TREATMENT OF CANCER USING A CD123 CHIMERIC ANTIGEN RECEPTOR

• US 20160068601A1
• Filed: 08/19/2015
• Published: 03/10/2016
• Est. Priority Date: 08/19/2014
• 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, Glass, David, Kenderian, Saad, Loew, Andreas, Mannick, Joan, Milone, Michael C., Murphy, Leon, Porter, David L., Ruella, Marco, Wang, Yongqiang, Wu, Qilong, Zhang, Jiquan
• Granted Patent: US 9,815,901 B2
• Time in Patent Office: Days
• 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 2039/5156 expressing foreign proteins
• 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 39/001119 Receptors for interleukins [IL]
• 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: A61K 39/464419 Receptors for interleukins [IL]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• 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 2317/622 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 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• 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
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The invention provides compositions and methods for treating diseases associated with expression of CD123. The invention also relates to chimeric antigen receptor (CAR) specific to CD123, vectors encoding the same, and recombinant cells comprising the CD123 CAR. The invention also includes methods of administering a genetically modified cell expressing a CAR that comprises a CD123 binding domain.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein said CD123 binding domain comprises:

(i) a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) of any CD123 heavy chain binding domain amino acid sequence listed in Table 9, Table 2, or Table 6; and

(ii) a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3) of any CD123 light chain binding domain amino acid sequence listed in Table 9, Table 2, or Table 6.

3. The isolated nucleic acid molecule of claim 1, wherein said LC CDR1, LC CDR2, and LC CDR3 are the LC CDR sequences listed in Tables 11, 13, 4 or 8.

4. The isolated nucleic acid molecule of claim 1, wherein said HC CDR1, HC CDR2, and HC CDR3 are the HC CDR sequences listed in Tables 10, 12, 3, or 7.

5. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising:

(i) the amino acid sequence of any light chain variable region listed in Table 9 or 2;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of any of the light chain variable regions provided in Table 9 or 2;

or

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any of the light chain variable regions provided in Table 9 or 2.

6. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising:

(i) the amino acid sequence of any heavy chain variable region listed in Table 9 or 2;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of any of the heavy chain variable regions provided in Table 9 or 2;

or

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any of the heavy chain variable regions provided in Table 9 or 2.

7. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising the amino acid sequence of any light chain variable region listed in Table 9 or 2, and the amino acid sequence of any heavy chain variable region listed Table 9 or 2.

8. The isolated nucleic acid molecule of claim 1, wherein the encoded CD123 binding domain comprises:

(i) an amino acid sequence selected from the group consisting of SEQ ID NO;

480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, and 275;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any of SEQ ID NO;

480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, or 275;

or

(iii) an amino acid sequence with 95-99% identity to any of SEQ ID NO;

480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, or 275.

9. The isolated nucleic acid molecule of claim 1, wherein the CD123 binding domain comprises a nucleotide sequence chosen from SEQ ID NO:

479, 481, 482, 484, or a nucleotide sequence with 95-99% identity thereof.

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

(i) 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, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154;

or

(ii) the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO;

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

6;

or

(iii) the nucleic acid sequence encoding the transmembrane domain comprises the nucleotide sequence of SEQ ID NO;

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

12. The isolated nucleic acid molecule of claim 1, wherein the encoded CD123 binding domain is connected to the transmembrane domain by a hinge region, wherein

(i) the encoded hinge region comprises the amino acid sequence of SEQ ID NO:

2, or an amino acid sequence comprising at least one, two, or three modifications, but no more than 20, 10, or 5 modifications of SEQ ID NO;

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

2;

or

(ii) the nucleic acid sequence encoding the hinge region comprises the nucleotide sequence of SEQ ID NO;

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

14. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain obtained derived from a protein selected from the group consisting of a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

15. The isolated nucleic acid molecule of claim 14, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:

7;

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

7.

16. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid sequence encoding the costimulatory domain comprises the nucleotide sequence of SEQ ID NO:

18, or a nucleotide 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 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10;

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 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10;

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

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

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

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

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

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

18, or a nucleotide sequence with 95-99% identity thereof, and/or the nucleotide sequence of SEQ ID NO;

20 or SEQ ID NO;

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

21. The isolated nucleic acid molecule of claim 1, further comprising a leader sequence which encodes the amino acid sequence of SEQ ID NO:

1.

22. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising:

(i) the amino acid sequence of any of SEQ ID NOs;

99, 100, 101, or 98;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any of SEQ ID NOs;

99, 100, 101, or 98;

or

(iii) an amino acid sequence with 95-99% identity to any of SEQ ID NOs;

99, 100, 101, or 98.

23. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence of any of SEQ ID NOs:

39, 40, 41, or 38, or a nucleotide sequence with 95-99% identity to any of SEQ ID NOs;

39, 40, 41, or 38.

46. A vector comprising the nucleic acid molecule of claim 1, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

49. An immune effector cell comprising the nucleic acid molecule of claim 1.

50. A method of making an immune effector cell, comprising transducing the immune effector cell with the vector of claim 46.

51. A method of generating a population of RNA-engineered cells, comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the nucleic acid molecule of claim 1.

52. A method of providing an anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell comprising the nucleic acid molecule of claim 1.

54. 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 comprising the nucleic acid molecule of claim 1.

57. The method of claim 54, wherein the disease is chosen from one or more of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia (B-cell acute lymphoid leukemia, BALL), acute lymphoblastic T-cell leukemia (T-cell acute lymphoid leukemia (TALL), B-cell prolymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin lymphoma, a histiocytic disorder, a mast cell disorder, a myelodysplasia, a myelodysplastic syndrome, a myeloproliferative neoplasm, a plasma cell myeloma, a plasmacytoid dendritic cell neoplasm, or a combination thereof.

58. The method of claim 54, wherein the cell is administered in combination with one or more of:

(i) an agent that increases the efficacy of the cell comprising the CAR nucleic acid or CAR polypeptide;

(ii) an agent that ameliorates one or more side effects associated with administration of the cell comprising the CAR nucleic acid or CAR polypeptide;

or

(iii) an agent that treats the disease associated with the expression of CD123.

59. The method of claim 54, wherein the cell comprises an mRNA encoding the CAR.

61. The method of claim 54, wherein the cell is administered in combination with second therapeutic agent or procedure chosen from one or more of chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, a cytokine, surgical procedure, a radiation procedure, an agonist of a costimulatory molecule, an inhibitor of an immune checkpoint molecule, a vaccine, or a second CAR-based immunotherapy.

62. The method of claim 61, wherein the cell is administered in combination with:

(i) an agonist of a costimulatory molecule chosen from one or more of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand;

or

(ii) an inhibitor of an immune checkpoint molecule chosen from one or more of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM-1, CEACAM-3, CEACAM-5, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GALS, adenosine, or TGFR.

64. The method of claim 61, wherein the cell is administered in combination with a PD-1 inhibitor, a TIM-3 inhibitor, a CEACAM-1 inhibitor, or a combination thereof.

70. The method of claim 54, wherein the cell is administered in combination with a CD19 inhibitor.

73. A method of preventing a CD19-negative relapse in a mammal, comprising administering to the mammal an effective amount of a cell comprising the nucleic acid of claim 1.

81. The method of claim 54, further comprising administering a T cell depleting agent after treatment with the cell, thereby depleting the cell.

83. The method of claim 81, wherein the T cell depleting agent is a CD52 inhibitor and is administered one, two, three, four, or five weeks after administration of the cell.

87. The method of claim 81, wherein the cell expresses a CD123CAR polypeptide and a target protein recognized by the T cell depleting agent, wherein the target protein is CD20 and the T cell depleting agent is an anti-CD20 antibody.

96. The cell of claim 49, further expressing a chimeric 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.

98. The method of claim 58, wherein the agent is an mTOR inhibitor and the subject is administered a low, immune enhancing, dose of an mTOR inhibitor.

100. A method of conditioning a subject prior to cell transplantation comprising administering to the subject an effective amount of the cell comprising the CAR nucleic acid molecule of claim 1.

