certain embodiments, a co-stimulatory ED may be absent. In certain embodiments, any number of various intracellular signaling EDs may be present.

[0330] In some embodiments of any of the CARs of the disclosure, the first polypeptide chain may comprise one or more of a first co-stimulatory ED. In some embodiments, the co-stimulatory ED may be absent from the first polypeptide such that the first polypeptide does not comprise a first co-stimulatory ED.Attorney Docket No: 252457.000033

[0331] In some embodiments of any of the CARs of the disclosure, the second polypeptide chain may comprise one or more of a second co-stimulatory ED. In some embodiments, the costimulatory ED may be absent from the second polypeptide such that the second polypeptide does not comprise a second co-stimulatory ED.

[0332] In some embodiments, the first co-stimulatory ED and the second co-stimulatory ED may be derived from the same co-stimulatory ED. As a non-limiting example, when the first co- stimulatory ED and the second co-stimulatory ED are derived from the same co-stimulatory ED, the co-stimulatory domain may be derived from 4-1BB (CD137). In some embodiments, the first co-stimulatory ED and the second co-stimulatory ED may be derived from different co-stimulatory domains. As a non-limiting example, when the first co-stimulatory ED and the second co- stimulatory ED are derived from different co-stimulatory EDs, the first co-stimulatory domain may be derived from 4- IBB (CD 137) and the second co-stimulatory ED may be derived from CD28. In some embodiments, the first and second co-stimulatory EDs of the first and second polypeptide may be the same. In some embodiments, the first and second co-stimulatory EDs of the first and second polypeptide may be different.

[0333] In some embodiments, the intracellular signaling ED may be absent from the first polypeptide such that the first polypeptide does not comprise an intracellular signaling ED. In some embodiments of any of the CARs of the disclosure, the second polypeptide chain may comprise one or more intracellular signaling EDs.

[0334] In certain embodiments, the one or more intracellular signaling ED domains and one or more co-stimulatory EDs may be placed in any order in the CAR. The cytoplasmic domain, which may comprise the intracellular signaling ED domain of the CAR of the disclosure, may be responsible for activation of at least one of various normal effector functions of the lymphocyte in which the CAR has been placed. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity, including, without limitation, the secretion of cytokines. Thus, the term “intracellular signaling ED domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling ED domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The termAttorney Docket No: 252457.000033 intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling ED or variant thereof sufficient to transduce the effector function signal.

[0335] Non-limiting examples of intracellular signaling EDs which may be used in the CARs of the disclosure include, e.g., intracellular signaling EDs derived from DAP10, DAP12, Fc epsilon receptor I gamma chain (FCER1G), FcR 0, CD38, CD3s, CD3y, CD3^ CD5, CD22, CD226, CD66d, CD79A, and CD79B. In some embodiments, the intracellular signaling ED of any of the CARs disclosed herein may comprise a signaling domain derived from CD3^ (CD3-zeta).

[0336] In certain embodiments, the intracellular signaling ED may be designed to comprise the signaling domain of CD3^. It is known that signals generated through the T cell receptor (TCR) alone may be insufficient for full activation of lymphocytes and that a secondary or co-stimulatory signal may also be required for such full activation. Thus, intracellular signaling ED can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling ED sequences, or intracellular signaling ED sequences, as discussed above) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0337] In various embodiments, the intracellular signaling ED may comprise a signaling ED that is derived from CD3^ (CD3 zeta) comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.

[0338] In certain embodiments, the CAR of the disclosure may comprise one or more co- stimulatory EDs. Non-limiting co-stimulatory EDs include, but are not limited to, 4-1BB (CD137), CD28, ICOS, CD 134 (OX-40), BTLA, CD27, CD30, GITR, CD226, and HVEM. In some embodiments, the one or more co-stimulatory EDs may be derived from 4- IBB.

[0339] In some embodiments, the CAR of the disclosure may comprise a first polypeptide chain comprising one or more of a first co-stimulatory ED. In some embodiments, the CAR of the disclosure may comprise a second polypeptide chain comprising one or more of a second co- stimulatory ED.

[0340] In various embodiments, the first and / or second co-stimulatory ED may comprise a co- stimulatory ED of 4-1BB (CD137) comprising, consisting of, or consisting essentially of an aminoAttorney Docket No: 252457.000033 acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.The first and / or second co-stimulatory endodomain (ED) of any of the CARs disclosed herein may be derived from 4-1BB (CD137), CD28, ICOS, CD134 (OX-40), BTLA, CD27, CD30, GITR, CD226, or HVEM. In some embodiments, the first co-stimulatory endodomains (ED) is derived from 4- IBB (CD 137). In some embodiments, the second co-stimulatory ED is derived from 4- 1BB (CD 137). In some embodiments, the first and / or second co-stimulatory EDs are derived from 4-1BB (CD137). In certain embodiments, the co-stimulatory ED derived from 4-1BB (CD137) may comprise SEQ ID NO: 14.Degron Sequences

[0341] In various aspects of the CARs of the present disclosure, the first polypeptide chain and / or second polypeptide chain may comprise a degron sequence.

[0342] In some embodiments, the degron sequence is located intracellularly. In some embodiments, the degron sequence is present on either of the first polypeptide chain and second polypeptide chain intracellularly. In some embodiments, the degron sequence is present on each of the first polypeptide chain and second polypeptide chain intracellularly. In some embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and second polypeptide chain. In some embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and between the co-stimulatory ED and the intracellular signaling ED in the second polypeptide chain. In some embodiments, the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and C- terminal to the intracellular signaling ED in the second polypeptide chain.

[0343] In some embodiments, the degron sequences present on each polypeptide chain of the CAR molecule are the same. In some embodiments, degron sequences present on each polypeptide chain of the CAR molecule are not the same. The degron sequence(s) present on the CAR molecule enhances sensitivity to ligand-induced degradation of the CAR molecule. For example, engineered zinc finger domains can be used as degron sequences to mediate degradation of the CAR molecules in the presence of the ligand. In some embodiments, the ligand mediates proteasomal degradation of the CAR by inducing interactions between the CRL4CRBNE3 ubiquitin ligase and a C2H2 (Cys2- His2) zinc finger degron motif (Sci Transl Med., 2021 January 06; 13(575), incorporated herein by reference in its entirety). In some embodiments, the degron sequence is an E3 ubiquitin ligaseAttorney Docket No: 252457.000033 dependent degron sequence. The degron sequence is able to bind to ubiquitin ligase. The addition of ubiquitin to a substrate protein (in this case, a chimeric antigen receptor (CAR)) is called ubiquitination and thereby marks the substrate protein for degradation. The degron sequences of the present disclosure may comprise degron sequences as disclosed in Jan et al., Sci Transl Med., 2021 January 06; 13(575) e.g., Figure 3D); and Sievers et al., 2018, Science 362, eaat0572 (e.g., Figure 3A), incorporated herein by reference in their entireties. In some embodiments, the degron sequence is derived from a transcription factor Aiolos (IKZF3). In some embodiments, the degron sequences comprises zinc figure motif or a fragment thereof. The zing finger motif may be a C2H2 zinc finger motif. In some embodiments, the degron sequences are composed from the matrix of 5 N-termini (ZN653, ZN827, ZFP91, ZN276, and / or IKZF3) with 7 C-termini (ZN787, ZN517, IKZF3, ZN654, PATZ1, E4F1, and / or ZKSC5). In some embodiments, the degron sequence comprises an amino acid sequence from IKZF3 or a fragment thereof. In some embodiments, the degron sequence comprises 136-180 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises 130-189 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises 236-249 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises 136-180 and 236-249 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises an amino acid sequence from ZFP91 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZN827 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZN653 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZN276 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZN787 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from PATZ1 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZN517 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from E4F1 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZN654 or a fragment thereof. In some embodiments, the degron sequence comprises an amino acid sequence from ZKSC5 or a fragment thereof. In some embodiments, the degron sequence comprises fragments of ZFP91 and IKZF3 (ZFP91-IKZF3). In some embodiments, the degron sequence comprises 146-156 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises 400-410 amino acid residues of ZFP91. InAttorney Docket No: 252457.000033 some embodiments, the degron sequence comprises 400-410 amino acid residues of ZFP91 and 146-156 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises 400-410 amino acid residues of ZFP91 and 157-168 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises 157-168 amino acid residues of IKZF3. In some embodiments, the degron sequence comprises fragments of IKZF3 and ZN787 (IKZF3-ZN787). In some embodiments, the degron sequence comprises fragments of ZFP91 and ZN787 (ZFP91- ZN787). In some embodiments, the degron sequence comprises fragments of ZN276 and ZN787 (ZN276-ZN787). In some embodiments, the degron sequence comprises ZN653-PATZ1. In some embodiments, the degron sequence comprises fragments of ZN827 and ZKSC5 (ZN827-ZKSC5).

[0344] In some embodiments, the degron sequence is selected from auxin inducible degron, B-LID degron, C-terminal phosphodegron, DD-degron, It-degron, LID-degron, PSD degron, TIPI degron, Ts-degron, Ura3-d degron, Ubi-R degron, Ubi-M degron, Ubi-Q degron, Ubi-E degron, and ZF1 degron.

[0345] In some embodiments, the degron sequence is ZFP91-IKZF3 (Jan et al, Sci Transl Med. 2021 Jan 6; 13(575): eabb6295, which is incorporated herein by reference in its entirety). In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 39. In some embodiments, the nucleic acid molecule encoding the degron sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 38.

[0346] In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 81. In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 82. In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQAttorney Docket No: 252457.000033ID NO: 83. In some embodiments, the nucleic acid molecule encoding the degron sequence comprises, consists of, or consists essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 90.

[0347] In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 84. In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 85. In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 86. In some embodiments, the degron sequence comprises, consists of, or consists essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence SEQ ID NO: 87.

[0348] In some embodiments, the first and / or second polypeptide chain of the CAR may further comprise one or more additional polypeptide sequences encoded by any of accessory genes. Exemplary additional polypeptide sequences include, but are not limited to, additional costimulatory endodomains (EDs), signal sequences (or leader sequences), separation sequences, epitope tags, and polypeptides that produce a detectable signal.Accessory Genes

[0349] In addition to the CAR construct, the CAR may further comprise one or more accessory gene that encodes one or more additional polypeptide. Examples of accessory genes can include a transduced host cell selection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene.

[0350] In certain embodiments, the accessory gene may increase the safety of the CAR. In certain embodiments, the CAR comprises at least one accessory gene. In certain embodiments, theAttorney Docket No: 252457.000033CAR comprises one accessory gene. In other embodiments, the CAR comprises two accessory genes. In yet another embodiment, the CAR comprises three accessory genes.

[0351] In some embodiments, the accessory gene may increase the effector function of any number of various host cells comprising a CAR of the present disclosure. Non-limiting examples of classes of accessory genes that can be used to increase the effector function of CAR containing host cells, include i) secretable cytokines (e.g., but not limited to, IL-7, IL-12, IL-15, IL-18), ii) membrane bound cytokines (e.g., but not limited to, IL-15), iii) chimeric cytokine receptors (e.g., but not limited to, IL-2 / IL-7, IL-4 / IL-7), iv) constitutive active cytokine receptors (e.g., but not limited to, C7R), v) dominant negative receptors (DNR; e.g., but not limited to TGFRII DNR), vi) ligands of costimulatory molecules (e.g., but not limited to, CD80, 4-1 BBL), vii) antibodies, including fragments thereof and bispecific antibodies (e.g., but not limited to, bispecific T-cell engagers (BiTEs)), or vii) a second CAR.

[0352] In certain embodiments, the accessory gene may be a suicide gene. A suicide gene is a recombinant gene that may cause the host cell that the gene is expressed in to undergo programmed cell death or antibody mediated clearance at a desired time. Suicide genes can function to increase the safety of the CAR. In another embodiment, the accessory gene is an inducible suicide gene. Non-limiting examples of suicide genes include i) molecules that are expressed on the cell surface and can be targeted with a clinical grade monoclonal antibody including CD20, EGFR or a fragment thereof, HER2 or a fragment thereof, and ii) inducible suicide genes (e.g., but not limited to inducible caspase 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; US Publ. No. 2011 / 0286980, each of which are incorporated herein by reference in their entirety for all purposes)).

