Treatment of cancer using humanized anti-EGFRvIII chimeric antigen receptor

• US 9,394,368 B2
• Filed: 02/20/2014
• Issued: 07/19/2016
• Est. Priority Date: 02/20/2013
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania), University of Pittsburgh of The Commonwealth System of Higher Education
• Original Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania), University of 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
• Primary Examiner(s): PAK, MICHAEL D
• Application Number:
  US14/184,924
• Publication Number:
  US 20140322275A1
• Time in Patent Office: 880 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/47 Brain; Nervous system
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/001102 Receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464404 Epidermal growth factor receptors [EGFR]
• CPC Class Codes: A61K 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/3053 Skin, nerves, brain
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/34 : Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

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.

Claims

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 greater than 99% identity thereof.

36. The isolated nucleic acid molecule of claim 1, wherein the encoded CAR comprises the amino acid sequence of SEQ ID NO:

73, or an amino acid sequence having greater than 99% identity thereof.

37. The isolated nucleic acid molecule of claim 1, wherein the encoded anti-EGFRvIII binding domain comprises the sequence of SEQ ID NO:

68.

38. The isolated nucleic acid molecule of claim 2, wherein the nucleic acid sequence encoding the anti-EGFRvIII binding domain comprises the nucleotide sequence of SEQ ID NO:

69.