Treatment of cancer using humanized anti-EGFRvIII chimeric antigen receptor
The invention provides compositions and methods for treating diseases associated with expression of EGFRvIII. The invention also relates to chimeric antigen receptor (CAR) specific to EGFRvIII, vectors encoding the same, and recombinant T cells comprising the anti-EGFRvIII CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises an anti-EGFRvIII binding domain.
1. A method of treating a mammal having a cancer associated with expression of EGFRvIII comprising administering to the mammal an effective amount of cells expressing a chimeric antigen receptor (CAR) comprising a humanized anti-EGFRvIII binding domain, a transmembrane domain, and an intracellular signaling domain, wherein said anti-EGFRvIII binding domain comprises a light chain variable region and a heavy chain variable region, wherein:
(a) the heavy chain variable region comprises:
(i) a CDR1 comprising the sequence DYYIH (SEQ ID NO: 22);
(ii) a CDR2 comprising the sequence RIDPENDETKYGPIFQG (SEQ ID NO: 23); and
(iii) a CDR3 comprising the sequence RGGVY (SEQ ID NO: 24); and
(b) the light chain variable region comprises:
(i) a CDR1 comprising the sequence KSSQSLLDSDGKTYLN (SEQ ID NO: 26);
(ii) a CDR2 comprising the sequence LVSKLDS (SEQ ID NO: 27); and
(iii) a CDR3 comprising the sequence WQGTHFPGT (SEQ ID NO: 28).
2. The method of claim 1 , wherein the cancer associated with expression of EGFRvIII is selected from the group consisting of glioblastoma multiforme (GBM), anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma, anaplastic oligodendroglioma, anaplastic ependymoma, choroid plexus carcinoma, anaplastic ganglioglioma, pineoblastoma, medulloepithelioma, ependymoblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, atypical teratoid/rhabdoid tumor, lung cancer, non-small cell lung carcinomas, breast, prostate, ovarian, colorectal and bladder carcinoma and any combination thereof, and metastases of any of the cancers.
3. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises:
(1) the amino acid sequence of SEQ ID NO:68, SEQ ID NO:50, or SEQ ID NO:80;
(2) an amino acid sequence with at least 95% sequence identity to SEQ ID NO:68, SEQ ID NO:50, or SEQ ID NO:80; or
(3) an amino acid sequence having at least one, two or three modifications but not more than 10 modifications of the amino acid sequence of SEQ ID NO:68, SEQ ID NO:50, or SEQ ID NO:80.
4. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 68.
5. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 99% identity to the amino acid sequence of SEQ ID NO: 68.
6. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence having at least one, two or three modifications but not more than 10 modifications of the amino acid sequence of SEQ ID NO: 68.
7. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO: 68.
8. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:50.
9. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO:50.
10. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence having at least one, two or three modifications but not more than 10 modifications of the amino acid sequence of SEQ ID NO:50.
11. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO:50.
12. The method of claim 1 , wherein the anti-EGFRvIII binding comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO:80.
13. The method of claim 1 , wherein the anti-EGFRvIII binding comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO:80.
14. The method of claim 1 , wherein the anti-EGFRvIII binding comprises an amino acid sequence having at least one, two or three modifications but not more than 10 modifications of the amino acid sequence of SEQ ID NO:80.
15. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO:80.
16. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an scFv domain or a Fab domain.
17. The method of claim 1 , 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, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
18. The method of claim 17 , wherein the transmembrane domain comprises an amino acid sequence having at least one modification but not more than 20 modifications of the amino acid sequence of SEQ ID NO:15, or a sequence with greater than 95% identity to the amino acid sequence of SEQ ID NO:15.
19. The method of claim 17 , wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:15.
20. The method of claim 1 , wherein the anti-EGFRvIII binding domain is connected to the transmembrane domain by a hinge region.
21. The method of claim 20 , wherein the encoded hinge region comprises SEQ ID NO:14, or a sequence with greater than 95% identity thereof.
22. The method of claim 1 , wherein the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both a primary signaling domain and a costimulatory domain.
23. The method of claim 22 , wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).
24. The method of claim 22 , wherein the costimulatory domain comprises an amino acid sequence having at least one modification but not more than 20 modifications of the amino acid sequence of SEQ ID NO:16, or a sequence with greater than 95% identity to the amino acid sequence of SEQ ID NO:16.
25. The method of claim 22 , wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:16.
26. The method of claim 1 , wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.
27. The method of claim 26 , wherein the intracellular signaling domain comprises an amino acid sequence having at least one modification but not more than 20 modifications of the amino acid sequence of SEQ ID NO:16 and/or the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:99, or a sequence with greater than 95% identity to the amino acid sequence of SEQ ID NO:16 and/or the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:99.
28. The method of claim 26 , wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and/or the sequence of SEQ ID NO:17.
29. The method of claim 26 , wherein intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and/or the sequence of SEQ ID NO:99.
30. The method of claim 26 , wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and the sequence of SEQ ID NO: 17 or SEQ ID NO:99, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.
31. The method of claim 1 , further comprising a leader sequence, wherein the leader sequence comprises an amino acid sequence of SEQ ID NO: 13, or a sequence with greater than 95% identity to an amino acid sequence of SEQ ID NO:13.
32. The method of claim 1 , wherein the cells are T cells or CD8+ T cells.
33. The method of claim 1 , wherein the cells are administered in combination with an agent that increases the efficacy of a cell expressing a CAR.
34. The method of claim 1 , wherein the cells are administered in combination with an agent that ameliorates one or more side effects associated with administration of the cell expressing a CAR.
35. The method of claim 1 , wherein the cells are administered in combination with an agent that treats the cancer associated with EGFRvIII.
36. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence having no more than 20 modifications of the light chain variable region and/or the heavy chain variable region of SEQ ID NO: 68.
37. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 95% sequence identity to the light chain variable region and/or the heavy chain variable region of SEQ ID NO: 68.
38. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 95% sequence identity to the light chain variable region and/or the heavy chain variable region of SEQ ID NO: 50.
39. The method of claim 1 , wherein the anti-EGFRvIII binding domain comprises an amino acid sequence with at least 95% sequence identity to the light chain variable region and/or the heavy chain variable region of SEQ ID NO: 80.