Effective targeting of primary human leukemia using anti-CD123 chimeric antigen receptor engineered T cells

• US 9,573,988 B2
• Filed: 02/20/2014
• Issued: 02/21/2017
• 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, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Brogdon, Jennifer, Gill, Saar, June, Carl H., Kalos, Michael D., Loew, Andreas, Scholler, John
• Primary Examiner(s): PAK, MICHAEL D
• Application Number:
  US14/184,895
• Publication Number:
  US 20140322212A1
• Time in Patent Office: 1,097 Days
• Field of Search: None
• US Class Current: 1/1
• 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 2039/5158 Antigen-pulsed cells, e.g. T-cells
• 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 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 a humanized anti-CD123 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, and

wherein said anti-CD123 binding domain comprises:

(a) a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

22, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

18;

or

(b) a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

30, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

26;

or

(c) a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

89, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

86.

2. The isolated nucleic acid molecule of claim 1, wherein the encoded anti-CD123 binding domain comprises the light chain variable region 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—

and the heavy chain variable region 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.

3. The isolated nucleic acid molecule of claim 1, wherein the anti-CD 123 binding domain is a scFv.

4. The isolated nucleic acid molecule of claim 1, wherein

(a) the encoded anti-CD123 binding domain comprises a light chain variable region comprising 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 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 to an amino acid sequence provided in 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;

(b) the encoded anti-CD123 binding domain comprises a heavy chain variable region comprising 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 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 to an amino acid sequence provided in 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

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

5. The isolated nucleic acid molecule of claim 1, wherein the anti-CD123 binding domain comprises a sequence selected from the 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.

6. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the anti-CD123 binding domain comprises a sequence selected from the 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.

7. 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.

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

5;

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

5;

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

5.

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

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

10. 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.

11. The isolated nucleic acid molecule of claim 10, wherein the encoded hinge region comprises the amino acid sequence of any of SEQ ID NO:

4, 104, 122 or 124, or a sequence with 95-99% identity thereof.

12. The isolated nucleic acid molecule of claim 10, wherein the nucleic acid sequence encoding the hinge region comprises the nucleic acid sequence of SEQ ID NO:

11, or a nucleic acid sequence with 95-99% identity thereof.

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

14. The isolated nucleic acid molecule of claim 13, 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).

15. The isolated nucleic acid molecule of claim 13, wherein the encoded costimulatory domain comprises the sequence of SEQ ID NO:

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23.

16. The isolated nucleic acid molecule of claim 13, wherein the nucleic acid sequence encoding the costimulatory domain comprises the nucleic acid sequence of SEQ ID NO:

13, SEQ ID NO;

27, or a nucleic acid sequence with 95-99% identity thereof.

17. 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.

18. The isolated nucleic acid molecule of claim 1, wherein:

the encoded costimulatory domain comprises the sequence of SEQ ID NO;

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23, and

the encoded primary signaling domain comprises the sequence of SEQ ID NO;

7 or SEQ ID NO;

98;

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

7 or SEQ ID NO;

98;

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

7 or SEQ ID NO;

98.

19. 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.

20. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises one or both of:

(a) the sequence of SEQ ID NO;

13 or SEQ ID NO;

27, or a sequence with 95-99% identity thereof;

or

(b) the sequence of SEQ ID NO;

14 or SEQ ID NO;

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

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

22. The isolated nucleic acid molecule of claim 21, wherein the leader sequence comprises SEQ ID NO:

3.

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

24. The isolated polypeptide molecule of claim 23, 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, or SEQ ID NO;

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

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

30. The vector of claim 29, where in the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, and a retrovirus vector.

31. The vector of claim 29, further comprising a promoter.

32. The vector of claim 31, wherein the promoter is an EF-1 promoter.

33. The vector of claim 32, wherein the EF-1 promoter comprises the sequence of SEQ ID NO:

97.

34. The vector of claim 29, wherein the vector is an in vitro transcribed vector.

35. The vector of claim 29, wherein the nucleic acid sequence in the vector further comprises a poly(A) tail.

36. The vector of claim 29, wherein the nucleic acid sequence in the vector further comprises a 3′

UTR.

37. An isolated cell or population of cells comprising the vector of claim 29.

38. The cell of claim 37, wherein the cell is a T cell.

39. The cell of claim 38, wherein the T cell is a CD8+ T cell.

40. The cell of claim 37, wherein the cell is a human cell.

41. A method of making a cell comprising transducing a T cell with the vector of claim 29.

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

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

45. The isolated nucleic acid molecule of claim 1, wherein said anti-CD 123 binding domain comprises:

a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

22, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

18.

