Anti-hk2 chimeric antigen receptor (CAR)

Abstract

This disclosure provides a chimeric antigen receptor (CAR) that specifically targets human bradykinin 2 (hK2), and immune-reactive cells containing such CARs, which are used to treat cancer.

Description

Technical field

Prior technology

Content of invention

Implementation

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain binds to a human kallikrein-2 (hK2) antigen, and wherein the extracellular antigen-binding domain comprises: a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 65, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 66; The heavy chain CDR2 of the amino acid sequence SEQ ID NO: 65, the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 66, and the light chain complementarity determination region 1 (CDR1) of the amino acid sequence SEQ ID NO: 67, the light chain CDR2 of the amino acid sequence SEQ ID NO: 69, and the light chain CDR3 of the amino acid sequence SEQ ID NO: 71; or the heavy chain complementarity determination region 1 (CDR1) of the amino acid sequence SEQ ID NO: 314, the heavy chain CDR2 of the amino acid sequence SEQ ID NO: 315, the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 311, and the light chain complementarity determination region 1 (CDR1) of the amino acid sequence SEQ ID NO: 312, the light chain CDR2 of the amino acid sequence SEQ ID NO: 313, and the light chain CDR3 of the amino acid sequence SEQ ID NO: 91; The transmembrane domain includes a CD8a transmembrane region (CD8a-TM) polypeptide, which contains the amino acid sequence of SEQ ID NO: 26; and the intracellular signal transduction domain includes a TNF receptor superfamily member 9 (CD137) component and a T cell surface glycoprotein CD3ζ chain (CD3z) component, which contains the amino acid sequence of SEQ ID NO: 27 and the CD3z component contains the amino acid sequence of SEQ ID NO:

28.

2. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain binds to human kallikrein-2 (hK2) antigen, and wherein the extracellular antigen-binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 ... The transmembrane domain comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 1; the light chain variable region of the amino acid sequence SEQ ID NO: 6; the light chain variable region of the amino acid sequence SEQ ID NO: 2 and the heavy chain variable region of the amino acid sequence SEQ ID NO: 5; the light chain variable region of the amino acid sequence SEQ ID NO: 74 and the heavy chain variable region of the amino acid sequence SEQ ID NO: 76; or the light chain variable region of the amino acid sequence SEQ ID NO: 75 and the heavy chain variable region of the amino acid sequence SEQ ID NO: 77; wherein the transmembrane domain comprises the CD8a transmembrane region (CD8a-TM) polypeptide, the CD8a-TM polypeptide comprising the amino acid sequence SEQ ID NO: 26; and wherein the intracellular signal transduction domain comprises TNF receptor superfamily member 9. The CD137 component and the CD3ζ chain (CD3z) component of the T cell surface glycoprotein, wherein the CD137 component contains the amino acid sequence of SEQ ID NO: 27 and the CD3z component contains the amino acid sequence of SEQ ID NO:

28.

3. The CAR as described in claim 1 or claim 2, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv), the scFv comprising a light chain variable region (VL) and a heavy chain variable region (VH).

4. The CAR as described in claim 3, wherein the scFv comprises a linker polypeptide between the light chain variable region (VL) and the heavy chain variable region (VH).

5. The CAR as claimed in claim 4, wherein the linker polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

6. The CAR as claimed in claim 3, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 to 17 and 340 to 343.

7. The CAR as described in claim 1 or claim 2, wherein the extracellular antigen-binding domain comprises a signaling polypeptide.

8. The CAR as claimed in claim 7, wherein the signaling polypeptide comprises the amino acid sequence of SEQ ID NO:

24.

9. The CAR as claimed in claim 1, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:

45.

10. The CAR as claimed in claim 9, wherein the intracellular signal transduction domain comprises a co-stimulatory domain containing the amino acid sequence of SEQ ID NO: 27 and a primary signal transduction domain containing the amino acid sequence of SEQ ID NO:

28.

11. The CAR as described in claim 1 or claim 2, further comprising a hinge region connecting the transmembrane domain to the extracellular antigen-binding domain.

12. The CAR as described in claim 11, wherein the hinge region is the CD8a hinge region.

13. The CAR as claimed in claim 12, wherein the CD8a hinge region contains an amino acid sequence of SEQ ID NO: 25, 269, 270, 271, or 272.

14. The CAR of claim 1 or claim 2, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to 38, 78 to 81, 273 to 282, and 289 to 292.

15. The CAR of claim 1 or claim 2, wherein the CAR contains an amino acid sequence selected from the group consisting of SEQ ID NO: 46 to 55 and 82 to 85.

16. A CAR that is isolated from lymphocytes and exhibits the characteristics described in any one of claims 1 to 15.

17. The isolated lymphocyte as described in claim 16, wherein the lymphocyte is a T lymphocyte.

18. Isolated lymphocytes as described in claim 16, wherein the lymphocytes are natural killer (NK) cells.

19. An isolated nucleic acid molecule encoding a CAR as described in any one of claims 1 to 15.

20. A vector comprising a nucleic acid molecule as described in claim 19.

21. A cell that exhibits nucleic acid molecules as described in claim 19.

22. A pharmaceutical composition comprising an effective amount of lymphocytes as described in any one of claims 16 to 18.

23. A pharmaceutical composition comprising an effective amount of lymphocytes as described in any one of claims 16 to 18 and a pharmaceutically acceptable excipient.

24. The CAR as described in claim 1 or claim 2, used in treatment.

25. A pharmaceutical composition as described in claim 22 or claim 23, used in treatment.

26. Use of a CAR as described in any one of claims 1 to 15 in the preparation of a medicament for treating a person suffering from cancer.

27. Use of a pharmaceutical composition as described in claim 22 or claim 23 in the preparation of a medicament for treating a person suffering from cancer.

28. Use of a therapeutically effective amount of any one of claims 16 to 18 in the preparation of a medicament for treating a person suffering from cancer, wherein the lymphocytes are administered to the person and thereby induce the killing of cancer cells in the person.

29. The use as described in any of claims 26 to 28, wherein the cancer is prostate cancer or breast cancer expressing androgen receptors.

30. An in vitro method for targeted killing of cancer cells, the method comprising: contacting the cancer cells with lymphocytes as described in any one of claims 16 to 18, wherein the lymphocytes induce the killing of the cancer cells.

31. The in vitro method as described in claim 30, wherein the cancer cells are prostate cancer cells or breast cancer cells expressing androgen receptors.

32. An in vitro method for detecting the presence of cancer in a subject, comprising: contacting a cell sample obtained from the subject with a CAR as described in any one of claims 1 to 15 to form a CAR-cell complex; and detecting the complex, wherein the detection of the complex indicates the presence of cancer in the subject.

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