TREATMENT OF CANCER USING HUMANIZED ANTI-EGFRvIII CHIMERIC ANTIGEN RECEPTOR

• US 20140322275A1
• Filed: 02/20/2014
• Published: 10/30/2014
• 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: University of Pittsburgh of The Commonwealth System of Higher Education
• Inventors: Brogdon, Jennifer, June, Carl H., Loew, Andreas, Maus, Marcela, Scholler, John, Okada, Hideho, Johnson, Laura Alexandra
• Granted Patent: US 9,394,368 B2
• Time in Patent Office: Days
• US Class Current: 424/277.1
• 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 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 39/4611 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, and wherein said anti-EGFRvIII binding domain comprises one or more of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of any anti-EGFRvIII light chain binding domain amino acid sequence listed in Table 2 or SEQ ID NO:

11, and one or more of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any anti-EGFRvIII heavy chain binding domain amino acid sequence listed in Table 2 or SEQ ID NO;

11.

2. The isolated nucleic acid molecule of claim 1, comprising (a) LC CDR1, LC CDR2 and LC CDR3 of any anti-EGFRvIII light chain binding domain amino acid sequence listed in Table 2 or SEQ ID NO:

11;

(b) HC CDR1, HC CDR2, and HC CDR3 of any anti-EGFRvIII heavy chain binding domain amino acid sequence listed in Table 2 or SEQ ID NO;

11;

or (c) LC CDR1, LC CDR2 and LC CDR3 of any anti-EGFRvIII light chain binding domain amino acid sequence listed in Table 2 or SEQ ID NO;

11, and HC CDR1, HC CDR2, and HC CDR3 of any anti-EGFRvIII heavy chain binding domain amino acid sequence listed in Table 2 or SEQ ID NO;

11.

5. The isolated nucleic acid molecule of claim 1, comprising (a) any light chain variable region listed in Table 2 or SEQ ID NO:

11;

(b) any heavy chain variable region listed in Table 2 or SEQ ID NO;

11;

or (c) any light chain variable region listed in Table 2 or SEQ ID NO;

11 and any heavy chain variable region listed Table 2 or SEQ ID NO;

11.

8. The isolated nucleic acid molecule of claim 1, wherein the EGFRvIII binding domain is a scFv.

9. The isolated nucleic acid molecule of claim 1, wherein (a) the light chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 2 or SEQ ID NO:

11, or a sequence with 95-99% identity to an amino acid sequence provided in Table 2 or SEQ ID NO;

11;

(b) wherein the heavy chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in Table 2 or SEQ ID NO;

11, or a sequence with 95-99% identity to an amino acid sequence provided in Table 2 or SEQ ID NO;

11;

or (c) both (a) and (b).

11. The isolated nucleic acid molecule of claim 1, wherein the anti-EGFRvIII binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

38, SEQ ID NO;

44, SEQ ID NO;

50, SEQ ID NO;

56, SEQ ID NO;

62, SEQ ID NO;

68, SEQ ID NO;

74, SEQ ID NO;

80, and SEQ ID NO;

86, or a sequence with 95-99% identify thereof.

12. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the anti-EGFRvIII binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

39, SEQ ID NO;

45, SEQ ID NO;

51, SEQ ID NO;

57, SEQ ID NO;

63, SEQ ID NO;

69, SEQ ID NO;

75, SEQ ID NO;

81 and SEQ ID NO;

98, or a sequence with 95-99% identify thereof.

13. 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.

14. The isolated nucleic acid molecule of claim 1, wherein the encoded transmembrane domain comprises a sequence of SEQ ID NO:

15, or an amino acid sequence comprises at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO;

15, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

15.

16. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the transmembrane domain comprises a sequence of SEQ ID NO:

8, or a sequence with 95-99% identify thereof.

17. 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.

18. The isolated nucleic acid molecule of claim 17, wherein the encoded hinge region comprises SEQ ID NO:

14, or a sequence with 95-99% identity thereof.

19. The isolated nucleic acid molecule of claim 17, wherein the nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO:

7, or a sequence with 95-99% identify thereof.

20. The isolated nucleic acid molecule of claim 1, further comprising a sequence encoding a costimulatory domain.

21. The isolated nucleic acid molecule of claim 20, wherein the encoded costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137).

22. The isolated nucleic acid molecule of claim 20, wherein the encoded costimulatory domain comprises a sequence of SEQ ID NO:

16, or an amino acid sequence which comprises at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO;

16, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

16.

