Altering gene expression in cart cells and uses thereof

Abstract

To provide a patient-specific modified T cell intended for treatment of cancer, methods for generating the modified T cell, and pharmaceutical compositions comprising the modified T cell.SOLUTION: The present invention provides a modified T cell comprising: a nucleic acid capable of downregulating expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, an 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. Also provided are methods for generating the modified T cell. Further provided are pharmaceutical compositions comprising the modified T cell for adoptive therapy and for treating a condition, such as an autoimmune disease.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 073,651, filed on October 31, 2014, under 35 U.S.C. § 119(e), the entire disclosure of which is incorporated herein by reference.

[0002] Statement Regarding Federally Sponsored Research or Development This invention was made with government support under CA120409, awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] Background of the Invention Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR) - modified T cells has been shown to be a promising strategy for the treatment of cancer (Louis et al., 2011, Blood 118:6050 - 6056 (Non - Patent Document 1); Kochenderfer et al., 2010, Blood 116:3875 - 3886 (Non - Patent Document 2); and Porter et al., 2011, N Engl J Med 365:725 - 733 (Non - Patent Document 3)).

[0004] Safety concerns associated with integration using lentiviral vectors or retroviral vectors are a major concern for the modification of cells used in ACT. Some advances have been made to avoid off-target or off-target unwanted side effects, such as RNA transfection of T cells by T cell receptor (TCR) or CAR RNA electroporation (Zhao, 2006, Mol Ther 13:151-159 (Non-Patent Document 4); Mitchell et al., Smits et al., 2004, Leukemia 18:1898-1902 (Non-Patent Document 5)). By minimizing the dosage of both RNA and T cells, such methods efficiently enable the introduction of multiple genes into cells. However, the main limitation of transient CAR expression is the suboptimal effector activity and functionality of RNA-transfected T cells. To improve the function of CAR, multiple T cell infusions and / or significant use of low-dose chemotherapy have been used (Barrett et al., 2013, Hum Gene Ther 24(8):717-27 (Non-Patent Document 6)).

[0005] To avoid the need for combination therapies and additional treatments while improving the effector activity and functionality of CAR, various attempts have been made. Increased RNA during the transfection process has an adverse effect on T cell function, particularly antitumor activity in vivo (Barrett et al., 2011, Hum Gene Ther 22:1575-1586 (Non-Patent Document 7)). Another construct in which an anti-CD3 antigen antibody fragment is fused to an anti-tumor antigen antibody fragment has also been tested in clinical trials for cancer treatment (Bargou et al., 2008, Science 321:974-977 (Non-Patent Document 8); Klinger et al., 2012, Blood 119:6226-6233 (Non-Patent Document 9)). Unfortunately, these constructs have been severely limited in function due to short half-life, poor accessibility to target cell sites, and lack of appropriate long-term signaling function.

[0006] In addition to the low expression levels of the transduced TCRs, TCR clinical studies have been hampered by mispairing of the α- and β-chains. When T cells transcribe the chains of two different TCRs (native α / β, exogenous α / β, and native / exogenous "mispairing" heterodimers), four types of TCRs can potentially be expressed on the cell surface, which is clearly a major obstacle in using this approach. Previous studies have clearly demonstrated through preclinical trials that TCR mispairing can induce harmful self-antigen recognition.

[0007] Initial clinical data on TCR and CAR T cells for cancer treatment have shown promising results, but the risks to patients are significant, and some patients' T cells do not have sufficient potency to be an effective treatment even after redirection of the TCR or CAR and forced modification of allogeneic donor-derived T cells. However, the endogenous αβ T cell receptor on the infused allogeneic T cells can recognize major and minor histocompatibility antigens in the recipient, potentially leading to graft-versus-host disease (GVHD). As a result, most current clinical trials using autologous CAR T cell infusion rely on immune tolerance to prevent harmful recognition via the TCR of normal tissues after adoptive cell transfer. This approach has achieved initial clinical success, but the time and cost of manufacturing patient-specific T cell products are limiting factors. Therefore, there is a need for a safer method of modifying T cells while avoiding the time and cost of manufacturing patient-specific T cell products.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

[0009] As described herein, the present invention relates to a composition and method for producing modified T cells, which have a nucleic acid capable of changing the gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1 and FAS, and further contain a nucleic acid encoding a chimeric antigen receptor (CAR).

[0010] One aspect of the present invention includes a nucleic acid capable of down-regulating the gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, 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 costimulatory molecule, and includes modified T cells.

[0011] In another aspect, the present invention includes the step of introducing into T cells a nucleic acid capable of down-regulating the gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1 and FAS; and the step of introducing a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain and a transmembrane domain, and includes a method for producing modified T cells.

[0012] In yet another aspect, the present invention includes the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells described herein, and includes a method for treating a disease or condition associated with immune hyperactivity in a subject.

[0013] In still another aspect, the present invention provides a method for treating a condition in a subject, which includes the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cells described herein.

[0014] In another aspect, the present invention includes the step of administering to a subject an effective amount of a pharmaceutical composition comprising the modified T cells described herein, and includes a method for stimulating a T cell-mediated immune response against target cells or tissues in a subject.

[0015] In yet another aspect, the present invention includes the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells described herein for preventing or treating an immune reaction harmful to the subject, and includes a method for adoptive cell transfer therapy.

[0016] In yet another aspect, the invention includes the use of the modified T cells described herein in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

[0017] In another aspect, the invention includes a composition comprising modified T cells produced according to the methods described herein.

[0018] In yet another aspect, the invention includes a pharmaceutical composition comprising modified T cells produced according to the methods described herein.

[0019] In various embodiments of the above and any other aspects of the invention described herein, the nucleic acids capable of downregulating gene expression are selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA, and CRISPR systems, such as the pAd5 / F35-CRISPR vector.

[0020] In one embodiment, the antigen-binding domain of the CAR comprises an antibody selected from the group consisting of monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, single-chain variable fragments, and antigen-binding fragments thereof. In another embodiment, the antigen-binding domain of the CAR specifically binds to an antigen on the target cell. In yet another embodiment, the intracellular domain of the CAR comprises a dual-signaling domain.

[0021] In another embodiment, the modified T cells described herein further comprise an exogenous nucleic acid encoding a costimulatory molecule such as CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In one embodiment, the method of making the modified T cells described herein further comprises the step of electroporating RNA encoding a costimulatory molecule into the T cells. In some embodiments where the costimulatory molecule is CD3, CD3 comprises at least two different CD3 chains, such as the CD3ζ chain and the CD3ε chain.

[0022] In another aspect, the T cells are obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, purified T cell populations, and T cell lines.

[0023] In yet another aspect, the method of making modified T cells as described herein further includes a step of expanding the T cells. In one aspect, the step of expanding the T cells includes culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0024] In still another aspect, the method of making modified T cells as described herein further includes a step of cryopreserving the T cells. In another aspect, the method described herein further includes a step of thawing the cryopreserved T cells before introducing the nucleic acid into the T cells.

[0025] In one aspect, 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.

[0026] In yet another aspect, the method described herein further includes a step of expressing Klf4, Oct3 / 4, and Sox2 in the T cells to induce pluripotency of the T cells.

[0027] In various aspects of the above and any other aspects of the invention described herein, the invention includes administering modified T cells to a subject. In one aspect, the subject has a condition such as an autoimmune disease. In some aspects, the autoimmune disease is acquired immunodeficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid antibody 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 pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Ménière's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), which is also known as systemic sclerosis (SS)), Sjögren'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 selected from the group consisting of.

[0028] In another aspect, the condition is cancer, for example, cancer selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and any combination thereof.

[0029] In another aspect, the methods described herein further include inducing lysis of target cells or tissues, such as antibody-dependent cell-mediated cytotoxicity (ADCC). [Invention 1001] A nucleic acid capable of down-regulating the gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, 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 costimulatory molecule A modified T cell comprising the same. [Invention 1002] The modified T cell of Invention 1001, wherein the nucleic acid capable of down-regulating gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA, and CRISPR systems. [Invention 1003] The modified T cell of Invention 1002, wherein the CRISPR system comprises a pAd5 / F35-CRISPR vector. [Invention 1004] The modified T cell of Invention 1001, wherein the antigen-binding domain of the CAR comprises an antibody selected from the group consisting of monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, single-chain variable fragments, and antigen-binding fragments thereof. [Invention 1005] The modified T cell of Invention 1001, wherein the antigen-binding domain of the CAR specifically binds to an antigen on the target cell. [Invention 1006] The modified T cell of Invention 1001, wherein the intracellular domain of the CAR comprises a dual signaling domain. [Invention 1007] The modified T cell of Invention 1001, further comprising an exogenous nucleic acid encoding a costimulatory molecule. [Invention 1008] The modified T cells of the present invention 1007, 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. [The present invention 1009] The modified T cells of the present invention 1008, wherein CD3 comprises at least two different CD3 chains. [The present invention 1010] The modified T cells of the present invention 1009, wherein the different CD3 chains are CD3ζ chain and CD3ε chain. [The present invention 1011] Introducing into T cells a nucleic acid capable of down-regulating the gene expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1 and FAS; and Introducing into T cells a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain and a transmembrane domain A method for producing modified T cells, comprising. [The present invention 1012] The method of the present invention 1011, wherein the nucleic acid capable of down-regulating gene expression is selected from the group consisting of antisense RNA, antagomir RNA, siRNA, shRNA, and CRISPR system. [The present invention 1013] The method of the present invention 1012, wherein the CRISPR system comprises a pAd5 / F35-CRISPR vector. [The present invention 1014] The method of the present invention 1011, wherein the antigen-binding domain of the CAR comprises an antibody selected from the group consisting of monoclonal antibody, polyclonal antibody, synthetic antibody, human antibody, humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof. [The present invention 1015] The method of the present invention 1011, wherein the antigen-binding domain of the CAR specifically binds to an antigen on the target cell. [The present invention 1016] The method of the present invention 1011, wherein the intracellular domain of the CAR comprises a dual signal transduction domain. [The present invention 1017] The method of the present invention 1011, wherein the T cells are obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, purified T cell populations, and T cell lines. [The present invention 1018] The method of the present invention 1011, further comprising the step of expanding the T cells. [The present invention 1019] The method of the present invention 1018, wherein the step of expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand. [The present invention 1020] The method of the present invention 1011, further comprising the step of cryopreserving the T cells. [The present invention 1021] The method of the present invention 1020, further comprising the step of thawing the cryopreserved T cells before introducing the nucleic acid into the T cells. [The present invention 1022] The method of the present invention 1011, wherein the step of 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. [The present invention 1023] The method of the present invention 1011, further comprising the step of electroporating RNA encoding a costimulatory molecule into the T cells. [The present invention 1024] The method of the present invention 1023, wherein the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. [The present invention 1025] The method of the present invention 1011, further comprising the step of expressing Klf4, Oct3 / 4, and Sox2 in the T cells to induce pluripotency of the T cells. [The present invention 1026] A method of treating a disease or condition associated with enhanced immunity in a subject, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells of the present invention 1001. [The present invention 1027] A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising modified T cells of the present invention 1001. [The present invention 1028] The method of the present invention 1027, wherein the condition is an autoimmune disease. [The present invention 1029] The autoimmune disease is selected from the group consisting of acquired immunodeficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid antibody 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 pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, dogo disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes, juvenile rheumatoid arthritis (Still's disease), juvenile rheumatoid arthritis, Ménière's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren'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. The method of the present invention 1028. [The present invention 1030] The method of the present invention 1027, wherein the condition is cancer. [The present invention 1031] The method of the present invention 1030, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and any combination thereof. [The present invention 1032] A method for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the modified T cells of the present invention 1001. [The present invention 1033] The method of the present invention 1032, further comprising inducing lysis of the target cell or tissue. [The present invention 1034] The method of the present invention 1033, wherein the induced lysis is antibody-dependent cell-mediated cytotoxicity (ADCC). [The present invention 1035] A method for adoptive cell transfer therapy, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells of the present invention 1001 for preventing or treating an immune reaction harmful to the subject. [The present invention 1036] Use of the modified T cells of the present invention 1001 in the manufacture of a medicament for treating an immune response in a subject in need thereof. [The present invention 1037] A composition comprising modified T cells produced according to the method of the present invention 1011. [The present invention 1038] A pharmaceutical composition comprising modified T cells produced according to the method of the present invention 1011 and a pharmaceutically acceptable carrier.

Brief Description of the Drawings

[0030] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the present invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and means of the embodiments shown in the drawings.

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[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are described herein. The following specialized terms are used in the description and claiming of the present invention. Definition It is also to be understood that the specialized terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0032]

[0033] ​The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0034] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass variations of ±20%, ±10%, more preferably ±5%, still more preferably ±1%, and even more preferably ±0.1% from a specified value, insofar as such variations are appropriate to carry out the disclosed method.

[0035] As used herein, "activation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers, inter alia, to a T cell that is undergoing cell division.

[0036] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody may be an intact immunoglobulin derived from a natural or recombinant source, or it may be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention include, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab') 2and can exist in various forms, including single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0037] The term "antibody fragment" refers to a portion of an intact antibody and to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0038] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an intact antibody molecule in its naturally occurring conformation.

[0039] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an intact antibody molecule in its naturally occurring conformation. Alpha and beta light chains refer to the two major antibody light chain isotypes.

[0040] As used herein, the term "synthetic antibody" means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage described herein. This term also refers to an antibody produced by the synthesis of an antibody protein or an antibody-encoding DNA molecule expressing the amino acid sequence defining the antibody, where the DNA sequence or amino acid sequence is obtained using available DNA sequence or amino acid sequence synthesis techniques well known in the art.

[0041] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can include either or both antibody production and activation of specific immunocompetent cells. One of ordinary skill in the art will understand that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Additionally, an antigen can be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will, therefore, encode an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Additionally, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be made, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0042] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in average life expectancy, or an improvement in various physiological symptoms associated with cancerous conditions. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in the prevention of the occurrence of tumors per se.

[0043] As used herein, the term "self-antigen" means, according to the present invention, any self-antigen that is recognized as foreign by the immune system. Self-antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cell lipids, nucleic acids, and glycoproteins, including cell surface receptors.

[0044] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

[0045] As used herein, the term "self" is intended to mean any material derived from the same individual that is later re-introduced into that individual.

[0046] "Allogeneic" refers to a graft derived from different animals of the same species.

[0047] "Xenogeneic" refers to a graft derived from animals of different species.

[0048] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In certain embodiments, the cancer is medullary thyroid cancer.

[0049] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor engineered to be expressed on immune effector cells and that specifically binds to an antigen. CARs can be used as a therapy involving adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific for a particular form of antigen. In some embodiments, the CAR is expressed, for example, with specificity for a tumor-associated antigen. A CAR can also include an extracellular domain that includes a cell activation domain, a transmembrane domain, and a tumor-associated antigen binding region. In some aspects, the CAR includes a fusion of a single-chain variable fragment (scFv)-derived monoclonal antibody fused to a CD3ζ transmembrane and intracellular domain. The specificity of the CAR design can be derived from the ligand (e.g., a peptide) of the receptor. In some embodiments, the CAR can target cancer by redirecting the specificity of T cells that express a CAR specific for a tumor-associated antigen.

[0050] The term "cleavage" refers to the cleavage of a covalent bond, such as in the backbone of a nucleic acid molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two different single-strand cleavage events. DNA cleavage can result in the generation of either blunt ends or sticky ends. In certain embodiments, a fusion polypeptide can be used for targeting the cleaved double-stranded DNA.

[0051] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody that includes an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for antigen binding ability using the functional assay methods described herein.

[0052] As used herein, the term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to the Toll ligand receptor, and a ligand that specifically binds to B7-H3. Costimulatory ligands can also include, inter alia, but are not limited to, an antibody that specifically binds to a costimulatory molecule present on a T cell, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0053] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, including, but not limited to, proliferation. Costimulatory molecules can include, but are not limited to, MHC class I molecules, BTLA, and the Toll ligand receptor.

[0054] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0055] The term "CRISPR / CAS", "clustered regularly interspaced short palindromic repeats system", or "CRISPR" refers to a DNA locus that contains short repeats of a nucleotide sequence. Following each repeat is a short segment of spacer DNA due to past exposure to a virus. Bacteria and archaea have developed an adaptive immune defense called the CRISPR-CRISPR associated (Cas) system that uses short RNAs to direct the degradation of foreign nucleic acids. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA by RNA-guided DNA cleavage.

[0056] In type II CRISPR / Cas systems, short segments of foreign DNA, named "spacers", are incorporated into the CRISPR genomic locus, transcribed, and processed into short CRISPR RNAs (crRNAs). These crRNAs anneal to trans-activating crRNAs (tracrRNAs) to direct sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins. Recent studies have shown that target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA binding region.

[0057] To direct Cas9 to cleave a sequence of interest, a crRNA-tracrRNA fusion transcript, hereafter referred to as "guide RNA" or "gRNA" in this specification, can be designed from the human U6 polymerase III promoter. Genome editing and regulation via CRISPR / CAS has shed light on its transformative potential for basic science, cell manipulation, and therapies.

[0058] The term "CRISPRi" refers to a CRISPR system for sequence-specific gene repression or inhibition of gene expression, such as at the transcriptional level.

[0059] A "disease" is a health condition of an animal in which the animal cannot maintain homeostasis and its health continues to deteriorate if the disease is not improved. In contrast, a "disorder" in an animal is a health condition in which the animal can maintain homeostasis, but the animal's health is in a less favorable state than in the absence of the disorder. A disorder does not necessarily cause a further decline in the animal's health if left untreated.

[0060] As used herein, the term "downregulation" refers to a decrease or loss of expression of one or more genes.

[0061] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results can include, but are not limited to, antitumor activity as determined by any suitable means in the art.

[0062] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, and the resulting biological property of serving as a template for the synthesis of other polymers and macromolecules in a biological process, having either a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is identical to the mRNA sequence and is usually the nucleotide sequence shown in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein, or other product of that gene or cDNA.

[0063] As used herein, "endogenous" refers to any material that is derived from within or produced within a living organism, cell, tissue, or system.

[0064] As used herein, the term "exogenous" refers to any material that is introduced from outside or produced outside a living organism, cell, tissue, or system.

[0065] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo increase in number compared to the number initially present in the culture. In another embodiment, T cells expanded ex vivo increase in number compared to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that are removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0066] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0067] An "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) into which the recombinant polynucleotide has been incorporated, and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0068] As used herein, "homology" refers to subunit sequence identity between two polymeric molecules, such as between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in both of the two molecules are occupied by the same monomeric subunit; for example, if the positions in each of two DNA molecules are occupied by adenine, they are homologous at that position. The homology between two sequences is a linear function of the number of positions that are identical or homologous; for example, if half of the positions in two sequences (e.g., 5 positions in a 10-subunit polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are identical or homologous, the two sequences are 90% homologous.