[0353] In some embodiments, the one or more accessory genes may encode one or more additional polypeptide sequences of the present disclosure. In various embodiments, the first and / or second polypeptides chains of any of the CARs disclosed herein may comprise one or more additional polypeptide sequences encoded by the one or more accessory genes. Exemplary additional polypeptide sequences include, but are not limited to, additional co-stimulatory endodomains (EDs), signal sequences (or leader sequences), separation sequences, epitope tags, and polypeptides that produce a detectable signal.

[0354] In some embodiments, the signal sequence may be derived from CD 8 a. In certain embodiments, the signal sequence may comprise a signal sequence derived from CD8aAttorney Docket No: 252457.000033 comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0355] In some embodiments, the epitope tag may be c-Myc. In certain embodiments, the epitope tag may comprise an epitope tag of c-Myc comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20.

[0356] A “separation sequence” as used herein refers to a peptide sequence that causes a ribosome to release the growing polypeptide chain that is being synthesized without dissociation from the mRNA. In this respect, the ribosome continues translating and therefore produces a second polypeptide. Non-limiting examples of separation sequences includes a T2A separation sequence.

[0357] In some embodiments, the separation sequence may be T2A. In certain embodiments, the separation sequence may comprise a separation sequence of T2A comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.

[0358] In some embodiments, the separation sequence may comprise Furin+T2A. In certain embodiments, the separation sequence may comprise a separation sequence of Furin+T2A comprising, consisting of, or consisting essentially of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 63.

[0359] In some embodiments, the separation sequence is a naturally occurring or synthetic sequence. In certain embodiments, the separation sequence includes the 2A consensus sequence D-X-E-X-NPGP (SEQ ID NO: 96), in which X is any amino acid residue such as but not limited to any natural amino acid.Polypeptide Chains

[0360] In one aspect, provided herein is a first polypeptide chain of any switchable chimeric antigen receptor (CAR) described herein.Attorney Docket No: 252457.000033

[0361] In another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first member of a dimerization pair; iii) a first linker region; iv) a first transmembrane (TM) region; v) a first co-stimulatory endodomain (ED); and vi) optionally, a degron sequence and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) a second member of the dimerization pair; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) a degron sequence; vi) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a targetbinding capacity, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

[0362] In yet another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first co-stimulatory endodomain (ED); v) a first member of a dimerization pair; and vi) optionally, a degron sequence, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a targetbinding capacity; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) a second member of the dimerization pair; vi) a degron sequence; and vii) an intracellular signaling ED, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0363] In still yet another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first member of a dimerization pair; and v) optionally, a first co-stimulatory endodomain (ED); and vi) optionally, a degron sequence, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second transmembrane (TM) region; iv) a second member of the dimerization pair; v) a second co-stimulatory ED; vi) a degron sequence; and vii) an intracellular signaling ED, wherein the first and second member ofAttorney Docket No: 252457.000033 the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR- mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0364] In another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first member of a dimerization pair; iii) a first linker region; iv) a first transmembrane (TM) region; v) a first co-stimulatory endodomain (ED); and vi) optionally, a degron sequence and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) a second member of the dimerization pair; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) an intracellular signaling ED; and vi) a degron sequence;, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a targetbinding capacity, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

[0365] In yet another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first co-stimulatory endodomain (ED); v) a first member of a dimerization pair; and vi) optionally, a degron sequence, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a targetbinding capacity; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) a second member of the dimerization pair; vi) an intracellular signaling ED; and vii) a degron sequence;, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0366] In still yet another aspect, provided herein is a switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first member of a dimerization pair; and v) optionally, a first co-stimulatory endodomain (ED); and vi) optionally, a degron sequence, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second transmembraneAttorney Docket No: 252457.000033(TM) region; iv) a second member of the dimerization pair; v) a second co-stimulatory ED; vi) an intracellular signaling ED; and vii) a degron sequence;, wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR- mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

[0367] Amino acid and Nucleotide sequences of exemplary first polypeptide chains, second polypeptide chains, and CAR constructs are presented with annotations in the Section -‘List of Sequences”. In the sequences, CD8 leader is indicated with wavy underline (e.g., CD8JLeader), scFv is indicated with bold letters (e.g., scFv), GS linker is indicated with italic letters (e.g., GS Linker), truncated Bcl2 mutant is indicated with underline, (e.g., tBc!2), CD8 Hinge region is indicated with bold and italic letters (e.g., CD8 Hinge), CD8 TM is indicated with double underline (e.g., CD8 TM), endodomain is indicated with bold letters and wavy underline (e.g., 41BB Endodomain), cMyc tag is indicated with bold letters and double underline (e.g., cMvc tag). CD3 zeta is indicated with italic letters and double underline (e.g., CDS eta). Furin+T2A separation sequence is indicated with bold and italic letters and with underline. (e.g., Furin+T2A) and degron sequence is indicated with bold letters and with underline (e.g., Degron). Restriction sites present in the amino acid and nucleotide sequences of the first polypeptide chains, second polypeptide chains, and / or CAR constructs are indicated with dot-dash underline (e.g., Gctagc). In the scFv sequences, heavy chain variable domain is indicated with bold letters and dotted underline (e.g., heavy chain vanable.domain), linker is indicated with italic letters and dot-dash underline (e.g., linker), and light chain variable domain is indicated with bold letters and dashed underline (e.g.,i£ht_chain_y ariable jdomam) •

[0368] In some embodiments, the chimeric antigen receptor (CAR) comprises a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, or SEQ ID NO: 92.

[0369] In one aspect is provided a second polypeptide chain of any chimeric antigen receptor (CAR) described herein.

[0370] In some embodiments, the chimeric antigen receptor (CAR) comprises a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Attorney Docket No: 252457.00003396%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 26, SEQ ID NO: 43, or SEQ ID NO: 45.

[0371] In one aspect is provided a chimeric antigen receptor (CAR) comprising a first polypeptide chain and a second polypeptide chain.

[0372] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 26.

[0373] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 43.

[0374] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 92, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 43.

[0375] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Attorney Docket No: 252457.00003396%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 45.

[0376] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 51, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 26.

[0377] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 55, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 26.

[0378] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 59, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 26.

[0379] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of anAttorney Docket No: 252457.000033 amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 28, wherein the anti-PSMA scFv sequence may be replaced with any antigen-binding polypeptide, including those disclosed herein.

[0380] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 47, wherein the anti-PSMA scFv sequence may be replaced with any antigen-binding polypeptide, including those disclosed herein.

[0381] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 94, wherein the anti-PSMA scFv sequence may be replaced with any antigen -binding polypeptide, including those disclosed herein.

[0382] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 49, wherein the anti-PSMA scFv sequence may be replaced with any antigen-binding polypeptide, including those disclosed herein.

[0383] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 53, wherein the anti-CD19 scFv sequence may be replaced with any antigen-binding polypeptide, including those disclosed herein.

[0384] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,Attorney Docket No: 252457.00003393%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 57, wherein the anti-EpCAM scFv sequence may be replaced with any antigenbinding polypeptide, including those disclosed herein.

[0385] In some embodiments, the chimeric antigen receptor (CAR) comprising a first and a second polypeptide chain disclosed herein may comprise, consist of, or consist essentially of an amino acid sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 61, wherein the anti-EPhA2 scFv sequence may be replaced with any antigen-binding polypeptide, including those disclosed herein.Nucleic Acid Molecules

[0386] In one aspect is provided a nucleic acid molecule encoding a first polypeptide chain of any chimeric antigen receptor (CAR) described herein.

[0387] In some embodiments, the first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 23, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 91.

[0388] In one aspect is provided a nucleic acid molecule encoding a second polypeptide chain of any chimeric antigen receptor (CAR) described herein.

[0389] In some embodiments, the second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 25, SEQ ID NO: 42, or SEQ ID NO: 44.

[0390] In one aspect is provided a chimeric antigen receptor (CAR) comprising a first nucleic acid molecule and a second nucleic acid molecule encoding the first polypeptide chain and a second polypeptide chain.

[0391] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%,Attorney Docket No: 252457.00003388%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 23 and b) a nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 25.

[0392] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 23 and b) a second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 42.

[0393] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 91 and b) a second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 42.

[0394] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 23 and b) a second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,Attorney Docket No: 252457.00003393%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 44.

[0395] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 50 and b) a second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 25.

[0396] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 54 and b) a second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 25.

[0397] In some embodiments, the chimeric antigen receptor (CAR) comprises: a) a first nucleic acid molecule encoding the first polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 58 and b) a second nucleic acid molecule encoding the second polypeptide chain of the CAR may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 25.

[0398] In one aspect is provided a nucleic acid molecule comprising a nucleotide sequence encoding any chimeric antigen receptor (CAR) described herein.Attorney Docket No: 252457.000033

[0399] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 27, wherein the anti-PSMA scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0400] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 46, wherein the anti-PSMA scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0401] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 95, wherein the anti-PSMA scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0402] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 93, wherein the anti-PSMA scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0403] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 48, wherein the anti-PSMA scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0404] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 52, wherein the anti-CD19 scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.Attorney Docket No: 252457.000033

[0405] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 56, wherein the anti-EpCAM scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0406] In a specific embodiment, the nucleic acid molecule may comprise, consist of, or consist essentially of a nucleotide sequence at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NO: 60, wherein the anti-EPhA2 scFv may be replaced with any nucleotide sequence encoding an antigen-binding polypeptide, including those disclosed herein.

[0407] In various embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter. In various embodiments, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter. In various embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter, and the first and second promoters are the same. In various embodiments, the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter, and the first and second promoters are different.

[0408] In various embodiments, the nucleotide sequences encoding the first and second polypeptide chains of the CAR are operably linked to a single promoter. In various embodiments, the first and / or second promoter is a T lymphocyte- specific promoter or an NK cell-specific promoter. In various embodiments, the nucleic acid molecule is a DNA molecule. In various embodiments, the nucleic acid molecule is an RNA molecule.Vectors

[0409] In one aspect is provided a recombinant vector comprising any nucleic acid molecule described herein, or any nucleic acid encoding any polypeptide described herein. In some embodiments, the recombinant vector is a viral vector. The vector may be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, aAttorney Docket No: 252457.000033 herpes virus vector, or a vaccinia virus vector. In some embodiments, the vector is a lentiviral vector. In various embodiments, the vector may be an in vitro transcribed vector.Host Cells

[0410] In another aspect is provided an isolated host cell comprising any CAR described herein. The isolated host cell may comprise any nucleic acid molecule described herein. The isolated host cell may comprise any vector described herein. The host cell may be a mammalian cell. Exemplary host cells include, but are not limited to, cytotoxic cells, T cells, stem cells, progenitor cells, and cells derived from a stem cell or a progenitor cell. The T cell may be a T- helper cell, a cytotoxic T-cell, a T-regulatory cell (Treg), or a gamma-delta T cell. The cytotoxic cell may be a cytotoxic T cell or a natural killer (NK) cell. The host cell may be activated ex vivo and / or expanded ex vivo. The host cell may be an allogeneic cell. The host cell may be an autologous cell. The host cell may be isolated from a subject having a disease. In various embodiments, the subject is human.

[0411] In some aspects, provided herein is a pharmaceutical composition comprising any of the host cells disclosed herein, and a pharmaceutically acceptable carrier and / or excipient.

[0412] Also provided is a method for producing any of the above host cells. The method may comprise genetically modifying the cell with any nucleic acid molecule or any vector described herein. The genetic modification may be conducted ex vivo. The method may further comprise activation and / or expansion of the cell ex vivo.

[0413] The polypeptides disclosed herein, or nucleic acids encoding such, may be introduced into the host cells using transfection and / or transduction techniques known in the art. The nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be a stable introduction. Transfection may be accomplished by a variety of means known in the art including, but not limited, to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell.Attorney Docket No: 252457.000033For example, a nucleic acid encoding a transmembrane polypeptide carried by a retroviral vector can be transduced into a cell through infection and pro virus integration.

[0414] In certain embodiments, the nucleic acid or viral vector is transferred via ex vivo transformation. Methods for transfecting vascular cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art. Thus, it is contemplated that cells or tissues may be removed and transfected ex vivo using the polynucleotides presented herein. In particular aspects, the transplanted cells or tissues may be placed into an organism. Thus, it is well within the knowledge of one skilled in the art to isolate antigen-presenting cells (e.g., T-cells or NK cells) from an animal (e.g., human), transfect the cells with the expression vector, and then, e.g., administer the transfected or transformed cells back to the animal.

