Altering Gene Expression in CART Cells and Uses Thereof
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.
1 . A modified T cell comprising:
a nucleic acid capable of downregulating gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, beta-2 microglobulin, a HLA molecule, CTLA-4, PD1, and FAS; and
a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain and an intracellular domain of a co-stimulatory molecule.
2 . The modified T cell of claim 1 , wherein the nucleic acid capable of downregulating gene expression is selected from the group consisting of an antisense RNA, antigomer RNA, siRNA, shRNA, and a CRISPR system.
3 . The modified T cell of claim 2 , wherein the CRISPR system comprises an pAd5/F35-CRISPR vector.
4 . The modified T cell of claim 1 , wherein the antigen binding domain of the CAR comprises an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, human antibody, humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof.
5 . The modified T cell of claim 1 , wherein the antigen binding domain of the CAR specifically binds an antigen on a target cell.
6 . The modified T cell of claim 1 , wherein the intracellular domain of the CAR comprises dual signaling domains.
7 . The modified T cell of claim 1 further comprising an exogenous nucleic acid encoding a costimulatory molecule.
8 . The modified T cell of claim 7 , wherein the co-stimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.
9 . The modified T cell of claim 8 , wherein the CD3 comprises at least two different CD3 chains.
10 . The modified T cell of claim 9 , wherein the different CD3 chains are CD3 zeta and CD3 epsilon chains.
11 . A method for generating a modified T cell comprising:
introducing a nucleic acid capable of downregulating gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, beta-2 microglobulin, a HLA molecule, CTLA-4, PD1, and FAS into a T cell; and
introducing a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain.
12 . The method of claim 11 , wherein the nucleic acid capable of downregulating gene expression is selected from the group consisting of an antisense RNA, antigomer RNA, siRNA, shRNA, and a CRISPR system.
13 . The method of claim 12 , wherein the CRISPR system comprises an pAd5/F35-CRISPR vector.
14 . The method of claim 11 , wherein the antigen binding domain of the CAR comprises an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, human antibody, humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof.
15 . The method of claim 11 , wherein the antigen binding domain of the CAR specifically binds an antigen on a target cell.
16 . The method of claim 11 , wherein the intracellular domain of the CAR comprises dual signaling domains.
17 . The method of claim 11 , wherein the T cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line.
18 . The method of claim 11 , wherein the method further comprises expanding the T cell.
19 . The method of claim 18 , wherein the step of expanding the T cell comprises culturing the T cell with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.
20 . The method of claim 11 further comprising cryopreserving the T cell.
21 . The method of claim 20 further comprising thawing the cryopreserved T cell prior to introducing the nucleic acid into the T cell.
22 . The method of claim 11 , wherein introducing the nucleic acid is selected from the group consisting of transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells.
23 . The method of claim 11 further comprising electroporating a RNA encoding a co-stimulatory molecule into the T cell.
24 . The method of claim 23 , wherein the co-stimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.
25 . The method of claim 11 further comprising expressing Klf4, Oct3/4 and Sox2 in the T cells to induce pluripotency of the T cell.
26 . A method of treating a disease or condition associated with enhanced immunity in a subject comprising administering an effective amount of a pharmaceutical composition comprising the modified T cell of claim 1 to a subject in need thereof.
27 . A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cell of claim 1 .
28 . The method of claim 27 , wherein the condition is an autoimmune disease.
29 . The method of claim 28 , wherein the autoimmune disease is selected from the group consisting of Acquired Immunodeficiency Syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernacious anemia, polyarteritis nodosa , polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo, Wegener's granulomatosis, and any combination thereof.
30 . The method of claim 27 , wherein the condition is a cancer.
31 . The method of claim 30 , 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 cancer, and any combination thereof.
32 . A method for stimulating a T cell-mediated immune response to a target cell or tissue in a subject comprising administering to a subject an effective amount of a pharmaceutical composition comprising the modified T cell of claim 1 .
33 . The method of claim 32 further comprising inducing lysis of the target cell or tissue.
34 . The method of claim 33 , wherein the induced lysis is antibody-dependent cell-mediated cytotoxicity (ADCC).
35 . A method for adoptive cell transfer therapy comprising administering an effective amount of a pharmaceutical composition comprising the modified T cell of claim 1 to a subject in need thereof to prevent or treat an immune reaction that is adverse to the subject.
36 . Use of the modified T cell of claim 1 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.
37 . A composition comprising the modified T cell generated according to the method of claim 11 .
38 . A pharmaceutical composition comprising the modified T cell generated according to the method of claim 11 and a pharmaceutically acceptable carrier.