Human mesothelin chimeric antigen receptors and uses thereof
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.
1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: i) a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain, and wherein said anti-mesothelin binding domain comprises:
(a) a light chain variable region comprising: a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 203; a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 227; and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 251; and
(b) a heavy chain variable region comprising: a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO:138; a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 156; and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 179, wherein the CAR is not a regulatable CAR.
2. The isolated nucleic acid molecule of claim 1 , wherein the anti-mesothelin binding domain comprises
the amino acid sequence of the light chain variable region of SEQ ID NO: 43; or
an amino acid sequence with 95-99% identity to the amino acid sequence of the light chain variable region of SEQ ID NO: 43.
3. The isolated nucleic acid molecule of claim 1 , wherein the anti-mesothelin binding domain comprises
the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43; or
an amino acid sequence with 95-99% identity to the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43.
4. The isolated nucleic acid molecule of claim 1 , wherein the anti-mesothelin binding domain comprises the amino acid sequence of the light chain variable region of SEQ ID NO: 43; and the heavy chain variable region of SEQ ID NO: 43.
5. The isolated nucleic acid molecule of claim 1 , wherein the anti-mesothelin binding domain is a scFv.
6. The isolated nucleic acid molecule of claim 1 , wherein
a) the encoded anti-mesothelin binding domain comprises the sequence SEQ ID NO: 43, or a sequence with 95-99% identify to the amino acid sequence SEQ ID NO: 43; or
b) the isolated nucleic acid sequence encoding the anti-mesothelin binding domain comprises the sequence of SEQ ID NO: 91, or a sequence with 95-99% identify to the nucleic acid sequence of SEQ ID NO: 91.
7. The isolated nucleic acid molecule of claim 1 , 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, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
8. The isolated nucleic acid molecule of claim 1 , wherein the encoded anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region, wherein
a) the encoded hinge region comprises the amino acid sequence of SEQ ID NO:2, or the encoded hinge region comprises a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:2, or
b) the encoded hinge region comprises the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:13, or the encoded hinge region comprises an amino acid sequence encoded by a sequence with 95-99% identity to the nucleic acid sequence of SEQ ID NO:13;
c) the encoded hinge region comprises the amino acid sequence of SEQ ID NO: 36, or the encoded hinge region comprises a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:36; or
d) the encoded hinge region comprises the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 37, or the encoded hinge region comprises an amino acid sequence encoded by a sequence with 95-99% identity to the nucleic acid sequence of SEQ ID NO: 37.
9. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a costimulatory domain, wherein the encoded costimulatory domain comprises a functional signaling domain derived from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137).
10. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.
11. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises
(i) the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10;
(ii) the amino acid sequence of SEQ ID NO:7, and an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or
(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:7, and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
12. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and the amino acid 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.
13. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the intracellular signaling domain comprises the nucleic acid sequence of SEQ ID NO: 18, or a sequence with 95-99% identity thereof, and the nucleic acid sequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequence with 95-99% identity thereof.
14. The isolated nucleic acid molecule of claim 1 , further comprising a leader sequence, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO: 1.
15. An isolated chimeric antigen receptor (CAR) molecule encoded by the nucleic acid molecule of claim 1 .
16. An isolated chimeric antigen receptor (CAR) molecule comprising: i) a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain, wherein the human anti-mesothelin binding domain comprises:
(a) a light chain variable region comprising: a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 203; a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 227; and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 251; and
(b) a heavy chain variable region comprising: a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO:138; a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 156; and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 179, wherein the CAR is not a regulatable CAR.
17. The isolated CAR molecule of claim 16 , wherein the anti-mesothelin binding domain is a scFv.
18. The isolated CAR molecule of claim 16 , wherein the anti-mesothelin binding domain comprises:
(i) the light chain variable region of the amino acid sequence of SEQ ID NO:43, and the heavy chain variable region of the amino acid sequence of SEQ ID NO: 43; or
(ii) a light chain variable region comprising an amino acid sequence with 95-99% identity with the amino acid sequence of SEQ ID NO: 43; and
a heavy chain variable region comprising an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 43.
19. The isolated CAR molecule of claim 16 , wherein the anti-mesothelin binding domain comprises the sequence of SEQ ID NO: 43, or a sequence with 95-99% identity thereof.
20. The isolated CAR molecule of claim 16 , wherein the transmembrane domain 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, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
21. The isolated CAR molecule of claim 16 , wherein the human anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:36, or a sequence with 95-99% identity thereof.
22. The isolated CAR molecule of claim 16 , wherein the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), CD278 (ICOS) and 4-1BB (CD137).
23. The isolated CAR molecule of claim 16 , wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7; or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 7.