[0069] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding portion sequences of an antibody) that contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species such as a mouse, rat, or rabbit (donor antibody) that have the desired specificity, affinity, and potency. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody can contain residues not found in either the recipient antibody or the introduced CDR or framework sequences. These modifications are made to further improve and optimize antibody performance. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. Also, a humanized antibody optimally contains at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0070] "Fully human" refers to an immunoglobulin, such as an antibody, in which the entire molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody.

[0071] As used herein, "identity" refers to subunit sequence identity between two polymer molecules, such as between two polypeptide molecules, particularly between two amino acid molecules. When two amino acid sequences have the same residue at the same position; for example, if the position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The degree or identity to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a linear function of the number of positions that match or are identical; for example, if half of the positions in two sequences (e.g., 5 positions in a 10 - amino acid - long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0072] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCR (B - cell receptor) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and urogenital tracts. IgG is the most common circulating blood antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination reactions, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is not known, but it may act as an antigen receptor. IgE is an immunoglobulin that mediates immediate - type hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to allergens.

[0073] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to remove the antigen.

[0074] As used herein, the term "induced pluripotent stem cell" or "iPS cell" refers to a pluripotent stem cell generated from an adult cell such as a T cell. Expression of reprogramming factors such as Klf4, Oct3 / 4, and Sox2 in adult cells converts the cells into pluripotent cells capable of proliferation and differentiation into multiple cell types.

[0075] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compound be used jointly by the recipient.

[0076] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its natural coexisting substances is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell.

[0077] As used herein, the term "knockdown" refers to a decrease in gene expression of one or more genes.

[0078] As used herein, the term "knockout" refers to the loss of gene expression of one or more genes.

[0079] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors because they can deliver a significant amount of genetic information into the DNA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0080] As used herein, the term "modified" means an altered state or structure of the molecule or cell of the present invention. Molecules can be modified in many ways, such as chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0081] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in the subject in the absence of treatment or compound and / or as compared to the level of response in a subject that is otherwise identical but not treated, and this term encompasses disturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0082] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0083] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding an RNA or protein" also includes that the nucleotide sequence encoding a protein may include introns to the extent that it may include introns depending on the type.

[0084] The term "functionally linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence if the first nucleic acid sequence is placed under the functional relationship with the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Generally, functionally linked DNA sequences are continuous and, when it is necessary to join two protein-coding regions, are in the same reading frame.

[0085] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate that the expression of a tumor antigen in cells from a disease area such as a solid tumor inside a specific tissue or organ of a patient is at an abnormal level compared to the level of expression in normal cells from that tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assay methods known in the art.

[0086] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection, or infusion methods.

[0087] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and that they can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any available means in the art, including recombinant means, i.e., recombinant libraries or cloning of nucleic acid sequences from cell genomes using conventional cloning techniques and PCR (trademark), and synthetic means.

[0088] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which are generally also referred to as peptides, oligopeptides and oligomers in the art, and long chains, which are generally referred to as proteins in the art, and there are many types among them. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0089] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell or an introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0090] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, cause the gene product to be expressed in a tissue-specific manner.

[0091] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly under most or all physiological conditions of the cell.

[0092] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced intracellularly only when, substantially, an inducer corresponding to the promoter is present intracellularly.

[0093] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced intracellularly only when, substantially, the cell is a cell of the tissue type corresponding to the promoter.

[0094] "Sendai virus" refers to a genus within the Paramyxoviridae family. Sendai virus is a negative-sense single-stranded RNA virus that does not integrate into the host genome or alter the host cell's genetic information. The host range of Sendai virus is remarkably broad, and it is not pathogenic to humans. Sendai virus is used as a recombinant viral vector and has the ability to transiently but strongly express genes.

[0095] "Signal transduction pathway" refers to the biochemical relationships among various signal transduction molecules that play a role in the transmission of signals from one part of a cell to another part of the cell. The term "cell surface receptor" includes molecules and molecular complexes that can receive signals and transmit them across the plasma membrane of the cell.

[0096] "Single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which the immunoglobulin heavy and light chain fragments are linked to the Fv region via engineered amino acid lengths. Various methods for producing single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:544-545; Skerra et al. (1988) Science 242:1038-1041.

[0097] As used herein with respect to an antibody, the term "specifically binds" means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may bind to that antigen from one or more species. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may bind to different genotypes of that antigen. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" may be used in the context of the interaction of an antibody, protein or peptide with a second chemical species, and may mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and that antibody.

[0098] The term "stimulate" means, when used herein, a primary response induced by a stimulatory molecule (e.g., the TCR / CD3 complex) binding to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as down-regulation of TGF-β, and / or rearrangement of the cytoskeletal structure.

[0099] As used herein, the term "stimulatory molecule" means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0100] As used herein, a "stimulatory ligand" means a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell and thereby mediate a primary response by the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0101] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be induced. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cows, pigs, dogs, cats, and murine mammals. Preferably, the subject is a human.

[0102] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. Also, a substantially purified cell refers to a cell that has been separated from other cell types that are normally associated with it in its natural state. In one example, a substantially purified population of cells refers to a homogeneous population of cells. In another example, the term simply refers to a cell that has been separated from cells that are normally associated with it in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0103] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0104] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. The TCR is composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γ / δ) chains. The TCR can exist in α / β and γ / δ forms that are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, namely a variable domain and a constant domain. In some embodiments, the TCR can be modified on any cell that includes the TCR (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells).

[0105] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0106] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This includes primary subject cells and their progeny.

[0107] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder that a subject is afflicted with.

[0108] As used herein, the terms "under transcriptional control" or "functionally linked" mean that the promoter is in the correct position and orientation with respect to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0109] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides complexed with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the uptake of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0110] Range: Throughout this disclosure, various aspects of the invention can be presented in range format. It should be understood that the description in range format is merely for convenience and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a description of a range should be considered to specifically disclose all the sub-ranges and individual numerical values within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0111] Explanation Universal T cells for avoiding graft-versus-host disease (GVHD) are highly desirable in clinical settings. However, the use of allogeneic T cells carries a risk of rejection by the host immune system through the recognition of HLA-A molecules. Targeting strategies for manipulating multiple genes are complex and have achieved only low efficiency in T cells, and have not been able to prevent GVHD and host-versus-graft reaction simultaneously.

[0112] The FAS receptor / FAS ligand (FAS / FASL) apoptosis signaling pathway negatively regulates T cell function. PD1 and CTLA4 are two major inhibitory signaling pathways in T cells. Enhancement of antitumor immunity resulting from antibody-mediated blockade of CTLA-4, PD-1, or PD-L1 suggests the possibility of improving the efficiency of immunotherapy by inhibiting these pathways. The present invention includes the production of modified T cells in which the TCR α-chain and β-chain, β-2 microglobulin, HLA molecule, CTLA-4, PD-1, and / or FAS are removed as a means for producing modified T cells with reduced immunogenicity.

[0113] The present invention includes methods and compositions for producing modified T cells by knocking down endogenous gene expression and expressing either a modified T cell receptor or a chimeric antigen receptor. In some embodiments, the present invention includes methods for producing modified T cells. Such modified T cells can be placed in a therapeutic composition and administered to a patient in need thereof.

[0114] Knockdown of endogenous gene expression The present invention includes downregulation of endogenous gene expression in T cells, such as downregulation of the α and / or β chains of the T cell receptor (TCR), β-2 microglobulin, CTLA-4, FAS, PD1, or major histocompatibility complex proteins, such as HLA molecules. In one aspect, T cells with downregulated gene expression have reduced immunogenicity in an allogeneic environment. In another aspect, T cells with reduced immunogenicity express a modified TCR or CAR for targeted effector activity.

[0115] In one aspect, the present invention is a method for producing modified T cells, comprising introducing into T cells a nucleic acid capable of downregulating endogenous gene expression, wherein the gene is selected from the group consisting of the TCR α chain, TCR β chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1, and FAS. By downregulating the expression of endogenous genes involved in causing an immune response against the cell, such as the TCR α chain, TCR β chain, β-2 microglobulin, or HLA molecule, the immune-mediated rejection of the modified T cells is reduced. For example, by downregulating the expression of the endogenous TCR, MHC, or β-2 microglobulin gene, the surface presentation of alloantigens on T cells that may cause rejection by the host immune system is removed. Also, by downregulating endogenous genes that regulate inhibitory signaling pathways in T cells, such as CTLA-4, PD1, and / or FAS, the antitumor efficacy of the modified T cells when exposed to an immunosuppressive microenvironment is enhanced.

[0116] In one aspect, a nucleic acid capable of downregulating endogenous gene expression is introduced into T cells by electroporation, transfection, or lentiviral or other viral transduction. In another aspect, the invention includes modified T cells comprising an electroporated nucleic acid capable of downregulating endogenous gene expression. In yet another aspect, the modified T cells comprise an electroporated nucleic acid capable of downregulating endogenous TCR gene expression. In another aspect, a composition comprising the modified T cells is produced according to the methods described herein. In yet another aspect, the invention includes a pharmaceutical composition comprising modified T cells produced according to the methods described herein or comprising modified T cells produced according to the methods described herein and a pharmaceutically acceptable carrier.

[0117] A nucleic acid capable of regulating endogenous gene expression can downregulate endogenous gene expression. In one embodiment, a nucleic acid capable of downregulating endogenous gene expression is selected from the group consisting of antisense RNA, antagomir RNA, siRNA, shRNA, and CRISPR systems. Endogenous gene expression can be downregulated, knocked down, decreased, and / or inhibited, for example, by antisense RNA, antagomir RNA, siRNA, shRNA, CRISPR systems, etc.

[0118] CRISPR / Cas The CRISPR / Cas system is a convenient and efficient system for inducing targeted genetic changes. Target recognition by the Cas9 protein requires a "seed" sequence within the guide RNA (gRNA) and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the gRNA binding region. The CRISPR / CAS system can therefore be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / CAS system can target multiple genomic loci simultaneously by co-expressing a single CAS9 protein with two or more gRNAs, making this system uniquely suitable for editing multiple genes or synergistic activation of target genes.

[0119] An example of the CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in US Patent Application Publication No. 2014 / 0068797. CRISPRi induces permanent gene disruption using an RNA-guided Cas9 endonuclease to introduce DNA double-strand breaks, which trigger error-prone repair pathways, resulting in frameshift mutations. Catalytically dead Cas9 lacks endonuclease activity. When co-expressed with guide RNA, a DNA recognition complex is generated that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently suppresses the expression of the targeted gene.

[0120] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific to the target gene and a Cas endonuclease are introduced into a cell, forming a complex that enables the Cas endonuclease to introduce a double-strand break at the location of the target gene. In one embodiment, the CRISPR system includes an expression vector, such as but not limited to, a pAd5F35-CRISPR vector. In one embodiment, the modified T cells are produced by introducing a Cas expression vector and a guide nucleic acid sequence specific to a gene into the T cells. In another embodiment, the Cas expression vector induces the expression of Cas9 endonuclease. Other endonucleases can also be used, including but not limited to T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, and any other nucleases known in the art, and any combination thereof.

[0121] In one embodiment, the induction of the Cas expression vector includes exposing the T cells to an agent that activates the inducible promoter in the Cas expression vector. In such an embodiment, the Cas expression vector includes an inducible promoter, such as one that can be induced by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, such as doxycycline). However, it should be understood that other inducible promoters can also be used. The inducer can be a selective condition (e.g., exposure to an agent, such as an antibiotic) that results in the induction of the inducible promoter. This results in the expression of the Cas expression vector.

[0122] The guide nucleic acid sequence is specific for a certain gene and targets that gene for double-strand breaks induced by the Cas endonuclease. The sequence of the guide nucleic acid sequence may be within the locus of that gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides in length or more.

[0123] The guide nucleic acid sequence may be specific for any sequence, such as a gene that is thought to reduce immunogenicity or reduce susceptibility to an immunosuppressive microenvironment. In one embodiment, the gene may contain a sequence specific for a T cell receptor (TCR) chain (such as an α, β, γ, and / or δ chain), β-2 microglobulin, FAS, PD1, a major histocompatibility complex protein (such as an HLA class I molecule and / or an HLA class II molecule), CTLA-4, or any combination thereof.

[0124] The guide nucleic acid sequence includes an RNA sequence, a DNA sequence, a combination thereof (RNA-DNA composite sequence), or a sequence having synthetic nucleotides. The guide nucleic acid sequence may be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

[0125] T cell receptor Adoptive immunotherapy with T cells bearing an antigen-specific TCR has therapeutic potential in the treatment of cancer and certain chronic viral infections. Genetic manipulation of T cells with a specific TCR has the advantage of redirecting T cells against intracellular antigens. Considering that most oncogenic proteins are intracellular, the development of a group of TCRs specific for oncogenic driver proteins is highly attractive.

[0126] The present invention also includes modified T cells in which gene expression is downregulated as described herein and which have an exogenous T cell receptor (TCR). In one aspect, the present invention provides a nucleic acid encoding a modified T cell receptor (TCR) comprising an affinity for a surface antigen on a target cell, and a nucleic acid capable of regulating the expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, PD1 and FAS, which is introduced into a T cell, A method for producing a modified T cell, comprising the step of allowing the T cell to express the modified TCR.

[0127] In another aspect, the present invention includes a modified T cell comprising a nucleic acid encoding a modified T cell receptor (TCR) comprising an affinity for a surface antigen on an exogenous target cell, and a nucleic acid capable of downregulating the expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, PD1 and FAS, wherein the T cell expresses the modified TCR and the endogenous gene expression is downregulated in the T cell. The present invention also includes a population of cells comprising the modified T cells described herein.

[0128] The T cell receptor is a complex of membrane proteins that is involved in the activation of T cells in response to antigen presentation. Stimulation of the TCR is induced by major histocompatibility complex molecules (MHC) on antigen-presenting cells that present antigen peptides to the T cells and bind to the TCR complex to induce a series of intracellular signaling cascades.

[0129] The TCR generally consists of six different membrane-bound chains that form a TCR heterodimer involved in ligand recognition. TCRs exist in α / β and γ / δ forms that are structurally similar but have different anatomical locations and functions. In one embodiment, the TCR comprises a TCR α chain and a TCR β chain, and thus the nucleic acid encoding the TCR comprises nucleic acids encoding the TCR α chain and the TCR β chain. In another embodiment, the TCR α chain and / or the TCR β chain comprises at least one N-deglycosylation.

[0130] Each chain is composed of two extracellular domains, namely a variable domain and a constant domain. In one embodiment, the TCR comprises at least one murine constant region. The constant domain is proximal to the cell membrane, followed by a transmembrane domain and a short cytoplasmic tail. In one embodiment, the modified TCR comprises a cytoplasmic domain that includes a co-stimulatory signaling domain, such as a 4-1BB co-stimulatory signaling domain. The variable domain contributes to the determination of the specific antigen and MHC molecule to which the TCR binds specifically. Next, the specificity of the T cell for a particular antigen-MHC complex resides in the specific TCR expressed by the T cell.

[0131] Each of the constant domain and the variable domain may contain an intrachain disulfide bond. In one embodiment, the TCR comprises at least one disulfide bond. The variable domain contains highly polymorphic loops similar to the complementarity-determining regions (CDRs) of antibodies. The diversity of TCR sequences is created through somatic recombination of the linked variable (V), diversity (D), joining (J), and constant region genes.

[0132] Functional α-chain and γ-chain polypeptides are formed by the rearranged V-J-C regions, while β-chains and δ-chains consist of V-D-J-C regions. The extracellular constant domain includes a membrane-proximal region and an immunoglobulin region.

[0133] In one aspect, the TCR includes wild-type TCR, high-affinity TCR, and chimeric TCR. When the TCR is modified, it may have a higher affinity for the target cell surface antigen than the wild-type TCR. In aspects where the TCR is a chimeric TCR, the TCR may include a chimeric domain, for example, the TCR includes a co-stimulatory signaling domain at the C-terminus of at least one chain. In other aspects, the TCR may include a modified chain, such as a modified α-chain or β-chain. Such modifications can include N-deglycosylation, altered domains (such as variable regions engineered to target specific antigens or increase affinity), addition of one or more disulfide bonds, whole or fragments of chains from different species, and any combination thereof, but are not limited thereto.

[0134] In one aspect, the TCR includes specificity for a target cell antigen. The target cell surface antigen can include any type of ligand that defines the surface of the target cell. For example, the target cell surface antigen can be selected to recognize a ligand that serves as a cell surface marker on the target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of the TCR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In one aspect, the target cell surface antigen includes any tumor-associated antigen (TAA) and viral antigen, disease cell-associated antigen, or any fragment thereof.

[0135] The target cell antigen can include any protein that can be processed and presented by the major histocompatibility complex. For example, the target antigen can be associated with a particular disease state. Thus, examples of cell markers that can act as targets for the TCR include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In one aspect, the target antigen includes either a tumor-associated antigen (TAA) and a viral antigen, or any fragment thereof.

[0136] In one aspect, the invention includes a nucleic acid encoding a modified T cell receptor (TCR) that includes an affinity for a surface antigen on a target cell, and a population of modified T cells that includes a nucleic acid capable of down-regulating the expression of an endogenous gene selected from the group consisting of a TCR α chain, a TCR β chain, β-2 microglobulin, an HLA molecule, CTLA-4, PD1, and FAS, wherein the T cells are capable of expressing the modified TCR.

[0137] Techniques for the manipulation and expression of T cell receptors include, without limitation, the production of TCR heterodimers that include native disulfide bridges that connect each subunit (Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S. Patent No. 6,080,840).

[0138] Chimeric antigen receptor (CAR) The invention also includes modified T cells in which gene expression is down-regulated as described herein and that have a CAR. Thus, the invention includes within its scope modified T cells that include a CAR or a nucleic acid encoding a CAR, wherein the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0139] In one aspect, the present invention provides a method for generating modified T cells, comprising introducing into T cells a nucleic acid capable of down-regulating the expression of an endogenous gene selected from the group consisting of the TCR α-chain, TCR β-chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1 and FAS, and introducing into T cells a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain and an intracellular domain of a costimulatory molecule.

[0140] In another aspect, the present invention provides modified T cells comprising a nucleic acid capable of down-regulating the expression of an endogenous gene and a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the endogenous gene whose expression is down-regulated is selected from the group consisting of the TCR α-chain, TCR β-chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1 and FAS, and the CAR comprises an antigen-binding domain, a transmembrane domain and an intracellular domain of a costimulatory molecule. In one embodiment, the modified T cells further comprise an exogenous nucleic acid encoding a modified TCR having affinity for a surface antigen on a target cell as described elsewhere herein. The present invention also provides a population of cells comprising the modified T cells described herein.

