CAR-EXPRESSING CELLS AGAINST MULTIPLE TUMOR ANTIGENS AND USES THEREOF

• US 20180044424A1
• Filed: 02/01/2016
• Published: 02/15/2018
• Est. Priority Date: 02/02/2015
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: June, Carl H., Powell, Jr., Daniel J.
• Granted Patent: US 11,161,907 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/507 Comprising a combination of two or more separate antibodies
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/58 Prostate
• CPC Class Codes: A61K 2239/59 Reproductive system, e.g. uterus, ovaries, cervix or testes
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/4632 T-cell receptors [TCR]; antibody T-cell receptor constructs
• CPC Class Codes: A61K 39/464402 Receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: A61K 39/464404 Epidermal growth factor receptors [EGFR]
• CPC Class Codes: A61K 39/464406 Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
• CPC Class Codes: A61K 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/5418 : IL-7
• CPC Class Codes: C07K 14/5443 : IL-15
• CPC Class Codes: C07K 14/55 : IL-2
• CPC Class Codes: C07K 14/7051 : T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70521 : CD28, CD152
• CPC Class Codes: C07K 14/70578 : NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/28 : against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 16/32 : against translation products of oncogenes
• CPC Class Codes: C07K 2317/22 : from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/31 : multispecific
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/569 : Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/02 : containing a signal sequence
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C12N 15/113 : Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/531 : Stem-loop; Hairpin
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

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.

Claims

1. A nucleic acid, e.g., an isolated nucleic acid, comprising:

(a) a nucleotide sequence encoding an agent, e.g., a polypeptide or nucleic acid, that enhances the immune response against a target cell, e.g., 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 nucleic acid of claim 1, further comprising:

(c) a nucleotide sequence encoding a second agent, e.g., a polypeptide or nucleic acid, that enhances the immune response against a target cell, e.g., a cancer cell, wherein the second agent is a CAR (second CAR);

(d) a second control region operatively linked to (c), wherein the second control region is other than an activation-conditional control region, e.g., the second control region comprises a constitutive control region.

4. The nucleic acid of claim 2 or 3, wherein the second control region comprises a constitutive control region, e.g., an elongation factor 1 alpha (EF1a) control region.

5. The nucleic acid of any of claims 2-4, wherein the second CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein the antigen binding domain binds to a cancer associated antigen selected from the group consisting of:

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, and IGLL1.

6. The nucleic acid of claim 5, wherein the antigen binding domain binds to mesothelin.

7. The nucleic acid of claim 6, wherein the mesothelin binding domain comprises 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.

8. The nucleic acid of claim 6 or 7, wherein the mesothelin binding domain comprises 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.

9. The nucleic acid of any of claims 6-8, wherein the mesothelin binding domain comprises an amino acid sequence of Table 2, e.g., 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 an amino acid sequence with at least 95-99% homology to an amino acid sequence provided in Table 2, e.g., 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.

10. The nucleic acid of claim 5, wherein the antigen binding domain binds to EGFRvIII.

11. The nucleic acid of any of claims 1-10, wherein (a) comprises a nucleotide sequence encoding a first CAR.

12. The nucleic acid of any of claims 3-10, wherein:

the first CAR comprises a first antigen binding domain that binds a first antigen; and

the second CAR comprises a second antigen binding domain that binds a second antigen.

13. The nucleic acid of claim 12, wherein the first antigen is different from the second antigen.

14. The nucleic acid of claim 12 or 13, wherein the first antigen and the second antigen are co-expressed on a cancer cell.

15. The nucleic acid of claim 12 or 13, wherein 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.

16. The nucleic acid of claim 12 or 13, wherein the second antigen is expressed on a cancer cell and wherein the first antigen is not expressed on the cancer cell.

17. The nucleic acid of claim 12 or 13, wherein the first antigen is selected from the group consisting of:

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, and IGLL1.

18. The nucleic acid of claim 12 or 13, wherein 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.

19. The nucleic acid of claim 12 or 13 wherein the second antigen is EGFRvIII and the first antigen is EGFR or a different variant thereof, EphA2, ErbB2 (Her2/neu), or IL-13Ra2.

