CHIMERIC ANTIGEN RECEPTOR AND METHODS OF USE THEREOF

• US 20160185862A1
• Filed: 03/09/2016
• Published: 06/30/2016
• 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 9,587,020 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 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 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. A nucleic acid comprising a nucleotide sequence encoding one or both polypeptide chains of a heterodimeric, conditionally active chimeric antigen receptor (CAR), wherein the CAR comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus;

i) an antigen-binding single-chain Fv (scFv);

ii) a transmembrane domain;

iii) a costimulatory polypeptide, wherein the co-stimulatory polypeptide is a 4-1BB polypeptide or an OX-40 polypeptide; and

iv) a first member of a dimerization pair; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus;

i) a transmembrane domain;

ii) a second member of the dimerization pair; and

iii) an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

38. The nucleic acid of claim 37, wherein the first polypeptide comprises a hinge region interposed between the scFv and the transmembrane domain.

39. The nucleic acid of claim 38, wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD8.

40. The nucleic acid of claim 37, wherein the intracellular signaling domain comprising an ITAM is selected from CD3-zeta and ZAP70.

41. The nucleic acid of claim 37, wherein the first and second members of the dimerization pair form a heterodimer in the presence of a small molecule dimerizer.

42. The nucleic acid of claim 37, wherein the first and second members of the dimerization pair form a homodimer in the presence of a small molecule dimerizer.

43. The nucleic acid of claim 41, wherein the first and second members of the dimerization pair are selected from:

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.

44. The nucleic acid of claim 42, wherein the first and second members of the dimerization pair are selected from:

a) FK506 binding protein (FKBP) and FKBP;

b) gyrase B (GyrB) and GyrB;

c) dihydrofolate reductase (DHFR) and DHFR; and

d) DmrB and DmrB;

45. The nucleic acid of claim 37, 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).

46. The nucleic acid of claim 37, wherein the first member of the dimerization pair is GID1, and wherein the second member of the dimerization pair is GAI.

47. The nucleic acid of claim 37, wherein the scFv binds an epitope present on a cancer cell.

48. The nucleic acid of claim 47, 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.

49. The nucleic acid of claim 37, wherein the nucleotide sequence is operably linked to a T lymphocyte-specific promoter.

50. The nucleic acid of claim 37, wherein the nucleotide sequence is operably linked to an NK cell-specific promoter.

51. The nucleic acid of claim 37, 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.

52. The nucleic acid of claim 37, 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.

53. The nucleic acid of claim 37, wherein the second polypeptide comprises a second costimulatory polypeptide between the transmembrane domain and the second member of the dimerization pair, wherein the second costimulatory polypeptide comprises an amino acid sequence that has at least 95% amino acid identity to the amino acid sequence of the costimulatory polypeptide in the first polypeptide.

54. The nucleic acid of claim 37, wherein the intracellular signaling domain comprising an 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.

55. The nucleic acid of claim 45, 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.

56. The nucleic acid of claim 46, 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.

57. The nucleic acid of claim 37, wherein:

a) the first polypeptide comprises, in order from N-terminus to C-terminus;

i) an scFv that binds an epitope present on a cancer cell;

ii) a transmembrane domain;

iii) a first 4-1BB polypeptide; and

iv) an FK506 binding protein (FKBP); and

wherein;

b) the second polypeptide comprises, in order from N-terminus to C-terminus;

i) a transmembrane domain;

ii) a second 4-1BB polypeptide;

iii) a FKBP-rapamycin associated protein (FRB); and

iv) a CD3-zeta polypeptide.

58. The nucleic acid of claim 57, wherein the first polypeptide comprises a hinge region derived from CD8 interposed between the scFv and the transmembrane domain.

59. The nucleic acid of claim 37, wherein:

a) the first polypeptide comprises, in order from N-terminus to C-terminus;

i) an scFv that binds an epitope present on a cancer cell;

ii) a transmembrane domain;

iii) a first OX-40 polypeptide; and

iv) an FK506 binding protein (FKBP); and

wherein;

b) the second polypeptide comprises, in order from N-terminus to C-terminus;

i) a transmembrane domain;

ii) a second OX-40 polypeptide;

iii) a FKBP-rapamycin associated protein (FRB); and

iv) a CD3-zeta polypeptide.

60. The nucleic acid of claim 59, wherein the first polypeptide comprises a hinge region derived from CD8 interposed between the scFv and the transmembrane domain.

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

62. The recombinant expression vector of claim 61, wherein the recombinant expression vector comprises a nucleotide sequence encoding both the first and the second polypeptides.

63. A T lymphocyte genetically modified to produce a heterodimeric, conditionally active chimeric antigen receptor (CAR), wherein the CAR comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus;

i) an antigen-binding single-chain Fv (scFv);

ii) a transmembrane domain;

iii) a costimulatory polypeptide, wherein the co-stimulatory polypeptide is a 4-1BB polypeptide or an OX-40 polypeptide; and

iv) a first member of a dimerization pair; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus;

i) a transmembrane domain;

ii) a second member of the dimerization pair; and

iii) an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

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

a) the genetically modified T lymphocyte of claim 63; and

b) an effective amount of a dimerizing agent that induces dimerization of the first and second members of the dimerization pair.

wherein, when the genetically modified T lymphocyte binds a target antigen on a cancer cell in the individual, in the presence of the dimerization agent, the genetically modified T lymphocyte is activated, and the cancer in the individual is treated.

65. The method of claim 64, comprising varying the dose of the dimerizing agent based on susceptibility of the individual to side effects to treatment.

66. The method of claim 64, wherein a single dose of the dimerizing agent is administered.