24. The isolated CAR molecule of claim 16 , wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.
25. The isolated CAR molecule of claim 24 , wherein the intracellular signaling domain comprises:
(i) the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO:9;
(ii) the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO: 10;
(iii) the amino acid sequence of SEQ ID NO:7 and an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 10; or
(iv) an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
26. The isolated CAR molecule of claim 25 , wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid 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.
27. The isolated CAR molecule of claim 16 , further comprising a leader sequence, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO: 1, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 1.
28. A human anti-mesothelin binding domain comprising a light chain variable region comprising 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 the anti-mesothelin binding domain sequence of SEQ ID NO: 43, and a heavy chain variable region comprising 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 the human anti-mesothelin binding domain sequence of SEQ ID NO: 43, wherein the anti-mesothelin binding domain is not part of a regulatable CAR.
29. The human anti-mesothelin binding domain of claim 28 , wherein the human anti-mesothelin binding domain:
(a) is a scFv comprising the light chain and the heavy chain of the amino acid sequence of SEQ ID NO: 43; or
(b) the human anti-mesothelin binding domain comprises:
(b)(i) a light chain variable region comprising an amino acid sequence having a sequence with 95-99% identity with the amino acid sequence of SEQ ID NO: 43; and
(b)(ii) a heavy chain variable region comprising an amino acid sequence having a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 43.
30. A vector comprising the nucleic acid molecule of claim 1 , 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.
31. An immune effector cell comprising the nucleic acid molecule of claim 1 .
32. A method of making an immune effector cell comprising transducing an immune effector cell with a vector of claim 30 .
33. A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the nucleic acid molecule of claim 1 .
34. A method of providing an anti-cancer immunity in a mammal comprising administering to the mammal an effective amount of a cell comprising a CAR nucleic acid molecule of claim 1 .
35. A method of treating a mammal having a disease or condition associated with cells that express mesothelin comprising administering to the mammal an effective amount of a cell comprising a CAR nucleic acid molecule of claim 1 .
36. The method of claim 35 , wherein the disease is a cancer 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 metastatic cancer, ovarian cancer, colorectal cancer and bladder cancer, or any combination thereof.
37. The cell of claim 31 , further expressing a polypeptide that comprises an extracellular portion of any of PD1, PD-L1, CEACAM, LAGS, CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4 or TIGIT, associated with a second polypeptide that comprises a costimulatory domain and/or a primary signaling domain.
38. The method of claim 35 , wherein:
the nucleic acid comprises an in vitro transcribed nucleic acid, and is introduced into the cell, 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, after the previous administration.
39. An isolated chimeric antigen receptor (CAR) molecule comprising i) a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain, wherein the human anti-mesothelin binding domain comprises:
a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 203, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 227, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 251, and
a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO:138, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 156, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 179, and
wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 67, or a sequence with 95-99% identity thereof, wherein the CAR is not a regulatable CAR.
40. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: i) a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain, and wherein said anti-mesothelin binding domain comprises:
(a) a light chain variable region comprising: a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 203; a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 227; and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 251; and
(b) a heavy chain variable region comprising: a heavy chain complementary determining region 1(HC CDR1) comprising the amino acid sequence of SEQ ID NO:138; a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 156; and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 179; and
wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 115, or a sequence with 95-99% identity thereof, wherein the CAR is not a regulatable CAR.
41. The isolated nucleic acid molecule of claim 40 , wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 115.
42. The isolated nucleic acid molecule of claim 40 , which encodes a CAR comprising the amino acid sequence of SEQ ID NO: 67, or a sequence with 95-99% identity thereof.
43. The isolated nucleic acid molecule of claim 40 , which encodes a CAR comprising the amino acid sequence of SEQ ID NO: 67.
44. The isolated nucleic acid molecule of claim 1 , wherein the encoded CAR comprises a transmembrane domain that comprises the amino acid sequence of SEQ ID NO: 6, or a transmembrane domain that comprises an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:6.
45. The isolated nucleic acid molecule of claim 1 , wherein the sequence encoding the transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 17; or a sequence with 95-99% identify to the nucleic acid sequence of SEQ ID NO:17.
46. The isolated nucleic acid molecule of claim 1 , wherein the encoded costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 7.
47. The isolated nucleic acid molecule of claim 1 , wherein the sequence encoding the costimulatory domain comprises the nucleic acid sequence of SEQ ID NO: 18, or a sequence with 95-99% identity to the nucleic acid sequence of SEQ ID NO: 18.
48. The isolated CAR molecule of claim 16 , wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 6.
49. The method of claim 38 , wherein the cell is a T cell or NK cell.