[0141] One or more domains, or fragments of domains, of the CAR may be of human origin. In one embodiment, the present invention provides a fully human CAR. Nucleic acid sequences encoding the desired domains can be obtained using recombinant methods known in the art, such as screening a library from cells expressing the gene, deriving the gene from a vector known to contain it, or directly isolating it from cells and tissues containing it. Alternatively, the gene of interest can be made synthetically, rather than as a cloned molecule.

[0142] Examples of CARs are described in U.S. Patent Nos. 8,911,993, 8,906,682, 8,975,071, 8,916,381, 9,102,760, 9,101,584, and 9,102,761, all of which are hereby incorporated by reference in their entirety.

[0143] Antigen-binding domain In one aspect, the CAR comprises an antigen-binding domain that binds to an antigen on the target cell. Examples of cell surface markers that can act as antigens that bind to the antigen-binding domain of the CAR include those associated with viral infections, bacterial infections, and parasitic infections, autoimmune diseases, and cancer cells.

[0144] The selection of the antigen-binding domain depends on the type and number of antigens present on the surface of the target cell. For example, the antigen-binding domain may be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state.

[0145] In one aspect, the antigen-binding domain binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In one aspect, the tumor antigen of the present invention comprises one or more antigenic cancer epitopes.

[0146] The antigen-binding domain can include any domain that binds to an antigen, which can include, without limitation, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and fragments thereof. Thus, in one aspect, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof.

[0147] The antigen-binding domain may bind to one or more antigens including, but not limited to, the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation antigen (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); protease-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostate acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prososome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100);Cancer gene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl); tyrosine kinase; ephrin type-A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycosphingolipid (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR γ-selective reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostain; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (Melan-A or MART1);Rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein) like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); regucalcin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0148] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as that in which the CAR will ultimately be used. For example, for use in humans, it is considered beneficial for the antigen-binding domain of the CAR to comprise a human antibody, a humanized antibody as described elsewhere herein, or a fragment thereof.

[0149] Also, it is beneficial for the antigen-binding domain to be functionally linked for expression in a cell to another domain of the CAR, such as, for example, a transmembrane domain or an intracellular domain, each of which is also described elsewhere herein. In one embodiment, a nucleic acid encoding the antigen-binding domain is functionally linked to a nucleic acid encoding the transmembrane domain and a nucleic acid encoding the intracellular domain.

[0150] Transmembrane domain With respect to the transmembrane domain, the CAR can be designed to include a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain. In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such a domain to the transmembrane domain of the same or a different surface membrane protein in order to minimize interaction with other members of the receptor complex.

[0151] The transmembrane domain can be derived from either a natural or a synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions that are particularly useful in the present invention can be derived from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., including at least their transmembrane regions). In some cases, various human hinges, including the human Ig (immunoglobulin) hinge, can also be used.

[0152] In one embodiment, the transmembrane domain may be synthetic, in which case it is thought to mainly contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0153] Intracellular domain The intracellular domain of the CAR, or in other words the cytoplasmic domain, causes activation of the cell in which the CAR is expressed. The term "intracellular domain" thus means including any part of the intracellular domain sufficient to transmit an activation signal. In one embodiment, the intracellular domain includes a domain that causes an effector function. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell would be cytolytic activity or helper activity including cytokine secretion.

[0154] In one embodiment, the intracellular domain of the CAR includes a domain that causes signal activation and / or transduction. The intracellular domain can transmit signal activation through protein-protein interactions, biochemical changes, or other responses that change the metabolism, shape, gene expression of the cell, or other cellular responses to the activation of chimeric intracellular signaling molecules.

[0155] Examples of intracellular domains for use in the present invention include the cytoplasmic portions of the T cell receptor (TCR) and any costimulatory molecule that act cooperatively to induce signal transduction after antigen receptor engagement, as well as any derivatives or variants of these elements having the same functional ability and any synthetic sequences. In one embodiment, the intracellular domain of the CAR includes a dual signal transduction domain. The dual signal transduction domain can include a fragment or domain from any of the molecules described herein.

[0156] Examples of intracellular domains include ligands that specifically bind to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivatives, variants or fragments thereof having the same functional ability, any synthetic sequences of costimulatory molecules, and fragments or domains derived from one or more molecules or receptors including, without limitation, any combination thereof.

[0157] In one embodiment, the intracellular domain of the CAR comprises any portion of a costimulatory molecule, such as at least one signaling domain derived from CD3, CD27, CD28, ICOS, 4-1BB, PD-1, the T cell receptor (TCR), any derivative or variant thereof having the same functional ability, any synthetic sequence thereof, and any combination thereof.

[0158] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to either the antigen-binding domain or the intracellular domain in a polypeptide chain. In one embodiment, the spacer domain is composed of up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. In another embodiment, a short oligopeptide linker or polypeptide linker, preferably one that is 2-10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular domain of the CAR. An example of a linker includes the glycine-serine doublet.

[0159] Human antibody When using the antigen-binding domains of bispecific antibodies or CARs, it is considered preferable to use human antibodies or fragments thereof. For the therapeutic treatment of human subjects, fully human antibodies are particularly desirable. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, in combination with modified methods of these techniques. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, each of which is incorporated herein by reference in its entirety. Bispecific antibodies can also include antibodies in which the heavy and light chains are encoded by nucleotide sequences derived from one or more sources of human DNA.

[0160] Alternatively, human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to the human heavy and light chain genes, human variable, constant, and diversity regions can also be introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of the human immunoglobulin locus by homologous recombination. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. These modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Subsequently, the chimeric mice are mated to produce homozygous progeny that express human antibodies. The transgenic mice are immunized in the normal manner with a selected antigen, for example, the whole or a part of the polypeptide of the present invention. Directional antibodies against the selected target can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutations. Thus, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies, including but not limited to IgG1 (γ1) and IgG3, using such techniques. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (Int. Rev. Immunol, 13:65-93 (1995)).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, for example, PCT Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is hereby incorporated by reference in its entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can enter into contracts to obtain human antibodies directed against selected antigens using techniques similar to those described above. For a specific discussion of the transfer of the human germline immunoglobulin gene array into germline mutant mice, which is thought to result in the production of human antibodies by antigen loading stimulation, see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol, 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0161] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire of non-immunized donors. According to this method, the antibody V domain gene is cloned in-frame into the gene of the major coat protein or minor coat protein of filamentous bacteriophage, such as M13 or fd, and presented as a functional antibody fragment on the surface of phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody that exhibits those properties. For this reason, phage mimics some of the properties of B cells. Phage display can be performed in various formats; for an overview, see Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice.Construct a repertoire of V genes from non-immunized human donors and isolate antibodies against diverse arrays of antigens (including autoantigens) essentially according to the methods described in Marks et al., J. Mol. Biol, 222:581-597 (1991), or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905, which are hereby incorporated by reference in their entirety.

[0162] Human antibodies can also be made by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, which are hereby incorporated by reference in their entirety). Human antibodies can also be made in vitro using hybridoma techniques such as those described by Roder et al. (Methods Enzymol, 121:140-167 (1986)), among others.

[0163] Humanized antibody Alternatively, in some embodiments, non-human antibodies can be humanized, in which case specific sequences or regions of the antibody are modified to enhance similarity to antibodies naturally produced in humans. For example, in the present invention, an antibody or a fragment thereof can include a non-human mammalian scFv. In one embodiment, the antigen-binding domain portion is humanized.

[0164] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to: CDR grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, Proc Natl Acad Sci USA 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. Patent No. 5,565,332.This is incorporated herein by reference in its entirety), as well as the methods disclosed in, for example, US Patent Application Publication No. US2005 / 0042664, US Patent Application Publication No. US2005 / 0048617, US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol, 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10(1994), and Pedersen et al., J. Mol. Biol, 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. In many cases, framework residues in the framework region are thought to be replaced by the corresponding residues from the CDR donor antibody, preferably to improve antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see, for example, Queen et al., US Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323. Each of these is incorporated herein by reference in its entirety).

[0165] Humanized antibodies have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically taken from the "import" variable domain. Thus, a humanized antibody comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Humanization of antibodies is well known in the art and can be essentially carried out according to the method of Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), that is, by substituting the corresponding sequences of human antibodies with rodent CDRs or CDR sequences, i.e., CDR grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640. These contents are hereby incorporated by reference in their entirety). In such humanized chimeric antibodies, the human variable domain that is not substantially intact is replaced by the corresponding sequences from non-human species. In practice, a humanized antibody is typically a human antibody in which some CDR residues and possibly some framework (FR) residues are replaced by residues from similar sites in rodent antibodies. Also, humanization of antibodies can be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), and these contents are hereby incorporated by reference in their entirety.

[0166] The selection of human variable domains of both the light and heavy chains, which is used in the production of humanized antibodies, aims to reduce antigenicity. According to the so-called "best fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable domain sequences. Subsequently, the human sequence closest to that of the rodent is accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol, 151:2296 (1993); Chothia et al., J. Mol. Biol, 196:901 (1987), the contents of which are hereby incorporated by reference in their entirety). In another method, a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of the light or heavy chain is used. The same framework can be used for several different humanized antibodies ((Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are hereby incorporated by reference in their entirety).

[0167] Antibodies can be humanized while maintaining high affinity for the target antigen and other advantageous biological properties. According to one aspect of the present invention, humanized antibodies are prepared by a process of analyzing the parental and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformations for a selected candidate immunoglobulin sequence. Examination of these displays allows for analysis of the putative role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences such that desired antibody properties, such as increased affinity for the target antigen, are achieved. Generally, CDR residues are most directly and substantially involved in affecting antigen binding.

[0168] Humanized antibodies retain antigen specificity similar to the original antibody. However, using certain humanization methods, the binding affinity and / or specificity of the antibody for the target antigen can be enhanced using methods of "directed evolution" as described by Wu et al., J. Mol. Biol, 294:151 (1999), the entire content of which is incorporated herein by reference.

[0169] Other molecules The present invention also includes the modified T cells described herein that further comprise a costimulatory molecule or a nucleic acid encoding a costimulatory molecule. In one aspect, the modified T cells of the present invention further comprise an exogenous nucleic acid encoding a costimulatory molecule such that the modified T cells express the costimulatory molecule. The nucleic acid can be introduced into the T cells by transducing the T cells, transfecting the T cells, or electroporating the T cells. In another aspect, the costimulatory molecule is selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In another aspect, the costimulatory molecule comprises CD3 and comprises at least two different CD3 chains, such as the CD3ζ chain and the CD3ε chain.

[0170] In another aspect, the modified T cells further comprise Klf4, Oct3 / 4 and / or Sox2, or a nucleic acid encoding Klf4, Oct3 / 4 and / or Sox2, for inducing the pluripotency of T cells. T cells can be induced to pluripotency by expressing Klf4, Oct3 / 4 and Sox2. Klf4, Oct3 / 4 and Sox2 can be expressed from a nucleic acid, a viral vector or an RNA molecule. In one aspect, a viral vector encoding Klf4, Oct3 / 4 and Sox2 is introduced into T cells to induce pluripotency. In another aspect, a Sendai viral vector is introduced into T cells to induce pluripotency, where the Sendai viral vector encodes Klf4, Oct3 / 4 and Sox2.

[0171] Introduction of nucleic acid Methods for introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides, such as RNA, into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. RNA can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.)), Multiporator (Eppendort, Hamburg Germany). RNA can also be introduced into cells using cationic liposome-mediated transfection using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using a microparticle gun particle delivery system such as a "gene gun" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0172] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example, human cells. Other viral vectors can be derived from, for example, lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0173] Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

[0174] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. "Liposome" is a general term encompassing various unilamellar and multilamellar lipid media formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization prior to the formation of the closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from normal vesicular structures in solution are also included. For example, the lipids can assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0175] Regardless of the method used to introduce exogenous nucleic acids into host cells or to expose cells to the inhibitors of the present invention by other means, various assay methods can be performed to confirm the presence of nucleic acids in host cells. Such assay methods include, for example, "molecular biology" assay methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assay methods such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blot) or by the assay methods described herein for identifying agents falling within the scope of the present invention.

[0176] In one embodiment, a nucleic acid encoding a T cell receptor (TCR) that includes an affinity for a surface antigen on a target cell is introduced into expanded T cells. The nucleic acid encoding the TCR may be the same or a separate nucleic acid from the nucleic acid that can down-regulate endogenous TCR gene expression. The nucleic acid encoding the TCR can be introduced into T cells simultaneously or sequentially with the nucleic acid that can down-regulate endogenous TCR gene expression. In one embodiment, the nucleic acid encoding the TCR is introduced prior to the nucleic acid that can down-regulate endogenous TCR gene expression.

[0177] Furthermore, the nucleic acid can be introduced by any means such as transduction of expanded T cells, transfection of expanded T cells, and electroporation of expanded T cells. One nucleic acid can be introduced by one method and another nucleic acid to be introduced into T cells can be introduced by a different method.

[0178] RNA In one embodiment, the nucleic acid introduced into the T cell is RNA. In another embodiment, the RNA is mRNA including in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences or any other suitable DNA source. The desired template for in vitro transcription is a chimeric membrane protein. By way of example, the template encodes an antibody, a fragment of an antibody or a portion of an antibody. As another example, the template includes an extracellular domain containing a single-chain variable domain of an antibody, such as anti-CD3, and an intracellular domain of a co-stimulatory molecule. In one embodiment, the template of the RNA chimeric membrane protein encodes a chimeric membrane protein including an extracellular domain containing an antigen-binding domain derived from an antibody against a co-stimulatory molecule and an intracellular domain derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0179] A template for in vitro mRNA transcription can be prepared using PCR, which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA that is used as the template for PCR. As used herein, "substantially complementary" refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize to the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions (reading frames) of genes that are normally transcribed in cells, including 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of human cDNA that includes all or a portion of the 5' and 3' UTRs. Primers useful for PCR are prepared by synthetic methods well known in the art. A "forward primer" is a primer that includes a region of nucleotides that is substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence to be amplified. As used herein, "upstream" refers to the 5'-side position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that includes a region of nucleotides that is substantially complementary to the double-stranded DNA template that is downstream of the DNA sequence to be amplified. As used herein, "downstream" refers to the 3'-side position of the DNA sequence to be amplified relative to the coding strand.

[0180] Chemical structures having the ability to promote RNA stability and / or translation efficiency may be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is from 0 to 3000 nucleotides in length. The lengths of the 5' and 3' UTR sequences added to the coding region can be varied by different methods including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can vary the lengths of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0181] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for altering the stability and / or translation efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to be able to decrease mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0182] In one aspect, the 5' UTR can include the Kozak sequence of an endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. The Kozak sequence can enhance the translation efficiency of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for the Kozak sequence for many mRNAs is known in the art. In other aspects, the 5' UTR can be derived from an RNA genome that is an intracellular stable RNA virus. In other aspects, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0183] To enable the synthesis of RNA from a DNA template without the need for gene cloning, the promoter for transcription should be added upstream of the sequence to be transcribed with respect to the DNA template. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the transcribed reading frame. In one aspect, the promoter is a T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0184] In one aspect, the mRNA has both a 5' end cap and a 3' poly(A) tail that determine ribosome binding, initiation of mRNA translation in the cell, and stability. For circular DNA templates, such as plasmid DNA, RNA polymerase produces long, linear products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in a normal-sized mRNA that is not effective in eukaryotic transfection even if polyadenylated post-transcriptionally.

[0185] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0186] The conventional method for incorporating a poly A / T stretch into a DNA template is molecular cloning. However, poly A / T sequences incorporated into plasmid DNA can cause plasmid instability because plasmid DNA templates obtained from bacterial cells are often highly damaged by deletions and other abnormalities. This makes the cloning procedure not only cumbersome and time-consuming but also often unreliable. This is why a method that enables the construction of DNA templates with a poly A / T 3' stretch without cloning is highly desirable.

[0187] The poly A / T segment of the transcribed DNA template can be produced during PCR by using a reverse primer containing a poly T tail, such as a 100T tail (the size can be 50 - 5000 T), or can be produced after PCR by any other method including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also confers stability to RNA and reduces RNA degradation. Generally, the length of the poly(A) tail has a positive correlation with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 - 5000 adenosines.

[0188] The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as E. coli poly A polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 - 400 nucleotides increases the translation efficiency of RNA by about two-fold. Further, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase the stability of RNA.

[0189] The 5' cap also confers stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein contains a 5' cap. 5' caps are known in the art and are provided using the techniques described herein (Cougot, et al., Trends in Biochem. Sci., 29:436 - 444 (2001); Stepinski, et al., RNA, 7:1468 - 95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958 - 966 (2005)).

[0190] RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to the mRNA and facilitates the start of translation. Any solute suitable for cell electroporation can be included, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0191] RNA transfection In some embodiments, RNA encoding a TCR is electroporated into cells. In one embodiment, the RNA encoding a TCR is RNA transcribed in vitro.

[0192] The disclosed methods can be applied to the regulation of T cell activity in basic research and therapy in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the evaluation of the ability of genetically engineered T cells to kill target cancer cells.

[0193] The method also provides the ability to control expression levels over a wide range, for example, by varying the promoter or the amount of input RNA, and allows for the individual regulation of expression levels. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0194] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and vector-free. The RNA-introduced gene can be delivered to lymphocytes as a minimal expression cassette without the need for any additional viral sequences and expressed therein after simple in vitro cell activation. Under these conditions, integration of the transgene into the host cell genome is unlikely to occur. Due to the efficiency of RNA transfection and the ability to uniformly modify the entire lymphocyte population, cell cloning is not necessary.

[0195] For the genetic modification of T cells using in vitro transcribed RNA (IVT-RNA), two different strategies are utilized, both of which have been sequentially tested in various animal models. The cells are transfected with in vitro transcribed RNA by lipofection or electroporation. To achieve long-term expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0196] Several IVT vectors are known in the literature and are utilized in a standardized manner as templates for in vitro transcription and are genetically engineered to produce stabilized RNA transcripts. Currently, the protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter that enables RNA transcription, followed by a gene of interest flanked on either the 3' and / or 5' side by an untranslated region (UTR), and a 3' polyadenylation cassette containing 50 - 70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and either extend or mask the poly(A) sequence at the 3' end. Whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs is not clear.

[0197] RNA has several advantages compared to more traditional plasmid or viral approaches. Gene expression from an RNA source does not require transcription, and protein products are produced rapidly after transfection. Furthermore, since RNA only needs to access the cytoplasm and not the nucleus, very high transfection rates can be obtained with typical transfection methods. Additionally, in plasmid-based approaches, the promoter driving the expression of the gene of interest needs to be active in the cells under study.

