Car-expressing cells against multiple tumor antigens and uses thereof

Abstract

The invention provides compositions and methods for treating cancer by using immune effector cells (e.g., T cells, NK cells) engineered to conditionally express an agent which enhances the immune effector response of an immune effector cell that expresses a Chimeric Antigen Receptor (CAR). The conditional agents described herein include agents that target a cancer associated antigen, e.g., a CAR, agents that inhibit one or more checkpoint inhibitors of the immune response, and a cytokine.

1 . An isolated nucleic acid, comprising:
 (a) a nucleotide sequence encoding an agent, wherein the agent is a polypeptide or nucleic acid that enhances the immune response against a target cell or a cancer cell, wherein the agent is chosen from a chimeric antigen receptor (first CAR), an inhibitor of a checkpoint inhibitor, or a cytokine; and   (b) a first activation-conditional control region operatively linked to (a).   
     
     
         2 . The isolated nucleic acid of  claim 1 , further comprising:
 (c) a nucleotide sequence encoding a second agent, wherein the second agent comprises a polypeptide or nucleic acid, that enhances the immune response against a target cell or a cancer cell, wherein the second agent is a CAR (second CAR); and   (d) a second control region operatively linked to (c), wherein the second control region is other than an activation-conditional control region or the second control region comprises a constitutive control region.   
     
     
         3 . An isolated nucleic acid comprising:
 (a) a nucleotide sequence encoding an agent, wherein the agent is a polypeptide or nucleic acid that enhances the immune response against a target cell, or a cancer cell, and wherein the agent is chosen from a chimeric antigen receptor (first CAR), an inhibitor of a checkpoint inhibitor, or a cytokine, wherein the first CAR comprises a first an antigen binding domain, a transmembrane domain, and an intracellular signaling domain;   (b) a first activation-conditional control region operatively linked to (a);   (c) a nucleotide sequence encoding a second CAR comprising a second antigen binding domain, a transmembrane domain, and an intracellular signaling domain; and   (d) a second control region operatively linked to (c), wherein the second control region is other than an activation-conditional control region and wherein the second control region comprises a constitutive control region.   
     
     
         4 . The isolated nucleic acid of  claim 3 , wherein the second control region comprises a constitutive control region, e.g., an elongation factor 1 alpha (EF1a) control region. 
     
     
         5 . The isolated nucleic acid of  claim 3 , wherein the first or second CARs comprise an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein the antigen binding domain binds to a cancer associated antigen chosen from: mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha (FRa), ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1. 
     
     
         6 . (canceled) 
     
     
         7 . The isolated nucleic acid of  claim 5 , wherein the mesothelin binding domain comprises:
 (i) a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3) of any mesothelin binding domain in Table 2; and a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) of a mesothelin binding domain in Table 2;   (ii) the LC CDR1, LC CDR2, and LC CDR3 of the LC CDR sequences listed in Table 4; and the HC CDR1, HC CDR2, and HC CDR3 of the HC CDR sequences listed in Table 3;   (iii) an amino acid sequence of Table 2 chosen from SEQ ID NO: 51, SEQ ID NO: 57, SEQ ID NO: 70, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; or   (iv) an amino acid sequence with at least 95-99% homology to an amino acid sequence provided in Table 2, chosen from SEQ ID NO: 51, SEQ ID NO: 57, SEQ ID NO: 70, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69.   
     
     
         8 .- 13 . (canceled) 
     
     
         14 . The isolated nucleic acid of  claim 3 , wherein:
 (i) the first antigen and the second antigen are co-expressed on a cancer cell;   (ii) the first antigen is expressed on a first population of cancer cells and the second antigen is expressed on a second population of cancer cells; or   (iii) the second antigen is expressed on a cancer cell and wherein the first antigen is not expressed on the cancer cell.   
     
     
         15 .- 16 . (canceled) 
     
     
         17 . The isolated nucleic acid of  claim 3 , wherein:
 (i) the first antigen is chosen from: Folate receptor alpha (FRa), ERBB2 (Her2/neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), Mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1; and/or   (ii) the second antigen is selected from the group consisting of: Mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor alpha (FRa), ERBB2 (Her2/neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.   
     
     
         18 .- 22 . (canceled) 
     
     
         23 . The isolated nucleic acid of  claim 3 , wherein the transmembrane domain of the first and/or second CAR comprises:
 (i) a transmembrane domain from a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154;   (ii) a transmembrane domain that comprises the amino acid sequence of SEQ ID NO: 12,   (iii) a transmembrane domain that comprises an amino acid sequence comprises at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:12, or   (iv) a transmembrane domain that comprises a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:12.   
     
