EFFECTIVE TARGETING OF PRIMARY HUMAN LEUKEMIA USING ANTI-CD123 CHIMERIC ANTIGEN RECEPTOR ENGINEERED T CELLS

• US 20140322212A1
• 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)
• Original Assignee: Novartis Ag
• Inventors: Brogdon, Jennifer, June, Carl H., Kalos, Michael D., Loew, Andreas, Scholler, John, Gill, Saar
• Granted Patent: US 9,573,988 B2
• Time in Patent Office: Days
• US Class Current: 424/134.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/507 Comprising a combination of two or more separate antibodies
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• 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 39/464429 Molecules with a "CD" designation not provided for elsewhere
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/53 Hinge
• 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 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• 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 leukemia, for example, acute myeloid leukemia (AML) and B-cell acute lymphoid leukemia (B-ALL). The invention also relates to at least one chimeric antigen receptor (CAR) specific to CD123, vectors comprising the same, and recombinant T cells comprising the CD123 CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a CD123 binding domain. The invention also includes methods of bone marrow ablation for use in treatments necessitating bone marrow reconditioning or transplant.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein said CAR comprises an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain, and wherein said anti-CD123 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-CD123 light chain binding domain amino acid sequence listed in Table 1, SEQ ID NO:

2 or SEQ ID NO;

101, 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-CD123 heavy chain binding domain amino acid sequence listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101.

2. The isolated nucleic acid molecule of claim 1, comprising (a) LC CDR1, LC CDR2 and LC CDR3 of any anti-CD123 light chain binding domain amino acid sequence listed in Table 1, SEQ ID NO:

2 or SEQ ID NO;

101;

(b) HC CDR1, HC CDR2, and HC CDR3 of any anti-CD123 heavy chain binding domain amino acid sequence listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101;

or (c) LC CDR1, LC CDR2 and LC CDR3 of any anti-CD123 light chain binding domain amino acid sequence listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101, and HC CDR1, HC CDR2, and HC CDR3 of any anti-CD123 heavy chain binding domain amino acid sequence listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101.

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

2 or SEQ ID NO;

101;

(b) any heavy chain variable region listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101;

or (c) any light chain variable region listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101 and any heavy chain variable region listed Table 1, SEQ ID NO;

2 or SEQ ID NO;

101.

8. The isolated nucleic acid molecule of claim 1, wherein the anti-CD123 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 1, SEQ ID NO:

2 or SEQ ID NO;

101, or a sequence with 95-99% identity to an amino acid sequence provided in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101;

(b) 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 1, SEQ ID NO;

2 or SEQ ID NO;

101, or a sequence with 95-99% identity to an amino acid sequence provided in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101;

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

11. The isolated nucleic acid molecule of claim 1, wherein the anti-CD123 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

36, SEQ ID NO;

42, SEQ ID NO;

48, SEQ ID NO;

54, SEQ ID NO;

60, SEQ ID NO;

66, SEQ ID NO;

72, and SEQ ID NO;

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

12. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the anti-CD123 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

37, SEQ ID NO;

43, SEQ ID NO;

49, SEQ ID NO;

55, SEQ ID NO;

61, SEQ ID NO;

67, SEQ ID NO;

73, and SEQ ID NO;

79, or a sequence with 95-99% identity 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:

5, or an amino acid sequence comprising 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;

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

5.

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:

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

17. The isolated nucleic acid molecule of claim 1, wherein the encoded anti-CD123 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 an amino acid sequence of any of SEQ ID NO:

4, 104, 122 or 124, 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:

11, or a sequence with 95-99% identity 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, CDS, 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:

6 or SEQ ID NO;

23, or an amino acid sequence comprising 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;

6 or SEQ ID NO;

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

6 or SEQ ID NO;

23.

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:

13, SEQ ID NO;

27, or a sequence with 95-99% identity 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:

6 or SEQ ID NO;

23 and/or the sequence of SEQ ID NO;

7 or SEQ ID NO;

98, 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;

6 or SEQ ID NO;

23 and an amino acid sequence of SEQ ID NO;

7 or SEQ ID NO;

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

6 or SEQ ID NO;

23 and an amino acid sequence of SEQ ID NO;

7 or SEQ ID NO;

98.

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

6 or SEQ ID NO;

23 and the sequence of SEQ ID NO;

7 or SEQ ID NO;

98, 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 one or both of a sequence of SEQ ID NO:

13 or SEQ ID NO;

27, or a sequence with 95-99% identity thereof, and a sequence of SEQ ID NO;

14 or SEQ ID NO;

99, 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:

3.

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:

41, SEQ ID NO;

47, SEQ ID NO;

53, SEQ ID NO;

59, SEQ ID NO;

65, SEQ ID NO;

71, SEQ ID NO;

77, and SEQ ID NO;

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

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

1, SEQ ID NO;

109, SEQ ID NO;

111, SEQ ID NO;

113, SEQ ID NO;

100, SEQ ID NO;

117, SEQ ID NO;

119, and SEQ ID NO;

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

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

74. The vector of claim 73, where in 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 CD123 comprising administering to the mammal an effective amount of a cell of claim 81.

92. The method of claim 91, wherein the disease associated with CD123 expression is acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), myelodysplastic syndrome, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, Hodgkin lymphoma, blastic plasmacytoid dendritic cell neoplasm, and any combination thereof.

103. A method of eradicating at least a portion of existing bone marrow in a subject, the method comprising administering to the subject an effective amount of a cell of claim 81.

3-4. -4. (canceled)

6-7. -7. (canceled)

10. (canceled)

15. (canceled)

23. (canceled)

25. (canceled)

28. (canceled)

36. (canceled)

37. An isolated chimeric antigen receptor (CAR) molecule comprising an anti-CD123 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-CD123 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-CD123 binding domain listed in Table 1, SEQ ID NO:

2 or SEQ ID NO;

101, 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-CD123 binding domain listed in Table 1, SEQ ID NO;

2 or SEQ ID NO;

101.

52. The isolated CAR molecule of claim 37, wherein the anti-CD123 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.

108. An in vitro transcribed vector encoding the CAR of claim 37.

39. (canceled)

41-51. -51. (canceled)

53-63. -63. (canceled)

65. (canceled)

66. An anti-CD123 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-CD123 binding domain in SEQ ID NO:

36, 42, 48, 54, 60, 66, 72 or 78, 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-CD123 binding domain in SEQ ID NO;

36, 42, 48, 54, 60, 66, 72 or 78.

72. The anti-CD123 binding domain of claim 66, comprising a sequence selected from a group consisting of SEQ ID NO:

36, SEQ ID NO;

42, SEQ ID NO;

48, SEQ ID NO;

54, SEQ ID NO;

60, SEQ ID NO;

66, SEQ ID NO;

72 or SEQ ID NO;

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

67-71. -71. (canceled)

88-90. -90. (canceled)

93-102. -102. (canceled)

104-107. -107. (canceled)

109-114. -114. (canceled)