[0415] In certain embodiments, the nucleic acid or viral vector is transferred via injection. In certain embodiments, a polynucleotide is introduced into an organelle, a cell, a tissue or an organism via electroporation. In certain embodiments, a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In certain embodiments, the polynucleotides encode any of the first and second transmembrane polypeptides described herein, and are inserted into a vector or vectors. The vector is a vehicle into which a polynucleotide encoding a protein may be covalently inserted so as to bring about the expression of that protein and / or the cloning of the polynucleotide. Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences coding for at least part of a gene product capable of being transcribed in a cell. In most cases, RNA molecules are then translated into a protein.

[0416] Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The expression vector may have additional sequence such as 6x-histidine (SEQ ID NO: 97), c-Myc, and FLAG tags which are incorporated into the expressed polypeptides. In certain embodiments, c-Myc may be incorporated into any of the expressed polypeptides of theAttorney Docket No: 252457.000033 present disclosure. As a non-limiting example, c-Myc may be incorporated into any domain of a first and / or second polypeptide chain disclosed herein. As a non-limiting example, c-Myc may be incorporated into any domain of a polypeptide chain that is a signaling chain (S-chain) disclosed herein. As another non-limiting example, c-Myc may be incorporated into any domain of a polypeptide chain that is a recognition chain (R-chain) disclosed herein.

[0417] In various embodiments, the vectors are plasmid, autonomously replicating sequences, and transposable elements.

[0418] In certain embodiments, the nucleic acids encoding the polypeptides of the present invention are provided in a viral vector. In certain embodiments, the viral vector is a retroviral vector or a lentiviral vector. The term “retroviral vector” refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a vector containing structural and functional genetic elements outside the long terminal repeats (LTRs) that are primarily derived from a lentivirus.

[0419] In certain embodiments, the present disclosure provides isolated host cells (e.g., T- cells) containing the vectors provided herein. The host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector.

[0420] In one aspect is provided a method for activating any of various CARs in any number of host cell(s) of the present disclosure. The method comprises contacting the host cell with an activator disclosed herein (e.g., venetoclax), wherein the activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling. The first member and the second member of the dimerization pair may be derived from a Bcl-2 protein (e.g., a truncated and / or a mutant Bcl-2 protein). The first member and the second member of the dimerization pair may be derived from a Bcl-xL protein (e.g., a truncated and / or a mutant Bcl-xL protein).Pharmaceutical Compositions

[0421] In another aspect is provided a pharmaceutical composition comprising any host cell described herein, and a pharmaceutically acceptable carrier and / or excipient. Exemplary carriers include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutionsAttorney Docket No: 252457.000033 are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin

[0422] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).

[0423] The pharmaceutical composition may be used in combination with other therapies. It is contemplated that when used to treat various diseases, the compositions and methods can be combined with other therapeutic agents suitable for the same or similar diseases. Also, two or more embodiments described herein may be also co- administered to generate additive or synergistic effects. When co-administered with a second therapeutic agent, the embodiment described herein and the second therapeutic agent may be simultaneously or sequentially (in any order). Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0424] As a non-limiting example, the methods described herein can be combined with other therapies that block inflammation e.g., via blockage of IL1, INFa / p, IL6, TNF, IL13, IL23, etc.).

[0425] In some embodiments, the compositions and methods disclosed herein are useful to enhance the efficacy of vaccines directed to tumors or infections. Thus, the compositions and methods described herein can be administered to a subject either simultaneously with or before (e.g., 1-30 days before) a reagent (including but not limited to small molecules, antibodies, or cellular reagents) that acts to elicit an immune response (e.g., to treat cancer or an infection) is administered to the subject.

[0426] The compositions and methods described herein can be also administered in combination with an anti-tumor antibody or an antibody directed at a pathogenic antigen or allergen.

[0427] The compositions and methods described herein can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GV AX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents thatAttorney Docket No: 252457.000033 enhance 41BB, 0X40, etc.). The inhibitory treatments described herein can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CDld, CDld-fusion proteins, CDld dimers or larger polymers of CDld either unloaded or loaded with antigens, CDld-chimeric antigen receptors (CDld-CAR), or any other of the five known CD1 isomers existing in humans (CDla, CDlb, CDlc, CDle), in any of the aforementioned forms or formulations, alone or in combination with each other or other agents.

[0428] Therapeutic methods described herein can be combined with additional immunotherapies and therapies. For example, when used for treating cancer, NKT cells described herein can be used in combination with conventional cancer therapies, such as, e.g., surgery, radiotherapy, chemotherapy or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors. In certain aspects, other therapeutic agents useful for combination cancer therapy with the inhibitors described herein include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, e.g., TNP-470, platelet factor 4, thrombospondin- 1 , tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT- 1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000). In some embodiments, the inhibitors described herein can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti -h VEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0429] The present invention provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary CAR described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined with or administered in combination with a CAR include, e.g., an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2 / ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds EGFRvIII), a cMET antagonist (e.g., an antiAttorney Docket No: 252457.000033 cMET antibody), an IGF1R antagonist (e.g., an anti-IGFIR antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PFX-4720), a PDGFR-a inhibitor (e.g., an anti-PDGFR-a antibody), a PDGFR-P inhibitor (e.g., an anti-PDGFR-P antibody), a VEGF antagonist (e.g., a VEGF-Trap, see, e.g., US 7,087,411 (also referred to herein as a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009 / 0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 2011 / 0027286 such as H1H685P), a FOLH1 (PSMA) antagonist, a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAPl antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), auroplakin antagonist (e.g., an anti-uroplakin antibody), etc. Other agents that may be beneficially administered in combination with a CAR include cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors. The pharmaceutical compositions of the present invention may also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from “ICE”: ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); “DHAP”: dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and “ESHAP”: etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP- 16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[0430] The present invention also includes therapeutic combinations comprising any of the antigen-binding molecules mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-a, PDGFR-0, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). The CAR may also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics,Attorney Docket No: 252457.000033 corticosteroids and / or NSAIDs. The antigen-binding molecules of the invention may also be administered as part of a treatment regimen that also includes radiation treatment and / or conventional chemotherapy.

[0431] Non-limiting examples of chemotherapeutic compounds which can be used in combination treatments include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0432] These chemotherapeutic compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites / anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,Attorney Docket No: 252457.000033 mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes- dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0433] For treatment of infections, a combined therapy may be used. The combined therapy can encompass co-administering compositions and methods described herein with an antibiotic, an anti-fungal drug, an anti-viral drug, an anti-parasitic drug, an anti-protozoal drug, or a combination thereof.

[0434] Non-limiting examples of useful antibiotics include lincosamides (clindomycin); chloramphenicols; tetracyclines (such as Tetracycline, Chlortetracycline, Demeclocycline, Methacycline, Doxycycline, Minocycline); aminoglycosides (such as Gentamicin, Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin, Neomycin); beta-lactams (such as penicillins,Attorney Docket No: 252457.000033 cephalosporins, Imipenem, Aztreonam); vancomycins; bacitracins; macrolides (erythromycins), amphotericins; sulfonamides (such as Sulfanilamide, Sulfamethoxazole, Sulfacetamide, Sulfadiazine, Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide, p-Aminobenzoic Acid, Trimethoprim-Sulfamethoxazole); Methenamin; Nitrofurantoin; Phenazopyridine; trimethoprim; rifampicins; metronidazoles; cefazolins; Lincomycin; Spectinomycin; mupirocins; quinolones (such as Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin, Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin); novobiocins; polymixins; gramicidins; and antipseudomonals (such as Carbenicillin, Carbenicillin Indanyl, Ticarcillin, Azlocillin, Mezlocillin, Piperacillin) or any salts or variants thereof. See also Physician's Desk Reference, 59thedition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy, 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J. Such antibiotics can be obtained commercially, e.g., from Daiichi Sankyo, Inc. (Parsipanny, N.J.), Merck (Whitehouse Station, N.J.), Pfizer (New York, N.Y.), Glaxo Smith Kline (Research Triangle Park, N.C.), Johnson & Johnson (New Brunswick, N.J.), AstraZeneca (Wilmington, Del.), Novartis (East Hanover, N.J.), and Sanofi-Aventis (Bridgewater, N.J.). The antibiotic used will depend on the type of bacterial infection.

[0435] Non-limiting examples of useful anti-fungal agents include imidazoles (such as griseofulvin, miconazole, terbinafine, fluconazole, ketoconazole, voriconazole, and itraconizole); polyenes (such as amphotericin B and nystatin); Flucytosines; and candicidin or any salts or variants thereof. See also Physician's Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0436] Non-limiting examples of useful anti-viral drugs include interferon alpha, beta or gamma, didanosine, lamivudine, zanamavir, lopanivir, nelfinavir, efavirenz, indinavir, valacyclovir, zidovudine, amantadine, rimantidine, ribavirin, ganciclovir, foscarnet, and acyclovir or any salts or variants thereof. See also Physician's Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice ofAttorney Docket No: 252457.000033Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0437] Non-limiting examples of useful anti-parasitic agents include chloroquine, mefloquine, quinine, primaquine, atovaquone, sulfasoxine, and pyrimethamine or any salts or variants thereof. See also Physician's Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY ; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0438] Non-limiting examples of useful anti-protozoal drugs include metronidazole, diloxanide, iodoquinol, trimethoprim, sufamethoxazole, pentamidine, clindamycin, primaquine, pyrimethamine, and sulfadiazine or any salts or variants thereof. See also Physician's Desk Reference, 59th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0439] The additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of any of the CARs disclosed herein (for purposes of the present disclosure, such administration regimens are considered the administration of a CAR “in combination with” an additional therapeutically active component).

[0440] The present disclosure includes pharmaceutical compositions in which a CAR is coformulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.Therapeutic Uses

[0441] The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising a CAR as described herein. The therapeutic composition can comprise any of the various CARs as disclosed herein and a pharmaceuticallyAttorney Docket No: 252457.000033 acceptable carrier or diluent. As used herein, the expression “a subject in need thereof’ means a human or non-human animal that exhibits one or more symptoms or indicia of an infection (e.g., a subject suffering from a bacterial or viral infection, including any of those mentioned herein) cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned herein), an autoimmune disorder (e.g. , a subject suffering from any of the autoimmune diseases or disorders mentioned herein), inflammatory diseases, or who otherwise would benefit from enhancement or suppression of T cell activity.

[0442] In another aspect, described herein is a method of treating a disorder in a subject in need thereof comprising administering to said subject an effective amount of a CAR described herein, wherein the CAR binds to an antigen-specific TCR and wherein the antigen recognized by the TCR is associated with the disorder.

[0443] The CARs of the invention (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation or suppression of an immune response (via T cell modulation) targeted against a specific antigen would be beneficial. In particular, the CARs of the present invention may be used for the treatment and prevention of infections, cancers or autoimmune disorders.

[0444] Where the CAR described herein includes a second molecule comprising a domain that specifically binds a T-cell immunomodulatory molecule that is an activating polypeptide, transduction of the T cell with the CAR activates the epitope-specific T cell. In some instances, the epitope-specific T cell is a T cell that is specific for an epitope present on a cancer cell, and contacting the epitope-specific T cell with the CAR increases cytotoxic activity of the T cell toward the cancer cell. In some embodiments, the epitope-specific T cell is a T cell that is specific for an epitope present on a cancer cell, and contacting the epitope-specific T cell with the CAR increases the number of the epitope-specific T cells.

[0445] In some embodiments, the epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and contacting the epitope-specific T cell with the CAR increases cytotoxic activity of the T cell toward the virus-infected cell. In some instances, the epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and contacting the epitope-specific T cell with the CAR increases the number of the epitope-specific T cells.Attorney Docket No: 252457.000033

[0446] The interaction of a T cell with the CARs described herein can result in, e.g., activation, induction of anergy, or death of a T cell that occurs when the TCR of the T cell is bound by a TCR- binding pMHC complex. “Activation of a T cell” refers to induction of signal transduction pathways in the T cell resulting in production of cellular products (e.g., interleukin-2) by that T cell. “Anergy” refers to the diminished reactivity by a T cell to an antigen. Activation and anergy can be measured by, for example, measuring the amount of IL-2 produced by a T cell after an pMHC complex has bound to the TcR. Anergic cells will have decreased IL-2 production when compared with stimulated T cells. Another method for measuring the diminished activity of anergic T cells includes measuring intracellular and / or extracellular calcium mobilization by a T cell upon engagement of its TCRs. “T cell death” refers to the permanent cessation of substantially all functions of the T cell.