20. The nucleic acid of any of claim 12 or 13, wherein the second antigen is mesothelin and the first antigen is FRa or ErbB2 (Her2/neu).

21. The nucleic acid of any of claims 12-17 or 20, wherein the first antigen binding domain that binds to FRa comprises 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 a FRa binding domain in Table 5;

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 FRa binding domain in Table 5.

22. The nucleic acid of claim 21, wherein the FRa binding domain comprises an amino acid sequence of Table 5, e.g., SEQ ID NO:

96 or SEQ ID NO;

98;

or an amino acid sequence with at least 95-99% homology to an amino acid sequence provided in Table 5, e.g., SEQ ID NO;

96 or SEQ ID NO;

98.

23. The nucleic acid of any of claims 12 to 22, wherein the transmembrane domain of the first and/or second CAR comprises 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.

24. The nucleic acid of any of claim 23, wherein the transmembrane domain comprises

(i) the amino acid sequence of SEQ ID NO:

12,

(ii) 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

(iii) a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

12.

25. The nucleic acid of any of claims 12 to 24, wherein the antigen binding domain of the first and/or second CAR is connected to the transmembrane domain by a hinge region.

26. The nucleic acid of claim 25, wherein the hinge region comprises SEQ ID NO:

4, or a sequence with 95-99% identity thereof.

27. The nucleic acid of any of claims 12 to 26, wherein the intracellular signaling domain of the first and/or second CAR comprises a costimulatory signaling domain comprising a functional signaling domain obtained from a protein selected from the group consisting of 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, and a ligand that specifically binds with CD83.

28. The nucleic acid of claim 27, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:

14, 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, or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

14.

29. The nucleic acid of any of claims 12-28, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

30. The nucleic acid of claim 29, wherein the intracellular signaling domain comprises 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.

31. The nucleic acid of claim 30, wherein the intracellular signaling domain comprises 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.

32. The nucleic acid of any of claims 12-31, wherein the first and/or second CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

2.

33. The nucleic acid of any of claims 12-32, wherein the first CAR comprises:

(i) the amino acid sequence of any amino acid sequence in Table 7, e.g., SEQ ID NO;

150 or 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 any amino acid sequence in Table 7, e.g., SEQ ID NO;

150 or SEQ ID NO;

152;

or

(iii) an amino acid sequence with 95-99% identity to any amino acid sequence in Table 7, e.g., SEQ ID NO;

150 or SEQ ID NO;

152.

34. The nucleic acid of claim 33, wherein the first CAR comprises:

(i) a nucleic acid sequence in Table 7, e.g., SEQ ID NO;

151 or SEQ ID NO;

153;

or

(ii) a nucleic acid sequence with 95-99% identity to a nucleic acid sequence in Table 76, e.g., SEQ ID NO;

151 or SEQ ID NO;

153.

35. The nucleic acid of any of claims 12-34, wherein the second CAR comprises:

(i) the amino acid sequence in Table 6, e.g., 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 any amino acid sequence in Table 6, e.g., 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;

or

(iii) an amino acid sequence with 95-99% identity to any amino acid sequence in Table 6, e.g., 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.

36. The nucleic acid of claim 35, wherein the second CAR comprises:

(i) a nucleic acid sequence in Table 6, e.g., 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

(ii) a nucleic acid sequence with 95-99% identity to a nucleic acid sequence in Table 6, e.g., 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.

37. The nucleic acid of any of claims 12-36, further comprising:

(e) a sequence encoding a third CAR; and

optionally,

(f) a second activation-conditional control region operatively linked to (e), wherein the activation-conditional control region is operatively linked to (f).

38. The nucleic acid of any of claims 1-2, or 4-10, wherein (a) comprises a nucleotide sequence encoding an inhibitor of a checkpoint inhibitor of the immune response.

39. The nucleic acid of claim 38, wherein 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, GAL9, adenosine, and TGFR beta, or a combination thereof.

