Chimeric antigen receptor and methods of use thereof

• US 9,587,020 B2
• Filed: 03/09/2016
• Issued: 03/07/2017
• 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.
• Primary Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US15/064,938
• Publication Number:
  US 20160185862A1
• Time in Patent Office: 363 Days
• Field of Search: 435/372.3, 435/328, 435/330
• 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 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. An isolated nucleic acid comprising a nucleotide sequence 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, in order from N-terminus to C-terminus;

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

ii) a transmembrane domain; and

iii) a first member of a dimerization pair; and

b) the second polypeptide comprises, 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 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 a costimulatory polypeptide interposed between the transmembrane domain and the member of the dimerization pair, and wherein the costimulatory polypeptide is selected from the group consisting of;

a 4-1BB polypeptide, a CD28 polypeptide and an OX-40 polypeptide.

2. The nucleic acid of claim 1, wherein the first polypeptide comprises a hinge region interposed between the scFv and the transmembrane domain.

3. The nucleic acid of claim 2, wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD8.

4. The nucleic acid of claim 1, wherein the intracellular signaling domain comprising the ITAM is selected from the group consisting of CD3-zeta and ZAP70.

5. The nucleic acid of claim 1, wherein the first and second members of the dimerization pair form a heterodimer in the presence of the small molecule dimerizer.

6. The nucleic acid of claim 1, wherein the first and second members of the dimerization pair form a homodimer in the presence of the small molecule dimerizer.

7. The nucleic acid of claim 5, 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.

8. The nucleic acid of claim 6, 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.

9. The nucleic acid of claim 1, 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).

10. The nucleic acid of claim 1, wherein the first member of the dimerization pair is GID1, and wherein the second member of the dimerization pair is GAI.

11. The nucleic acid of claim 1, wherein the scFv binds an epitope present on a cancer cell.

12. The nucleic acid of claim 11, 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.

13. The nucleic acid of claim 1, wherein the nucleotide sequence is operably linked to a T lymphocyte-specific promoter.

14. The nucleic acid of claim 1, wherein the nucleotide sequence is operably linked to an NK cell-specific promoter.

15. The nucleic acid of claim 1, 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.

16. The nucleic acid of claim 1, 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.

17. The nucleic acid of claim 1, 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.

18. The nucleic acid of claim 1, 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.

19. The nucleic acid of claim 9, 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.

20. The nucleic acid of claim 10, 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.

21. The nucleic acid of claim 1, wherein:

a) the scFv binds an epitope present on a cancer cell;

b) the first member of the dimerization pair is an FK506binding protein (FKBP);

c) the second member of the dimerization pair is a FKBP-rapamycin associated protein (FRB);

d) the first polypeptide comprises a first costimulatory polypeptide comprising a 4-1BB polypeptide;

e) the second polypeptide comprises a second costimulatory polypeptide comprising a 4-1BB polypeptide; and

f) the intracellular signaling domain is a CD3-zeta polypeptide.

22. The nucleic acid of claim 21, wherein the first polypeptide comprises a hinge region derived from CD8 interposed between the scFv and the transmembrane domain.

23. The nucleic acid of claim 1, wherein:

a) the scFv binds an epitope present on a cancer cell;

b) the first member of the dimerization pair is an FK506 binding protein (FKBP);

c) the second member of the dimerization pair is a FKBP-rapamycin associated protein (FRB);

d) the first polypeptide comprises a first costimulatory polypeptide comprising an OX-40 polypeptide;

e) the second polypeptide comprises a second costimulatory polypeptide comprising an OX-40 polypeptide; and

f) the intracellular signaling domain is a CD3-zeta polypeptide.

24. The nucleic acid of claim 23, wherein the first polypeptide comprises a hinge region derived from CD8 interposed between the scFv and the transmembrane domain.

25. A recombinant expression vector comprising the nucleic acid of claim 1.

26. A T lymphocyte comprising the recombinant expression vector of claim 25.