[0447] T-cell phenotypes may be evaluated using well-known methods, e.g., by measuring changes in the level of expression of cytokines and / or T cell activation markers, and / or the induction of antigen-specific proliferating cells. Techniques known to those of skill in the art, include, but not limited to, immunoprecipitation followed by Western blot analysis, ELISAs, flow cytometry, Northern blot analysis, and RT-PCR can be used to measure the expression cytokines and T cell activation markers. Cytokine release may be measured by measuring secretion of cytokines including but not limited to Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL- 6), Interleukin- 12 (IL-12), Interleukin- 16 (IL-16), PDGF, TGF-a, TGF-0, TNF-a, TNF-0, GCSF, GM-CSF, MCSF, IFN-a, IFN-J3, IFN-y, TFN-y, IGF-I, and IGF-II (see, e.g., Isaacs et al., 2001, Rheumatology, 40: 724-738; Soubrane et al., 1993, Blood, 81(1): 15-19).

[0448] T cell modulation may also be evaluated by measuring (e.g., proliferation) by, for example, 3H-thymidine incorporation, trypan blue cell counts, and fluorescence activated cell sorting (FACS).

[0449] The anti-tumor responses of T cells after exposure to the CAR may be determined in xenograft tumor models. Tumors may be established using any human cancer cell line expressing the tumor associated antigen presented by the CAR. The endpoint of the xenograft tumor models can be determined based on the size of the tumors, weight of animals, survival time and histochemical and histopathological examination of the cancer, using methods known to one skilled in the art.Attorney Docket No: 252457.000033

[0450] Non-limiting examples of useful animal models for analyzing the effect of the exposure of T cells to the CARs described herein on inflammatory diseases include adjuvant-induced arthritis rat models, collagen-induced arthritis rat and mouse models and antigen-induced arthritis rat, rabbit and hamster models (see, e.g., Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritis and Allied Conditions: A Textbook of Rheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993); Trenthorn et al., 1977, J. Exp. Med. 146:857; Courtenay et al., 1980, Nature 283:665; Cathcart et at, 1986, Lab. Invest. 54:26; Holmdahl, R., 1999, Curr. Biol. 15:R528-530). Other useful animal models of inflammatory diseases include animal models of inflammatory bowel disease, ulcerative colitis and Crohn's disease induced, e.g., by sulfated polysaccharides (e.g., amylopectin, carrageen, amylopectin sulfate, dextran sulfate) or chemical irritants (e.g., trinitrobenzenesulphonic acid (TNBS) or acetic acid). See, e.g., Kim et al., 1992, Scand. J. Gastroentrol. 27:529-537; Strober, 1985, Dig. Dis. Sci. 30(12 Suppl):3S-10S).

[0451] Additional useful models are animal models for asthma such as, e.g., adoptive transfer model in which aeroallergen provocation of TH1 or TH2 recipient mice results in TH effector cell migration to the airways and is associated with an intense neutrophilic (TH1) and eosinophilic (TH2) lung mucosal inflammatory response (see, e.g., Cohn et al., 1997, J. Exp. Med. 1861737- 1747). Useful animal models of studying the effect of the CARs of the invention on multiple sclerosis (MS) include an experimental allergic encephalomyelitis (EAE) model (see, e.g., Zamvil et al, 1990, Ann. Rev, Immunol. 8:579). Animal models which can be used for analyzing the effect of the CARs of the invention on autoimmune disorders such as type 1 diabetes, thyroid autoimmunity, systemic lupus erythematosus, and glomerulonephritis have been also developed (see, e.g., Bluestone et al., 2004, PNAS 101:14622-14626; Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19: 12-24).

[0452] Efficacy of a CAR to downregulate immune responses in treating an autoimmune disorder may be evaluated, e.g., by detecting their ability to reduce one or more symptoms of the autoimmune disorder, to reduce mean absolute lymphocyte counts, to decrease T cell activation, to decrease T cell proliferation, to reduce cytokine production, or to modulate one or more particular cytokine profiles (e.g., Interleukin-2 (IL-2). Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin- 12 (IL-12), Interleukin- 16 (IL-16), PDGF, TGF-a, TGF-0, TNF-a, TNF-0, GCSF,Attorney Docket No: 252457.000033GM-CSF, MCSF, IFN-a, IFN-0, IFN-y, TFN-y, IGF-I, and IGF-II) (see, e.g., Isaacs et al., 2001, Rheumatology, 40: 724-738; Soubrane et al., 1993, Blood, 81(1): 15-19).

[0453] Efficacy of the CARs for use in treating diabetes may be evaluated, e.g., by the ability of the CARs to reduce one or more symptoms of diabetes, to preserve the C-peptide response to MMTT, to reduce the level HA1 or HAlc, to reduce the daily requirement for insulin, or to decrease T cell activation in pancreatic islet tissue. Efficacy in treating arthritis may be assessed through tender and swollen joint counts, determination of a global scores for pain and disease activity, ESRICRP, determination of progression of structural joint damage (e.g., by quantitative scoring of X-rays of hands, wrists, and feet (Sharp method)), determination of changes in functional status (e.g., evaluated using the Health Assessment Questionnaire (HAQ)), or determination of quality of life changes (assessed, e.g., using SF-36).

[0454] In a related aspect, disclosed herein is a method of treating a disorder in a subject in need thereof comprising administering to said subject an effective amount of the CAR, wherein the CAR binds to an antigen-specific TCR and wherein the antigen is associated with the disorder. In some embodiments, the disorder is an inflammatory or an autoimmune disorder, and the administration results in a downregulation of an inflammatory or autoimmune response. In one specific embodiment, the disorder is celiac disease or gluten sensitivity. In one specific embodiment, the antigen comprises a gliadin or a fragment thereof (e.g., (i) a-gliadin fragment corresponding to amino acids 57-73 or (ii) y-gliadin fragment corresponding to amino acids 139-153 or (iii) co-gliadin fragment corresponding to amino acids 102-118). In one specific embodiment, the CAR presents a peptide derived from the antigen in the context of a class II MHC. In some embodiments, the disorder is a tumor and the administration results in an upregulation of an anti-tumor immune response.

[0455] CAR T cells comprising the CARs described herein can eliminate auto-reactive B cells. CAR T cells comprising the CARs described herein can be used to dampen immune responses, which may be useful in the context of GVHD, autoimmunity or transplantation tolerance.

[0456] In another embodiment, the disorder is an infection caused by an infectious agent and the administration results in an upregulation of an immune response against the infectious agent. In one specific embodiment, the infectious agent is selected from the group consisting of a virus, a bacterium, a fungus, a protozoa, a parasite, a helminth, and an ectoparasite.Attorney Docket No: 252457.000033

[0457] According to certain aspects, a CAR may be used to treat a cancer in which the tumor cells express a tumor-associated antigen, for example, a tumor-associated antigen selected from the group consisting of adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTCI, ALK, BAGE proteins (e.g., BAGE-1), BIRC5 (survivin), BIRC7, P-catenin, BRCA1, BORIS, B-RAF, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA- 125, CALCA, carcinoembryonic antigen (“CEA”), CAGE-1 to 8, CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin DI, Cyclin-Al, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, cyclin-Bl, CYP1B1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), EphA3, epithelial tumor antigen (“ETA”), EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML1 fusion protein, EpCAM, EphA2, EZH2, FGF5, FLT3-ITD, FN1, Fra-1, FOLR1, G250 / MN / CAIX, GAGE proteins (e.g., GAGE-1- 8), GD2, GD3, GloboH, glypican-3, GM3, gplOO, GAS7, GnTV, gplOO / Pmel 17, GPNMB, GnTV, HAUS3, Hepsin, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, HPV E2, HPV E6, HPV E7, HPV EG, Her2 / neu, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein, Lengsin, LMP2, M-CSF, MAGE proteins (e.g., MAGE-A1, -A2, -A3, -A4, -A6, - A9, -A10, -A12, -Cl, and -C2), malic enzyme, mammaglobin-A, MART-1, MART-2, MATN, MC1R, MCSP, mdm-2, MEI, Melan-A / MART- 1 , Meloe, Midkine, MMP-2, MMP-7, mesothelin, ML-IAP, Mucl, Muc2, Muc3, Muc4, Muc5, Mucl6 (CA-125), MUC5AC, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NA17, NA-88, NY-BR1, NY-BR62, NY-BR85, NY-ESO1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, pI5, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, RAGE proteins (e.g., RAGE-1), Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, SAGE, secernin 1, SIRT2, SNRPD1, SOXIO, Spl7, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, TAG-72, TGF-0, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-l / gp75, TRP-2, TRP2-INT2, tyrosinase, Telomerase, TPBG, TRAG-3, Triosephosphate isomerase, uroplakin-3, VEGF, XAGE-lb / GAGED2a, WT-1, NeuGcGM3, N-glycolyl GM3 ganglioside,Attorney Docket No: 252457.000033Neu5Gc, GM3 -Ganglioside, GD3, GM2, carbohydrate antigens, ganglioside antigens, Lewis Y, and Lewis B, CD 123 and Kappa chain of immunoglobulin. In some embodiments, the peptide is a neo-antigen. In some embodiments, the peptide is a tumor specific antigen.

[0458] Specific cancers / tumors treatable by the methods and CARs of the present invention include, without limitation, various solid malignancies, carcinomas, lymphomas, sarcomas, blastomas, and leukemias. Non-limiting specific examples, include, for example, breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, renal cancer, bladder cancer, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancers of all histopathologic types, angiosarcoma, hemangiosarcoma, bone sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, Ewing's tumor, leiomyosarcoma, Ewing’s sarcoma, rhabdomyosarcoma, carcinoma of unknown primary (CUP), squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroom's macroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma, bronchogenic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, lung carcinoma, epithelial carcinoma, cervical cancer, testicular tumor, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, leukemia, neuroblastoma, small cell lung carcinoma, bladder carcinoma, lymphoma, multiple myeloma, medullary carcinoma, B cell lymphoma, T cell lymphoma, NK cell lymphoma, large granular lymphocytic lymphoma or leukemia, gamma-delta T cell lymphoma or gamma-delta T cell leukemia, mantle cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, hematopoietic neoplasias, thymoma, sarcoma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, Epstein-Barr virus (EBV) induced malignancies of all types including but not limited to EBV-associated Hodgkin’s and nonHodgkin’s lymphoma, all forms of post-transplant lymphomas including post-transplant lymphoproliferative disorder (PTLD), uterine cancer, renal cell carcinoma, hepatoma, hepatoblastoma, Cancers that may treated by methods and compositions described herein include,Attorney Docket No: 252457.000033 but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;Attorney Docket No: 252457.000033 struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0459] The present invention also includes methods for treating residual cancer in a subject. As used herein, the term “residual cancer” means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.

[0460] Non-limiting examples of the inflammatory and autoimmune diseases include, but are not limited to, inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn’s disease, diabetes (e.g., diabetes mellitus type 1), multiple sclerosis, arthritis (e.g., rheumatoid arthritis), Graves' disease, lupus erythematosus, ankylosing spondylitis, psoriasis, Behcet's disease, autistic enterocolitis, Guillain-Barre Syndrome, myasthenia gravis, pemphigus vulgaris, acute disseminated encephalomyelitis (ADEM), transverse myelitis autoimmune cardiomyopathy, Celiac disease, dermatomyositis, Wegener's granulomatosis, allergy, asthma, contact dermatitis, atherosclerosis (or any other inflammatory condition affecting the heart or vascular system), autoimmune uveitis, as well as other autoimmune skin conditions, autoimmune kidney, lung, or liver conditions, autoimmune neuropathies, asthma, allergy, celiac disease, systemic lupus erythematosis (SLE), scleroderma, sarcoidosis, thyroiditis, multiple sclerosis, spondylitis,Attorney Docket No: 252457.000033 periarteritis, eczema, atopic dermatitis, myasthenia gravis, insulin-dependent diabetes mellitus, Crohn's disease, Guillain-Barre syndrome, Graves' disease, glomerulonephritis, ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, psoriasis, acute or chronic immune disease associated with organ transplantation, an inflammatory disease, skin or organ transplantation rejection, graft-versus-host disease (GVHD), or autoimmune diseases, comprising administering to a subject a pharmaceutical composition described herein (e.g., a pharmaceutic composition comprising a CAR described herein. Examples of autoimmune diseases include, for example, glomerular nephritis, arthritis, dilated cardiomyopathy-like disease, ulcerous colitis, Sjogren syndrome, Crohn’s disease, systemic erythematodes, chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyosiis, pachyderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulin dependent diabetes mellitus, Behcet disease, Hashimoto disease, Addison disease, dermatomyositis, myasthenia gravis, Reiter syndrome, Graves' disease, anaemia perniciosa, sterility disease, chronic active hepatitis, pemphigus, autoimmune thrombopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, anti-phospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydra autoimmune, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus, erythematosis, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis, some embodiments of lymphopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anema, phacogenic uveitis, polyarteritis nodosa, polyglandular autosyndromes, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, limited scleroderma (or crest syndrome), sympathetic ophthalmia, systemic lupus erythematosis, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type b insulin resistance, ulcerative colitis and Wegener's granulomatosis.