[0198] In another aspect, the RNA construct is delivered to cells by electroporation. For example, reference is made to the formulations and methodologies for electroporation of nucleic acid constructs into mammalian cells as taught in U.S. Patent No. 2004 / 0014645, U.S. Patent No. 2005 / 0052630A1, U.S. Patent No. 2005 / 0070841A1, U.S. Patent No. 2004 / 0059285A1, and U.S. Patent No. 2004 / 0092907A1. A variety of parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant research literature as well as in numerous patents and applications in the art. See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264, and U.S. Patent No. 7,173,116. Devices for the therapeutic application of electroporation are commercially available, such as, for example, the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Patent No. 6,567,694; U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701, and U.S. Patent No. 6,233,482; electroporation can also be used for transfection of cells in vitro, as described, for example, in U.S. Patent No. 20070128708A1. Electroporation can also be utilized to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs that utilize any of a number of available devices and electroporation systems known to those of skill in the art, represents an excellent new means for delivering the RNA of interest to target cells.

[0199] In one aspect, the method includes the step of electroporating RNA encoding the TCR α and β chains. The TCR α and β chains can be encoded on the same or separate RNAs. If α and β are encoded by separate RNAs, the RNAs can be co-electroporated.

[0200] In another aspect, the method can further include the step of electroporating a nucleic acid encoding a co-stimulatory molecule. The co-stimulatory molecule nucleic acid can be co-electroporated with the TCR RNA.

[0201] Source of T cells Prior to expansion, a source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is human. T cells can be obtained from several sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain aspects, any number of T cell lines available in the art can be used. In certain aspects, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as Ficoll separation. In one aspect, cells from an individual's circulating blood are obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis are washed to remove the plasma fraction and placed in a washing solution that may lack calcium and / or magnesium or may lack many, but not all, divalent cations, such as phosphate buffered saline (PBS), for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers, such as calcium-free, magnesium-free PBS. Alternatively, unwanted components of the apheresis sample are removed and the cells can be resuspended directly in media.

[0202] In another aspect, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In any case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0203] The thus isolated cord blood mononuclear cells can deplete cells expressing specific antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be achieved using isolated antibodies, biological samples containing antibodies such as ascites, antibodies conjugated to a physical support, and cell-bound antibodies.

[0204] Enrichment of the T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers specific to the negatively selected cells. A preferred method is negative magnetic immunoadhesion or cell sorting and / or selection by flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0205] For the isolation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the beads and cells. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are used. In a further embodiment, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 75, 80, 85, 90, 95, or 100 million cells / ml are used. In a further embodiment, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0206] T cells do not require a monocyte removal step and can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and to some extent monocytes from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this context, but in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen at a rate of 1°C per minute to -80°C and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as instantaneous uncontrolled freezing at -20°C or in liquid nitrogen, can be used.

[0207] In one embodiment, the population of T cells is contained within cells such as peripheral blood mononuclear cells, cord blood cells, purified T cell populations, and T cell lines. In another embodiment, the peripheral blood mononuclear cells contain a population of T cells. In yet another embodiment, the purified T cells contain a population of T cells.

[0208] Chimeric membrane protein Generally, T cells expand by contact with a surface to which an agent that stimulates CD3 / TCR complex-related signals and a ligand that stimulates costimulatory molecules on the surface of the T cells are attached. The present invention provides a novel method for expanding a population of T cells, comprising the steps of electroporating T cells with RNA encoding a chimeric membrane protein, and culturing the electroporated T cells, wherein the electroporated T cells in the population expand at least 10-fold. The chimeric membrane protein of the present invention comprises an extracellular domain and an intracellular domain. The extracellular domain contains a target-specific binding element, such as an antibody. In one embodiment, the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3, and an intracellular domain derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0209] Expression of the chimeric membrane protein enables interaction with other cells in the population, such as cells expressing CD3, and as a result, expansion and activation of the electroporated T cells are stimulated. Without wishing to be bound by any particular theory, it is contemplated that cells expressing CD3 contact and bind to the chimeric membrane protein expressed on the surface of the electroporated T cells. At least one T cell expressing the chimeric membrane protein interacts with another cell expressing CD3. This interaction stimulates expansion of the electroporated T cells.

[0210] In one embodiment, the T cells are expanded prior to down-regulation of the endogenous gene. In another embodiment, modified T cells are expanded.

[0211] Extracellular domain The present invention includes an extracellular domain comprising an antigen-binding domain derived from an antibody directed against a costimulatory molecule. The costimulatory molecule includes any molecule that costimulates T cells, such as, but not limited to, CD3, CD28, or combinations thereof. In one embodiment, the extracellular domain may include an antigen-binding domain derived from anti-CD3, anti-CD28, or combinations thereof. In another embodiment, the extracellular domain includes a single-chain variable fragment (scFv) against CD3.

[0212] In another embodiment, the extracellular domain may include any portion of an antibody that binds to an antigen, including, but not limited to, synthetic antibodies, human antibodies, humanized antibodies, single-domain antibodies, antigen-binding domains of single-chain variable fragments, and fragments thereof. Optionally, it may be beneficial for the extracellular domain to be derived from the same species as that in which the chimeric membrane protein will ultimately be used. For example, for use in humans, it is considered beneficial for the extracellular domain of the chimeric membrane protein to include a human antibody or fragment thereof. Thus, in one embodiment, the extracellular domain portion includes a human antibody or fragment thereof as described elsewhere herein. Alternatively, in some embodiments, the extracellular domain portion includes a humanized non-human antibody as described elsewhere herein.

[0213] Intracellular domain The intracellular domain or cytoplasmic domain includes a costimulatory signaling region. The costimulatory signaling region refers to the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to an antigen.

[0214] The cytoplasmic domain or intracellular signaling domain of the chimeric membrane protein is responsible for the activation of at least one of the effector functions of T cells. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. To the extent that a shortened portion of the intracellular signaling domain is used, such a shortened portion can be used in place of the intact chain as long as it transmits the effector function signal. The intracellular signaling domain, therefore, includes any shortened portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0215] Non-limiting examples of intracellular signaling domains for use in chimeric membrane proteins include CD28, 4-1BB, the T cell receptor (TCR), any portion of the intracellular domain of a costimulatory molecule, derivatives or variants of these sequences having the same functional capacity, any synthetic sequence, and any combination thereof. In one embodiment, the intracellular domain includes a portion of the intracellular domains of CD28 and 4-1BB.

[0216] Other domains of chimeric membrane protein A spacer domain, such as an oligopeptide or polypeptide, that serves to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain in the polypeptide chain may be incorporated between the extracellular domain and the transmembrane domain of the chimeric membrane protein, or between the cytoplasmic domain and the transmembrane domain of the chimeric membrane protein. The spacer domain is composed of up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0217] In some embodiments, the chimeric membrane protein further includes a transmembrane domain. In some embodiments, the chimeric membrane protein further includes a hinge domain. In one embodiment, the RNA encoding the chimeric membrane protein further includes a transmembrane domain and a hinge domain, such as a CD28 transmembrane domain and a CD8-α hinge domain.

[0218] Expansion of T cells As demonstrated by the data disclosed herein, by the step of expanding T cells by the method disclosed herein, it can be increased by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and any and all whole and partial integer multiples therebetween. In one embodiment, the T cells are expanded in the range of about 20-fold to about 50-fold.

[0219] After culturing, the T cells are incubated in a cell culture medium in a culture device for a period of time or until the cells reach a confluent or high cell density for optimal passage, and then passaged to another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. Preferably, the confluent level before passage of the cells to another culture device is 70% or more. More preferably, the confluent level is 90% or more. The period may be any time suitable for culturing cells in vitro. The replacement of the T cell culture medium may be performed at any point during the culture of the T cells. Preferably, the T cell culture medium is replaced every about 2 to 3 days. Subsequently, the T cells can be harvested from the culture device and used immediately thereon or cryopreserved for storage for later use. In one embodiment, the present invention includes the step of cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing nucleic acid into the T cells.

[0220] In another aspect, the method includes the steps of isolating T cells and expanding the T cells. In another aspect, the invention further includes the step of cryopreserving the T cells prior to expansion. In yet another aspect, the cryopreserved T cells are thawed for electroporation with RNA encoding a chimeric membrane protein.

[0221] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (which is incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 may be an alternative or may be in addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells involves the addition of cell growth factors such as those described in U.S. Patent No. 5,199,942, or the addition of other factors such as flt3-L, IL-1, IL-3, and c-kit ligand. In one aspect, the step of expanding the T cells includes culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0222] The culture step described herein (after contact or electroporation with the agents described herein) may be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culture step further described herein (contact with the agents described herein) may be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

[0223] Various terms are used to describe cultured cells. Cell culture generally refers to cells that are taken from an organism and grown under controlled conditions. Primary cell culture is the culture of cells, tissues, or organs that are taken directly from an organism and before the first subculture. Cells are expanded in culture to yield a larger cell population when placed in a growth medium under conditions that promote cell growth and / or division. When cells are expanded in culture, the rate of cell growth is typically measured by the amount of time it takes for the cells to double, which is also known as the doubling time.

[0224] Each round of subculture is referred to as a passage. When cells are subcultured, they are said to have been passaged. A particular cell population, or cell line, may also be referred to or characterized by the number of passages it has undergone. For example, a cultured cell population that has been passaged 10 times may be referred to as a P10 culture. The primary culture, i.e., the first culture after cell isolation from the tissue, is designated as P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there can be many population doublings during a passage; therefore, the number of population doublings in a culture is greater than the number of passages. The expansion of cells (i.e., the number of population doublings) during the period between passages depends on many factors including, but not limited to, seeding density, substrate, medium, and the time between passages.

[0225] In one embodiment, the cells can be cultured for several hours (about 3 hours) to about 14 days, or any integer value of time units therebetween. Conditions suitable for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or, X-vivo 15, (Lonza)) containing factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additive known to those skilled in the art for cell proliferation. Other additives for cell proliferation include, but are not limited to, surfactants, plasma expanders, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can be RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer supplemented with amino acids, sodium pyruvate, and vitamins, and can be serum-free or supplemented with an appropriate amount of serum (or plasma) or defined hormones, and / or cytokines in an amount sufficient for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be injected into a subject. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., 5% CO 2 ) in addition to air).

[0226] The medium used for culturing T cells may contain agents capable of co-stimulating T cells. For example, an agent capable of stimulating CD3 is an antibody against CD3, and an agent capable of stimulating CD28 is an antibody against CD28. This is because, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can be expanded by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one embodiment, T cells are expanded within a range of about 20-fold to about 50-fold, or more, by culturing an electroporated population.

[0227] In one embodiment, the method includes introducing a nucleic acid encoding a T cell receptor (TCR) having affinity for a surface antigen on a target cell into the expanded T cells, and electroporating RNA encoding a co-stimulatory molecule into the T cells, wherein the electroporated T cells can express the TCR and the co-stimulatory molecule.

[0228] In another embodiment, the method further includes stimulating the expanded T cells with at least one co-stimulatory molecule selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L. Stimulation may include co-electroporation with RNA encoding the co-stimulatory molecule. In such an embodiment, the expanded T cells are further electroporated or co-electroporated with RNA encoding CD3. CD3 includes at least two different CD3 chains, such as the CD3ζ chain and the CD3ε chain.

[0229] In another aspect, the method of expanding T cells can further include the step of isolating the expanded T cells for further application. In yet another aspect, the method of expanding can further include subsequent electroporation of the expanded T cells prior to the culturing step. The subsequent electroporation can include introducing a nucleic acid encoding an agent into the expanded T cell population, such as transducing the expanded T cells with a nucleic acid encoding a TCR, transfecting the expanded T cells, or performing electroporation on the expanded T cells, where the agent further stimulates the T cells. The agent can stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function. In one aspect, the agent nucleic acid is co-electroporated with chimeric membrane protein RNA. In another aspect, the agent nucleic acid, such as TCR RNA, is electroporated after culturing the electroporated population. In a further aspect, the agent nucleic acid, such as TCR RNA, is electroporated into the expanded T cells that had been cryopreserved.

[0230] Treatment The modified T cells described herein can be included in a composition for treatment. The composition includes a pharmaceutical composition and can further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells can be administered.

[0231] In one aspect, the invention includes a method for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising administering to the subject an effective amount of modified T cells. In this aspect, the T cells are modified as described elsewhere herein. The modified T cells can be administered to induce lysis of the target cell or tissue, such as when the induced lysis is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0232] In another aspect, the present invention includes the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells described herein to prevent or treat an immune reaction harmful to the subject, the method for adoptive cell transfer therapy.

[0233] In yet another aspect, a method of treating a disease or condition associated with enhanced immunity in a subject includes the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells described herein.

[0234] The modified T cells produced as described herein can be homogeneous and retain T cell function. Further, the modified T cells can be administered to an animal, preferably a mammal, more preferably a human, to suppress immune responses such as those common to autoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhanced induction of allograft tolerance, graft rejection, etc. Further, the cells of the present invention can be used to treat any condition where a diminished or otherwise inhibited immune response, particularly a cell-mediated immune response, is desirable to treat or alleviate a disease. In one aspect, the present invention includes treating a condition such as an autoimmune disease in a subject, the method including the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cells described herein.

[0235] Examples of autoimmune diseases include acquired immunodeficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac disease - dermatitis herpetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, Graves' disease, Guillain - Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin - dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Ménière's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), which is also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff - man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo as well as Wegener's granulomatosis, but are not limited thereto.

[0236] The T cells produced as described herein can also be modified and used to treat inflammatory disorders. Examples of inflammatory disorders include, but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft - versus - host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy as well as ventilator - induced lung injury.

[0237] In another aspect, the modified T cells described herein can be used in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

[0238] The cells of the present invention can be administered at a dosage, route, and time determined in appropriate preclinical and clinical experiments and tests. The cell composition can be administered multiple times at dosages within these ranges. Administration of the cells of the present invention may be combined with other methods useful for treating a desired disease or condition determined by those skilled in the art.

[0239] The cells of the present invention administered can be autologous, allogeneic or xenogeneic with respect to the subject being treated.

[0240] Administration of the cells of the present invention can be performed in any convenient manner known to those skilled in the art. The cells of the present invention can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other examples, the cells of the present invention are directly injected into an inflammatory site in a subject, a local disease site in a subject, a lymph node, an organ, a tumor, etc.

[0241] The cells described herein can also be administered using a number of matrices. In the present invention, such matrices are utilized in the new context of acting as artificial lymphoid organs, typically to support, maintain or regulate the immune system by the regulation of T cells. Thus, the present invention can utilize matrix compositions and formulations that have been demonstrated to be useful in tissue engineering. Thus, the types of matrices that can be used in the compositions, devices and methods of the present invention are virtually infinite and can include both biological and synthetic matrices. In one particular example, the compositions and devices described by U.S. Patent Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized, and accordingly these patents are hereby incorporated by reference in their entirety. Matrices generally relate to features that are biocompatible when administered to a mammalian host. The matrix may be formed from natural materials and / or synthetic materials. The matrix may be non-biodegradable, if it is desirable to leave a permanent or removable structure in the body of the animal, such as an implant; or it may be biodegradable. The matrix can take the form of a sponge, implant, tube, telfa pad, fiber, hollow fiber, lyophilized component, gel, powder, porous composition, or nanoparticles. Further, the matrix can be designed to allow for the sustained release of seeded cells or produced cytokines or other active agents. In certain embodiments, the matrix of the present invention is flexible and stretchable and can be described as a semi-solid scaffold that permits the passage of substances such as inorganic salts, aqueous liquids and dissolved gaseous agents including oxygen.

[0242] In this specification, a matrix is used as an example of a biocompatible substance. However, since the present invention is not limited to a matrix, when the term matrix appears, all of these terms allow retention or traversal of cells, are biocompatible, and allow polymers to directly traverse through the substance, so the substance itself is a semipermeable membrane or can be used in combination with a specific semipermeable substance, and should be read to include devices and other substances.

[0243] Pharmaceutical composition The pharmaceutical composition of the present invention may comprise the modified T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0244] The pharmaceutical composition of the present invention may be administered in a manner appropriate for the disease to be treated (or prevented). Although the appropriate dosage can be determined by clinical trials, the amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease.

[0245] When an "immunologically effective amount", "amount effective against an immune response", "effective amount to inhibit an immune response" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the age, weight, immune response, and condition of the patient (subject). The pharmaceutical composition comprising the modified T cells described herein is 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6It can generally be referred to as administrable at a dosage of cells per kg body weight, including all integer values within those ranges. The T cell composition can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0246] In certain embodiments, it may be desirable to administer activated T cells to a subject, then re - draw blood (or perform apheresis), activate the T cells therefrom according to the present invention, and reinject these activated and expanded T cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, the T cells can be activated from a blood draw of 10 ml to 400 ml. In certain embodiments, the T cells are activated from a blood draw of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml or 100 ml. Without being bound by theory, a particular population of T cells can be selected using this multiple blood draw / multiple reinfusion protocol.

[0247] In certain embodiments of the invention, cells expanded and modified using the methods described herein, or other methods known in the art by which T cells are expanded to therapeutic levels, are administered to a patient (e.g., before, at the same time as, or after) in conjunction with many related treatment regimens including, but not limited to, antiviral therapies, agents such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies, or other immunodepleting agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), or inhibit the p70S6 kinase important for signal transduction induced by growth factors (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In further embodiments, the cell compositions of the invention are administered to a patient (e.g., before, at the same time as, or after) in conjunction with bone marrow transplantation, chemotherapeutic agents such as fludarabine for T cell depletion therapy, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the invention are administered after B cell depletion therapy with an agent that reacts with CD20, such as rituxan. For example, in one embodiment, a subject may undergo peripheral blood stem cell transplantation after standard treatment with high-dose chemotherapy.In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In further embodiments, the expanded cells are administered before or after surgery.

[0248] The dosage of the treatment administered to a patient will vary depending on the condition being treated and the exact nature of the recipient of the treatment. The scaling of dosages for human administration can be done according to practices recognized in the art. The dosage of CAMPATH, for example, generally ranges from 1 to about 100 mg for adult patients and is usually administered daily for 1 to 30 days. A preferred daily dosage is 1 to 10 mg / day, although in some cases higher dosages up to 40 mg / day may be used (as described in U.S. Patent No. 6,120,766).

[0249] It should be understood that the methods and compositions useful in the present invention are not limited to the specific formulations described in the examples. The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0250] In the practice of the present invention, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are utilized, which are well within the purview of those skilled in the art. Such techniques are well described in the literature, such as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and are thus contemplated in the making and practice of the present invention. Techniques particularly useful for certain embodiments are discussed in the following sections.

Examples

[0251] Experimental Examples The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting, unless otherwise specified. Accordingly, the present invention should not be construed as limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

[0252] Without further elaboration, it is believed that one of ordinary skill in the art can make and use the compounds of the present invention and practice the methods described in the claims using the above description and the following examples. Therefore, the following examples are illustrative of the preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0253] The materials and methods used in these experiments are described below.

