TREATMENT OF CANCER USING HUMANIZED ANTI-CD19 CHIMERIC ANTIGEN RECEPTOR

• US 20140271635A1
• Filed: 03/15/2014
• Published: 09/18/2014
• Est. Priority Date: 03/16/2013
• 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: June, Carl H., Loew, Andreas, Brogdon, Jennifer, Maus, Marcela, Scholler, John
• Granted Patent: US 10,221,245 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• 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 2239/48 Blood cells, e.g. leukemia or lymphoma
• 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 39/464412 CD19 or B4
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/70503 Immunoglobulin superfamily
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/3061 Blood cells
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/565 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 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C12N 2510/02 : Cells for production
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The invention provides compositions and methods for treating diseases associated with expression of CD19. The invention also relates to chimeric antigen receptor (CAR) specific to CD19, vectors encoding the same, and recombinant T cells comprising the CD19 CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a CD19 binding domain.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment which includes a humanized anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein said anti-CD19 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-CD19 light chain binding domain amino acid sequence listed in Table 3, 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-CD19 heavy chain binding domain amino acid sequence listed in Table 3.

2. The isolated nucleic acid molecule of claim 1, comprising a LC CDR1, LC CDR2 and LC CDR3 of any anti-CD19 light chain binding domain amino acid sequence listed in Table 3.

3. The isolated nucleic acid molecule of claim 1, comprising a HC CDR1, HC CDR2 and HC CDR3 of any anti-CD19 light chain binding domain amino acid sequence listed in Table 3.

4. The isolated nucleic acid molecule of claim 1, comprising LC CDR1, LC CDR2 and LC CDR3 of any anti-CD19 light chain binding domain amino acid sequence listed in Table 3, and HC CDR1, HC CDR2, and HC CDR3 of any anti-CD19 heavy chain binding domain amino acid sequence listed in Table 3.

5. The isolated nucleic acid molecule of claim 1, comprising any light chain variable region listed in Table 3.

6. The isolated nucleic acid molecule of claim 1, comprising any heavy chain variable region listed in Table 3.

7. The isolated nucleic acid molecule of claim 1, comprising any light chain variable region listed in Table 3 and any heavy chain variable region listed Table 3.

8. The isolated nucleic acid molecule of any of the preceding claims, wherein the CD19 binding domain is a scFv.

9. The isolated nucleic acid molecule of any of the preceding claims, wherein 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 3, or a sequence with 95-99% identity to an amino acid sequence provided in Table 3.

10. The isolated nucleic acid molecule of any of the preceding claims, 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 3, or a sequence with 95-99% identity to an amino acid sequence provided in Table 3.

11. The isolated nucleic acid molecule of any of the preceding claims, wherein the anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

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

12. The isolated nucleic acid molecule of any of the preceding claims, wherein the nucleic acid sequence encoding the anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

61, SEQ ID NO;

62, SEQ ID NO;

63, SEQ ID NO;

64, SEQ ID NO;

65, SEQ ID NO;

66, SEQ ID NO;

67, SEQ ID NO;

68, SEQ ID NO;

70, SEQ ID NO;

71 and SEQ ID NO;

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

13. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded CAR includes 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 any of the preceding claims, wherein the encoded transmembrane domain comprises a sequence of SEQ ID NO:

15.

15. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded transmembrane domain comprises 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 any of the preceding claims, wherein the, the nucleic acid sequence encoding the transmembrane domain comprises a sequence of SEQ ID NO:

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

17. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded anti-CD19 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:

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

20. The isolated nucleic acid molecule of any of the preceding claims, further comprising a sequence encoding a costimulatory domain.

21. The isolated nucleic acid molecule of claim 20, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137).

22. The isolated nucleic acid molecule of claim 20 or 21, wherein the encoded costimulatory domain comprises a sequence of SEQ ID NO:

16.

23. The isolated nucleic acid molecule of claim 20 or claim 21, wherein the encoded costimulatory domain comprises 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, 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 or 21, wherein the nucleic acid sequence encoding the costimulatory domain comprises a sequence of SEQ ID NO:

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

25. The isolated nucleic acid molecule of any of the preceding claims, further comprising a sequence encoding an intracellular signaling domain.

26. The isolated nucleic acid molecule of any of the preceding claims, wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

27. The isolated nucleic acid molecule of any of the preceding claims, 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;

43.

