HUMAN MESOTHELIN CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

• US 20160311917A1
• Filed: 12/19/2014
• Published: 10/27/2016
• Est. Priority Date: 12/19/2013
• 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, Idamakanti, Neeraja, Engels, Boris, June, Carl H., Loew, Andreas, Song, Huijuan, Wu, Qilong
• Granted Patent: US 10,640,569 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising 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 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 2239/54 Pancreas
• CPC Class Codes: A61K 2239/59 Reproductive system, e.g. uterus, ovaries, cervix or testes
• CPC Class Codes: A61K 31/436 the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• 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 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• CPC Class Codes: A61K 38/1793 for cytokines; for lymphokines; for interferons
• 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 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/10 of the bladder
• CPC Class Codes: A61P 15/00 : Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/04 : specific for metastasis
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/7051 : T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 : CD8
• CPC Class Codes: C07K 14/7151 : for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 16/303 : Liver or Pancreas
• CPC Class Codes: C07K 16/3061 : Blood cells
• CPC Class Codes: C07K 16/3069 : Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• 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: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 15/86 : Viral vectors
• CPC Class Codes: C12N 2740/15041 : Use of virus, viral particle or viral elements as a vector
• CPC Class Codes: C12N 5/0638 : Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]

Abstract

Provided are compositions and methods for treating diseases associated with expression of mesothelin. Also provided are a chimeric antigen receptor (CAR) specific to mesothelin, vectors encoding the same, and recombinant T cells comprising the mesothelin CAR. Further provided are methods of administering a genetically modified T cell expressing a CAR that comprises a mesothelin binding domain.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises:

i) an antibody or antibody fragment comprising a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain comprising a stimulatory domain, and wherein said anti-mesothelin binding domain comprises one or more of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) comprising an amino acid sequence listed in Table 5 and one or more of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) comprising an amino acid sequence listed in Table 4.

2. The isolated nucleic acid molecule of claim 1, comprising a LC CDR1, LC CDR2 and LC CDR3, wherein LC CDR1, LC CDR2 and LC CDR3 comprise an amino acid sequence listed in Table 5.

3. The isolated nucleic acid molecule of claim 1, comprising a HC CDR1, HC CDR2 and HC CDR3, wherein HC CDR1, HC CDR2 and HC CDR3 comprise an amino acid sequence listed in Table 4.

4. The isolated nucleic acid molecule of claim 1, comprising LC CDR1, LC CDR2 and LC CDR3, wherein LC CDR1, LC CDR2 and LC CDR3 comprise an amino acid sequence listed in Table 5, and HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1, HC CDR2 and HC CDR3 comprise an amino acid sequence listed in Table 4.

5. The isolated nucleic acid molecule of claim 1, wherein the anti-mesothlin binding domain comprises an amino acid sequence of any one of the light chain variable regions listed in Table 2.

6. The isolated nucleic acid molecule of claim 1, wherein the anti-mesothlin binding domain comprises an amino acid sequence of any one of the heavy chain variable region listed in Table 2.

7. The isolated nucleic acid molecule of claim 1, wherein the anti-mesothlin binding domain comprises an amino acid sequence of any light chain variable region listed in Table 2 and any heavy chain variable region listed Table 2.

8. The isolated nucleic acid molecule of any of the preceding claims, wherein the anti-mesothelin binding domain is a scFv.

9. The isolated nucleic acid molecule of any of the preceding claims, wherein the anti-mesothelin binding domain comprises a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 2, or a sequence with 95-99% identity to an amino acid sequence provided in Table 2.

10. The isolated nucleic acid molecule of any of the preceding claims, wherein the anti-mesothelin binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in Table 2, or a sequence with 95-99% identity to an amino acid sequence provided in Table 2.

11. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded anti-mesothelin binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

39;

SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, and SEQ ID NO;

62, or a sequence with 95-99% identify thereof.