[0461] In another embodiment, the methods described herein are used for treating or preventing a transplantation-related condition. In another embodiment, the methods described herein are used for treating or preventing graft-versus-host disease. In another embodiment, the methods described herein are used for treating or preventing a post-transplant lymphoproliferative disorder.Attorney Docket No: 252457.000033

[0462] According to certain aspects, the CAR may be used to treat an infection, such as a bacterial infection (e.g. a bacterial infection resistant to conventional antibiotics) or a viral infection. In particular embodiments, the CAR is designed to present a peptide derived from a viral antigen or a bacterial antigen. In some embodiments, the viral antigen is derived from a virus selected from the group consisting of adenovirus, astrovirus, chikungunya, cytomegalovirus, dengue, ebola, EBV, hantavirus, HBsAg, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, lassa, measles, metapneumovirus, mumps, norovirus, oropauche, HPV, parvovirus, rotavirus, RSV, rubella, SARS, TBEV, usutu, vaccina, varicella, West Nile, yellow fever, and zika. In some embodiments, the bacterial antigen is derived from a bacterium selected from the group consisting of methicillin- resistant Staphylococcus Aureus (MRSA), Clostridium Difficile, carbapenum-resistant Enterobacteriaceae, drug-resistant Neisseria Gonorrhoeae, multidrug-resistant Acinetobacter, drug-resistant Campylobacter, Fluconazole -resistant Candida, extended-spectrum P-lactamase producing bacteria, Vancomycin-resistant enterococcus, multidrug -resistant pseudomonas Aeruginosa, drug-resistant non-typhoidal Salmonella, drug-resistant Salmonella serotype typhi, drug-resistant Shigella, drug-resistant Streptococcus Pneumoniae, drug-resistant tuberculosis, Vancomycin -resistant Staphylococcus Aureus, Erythomycin-resistant group A Streptococcus, and Clindamycin-resistant group B Streptococcus.

[0463] Activatable or Switchable CARs designed to treat cancer or an infection may include an antigen-binding domain (e.g., a one-arm antibody) on the second binding molecule that specifically binds a T-cell co-stimulatory molecule (e.g., CD28) to induce activation, proliferation (e.g., clonal expansion) and / or survival of T cells (e.g., CD8+ T cells) specific for the peptide presented on the first binding molecule. In some embodiments, T cell activation is revived. In some embodiments, naive T-cells are activated or caused to proliferate. Such T cells can enhance or stimulate an immune response against cells (e.g., tumor cells or infected cells) expressing a protein comprising the peptide presented on the first binding molecule of the CAR. In various embodiments, the CARs do not induce proliferation of non-specific T cells (i.e., T cells that are not specific for the peptide presented on the first binding molecule).

[0464] According to certain aspects, the CAR may be used to treat, prevent, or ameliorate an autoimmune disease or disorder by targeting the activity of T cells with specificity for a peptide corresponding to an antigen associated with the autoimmune disease or disorder. For example, theAttorney Docket No: 252457.000033 antigen may be selected from the group consisting of gliadin (celiac disease; e.g., (i) a-gliadin fragment corresponding to amino acids 57-73 or (ii) y-gliadin fragment corresponding to amino acids 139 153 or (iii) co-gliadin fragment corresponding to amino acids 102-118), GAD 65, IA-2 and insulin B chain (for type 1 -diabetes), glatiramer acetate (GA) (for multiple sclerosis), acetylcholine receptor (AChR) (for myasthenia gravis), p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP1, and myelin antigens (including myelin basic protein (MBP) and proteolipid protein (PLP)). In some embodiments, the antigen may be IL-4R, IL-6R, or DLL4.

[0465] The present invention also includes use of the switchable CARs herein in the manufacture of a medicament for preventing, treating and / or ameliorating an infection, a cancer, or an autoimmune disorder (e.g., as discussed herein).

[0466] In one aspect is provided a method for stimulating elimination of a cell comprising an antigen in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to said antigen and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR- mediated signaling.

[0467] The antigen may be a tumor-associated antigen, an infection-associated antigen or an auto-antigen. The antigen may be a tumor-associated antigen. The tumor-associated antigen may be associated with a solid tumor. The tumor cell associated antigen may be a prostate-specific membrane antigen (PSMA). The antigen may be an infection-associated antigen. The antigen may be an auto-antigen. The antigen may be CD 19. The antigen may be NeuGcGM3 or N-glycolyl GM3 ganglioside. The antigen may be EpCAM. The antigen may be EPhA2.

[0468] In another aspect is provided a method for stimulating elimination of a cell comprising prostate-specific membrane antigen (PSMA) in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0469] In another aspect is provided a method for stimulating elimination of a cell comprising Cluster of Differentiation 19 (CD 19) in a subject in need thereof. The method comprisesAttorney Docket No: 252457.000033 administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0470] In another aspect is provided a method for stimulating elimination of a cell comprising epithelial cellular adhesion molecule (EpCAM) in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0471] In another aspect is provided a method for stimulating elimination of a cell comprising Ephrin type-A receptor 2 (EPhA2) in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0472] In another aspect is provided a method for treating a cancer in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CAR disclosed herein, and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said cancer and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling. The cancer may be from a solid tumor. The cancer may be carcinoma, melanoma, prostate cancer, sarcoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, and retinoblastoma. The cancer may be a leukemia or a lymphoma.

[0473] In another aspect is provided a method for treating prostate cancer in a subject in need thereof. The method comprises administering to the subject (i) an effective amount cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and theAttorney Docket No: 252457.000033 second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

[0474] In another aspect is provided a method for treating an infection in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising any of various CARs disclosed herein, and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said infection and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling

[0475] In another aspect is provided a method for treating an inflammatory condition or an autoimmune disease in a subject in need thereof. The method comprises administering to the subject (i) an effective amount of T-helper cells or Treg cells comprising any of various switchable CARs disclosed herein, and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said inflammatory condition or an autoimmune disease, and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR- mediated signaling. The extracellular region of the CAR binds to an antigen associated with said inflammatory condition or an autoimmune disease. The method may result in reducing an immune response to a transplanted organ or tissue.

[0476] The method may comprise a) isolating T cells or NK cells from the subject; b) genetically modifying said T cells or NK cells ex vivo with any nucleic acid molecule or any vector described herein. The T cells or NK cells may be expanded or activated before, after or during step (b). The genetically modified T cells or NK cells are introduced into the subject.

[0477] The above methods may further comprise stimulating the activity of the CAR by administering to the subject an effective amount of an activator molecule, e.g. a dimerizer drug, that promotes the dimerization of the first and second member of the dimerization pair within the CAR resulting in stimulation of CAR-mediated signaling.

[0478] In various embodiments, the subject is human.

[0479] In yet another aspect is provided a method for activating any of various switchable CARs in any number of host cell(s) of the present disclosure. The method comprises contacting the host cell with an activator disclosed herein (e.g., venetoclax), wherein the activator promotesAttorney Docket No: 252457.000033 the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling. The first member and / or the second member of the dimerization pair may be derived from a Bcl-2 protein. In some embodiments, the first and / or second member of the dimerization pair comprise a truncated and / or mutant Bcl-2 protein disclosed herein. The first member and / or the second member of the dimerization pair may be derived from a Bcl-xL protein. In some embodiments, the first and / or second member of the dimerization pair comprise a truncated and / or mutant Bcl-xL protein disclosed herein.

[0480] In further aspect is provided a method for downregulating any of various switchable CARs in any number of host cell(s) of the present disclosure. The method comprises contacting the host cell with a ligand disclosed herein (e.g., lenalidomide), wherein the ligand mediates proteasomal degradation of the degradable CAR, resulting in downregulation of CAR-mediated signaling. The ligand mediates proteasomal degradation of the CAR by inducing interactions between the E3 ubiquitin ligase and a degron sequence present on the CAR molecule.

[0481] According to certain embodiments of the present invention, multiple doses of a CAR, an activator, and / or a ligand disclosed herein may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of a CAR, an activator, and / or a ligand of the invention. As used herein, “sequentially administering” means that each dose of a CAR, an activator, and / or a ligand is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of a CAR, activator, and / or ligand, followed by one or more secondary doses of the CAR, activator, and / or ligand, and optionally followed by one or more tertiary doses of the CAR, activator, and / or ligand.

[0482] The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the CAR, activator, and / or ligand. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the CAR, activator, and / or ligand, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of CAR , activator and / or ligand contained in the initial,Attorney Docket No: 252457.000033 secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).

[0483] In one exemplary embodiment of the present invention, each secondary and / or tertiary dose is administered 1 to 26 (e.g., 1, 116, 2, 216, 3, 316, 4, 416, 5, 516, 6, 616, 7, 716, 8, 816, 9, 916, 10, 1016, 11, 1116, 12, 1216, 13, 1316, 14, 1416, 15, 1516, 16, 1616, 17, 1716, 18, 1816, 19, 1916, 20, 2016, 21, 2116, 22, 2216, 23, 2316, 24, 2416, 25, 2516, 26, 2616, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of a CAR, activator, and / or a ligand which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0484] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and / or tertiary doses of a CAR, activator, and / or a ligand. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0485] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

[0486] According to certain embodiments of the present invention, an activator disclosed herein may be administered at a dose of about 1 to 10, about 10 to 20, about 20 to 30, about 30 toAttorney Docket No: 252457.00003340, about 40 to 50, about 50 to 60, about 60 to 70, about 70 to 80, about 80 to 90, about 90 to 100, about 100 to 110, about 110 to 120, about 120 to 130, about 130 to 140, about 140 to 150, about 150 to 160, about 160 to 170, about 170 to 180, about 180 to 190, about 190 to 200, about 200 to 210, about 210 to 220, about 220 to 230, about 230 to 240, about 240 to 250, about 250 to 260, about 260 to 270, about 270 to 280, about 280 to 290, about 290 to 300, about 300 to 310, about 310 to 320, about 320 to 330, about 330 to 340, about 340 to 350, about 350 to 360, about 360 to 370, about 370 to 380, about 380 to 390, or about 390 to 400 mg / kg.

[0487] In some embodiments, the activator may be administered at a dose of about 1 to 50 mg / kg. In some embodiments, the activator may be administered at a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / kg.

[0488] In various embodiments, an activator disclosed herein may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / kg. In some embodiments, the activator may be administered at about 2.5 mg / kg.

[0489] In some embodiments, the activator may be administered at a dose not to exceed about 50 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 100 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 150 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 200 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 250 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 300 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 350 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 400 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 500 mg per day. In some embodiments, the activator may be administered at a dose not to exceed about 600 mg per day.

[0490] In some embodiments, the activator may be administered at a frequency of every 8 hours, every 12 hours, every 16 hours, every 24 hours, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 8 days, every 10 days, once a week,Attorney Docket No: 252457.000033 twice a week, biweekly, once a month, twice a month, 3 times a month, 4 times a month, or 5 times a month

[0491] In some embodiments, the activator may be administered at a sub-chemotherapeutic dose.

[0492] According to certain embodiments of the present invention, a ligand disclosed herein may be administered at a dose of about 1 to 10, about 10 to 20, about 20 to 30, about 30 to 40, about 40 to 50, about 50 to 60, about 60 to 70, about 70 to 80, about 80 to 90, about 90 to 100, about 100 to 110, about 110 to 120, about 120 to 130, about 130 to 140, about 140 to 150, about 150 to 160, about 160 to 170, about 170 to 180, about 180 to 190, about 190 to 200, about 200 to 210, about 210 to 220, about 220 to 230, about 230 to 240, about 240 to 250, about 250 to 260, about 260 to 270, about 270 to 280, about 280 to 290, about 290 to 300, about 300 to 310, about 310 to 320, about 320 to 330, about 330 to 340, about 340 to 350, about 350 to 360, about 360 to 370, about 370 to 380, about 380 to 390, or about 390 to 400 mg / kg.

[0493] In some embodiments, the ligand may be administered at a dose of about 1 to 50 mg / kg. In some embodiments, the ligand may be administered at a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / kg.

[0494] In various embodiments, a ligand disclosed herein may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg / kg. In some embodiments, the ligand may be administered at about 2.5 mg / kg.