24. The isolated nucleic acid molecule of claim 20, wherein the nucleic acid sequence encoding the costimulatory domain comprises a sequence of SEQ ID NO:

9, or a sequence with 95-99% identify thereof.

26. 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.

27. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO:

16 and/or the sequence of SEQ ID NO;

17 or SEQ ID NO;

99, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO;

16 and/or an amino acid sequence of SEQ ID NO;

17 or SEQ ID NO;

99, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

16 and/or an amino acid sequence of SEQ ID NO;

17 or SEQ ID NO;

99.

29. 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.

30. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence of SEQ ID NO:

9, or a sequence with 95-99% identify thereof, and/or a sequence of SEQ ID NO;

10 or SEQ ID NO;

100, or a sequence with 95-99% identity thereof.

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

32. The isolated nucleic acid molecule of claim 31, wherein the leader sequence comprises SEQ ID NO:

13.

33. An isolated polypeptide molecule encoded by the nucleic acid molecule of claim 1.

34. The isolated polypeptide molecule of claim 33 comprising a sequence selected from the group consisting of SEQ ID NO:

43, SEQ ID NO;

49, SEQ ID NO;

55, SEQ ID NO;

61, SEQ ID NO;

67, SEQ ID NO;

73, SEQ ID NO;

79, SEQ ID NO;

85 and SEQ ID NO;

90, or a sequence with 95-99% identify thereof.

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

74. The vector of claim 73, wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

75. The vector of claim 73, further comprising a promoter.

76. The vector of claim 75, wherein the promoter is an EF-1 promoter.

77. The vector of claim 76, wherein the EF-1 promoter comprises a sequence of SEQ ID NO:

97.

78. The vector of claim 73, wherein the vector is an in vitro transcribed vector.

79. The vector of claim 73, wherein the nucleic acid sequence in the vector further comprises a poly(A) tail.

80. The vector of claim 73, wherein the nucleic acid sequence in the vector further comprises a 3′

UTR.

81. A cell comprising the vector of claim 73.

82. The cell of claim 81, wherein the cell is a T cell.

83. The cell of claim 82, wherein the T cell is a CD8+ T cell.

84. The cell of claim 81, wherein the cell is a human cell.

85. A method of making a cell comprising transducing a T cell with a vector of claim 73.

86. 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 a nucleic acid molecule of claim 1.

87. A method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of a cell of claim 81.

91. A method of treating a mammal having a disease associated with expression of EGFRvIII comprising administering to the mammal an effective amount of the cell of claim 81.

93. The method of claim 91, wherein the disease associated with expression of EGFRvIII is a cancer 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 (e.g., non-small cell lung carcinomas) breast, prostate, ovarian, colorectal and bladder carcinoma and any combination thereof, and metastases of any of the cancers.

3-4. -4. (canceled)

6-7. -7. (canceled)

10. (canceled)

15. (canceled)

23. (canceled)

25. (canceled)

28. (canceled)

35-36. -36. (canceled)

37. An isolated chimeric antigen receptor (CAR) molecule comprising an anti-EGFRvIII binding domain, a transmembrane domain, and an intracellular signaling domain.

38. The isolated CAR molecule of claim 37, wherein the intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

40. The isolated CAR molecule of claim 37, wherein the anti-EGFRvIII binding domain comprises one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of any anti-EGFRvIII binding domain listed in Table 2 or SEQ ID NO:

11, and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any anti-EGFRvIII binding domain listed in Table 2 or SEQ ID NO;

11.

52. The isolated CAR molecule of claim 37, wherein the anti-EGFRvIII binding domain is connected to the transmembrane domain by a hinge region.

64. The isolated CAR molecule of claim 37, further comprising a leader sequence.

39. (canceled)

41-51. -51. (canceled)

53-63. -63. (canceled)

65. (canceled)

66. An anti-EGFRvIII binding domain comprising one or more light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of an anti-EGFRvIII binding domain in SEQ ID NO:

38, 44, 50, 56, 62, 68, 74 or 80, and one or more heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-EGFRvIII binding domain in SEQ ID NO;

38, 44, 50, 56, 62, 68, 74 or 80.

72. The anti-EGFRvIII binding domain of claim 66, comprising a sequence selected from a group consisting of SEQ ID NO:

38, SEQ ID NO;

44, SEQ ID NO;

50, SEQ ID NO;

56, SEQ ID NO;

62, SEQ ID NO;

68, SEQ ID NO;

74, SEQ ID NO;

80, and SEQ ID NO;

86, or a sequence with 95-99% identify thereof.

67-71. -71. (canceled)

88-90. -90. (canceled)

92. (canceled)

94-98. -98. (canceled)