103. 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, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD278, Fcε

RI, DAP10, DAP12, or CD66d.

104. The isolated nucleic acid molecule of claim 103, 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:

9 or SEQ ID NO;

10, 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;

9 or SEQ ID NO;

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

9 or SEQ ID NO;

10.

2. (canceled)

11. (canceled)

13. (canceled)

24. (canceled)

25. An isolated chimeric antigen receptor (CAR) polypeptide, wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein said CD123 binding domain comprises:

(i) a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) of any CD123 heavy chain binding domain amino acid sequences listed in Table 9, 2 or 6; and

(ii) a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3) of any CD123 light chain binding domain amino acid sequences listed in Table 9, 2 or 6.

27. The isolated CAR polypeptide of claim 25, wherein said LC CDR1, LC CDR2, and LC CDR3 are the LC CDR sequences listed in Tables 11, 13, 4 or 8.

28. The isolated CAR polypeptide of claim 25, wherein said HC CDR1, HC CDR2, and HC CDR3 are the HC CDR sequences listed in Tables 10, 12, 3, or 7.

29. The isolated CAR polypeptide of claim 25, comprising:

(i) the amino acid sequence of any light chain variable region listed in Table 9 or 2;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of any of the light chain variable regions provided in Table 9 or 2;

or

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any of the light chain variable regions provided in Table 9 or 2.

30. The isolated CAR polypeptide of claim 25, comprising:

(i) the amino acid sequence of any heavy chain variable region listed in Table 2 or 9;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of any of the heavy chain variable regions provided in Table 2 or 9;

or

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of any of the heavy chain variable regions provided in Table 2 or 9.

31. The isolated CAR polypeptide of claim 25, comprising the amino acid sequence of any light chain variable region listed in Table 2 or 9, and the amino acid sequence of any heavy chain variable region listed Table 2 or 9.

32. The isolated CAR polypeptide of claim 25, comprising:

(i) an amino acid sequence selected from the group consisting of SEQ ID NO;

480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, and 275;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any of SEQ ID NO;

480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, or 275;

or

(iii) an amino acid sequence with 95-99% identity to any of SEQ ID NO;

480, 483, 485, 478, 158, 159, 160, 157, 217, 218, 219, 216, 276, 277, 278, or 275.

33. The isolated CAR polypeptide of claim 25, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

34. The isolated CAR polypeptide of claim 25, wherein

the transmembrane domain comprises:

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

6,

(ii) 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

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

6.

35. The isolated CAR polypeptide of claim 25, wherein the CD123 binding domain is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises SEQ ID NO:

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

37. The isolated CAR polypeptide of claim 25, wherein the encoded intracellular domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain derived from a protein selected from the group consisting of a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

38. The isolated CAR polypeptide of claim 25, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:

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

7.

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

40. The isolated CAR polypeptide of claim 25, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10;

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 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10;

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

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

43. The isolated CAR polypeptide of claim 25, comprising:

(i) the amino acid sequence of any of SEQ ID NOs;

99, 100, 101, or 98;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any of SEQ ID NOs;

99, 100, 101, or 98;

or

(iii) an amino acid sequence with 95-99% identity to any of SEQ ID NOs;

99, 100, 101, or 98.

105. The isolated CAR polypeptide of claim 25, wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD278, Fcε

RI, DAP10, DAP12, or CD66d.

106. The isolated CAR polypeptide of claim 105, 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:

9 or SEQ ID NO;

10, 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;

9 or SEQ ID NO;

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

9 or SEQ ID NO;

10.

26. (canceled)

36. (canceled)

41-42. -42. (canceled)

44-45. -45. (canceled)

47-48. -48. (canceled)

53. (canceled)

55-56. -56. (canceled)

60. (canceled)

63. (canceled)

65-69. -69. (canceled)

71-72. -72. (canceled)

74-80. -80. (canceled)

82. (canceled)

84-86. -86. (canceled)

88-95. -95. (canceled)

97. (canceled)

99. (canceled)

101-102. -102. (canceled)