Mesothelin-targeted chimeric antigen receptors and uses thereof

Patent metadata

Field	Value
Publication number	US-10633441-B2
Application number	US-201615368278-A
Country	US
Kind code	B2
Filing date	Dec 2, 2016
Priority date	Jun 6, 2014
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

The presently disclosed subject matter provides for methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to chimeric antigen receptors (CARs) that specifically target human mesothelin, and immunoresponsive cells comprising such CARs. The presently disclosed mesothelin-targeted CARs have enhanced immune-activating properties, including anti-tumor activity.

First claim

What is claimed is:

1. A chimeric antigen receptor (CAR), comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv) that specifically binds to human mesothelin, wherein the scFv comprises:
(a) a heavy chain variable region comprising an amino acid sequence that is at least about 95% identical to amino acids 1-119 of SEQ ID NO:1, wherein SEQ ID NOS: 11, 12, and 13 are invariable; and
(b) a light chain variable region comprising an amino acid sequence that is at least about 95% identical to amino acids 1-107 of SEQ ID NO:3, wherein SEQ ID NOS: 14, 15, and 16 are invariable.

2. The CAR of claim 1 , wherein the scFv is a human scFv.

3. The CAR of claim 1 , wherein the scFv is comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

4. The CAR of claim 1 , wherein the extracellular antigen-binding domain of the CAR recognizes human mesothelin with a mesothelin expression level of about 1,000 or more mesothelin binding sites/cell.

5. The CAR of claim 1 , wherein the heavy chain variable region of the scFv comprises amino acids 1-119 of SEQ ID NO:1.

6. The CAR of claim 1 , wherein the light chain variable region of the scFv comprises amino acids 1-107 of SEQ ID NO:5.

7. The CAR of claim 1 , wherein the light chain variable region of the scFv comprises amino acids 1-107 of SEQ ID NO:3.

8. The CAR of claim 1 , wherein the heavy chain variable region of the scFv comprises amino acids 1-119 of SEQ ID NO:1; and the light chain variable region of the scFv comprises amino acids 1-107 of SEQ ID NO:5.

9. The CAR of claim 1 , wherein the heavy chain variable region of the scFv comprises amino acids 1-119 of SEQ ID NO:1; and the light chain variable region of the scFv comprises amino acids 1-107 of SEQ ID NO:3.

10. The CAR of claim 1 , wherein the scFv comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13.

11. The CAR of claim 1 , wherein the scFv comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16.

12. The CAR of claim 1 , wherein the scFv comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16.

13. The CAR of claim 1 , wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region.

14. The CAR of claim 1 , wherein the extracellular antigen-binding domain comprises a leader that is covalently joined to the N-terminus of the extracellular antigen-binding domain.

15. The CAR of claim 14 , wherein the leader comprises a CD8 polypeptide.

16. The CAR of claim 1 , wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof.

17. The CAR of claim 16 , wherein the transmembrane domain comprises a CD8 polypeptide.

18. The CAR of claim 16 , wherein the transmembrane domain comprises a CD28 polypeptide.

19. The CAR of claim 1 , wherein the intracellular domain comprises a CD3ζ polypeptide.

20. The CAR of claim 1 , wherein the intracellular domain further comprises at least one co-stimulatory signaling region.

21. The CAR of claim 20 , wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, or a combination thereof.

22. The CAR of claim 1 , wherein the transmembrane domain comprises a CD8 polypeptide and the intracellular domain comprises a CD3ζ polypeptide.

23. The CAR of claim 1 , wherein the transmembrane domain comprises a CD28 polypeptide and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling domain comprising a CD28 polypeptide.

24. The CAR of claim 1 , wherein the transmembrane domain comprises a CD8 polypeptide and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling domain comprising a 4-1BB polypeptide.

25. The CAR of claim 1 , wherein the CAR is Mz.

26. The CAR of claim 25 , wherein the transmembrane domain of Mz comprises a CD8 polypeptide, and the intracellular domain of Mz comprises a CD3ζ polypeptide.

27. The CAR of claim 1 , wherein the CAR is M28z.

28. The CAR of claim 27 , wherein the transmembrane domain M28z comprises a CD28 polypeptide, and the intracellular domain of M28z comprises a CD3ζ polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide.

29. The CAR of claim 1 , wherein the CAR is MBBz.

30. The CAR of claim 29 , wherein the transmembrane domain of MBBz comprises a CD8 polypeptide, and the intracellular domain of MBBz comprises a CD3ζ polypeptide and a co-stimulatory signaling region comprising a 4-1BB polypeptide.

31. The CAR of claim 1 , wherein the CAR is recombinantly expressed.

32. The CAR of claim 1 , wherein the CAR is expressed from a vector.

33. The CAR of claim 32 , wherein the vector is a y-retroviral rector.

34. An immunoresponsive cell comprising the CAR of claim 1 .

35. The immunoresponsive cell of claim 34 , wherein the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent stem cell from which lymphoid cells may be differentiated.

36. The immunoresponsive cell of claim 34 , wherein the isolated immunoresponsive cell expresses from about 1 to about 4 vector copy numbers/cell of the CAR.

37. A method for producing an immunoresponsive cell that binds to human mesothelin, comprising introducing into the immunoresponsive cell
a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv) that specifically binds to human mesothelin, wherein the scFv comprises:
(a) a heavy chain variable region comprising an amino acid sequence that is at least about 95% ide

Assignees

• Memorial Sloan Kettering Cancer Center
• Us Health

Inventors

• Adusumilli Prasad S
• Sadelain Michel
• Dimitrov Dimiter S
• Feng Yang

Classifications

• A61K2039/505

  comprising antibodies · CPC title

• C07K14/70517

  CD8 · CPC title

• C07K2317/21

  from primates, e.g. man · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title