[0254] Primary human lymphocytes: Primary lymphocytes were stimulated with microbeads coated with CD3 and CD28 stimulating antibodies (Life Technologies, Grand Island, NY, catalog) as described (Human gene therapy 2011, 22(12):1575-1586). On day 10, T cells were frozen at 1×10 8 cells / vial in a solution of 90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO).

[0255] NALM-6 was purchased from the German DSMZ Cell Collection (DSMZ catalog code: ACC 128). K562 and PC3 were purchased from the American Type Culture Collection. The 624mel melanoma cell line was purchased from the Surgery Branch (NCI / NIH). All cell lines were cultured as directed and regularly tested for mycoplasma contamination to confirm negativity.

[0256] Generation of TCR constructs for mRNA electroporation and lentiviral transduction: Based on sequencing information obtained from relevant publications, synthesize and / or amplify a 1G4 NY-ESO-1 TCR having various mutations (1G4 and 8F) and CAR (PSCA or CD19) by PCR (The Journal of experimental medicine 2005, 201(8):1243-1255; J Immunol 2008, 180(9):6116-6131), and subclone it into a pGEM.64A RNA-based vector or a pTRPE lentiviral vector.

[0257] Preparation of human primary T cells: Primary human CD4 T cells and CD8 T cells were isolated from healthy volunteer donors by leukapheresis followed by negative selection using the RosetteSep kit (Stem Cell Technologies, Vancouver BC, Canada). All specimens were collected in accordance with a protocol approved by the University Institutional Review Board, and written informed consent was obtained from each donor.

[0258] Design and construction of CRISPR: Cas9 DNA was synthesized by PCR and subsequently inserted into the PGEM vector. gRNAs were selected using the NGG PAM site by GN19, and some were selected using the NGG PAM site from N20. All gRNAs contained complementary sequences composed of mismatches exceeding 13 base pairs, and potential sites for off-target mRNA sites were excluded (Table 1). gRNAs were designed as shown in Figure 1A and synthesized by overlap PCR. All gRNA PCR products were ligated into the MSGV vector. In vitro transcribed CAS9 and gRNAs targeted the constant regions of TCR α, β chains, and β-2 microglobulin. gRNAs were designed to target either the sequence within exon 1 of the TCR α constant region, the consensus sequence common to exon 1 of both TCR β constant regions 1 and 2, β-2 microglobulin, or PD1. The sequences encoding gRNAs were assembled using overlap PCR and cloned into the MSGV vector containing the T7 promoter. These plasmids were linearized with EcoRI. gRNAs were transcribed in vitro. Cas9 mRNA was transcribed in vitro using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, Carlsbad, CA). The mRNA was stored at -80 °C in nuclease-free vials for single use. The gRNA targeting sequences used for animal studies were as follows: TIFF2025089344000002.tif44165

[0259] Flow cytometry: The following monoclonal antibodies and reagents with specific labeling were used together with appropriate isotype controls. From BD Biosciences (San Jose, CA): APC-conjugated anti-CD3 (555335), FITC-anti-CD8 (555366), PE-anti-CD8 (555635), FITC-anti-CD27 (555440), PE-anti-CD107 (555801), PE-anti-β2-microglobulin (551337), FITC-anti-HLA (555552); from Biolegend (San Diego, CA): FITC-anti-CD45RO (304204), APC-anti-CD62L (304814), APC-anti-CCR7 (353214); and from Beckman Coulter (Pasadena, CA): PE-anti-Vb13.1 (IM2021U). Data were acquired using CellQuest version 3.3 (BD Biosciences, San Jose, CA) on a FACS Accuri (BD Biosciences, San Jose, CA) and analyzed using FCS Express version 3.00 (De Novo Software, Los Angeles, CA) or FlowJo version 7.6.1 (Tree Star, Inc., Ashland, OR).

[0260] Proliferation of primary T cells: Primary human T cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, and 10 mM Hepes and stimulated at a cell:bead ratio of 1:3 with magnetic beads coated with anti-CD3 / anti-CD28. Cells were counted and nutrients were added every 2 days, and when the T cells were thought to have entered a resting state, as judged by both a decline in the growth kinetics and cell size, the T cells were used for functional assays or cryopreserved.

[0261] CD3 neg Generation of T cells: The DNA supercoiled plasmid was linearized by SpeI and EcoRI respectively. gRNA was in vitro transcribed by the T7 mScript™ Standard mRNA Production System (Cambio, C-MSC100625, Cambridge, England). All mRNAs (Cas9, TCR α, TCR β and CAR) were in vitro transcribed using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, AM1345, Carlsbad, CA). Prior to electroporation, T cells were stimulated by CD3 / CD28 Dynabeads for 3 days. 10 million primary T cells were bead-removed, and then 20 μg of Cas9 and 10 μg of gRNA species were electroporated into the cells by BTX830 at 360 V, 1 ms parameters, and then the second and / or third electroporation of 10 μg of gRNA was performed. Furthermore, the T cells were washed with 3-fold OPTI-MEM and resuspended in OPTI-MEM (Invitrogen) at a final concentration of 1 - 3×10 8 cells / ml. Then, 0.1 ml of the cells were mixed with 10 μg of IVT RNA (or as specified) in a 2 mm cuvette and electroporation was performed. 10 million primary T cells were bead-removed, and then 20 μg of Cas9 and 10 μg of gRNA species were electroporated into the cells using BTX830 (Harvard Apparatus BTX) at 360 V and 1 ms; following this process, 12 - 24 hours later, the second and third electroporation of 5 μg of gRNA was performed.

[0262] After electroporation, the cells were immediately placed into 2 mL of pre-warmed medium and cultured at 37°C, 5% CO 2 or cultured at 32°C, 5% CO 2 for 1 day and then returned to 37°C, 5% CO 2 .

[0263] TCR α and β double disruption, or TRAC, TRBC, and B2M triple disruption: To generate TCR α and β double knockout T cells, co - electroporation of Cas9 mRNA and two different gRNAs targeting the TCR α chain (TRAC) and TCR β chain (TRBC) was performed. TCR α and β double knockout T cells could be purified in two steps: 1) removal of TCR - positive cells and α - chain single knockout cells by anti - CD3 microbeads after electroporation of 1G4 TCR α - chain RNA, and 2) removal of TCR β - chain single knockout cells by anti - CD3 microbeads after electroporation of TCR β - chain RNA. For TRAC, TRBC, and B2M triple disruption, T cells were electroporated with Cas9 mRNA and gRNAs targeting the TCR α and β chains and β - 2 microglobulin 3 days after anti - CD3 / CD28 bead stimulation. The HLA - I negative cell population was enriched on day 9 and electroporated with TCR α - chain RNA. The TCR - negative population was enriched on day 10. Five days later, these cells were electroporated with TCR β - chain RNA, and the TCR - negative cell population was sorted the next day to obtain universal T cells. On day 18, the universal T cells were electroporated with TCR or CAR RNA to generate universal effector cells. The expression of TCR and HLA - I molecules was confirmed at each step.

[0264] Generation of universal CAR T cells: Universal CART cells were generated by combining lentiviral transduction of CD19 or PSCA CAR with RNA electroporation of CRISPR / gRNA. One day after anti-CD3 / CD28 bead stimulation, T cells were transduced with lentiviral-CD19 or PSCA CAR. Two days later, Cas9 and gRNAs targeting TCR α, β chains, B2M, and PD1 were introduced into T cells by electroporation. Six days after CRISPR delivery, T cells negative for CD3, HLA-I, and PD1 were sorted by microbead depletion.

[0265] CD3 neg Concentration of T cells: Cells washed with Auto MACS buffer were incubated with CD3 microbeads (Miltenyi Biotec, 130 - 050 - 101, Auburn, CA) at 4°C for 30 minutes. After washing twice, the cells were passed through an LD column (Miltenyi Biotec, 130 - 042 - 901, Auburn, CA), and the flow-through fraction was collected for further use. CD3 neg CD3 expression of T cells was restored by co-electroporation of 1G4 TCR α and β mRNAs, and the cells were expanded by a single Rapid Expansion Protocol (REP), CD3 / CD28 Dynabeads, or K562-based aAPC.

[0266] CD3 neg Generation and proliferation of T cells: CD3 neg In T cells, CD3 expression was restored by electrotransfer of in vitro-transcribed mRNAs (5 μg for each chain) of exogenous 1G4 TCR α and β chains. These cells were expanded using a single Rapid Expansion Protocol (REP). PBMCs from three different donors: ND052 105×10 6 cells, ND405 83×10 6 cells, ND410 136×10 6Irradiated each one, and then mixed them to obtain a total of 324×10 6 PBMCs. The PBMCs were resuspended to a final volume of 90 ml, then R10 was added to make it 300 ml and mixed, and then divided into two T150 ml flasks. OKT was added at a final concentration of 30 ng / ml. On the second day, IL-2 was added at 50 CU / ml. From the fifth day, cell counting and nutrient addition were performed every two days. When the T cells were considered to have entered a resting state based on both the decline in growth dynamics and cell size, they were used for functional assays or cryopreserved.

[0267] Sanger sequencing: The levels of genomic disruption of TCR α-chain (TRAC), TCR β-chain 1 (TRBC1), and TCR β-chain 2 (TRBC2) in T cells were determined by the Surveyor Nuclease assay (Transgenomics, Omaha, NE). The target disruption rate was quantified by concentration measurement. The PCR primers used for amplification of the target locus were as follows: TIFF2025089344000003.tif33169

[0268] The PCR products were purified and ligated into a TOPO cloning vector (Invitrogen), and then transformed into Escherichia coli (E.coli). Single clones were picked out and sequenced to calculate indels.

[0269] Preparation of siRNA and CRISPRi for electroporation: RNA duplexes targeting the TCR constant regions related to either TIFF2025089344000004.tif34164 were designed using the Custom RNAi Design Tool (Integrated DNA Technologies, Coralville, IA), and siRNAs were synthesized (Integrated DNA Technologies, Coralville, IA). siRNAs against both TCR α and β were mixed and electroporation into stimulated T cells was performed for endogenous TCR knockdown.

[0270] mRNA in vitro transcription and T cell electroporation: In vitro transcribed (IVT) RNA was generated using the T7 mscript systems kit (CellScript). Electroporation with IVT RNA was performed on T cells stimulated with CD3 / CD28 beads as previously described (Cancer research 2010, 70(22):9053 - 9061) using BTX EM830 (Harvard Apparatus BTX). Briefly, T cells were washed three times and resuspended in OPTI - MEM (Invitrogen) at a final concentration of 1 - 3×10 8 cells / ml. Subsequently, 0.1 ml of cells was mixed with 10 μg of IVT RNA (or as indicated) and electroporation was performed in a 2 mm cuvette.

[0271] ELISA assay: Different tumor cell lines expressing CD19, the target cells, were washed and suspended in R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum; Invitrogen) at 1×10 6 cells / ml. 100 μl of each target cell type was added in duplicate to a 96 - well round - bottom plate (Corning). Effector T cells were washed and resuspended in R10 medium at 1×10 6After resuspending at cells / ml, 100 μl of T cells were combined with the target cells in the indicated wells. Wells containing only T cells were prepared as controls. The plate was incubated at 37 °C for 18 - 20 hours. After incubation, the supernatant was collected and subjected to an ELISA assay (eBioscience).

[0272] CD107a staining: In a 96-well plate, cells were seeded at an effector cell:T cell ratio of 1:1 (1×10 5 effector per 1×10 5 targets) in 160 μl of complete RPMI medium. 20 μl of phycoerythrin-labeled anti-CD107a antibody (BD Biosciences, 555801) was added and the plate was incubated at 37 °C for 1 hour. Then Golgi Stop (2 μl of Golgi Stop in 3 ml of RPMI medium, 20 μl / well; BD Biosciences, 51-2092KZ) was added and the plate was incubated for an additional 2.5 hours. Next, 5 μl of FITC-anti-CD8 and 5 μl of APC-anti-CD3 were added and incubated at 37 °C for 30 minutes. After incubation, the samples were washed with FACS buffer and analyzed by flow cytometry.

[0273] Luciferase-based CTL assay: Naml6-CBG tumor cells were generated and used in a modified version of the luciferase-based cytotoxic T lymphocyte assay. Briefly, click beetle green luciferase (CBG) was cloned into the pELNS vector, packaged into lentivirus, transduced into Naml6 tumor cells, and selected for CBG expression. The resulting Naml6-CBG cells were washed and resuspended at 1×10 5Resuspended in cells / ml, 100 μl of CBG-labeled cells were incubated overnight at 37 °C with T cells at different ratios (e.g., 30:1, 15:1, etc.). 100 μl of the mixture was transferred to a 96-well white luminometer plate. 100 μl of substrate was added to the cells, and luminescence was measured immediately. The results were reported as the percentage of killing based on luciferase activity in wells containing tumor cells but no T cells (% killing = 100 - ((RLU from wells containing effector and target cell cultures) / (RLU from wells containing target cells) × 100)).

[0274] Mouse xenograft study: The study was performed with some modifications as previously described (Human gene therapy 2011, 22(12):1575 - 1586; Proceedings of the National Academy of Sciences of the United States of America 2009, 106(9):3360 - 3365). Briefly, on day 0, 1 × 10 6 PC3-CBG tumor cells were subcutaneously injected into the right flank of 6 - to 10-week-old NOD / SCIDγ (NSG) mice, and on day 5, SK-OV3-CBG tumor cells (5 × 10 6 cells / mouse, subcutaneous) were administered to the left flank of the same mice. The mice were treated with T cells via the tail vein on day 23 after inoculation of PC3-CBG tumors, and both tumors reached a volume of approximately 200 mm 3 T cells transduced with lentivirus were administered at 1 × 10 7 cells / mouse (10M) or 3 × 10 6 cells / mouse (3M). Briefly, for the Nalm6 tumor model, 1 × 10 6Individuals of the mealworm green protein (CBG) transfected Nalm6 (Nalm6-CBG) cells were injected via the tail vein on day 0. T cell administration was initiated on day 7 after tumor inoculation. For the PC3-PDL1 solid tumor model, 1×10 6 Individuals of PSCA, PD-L1, and CBG transfected PC3 (PC3-PSCA-PDL1-CBG) tumor cells were subcutaneously injected into the right abdomen on day 0. On day 22 after PC3-PDL1-CBG tumor inoculation, T cells were administered to the mice via the tail vein to make the tumor reach approximately 200 mm 3 in volume. T cells were administered at 2×10 6 individuals / mouse (2M). The animals were randomized and divided into groups based on the baseline tumor size. All animals were included in the experiment, and tumor evaluations were performed blindly for all animal experiments conducted.

[0275] Procedures for T cell stimulation, lentiviral transfection, and CRISPR electroporation: Figure 84 shows the procedures used for T cell stimulation, lentiviral transfection, and CRISPR electroporation. On day 0, T cells were obtained from three donors (100×10 6 individuals / donor). The cells were stimulated with anti-CD3 / anti-CD28 beads at a T cell:bead ratio of 1:3. The cell concentration was adjusted to 0.5×10 6 individuals / ml and 100 mL / flask. On day 1, the stimulated T cells were transfected with CD19 CAR lentivirus at a multiplicity of infection (MOI) of 2. 50 mL (25×10 6 individuals) of T cells were reserved as non-modified T cells (group 9). On day 3, the beads were removed and the cells were washed twice in Opti-MEM medium, and the transfected T cells from each donor were divided into CART / mock EP (10 mL, 50×10 6 individuals / mL) and CART / CRISPR (10 mL, 50×10 6The cells were divided into two groups (at a density of 6 cells / mL). Subsequently, the cells were subjected to electroporation with 120 μg of CAS9 RNA / 400 μL of T cells at 500 V / 1 ms for CAS9 RNA (first EP). After electroporation, the cells in the 1st, 3rd, 5th, and 7th groups were subsequently divided by culturing half of the T cells in fresh medium and half in conditioned medium. On the 4th day, the cells were washed twice and resuspended in Opti-MEM at 50×10 6 cells / mL. Electroporation of 20 μg of TRBC4 and B2M gRNA into 400 μL of T cells was performed. After electroporation, the cells were cultured at 1×10 6 cells / mL, with half in fresh medium and half in conditioned medium. On the 5th and 7th days, the cells were divided and resuspended, half in fresh medium and half in conditioned medium. On the 8th day, CD3+ cells were removed from the 2nd, 4th, and 6th groups using a low-density column. The CD3− T cells were resuspended at 0.5 - 1×10 6 cells / mL, half in fresh medium and half in conditioned medium, and cultured to expand the cells. On the 11th day, the T cells were harvested and 25×10 5 cells from three donors were transported for karyotype analysis. The remaining cells were aliquoted and frozen.

[0276] The results of the experiment are described below.

[0277] Example 1: Disruption of the TCR-CD3 complex on T cells using CRISPR We developed 13 types of gRNAs targeting the constant region of the TCR α-chain, 10 types of gRNAs targeting the constant region of the TCR β-chain, and 10 types of RNAs targeting the β-2 microglobulin gene (Figs. 1A-1C and 9A-9D) and examined them in 293T cells. Primary human T cells were expanded ex vivo for 3 days with anti-CD3 / anti-CD28 Dynabeads for 3 days. Since transient expression of CRISPR is sufficient to mediate gene knockout, we developed a "hit-and-run" delivery strategy to transiently express CRISPR by using electroporation of in vitro-transcribed RNAs encoding CAS9 and gRNA (Fig. 2C).

[0278] To measure TCR expression, we used a mAb specific for CD3 that is present on the cell surface only when the TCR antibody is expressed. Six days after electroporation, flow cytometry analysis revealed that CRISPR targeting TRBC eliminated CD3 expression on primary T cells in donor ND147 at a level of 13.7 (Fig. 2D). The efficiency of TCR knockout correlated with the amount of electroporated mRNA (Fig. 2D). Electroporation of RNA in primary T cells was well tolerated, although a slight decrease in cell viability was observed, which correlated with an increase in the amount of introduced RNA. Gene disruption mediated by ZFN and TALEN has been reported to be more efficient when cells are transiently exposed to mild hypothermia. The same phenomenon was also observed in this CRISPR system.

[0279] T cells were cultured at 32 °C for 1 day after electroporation. CRISPR-mediated disruption of CD3 was up to 2.5-fold better when T cells that had been electroporated were cultured at 32 °C compared to 37 °C. Using this approach, with CRISPR targeting TRAC and TRBC, CD3 expression was lost in 5.43% and 16.7%, respectively, of the electroporated T cells (Figure 2D, lower panel). There was no change in the level of CD3-negative cells in the CAS9 mock sample, and no appreciable decrease in viability (measured by trypan blue) was observed.

