TREATMENT OF CANCER USING A CD33 CHIMERIC ANTIGEN RECEPTOR

• US 20160096892A1
• Filed: 07/21/2015
• Published: 04/07/2016
• Est. Priority Date: 07/21/2014
• 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, Ebersbach, Hilmar Erhard, Gill, Saar, Glass, David, Huber, Thomas, Jascur, Julia, Kenderian, Saad, Mannick, Joan, Milone, Michael C., Murphy, Leon, Richardson, Celeste, Singh, Reshma, Wu, Qilong, Zhang, Jiquan, Song, Huijuan
• Granted Patent: US 9,777,061 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising 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 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/436 the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• CPC Class Codes: A61K 31/711 Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• 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 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• CPC Class Codes: A61K 38/1793 for cytokines; for lymphokines; for interferons
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• 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 39/464411 Immunoglobulin superfamily
• CPC Class Codes: A61K 39/464419 Receptors for interleukins [IL]
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/04 : Immunostimulants
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/7051 : T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 : CD8
• CPC Class Codes: C07K 14/7056 : Lectin superfamily, e.g. CD23, CD72
• 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 14/7151 : for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C07K 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2851 : against the lectin superfamily, e.g. CD23, CD72
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 16/3061 : Blood cells
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/33 : Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/53 : Hinge
• 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 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/70 : containing domain for protein-protein interaction

Abstract

The invention provides compositions and methods for treating diseases associated with expression of CD33. The invention also relates to chimeric antigen receptor (CAR) specific to CD33, vectors encoding the same, and recombinant T cells comprising the CD33 CAR. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises a CD33 binding domain.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a CD33 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein said CD33 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 CD33 heavy chain binding domain amino acid sequences listed in Table 2 or 9; 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 CD33 heavy chain binding domain amino acid sequences listed in Table 2 or 9.

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 Table 11, 13 or 4.

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 Table 10, 12 or 3.

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 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 light 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 light chain variable regions provided in Table 2 or 9.

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 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.

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 2 or 9, and the amino acid sequence of any heavy chain variable region listed Table 2 or 9.

8. The isolated nucleic acid molecule of claim 1, wherein the encoded CD33 binding domain comprises:

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

39-47, 57-65, 66-74, and 262-268;

(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;

39-47, 57-65, 66-74, or 262-268;

or

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

39-47, 57-65, 66-74, or 262-268.

9. The isolated nucleic acid molecule of claim 1, wherein the CD33 binding domain comprises a nucleotide sequence selected from a group consisting of SEQ ID NO:

255-261, or a 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, CD5, 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 a 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 CD33 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 a sequence with 95-99% identity thereof;

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

13, or a 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 derived from a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, 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 encoded 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.

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 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 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 a 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 sequence of SEQ ID NO:

7 and the 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 sequence of SEQ ID NO:

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

20 or SEQ ID NO;

21, or a 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;

48-56;

(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;

48-56;

or

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

48-56.

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

75-83, or a nucleotide sequence with 95-99% identity to any of SEQ ID NOs;

75-83.

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

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

47. A method of making an immune effector cell, comprising transducing the immune effector cell with the vector of claim 44.

48. 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 the nucleic acid molecule of claim 1.

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

51. A method of treating a mammal having a disease associated with expression of CD33, comprising administering to the mammal an effective amount of a cell comprising the CAR nucleic acid molecule of claim 1.

52. The method of claim 51, wherein the disease associated with CD33 expression is:

(i) a cancer or malignancy, or a precancerous condition chosen from one or more of a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or

(ii) a non-cancer related indication associated with expression of CD33.

54. The method of claim 51, wherein the disease is an acute leukemia chosen from one or more of acute myeloid leukemia (AML), myelodysplastic syndrome, myeloproliferative neoplasms, chronic myeloid leukemia (CML), or Blastic plasmacytoid dendritic cell neoplasm, or a combination thereof.

55. The method of claim 51, 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 CD33.

58. The cell of claim 46, further expressing a 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.

60. The method of claim 55, wherein the agent is an mTOR inhibitor and the subject is administered a low, immune enhancing, dose of an mTOR inhibitor.

62. A method of conditioning a subject prior to cell transplantation comprising administering to the subject an effective amount of the cell of claim 46.

65. 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, CD5, CD22, CD79a, CD79b, CD278 (also known as “

ICOS”

), Fcε

RI, DAP10, DAP12, or CD66d.

66. The isolated nucleic acid molecule of claim 65, wherein the encoded 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.

69. The isolated nucleic acid molecule of claim 1, wherein the encoded CAR is expressed in frame and as a single polypeptide chain.

2. (canceled)

11. (canceled)

13. (canceled)

24. (canceled)

25. An isolated chimeric antigen receptor (CAR) polypeptide, wherein the CAR comprises a CD33 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain, and wherein said CD33 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 CD33 heavy chain binding domain amino acid sequence listed in Table 2 or 9; 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 CD33 heavy chain binding domain amino acid sequence listed in Table 2 or 9.

27. The isolated CAR polypeptide of claim 25, wherein:

(i) said LC CDR1, LC CDR2, and LC CDR3 are the LC CDR sequences listed in Table 11, 13 or 4;

(ii) said HC CDR1, HC CDR2, and HC CDR3 are the HC CDR sequences listed in Table 10, 12 or 3;

or

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

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

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

(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 2 or 9;

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

(v) 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;

(vi) 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;

or

(vii) any of (i)-(iii) and any of (iv)-(vi).

31. The isolated CAR polypeptide of any of claims 25-30, 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;

39-47, 57-65, 66-74, and 262-268;

(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;

39-47, 57-65, 66-74, or 262-268;

or

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

39-47, 57-65, 66-74, or 262-268.

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, CD5, 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 CD33 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 MHC class I molecules, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, 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 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 a 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;

48-56;

(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;

48-56;

or

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

48-56.

67. 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, CD5, CD22, CD79a, CD79b, CD278 (also known as “

ICOS”

), Fcε

RI, DAP10, DAP12, or CD66d.

68. The isolated CAR polypeptide of claim 67, 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)

28. (canceled)

30. (canceled)

36. (canceled)

41-42. -42. (canceled)

45. (canceled)

50. (canceled)

53. (canceled)

56-57. -57. (canceled)

59. (canceled)

61. (canceled)

63-64. -64. (canceled)