[0495] In some embodiments, the ligand may be administered at a dose not to exceed about 50 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 100 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 150 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 200 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 250 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 300 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 350 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 400 mg per day. In some embodiments, the ligand may beAttorney Docket No: 252457.000033 administered at a dose not to exceed about 500 mg per day. In some embodiments, the ligand may be administered at a dose not to exceed about 600 mg per day.

[0496] In some embodiments, the ligand may be administered at a frequency of every 8 hours, every 12 hours, every 16 hours, every 24 hours, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 8 days, every 10 days, once a week, twice a week, biweekly, once a month, twice a month, 3 times a month, 4 times a month, or 5 times a month

[0497] In some embodiments, the ligand may be administered at a sub-chemotherapeutic dose.

[0498] In some embodiments, a CAR, an activator, and / or a ligand of the present invention may be delivered to a cell, tissue, or organism, including a subject in need thereof, via parenteral administration such as, but not limited to, intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural and intramuscular administration.

[0499] In some embodiments, a CAR, an activator, and / or a ligand of the present invention may be delivered to a cell, tissue, or organism, including a subject in need thereof, via one or more injections (e.g., a needle injection). Non-limiting methods of injection include injection of a composition (e.g., a saline based composition). Non-limiting sites of injection include, without limitation subcutaneous, intradermal, intramuscular, intravenous, intraprostatic, intratumoral, peritumoral, intralymphatic and intraperitoneal.

[0500] In some embodiments, the activator may be administered in accordance with any of the dosing regimens as described in Kholhapp et al., 202110, the contents of which is incorporated herein in its entirety for all purposes. An exemplary dosing regimen may comprise oral administration of the activator once a day for 14 days at 50 mg / kg / day.

[0501] In some embodiments, the activator may be navitoclax or venetoclax.

[0502] In some embodiments, the ligand may be lenalidomide.Activation of CAR-mediated Signaling

[0503] Also provided herein is a method for stimulating the activity of the activatable chimeric antigen receptor (CAR) in any host cell described herein. The method comprises contacting the host cell with an activator that promotes the dimer formed by the first and second member of the dimerization pair within the activatable CAR, resulting in activation of CAR-mediated signaling. The activator may be a small molecule or a polypeptide. In some embodiments, the activator bindsAttorney Docket No: 252457.000033 to the first member of the dimerization pair. In some embodiments, the activator binds to the second member of the dimerization pair. In some embodiments, the activator binds to the first and the second member of the dimerization pair.

[0504] In various embodiments, the activator is navitoclax, A-1331852, A-l 155463, venetoclax, ABT-199 (GDC-0199), obatoclax mesylate (GX15-070), HA14-1, ABT-737, TW-37, AT101, sabutoclax, gambogic acid, ARRY 520 trifluoroacetate, iMAC2, maritoclax, methylprednisolone, MIMI, ML 311, glossypol, BH3I-1, or 2-methoxy-antimycin A3 or derivatives thereof.

[0505] In some embodiments, the Bcl-2 or mutant thereof activator is venetoclax or navitoclax or derivatives thereof. In some embodiments, the Bcl-2 or mutant thereof activator is A-1331852 or A-l 155463 or derivatives thereof. In various embodiments, the Bcl-2 or mutant thereof activator is navitoclax, A-1331852, A-l 155463, venetoclax, ABT-199 (GDC-0199), obatoclax mesylate (GX15-070), HA14-1, ABT-737, TW-37, AT101, sabutoclax, gambogic acid, ARRY 520 trifluoroacetate, iMAC2, maritoclax, methylprednisolone, MIMI, ML 311, glossypol, BH3L1, or 2-methoxy-antimycin A3 or derivatives thereof.

[0506] In some embodiments, when the first and / or second member of the dimerization pair of any of the activatable CARs of the disclosure is derived from a Bcl-2 protein (e.g., a truncated and / or mutant Bcl-2 protein), the activator may be selected from, e.g., navitoclax or venetoclax. In some embodiments, the activator is navitoclax or derivatives thereof. In some embodiments, the activator is venetoclax or derivatives thereof.

[0507] In some embodiments, the Bcl-xL or mutant thereof activator is A-l 155463 or derivatives thereof. In various embodiments, the Bcl-xL or mutant thereof activator is A-l 155463, navitoclax, A-1331852, venetoclax, ABT-199 (GDC-0199), obatoclax mesylate (GX15-070), HA14-1, ABT-737, TW-37, AT101, sabutoclax, gambogic acid, ARRY 520 trifluoroacetate, iMAC2, maritoclax, methylprednisolone, MIMI, ML 311, glossypol, BH3I-1, or 2-methoxy- antimycin A3 or derivatives thereof. In some embodiments, when the first and / or second member of the dimerization pair of any of the activatable CARs of the disclosure is derived from a Bcl-xL protein (e.g., a truncated and / or mutant Bcl-xL protein), the activator may be selected from, e.g., A-l 155463. In some embodiments, the activator is A-l 155463 or derivatives thereof.

[0508] In some embodiments, the activator is venetoclax or derivatives thereof. Venetoclax is an orally bioavailable, selective small molecule inhibitor of the anti- apopto tic protein Bcl-2, withAttorney Docket No: 252457.000033 potential antineoplastic activity. Venetoclax is an antineoplastic agent used in the therapy of refractory chronic lymphocytic leukemia (CLL). The IUPAC name for venetoclax is 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen- 1 -yl]methyl]piperazin- 1 -yl]-N-[3-nitro-4-(oxan-4- ylmethylamino)phenyl]sulfonyl-2-(lH-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide. The chemical structure of venetoclax is as follows:

[0509] Without wishing to be bound by theory, venetoclax mimics BH3-only proteins, the native ligands of Bcl-2 and apoptosis activators, by binding to the hydrophobic groove of Bcl-2 proteins thereby repressing Bcl-2 activity and restoring apoptotic processes in tumor cells. Bcl2 protein is overexpressed in some cancers and plays an important role in the regulation of apoptosis; its expression is associated with increased drug resistance and tumor cell survival.

[0510] Effective doses of venetoclax may be determined using methods known to one skilled in the art. Effective doses may be determined, preferably in vitro, in order to identify the optimal dose range using methods known in the art and any of the various methods described herein. The dose and dose interval of venetoclax can be optimized depending on the disease to be targeted and combination therapy involved in the treatment. In some embodiments, venetoclax is administered at about 1-50 mg / kg. In some embodiments, venetoclax is administered at about 2.5 mg / kg. In certain embodiments, venetoclax is administered at about 2.5-5 mg / kg every 48 hours. In some embodiments, venetoclax is administered at a dose not to exceed about 100 mg / day -600 mg / day.Attorney Docket No: 252457.000033In some embodiments, venetoclax is administered at a dose not to exceed about 400 mg / day. In some embodiments, venetoclax is administered at sub-chemotherapeutic dose. The sub- chemotherapeutic dose of venetoclax may include about 2.5-5 mg / kg every 48 hours.

[0511] In some embodiments, the activator is navitoclax or derivatives thereof. Navitoclax is an orally active, synthetic small molecule and an antagonist of a subset of the B-cell leukemia 2 (Bcl-2) family of proteins with potential antineoplastic activity. Navitoclax selectively binds to apoptosis suppressor proteins Bcl-2, Bcl-xL, and Bcl-w, which are frequently overexpressed in a wide variety of cancers, including those of the lymph, breast, lung, prostate, and colon, and are linked to tumor drug resistance. Inhibition of these apoptosis suppressors prevents their binding to the apoptotic effectors Bax and Bak proteins, thereby triggering apoptotic processes in cells overexpressing Bcl-2, Bcl-xL, and Bcl-w. This eventually reduces tumor cell proliferation. The IUPAC name of navitoclax is 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-l- yl]methyl]piperazin- 1 -yl] -N- [4- [ [(2R)-4-morpholin-4-yl- 1 -phenylsulf anylbutan-2-yl] amino] -3 - (trifluoromethylsulfonyl)phenyl] sulfonylbenzamide. The chemical structure of navitoclax is as follows:

[0512] In some embodiments, the activator is A- 1155463 or derivatives thereof. A- 1155463 is an orally bioavailable, highly potent and selective Bcl-xL inhibitor with an ECso of 70 nM in Molt- 4 cell. The IUPAC name of A-l 155463 is 2-[8-(l,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-lH- isoquinolin-2-yl] -5 - [3- [4- [3-(dimethylamino)prop- 1 -ynyl] -2-fluorophenoxy ]propyl] -1,3- thiazole-4-carboxylic acid. The chemical structure of A-l 155463 is as follows:Attorney Docket No: 252457.000033

[0513] Effective doses of A- 1155463 may be determined using methods known to one skilled in the art. Effective doses may be determined, for example in vivo, in order to identify the optimal dose range using methods known in the art and any of the various methods described herein. The dose and dose interval of A- 1155463 can be optimized depending on the disease to be targeted and combination therapy involved in the treatment. In some embodiments, A- 1155463 is administered at about 1-50 mg / kg. In some embodiments, A-l 155463 is administered at about 2.5 mg / kg. In certain embodiments, A-l 155463 is administered at about 2.5-5 mg / kg every 24 hours. In some embodiments, A-l 155463 is administered at a dose not to exceed about 100 mg / day-600 mg / day. In some embodiments, A-l 155463 is administered at a dose not to exceed about 400 mg / day. In some embodiments, A-l 155463 is administered at sub-chemotherapeutic dose. The sub- chemotherapeutic dose of A-l 155463 may include about 2.5-5 mg / kg every 24 hours.Downregulation of CAR-mediated Signaling

[0514] Also provided herein is a method for downregulating the activity of the degradable chimeric antigen receptor (CAR) in any host cell described herein. The method comprises contacting the host cell with a ligand that mediates proteasomal degradation of the degradable CAR, resulting in downregulation of CAR-mediated signaling.

[0515] Degron sequence(s) present on the CAR molecule enhances sensitivity to ligand- induced degradation of the CAR molecule. An engineered zinc finger domains can be used as degron sequences to mediate degradation of the CAR molecules in the presence of the ligand. In some embodiments, the ligand mediates proteasomal degradation of the CAR by inducing interactions between the CRL4CRBNE3 ubiquitin ligase and a C2H2 zinc finger degron motif (Sci Transl Med., 2021 January 06; 13(575)). In some embodiments, the degrons are composed fromAttorney Docket No: 252457.000033 the matrix of 5 N-termini (ZN653, ZN827, ZFP91, ZN276, and / or IKZF3) with 7 C-termini (ZN787, ZN517, IKZF3, ZN654, PATZ1, E4F1, and / or ZKSC5). In some embodiments, the degron sequence is IKZF3. In some embodiments, the degron sequence is ZFP91. In some embodiments, the degron sequence is ZN827. In some embodiments, the degron sequence is ZN653. In some embodiments, the degron sequence is ZN276. In some embodiments, the degron sequence is ZFP91-IKZF3. In some embodiments, the degron sequence is IKZF3-ZN787. In some embodiments, the degron sequence is ZFP91-ZN787. In some embodiments, the degron sequence is ZN276-ZN787. In some embodiments, the degron sequence is ZN653-PATZ1. In some embodiments, the degron sequence is ZN827-ZKSC5.

[0516] The ligand may be a small molecule or a polypeptide. In some embodiments, the ligand binds to the degron sequence of the first polypeptide chain. In some embodiments, the ligand binds to the degron sequence of the second polypeptide chain. In some embodiments, the ligand binds to the degron sequence of the first polypeptide chain and the second polypeptide chain.

[0517] In various embodiments, the ligand is lenalidomide, thalidomide, pomalidomide, avadomide, or iberdomide, or derivatives thereof.

[0518] In various embodiments, the ligand of the degron sequence is lenalidomide, thalidomide, pomalidomide, avadomide, or iberdomide, or derivatives thereof. In various embodiments, the ligand of the degron sequence is lenalidomide or derivative thereof.

[0519] In some embodiments, when the degron sequence of the first polypeptide chain and / or second polypeptide chain of any of the CARs of the disclosure is derived from degron sequence comprising an amino acid sequence set forth in SEQ ID NO: 39 (e.g., SEQ ID NO: 39, a functional fragment or a derivative thereof), the ligand may be selected from, e.g., lenalidomide, thalidomide, pomalidomide, avadomide, or iberdomide. In some embodiments, the ligand is lenalidomide or derivative thereof. In some embodiments, the ligand is thalidomide or derivative thereof. In some embodiments, the ligand is pomalidomide or derivative thereof. In some embodiments, the ligand is avadomide or derivative thereof. In some embodimen...