28. The isolated nucleic acid molecule of any of the preceding claims, wherein the intracellular signaling domain comprises 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;

43, 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;

43.

29. The isolated nucleic acid molecule of any of the preceding claims, 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;

43, 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 any of the preceding claims, wherein the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence of SEQ ID NO:

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

101 or SEQ ID NO;

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

31. The isolated nucleic acid molecule of any of the preceding claims, 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 any one of claims 1-32.

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

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, SEQ ID NO;

40, SEQ ID NO;

41, and SEQ ID NO;

42.

35. The isolated polypeptide of claim 34, comprising a sequence of SEQ ID NO:

31.

36. The isolated polypeptide of claim 34, comprising a sequence of SEQ ID NO:

32.

37. The isolated polypeptide of claim 34, comprising a sequence of SEQ ID NO:

35.

38. The isolated polypeptide of claim 34, comprising a sequence of SEQ ID NO:

36.

39. The isolated polypeptide of claim 34, comprising a sequence of SEQ ID NO:

37.

71. A vector comprising a nucleic acid molecule encoding a CAR of any of the preceding claims.

72. The vector of claim 71, 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.

73. The vector of claim 71 or 72, further comprises a promoter.

74. The vector of claim 73, wherein the promoter is an EF-1 promoter.

75. The vector of claim 74, wherein the EF-1 promoter comprises a sequence of SEQ ID NO:

100.

76. The vector of any of claims 71-75, wherein the vector is an in vitro transcribed vector.

77. The vector of any of claims 71-76, wherein the, the nucleic acid sequence in the vector further comprises a poly(A) tail.

78. The vector of any of claims 71-76, wherein the nucleic acid sequence in the vector further comprises a 3′

UTR.

79. A cell comprising the vector of any of claims 71-78.

80. The cell of claim 79, wherein the cell is a human T cell.

81. The cell of claim 80, wherein the T cell is a CD8+ T cell.

82. A method of making a cell comprising transducing a T cell with a vector of any of claims 71-78.

83. 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 encoding a CAR molecule of any of the preceding claims.

84. A method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of a cell expressing a CAR molecule of any of the preceding claims.

85. The method of claim 84, wherein the cell is an autologous T cell.

86. The method of claim 84 or 85, wherein the cell is an allogeneic T cell.

87. The method of any of claims 84-86, wherein the mammal is a human.

88. A method of treating a mammal having a disease associated with expression of CD19 comprising administering to the mammal an effective amount of the cell of comprising a CAR molecule of any of the preceding claims.

89. The method of claim 88, wherein the disease associated with CD19 expression is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of CD19.

90. The method of claim 88 or 89, wherein the disease is a hematologic cancer selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia (“

BALL”

), T-cell acute lymphoid leukemia (“

TALL”

), acute lymphoid leukemia (ALL);

one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL);

additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt'"'"'s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin'"'"'s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “

preleukemia”

which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with CD19 expression include, but not limited to atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing CD19; and

combinations thereof.

91. The method of any of claims 88-90, wherein the cells expressing a CAR molecule are administered in combination with an agent that increases the efficacy of a cell expressing a CAR molecule.

92. The method of any of claims 88-91, wherein the cells expressing a CAR molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a CAR molecule.

93. The method of any of claims 88-92, wherein the cells expressing a CAR molecule are administered in combination with an agent that treats the disease associated with CD19.

94. The isolated nucleic acid molecule of any of claims 1-32, the isolated polypeptide molecule of any of claims 33-39, the isolated CAR of any of claims 40-67, the anti-CD19 binding domain of any of claims 68-70, the vector of any of claims 71-78 or the cell of any of claims 79-81 for use use as a medicament.

95. The isolated nucleic acid molecule of any of claims 1-32, the isolated polypeptide molecule of any of claims 33-39, the isolated CAR of any of claims 40-68, the anti-CD19 binding domain of any of claims 69-71, the vector of any of claims 72-79 or the cell of any of claims 80-82 for use use in the treatment of a disease expressing CD19.