[0280] When electroporation of gRNA was performed two and three times, the level of efficiency at which CD3 expression on primary T cells was eliminated was greatly improved. · Targeting of TRAC: After three electroporations of gRNA, it reached a level of 77% (Figure 4A), · For targeting of TRAC or TRBC, after the second electroporation of gRNA, it reached levels of 64.5% or 57.5%, respectively, with a slight decrease in viability being observed (Figure 4C).

[0281] To confirm that electroporated T cells were genetically modified at the intended gRNA target sites (TCR α or β loci), Sanger sequencing was performed using specific oligonucleotide primers adjacent to the target sites within TRAC, TRBC1, or TRBC2. Multiple peaks in the designated PCR products starting from the target sites were present only after electroporation of CRISPR, and the disruption rate correlated with the loss of cell surface CD3 expression (Figures 1C and 3B). These experiments in primary T cells confirmed that CRISPR designed to target TRAC or TRBC caused a permanent disruption of αβ TCR expression, as evaluated by Sanger sequencing and confirmed by flow cytometry analysis of CD3.

[0282] Example 2: Enrichment of TCR αβ-negative T cells For future clinical applications, rapid and robust methods can be utilized to isolate sources of populations with disrupted TCRs. To begin addressing this issue, TCR / CD3 was enriched by negative selection using clinically approved paramagnetic beads and depletion columns. neg population. neg The CD3 population was enriched by more than 99% (Figure 3A). The CD3 population could not be enriched from non-transfected control cells. In sequential depletion steps, enrichment of more than 99% was obtained, and no skewing towards CD4 or CD8 T cell subsets was seen (Figure 3C). Sequencing results also showed that deletions and insertions were introduced into the TCR α and β loci after CRISPR modification (Figure 3D). neg

[0283] Example 3: Generation of HLA-Class I neg T cells using CRISPR To examine the ability of CRISPR to knock out HLA-Class I expression from allogeneic T cells, gRNAs targeting β-2 microglobulin were designed. In 293 T cells, the β-2 microglobulin locus could be manipulated by CRISPR (Figure 9A). Evidence showed that disruption of β-2 microglobulin led to the loss of HLA-Class I expression on the T cell surface (Figure 9B).

[0284] IFN-γ improved the targeting efficiency of β-2 microglobulin in T cells by approximately 10-fold (Figure 9C). Multiple electroporations of β-2 microglobulin gRNA resulted in a population where 66% were β-2 microglobulin negative (Figure 11A).

[0285] ​For future clinical applications of allotransplantation, a rapid and robust method for isolating HLA-class I null populations is thought to be necessary. To begin addressing this problem, cells were labeled with PE-anti-β2 microglobulin antibody and HLA-class I was enriched by negative selection using clinically approved paramagnetic anti-PE microbeads and a depletion column. neg By a single depletion step, the HLA-class I neg population was enriched by over 99%. The HLA-class I neg population could not be enriched from non-transfected control cells. Analysis of the HLA-class I repertoire in the enriched HLA-class I neg T cells via flow cytometry demonstrated elimination of HLA-class I expression from the cell surface (Figure 9D).

[0286] Example 4: CD3 neg CD3 T cells can be expanded by various methods neg CD3 6 T cells regained CD3 expression after electroporation of mRNA (5 μg each) of exogenously in vitro transcribed 1G4-TCR α and β chains. These cells were expanded by (1) a single rapid expansion protocol (REP) and subsequently examined for activity and specificity. PBMCs were obtained from three different donors: ND052 105×10 6 cells, ND405 83×10 6 cells, ND410 136×10 6 cells. The cells were irradiated and subsequently mixed to obtain a total of 324×10 6 PBMCs. Electroporation with RNA was performed on 2×10 negT was resuspended to a final volume of 90 ml, and R10 medium was added to make a total volume of 300 ml. The cells were divided into two 150-ml T flasks. OKT was added to a final concentration of 30 ng / ml. On the second day, IL-2 was added to 50 CU / ml. From the fifth day, the cells were counted and nutrients were added every two days, and when the T cells were thought to have entered a resting state as judged by both a decrease in the growth rate and cell size, they were used for functional assays or cryopreserved.

[0287] After a single REP, CD3 neg T cells were expanded until the number increased 500-fold. These cells were expanded by stimulating them with anti-CD3 / anti-CD28-coated magnetic beads at a cell-bead ratio of 1:3.

[0288] After a single REP, CD3 neg T cells were expanded until the number increased 500-fold. These cells were expanded by co-culturing them with an equal volume mixture of irradiated K562-CD19 and K562 / 86 / 64 / A2 (2D11) at a concentration of 1×10 6 cells / ml.

[0289] After a single REP, CD3 neg T cells were expanded until the number increased 500-fold. These cells were expanded by co-culturing them with an equal volume mixture of irradiated K562-CD19 and K562 / 86 / 64 / A2 (2D11) at a concentration of 1×10 6 cells / ml and 30 ng / ml OKT.

[0290] After a single REP, CD3 neg T cells were expanded until the number increased 500-fold. These cells were expanded by co-culturing them with an equal volume mixture of irradiated K562-CD19 and K562 / 86 / 64 / A2 (2D11) at a concentration of 1×10 6 cells / ml and 1 mg / ml NY-ESO peptide.

[0291] Example 5: TCR by Electrotransfer of TCR neg Redirection of T cells TCR neg To examine the function of T cells, these cells were redirected by electrotransfer of TCR. By introducing the TCR α-chain and TCR β-chain, these cells expressed high levels of TCR. The expression of Vb13.1 was much higher in electrotransferred TCR T cells compared to the CAS9 mock control. (Figure 7A). When the cells were co-cultured with the Nalm-6 NY-ESO leukemia cell line, which is positive for both HLA-A2 and NY-ESO, the cells showed high levels of 107a, indicating an increase in degranulation activity (Figure 7B). A strong cytotoxicity against this cell line was also shown from the killing assay (Figure 7C). This indicates that these cells are considered not to induce GVHD and are potentially safer than conventional clinical trials using T cells expressing CAR and TCR, as they have less mismatched cytotoxicity than normal T cells treated with TCR. neg Several reports have shown that T cells can be genetically edited by ZFN or TALEN to eliminate the expression of endogenous αβ TCR. The methods and compositions described herein for selectively removing T cells expressing unwanted αβ TCR include incomplete knockout of endogenous TCR to treat GVHD and to inhibit the endogenous TCR from having a harmful effect on CAR function (e.g., through competition with transcription factors). Therefore, a genetic approach was designed using designer ZFNs to permanently disrupt the α and β constant region sequences in T cells, thereby eliminating TCR expression.

[0292]

[0293] ​ZFNs and TALENs are artificial restriction enzymes created by fusing a DNA-binding domain to a DNA cleavage domain. When ZFNs and TALENs do not work efficiently, it is often difficult to determine the cause. The failure may reflect a problem with the design, the accessibility of the target sequence, or the delivery. At the same time, the targeting efficiency of ZFNs in T cells is low, making it difficult to manipulate multiple genes simultaneously.

[0294] A CRISPR / Cas system, different from ZFNs and TALENs, has recently emerged as a potentially convenient and efficient alternative to ZFNs and TALENs for inducing targeted genetic changes. Recent studies have shown that target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region. The CRISPR / CAS system can therefore be re-targeted to cleave virtually any DNA sequence by redesigning the crRNA. The data disclosed herein demonstrate the potential of CRISPR / CAS for gene editing in 293T cells and primary T cells. The CRISPR / CAS system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suitable for multiplex gene editing or synergistic activation of target genes. By administering various gRNAs together with Cas9, multiple genes in T cells can be disrupted simultaneously.

[0295] Example 6: Triple Knockout of HLA CLASS I and TCR α, β Chains by CRISPR To work towards "off-the-shelf" allogeneic T cell therapies for malignant tumors and infectious diseases, a cell therapy by infusion of T cells was designed to reconstruct immunity against pathogens and malignant tumors. The time required to produce a sufficient number of T cells with desired characteristics ex vivo often does not match the time frame for patients. Moreover, autologous T cells from patients with progressive diseases may be functionally impaired and may be tolerant to the desired antigens.

[0296] To address this, allogeneic T cells can be infused into patients for the purpose of avoiding immune-mediated rejection reactions caused by host T cells that recognize different major histocompatibility antigens or minor histocompatibility antigens on the infused cells. To expand the use of T cell therapies and for future allotransplantation, a rapid and robust method can be created to isolate a source of a population with disrupted TCR and HLA-class I.

[0297] ZFNs and TALENs contain zinc finger DNA binding domains designed to bind to specific DNA sequences fused to the cleavage domain of the Fokl endonuclease. When multiple genes need to be manipulated, the design and construction of ZFNs and TALENs are very complex and time-consuming because the genes have to be targeted individually. By using the CRISPR system described herein, gene disruption efficiency and a shortened time course can be obtained.

[0298] To address this issue, electroporation of CAS9 was performed with three different gRNAs targeting TRAC, TRBC, and β-2 microglobulin. Cells were labeled with a PE-anti-β-2 microglobulin antibody and HLA-class I was enriched by negative selection using clinically approved paramagnetic anti-PE microbeads and a depletion column. neg By a single depletion step, HLA-class I negThe population was enriched to greater than 99% (Figure 9D). Subsequently, the TCR α chain was reintroduced into the cells, and HLA-class I neg CD3 neg The population was enriched by microbeads (Figure 11). Five days later, the TCR β chain was reintroduced into the cells, and HLA-class I neg CD3 neg The population was enriched again by microbeads. Two days later, electrotransfer of TCRs into these triple knockout cells was performed. The day after the electrotransformation, the cells were stimulated with CD3 / CD28 Dynabeads. Subsequently, lentiviral delivery of the antigen-specific TCR into the cells was performed the next day, and culture expansion was carried out.

[0299] Example 7: FAS, PD1, CTLA4, PPP2R2D Knockout by CRISPR The FAS receptor / FAS ligand (FAS / FASL) apoptosis signaling pathway has been widely studied and characterized in T cells. PD1 and CTLA4 are two major inhibitory signaling pathways in T cells, and these have also been studied in detail. Direct evidence regarding the potential impact of targeting these pathways on therapy has been obtained from studies using preclinical mouse tumor models, which demonstrate enhanced antitumor immunity after antibody-mediated blockade of CTLA-4, PD-1, or PD-L1. Similar antibodies for use in humans have been developed, and promising results have been shown from initial clinical data. Ppp2r2d knockdown can inhibit T cell apoptosis, enhance T cell proliferation, and further enhance cytokine production. Ppp2r2d may have potential as a target for improving the function of human T cells.

[0300] To address this problem, electrotransfer of CAS9 and three different gRNAs targeting FAS, PD1, CTLA4, and PPP2r2d into T cells was performed. Sanger sequencing data showed that the designated loci of FAS, PD1, CTLA4, and PPP2r2d were modified by CRISPR. FAS was further replaced with GFP by homologous recombination induced by CRISPR. FACS data showed that the surface expression of FAS and PD1 disappeared.

[0301] Example 8: Generation of iPS cells from genetically modified primary cells and T cells The progress of adoptive T cell therapy for cancer and infectious diseases has been hampered by the lack of readily available and antigen-specific human T lymphocytes. Pluripotent stem cells can be an unlimited source of T lymphocytes. To address this problem, the expression of FAS, PD1, CTLA4, and PPP2r2d was disrupted in primary cells and T cells.

[0302] Sendai virus was used for the reprogramming of primary cells and T cells. There are many methods for generating iPSCs, including viral-mediated gene transduction and chemical induction. Lentiviral vectors and retroviral vectors require integration into the host chromosome to express reprogramming genes, while DNA-based vectors, such as adenoviral vectors, adeno-associated viral vectors, and plasmid vectors, exist episomally and do not require integration. However, they are still integrated into the host chromosome at a certain frequency, and the reprogramming efficiency is relatively low. Similarly, mRNA-based reprogramming is complex and has been shown to be extremely inefficient.

[0303] Unlike these methods, Sendai virus neither integrates into the host genome nor changes the genetic information of host cells. Sendai virus also has reprogramming ability equivalent to lentivirus- and retrovirus-based gene transduction.

[0304] Into each well in a 24-well plate, 100,000 wild-type, FAS neg , CD3 neg TCR α-chain and TCR β-chain knockout T cells were seeded. The cells were stimulated with CD3 / CD28 beads. On the 3rd day after stimulation, the beads were removed, and the cells were resuspended in 1 mL of pre-warmed complete T cell medium, and then incubated with a calculated volume of CytoTune Sendai virus (Life Technologies, Carlsbad, CA) containing a polycistronic vector for the expression of hKlf4, hOct3 / 4, and hSox2 in the cells. The treated T cells were seeded into a 24-well plate and centrifuged at room temperature at 2250 rpm for 90 minutes. An additional 1 mL of complete T cell medium was added to each well, and the plate was incubated overnight at 37 °C in a humidified atmosphere of 5% CO2.

[0305] On the day after transduction, the Sendai virus was removed by washing the T cells with fresh complete medium, and the cells were cultured for 2 days. Half of the medium was changed daily. On the 3rd day after infection, the cells were transferred to a MEF feeder plate and cultured in T cell medium without adding cytokines. Four days after infection, the cells were cultured in standard hES medium. The medium was changed daily. ES-like colonies were observed approximately on the 7th day. From the 15th day, the cells were cultured in conditioned hES medium, and the culture was continued for another 10 days. The colonies were removed approximately 25 - 30 days after transduction.

[0306] At approximately the 4th day, cell clusters were formed on top of the feeder cells, which indicated the start of the reprogramming process. The T cells underwent dramatic morphological changes during the process of reprogramming into iPSCs. At approximately the 12th day, large cell clusters with loose edges began to appear. At approximately the 18th day, the T cells transformed into typical ES-like colonies with distinct edges. Typical embryonic stem cell morphology was observed, which was FAS neg , CD3 negIndicates that TCR α-chain and TCR β-chain knockout T cells were induced into a pluripotent state under predetermined reprogramming conditions (Figures 17A and 18A).

[0307] FAS neg T cells were more easily reprogrammed into iPSCs, with an efficiency approximately five times that of their wild-type counterparts (Figure 17B). Similarly, CD3 neg The efficiency of T cell reprogramming was approximately five times higher than that of their wild-type counterparts (Figure 18B). p53-deficient cell lines have been reported to be easily reprogrammed because the apoptotic pathway is disrupted. FAS knockout further induces apoptosis resistance. Loss of TCR expression makes T cells more defective, indicating that apoptosis plays an important role in the reprogramming process.

[0308] Example 9: Knockdown of TCR in T cells by siRNA Figure 19 is a graph showing IFN-γ production of wild-type NY-ESO-1 TCR (wt) or modified NY-ESO-1 TCR with a second disulfide bond and deglycosylation in the β-chain (S / SD). T cells were transfected with RNA after endogenous T cell receptor (TCR) was knocked down by siRNA. IFN-γ was detected by ELISA after stimulation of T cells with an HLA-A2 positive cell line pulsed with the NY-ESO-1 specific peptide p156-165 for 18 hours.

[0309] Figure 20, composed of Figures 20A and 20B, shows TCR α knockdown by co-electroporation of CAS9 RNA and gRNA. Six days after electroporation, cells were analyzed for TCR expression by evaluating CD3.

[0310] Figure 21 shows Sanger sequencing. The results show multiple peaks in CD3-negative enriched T cells when either CAS9 mRNA and gRNA were electroporated to knockdown TCR α (TRAC-5) or TCR β (TRBC-7).

[0311] Figure 22 is a set of graphs showing that CD3-negative T cells that received knockdown of endogenous TCR β (TRB-7) re-expressed CD3 4 hours after electroporation of NY-ESO-1 TCR α and β (1G4LY95 TCR) RNA. Normal T cells (ND424 beads) were used as a control, which showed that almost 100% were CD3-positive and the endogenous TCR vb13.1 expression was 5.25%.

[0312] Figure 23, composed of Figures 23A - 23D, is a set of graphs showing that knockdown of endogenous TCR enhanced both transgene expression and function in TCR RNA electroporated T cells. Figure 23A shows TCR expression in T cells electroporated with TCR siRNA (solid white histograms), T cells electroporated with control siRNA (dashed white histograms), and T cells without any siRNA (filled histograms). Figure 23B shows transgene (TCR vb13.1) expression in modified T cells electroporated with wild-type NY-ESO-1 TCR (wt) RNA or TCR (SD) RNA, with or without TCR siRNA, control siRNA, or no siRNA. Figure 23C shows NY-ESO-1 tetramer staining in modified T cells electroporated with wild-type NY-ESO-1 TCR (wt) RNA or TCR (SD) RNA, with or without TCR siRNA, control siRNA, or no siRNA. Figure 23D shows specific lysis of HLA-A2 / NY-ESO-1 positive tumor lines by TCR siRNA knockdown, wild-type NY-ESO-1 TCR RNA electroporated T cells.

[0313] Figure 24 is a graph showing the fluorescence of tumor cells after injection of T cells into a mouse model. 10 million Nalm6-CBG-ESO-GFP tumor cells expressing both NY-ESO-1 and GFP (green fluorescent protein of firefly) were intravenously injected into NOD / SCID mice. Five days after tumor inoculation, T cells that had been transduced with CBR (click beetle red) and electroporated with RNA were injected into various groups as indicated, and tumor growth was monitored by bioluminescence imaging (BLI).

[0314] Figure 25 shows bioluminescence images of two groups of mice administered CD19BBZ CAR RNA T cells or modified NY-ESO-1 TCR RNA at various time points.

[0315] Example 10: Universal CAR19 T cells produced by a combination of lentiviral transduction and disruption of the TCR-CD3 complex on T cells using CRISPR As shown in Figure 26, on day 0, primary T cells were stimulated with anti-CD3 / anti-CD28 beads and subsequently transduced with lentivirus-CAR19. By detection using flow cytometry, more than 70% of the cells were positive for CAR19. Since transient expression of CRISPR is sufficient to mediate gene knockout, a "hit-and-end-run" delivery strategy was developed to transiently express CRISPR by using in vitro transcribed RNA of CAS9 and gRNA targeting the constant regions of the TCR α chain, TCR β chain, and β-2 microglobulin gene on day 3. After electroporation, the T cells were cultured at 32°C for 24 hours and then returned to normal conditions.

[0316] To measure TCR expression, a monoclonal antibody specific for CD3 was used. The reason for selecting CD3 is that CD3 is present on the cell surface only when TCR is expressed. Primary T cells were electroporated with the CRISPR construct (Figure 26). TCR single-negative cells and TCR / HLA-A double-negative cells were expanded by exposure to CD19-presenting K562 cells, and the expansion rate exceeded 100 (Figure 27).

