Altering Gene Expression in CART Cells and Uses Thereof

Patent metadata

Field	Value
Publication number	US-2020407728-A1
Application number	US-202016943335-A
Country	US
Kind code	A1
Filing date	Jul 30, 2020
Priority date	Oct 31, 2014
Publication date	Dec 31, 2020
Grant date	—

Abstract

The present invention relates to compositions and methods for generating a modified T cell with a nucleic acid capable of downregulating endogenous gene expression selected from the group consisting of TCR α chain, TCR β chain, beta-2 microglobulin, a HLA molecule, CTLA-4, PD1, and FAS and further comprising a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for a surface antigen on a target cell or an electroporated nucleic acid encoding a chimeric antigen receptor (CAR). Also included are methods and pharmaceutical compositions comprising the modified T cell for adoptive therapy and treating a condition, such as an autoimmune disease.

First claim

What is claimed:

1 . A CRISPR-modified T cell comprising:
(i) a CRISPR-mediated insertion or deletion in a TCR α chain (TRAC) and/or a TCR β (TRBC) chain gene locus causing downregulated gene expression of an endogenous TCR α chain and/or a TCR β chain gene; (ii) a CRISPR-mediated insertion or deletion in a beta 2-microglobulin (B2M) gene locus causing downregulated gene expression of an endogenous beta 2-microglobulin gene; and (iii) a nucleic acid encoding a chimeric antigen receptor (CAR) comprising affinity for a tumor associated-antigen (TAA) on a target cell.

2 . The CRISPR-modified T cell of claim 1 , further comprising:
(iv) a CRISPR-mediated insertion or deletion in a gene locus causing downregulated gene expression of a HLA molecule, wherein the HLA molecule is not a class I HLA molecule.

3 . The CRISPR-modified T cell of claim 1 , wherein the antigen binding domain of the CAR:
(a) comprises an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof; and/or (b) specifically binds an antigen on a target cell.

4 . The CRISPR-modified T cell of claim 3 , wherein the antigen comprises:
(a) CD19; and/or (b) prostate-specific membrane antigen (PSMA) and/or prostate stem cell antigen (PSCA).

5 . The CRISPR-modified T cell of claim 1 , wherein:
(a) the CAR further comprises a hinge region; and/or (b) the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

6 . The CRISPR-modified T cell of claim 1 , wherein the intracellular domain comprises:
(a) a costimulatory signaling domain and an intracellular signaling domain; and/or (b) one or more of a costimulatory domain of a protein selected from the group consisting of CD3, CD83, CD86, CD27, CD28, 4-1BB (CD137), CD127, 4-1BBL, CD134, PD-1, PD-1L, CD7, LIGHT, DAP10, DAP12, CD2, ICAM-1, LFA-1, lymphocyte-specific protein tyrosine kinase (LCK), TNFR2, CD30, CD40, ICOS (CD278), NKG2C, B7-H3, or a variant thereof; and/or (c) an intracellular domain selected from the group consisting of cytoplasmic signaling domains of TCR, CD3 zeta chain (CD3ζ), common FcRγ, FcγRIIa, FcεRIβ, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

7 . The CRISPR-modified T cell of claim 1 , wherein the CRISPR-mediated insertion or deletion comprises a CRISPR-associated (Cas) nuclease and a guide RNA, wherein the guide RNA comprises a guide sequence that is complementary with a sequence within the TCR α chain, TCR β chain, or beta 2-microglobulin gene locus.

8 . The CRISPR-modified T cell of claim 7 , wherein:
(a) the sequence is within the TCR α chain gene locus and the guide RNA comprises a nucleic acid sequence encoded by SEQ ID NO: 1; or (b) the sequence is within the TCR β chain gene locus and the guide RNA comprises a nucleic acid sequence encoded by SEQ ID NO: 2; or (c) the sequence is within the beta 2-microglobulin gene locus and the guide RNA comprises a nucleic acid sequence encoded by SEQ ID NO: 3.

9 . A method of generating a CRISPR-modified T cell, the method comprising:
(i) introducing into a T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous TCR α chain and/or TCR β chain in the T cell; (ii) introducing into the T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous beta-2 microglobulin (B2M) gene locus in the T cell; and (iii) introducing into the T cell a nucleic acid encoding a chimeric antigen receptor (CAR) comprising affinity for a tumor associated-antigen (TAA) on a target cell, thereby generating the CRISPR-modified T cell.

10 . The method of claim 9 , further comprising:
(iv) introducing into the T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous HLA gene locus in the T cell, wherein the HLA molecule is not a class I HLA molecule.

11 . The method of claim 9 , wherein:
(a) introduction of a CRISPR system into a T cell comprises electroporation of the CRISPR system into the T cell; and/or (b) the T cell is obtained from the group consisting of peripheral blood mononuclear cells, umbilical cord blood cells, bone marrow, lymph node tissue, spleen tissue, blood, a purified population of T cells, and a T cell line; and/or (c) the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of a co-stimulatory molecule; and/or (d) the CAR further comprises a hinge region.

12 . The method of claim 11 , wherein:
(a) the antigen binding domain comprises a full-length antibody or antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody; and/or (b) the antigen comprises CD19; and/or (c) the antigen comprises prostate-specific membrane antigen (PSMA) and/or prostate stem cell antigen (PSCA).

13 . The method of claim 11 , wherein:
(a) the transmembrane domain is selected from the group consisting of a synthetic hydrophobic sequence, a transmembrane domain of any membrane-bound or transmembrane protein, a transmembrane domain of an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; and/or (b) the intracellular domain of the CAR comprises dual signaling domains; and/or (c) the intracellular domain comprises one or more of a costimulatory domain of a protein selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD-1 and PDL1.

14 . The method of claim 13 , wherein:
(a) the costimulatory domain comprises 4-1BB; and/or (b) the CD3 is a CD3 zeta; and/or (c) the dual signaling domains comprise the costimulatory domain of 4-1BB and the costimulatory domain of CD3z zeta.

15 . The method of claim 9 , wherein the CRISPR system comprises a CRISPR-associated (Cas) nuclease and a guide RNA, wherein the guide RNA comprises a guide sequence that is complementary with a sequence within the TCR α chain, TCR β chain, or beta 2-microglobulin gene locus.

16 . The method of claim 15 , wherein:
(a) the sequence is within the TCR α chain gene locus and the guide RNA comprises a nucleic acid sequence encoded by SEQ ID NO: 1; or (b) the sequence is within the TCR β chain gene locus and the guide RNA comprises a nucleic acid sequence encoded by SEQ ID NO: 2; or (c) the sequence is within the beta 2-microglobulin gene locus and the guide RNA comprises a nucleic acid sequence encoded by SEQ ID NO: 3.

17 . A pharmaceutical composition comprising the modified T cell generated according to the method of claim 9 and a pharmaceutically acceptable carrier.

18 . A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cell of claim 1 .

19 . The method of claim 18 , wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung c

Assignees

• Univ Pennsylvania

Inventors

• Zhao Yangbing
• Ren Jiangtao
• Liu Xiaojun
• June Carl H

Classifications

• A61K40/32

  T-cell receptors [TCR] · CPC title

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title

• A61K40/50

  characterised by the use of allogeneic cells · CPC title

• A61K40/4274

  Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR] · CPC title

• A61K40/4269

  NY-ESO · CPC title