12. The isolated nucleic acid molecule of any of the preceding claims, wherein the nucleic acid sequence encoding the anti-mesothelin binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

87;

SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

90, SEQ ID NO;

91, SEQ ID NO;

92, SEQ ID NO;

93, SEQ ID NO;

94, SEQ ID NO;

95, SEQ ID NO;

96, SEQ ID NO;

97, SEQ ID NO;

98, SEQ ID NO;

99, SEQ ID NO;

100, SEQ ID NO;

101, SEQ ID NO;

102, SEQ ID NO;

103, SEQ ID NO;

104, SEQ ID NO;

105, SEQ ID NO;

106, SEQ ID NO;

107, SEQ ID NO;

108, SEQ ID NO;

109, and SEQ ID NO;

110, or a sequence with 95-99% identify thereof.

13. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded CAR includes a transmembrane domain that comprises a transmembrane domain of 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.

14. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded transmembrane domain comprises a sequence of SEQ ID NO:

6.

15. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded transmembrane domain comprises an amino acid sequence that comprises at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

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

6.

16. The isolated nucleic acid molecule of any of the preceding claims, wherein the nucleic acid sequence encoding the transmembrane domain comprises a sequence of SEQ ID NO:

17, or a sequence with 95-99% identify thereof.

17. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region.

18. The isolated nucleic acid molecule of claim 17, wherein the encoded hinge region comprises SEQ ID NO:

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

19. The isolated nucleic acid molecule of claim 17, wherein the nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO:

13, or a sequence with 95-99% identify thereof.

20. The isolated nucleic acid molecule of any of the preceding claims, further comprising a sequence encoding a costimulatory domain.

21. The isolated nucleic acid molecule of claim 20, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137).

22. The isolated nucleic acid molecule of claim 20 or 21, wherein the encoded costimulatory domain comprises a sequence of SEQ ID NO:

7.

23. The isolated nucleic acid molecule of claim 20 or claim 21, wherein the encoded costimulatory domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

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

7.

24. The isolated nucleic acid molecule of claim 20 or 21, wherein the nucleic acid sequence encoding the costimulatory domain comprises a sequence of SEQ ID NO:

18, or a sequence with 95-99% identify thereof.

25. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

26. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:

7 and/or the sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

27. The isolated nucleic acid molecule of any of the preceding claims, wherein the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

7 and/or an amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

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

7 and/or an amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

28. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:

7 and the sequence of SEQ ID NO;

9 or SEQ ID NO;

10, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

29. The isolated nucleic acid molecule of any of the preceding claims, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence of SEQ ID NO:

18, or a sequence with 95-99% identify thereof, and/or a sequence of SEQ ID NO;

20 or SEQ ID NO;

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

30. The isolated nucleic acid molecule of any of the preceding claims, further comprising a leader sequence.

31. The isolated nucleic acid molecule of claim 32, wherein the leader sequence comprises SEQ ID NO:

1.

32. An isolated polypeptide molecule encoded by the nucleic acid molecule of any one of claims 1-32.

33. The isolated polypeptide of claim 32, comprising a sequence selected from the group consisting of SEQ ID NO:

63;

SEQ ID NO;

64, SEQ ID NO;

65, SEQ ID NO;

66, SEQ ID NO;

67, SEQ ID NO;

68, SEQ ID NO;

69, SEQ ID NO;

70, SEQ ID NO;

71, SEQ ID NO;

72, SEQ ID NO;

73, SEQ ID NO;

74, SEQ ID NO;

75, SEQ ID NO;

76, SEQ ID NO;

77, SEQ ID NO;

78, SEQ ID NO;

79, SEQ ID NO;

80, SEQ ID NO;

81, SEQ ID NO;

82, SEQ ID NO;

83, SEQ ID NO;

84, SEQ ID NO;

85, and SEQ ID NO;

86.

76. A vector comprising a nucleic acid molecule encoding a CAR of any of the preceding claims.

77. The vector of claim 76, wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

78. The vector of claim 76 or 77, further comprising a promoter.

79. The vector of claim 78, wherein the promoter is an EF-1 promoter.

80. The vector of claim 79, wherein the EF-1 promoter comprises a sequence of SEQ ID NO:

11.