[0317] After expansion, the cells maintained TCR single-negative or TCR / HLA-A double-negative, and the CAR19-positive population was enriched. Endogenous TCR expression was maintained negative in TCR single-negative cells, while in TCR / HLA-A double-negative T cells, TCR and HLA-A expression were maintained negative after expansion stimulated by K562-CD19 (Figure 28A). Expansion stimulated by K562-CD19 enriched CAR19-positive cells (Figure 28B).

[0318] Most of the expanded universal T cells were CD45RO-positive (Figure 29A), maintaining high levels of CD62L expression (Figure 29B), moderate levels of CD28 expression (Figure 29A), and low levels of CCR7 expression (Figure 29B).

[0319] CRISPR gene editing did not affect the anti-tumor activity of universal CAR19 T cells in vitro (Figure 30A). Removal of TCR or TCR / HLA-A had only a slight effect on CAR19 expression and anti-tumor activity (Figures 30B and 30C). TCR single-negative and TCR / HLA-A double-negative CAR19 T showed strong lysis ability when stimulated by Nalm6 tumor cells (Figure 30B). Strong anti-tumor activity in universal cells was also shown from CD107a release and cytokine secretion (Figure 30C). TCR single ablation or TCR and HLA-A double ablation CAR19 T cells showed similar growth kinetics after stimulation by CD19-expressing cells (Figure 30D).

[0320] To investigate the antitumor activity of CAR19 T cells edited by CRISPR / CAS9, TCR single-negative, TCR and HLA-A double-negative CAR19 T cells were injected into NSG mice bearing Nalm6 tumor cells. Mice administered with unmanipulated T cells and mice injected with wild-type T cells that received lentiviral GFP transduction all died within 3 weeks after tumor cell injection. Objective tumor regression was observed in mice administered with CAR19 T cells (Figure 6). CRISPR / CAS9 did not affect the in vivo tumor killing activity of CAR19 T cells, thus confirming the advantage of combining lentiviral gene transfer and CRISPR / CAS9 for T cell therapy.

[0321] Complete ablation of TCR α and β chains and HLA-A molecules on T cells completely eliminated non-specific killing when the cells were challenged with HLA-mismatched tumor cell lines (Figure 32A). Elimination of HLA-A molecules activated NK cells after long-term co-culture (5 days). No off-target activity was observed after 24 hours of challenging these cells with allogeneic whole blood PBMCs in an IFNγ Elispot assay. The absence of off-target activity suggests that T cells can play a major role in the acute immune response after encounter with allogeneic cells. All these results suggest that TCR α and β chains and HLA-A molecule (triple-negative) T cells edited by CRISPR / CAS9 can serve as a source of versatile effector donor cells.

[0322] Electroporation of CAS9 and various gRNAs targeting FAS into T cells was performed. FASneg cells were sorted and subsequently transduced with lentiviral CAR19. Flow cytometry and Sanger sequencing data showed that FAS was modified by CRISPR (Figure 33). CAR19 gene expression in FASneg T cells was equivalent to that of the wild type. CD107a expression in FASneg CAR19 T cells was significantly enhanced compared to the wild-type counterpart even after a short incubation with Nalm6 tumor cells, even in co-culture within 4 hours.

[0323] Several reports have shown that even weak antigen stimulation induces FAS activation and promotes T cell proliferation (Rethi, et al, Blood, vol. 112(4):1195-1204, 2008). Interestingly, FASneg CAR19 T cells expanded much faster than wild-type CAR19 T cells when the cells were stimulated with high levels of CD19+ K562 cells. This suggests that under high-level antigen conditions, FAS / FASL induced apoptosis rather than activation (Figure 34A). Measurement by annexin V staining also further showed a decrease in the apoptosis level of FASneg CAR19 T cells (Figure 34B).

[0324] As observed in vitro, FASneg T cells showed enhanced proliferation compared to wild-type T cells. When the True Count assay of CAR19 T cells was performed after injection of the cells into Nalm6-bearing mice, similar proliferation results were observed. The FASneg CAR19 group showed better antitumor activity compared to the wild-type group (Figure 35B). This difference is illustrated in Figure 35C, which shows the bioluminescence data between these two groups. These data indicate that ablation of FAS in CART cells enhanced its antitumor activity.

[0325] Electroporation of CAS9 and various gRNAs targeting PD1 into T cells was performed after lentiviral transduction with PSCA-CAR. PD1 knockout cells were confirmed by negative surface PD1 expression after CD3 / CD28 bead stimulation (Figure 36). PD1-negative cells were enriched by microbead depletion and subsequently stimulated with PSCA antigen-presenting PC3 tumor cells. PSCA-CAR-positive cells were enriched in both the wild-type group and the PD1-negative group. After incubation with PC3-PSCA-PDL1 tumor cells, PD1 expression was rapidly upregulated on the surface of wild-type PSCA-CAR T cells, while PD1 expression detected on PD1-negative PSCA-CAR T cells was at extremely low levels (Figure 37). PD1-negative PSCA-CAR T cells also showed a greatly enhanced and sustained high level of expression of CD137, a marker of T cell activation (Figure 37), indicating that the PD1 / PDL1 inhibitory signaling pathway was blocked.

[0326] When examined in an in vivo PC3-PSCA-PDL1 NSG model, a significant enhancement of antitumor activity was detected in the PD1-negative PSCA-CAR T cell group compared to the wild-type group (Figures 38A and 38B), suggesting that PD1 ablation has therapeutic value for CAR T cell therapy.

[0327] To examine the effect of CRISPR-engineered universal CART cells on graft-versus-host disease (GVHD), high-dose T cells were administered to NSG mice with Nalm6 leukemia. When double or triple knockout CART cells were administered to the mice, no signs of developing GVHD were shown. In contrast, 3 out of 4 mice in the wild-type CD19 CART group developed GVHD by day 65, which was confirmed by histological examination of various organs (Figure 39).

[0328] In another experiment, cells were resuspended in FBS and intravenously injected after sublethal irradiation of mice. Clinical GVHD was monitored 2 - 3 times a week. Four out of five mice that received wild-type T cell administration died during the 60-day test, while the PBS administration group, and the groups receiving T cells with single TCR ablation and TCR / HLA-I double ablation showed no signs of GVHD. Mice that received wild-type T cell administration developed weight loss. However, in the PBS administration group, and the groups receiving T cells with single TCR ablation and TCR / HLA-I double ablation, the body weight increased slightly during the test period (Figures 40A and 40B).

[0329] After lentiviral CD19-CAR transduction, T cells were treated with Cas9 and gRNAs targeting CD3, B2M, and PD1 or Fas. Triple knockout universal CART cells were injected into mice bearing Nalm6-PDL1 tumors. Better antitumor activity was observed in mice that received PD1 / CD3 / HLA-I triple knockout cell administration compared to those that received CD3 / HLA-I double knockout cells, further indicating the therapeutic value of blocking the PD1 signaling pathway (Figures 41A and 41B). These data provide a way to enhance the treatment of universal CART cells by CRISPR / Cas9.

[0330] Since gRNA is easily degraded, a simplified one-shot method for generating universal CART cells was developed. The gRNA was constitutively expressed together with the CAR in a single lentiviral vector. Naive T cells were transduced with lentiviruses encoding the gRNA and the CAR 1 day after stimulation with CD3 / CD28 Dynabeads. On day 3, the cells were electroporated with Cas9 mRNA (Figure 42). This system enables the manipulation of several genes with a single vector (Figure 42). CD3 expression was confirmed by flow cytometry on day 6. T cells treated with this one-shot system showed consistent gene ablation at approximately 90% in each of the various Cas9 mRNA groups (Figure 43).

[0331] The progress of adoptive T cell therapy for cancer and infectious diseases has been hampered by the lack of readily available and antigen-specific human T lymphocytes. Pluripotent stem cells can be an unlimited source of T lymphocytes. To address this problem, the expression of FAS, PD1, CTLA4, and PPP2r2d was disrupted in primary cells and T cells.

[0332] Sendai virus was used for the reprogramming of primary cells and T cells. There are many methods for generating iPSCs, including virus-mediated gene transduction and chemical induction. Lentiviral vectors and retroviral vectors require integration into the host chromosome to express reprogramming genes, while DNA-based vectors, such as adenoviral vectors, adeno-associated viral vectors, and plasmid vectors, exist episomally and do not require integration, but they are still integrated into the host chromosome at a certain frequency and the reprogramming efficiency is relatively low. Similarly, mRNA-based reprogramming is complex and has been shown to be extremely inefficient.

[0333] In contrast, Sendai virus neither integrates into the host genome nor alters the genetic information of the host cell. Sendai virus also has reprogramming ability equivalent to lentivirus- and retrovirus-based gene transduction.

[0334] Each well in a 24-well plate was seeded with 100,000 wild-type, FASneg, CD3neg TCR α-chain and TCR β-chain knockout T cells. The cells were stimulated with CD3 / CD28 beads. On the third day after stimulation, the beads were removed and the cells were resuspended in 1 mL of pre-warmed complete T cell medium, and then incubated with a calculated volume of CytoTune Sendai virus (Life Technologies, Carlsbad, CA) containing a multicistronic vector for the expression of hKlf4, hOct3 / 4 and hSox2 in the cells. The treated T cells were seeded in a 24-well plate and centrifuged at room temperature at 2250 rpm for 90 minutes. An additional 1 mL of complete T cell medium was added to each well and the plate was incubated overnight at 37 °C in a humidified atmosphere of 5% CO2.

[0335] On the day after transduction, the Sendai virus was removed by washing the T cells with fresh complete medium and the cells were cultured for 2 days. Half of the medium was changed daily. On the third day after infection, the cells were transferred to a MEF feeder plate and cultured in T cell medium without adding cytokines. Four days after infection, the cells were cultured in standard hES medium. The medium was changed daily. ES-like colonies were observed approximately on the seventh day. From the 15th day, the cells were cultured in conditioned hES medium and the culture was continued for an additional 10 days. Colonies were picked approximately 25 - 30 days after transduction.

[0336] At approximately day 4, cell clusters formed on top of the feeder cells, indicating the initiation of the reprogramming process. T cells underwent dramatic morphological changes during the reprogramming process to iPSCs (Figure 44A). At approximately day 12, large cell clusters with loose edges began to appear. At approximately day 18, T cells transformed into typical ES-like colonies with distinct edges. FASneg T cells were reprogrammed into iPSCs with approximately five-fold higher efficiency than the wild-type counterparts (Figure 44B). p53-deficient cell lines have been reported to be more amenable to reprogramming because the apoptotic pathway is blocked. FAS knockout can facilitate the reprogramming process by a similar mechanism.

[0337] ES-like morphology of iPSCs reprogrammed from CD3neg TCR α or β chain knockout T cells was observed (Figure 45A). This morphology was maintained consistently after several passages. The reprogramming efficiency of CD3neg T cells was approximately one-fifth that of the wild-type counterparts (Figure 45B), suggesting that TCR knockout plays a role in the process of T cell reprogramming or affects cell viability after Sendai virus infection. Figure 45C is a group of images showing phosphatase staining of CD3neg iPSC cells.

[0338] Typical embryonic stem cell morphology was observed, indicating that FASneg, CD3neg TCR α chain and β chain knockout T cells were induced into a pluripotent state under the given reprogramming conditions. Loss of TCR expression makes T cells more defective, but the data described herein indicate that apoptosis plays an important role in the reprogramming process.

[0339] The induction of endogenous pluripotent stem cell genes was also detected in various T-iPSC cell lines (Figure 46). Immunostaining for the expression of Tra-1-60 and SSEA4 further indicated the stem cell phenotype of T-iPSC cells (Figure 47A). The Fas knockout in T-iPSC was confirmed by Sanger sequencing (Figure 47B).

[0340] dCas9 and FokI-Cas9 have been reported to have relatively weak off-target activities. T cells were evaluated for whether they could be edited by the modified versions of the CRISPR / dCAS9 and CRISPR / FokI-CAS9 systems (Figure 48A). Flow cytometry data showed that primary T cells were edited by both CRISPR / dCAS9 and CRISPR / FokI-CAS9 (Figure 48B). The CRISPR / dCAS9 gene knockout system showed enhanced specificity with at least one pair of gRNAs, making the knockout events more accurate and specific.

[0341] To examine the off-target events of CRISPR / CAS9 in T cells, a Surveyor assay at the off-target sites was performed. No obvious cleavage was observed at the genomic loci for the genes examined (Figure 48C).

[0342] Example 11: Multiplex Genome Editing CART cells were generated by using a CRISPR / Cas9 system that simultaneously disrupts multiple genomic loci. These CART cells are deficient in the expression of endogenous TCR molecules and HLA class I (HLA-I) molecules for use as allogeneic universal CART cells. The genes for the T cell receptor (TCR) α chain, TCR β chain, and β-2 microglobulin (B2M) were disrupted with high efficiency by co-electroporation of gRNAs targeting these genes and mRNA encoding Cas9. Universal TCR or CART cells were generated by combining lentiviral (LV) delivery of the CAR and CRISPR RNA electroporation for simultaneous disruption of the endogenous TCR and B2M genes. Furthermore, disruption of endogenous PD1 also enhanced the efficacy of CAR therapy in solid tumor models.

[0343] Multiple genes in human primary T cells are disrupted with high efficiency without impairing effector function by multiplex delivery of gRNAs Efficient multiplex genome editing is required to generate universal T cells deficient in TCR, HLA, and other genes. To achieve efficient gene disruption in T cells, CRISPR / gRNA RNA electroporation was optimized. First, co-electroporation of Cas9 and gRNAs with RNA generated using an in vitro transcription system was performed (Figure 49, left), and a "hit-end-run" delivery strategy for transient delivery of Cas9 mRNA and gRNAs to T cells by electroporation was developed (Figure 49, right).

[0344] In initial experiments targeting the TCR α constant region (TRAC) or β constant region (TRBC) using a single electroporation, 1% - 3% of CD3 negative (CD3 neg) T cells were obtained (Figure 50A, upper graph). To determine whether transient exposure to mild hypothermia enabled more efficient gene disruption, cell editing was performed at 37°C or 32°C. CRISPR-mediated disruption of TRAC and TRB increased four-fold when T cells were cultured at 32°C for 24 hours after Cas9 / gRNA co-electroporation (Figure 50A, lower graph). The optimal molar ratio of Cas9:gRNA for maximum disruption efficiency was 1:1 to 2:1, and the gene disruption efficiency correlated with the amount of electroporated mRNA (Figure 51A).

[0345] gRNA is more susceptible to rapid degradation compared to mRNA, which may limit its targeting efficiency. Therefore, multiple sequential electroporations of gRNA were examined after the initial Cas9 / gRNA electroporation. A significant increase in disruption frequency was observed at the protein level, and 82.4% of the cells were CD3 neg after the third gRNA electroporation (Figure 50B). Clone sequencing showed that the genomic targeting efficiency reached 89.4% after the third gRNA electroporation (Figure 51B). The Surveyor assay confirmed that the cleavage rates at the genomic loci of TRAC and TRBC were 81.7% and 49.3%, respectively, after the third gRNA electroporation (Figure 52). Multiple peaks in the Sanger sequencing data adjacent to the TRAC and TRBC target sites confirmed that the genomic reading frame had shifted downstream of the target site (Figure 53A). The occurrence of insertions or deletions (indels) caused by CRISPR / Cas9-mediated NHEJ was confirmed by clone sequencing (Figure 53B). TCR enriched the TCR / CD3 neg population by more than 99% (99.70 ± 0.20%) through a single-step CD3 negative selection (Figure 54).

[0346] TCR / CD3 neg To develop a method for expanding TCR / CD3neg T cells were co-electroporated with HLA-A2-restricted 1G4 NY-ESO-1 TCR (α+β) RNA to restore CD3 expression (Figure 55, left panel). After the T cell stimulation / enlargement method, the following were compared: (1) a rapid T cell expansion protocol (REP) using PBMC as feeder cells, (2) anti-CD3 / CD28 Dynabeads (beads), or (3) OKT3 loaded with K562-based artificial antigen-presenting cells (K562 aAPC) expressing the ligands for CD28 and 4-1BB. TCR / CD3 neg T cells were also electroporated with CD19 CAR RNA (Figure 55, right panel) and subsequently stimulated with irradiated K562 aAPC (K562-CD19) expressing CD19. After single stimulation for 10 days, expansion magnification values of 751.0±217.1, 35.7±9.3, 46.3±8.5, and 57.5±5.0 were achieved for REP, beads, K562 aAPC, and K562-CD19, respectively (Figure 56).

[0347] To examine whether CRISPR / Cas9 gene editing affects the phenotype and function of T cells, TCR / CD3 expanded by various methods neg When examining the phenotype of the T cells, all the expanded cells maintained CD3 negativity and most maintained a high level of CD27 (79.8% - 93.4%), indicating that they were consistent with the phenotype of central memory cells (Figure 57). The expanded TCR / CD3 neg T cells were subjected to a second electroporation with CD19 CAR mRNA to examine their anti-tumor activity. TCR / CD3 neg The surface CAR expression of the T cells was equivalent to that of the control group (Figure 58). TCR / CD3 neg CD19 CAR T cells + When stimulated with Nalm6 leukemia cells, CD19 CAR + TCR / CD3 negCD107a upregulation (Figure 59A), cytokine secretion (Figure 59C), and killing activity (Figure 59B) of the T cells were equivalent to those of wild-type control cells. CD19 CAR TCR / CD3 neg The TCR α, β, and B2M triple-disrupted T cells were injected into Nalm6-bearing NSG mice to examine their in vivo antitumor activity. Tumor regression was evident, and the efficacy was equivalent to that regarding CART19 wild-type counterpart cells (Figures 59D and 59E). These results indicate that CRISPR / Cas9 editing of the endogenous TCR does not have a detrimental effect on the functions related to adoptive immunotherapy of primary T cells.

[0348] Reduction of alloreactivity of TCR α, β, and B2M triple-disrupted T cells To prevent TCR mismatch-related toxicity in adoptive immunotherapy of T cells redirected for TCR, it is necessary to disrupt both the TCR α-chain and β-chain, and B2M is essential for the assembly and expression of the HLA-I complex. Considering this point, TCR α-chain, β-chain, and B2M triple disruption was developed to generate universal T cells. First, it was examined whether HLA-I expression on T cells could be eliminated by disrupting B2M. T cells were subjected to electroporation of Cas9 / gRNA RNA targeting B2M. As a result, a B2M- and HLA-I double-negative population of 79.9% was generated. The HLA-I neg population could be further enriched by negative selection (Figure 60).