Claims

Attorney Docket No: 252457.000033Claims1. A switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first polypeptide chain and the second polypeptide chain comprise a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR- mediated signaling.

2. The CAR of claim 1 , wherein the first and second member of the dimerization pair are located extracellularly.

3. The CAR of claim 2, wherein the first member of the dimerization pair is located in the first polypeptide chain between the extracellular region and the first TM region; and the second member of the dimerization pair is located in the second polypeptide chain N-terminal to the second TM region.

4. The CAR of claim 1 , wherein the first and second member of the dimerization pair are located intracellularly.

5. The CAR of claim 4, wherein the first member of the dimerization pair is located in the first polypeptide chain C-terminal to the first TM region, and the second member of the dimerizationAttorney Docket No: 252457.000033 pair is located in the second polypeptide chain between the second TM region and the intracellular signaling ED.

6. The CAR of claim 5, wherein the first polypeptide chain comprises a first co-stimulatory ED and the second polypeptide chain comprises a second co-stimulatory ED, and wherein the first member of the dimerization pair is located in the first polypeptide chain C-terminal to the first co- stimulatory ED, and the second member of the dimerization pair is located in the second polypeptide chain between the second co-stimulatory ED and the intracellular signaling ED.

7. The CAR of claim 5, wherein the first polypeptide chain comprises a first co-stimulatory ED, the second polypeptide chain comprises a second co-stimulatory ED, and wherein the first member of the dimerization pair is located in the first polypeptide chain C-terminal to the first TM region and N-terminal to the first co-stimulatory ED, and the second member of the dimerization pair is located in the second polypeptide chain between the second TM region and the second co- stimulatory ED.

8. The CAR of any one of claims 1-7, wherein the second polypeptide chain comprises an extracellular region which does not comprise a target-binding capacity.

9. A switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first member of a dimerization pair; iii) a first linker region; iv) a first transmembrane (TM) region; and v) a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) a second member of the dimerization pair; ii) a second linker region; iii) a second TM region;Attorney Docket No: 252457.000033 iv) a second co-stimulatory ED; and v) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

10. A switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region; iv) a first co-stimulatory endodomain (ED); and v) a first member of a dimerization pair, and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second TM region; iv) a second co-stimulatory ED; v) a second member of the dimerization pair; and vi) an intracellular signaling ED, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

11. A switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region; ii) a first linker region; iii) a first transmembrane (TM) region;Attorney Docket No: 252457.000033 iv) a first member of a dimerization pair; and v) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising in the direction from the N terminus to the C terminus: i) an extracellular region which does not comprise a target-binding capacity; ii) a second linker region; iii) a second TM region; iv) a second member of the dimerization pair; v) a second co-stimulatory ED; and vi) an intracellular signaling ED, and wherein the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling and the first polypeptide chain does not comprise an intracellular signaling ED.

12. The CAR of any one of claims 1-11, wherein the first member and the second member of the dimerization pair are derived from a Bcl-2 protein or a Bcl-xL protein.

13. The CAR of claim 12, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein.

14. The CAR of claim 13, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking the transmembrane domain.

15. The CAR of claim 14, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking both the transmembrane domain and the flexible loop do-main (FLD) between the first and second protein helices.

16. The CAR of claim 12, wherein the first and / or second member of the dimerization pair comprises the amino acid sequenceMAHPGRTGYDNREIVMKYIHYKLSQRGYEWDAGDDVEENRTEAPEGTESEVVHLTLR QAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGV MCVESVNREMSPEVDNIAEWMTEYENRHEHTWIQDNGGWDAFVEEYGP (SEQ ID NO: 8), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8.Attorney Docket No: 252457.00003317.The CAR of claim 12, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein.

18. The CAR of claim 17, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking the transmembrane domain.

19. The CAR of claim 18, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking both the transmembrane domain and the flexible loop do-main (FLD) between the first and second protein helices.

20. The CAR of claim 12, wherein the first and / or second member of the dimerization pair comprises the amino acid sequenceMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFEL RYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKE MQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERED (SEQ ID NO:74), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 74.

21. The CAR of any one of claims 1 -20, wherein the activator is a small molecule or a polypeptide.

22. The CAR of any one of claims 1-16 and 21, wherein the first member and the second member of the dimerization pair are derived from a Bcl-2 protein and said activator is navitoclax or venetoclax.

23. The CAR of claim 22, wherein said activator is venetoclax.

24. The CAR of any one of claims 1-12 and 17-21, wherein the first member and the second member of the dimerization pair are derived from a Bcl-xL protein and said activator is A- 1155463.

25. The CAR of any one of claims 1-24, wherein the first polypeptide chain and / or the second polypeptide chain further comprises a degron sequence that mediates proteasomal degradation ofAttorney Docket No: 252457.000033 the CAR in the presence of a ligand.

26. The CAR of claim 25, wherein the degron sequence is located intracellularly within the first polypeptide chain and / or the second polypeptide chain.

27. The CAR of claim 26, wherein the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and / or second polypeptide chain.

28. The CAR of claim 27, wherein the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and / or between the co-stimulatory ED and the intracellular signaling ED in the second polypeptide chain.

29. The CAR of claim 26, wherein the degron sequence is located C-terminal to the co-stimulatory ED in the first polypeptide chain and / or C-terminal to the intracellular signaling ED in the second polypeptide chain.

30. The CAR of any one of claims 25-29, wherein the degron sequence is a Cereblon (CRBN) binding moiety and the ligand is lenalidomide.

31. The CAR of claim 30, wherein the degron sequence comprises LQCEICGFTCRQKGNLLRHIKLH (SEQ ID NO: 39), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 39.

32. The CAR of any one of claims 2, 3, 9, and 12-31 , wherein the first polypeptide chain comprises a first linker region interposed between the first member of a dimerization pair and the first TM region.

33. The CAR of any one of claims 2, 3, 9, and 12-32, wherein the second polypeptide chain comprises a second linker region interposed between the second member of the dimerization pair and the second TM region.Attorney Docket No: 252457.00003334. The CAR of any one of claims 4-8, and 12-31, wherein the first polypeptide chain comprises a first linker region interposed between the extracellular region and the first TM region.

35. The CAR of any one of claims 4-8, 10-31, and 34, wherein the second polypeptide chain comprises a second linker region N-terminal to the second TM region.

36. The CAR of any one of claims 32-35, wherein the first and / or second linker region is an immunoglobulin hinge region or a hinge region derived from CD8, CD8a, or CD28.

37. The CAR of claim 36, wherein the first and / or second linker region is a hinge region derived from CD8a.

38. The CAR of any one of claims 1-37, wherein the first polypeptide chain comprises a targetbinding capacity in the extracellular region.

39. The CAR of claim 38, wherein the target-binding capacity is provided by an antigen-binding polypeptide, a receptor, or a natural ligand for a target cell antigen or receptor.

40. The CAR of claim 39, wherein the target-binding capacity is provided by an antigen-binding polypeptide.

41. The CAR of claim 40, wherein the antigen-binding polypeptide is an antibody or an antibody fragment.

42. The CAR of claim 40 or claim 41, wherein the antigen-binding polypeptide is a single chain antibody.

43. The CAR of any one of claims 39-41, wherein the antigen recognized by the antigen-binding polypeptide is selected from a tumor-associated antigen, an infection-associated antigen and an auto-antigen.Attorney Docket No: 252457.00003344. The CAR of claim 43, wherein the tumor-associated antigen is associated with a solid tumor.

45. The CAR of any one of claims 39-41, wherein the antigen recognized by the antigen-binding polypeptide is selected from CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CA125, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, EpCAM, EPhA2, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF-I), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1 , MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleophosmin, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, NeuGcGM3, N-glycolyl GM3 ganglioside, Neu5Gc, GM3- Ganglioside, GD3, GM2, carbohydrate antigens, ganglioside antigens, Lewis Y, Lewis B, CD 123, and Kappa chain of immunoglobulin.

46. The CAR of claim 45, wherein the antigen recognized by the antigen-binding polypeptide is PSMA.

47. The CAR of claim 45, wherein the antigen recognized by the antigen-binding polypeptide is CD 19.

48. The CAR of claim 45, wherein the antigen recognized by the antigen-binding polypeptide is EpCAM.Attorney Docket No: 252457.00003349. The CAR of claim 45, wherein the antigen recognized by the antigen-binding polypeptide is EPhA2.

50. The CAR of claim 39, wherein the extracellular region of the first polypeptide chain comprises a natural ligand for a target cell antigen or receptor.

51. The CAR of claim 50, wherein the natural ligand for a target cell antigen or receptor is an NKG2D ectodomain.

52. The CAR of claim 39, wherein the extracellular region of the first polypeptide chain comprises a T-cell receptor (TCR) based recognition domain.

53. The CAR of claim 52, wherein the TCR based recognition domain is a single chain TCR.

54. The CAR of any one of claims 1-53, wherein the first and / or second TM region is derived from CD8, CD8a, CD4, CD3-zeta, CD3-epsilon, CD28, CD45, CD4, CD5, CD7, CD9, CD 16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (OX-40), CD137, CD154, DAP10, or DAP 12.

55. The CAR of any one of claims 1-54, wherein the first and second TM regions are the same.

56. The CAR of claim 54, wherein the first and second TM regions are derived from CD8a.

57. The CAR of any one of claims 8 and 10-56, wherein the extracellular region of the second polypeptide chain comprises a stabilizing domain.

58. The CAR of any one of claims 8 and 10-57, wherein the extracellular region of the second polypeptide chain comprises an extracellular region derived from DAP 10 or DAP 12.

59. The CAR of any one of claims 1-58, wherein the first and / or second co-stimulatory ED isAttorney Docket No: 252457.000033 derived from 4-1BB (CD137), CD28, ICOS, CD134 (OX-40), BTLA, CD27, CD30, GITR, CD226, or HVEM.

60. The CAR of claim 59, wherein the first and / or second co-stimulatory EDs are derived from 4- 1BB (CD137).

61. The CAR of claim 59 or claim 60, wherein the first and the second co-stimulatory EDs are different.

62. The CAR of any one of claims 1-61, wherein the intracellular signaling ED is derived from DAP10, DAP12, Fc epsilon receptor I gamma chain (FCER1G), FcR beta CD3-delta, CD3- epsilon, CD3-gamma, CD3-zeta, CD226, CD66d, CD79A, or CD79B.

63. The CAR of claim 62, wherein the intracellular signaling ED is derived from CD3-zeta.

64. The CAR of any one of claims 1-63, wherein the first and / or second polypeptide chain further comprises one or more additional polypeptide sequences.

65. The CAR of claim 64, wherein said one or more additional polypeptide sequences are selected from one or more additional co-stimulatory EDs, signal sequences, separation sequences, epitope tags, and polypeptides that produce a detectable signal.

66. The CAR of claim 65, wherein the signal sequence is derived from CD8a.

67. The CAR of claim 65, wherein the epitope tag is cMyc.

68. The CAR of claim 65, wherein the separation sequence is T2A.

69. The CAR of any one of claims 1-45 and 54-68, wherein the extracellular region of the first polypeptide chain comprises the amino acid sequenceVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNAttorney Docket No: 252457.000033QKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR (SEQ ID NO: 4),DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 33),QVKLQQSGAELVRPGASVKLSCKASGYTFTNYWINWVKQRPGQGLEWIGNIYPSYIYTNYNQEFKDKVTLTVDESSSTAYMQLSSPTSEDSAVYYCTRSPYGYDEYGLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLTVTAGEKVTMNCKSSQSLLNSRNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYVYPLTFGAGTKLEIKR (SEQ ID NO: 35), orQVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGQALEWMGTISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREAIFTYWGRGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKASQDINNYLSWYQQKPGQAPRLLIYRANRLVDGVPDRFSGSGYGTDFTLTINNIESEDAAYYFCLKYDVFPYTFGQGTKVE IK (SEQ ID NO: 37).

70. The CAR of any one of claims 1-69, wherein the intracellular signaling ED comprises the amino acid sequenceRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 22).

71. The CAR of any one of claims 9-70, wherein the first and / or second linker region comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 10).

72. The CAR of any one of claims 1-71, wherein the first and / or second TM region comprises theAttorney Docket No: 252457.000033 amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12).

73. The CAR of any one of claims 1-72, wherein the first and / or second co-stimulatory ED comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14).

74. The CAR of any one of claims 1-3, 8-9, 12-45, and 54-73 wherein the first polypeptide chain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 51, SEQ ID NO: 55, or SEQ ID NO: 59.

