TARGETING CYTOTOXIC CELLS WITH CHIMERIC RECEPTORS FOR ADOPTIVE IMMUNOTHERAPY

• US 20170260268A1
• Filed: 09/17/2015
• Published: 09/14/2017
• Est. Priority Date: 09/17/2014
• 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: Beatty, Gregory, Engels, Boris, Idamakanti, Neeraja, June, Carl H., Loew, Andreas, Milone, Michael, Song, Huijuan, Wang, Enxiu, Wu, Qilong
• Granted Patent: US 10,577,417 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2239/28 Expressing multiple CARs, TCRs or antigens
• 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 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• 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 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/70503 Immunoglobulin superfamily
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70535 Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 14/7056 Lectin superfamily, e.g. CD23, CD72
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/74 : Inducing cell proliferation
• CPC Class Codes: C07K 2319/02 : containing a signal sequence
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/70 : containing domain for protein-protein interaction
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The present invention provides compositions and methods for regulating the specificity and activity of immune effector cells for use in immunotherapy. In one embodiment, the invention provides a type of chimeric antigen receptor (CAR) wherein the CAR is termed a “NKR-CAR” which is a CAR design comprising a component of a receptor naturally found on natural killer (NK) cells. In one embodiment, the NK receptor includes but is not limited to a naturally occurring activating and inhibitory receptor of NK cells known as a killer cell immunoglobulin-like receptor (KIR).

Claims

1. An isolated nucleic acid molecule encoding a natural killer cell immune-function receptor-chimeric antigen receptor (NKR-CAR), wherein the encoded NKR-CAR comprises

an extracellular non-murine antigen binding domain that binds to mesothelin, and one or both of:

a transmembrane domain, e.g., a NKR transmembrane domain;

or

a cytoplasmic domain, e.g., a NKR cytoplasmic domain.

2. The isolated nucleic acid molecule of claim 1, wherein the encoded NKR-CAR comprises an extracellular non-murine antigen binding domain that binds mesothelin, a transmembrane domain and a NKR cytoplasmic domain.

3. The isolated nucleic acid molecule of claim 1 or 2, wherein the extracellular non-murine antigen binding domain that binds mesothelin is a human or humanized antigen binding domain that binds mesothelin.

4. The isolated nucleic acid molecule of claim 3, wherein the encoded human antigen binding domain that binds mesothelin comprises:

a heavy chain complementarity 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 any human anti-mesothelin heavy chain amino acid sequence listed in Table 4; and

/or a light chain complementarity 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 human anti-mesothelin light chain amino acid sequence listed in Table 4.

5. The isolated nucleic acid molecule of claim 3 or 4, wherein the human antigen binding domain that binds mesothelin comprises:

a heavy chain variable region comprising;

i) the amino acid sequence of any human anti-mesothelin heavy chain variable region listed in Table 4;

ii) an amino acid sequence having at least at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any human anti-mesothelin heavy chain variable region listed in Table 4;

or

iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any human anti-mesothelin heavy chain variable region listed in Table 4;

and/or a light chain variable region comprising;

i) the amino acid sequence of any human anti-mesothelin light chain variable region listed in Table 4;

ii) an amino acid sequence having at least at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any human anti-mesothelin light chain variable region listed in Table 4;

or

iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any human anti-mesothelin light chain variable region listed in Table 4.

6. The isolated nucleic acid molecule of any of claims 3-5, wherein the human antigen binding domain that binds mesothelin comprises:

i) the amino acid sequence of any of SEQ ID NOs;

234, 240, 230-233, 235-239, and 241-253;

ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any of SEQ ID NOs;

234, 240, 230-233, 235-239, and 241-253;

or

iii) an amino acid sequence with 95-99% identity to any of SEQ ID NOs;

234, 240, 230-233, 235-239, and 241-253.

7. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded NKR-CAR comprises:

a killer cell immunoglobulin-like receptor chimeric antigen receptor (KIR-CAR), wherein the KIR-CAR comprises one or both of a transmembrane domain from a KIR (a KIR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a KIR (a KIR cytoplasmic domain);

a natural cytotoxicity receptor chimeric antigen receptor (NCR-CAR), wherein the NCR-CAR comprises one or both of a transmembrane domain from a NCR (a NCR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a NCR (a NCR cytoplasmic domain);

a signaling lymphocyte activation molecule family chimeric antigen receptor (SLAMF-CAR), wherein the SLAMF-CAR comprises one or both of a transmembrane domain from a SLAMF (a SLAMF transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a SLAMF (a SLAMF cytoplasmic domain);

a Fc receptor chimeric antigen receptor (FcR-CAR), wherein the FcR-CAR comprises one or both of a transmembrane domain from a FcR selected from CD16 or CD64, or a cytoplasmic domain comprising a functional signaling domain from a FcR selected from CD16 or CD64;

or

a Ly49 receptor chimeric antigen receptor (Ly49-CAR), wherein the Ly49-CAR comprises one or both of a transmembrane domain from Ly49 (a Ly49 transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from Ly49 (a Ly49 cytoplasmic domain).