[0349] To generate triple knockout T cells lacking TCR α and β chains and B2M, co-electroporation was performed with Cas9 mRNA and three different gRNAs targeting TRAC, TRBC, and B2M. As a result, the CD3 and HLA-I double-negative cell population was 65.4% (Figure 61). After enrichment of double- and triple-knockout cells, non-specific killing of HLA-mismatched tumor cell lines was abolished in TCR α-chain and β-chain and B2M triple knockout T cells (Figure 62). When these cells were stimulated with allogeneic whole blood-irradiated PBMC in the IFNγ Elispot assay, no response was observed (Figure 63, left panel). Ablation of HLA-I molecules also significantly reduced alloreactivity, as confirmed by co-culture of allogeneic PBMC with irradiated B2M-disrupted cells (Figure 63, right panel). These results suggest that triple-negative T cells lacking TCR α and β chains and B2M may serve as a universal T cell source for adoptive immunotherapy, which can resist rejection by the host immune system while simultaneously making it impossible to cause graft-versus-host disease.

[0350] Improvement of antitumor activity of endogenous TCR-disrupted T cells with TCR redirection T cells with TCR α and β chains disrupted by CRISPR / Cas9 showed increased transgenic TCR expression on the cell surface after redirection with NY-ESO-1 TCR (1G4). Transgenic TCR expression was 67.6%, 78.8%, or 94.3% in single knockout of TCR α-chain or β-chain or α / β double knockout, respectively, compared with 46.8% in wild-type T cells. The improved transgenic TCR expression led to enhanced T cell function, as evidenced by increased antigen-specific CD107a expression (Figure 65A) and enhanced cytotoxicity (Figure 65B), particularly in α / β double knockout T cells.

[0351] In another experiment, α / β double knockout T cells were transfected with different NY-ESO-1 TCRs (8F). Compared to the 1G4 TCR, this 8F TCR showed a highly significant improvement in both transgenic TCR expression (Figure 66; TCR / CD3 neg showed 60.1% in transgenic TCR expression, compared to 44.7% in wild-type T cells (Cas9 mock T cells) (with an endogenous TCR Vβ8 background of approximately 5%)) and function (CD107a expression in Figure 67A and cytokine production in Figure 67B). These results strongly indicate that the endogenous TCR has different effects on the expression and function of transgenic TCRs.

[0352] Universal CART cells maintain anti-tumor efficacy and do not cause GVHD Universal CD19 CART cells were generated by combining LV transduction of CD19 CAR and RNA electroporation of Cas9 / gRNA (Figure 68). When the cells were expanded, the remaining CD3 neg cells had high levels of CD19 CAR expression (Figure 69). The majority of the expanded T cells were CD45RO positive, maintaining high levels of CD62L expression and moderate levels of CD28 expression, which is consistent with the central memory cell state (Figure 70). The expanded TCR / HLA-I double-negative CD19 CARTs showed robust in vitro anti-tumor activities such as CD107a release (Figure 71), cytokine secretion (Figure 72), lysis ability (Figure 73), and proliferation (Figure 74), and these were as potent as wild-type CD19 CART cells.

[0353] T cells were injected into NSG mice bearing disseminated Nalm6 leukemia. CART cells with disrupted endogenous TCR (LV-CD19 CAR TCR neg ) or CART cells with simultaneous disruption of TCR and HLA-I (LV-CD19 CAR TCR / HLA-I negMice administered with ( ) showed tumor regression comparable to that of mice administered with wild-type CD19 CART cells (LV-CD19 CAR) (Figures 75A and 75B), suggesting that disruption of TCR alone or disruption of both TCR and B2M does not affect the anti-tumor activity of CART cells.

[0354] To examine the effect of engineered T cells on GVHD, high-dose T cells (20×10 6 cells / mouse) were administered to NSG mice with Nalm6 leukemia. As shown in Figure 76, mice treated with CD19 CART cells with TCR disruption only (LV-CD19 CAR TCR / CD3 neg ) or CD19 CART cells with simultaneous disruption of TCR and B2M (LV-CD19 CAR TCR / HLA-I neg ) showed tumor regression comparable to that of mice treated with wild-type CD19 CAR T cells (LV-CD19 CAR). Mice treated with double or triple knockout CAR T cells did not develop any signs of GVHD. In contrast, 3 out of 4 mice in the wild-type CD19 CART (LV-CD19 CAR) group developed GVHD by day 65, which was confirmed by histological examination of various organs. Thus, disruption of TCR alone or disruption of TCR and HLA-I does not affect the in vivo anti-tumor activity of CART cells, and at the same time alloreactivity is eliminated.

[0355] Adenoviral CRISPR delivery to primary T cells The CRISPR / Cas9 system has rapidly become utilized for gene regulation and gene editing purposes in model organisms and cell lines. Viral vectors are considered particularly suitable for expanding the applicability of CRISPR to other cell types, including dividing cells and quiescent primary cells. Adenoviruses encoding Cas9 and a single guide RNA (gRNA) molecule, i.e., second-generation fiber-modified adenoviruses, were used to deliver Cas9 nuclease to the PD1, Fas, and TRAC loci (Figure 77). Adenovirus-mediated CRISPR transduction into tumor cells (Figure 78) resulted in targeted mutagenesis at a high rate of up to approximately 71% (Figures 79A and 79B). Adenoviruses are a useful platform for introducing CRISPR into human T cells even when they are in a quiescent state. This approach is thought to be useful for investigating the potential of CRISPR for gene regulation and editing in numerous experimental settings.

[0356] Optimization of Electroporation The knockout efficiencies of CD3 and B2M and T cell expansion were evaluated after electroporation (EP) of Cas9 and gRNA in 4 mm cuvettes and 2 mm cuvettes. Under standard EP conditions (360 v / 1 ms, first EP - 20 μg Cas9 RNA + 10 μg gRNA / 100 μl T cells, second EP - 5 μg gRNA / 100 μl T cells) using 2 mm cuvettes, the highest knockout rates of CD3 and B2M were shown to be 81.8% and 88.6% respectively at the time of approximately 2.7-fold T cell expansion (EP#1), whereas the expansion fold of control EP T cells (EP#12) was approximately 18.8. When the gRNA dosage was decreased (EP#2 - 5), T cell expansion increased dramatically, but there was only a slight effect on the knockout efficiencies of CD3 and B2M. See Figure 80. Under standard EP conditions using 4 mm cuvettes, the knockout efficiencies of CD3 and B2M were significantly reduced (EP#8), suggesting that the EP conditions (potential or / and pulse width) need to be further optimized for using 4 mm cuvettes.

[0357] Compared with the standard electroporation (EP) conditions in 2 mm cuvettes (EP#10 - 13) or 4 mm cuvettes, high CD3 / B2M knockout efficiency was observed, accompanied by an improvement in the T cell expansion ratio (EP#1 and 5). See Figure 81.

[0358] To further optimize the EP conditions to achieve the maximum T cell expansion ratio with a CD3 / B2M knockout efficiency exceeding 60%, various EP conditions and RNA amounts were investigated. As a result, for EP#4 where EP#1 is (400 v / 2 ms / 120 μg CAS9 RNA) and EP#2 is (500 v / 1 ms / 20 μg gRNA), an improvement in the expansion ratio was shown with relatively high CD3 / B2M knockout efficiency (63.5% for CD3 and 84.8% for B2M). See Figure 82.

[0359] Further experiments were conducted to optimize the EP conditions. As a result, when using 500 v / 1 ms / 120 μg CAS9 RNA (EP#1) and 500 v / 1 ms / 20 μg gRNA (EP#2), an increase in the CD3 / B2M knockout efficiency and T cell expansion occurred compared to the most favorable conditions examined (EP#1 in Figure 82). See Figure 83.

[0360] Large-scale electroporation and expansion Experiments were conducted to clarify whether high knockout efficiency and expansion efficiency can be obtained by large-scale electroporation. On day 0, T cells obtained from three donors (100×10 6 cells / donor, concentrated to 0.5×10 6 / ml) were stimulated using anti-CD3 / anti-CD28 beads. On day 1, the stimulated T cells were transduced with CD19 CAR lentivirus. 50 mL (25×10 6 cells) of T cells were secured as non-modified T cells (Group 9). On day 3, the beads were removed, and the transduced T cells from each donor were subjected to CART / mock EP (10 mL, 5×106 )(pieces) and CART / CRISPR (10 mL, 50×10 6 were divided into two groups. Subsequently, electroporation with CAS9 RNA was performed on the cells (the first EP), and the cells in the 1st, 3rd, 5th, and 7th groups were separated. On the 4th day, electroporation of gRNA into T cells was carried out, and the cells were cultured at 1×10 6 cells / mL. On the 5th and 7th days, the cells were separated. On the 8th day, CD3+ cells were removed from the 2nd, 4th, and 6th groups. On the 11th day, T cells were collected, and 25×10 5 cells from three donors were transported for karyotype analysis.

[0361] (Table 1) Experimental groups TIFF2025089344000005.tif79165

[0362] The T cell number (the upper chart in Figure 85) and magnification (the lower chart in Figure 85) were evaluated after the electroporation and culture procedures. The magnification of T cells transduced with CD19 CAR only (TD only), or T cells transduced with CD19 CAR and edited by CRISPR (TD / KO) is shown in the left graph of Figure 86, and the magnification of T cells at the 10th day is shown in the right graph of Figure 86. By optimizing the electroporation conditions and CAS9 / gRNA dosage, a CD3 / B2M knockdown efficiency of approximately 60 - 70% and a T cell expansion of approximately 30-fold were observed 10 days later (Figure 87 shows the CD3 / B2M / CAR expression at the 10th day).

[0363] Eight days after CD3 / CD28 bead stimulation and CRISPR RNA electroporation, CD3-positive T cells were removed. Figure 88 shows CD3 / B2M expression in a three-donor population at the 8-day time point. On day 11, the T cells were subjected to FACS staining to detect the expression of CD3, B2M, and CAR. Non-transduced ND463 (NOTD) was used as a negative control. Figure 89 shows CD3 and B2M expression in CD3-depleted T cells that underwent CD19 CAR TD (transduced) / CRISPR electroporation; T cells that underwent CD19 CAR TD / CRISPR electroporation; and CD19 CAR TD T cells. Figure 90 shows CAR expression in CD3-depleted T cells that underwent CD19 CAR TD / CRISPR electroporation; T cells that underwent CD19 CAR TD / CRISPR electroporation; and CD19 CAR TD T cells. Figure 91 shows CD3 / B2M / CAR expression at the 11-day time point in CD3-depleted T cells that underwent CD19 CAR TD (transduced) / CRISPR electroporation; T cells that underwent CD19 CAR TD / CRISPR electroporation; and CD19 CAR TD T cells. Figure 92 summarizes CD3 / B2M / CAR expression in various T cell populations.

[0364] On day 11, various T cell populations were stimulated with Raji or Nalm6, CD19-positive cell lines, as indicated in the legend of Figure 93. K562 was used as a CD19-negative control. After 4 hours of co-culture, CD107a upregulation was detected in each of the T cell populations except for the negative control.

[0365] On day 11, the killing ability of the T cells was examined using a luminescent cytotoxic lymphocyte (CTL) assay after co-culture of the T cells with Nalm6-CBG, a CD19-positive target cell, as indicated in the legend of Figure 94. Also on day 11, the cytokine production of the T cells was analyzed by stimulating the T cell populations with Nalm6 target cells. See Figure 95.

[0366] T cells were cultured for up to 26 days in a medium containing 100 U / ml of IL-2. The results shown in Figure 96 indicate that no abnormal T cell proliferation was observed for CRISPR-edited T cells derived from three donors.

[0367] As one of the most attractive applications of the CRISPR / Cas9 system, multiplex genome editing is highly expected to advance T cell-based adoptive immunotherapy. However, the low targeting efficiency of DNA transfection has restricted the use of multiplex genome manipulation in primary T cells. A "hit-and-run" delivery strategy was developed to introduce CRISPR into T cells via co-electroporation of Cas9 mRNA and gRNA. By combining up to three gRNA electroporations with a transient exposure to mild hypothermia, targeting efficiencies exceeding 80% at the protein level were always achieved for single gene disruption. More promisingly, triple gene disruption of TRAC, TRBC, and B2M resulted in approximately 65% double negative CD3 and HLA-I without any purification and selection. These results also demonstrated that enrichment of gene-disrupted T cells to a purity exceeding 99% could be easily achieved using clinically approved paramagnetic beads, and that purified T cells could expand up to 500-fold in 10 days. The expanded T cells maintained their gene-disrupted phenotypes and exhibited characteristics consistent with central memory T cells. The disrupted T cells did not cause GVHD, suggesting that they could be used as allogeneic CAR T cells. Importantly, the gene-edited T cells showed anti-tumor activity both in vitro and in various tumor mouse models, and they had equal or greater efficacy compared to non-gene-edited T cells. Therefore, the processes described herein for generating synthetic cells are considered to be easily convertible into manufacturing procedures compliant with current GMP.

[0368] The data described herein demonstrate that CRISPR / Cas9 is a powerful multiplex genome editing tool in primary human T cells. Previous reports have shown that T cells can be genetically edited by ZFNs or TALENs for the purpose of eliminating the expression of endogenous TCR α- and β-chains to avoid GVHD. Due to the complexity of targeting strategies for manipulating multiple genes by zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) in T cells, previous studies have not been able to simultaneously prevent GVHD and host-versus-graft reactions in preclinical animal models. Also, NK cell activation can be disrupted by CRISPR / Cas9 or by removing stimulatory NK ligands by expression of non-classical HLA class I molecules such as HLA-E, which is thought to potentially protect allogeneic T cells from NK cell-mediated rejection.

[0369] In summary, clinically-scaled allogeneic CAR T cells with potent anti-tumor activity and reduced alloreactivity were efficiently generated using multiplex CRISPR technology. This approach can be incorporated into current GMP-compliant manufacturing procedures and is likely to be translated to the clinic considering the successful clinical translation of ZFN-based adoptive therapy for HIV / AIDS. Allogeneic CAR T cells and TCR T cells may represent an alternative to autologous T cells. Moreover, allogeneic CAR T cells and TCR T cells with checkpoint molecules disabled are thought to be more effective and have a broader utility than current CAR T therapies using autologous T cells for cancer and infectious diseases.

[0370] Other aspects The recitation of a list of elements in a definition of a variable herein includes that variable as either a single element or a combination (or partial combination) of listed elements. The recitation of an aspect herein includes that aspect as either a single aspect or in combination with other aspects or portions thereof.

[0371] The disclosures of each patent, patent application, and publication cited in this specification are hereby incorporated by reference in their entirety. Although the invention has been disclosed in connection with certain embodiments, it is apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0372] Array information SEQUENCE LISTING <110> The Trustees of the University of Pennsylvania <120> ALTERING GENE EXPRESSION IN CART CELLS AND USES THEREOF <150> US 62 / 073,651 <151> 2014-10-31 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 1 tgtgctagac atgaggtcta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 2 gcagtatctg gagtcattga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 3 cgcgagcaca gctaaggcca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 4 ggcgccctgg ccagtcgtct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 5 gagggtccag atgcccagca 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 6 tcatgtccta accctgatcc tctt 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 7 ttggactttt cccagctgac aga 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 8 taccaggacc agacagctct taga 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 9 tctcacctaa tctcctccag gcat 24 <210> 10 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 10 rargrgrarg rgrarururc rgrgrararc rcrcrararu rcrarcrurg rarc 54 <210> 11 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 11 rcrargrurg rarururgrg rgrururcrc rgrararurc rcrurcct 48 <210> 12 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 12 rarcrcrurc rcrururcrc rcrarururc rarcrcrcra rcrcrargrc rurc 54 <210> 13 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 13 rgrcrurgrg rurgrgrgru rgrararurg rgrgrararg rgrarggt 48

Claims

1. A nucleic acid capable of downregulating gene expression of an endogenous gene selected from the group consisting of TCR alpha chain, TCR beta chain, beta-2 microglobulin, 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 costimulatory molecule. A modified T cell comprising:

2. The modified T cell of claim 1, wherein the nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA, and a CRISPR system.

3. The modified T cell of claim 2, wherein the CRISPR system comprises a 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, a human antibody, a humanized antibody, a single domain antibody, a single chain variable fragment, and an antigen-binding fragment thereof.

5. The modified T cell of claim 1, wherein the antigen-binding domain of the CAR specifically binds to an antigen on a target cell.

6. The modified T cell of claim 1, wherein the intracellular domain of the CAR comprises a dual signaling domain.

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 costimulatory 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 a CD3ζ chain and a CD3ε chain.

11. Introducing into the T cell a nucleic acid capable of downregulating gene expression of an endogenous gene selected from the group consisting of TCR alpha chain, TCR beta chain, beta-2 microglobulin, HLA molecule, CTLA-4, PD1 and FAS; and Introducing a nucleic acid encoding a chimeric antigen receptor (CAR) containing an antigen-binding domain and a transmembrane domain into a T cell. A method for producing a modified T cell, comprising:

12. 12. The method of claim 11, wherein the nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antigenomic RNA, siRNA, shRNA, and CRISPR systems.

13. The method of claim 12, wherein the CRISPR system comprises a 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, a human antibody, a humanized antibody, a single domain antibody, a single-chain variable fragment, and an antigen-binding fragment thereof.

15. The method of claim 11, wherein the antigen-binding domain of the CAR specifically binds to an antigen on a target cell.

16. The method of claim 11, wherein the intracellular domain of the CAR comprises a dual signaling domain.

17. The method of claim 11, wherein the T cells are obtained from the group consisting of peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines.

18. The method of claim 11, further comprising expanding the T cells.

19. 20. The method of claim 18, wherein the step of expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

20. 12. The method of claim 11, further comprising cryopreserving the T cells.

21. 21. The method of claim 20, further comprising thawing cryopreserved T cells prior to introducing said nucleic acid into the T cells.

22. 12. The method of claim 11, wherein the step of 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 RNA encoding a costimulatory molecule into the T cell.

24. 24. The method of claim 23, wherein the costimulatory 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 cell to induce pluripotency of the T cell.

26. 13. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cell of claim 1.

27. 13. 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. 28. The method of claim 27, wherein the condition is an autoimmune disease.

29. Autoimmune diseases include acquired immune deficiency 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), Behçet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis); chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic 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, pernicious anemia, polyarteritis nodosa, 29. The method of claim 28, wherein the patient is selected from the group consisting of polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, 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's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, Wegener's granulomatosis, and any combination thereof.

30. 28. The method of claim 27, wherein the condition is cancer.

31. 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, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and any combination thereof.

32. 13. A method for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the modified T cell of claim 1.

33. 33. The method of claim 32, further comprising inducing lysis of the target cell or tissue.

34. 34. The method of claim 33, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

35. 13. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells of claim 1 to prevent or treat an immune response that is deleterious to the subject.

36. 2. Use of the modified T cell of claim 1 in the manufacture of a medicament for treating an immune response in a subject in need thereof.

37. 12. A composition comprising modified T cells produced according to the method of claim 11.

38. 12. A pharmaceutical composition comprising the modified T cells produced according to the method of claim 11 and a pharma- ceutically acceptable carrier.

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