     
         24 . (canceled) 
     
     
         25 . The isolated nucleic acid of  claim 3 , wherein the antigen binding domain of the first and/or second CAR is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises SEQ ID NO:4, or a sequence with 95-99% identity thereof. 
     
     
         26 . (canceled) 
     
     
         27 . The isolated nucleic acid of  claim 3 , wherein the intracellular signaling domain of the first and/or second CAR comprises a costimulatory signaling domain comprising:
 (i) a functional signaling domain obtained from a protein chosen from a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, or a ligand that specifically binds with CD83;   (ii) the amino acid sequence of SEQ ID NO:14;   (iii) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:14, or   (iv) an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:14.   
     
     
         28 . (canceled) 
     
     
         29 . The isolated nucleic acid of  claim 3 , wherein the intracellular signaling domain comprises:
 (i) a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta;   (ii) the amino acid sequence of SEQ ID NO: 14 and/or the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20; or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:14 and/or the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20; or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:14 and/or the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20; or   (iii) the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:20, wherein the amino acid sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.   
     
     
         30 .- 32 . (canceled) 
     
     
         33 . The isolated nucleic acid of  claim 3 , wherein the first CAR comprises:
 (i) the amino acid sequence of: an amino acid sequence in Table 7, the amino acid sequence of SEQ ID NO: 150, or the amino acid sequence of SEQ ID NO: 152;   (ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to: an amino acid sequence in Table 7, the amino acid sequence of SEQ ID NO: 150, or the amino acid sequence of SEQ ID NO: 152;   (iii) an amino acid sequence with 95-99% identity to: an amino acid sequence in Table 7, the amino acid sequence of SEQ ID NO: 150, or the amino acid sequence of SEQ ID NO: 152;   (iv) a nucleic acid sequence in Table 7, the nucleic acid sequence of SEQ ID NO: 151 or the nucleic acid sequence of SEQ ID NO: 153; or   (v) a nucleic acid sequence with 95-99% identity to: a nucleic acid sequence in Table 7, or the nucleic acid sequence of SEQ ID NO: 151 or the nucleic acid sequence SEQ ID NO: 153.   
     
     
         34 . (canceled) 
     
     
         35 . The isolated nucleic acid  claim 3 , wherein the second CAR comprises:
 (i) an amino acid sequence in Table 6chosen from SEQ ID NO: 104, SEQ ID NO: 110, SEQ ID NO: 124, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123;   (ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to an amino acid sequence in Table 6, chosen from SEQ ID NO: 104, SEQ ID NO: 110, SEQ ID NO: 124, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123;   (iii) an amino acid sequence with 95-99% identity to an amino acid sequence in Table 6, chosen from SEQ ID NO: 104, SEQ ID NO: 110, SEQ ID NO: 124, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123;   (iv) a nucleic acid sequence in Table 6 chosen from SEQ ID NO: 129, SEQ ID NO: 135, SEQ ID NO: 149, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO: 148; or   (v) a nucleic acid sequence with 95-99% identity to a nucleic acid sequence in Table 6 chosen from SEQ ID NO: 129, SEQ ID NO: 135, SEQ ID NO: 149, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO: 148.   
     
     
         36 . (canceled) 
     
     
         37 . The isolated nucleic acid of  claim 3 , further comprising:
 (e) a sequence encoding a third CAR; and/or,   (f) a second activation-conditional control region operatively linked to (e), wherein the activation-conditional control region is operatively linked to (f).   
     
     
         38 . The isolated nucleic acid of  claim 3 , wherein (a) comprises:
 (i) a nucleotide sequence encoding an inhibitor of a checkpoint inhibitor of the immune response, wherein:
 (a) the checkpoint inhibitor of the immune response is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GALS, adenosine, and TGFR beta, or a combination thereof; 
 (b) the nucleotide sequence encodes an RNA-based inhibitor, e.g., an shRNA; or 
 (c) the nucleotide sequence encodes an antibody molecule; and/or 
   (ii) a nucleotide sequence encoding a cytokine, wherein the cytokine comprises IL-2, IL-7, IL-15, or IL-21.   
     