8. The isolated nucleic acid molecule of claim 7, wherein the KIR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

9. The isolated nucleic acid molecule of claim 7, wherein the KIR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

10. The isolated nucleic acid molecule of any of claims 7-9, wherein the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain, and/or a KIR D2 domain.

11. The isolated nucleic acid molecule of claim 7, wherein the NCR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

NKp46, NKp30, and NKp44.

12. The isolated nucleic acid molecule claim 7, wherein the NCR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

NKp46, NKp30, and NKp44.

13. The isolated nucleic acid molecule of claim 7, wherein the SLAMF transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10.

14. The isolated nucleic acid molecule of claim 7, wherein the SLAMF cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10.

15. The isolated nucleic acid molecule of claim 7, wherein the Ly49 transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

Ly49A, Ly49C, Ly49H, and Ly49D.

16. The isolated nucleic acid molecule claim 7, wherein the Ly49 cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

Ly49A, Ly49C, Ly49H, and Ly49D.

17. The isolated nucleic acid molecule of any of claims 1-6, wherein the encoded transmembrane domain comprises an NKR transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, a killer cell immunoglobulin-like receptor (KIR), a natural cytotoxicity receptor (NCR), a signaling lymphocyte activation molecule family (SLAMF), a Fc receptor (FcR), and a Ly49 receptor (Ly49).

18. The isolated nucleic acid molecule of claim 17, wherein

i) the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NOs:

357, 358, or 359;

an amino acid sequence comprising at least one, two, or three modifications but not more than 5 modifications of the amino acid sequence of SEQ ID NOs;

357, 358, or 359;

or an amino acid sequence with 95-99% sequence identity to SEQ ID NOs;

357, 358, or 359;

or

ii) the nucleic acid sequence encoding the transmembrane domain comprises nucleotides 771-830 of SEQ ID NO;

343, nucleotides 773-832 of SEQ ID NO;

345, or nucleotides 803-875 of SEQ ID NO;

347, or a nucleic acid sequence with 95-99% sequence identity thereof.

19. The isolated nucleic acid molecule of any claims 1-6 and 17-18, wherein the encoded cytoplasmic domain comprises a NKR cytoplasmic domain comprising one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, DAP12, a KIR, a NCR, a SLAMF, a FcR, and a Ly49.

20. The isolated nucleic acid molecule of claim 19, wherein

i) the encoded cytoplasmic domain comprises the amino acid sequence of SEQ ID NOs:

360, 361, or 362;

an amino acid sequence comprising at least one, two, or three modifications but not more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NOs;

360, 361, or 362;

or an amino acid sequence with 95-99% sequence identity to SEQ ID NOs;

360, 361, or 362;

or

ii) the nucleic acid sequence encoding the cytoplasmic domain comprises nucleotides 831-947 of SEQ ID NO;

343, nucleotides 833-1060 of SEQ ID NO;

345, or nucleotides 876-949 of SEQ ID NO;

347, or a nucleic acid sequence with 95-99% sequence identity thereof.

21. The isolated nucleic acid molecule of any of the preceding claims, further comprising a leader sequence which encodes the amino acid sequence of SEQ ID NO:

1.

22. The isolated nucleic acid molecule of any of the preceding claims, wherein the extracellular non-murine antigen binding domain that binds mesothelin is connected to the transmembrane domain by a hinge domain.

23. The isolated nucleic acid molecule of claim 22, wherein the encoded hinge domain is selected from the group consisting of a CD8 hinge, a GS hinge, an IgG4 hinge, an IgD hinge, a KIR2DS2 hinge, a KIR hinge, a NCR hinge, a SLAMF hinge, a FcR hinge, and a LY49 hinge.