81. The vector of any of claims 76-78, wherein the vector is an in vitro transcribed vector.

82. The vector of claim 81, wherein the, the nucleic acid sequence in the vector further comprises a poly(A) tail.

83. The vector of any of claim 81, wherein the nucleic acid sequence in the vector further comprises a 3′

UTR.

84. A cell comprising the vector of any of claims 76-83.

85. The cell of claim 84, wherein the cell is a human T cell.

86. The cell of claim 85, wherein the T cell is a CD8+ T cell.

87. A method of making a cell comprising transducing a T cell with a vector of any of claims 76-83.

88. A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding a CAR molecule of any of the preceding claims.

89. A method of providing an anti-cancer immunity in a mammal comprising administering to the mammal an effective amount of a cell expressing a CAR molecule of any of the preceding claims.

90. The method of claim 89, wherein the cell is an autologous T cell.

91. The method of claim 89 or 90, wherein the cell is an allogeneic T cell.

92. The method of any of claims 89-91, wherein the mammal is a human.

93. A method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of a cell comprising a CAR molecule of any of the preceding claims.

94. The method of claim 93, wherein the disease associated with mesothelin expression is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition, or is a non-cancer related indication associated with expression of mesothelin.

95. The method of claim 93-94, wherein the disease is a cancer associated with mesothelin selected from the group consisting of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic metatstatic, ovarian cancer, colorectal cancer and bladder cancer, or any combination thereof.

96. The method of any of claims 93-95, wherein the cells expressing a CAR molecule are administered in combination with an agent that increases the efficacy of a cell expressing a CAR molecule.

97. The method of any of claims 93-96, wherein the cells expressing a CAR molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a CAR molecule.

98. The method of any of claims 93-97, wherein the cells expressing a CAR molecule are administered in combination with an agent that treats the disease associated with mesothelin

99. The isolated nucleic acid molecule of any of claims 1-31, the isolated polypeptide molecule of any of claims 32-33, the isolated CAR of any of claims 34-66, the anti-mesothelin binding domain of any of claims 67-75, the vector of any of claims 76-83 or the cell of any of claims 84-86 for use as a medicament.

100. The isolated nucleic acid molecule of any of claims 1-31, the isolated polypeptide molecule of any of claims 32-33, the isolated CAR of any of claims 34-66, the anti-mesothelin binding domain of any of claims 67-75, the vector of any of claims 76-83 or the cell of any of claims 84-86 for use in the treatment of a disease expressing mesothelin.

101. A cell of any of claims 84-86, further expressing an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.

102. The cell of claim 101, wherein the first polypeptide comprises at least a portion of PD1 and the second polypeptide comprises a costimulatory domain and a intracellular signaling domain.

103. The method of claim 96, wherein the agent is an mTOR inhibitor and the subject is administered a low, immune enhancing, dose of an mTOR inhibitor, e.g., RAD001 or rapamycin.

104. The method of claim 103, wherein the mTOR inhibitor is a RAD001.

105. The method of claim 103, wherein the dose comprises an allosteric and a catalytic mTOR inhibitor.

107. The method of claim 103, wherein the immune effector cell, e.g., T cell, to be engineered to express a CAR, is harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject has been, at least transiently, increased.

108. The method of claim 103, wherein the dose of an mTOR inhibitor is associated with mTOR inhibition of at least 5 but no more than 90%, e.g., as measured by p70 S6 kinase inhibition.

109. The method of claim 103, wherein the dose of an mTOR inhibitor is associated with mTOR inhibition of at least 10% but no more than 40%, e.g., as measured by p70 S6 kinase inhibition.

110. A method of treating a subject having a disease associated with expression of mesothelin comprising administering to the subject an effective amount of cells comprising a nucleic acid of any of claims 1-31, wherein the nucleic acid is introduced into T cells or NK cells using in vitro transcription, and the subject receives an initial administration of the cells comprising the nucleic acid, and one or more subsequent administrations of cells comprising the nucleic acid, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.