40. The nucleic acid of claim 38 or 39, wherein the nucleotide sequence encodes an RNA-based inhibitor, e.g., an shRNA.

41. The nucleic acid of claim 38 or 39, wherein the nucleotide sequence encodes an antibody molecule.

42. The nucleic acid of any of claims 1-2 or 4-10, wherein (a) comprises a nucleotide sequence encoding a cytokine.

43. The nucleic acid of claim 42, wherein the cytokine comprises IL-2, IL-7, IL-15, or IL-21.

44. The nucleic acid of any of claims 1-43, wherein the activation-conditional control region comprises a nucleotide sequence that induces expression of (a) upon immune effector cell activation.

45. The nucleic acid of claim 44, wherein the activation-conditional control region comprises a promoter of a gene that is induced upon immune effector cell activation.

46. The nucleic acid of claim 44 or 45, wherein the activation-conditional control region comprises 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.

47. The nucleic acid of any of claims 44-46, wherein the activation-conditional control region comprises one or more binding sites for a transcription modulator, e.g., a transcription factor, that induces gene expression upon immune effector cell activation.

48. The nucleic acid of any of claims 44-47, wherein the activation-conditional control region comprises one or more NFAT binding sides.

49. The nucleic acid of any of claims 44-48, wherein the activation-conditional control region comprises one or more sequences selected from GGAAA, GGGACT, SEQ ID NO:

261, or SEQ ID NO;

262.

50. The nucleic acid of any of claims 2-49, wherein the second control region that is other than an activation-conditional control region is a constitutive control region.

51. The nucleic acid of claim 50, wherein the constitutive control region comprises a constitutive promoter.

52. The nucleic acid of claim 51, wherein the constitutive promoter is an EF1alpha promoter.

53. The nucleic acid of claim 51, wherein the constitutive promoter comprises the nucleic acid sequence of SEQ ID NO:

1.

54. The nucleic acid of any of claims 1 to 53, wherein:

(a) and (b) are disposed on a single nucleic acid molecule, e.g., a viral vector, e.g., a lentivirus vector.

55. The nucleic acid of any of claims 2 to 54, wherein:

(a) and (c) are disposed on a single nucleic acid molecule, e.g., a viral vector, e.g., a lentivirus vector.

56. The nucleic acid of any of claims 3-54, wherein:

(a) the nucleotide sequence encoding the first CAR;

(b) the activation-conditional control region operatively linked to (a);

(c) the sequence encoding the second CAR; and

,

(d) the second control region operatively linked to (c), wherein the second control region comprises a constitutive control region,

are all disposed on a single nucleic acid molecule, e.g., a viral vector, e.g., a lentivirus vector.

57. The nucleic acid of any of claims 54-56, comprising a bicistronic viral vector.

58. The nucleic acid of claim 57 comprising a bicistronic lentivirus vector.

59. The nucleic acid of any of claims 56-58, wherein (a) is translated as a first RNA and (c) is translated as a second RNA.

60. The nucleic acid of any of claims 2-54, wherein:

(a) is disposed on a first nucleic acid molecule, e.g., a first viral vector, e.g., a first lentivirus vector; and

(c) is disposed on a second nucleic acid molecule, e.g., a second viral vector, e.g., a second lentivirus vector.

61. The nucleic acid of claim 3-54, wherein:

(a) the nucleotide sequence encoding the first CAR, and

(b) the activation-conditional control region operatively linked to (a), are disposed on a first nucleic acid molecule, e.g., a first viral vector, e.g., a first lentivirus vector; and

(c) the sequence encoding the second CAR, and,

(d) the second control region operatively linked to (c), wherein the second control region comprises a constitutive control region, are disposed on a second nucleic acid molecule, e.g., a second viral vector, e.g., a second lentivirus vector

62. The nucleic acid of any of claims 1-61, further comprising a sequence encoding a shRNA targeting an inhibitory molecule, e.g., a checkpoint inhibitor.

63. A vector system, e.g., a vector system comprising one or more vectors, comprising a nucleic acid of any of claims 1 to 62.

64. A cell, e.g., an immune effector cell, e.g., a T cell or NK cell, comprising:

a nucleic acid of any of claims 1 to 62;

or

a vector system comprising nucleic acid of any of claims 1 to 62, e.g., vector system of any of claim 63.