24. The isolated nucleic acid molecule of claim 23, wherein:

i) the encoded hinge domain comprises the amino acid sequence of SEQ ID NOs;

5, 2, 3, or 4;

an amino acid sequence comprising at least one, two, or three modifications but not more than 5 modifications of the amino acid sequence of SEQ ID NOs;

5, 2, 3, or 4;

or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NOs;

5, 2, 3, or 4;

or

ii) the nucleic acid sequence encoding the hinge domain comprises the nucleic acid sequence of SEQ ID NO;

356, 16, 13, 14, or 15, or a nucleic acid sequence with 95-99% identity thereof.

25. The isolated nucleic acid molecule of claim 6, wherein:

i) the encoded transmembrane domain and cytoplasmic domain collectively comprise;

the amino acid sequence of amino acids 413-487 of SEQ ID NO;

333 or amino acids 386-454 of SEQ ID NO;

335;

an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to amino acids 413-487 of SEQ ID NO;

333 or amino acids 386-454 of SEQ ID NO;

335;

or

an amino acid sequence with 95-99% identity to amino acids 413-487 of SEQ ID NO;

333 or amino acids 386-454 of SEQ ID NO;

335;

or

ii) the nucleic acid sequence encoding the transmembrane domain and the cytoplasmic domain comprises nucleotides 1237-1464 of SEQ ID NO;

332 or nucleotides 1156-1365 of SEQ ID NO;

334, or a sequence having 95-99% identity thereof.

26. The isolated nucleic acid molecule of any of the preceding claims, further comprising a nucleic acid sequence encoding an adaptor molecule.

27. The isolated nucleic acid molecule of claim 26, wherein the encoded adaptor molecule comprises a functional signaling domain of DAP12 or Fc epsilon receptor gamma (Fcε

Ry).

28. The isolated nucleic acid molecule of claim 26 or 27, wherein:

i) the encoded adaptor molecule comprises the amino acid sequence of amino acids 1-113 of SEQ ID NO;

333 or amino acids 1-86 of SEQ ID NO;

335;

an amino acid sequence having at least one, two or three modifications but not more than 20, 10, or 5 modifications to amino acids 1-113 of SEQ ID NO;

333 or amino acids 1-86 of SEQ ID NO;

335;

or

an amino acid sequence with 95-99% identity to amino acids 1-113 of SEQ ID NO;

333 or amino acids 1-86 of SEQ ID NO;

335;

or

ii) the nucleic acid sequence encoding the adaptor molecule comprises nucleotides comprises nucleotides 1-339 of SEQ ID NO;

332 or nucleotides 1-258 of SEQ ID NO;

334.

29. The isolated nucleic acid molecule of any of claims 26-28, further comprising a nucleic acid sequence encoding a peptide cleavage site selected from the group consisting of T2A, P2A, E2A, and F2A, and wherein the nucleic acid encoding the peptide cleavage site links the nucleic acid sequence encoding the NKR-CAR to the nucleic acid sequence encoding the adaptor molecule.

30. The isolated nucleic acid molecule of claim 29, wherein the nucleic acid sequence encoding the peptide cleavage site encodes an amino acid sequence of SEQ ID NOs:

57, 58, 59, or 60;

or an amino acid sequence having 95-99% sequence identity thereto.

31. The isolated nucleic acid molecule of any of the preceding claims, further comprising a nucleic acid sequence encoding a TCAR or a second NKR-CAR.

32. The isolated nucleic acid molecule of claim 31, wherein the encoded TCAR or the second NKR-CAR comprises an antigen binding domain that binds to a target antigen that is not mesothelin.

33. A purified or non-naturally occurring polypeptide encoded by the nucleic acid molecule of any of claims 1-32.

36. The isolated NKR-CAR polypeptide of claim 33 or 34, wherein the extracellular non-murine antigen binding domain that binds mesothelin is a human or humanized antigen binding domain that binds mesothelin.

37. The isolated NKR-CAR polypeptide of claim 36, wherein the human antigen binding domain that binds mesothelin comprises:

a heavy chain complementarity 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 any human anti-mesothelin heavy chain amino acid sequence listed in Table 4; and

/or a light chain complementarity 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 human anti-mesothelin light chain amino acid sequence listed in Table 4.