34. An isolated chimeric antigen receptor (CAR) molecule comprising a human anti-mesothelin binding domain, a transmembrane domain, and an intracellular signaling domain.

35. The isolated CAR molecule of claim 34, comprising i) an antibody or antibody fragment which includes a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain.

36. The isolated CAR molecule of any of claim 34 or 35, wherein the anti-mesothelin binding domain does not compete for binding to human mesothelin with an antigen binding domain comprising a sequence comprising SEQ ID NO:

279.

37. The isolated CAR molecule of any of claims 34-36, wherein the anti-mesothelin binding domain competes with an antigen binding domain comprising:

a) one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49, and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49;

b) a LC CDR1, LC CDR2 and LC CDR3 of an anti-mesothelin light chain amino acid sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49, and an HC CDR1, HC CDR2, and HC CDR3 of an anti-mesothelin heavy chain amino acid sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49;

or

c) a sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49.

38. The isolated CAR molecule of any of claims 34-37, wherein the anti-mesothelin binding domain binds to a different epitope of human mesothelin than the epitope of human mesothelin targeted by the antigen binding domain comprising a sequence comprising SEQ ID NO:

279.

39. The isolated CAR molecule of any of claims 34-38, wherein the anti-mesothelin binding domain binds to the C-terminus of human mesothelin.

40. The isolated CAR molecule of claim 39, wherein the anti-mesothelin binding domain binds an epitope within amino acids 450-588 of SEQ ID NO:

278.

41. The isolated CAR molecule of claim 40, wherein the epitope comprises amino acids 485-490, 498-507, 532-537, or 545-572 of SEQ ID NO:

278, or any subset or a combination thereof.

42. The isolated CAR molecule of any of claims 34-41, wherein the human anti-mesothelin binding domain comprises one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) comprising an amino acid sequence listed in Table 5, and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) comprising an amino acid sequence listed in Table 4.

43. The isolated CAR molecule of any of claims 34-42, comprising LC CDR1, LC CDR2 and LC CDR3 comprising an amino acid sequence listed in Table 5.

44. The isolated CAR molecule of any of claims 34-43, comprising HC CDR1, HC CDR2, and HC CDR3 comprising an amino acid sequence listed in Table 4.

45. The isolated CAR molecule of any of claims 34-44, comprising LC CDR1, LC CDR2 and LC CDR3 comprising an amino acid sequence listed in Table 5, and HC CDR1, HC CDR2, and HC CDR3 comprising an amino acid sequence listed in Table 4.

46. The isolated CAR molecule of any of claims 59-45, wherein the anti-mesothelin binding domain is a scFv.

47. The isolated CAR molecule of any of claims 34-46, wherein the anti-mesothelin binding domain comprises a light chain and a heavy chain of an amino acid sequence listed in Table 2.

48. The isolated CAR molecule of any of claims 34-47, wherein the anti-mesothelin binding domain comprises:

a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 2, or a sequence with 95-99% identity with an amino acid sequence provided in Table 2.

49. The isolated CAR molecule of any of claims 34-48, wherein the anti-mesothelin binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in Table 2, or a sequence with 95-99% identity to an amino acid sequence provided in Table 2.

50. The isolated CAR molecule of any of claims 34-49, wherein the anti-mesothelin binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, and SEQ ID NO;

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

51. The isolated CAR molecule of any of claims 34-50, wherein the transmembrane comprises a transmembrane domain of 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.

52. The isolated CAR molecule of claim 51, wherein the transmembrane domain comprises a sequence of SEQ ID NO:

6.

53. The isolated CAR molecule of claim 51, wherein the transmembrane domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

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

6.

54. The isolated CAR molecule of any one of claims 34-53, wherein the human anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region.

55. The isolated CAR molecule of claim 54, wherein the hinge region comprises SEQ ID NO:

2 or SEQ ID NO;

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

56. The isolated CAR molecule of any of claims 34-55, wherein the intracellular signaling domain comprises a sequence encoding a costimulatory domain.