46. The isolated nucleic acid molecule of claim 45, wherein the anti-CD123 binding domain comprises the light chain variable region 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 and the heavy chain variable region 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.

47. The isolated nucleic acid molecule of claim 45, wherein the encoded intracellular signaling domain comprises:

(i) a costimulatory domain comprising a functional signaling domain of a protein chosen from OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or 4-1BB (CD137);

(ii) a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, CD66d, DAP10, or DAP12;

or

(iii) both (i) and (ii).

48. The isolated nucleic acid molecule of claim 45, wherein the encoded intracellular signaling domain comprises:

(i) the amino acid sequence of SEQ ID NO;

6 or SEQ ID NO;

23;

(ii) the amino acid sequence of SEQ ID NO;

7 or SEQ ID NO;

98;

or

(iii) both (i) and (ii).

49. The isolated nucleic acid molecule of claim 47, wherein the encoded 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.

50. The isolated nucleic acid molecule of claim 45, wherein the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO:

5.

51. The isolated nucleic acid molecule of claim 45, wherein the anti-CD 123 binding domain is connected to the encoded transmembrane domain by a hinge region, and wherein the hinge region comprises the amino acid sequence of any of SEQ ID NO:

4, 104, 122, or 124.

52. The isolated nucleic acid molecule of claim 1, wherein said anti-CD 123 binding domain comprises:

a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

30, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

26.

53. The isolated nucleic acid molecule of claim 1, wherein said anti-CD 123 binding domain comprises:

a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

89, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

86.

54. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, CD66d, DAP10, or DAP12.

55. The isolated nucleic acid molecule of claim 54, wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta, wherein the CD3 zeta comprises the amino acid 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 the amino acid sequence of SEQ ID NO;

7 or SEQ ID NO;

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

7 or SEQ ID NO;

98.

58. An isolated polypeptide molecule encoded by the nucleic acid molecule of claim 45.

59. The isolated polypeptide molecule of claim 58, comprising the light chain variable region 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, and the heavy chain variable region 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.

60. The isolated polypeptide molecule of claim 59, comprising an intracellular signaling domain comprising:

(i) the amino acid sequence of SEQ ID NO;

6 or SEQ ID NO;

23;

(ii) the amino acid sequence of SEQ ID NO;

7 or SEQ ID NO;

98;

or

(iii) both (i) and (ii).

61. The isolated polypeptide molecule of claim 60, comprising a transmembrane domain comprising the amino acid sequence of SEQ ID NO:

5.

62. The isolated polypeptide molecule of claim 61, further comprising a hinge region, wherein the hinge region comprises the amino acid sequence of any of SEQ ID NO:

4, 104, 122, or 124.

76. The isolated nucleic acid molecule of claim 5, wherein the anti-CD123 binding domain comprises a sequence selected from the 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.

77. The isolated nucleic acid molecule of claim 6, wherein the nucleic acid sequence encoding the anti-CD123 binding domain comprises a sequence selected from the 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.

78. The isolated nucleic acid molecule of claim 8, wherein the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO:

5.

79. The isolated nucleic acid molecule of claim 9, wherein the nucleic acid sequence encoding the transmembrane domain comprises the nucleic acid sequence of SEQ ID NO:

12.

80. The isolated nucleic acid molecule of claim 11, wherein the encoded hinge region comprises the amino acid sequence of any of SEQ ID NO:

4, 104, 122 or 124.

81. The isolated nucleic acid molecule of claim 12, wherein the nucleic acid sequence encoding the hinge region comprises the nucleic acid sequence of SEQ ID NO:

11.

82. The isolated nucleic acid molecule of claim 15, wherein the encoded costimulatory domain comprises the sequence of SEQ ID NO:

6 or SEQ ID NO;

23.

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

13 or SEQ ID NO;

27.

84. The isolated nucleic acid molecule of claim 20, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises one or both of:

(a) the sequence of SEQ ID NO;

13 or SEQ ID NO;

27;

or

(b) the sequence of SEQ ID NO;

14 or SEQ ID NO;

99.

85. The isolated polypeptide molecule of claim 24, 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, or SEQ ID NO;

83.

86. The isolated nucleic acid molecule of claim 1, comprising a sequence selected from the group consisting of SEQ ID NO:

40, SEQ ID NO;

46, SEQ ID NO;

52, SEQ ID NO;

58, SEQ ID NO;

64, SEQ ID NO;

70, SEQ ID NO;

76, or SEQ ID NO;

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

87. The isolated nucleic acid molecule of claim 24, comprising a sequence selected from the group consisting of SEQ ID NO:

40, SEQ ID NO;

46, SEQ ID NO;

52, SEQ ID NO;

58, SEQ ID NO;

64, SEQ ID NO;

70, SEQ ID NO;

76, or SEQ ID NO;

82.