38. The isolated NKR-CAR polypeptide of claim 36 or 37, wherein the human antigen binding domain that binds mesothelin comprises:

a heavy chain variable region comprising;

i) the amino acid sequence of any human anti-mesothelin heavy chain variable region listed in Table 4;

ii) an amino acid sequence having at least at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any human anti-mesothelin heavy chain variable region listed in Table 4;

or

iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any human anti-mesothelin heavy chain variable region listed in Table 4;

and/or a light chain variable region comprising;

i) the amino acid sequence of any human anti-mesothelin light chain variable region listed in Table 4;

ii) an amino acid sequence having at least at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any human anti-mesothelin light chain variable region listed in Table 4;

or

iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any human anti-mesothelin light chain variable region listed in Table 4.

39. The isolated NKR-CAR polypeptide of any of claims 36-38, wherein the human antigen binding domain that binds mesothelin comprises:

i) the amino acid sequence of any of SEQ ID NOs;

234, 240, 230-233, 235-239, and 241-253;

ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any of SEQ ID NOs;

234, 240, 230-233, 235-239, and 241-253;

or

iii) an amino acid sequence with 95-99% identity to any of SEQ ID NOs;

234, 240, 230-233, 235-239, and 241-253.

58. The isolated NKR-CAR polypeptide of claim 39, wherein the transmembrane domain and cytoplasmic domain collectively comprise:

i) the amino acid sequence of amino acids 413-487 of SEQ ID NO;

333 or amino acids 386-454 of SEQ ID NO;

335;

ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to amino acids 413-487 of SEQ ID NO;

333 or amino acids 386-454 of SEQ ID NO;

335;

or

iii) an amino acid sequence with 95-99% identity to amino acids 413-487 of SEQ ID NO;

333 or amino acids 386-454 of SEQ ID NO;

335

59. A NKR-CAR complex, comprising a NKR-CAR encoded by the nucleic acid of any of claims 1-32 or a NKR-CAR of any of claims 33-58, and an adaptor molecule.

60. The NKR-CAR complex of claim 59, wherein the adaptor molecule is DAP12 or Fcε

Rγ

.

61. The NKR-CAR complex of claim 59 or 60, wherein the NKR-CAR interacts with the adaptor molecule upon binding of the extracellular non-murine antigen binding domain of the NKR-CAR to mesothelin.

62. The nucleic acid molecule of any of claims 1-32, wherein the nucleic acid molecule is a DNA molecule, an RNA molecule, e.g., an mRNA molecule, or a combination thereof.

63. A vector comprising the nucleic acid molecule of any of claims 1-32, wherein the vector is a DNA vector or an RNA vector.

64. The vector of claim 63, which is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.

65. The vector of claim 63 or 64, further comprising an EF-1 promoter comprising the sequence of SEQ ID NO:

11.

66. A cell, e.g., an immune effector cell, comprising the nucleic acid molecule of any of claims 1-32, the NKR-CAR polypeptide of any of claims 33-58, the vector of any of claims 63-65, or the NKR-CAR complex of any of claims 59-61.

67. The cell of claim 65, wherein the cell is a cytotoxic cell, e.g., a T cell or a NK cell.

68. A method of making a cell, e.g., an immune effector cell, comprising introducing into the immune effector cell the nucleic acid molecule of any of claims 1-32 or the vector of any of claims 63-65.

69. A method of providing anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of a cell comprising the nucleic acid molecule of any of claims 1-32, the NKR-CAR polypeptide of any of claims 33-58, the vector of any of claims 63-65, or the NKR-CAR complex of any of claims 59-61.

70. A method of treating a mammal having a disease or disorder comprising administering to the mammal an effective amount of a cell, e.g., a population of immune effector cells, comprising the nucleic acid molecule of any of claims 1-32, o the NKR-CAR polypeptide of any of claims 33-58, the vector of any of claims 63-65, or the NKR-CAR complex of any of claims 59-61.

71. The method of claim 70, wherein the disease or disorder is associated with the expression of mesothelin.

72. The method of claim 70 or 71, wherein the disease or disorder is selected from the group consisting of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic metastasis, ovarian cancer, colorectal cancer and bladder cancer, or any combination thereof.

34. An isolated NKR-CAR polypeptide comprising

an extracellular non-murine antigen binding domain that binds to mesothelin, and one or both of:

a transmembrane domain, e.g., an NKR transmembrane domain;

or

a cytoplasmic domain, e.g., an NKR cytoplasmic domain.

35. The isolated NKR-CAR polypeptide of claim 34, wherein the NKR-CAR comprises:

an extracellular non-murine antigen binding domain that binds mesothelin;

a transmembrane domain and an NKR cytoplasmic domain.