57. The isolated CAR molecule of claim 56, wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), CD278 (also known as “

ICOS”

) and 4-1BB (CD137).

58. The isolated CAR molecule of claim 56 or 57, wherein the costimulatory domain comprises a sequence of SEQ ID NO:

7.

59. The isolated CAR molecule of claim 56 or 57, wherein the costimulatory domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

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

7.

60. The isolated CAR molecule of claim 34-59, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

61. The isolated CAR molecule of claim 60, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO:

7 and/or the sequence of SEQ ID NO;

9.

62. The isolated CAR molecule of claim 60, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO:

7 and/or the sequence of SEQ ID NO;

10.

63. The isolated CAR molecule of any of claims 34-55, wherein the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

7 and/or an amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

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

7 and/or an amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

64. The isolated CAR molecule of any of claims 34-55, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO:

7 and the sequence of SEQ ID NO;

9 or SEQ ID NO;

10, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

65. The isolated CAR molecule of any of claims 59-64, further comprising a leader sequence.

66. The isolated CAR molecule of claim 65, wherein the leader sequence comprises an amino acid sequence of SEQ ID NO:

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

1.

67. A human anti-mesothelin binding domain comprising one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of any anti-mesothelin binding domain in SEQ ID NO:

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, and SEQ ID NO;

62, and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any human anti-mesothelin binding domain in SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, and SEQ ID NO;

62.

68. The human anti-mesothelin binding domain of claim 67, wherein the human anti-mesothelin binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence of SEQ ID NO:

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, and SEQ ID NO;

62.

69. The human anti-mesothelin binding domain of claim 67 or 68, wherein the human anti-mesothelin binding domain comprises:

a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, or SEQ ID NO;

62, or a sequence with 95-99% identity with an amino acid sequence of SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, or SEQ ID NO;

62; and

/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, or SEQ ID NO;

62;

or a sequence with 95-99% identity to an amino acid sequence in SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, SEQ ID NO;

42, SEQ ID NO;

43, SEQ ID NO;

44, SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, or SEQ ID NO;

62.

70. An human anti-mesothelin binding domain, wherein the anti-mesothelin binding domain does not compete for binding to human mesothelin with an antigen binding domain comprising a sequence comprising SEQ ID NO:

279.

71. The human anti-mesothelin binding domain of claim 70, wherein the anti-mesothelin binding domain competes with an antigen binding domain comprising:

a) one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49 and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49 b) a LC CDR1, LC CDR2 and LC CDR3 of an anti-mesothelin light chain amino acid sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49 and an HC CDR1, HC CDR2, and HC CDR3 of an anti-mesothelin heavy chain amino acid sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49;

or

c) a sequence selected from SEQ ID NO;

43 and SEQ ID NO;

49.

72. The human anti-mesothelin binding domain any of claim 70 or 71, wherein the anti-mesothelin binding domain binds to a different epitope of human mesothelin than the epitope of human mesothelin targeted by an antigen binding domain comprising a sequence comprising SEQ ID NO:

279.

73. The human anti-mesothelin binding domain of any of claims 70-72, wherein the anti-mesothelin binding domain binds to the C-terminus of human mesothelin.

74. The human anti-mesothelin binding domain of claim 73, wherein the anti-mesothelin binding domain binds an epitope within amino acids 450-588 of SEQ ID NO:

278.

75. The human anti-mesothelin binding domain of claim 74, wherein the epitope comprises amino acids 485-490, 498-507, 532-537, or 545-572 of SEQ ID NO:

278, or any subset or a combination thereof.

106. The method of 103, wherein the mTOR inhibitor is administered for an amount of time sufficient to decrease the proportion of PD-1 positive T cells, increase the proportion of PD-1 negative T cells, or increase the ratio of PD-1 negative T cells/PD-1 positive T cells, in the peripheral blood of the subject, or in a preparation of T cells isolated from the subject.