Treatment of cancer using humanized anti-EGFRvIII chimeric antigen receptor

Patent metadata

Field	Value
Publication number	US-9394368-B2
Application number	US-201414184924-A
Country	US
Kind code	B2
Filing date	Feb 20, 2014
Priority date	Feb 20, 2013
Publication date	Jul 19, 2016
Grant date	Jul 19, 2016

Abstract

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.

First claim

What is claimed is:

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein said CAR comprises an anti-EGFRvIII binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain, a costimulatory domain, or both a primary signaling domain and a costimulatory domain, wherein the encoded anti-EGFRvIII binding domain comprises an amino acid sequence with greater than 99% sequence identity to SEQ ID NO:68 comprising:
(a) a heavy chain immunoglobulin variable region comprising:
(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) a light chain immunoglobulin variable region comprising:
(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 isolated nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the anti-EGFRvIII binding domain comprises a nucleotide sequence with greater than 99% sequence identity to SEQ ID NO: 69.

3. The isolated nucleic acid molecule of claim 1 , wherein the encoded CAR comprises 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.

4. The isolated nucleic acid molecule of claim 1 , wherein the encoded transmembrane domain comprises the sequence of SEQ ID NO: 15; an amino acid sequence comprises 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.

5. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the transmembrane domain comprises the sequence of SEQ ID NO:8, or a sequence with greater than 95% identity thereof.

6. The isolated nucleic acid molecule of claim 1 , wherein the encoded anti-EGFRvIII binding domain is connected to the transmembrane domain by a hinge region.

7. The isolated nucleic acid molecule of claim 6 , wherein the encoded hinge region comprises SEQ ID NO:14, or a sequence with greater than 95% identity thereof.

8. The isolated nucleic acid molecule of claim 6 , wherein the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO:7, or a sequence with greater than 95% identity thereof.

9. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

10. The isolated nucleic acid molecule of claim 9 , wherein the encoded 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), ICOS (CD278), and 4-1BB (CD137).

11. The isolated nucleic acid molecule of claim 9 , wherein the encoded costimulatory domain comprises the sequence of SEQ ID NO:16; an amino acid sequence which comprises 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.

12. The isolated nucleic acid molecule of claim 9 , wherein the nucleic acid sequence encoding the costimulatory domain comprises the sequence of SEQ ID NO:9, or a sequence with greater than 95% identity thereof.

13. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.

14. The isolated nucleic acid molecule of claim 1 , wherein:
the encoded costimulatory domain comprises the sequence of SEQ ID NO: 16; 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, and
the encoded primary signaling domain comprises the sequence of SEQ ID NO: 17 or SEQ ID NO:99; an amino acid sequence having at least one modification but not more than 20 modifications of 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:17 or SEQ ID NO:99.

15. The isolated nucleic acid molecule of claim 1 , wherein the encoded 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.

16. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO:9, or a sequence with greater than 95% identify thereof, and/or the sequence of SEQ ID NO:10 or SEQ ID NO:100, or a sequence with greater than 95% identity thereof.

17. The isolated nucleic acid molecule of claim 1 , further comprising a leader sequence.

18. The isolated nucleic acid molecule of claim 17 , wherein the leader sequence comprises SEQ ID NO: 13.

19. A vector comprising a nucleic acid molecule of claim 1 .

20. The vector of claim 19 , wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, and a retrovirus vector.

21. The vector of claim 19 , further comprising a promoter.

22. The vector of claim 21 , wherein the promoter is an EF-1 promoter.

23. The vector of claim 22 , wherein the EF-1 promoter comprises the sequence of SEQ ID NO: 97.

24. The vector of claim 19 , wherein the vector is an in vitro transcribed vector.

25. The vector of claim 19 , wherein the nucleic acid sequence in the vector further comprises a poly(A) tail.

26. The vector of claim 19 , wherein the nucleic acid sequence in the vector further comprises a 3'UTR.

27. An isolated cell comprising the vector of claim 19 .

28. The cell of claim 27 , wherein the cell is a T cell.

29. The cell of claim 28 , wherein the T cell is a CD8+T cell.

30. The cell of claim 27 , wherein the cell is a human cell.

31. A method of making a cell comprising transducing a T cell with a vector of claim 19 .

32. 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 the nucleic acid molecule of claim 1 .

33. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a primary signaling domain.

34. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a costimulatory domain.

35. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the CAR comprises the nucleotide sequence of SEQ ID NO:72, or a nucleic acid sequence having great

Assignees

• Brogdon Jennifer
• Johnson Laura Alexandra
• June Carl H
• Loew Andreas
• Maus Marcela
• Scholler John
• Okada Hideho
• Novartis Ag
• Univ Pennsylvania
• Univ Pittsburgh—Of The Commonwealth System Of Higher Education

Inventors

• Brogdon Jennifer
• Johnson Laura Alexandra
• June Carl H
• Loew Andreas
• Maus Marcela
• Scholler John
• Okada Hideho

Classifications

• C07K2319/33

  fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• C07K14/7051

  T-cell receptor (TcR)-CD3 complex · CPC title

• A61P35/04

  specific for metastasis · CPC title

• A61P35/00

  Antineoplastic agents · CPC title