40. The isolated NKR-CAR polypeptide of any of the claims 34-39, wherein the NKR-CAR comprises:

a KIR-CAR, wherein the KIR-CAR comprises one or both of a transmembrane domain from a KIR (a KIR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a KIR (a KIR cytoplasmic domain);

a NCR-CAR, wherein the NCR-CAR comprises one or both of a transmembrane domain from a NCR (a NCR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a NCR (a NCR cytoplasmic domain);

a SLAMF-CAR, wherein the SLAMF-CAR comprises one or both of a transmembrane domain from a SLAMF (a SLAMF transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a SLAMF (a SLAMF cytoplasmic domain);

a FcR-CAR, wherein the FcR-CAR comprises one or both of a transmembrane domain from a FcR selected from CD16 or CD64, or a cytoplasmic domain comprising a functional signaling domain from a FcR selected from CD16 or CD64;

or

a Ly49-CAR, wherein the Ly49-CAR comprises one or both of a transmembrane domain from Ly49 (a Ly49 transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from Ly49 (a Ly49 cytoplasmic domain).

41. The isolated NKR-CAR polypeptide of claim 40, wherein the KIR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

42. The isolated NKR-CAR of claim 40, wherein the KIR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

43. The isolated NKR-CAR polypeptide of any of claims 40-42, wherein the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain, and/or a KIR D2 domain.

44. The isolated NKR-CAR polypeptide of claim 40, wherein the NCR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

NKp46, NKp30, and NKp44.

45. The isolated NKR-CAR polypeptide claim 40, wherein the NCR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

NKp46, NKp30, and NKp44.

46. The isolated NKR-CAR polypeptide of claim 40, wherein the SLAMF transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10.

47. The isolated NKR-CAR polypeptide of claim 40, wherein the SLAMF cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10.

48. The isolated NKR-CAR polypeptide of claim 40, wherein the Ly49 transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of:

Ly49A, Ly49C, Ly49H, and Ly49D.

49. The isolated NKR-CAR polypeptide claim 40, wherein the Ly49 cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of:

Ly49A, Ly49C, Ly49H, and Ly49D.

50. The isolated NKR-CAR polypeptide of any of claims 34-39, wherein the transmembrane domain comprises a NKR transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, a KIR, a NCR, a SLAMF, a FcR, and a Ly49.

51. The isolated NKR-CAR polypeptide of claim 50, wherein the transmembrane domain comprises:

i) the amino acid sequence of SEQ ID NOs;

357, 358, or 359;

ii) an amino acid sequence comprising at least one, two, or three modifications but not more than 5 modifications of the amino acid sequence of SEQ ID NOs;

357, 358, or 359;

or

iii) an amino acid sequence with 95-99% sequence identity to SEQ ID NOs;

357, 358, or 359.

52. The isolated NKR-CAR polypeptide of any of claims 34-39 and 50-51, wherein the cytoplasmic domain comprises a NKR cytoplasmic domain comprising one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, DAP12, a KIR, a NCR, a SLAMF, a FcR, and a Ly49.

53. The isolated NKR-CAR polypeptide of claim 52, wherein the cytoplasmic domain comprises:

i) the amino acid sequence of SEQ ID NOs;

360, 361, or 362;

ii) an amino acid sequence comprising at least one, two, or three modifications but not more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NOs;

360, 361, or 362;

or

iii) an amino acid sequence with 95-99% sequence identity to SEQ ID NOs;

360, 361, or 362.

54. The isolated NKR-CAR polypeptide of any of the claims 34-53, further comprising a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

55. The isolated NKR-CAR polypeptide of any of the claims 34-54, wherein the extracellular non-murine antigen binding domain that binds mesothelin is connected to the transmembrane domain by a hinge domain.

56. The isolated NKR-CAR polypeptide of claim 55, wherein the hinge domain is selected from the group consisting of a CD8 hinge, a GS hinge, an IgG4 hinge, an IgD hinge, a KIR2DS2 hinge, a KIR hinge, a NCR hinge, a SLAMF hinge, a CD16 hinge, a CD64 hinge, and a LY49 hinge.

57. The isolated NKR-CAR polypeptide of claim 56, wherein the hinge domain comprises:

i) the amino acid sequence of SEQ ID NOs;

5, 2, 3, or 4;

ii) an amino acid sequence comprising at least one, two, or three modifications but not more than 5 modifications of the amino acid sequence of SEQ ID NOs;

5, 2, 3, or 4;

or

iii) an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NOs;

5, 2, 3, or 4.