65. The cell of claim 64, comprising a nucleic acid of any of claims 11 to 32.

66. The cell of any of claim 64 or 65, wherein the cell is a human cell.

67. The cell of any of claims 64 to 66, wherein said cell is a T cell.

68. The cell of any of claims 64 to 66, wherein said cell is a NK cell.

69. A method of making a cell of any of claims 64-68, e.g., a CAR cell, comprising introducing into a cell,

a nucleic acid of claims any of 1 to 62;

or

a vector system comprising nucleic acid of any of claims 1 to 62, e.g., vector system of any of claim 63,

thereby making a cell, e.g., a CAR cell.

70. The method of claim 69, wherein the nucleic acid comprises a nucleic acid of any of claims 1-62.

71. The method of claim 69 or 70, wherein the cell is a human cell.

72. The method of any of claims 69 to 71, wherein said cell is a T cell.

73. The method of any of claims 69 to 71, wherein said cell is a NK cell.

74. A composition comprising an immune effector cell (e.g., a population of immune effector cells) comprising a chimeric antigen receptor (CAR) molecule for use in the treatment of a subject having a cancer, wherein the immune effector cell is a cell of any of claims 64-68, or a cell comprising a nucleic acid of any of claims 1-62, or a vector system of claim 63.

75. A method of treating a subject having a cancer comprising administering to the subject an effective amount of a cell of any of claims 64-68, or a cell comprising:

a nucleic acid of any of claims 1 to 62;

or

a vector system of claim 63

thereby treating a subject.

76. The use or method of claim 74 or 75, wherein the cell is a human cell.

77. The use or method of any of claims 74 to 76, wherein said cell is a T cell.

78. The use or method of any of claims 74 to 76, wherein said cell is a NK cell.

79. The use or method of claim 77, wherein the cell is an autologous T cell.

80. The use or method of claim 77, wherein the cell is an allogeneic T cell.

81. The use or method of any of claim 78, wherein the cell is an autologous NK cell.

82. The use or method of claim 78, wherein the cell is an allogeneic NK cell.

83. The use or method of any of claims 74-82, wherein the cancer is associated with expression of a cancer associated antigen selected from the group consisting of 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, and IGLL1.

84. The use or method of any of claims 74-83 wherein the cancer is a solid cancer selected from the group consisting of 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, combinations of said cancers, and metastatic lesions of said cancers.

85. The use or method of any of claims 74-83, wherein the cancer is 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.

86. The use or method of any of claims 74-85, 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.

87. The use or method of claim 86, wherein the second CAR comprises an antigen binding domain that binds to mesothelin.

88. The use or method claim 87, wherein the first CAR comprises an antigen binding domain that binds to FRa or ErbB2 (Her2/neu).

89. The use or method of claim 86, wherein the second CAR comprises an antigen binding domain that binds to EGFRvIII.

90. The use or method of claim 89, wherein the first CAR comprises an antigen binding domain that binds to EGFR or other variants thereof, ErbB2 (Her2/neu), EphA2, or IL-13Ra2.

91. The method of any of claims 74-90, wherein the method further comprises administering an additional therapeutic agent, e.g., an anti-cancer agent.

92. A method of providing a cell, e.g., a CAR cell, of any of claims 64-68, comprising:

providing an immune effector cell, e.g., a T cell from a human, to a recipient entity, e.g., a laboratory or hospital; and

receiving from said entity, a cell described herein, e.g., a CAR cell, derived from said immune effector cell, or a daughter cell thereof.

93. The method of claim 92, wherein said entity inserted a nucleic acid encoding of any of claims 1 to 62, into said immune effector cell or a daughter cell thereof.

94. The method of any of claim 92 or 93, further comprising administering the cell to a human.

95. A method of providing a cell, e.g., a CAR cell, of any of claims 64-68, comprising:

receiving from an entity, e.g., a health care provider, an immune effector cell, e.g., a T cell, from a human;

inserting a nucleic acid of any of claims 1 to 62, into said immune effector cell, or a daughter cell thereof, to form a the cell; and

, optionally, providing the cell to the entity.

3. A nucleic acid, e.g., an isolated nucleic acid, comprising

(a) a nucleotide sequence encoding a first CAR comprising 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 an 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, e.g., the second control region comprises a constitutive control region.