96. A cell of any of claims 79-81, further expressing an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.

97. The cell of claim 96, wherein the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

40. An isolated chimeric antigen receptor (CAR) molecule comprising a humanized anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

41. The isolated CAR molecule of claim 40, comprising an antibody or antibody fragment which includes a humanized anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

42. The isolated CAR molecule of claim 40 or 41, wherein the humanized anti-CD19 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 humanized anti-CD19 binding domain listed in Table 3, 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 humanized anti-CD19 binding domain listed in Table 3.

43. The isolated CAR molecule of any of claims 40-42, comprising LC CDR1, LC CDR2 and LC CDR3 of any anti-CD19 light chain binding domain amino acid sequence listed in Table 3.

44. The isolated CAR molecule of any of claims 40-43, comprising HC CDR1, HC CDR2, and HC CDR3 of any anti-CD19 heavy chain binding domain amino acid sequence listed in Table 3.

45. The isolated CAR molecule of any of claims 40-44, comprising LC CDR1, LC CDR2 and LC CDR3 of any anti-CD19 light chain binding domain amino acid sequence listed in Table 3, and HC CDR1, HC CDR2, and HC CDR3 of any anti-CD19 heavy chain binding domain amino acid sequence listed in Table 3.

46. The isolated CAR molecule of any of claims 40-45, wherein the anti-CD19 binding domain is a scFv.

47. The isolated CAR molecule of any of claims 40-46, wherein the anti-CD19 binding domain comprises a light chain and a heavy chain of an amino acid sequence listed in Table 3.

48. The isolated CAR molecule of any of claims 40-47, wherein the anti-CD19 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 Table 3, or a sequence with 95-99% identity with an amino acid sequence provided in Table 3.

49. The isolated CAR molecule of any of claims 40-48, wherein the anti-CD19 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 Table 3, or a sequence with 95-99% identity to an amino acid sequence provided in Table 3.

50. The isolated CAR molecule of any of claims 40-49, wherein the anti-CD19 binding domain comprises a sequence selected from a group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11 and SEQ ID NO;

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

51. The isolated CAR molecule of any of claims 40-50, further comprising 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.

52. The isolated CAR molecule of claim 51, wherein the transmembrane domain comprises a sequence of SEQ ID NO:

15.

53. The isolated CAR molecule of claim 51 or 52, wherein the transmembrane domain comprises 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:

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

15.

54. The isolated CAR molecule of any one of claims 40-53, wherein the humanized anti-CD19 binding domain is connected to the transmembrane domain by a hinge region.

55. The isolated CAR molecule of claim 54, wherein the encoded hinge region comprises SEQ ID NO:

14 or SEQ ID NO;

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

56. The isolated CAR molecule of any of claims 40-55, further comprising a sequence encoding a costimulatory domain.

57. The isolated CAR molecule of claim 56, wherein the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).

58. The isolated CAR molecule of claim 56 or 57, wherein the costimulatory domain comprises a sequence of SEQ ID NO:

16.

59. The isolated CAR molecule of claim 56 or 57, wherein the costimulatory domain comprises 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, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

16.

60. The isolated CAR molecule of any one of claims 40-59, further comprising a sequence encoding an intracellular signaling domain.

61. The isolated CAR molecule of claim 60, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

62. The isolated CAR molecule of claim 60 or 61, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO:

16 and/or the sequence of SEQ ID NO;

17.

63. The isolated CAR molecule of claim 60 or 61, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO:

16 and/or the sequence of SEQ ID NO;

43.

64. The isolated CAR molecule of any of claims 60-63, wherein the intracellular signaling domain comprises 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;

43, 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;

43.

65. The isolated CAR molecule of any of claims 60-64, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO:

16 and the sequence of SEQ ID NO;

17 or SEQ ID NO;

43, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

66. The isolated CAR molecule of any of claims 40-65, further comprising a leader sequence.

67. The isolated CAR molecule of claim 66, wherein the leader sequence comprises an amino acid sequence of SEQ ID NO:

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

13.

68. A humanized anti-CD19 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 any anti-CD19 binding domain in SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12, 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 humanized anti-CD19 binding domain in SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12.

69. The humanized anti-CD19 binding domain of claim 68, wherein the humanized anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, or SEQ ID NO;

12.

70. The humanized anti-CD19 binding domain of claim 68 or 69, wherein the humanized anti-CD19 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;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12 or a sequence with 95-99% identity with an amino acid sequence in SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12; and

/or 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 SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

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

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, and SEQ ID NO;

12.