     
         39 .- 43 . (canceled) 
     
     
         44 . The isolated nucleic acid of  claim 3 , wherein the activation-conditional control region comprises:
 (i) a nucleotide sequence that induces expression of (a) upon immune effector cell activation;   (ii) a promoter of a gene that is induced upon immune effector cell activation;   (iii) a nuclear factor of activated T cells (NFAT) promoter, an NF-kB promoter, an IL-2 promoter, or an IL-2 receptor (IL-2R) promoter;   (iv) one or more binding sites for a transcription modulator, e.g., a transcription factor, that induces gene expression upon immune effector cell activation;   (v) one or more NFAT binding sides;   (vi) comprises one or more sequences selected from GGAAA, GGGACT, SEQ ID NO: 261, or SEQ ID NO: 262.   
     
     
         45 .- 49 . (canceled) 
     
     
         50 . The isolated nucleic acid of  claim 3 , wherein the second control region that is other than an activation-conditional control region is a constitutive control region, wherein the constitutive control region comprises a constitutive promoter, an EF1alpha promoter, or the nucleic acid sequence of SEQ ID NO: 1. 
     
     
         51 .- 53 . (canceled) 
     
     
         54 . The isolated nucleic acid of  claim 3 , wherein:
 (i) (a) and (b) are disposed on a single nucleic acid molecule, a viral vector, or a lentivirus vector; or (a) and (c) are disposed on a single nucleic acid molecule, a viral vector, or a lentivirus vector;   (ii) (a), (b), (c) and (d) are all disposed on a single nucleic acid molecule, a viral vector, or a lentivirus vector;   (iii) (a) is disposed on a first nucleic acid molecule, or a first viral vector, or a first lentivirus vector; and (c) is disposed on a second nucleic acid molecule, or a second viral vector, or a second lentivirus vector; or (a) and (b) are disposed on a first nucleic acid molecule, or a first viral vector or a first lentivirus vector; and (c) and (d) are disposed on a second nucleic acid molecule, or a second viral vector, or a second lentivirus vector;   (iv) the isolated nucleic acid comprises a bicistronic viral vector, or a bicistronic lentivirus vector; or   (v) (a) is translated as a first RNA and (c) is translated as a second RNA.   
     
     
         55 .- 62 . (canceled) 
     
     
         63 . A vector system comprising the isolated nucleic acid of  claim 3 . 
     
     
         64 . A cell or an immune effector cell comprising the isolated nucleic acid of  claim 3 , optionally wherein the cell is a human cell, a T cell, or an NK cell. 
     
     
         65 .- 68 . (canceled) 
     
     
         69 . A method of making a CAR cell, comprising introducing into a cell, the isolated nucleic acid of  claim 3 ,
 thereby making the CAR cell.   
     
     
         70 .- 74 . (canceled) 
     
     
         75 . A method of treating a subject having a cancer comprising administering to the subject an effective amount of the cell of  claim 64 ,
 thereby treating a subject.   
     
     
         76 .- 82 . (canceled) 
     
     
         83 . The method of  claim 75 , wherein the cancer is:
 (i) associated with expression of a cancer associated antigen chosen from Mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1;   (ii) a solid cancer chosen from colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, or combinations of said cancers, and metastatic lesions of said cancers; or   (iii) a hematologic cancer chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or pre-leukemia.   
     
     
         84 .- 85 . (canceled) 
     
     
         86 . The method of  claim 75 , wherein the cell comprises a second CAR under control of a constitutive control region, and a first CAR under control of an activation-conditional control region, wherein the first CAR is expressed upon binding of the second CAR to a cancer associated antigen expressed on a cancer cell, optionally wherein:
 (i) the second CAR comprises an antigen binding domain that binds to mesothelin; and the first CAR comprises an antigen binding domain that binds to FRa or ErbB2 (Her2/neu); or   (ii) the second CAR comprises an antigen binding domain that binds to EGFRvIII; and the first CAR comprises an antigen binding domain that binds to EGFR or other variants thereof, ErbB2 (Her2/neu), EphA2, or IL-13Ra2.   
     
     
         87 .- 91 . (canceled) 
     
     
         92 . A method of providing a CAR cell of  claim 64 , comprising:
 (i) providing an immune effector cell or a T cell from a human, to a recipient entity, wherein the recipient entity is a laboratory or hospital; and   (ii) receiving from said entity, a CAR cell, derived from said immune effector cell, or a daughter cell thereof.   
     
     
         93 .- 94 . (canceled) 
     
     
         95 . A method of providing a CAR cell, comprising:
 (i) receiving from an entity, or a health care provider, an immune effector cell, or a T cell, from a human;   (ii) inserting a nucleic acid of  claim 3 , into said immune effector cell, or a daughter cell thereof, to form the CAR cell; and/or   (iii) providing the cell to the entity.