75. The CAR of any one of claims 1-3, 8-9, 12-46, and 54-73, wherein the second polypeptide chain comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 26, SEQ ID NO: 43, or SEQ ID NO: 45.

76. A switchable chimeric antigen receptor (CAR) comprising:(i) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 26;(ii) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:43;(iii) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 24, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:45;(iv) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 51, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:26;Attorney Docket No: 252457.000033(v) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 55, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:26; or(vi) a) a first polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 59, and b) a second polypeptide chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:26.

77. A nucleic acid molecule encoding the switchable CAR of any one of claims 1-76.

78. The nucleic acid molecule of claim 77, comprising(i) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 23; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25;(ii) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 23; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 42;(iii) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 23; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 44;(iv) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 50; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25;(v) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 54; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, orAttorney Docket No: 252457.000033 consisting essentially of the sequence SEQ ID NO: 25; or(vi) a nucleotide sequence encoding the first polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 58; and b) a nucleotide sequence encoding the second polypeptide chain of the CAR comprising, consisting of, or consisting essentially of the sequence SEQ ID NO: 25.

79. A nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the switchable CAR of any one of claims 1-76.

80. The nucleic acid molecule of claim 79, wherein the nucleotide sequence encoding the first polypeptide chain of the CAR is SEQ ID NO: 23, SEQ ID NO: 50, SEQ ID NO: 54, or SEQ ID NO: 58.

81. A nucleic acid molecule comprising a nucleotide sequence encoding the second polypeptide chain of the switchable CAR of any one of claims 1-76.

82. The nucleic acid molecule of claim 81 , wherein the nucleotide sequence encoding the second polypeptide chain of the CAR is SEQ ID NO: 25, SEQ ID NO: 42, or SEQ ID NO: 44.

83. The nucleic acid molecule of any one of claims 77-80, wherein the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter.

84. The nucleic acid molecule of any one of claims claim 77-78 and 81-82, wherein the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter.

85. The nucleic acid molecule of claim 78, wherein the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter, and the first and second promoters are the same.Attorney Docket No: 252457.00003386. The nucleic acid molecule of claim 78, wherein the nucleotide sequence encoding the first polypeptide chain of the CAR is operably linked to a first promoter, the nucleotide sequence encoding the second polypeptide chain of the CAR is operably linked to a second promoter, and the first and second promoters are different.

87. The nucleic acid molecule of claim 78, wherein the nucleotide sequences encoding the first and second polypeptide chains of the CAR are operably linked to a single promoter.

88. The nucleic acid molecule of any one of claims 83-87, wherein the first and / or second promoter is a T lymphocyte-specific promoter or an NK cell-specific promoter.

89. The nucleic acid molecule of any one of claims 77-88 which is a DNA molecule.

90. The nucleic acid molecule of any one of claims 77-88 which is an RNA molecule.

91. An expression system comprising a first nucleotide sequence encoding the first polypeptide chain of the switchable CAR of any one of claims 1-76 and a second nucleotide sequence encoding the second polypeptide chain of the switchable CAR of any one of claims 1-76.

92. A recombinant vector comprising the nucleic acid molecule of any one of claims 77-90.

93. The vector of claim 92 which is a viral vector.

94. The vector of claim 93, wherein the vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, and a vaccinia virus vector.

95. The vector of claim 94, wherein the vector is a lentiviral vector.

96. An isolated host cell comprising the switchable CAR of any one of claims 1-76.Attorney Docket No: 252457.00003397. An isolated host cell comprising the nucleic acid molecule of any one of claims 77-90.

98. An isolated host cell comprising the vector of any one of claims 92-95.

99. The host cell of any one of claims 96-98, which is a mammalian cell.

100. The host cell of any one of claims 96-99, which is selected from a cytotoxic cell, a T cell, a stem cell, a progenitor cell, and a cell derived from a stem cell or a progenitor cell.

101. The host cell of claim 100, wherein the T cell is selected from T-helper cells, cytotoxic T- cells, T-regulatory cells (Treg), and gamma-delta T cells.

102. The host cell of claim 101, wherein the cytotoxic cell is a cytotoxic T cell or a NK cell.

103. The host cell of any one of claims 96-102, wherein the host cell has been activated and / or expanded ex vivo.

104. The host cell of any one of claims 96-102, wherein the host cell is an allogeneic cell.

105. The host cell of any one of claims 96-102, wherein the host cell is an autologous cell.

106. The host cell of claim 96, wherein the host cell has been isolated from a subject having a disease.

107. The host cell of claim 96, wherein the subject is human.

108. A pharmaceutical composition comprising the host cell of any one of claims 96-107 and a pharmaceutically acceptable carrier and / or excipient.

109. A method for producing the host cell of any one of claims 96-107 comprising genetically modifying said cell with the nucleic acid molecule of any one of claims 77-90, the expressionAttorney Docket No: 252457.000033 system of claim 91, or the vector of any one of claims 92-95.

110. The method of claim 109, wherein the genetic modification is conducted ex vivo.

111. The method of claim 109 or claim 110, wherein the method further comprises activation and / or expansion of the cell ex vivo.

112. A method for stimulating elimination of a cell comprising an antigen in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 1-76 and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to said antigen and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

113. The method of claim 112, wherein the antigen is selected from a tumor- associated antigen, an infection-associated antigen and an auto-antigen.

114. The method of claim 113, wherein the antigen is a tumor-associated antigen.

115. The method of claim 114, wherein the tumor-associated antigen is associated with a solid tumor.

116. The method of claim 114, wherein the tumor-associated antigen is PSMA.

117. The method of claim 113, wherein the antigen is an infection-associated antigen.

118. The method of claim 113, wherein the antigen is an auto-antigen.

119. The method of any one of claims 112-115 or 117, wherein the antigen is CD19.

120. The method of any one of claims 112-115, wherein the antigen is EpCAM.Attorney Docket No: 252457.000033121. The method of any one of claims 112-115, wherein the antigen is EPhA2.

122. A method for stimulating elimination of a cell comprising PSMA in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 46 and 54-73, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR- mediated signaling.

123. A method for stimulating elimination of a cell comprising CD 19 in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 47 and 54-73, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR- mediated signaling.

124. A method for stimulating elimination of a cell comprising EpCAM in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 48 and 54-73, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

125. A method for stimulating elimination of a cell comprising EPhA2 in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 49 and 54-73, and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR- mediated signaling.Attorney Docket No: 252457.000033126. A method for treating a cancer in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 1-76 and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said cancer and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

127. The method of claim 126, wherein the cancer is from a solid tumor.

128. The method of claim 126, wherein the cancer is selected from carcinoma, melanoma, prostate cancer, sarcoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, and retinoblastoma.

129. The method of claim 126, wherein the cancer is a leukemia or a lymphoma.

130. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to the subject (i) an effective amount cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 46 and 54-73 and (ii) an effective amount of an activator, wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

131. A method for treating an infection in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of cytotoxic T cells or NK cells comprising the switchable CAR of any one of claims 45 and 54-73 and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said infection and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

132. A method for treating an inflammatory condition or an autoimmune disease in a subject in need thereof, said method comprising administering to the subject (i) an effective amount of T-Attorney Docket No: 252457.000033 helper cells or Treg cells comprising the switchable CAR of any one of claims 45 and 54-73 and (ii) an effective amount of an activator, wherein the extracellular region of said CAR binds to an antigen associated with said inflammatory condition or an autoimmune disease, and wherein said activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

133. The method of claim 132, wherein the method results in reducing an immune response to a transplanted organ or tissue.

134. The method of any one of claims 112-133, said method comprising: a) isolating T cells or NK cells from the subject; b) genetically modifying said T cells or NK cells ex vivo with the nucleic acid molecule of any one of claims 77-90, the expression system of claim 91, or the vector of any one of claims 92-95; c) optionally, expanding and / or activating said T cells or NK cells before, after or during step (b); and d) introducing the genetically modified T cells or NK cells into the subject.

135. The method of any one of claims 112-134, wherein the subject is human.

136. A method for activating the switchable CAR in the host cell of any one of claims 96-107, comprising contacting the host cell with an activator, wherein the activator promotes the dimerization of the first member and the second member of the dimerization pair within said CAR resulting in activation of CAR-mediated signaling.

137. The method of claim 136, wherein the first member and the second member of the dimerization pair are derived from a Bcl-2 protein or a Bcl-xL protein.

138. The method of claim 137, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein.Attorney Docket No: 252457.000033139. The method of claim 138, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking the transmembrane domain.

140. The CAR of claim 139, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-2 protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.141.The method of claim 137, wherein the first and / or second member of the dimerization pair comprises the amino acid sequenceMAHPGRTGYDNREIVMKYIHYKLSQRGYEWDAGDDVEENRTEAPEGTESEVVHLTLR QAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGV MCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGP (SEQ ID NO: 8), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8.142.The method of claim 136, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein.143.The method of claim 142, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking the transmembrane domain.

144. The CAR of claim 143, wherein the first and / or second member of the dimerization pair comprises a truncated and / or mutant Bcl-xL protein lacking both the transmembrane domain and the flexible loop domain (FLD) between the first and second protein helices.

145. The method of claim 137, wherein the first and / or second member of the dimerization pair comprises the amino acid sequenceMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEAVKQALREAGDEFEL RYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKE MQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERED (SEQ ID NO: 74), or a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 74.Attorney Docket No: 252457.000033146. The method of any one of claims 136-145, wherein the activator is a small molecule or a polypeptide.

147. The method of 146, wherein said activator is selected from navitoclax, venetoclax or A- 1155463.

148. The method of claim 147, wherein said activator is venetoclax.

149. The method of claim 148, wherein venetoclax is administered at about 1 - 50 mg / kg.

150. The method of claim 149, wherein venetoclax is administered at about 2.5 - 5 mg / kg.

151. The method of claim 150, wherein venetoclax is administered at a dose not to exceed about100-600 mg / day.

152. The method of claim 151, wherein venetoclax is administered at a dose not to exceed about 400 mg / day.

153. The method of claim 149, 151, or 152, wherein the venetoclax is administered at sub- chemotherapeutic dose.

154. The method of any one of claims 148-153, wherein venetoclax is administered orally, intravenously, subcutaneously, via intratumoral injection, or via peritumoral injection.

155. The method of any one of claims 136-154, further comprising contacting the host cell of any one of claims 96-107 with a ligand, wherein the ligand that mediates proteasomal degradation of the degradable CAR, resulting in downregulation of CAR-mediated signaling.

156. The method of claim 155, wherein the ligand is lenalidomide.

157. The method of claim 156, wherein lenalidomide is administered at about 1-25 mg / kg.Attorney Docket No: 252457.000033158. The method of claim 157, wherein lenalidomide is administered at about 2-5 mg / kg.

159. The method of claim 156, wherein lenalidomide is administered at a dose not to exceed about 50 mg / day-100 mg / day.

160. The method of claim 156, wherein lenalidomide is administered at sub-chemotherapeutic dose.

161. A switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region comprising a binding means for specifically interacting with a target; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region; ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, and wherein the first polypeptide chain and the second polypeptide chain comprise a means for dimerizing the two polypeptides in the presence of an activator resulting in activation of CAR- mediated signaling.

162. A switchable chimeric antigen receptor (CAR) comprising: a) a first polypeptide chain comprising: i) an extracellular region; ii) a first transmembrane (TM) region; and iii) optionally, a first co-stimulatory endodomain (ED), and b) a second polypeptide chain comprising: i) a second TM region;Attorney Docket No: 252457.000033 ii) optionally, a second co-stimulatory ED; and iii) an intracellular signaling ED, and wherein the first polypeptide chain does not comprise an intracellular signaling ED, wherein the second polypeptide chain does not comprise a target-binding capacity, the improvement comprising: said first polypeptide chain and said second polypeptide chain comprises a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

163. A switchable chimeric antigen receptor (CAR) comprising a first polypeptide chain and a second polypeptide chain, the improvement comprising: said first polypeptide chain does not comprise an intracellular signaling ED, said second polypeptide chain does not comprise a targetbinding capacity, and said first polypeptide chain and said second polypeptide chain comprises a first member and a second member of a dimerization pair, respectively, and the first and second member of the dimerization pair form a dimer in the presence of an activator resulting in activation of CAR-mediated signaling.

164. The CAR of any one of claims 157, 162, or 163, wherein each of the first polypeptide chain and the second polypeptide chain further comprises a degron sequence that mediates proteasomal degradation of the CAR in the presence of a ligand.

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