88. The isolated nucleic acid molecule of claim 1, wherein the anti-CD123 binding domain comprises the amino acid sequence of SEQ ID NO:

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

101.

89. The isolated nucleic acid molecule of claim 88, wherein the anti-CD123 binding domain comprises the amino acid sequence of SEQ ID NO:

101.

90. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the anti-CD123 binding domain comprises the nucleic acid sequence of SEQ ID NO:

103 or a sequence with 95-99% identity to SEQ ID NO;

103.

91. The isolated nucleic acid molecule of claim 90, wherein the nucleic acid sequence encoding the anti-CD123 binding domain comprises the nucleic acid sequence of SEQ ID NO:

103.

92. The isolated polypeptide molecule of claim 23, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

97 or SEQ ID NO;

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

93. The isolated polypeptide molecule of claim 92, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

97 or SEQ ID NO;

100.

94. The isolated nucleic acid molecule of claim 1, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

96 or SEQ ID NO;

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

25. An isolated chimeric antigen receptor (CAR) molecule comprising a humanized anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the anti-CD123 binding domain comprises:

(a) a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

22, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

18;

(b) a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

30, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

26;

or

(c) a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

89, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

86.

26. The isolated CAR molecule of claim 25, wherein the intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

27. The isolated CAR molecule of claim 25, wherein the anti-CD123 binding domain is connected to the transmembrane domain by a hinge region.

28. The isolated CAR molecule of claim 25, further comprising a leader sequence.

42. An in vitro transcribed vector encoding the CAR of claim 25.

56. The isolated CAR molecule of claim 25, wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, CD66d, DAP10, or DAP12.

57. The isolated CAR molecule of claim 56, wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta, wherein the CD3 zeta comprises the amino acid 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 the amino acid sequence of SEQ ID NO;

7 or SEQ ID NO;

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

7 or SEQ ID NO;

98.

63. The isolated CAR molecule of claim 25, comprising the light chain variable region 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 and the heavy chain variable region 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.

64. The isolated CAR molecule of claim 25, 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 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 to an amino acid sequence provided in 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;

(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 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 to an amino acid sequence provided in 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

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

65. The isolated CAR molecule of claim 25, wherein the anti-CD123 binding domain comprises a sequence selected from the 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.

66. The isolated CAR molecule of claim 25, 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.

67. The isolated CAR molecule of claim 25, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

5;

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

5;

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

5.

68. The isolated CAR molecule of claim 25, wherein the anti-CD123 binding domain is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises the amino acid sequence of any of SEQ ID NO:

4, 104, 122, or 124, or a sequence with 95-99% identity thereof.

69. The isolated CAR molecule of claim 26, 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), ICOS (CD278), and 4-1BB (CD137).

70. The isolated CAR molecule of claim 26, wherein the costimulatory domain comprises the sequence of SEQ ID NO:

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23.

71. The isolated CAR molecule of claim 25, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.

72. The isolated CAR molecule of claim 26, wherein:

the costimulatory domain comprises the sequence of SEQ ID NO;

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23;

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

6 or SEQ ID NO;

23, and

the primary signaling domain comprises the sequence of SEQ ID NO;

7 or SEQ ID NO;

98;

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

7 or SEQ ID NO;

98;

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

7 or SEQ ID NO;

98.

73. The isolated CAR molecule of claim 25, wherein the anti-CD123 binding domain comprises:

a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

20, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

21, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

22, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

16, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

17, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

18.

74. The isolated CAR molecule of claim 25, wherein the anti-CD123 binding domain comprises:

a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

28, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

29, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

30, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

24, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

25, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

26.

75. The isolated CAR molecule of claim 25, wherein the anti-CD123 binding domain comprises:

a light chain variable region comprising;

a light chain complementarity determining region 1 (LC CDR1) comprising the sequence of SEQ ID NO;

87, a light chain complementarity determining region 2 (LC CDR2) comprising the sequence of SEQ ID NO;

88, and a light chain complementarity determining region 3 (LC CDR3) comprising the sequence of SEQ ID NO;

89, and

a heavy chain variable region comprising;

a heavy chain complementarity determining region 1 (HC CDR1) comprising the sequence of SEQ ID NO;

84, a heavy chain complementarity determining region 2 (HC CDR2) comprising the sequence of SEQ ID NO;

85, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the sequence of SEQ ID NO;

86.