CHIMERIC ANTIGEN RECEPTOR AND METHODS OF USE THEREOF

• US 20170143765A1
• Filed: 01/30/2017
• Published: 05/25/2017
• Est. Priority Date: 02/15/2013
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Wu, Chia-Yung, Onuffer, James, Lim, Wendell A.
• Granted Patent: US 9,821,012 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2239/23 On/off switch
• CPC Class Codes: A61K 2239/24 Dimerizable CARs; CARs with adapter
• 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 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 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61K 47/6891 Pre-targeting systems involving an antibody for targeting specific cells
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• 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 16/18 against material from animals or humans
• 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 16/2878 : against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/20 : containing a tag with affinity for a non-protein ligand
• 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
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 9/14 : Hydrolases (3)
• CPC Class Codes: C12N 9/90 : Isomerases (5.)
• CPC Class Codes: C12Y 502/01008 : Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin

Abstract

The present disclosure provides a heterodimeric, conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides cells genetically modified to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

Claims

1-36. -36. (canceled)

37. One or more isolated nucleic acids comprising one or more nucleotide sequences encoding a first polypeptide and a second polypeptide of a heterodimeric chimeric antigen receptor (CAR), wherein the first and the second polypeptides are two separate polypeptides, such that when present in a eukaryotic cell membrane, the CAR dimerizes in the presence of a small molecule dimerizer and an antigen to which the first polypeptide of the CAR binds to produce titratable activity, wherein:

a) the first polypeptide comprises;

i) an extracellular antigen binding domain;

ii) a transmembrane domain; and

iii) a first member of a dimerization pair; and

b) the second polypeptide comprises;

i) a transmembrane domain;

ii) a second member of the dimerization pair; and

iii) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the intracellular signaling domain provides signal transduction activity,

wherein the second polypeptide does not comprise an antigen-binding domain,

wherein the first polypeptide, the second polypeptide or both the first and second polypeptides comprise an intracellular costimulatory polypeptide.

38. The one or more isolated nucleic acids according to claim 37, wherein the one or more isolated nucleic acids is a single nucleic acid comprising nucleotide sequences encoding the first and the second polypeptides of the heterodimeric CAR.

39. The one or more isolated nucleic acids according to claim 37, wherein the one or more isolated nucleic acids comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a first nucleotide sequence encoding the first polypeptide of the heterodimeric CAR and the second nucleic acid comprises a second nucleotide sequence encoding the second polypeptide of the heterodimeric CAR.

40. The one or more isolated nucleic acids according to claim 37, wherein the extracellular antigen binding domain comprises an antigen-binding portion of an antibody.

41. The one or more isolated nucleic acids according to claim 37, wherein the first polypeptide comprises a hinge region interposed between the extracellular antigen binding domain and the transmembrane domain.

42. The one or more isolated nucleic acids according to claim 41, wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD8.

43. The one or more isolated nucleic acids according to claim 37, wherein the intracellular signaling domain comprising the ITAM is selected from the group consisting of CD3-zeta and ZAP70.

44. The one or more isolated nucleic acids according to claim 37, wherein the first and second members of the dimerization pair form a heterodimer in the presence of the small molecule dimerizer.

45. The one or more isolated nucleic acids according to claim 37, wherein the first and second members of the dimerization pair form a homodimer in the presence of the small molecule dimerizer.

46. The one or more isolated nucleic acids according to claim 44, wherein the first and second members of the dimerization pair are selected from the group consisting of:

a) FK506 binding protein (FKBP) and FKBP-rapamycin associated protein (FRB);

b) a Gibberellic Acid Insensitive (GAI) protein and a gibberellin receptor (GID1) protein;

c) FKBP and calcineurin catalytic subunit A (CnA);

d) an abscisic acid receptor (PYL) protein and an abscissic acid insensitive (ABI) protein;

e) a cryptochrome 2 (Cry2) protein and a transcription factor bHLH63 (CIB1) protein; and

f) FKBP and cyclophilin.

47. The one or more isolated nucleic acids according to claim 45, wherein the first and second members of the dimerization pair are selected from the group consisting of:

a) FK506 binding protein (FKBP) and FKBP;

b) gyrase B (GyrB) and GyrB;

c) dihydrofolate reductase (DHFR) and DHFR; and

d) DmrB and DmrB;

48. The one or more isolated nucleic acids according to claim 46, wherein the first member of the dimerization pair is an FK506 binding protein (FKBP), and wherein the second member of the dimerization pair is a FKBP-rapamycin associated protein (FRB).

49. The one or more isolated nucleic acids according to claim 48, wherein the FKBP comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

12, and wherein the FRB comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO;

14.

50. The one or more isolated nucleic acids according to claim 46, wherein the first member of the dimerization pair is GID1, and wherein the second member of the dimerization pair is GAI.

51. The one or more isolated nucleic acids according to claim 50, wherein the GID1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:

95-97, and wherein the GAI polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO;

94.

52. The one or more isolated nucleic acids according to claim 37, wherein the extracellular antigen binding domain binds an epitope present on a cancer cell.

53. The one or more isolated nucleic acids according to claim 52, wherein the cancer cell is a breast cancer cell, a B cell lymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, or a colorectal cancer cell.

54. The one or more isolated nucleic acids according to claim 37, wherein the one or more nucleotide sequences are operably linked to one or more T lymphocyte-specific promoters.

55. The one or more isolated nucleic acids according to claim 37, wherein the one or more nucleotide sequences are operably linked to one or more NK cell-specific promoters.

56. The one or more isolated nucleic acids according to claim 37, wherein the intracellular costimulatory polypeptide is selected from the group consisting of:

4-1BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

57. The one or more isolated nucleic acids according to claim 56, wherein the 4-1BB polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

24.

58. The one or more isolated nucleic acids according to claim 56, wherein the OX-40 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

65.

59. The one or more isolated nucleic acids according to claim 56, wherein the CD28 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

63.

60. The one or more isolated nucleic acids according to claim 37, wherein the first polypeptide comprises a first costimulatory polypeptide comprising an amino acid sequence and the second polypeptide comprises a second costimulatory polypeptide comprising an amino acid sequence that has at least 95% amino acid identity to the amino acid sequence of the first costimulatory polypeptide in the first polypeptide.

61. The one or more isolated nucleic acids according to claim 37, wherein the intracellular signaling domain comprising the ITAM comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:

98-119.

62. A recombinant expression vector comprising the nucleic acid of claim 37.

63. A T lymphocyte comprising the recombinant expression vector of claim 62.

64. A method of treating a cancer in an individual, the method comprising:

a) introducing the T lymphocyte of claim 63 into the individual, wherein the antigen-binding domain of the heterodimeric, conditionally active CAR is specific for an epitope on a cancer cell in the individual; and

b) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of the heterodimeric, conditionally active CAR, wherein said dimerization provides for activation of the T lymphocyte thereby treating the cancer.

65. The method according to claim 64, wherein the method further comprises administering to the individual a biological response modifier selected from the group consisting of:

an inhibitor of tyrosine kinase (RTK) activity, an inhibitor of serine/threonine kinase activity, a tumor-associated antigen antagonist, an apoptosis receptor agonist, interleukin-2, interferon-α

, interferon-γ

, a colony-stimulating factor, an inhibitor of angiogenesis, and an antagonist of tumor necrosis factor.