Modification of gene expression in CART cells and uses thereof

Abstract

Provided are patient-specific modified T cells for the treatment of cancer, methods for producing the modified T cells, and pharmaceutical compositions comprising the modified T cells. The present invention provides modified T cells comprising a nucleic acid capable of downregulating 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, 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 method for producing the modified T cells, and pharmaceutical compositions comprising the modified T cells for adoptive therapy and for treating conditions such as autoimmune diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 073,651, filed October 31, 2014, which is incorporated by reference in its entirety herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under grant CA120409 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. 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 related to integration using lentiviral or retroviral vectors are a major concern for the modification of cells used in ACT. Some progress has been made to avoid on-target or off-target undesirable side effects, such as RNA transfection of T cells with 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 allow the introduction of multiple genes into cells. However, the main limitation of transient expression of CAR is the suboptimal effector activity and functionality of RNA-transfected T cells. To improve CAR function, 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).

[0005] Various attempts have been made to improve the effector activity and functionality of CAR while avoiding the need for combination therapy and additional treatment. Increased RNA during the transfection process adversely affects T cell function, especially antitumor activity in vivo (Barrett et al., 2011, Hum Gene Ther 22:1575-1586). Another construct that fuses anti-CD3 antigen antibody fragments to antitumor antigen antibody fragments has also been tested in clinical trials for cancer treatment (Bargou et al., 2008, Science 321:974-977; Klinger et al., 2012, Blood 119:6226-6233). Unfortunately, these constructs have been significantly limited in function due to short half-life, poor accessibility to target cell sites, and lack of proper long-term signaling function.

[0006] Clinical studies of TCRs have been hampered by low expression levels of transduced TCRs as well as α-chain and β-chain mispairing. Clearly, the potential for four TCRs to be expressed on the cell surface when T cells transcribe two different TCR chains (native α / β, exogenous α / β, and native / exogenous “mispaired” heterodimer) represents a significant barrier to the use of this approach. Previous studies have clearly demonstrated in preclinical studies that TCR mispairing may induce harmful self-antigen recognition.

[0007] Although early clinical data on TCR and CAR T cells for the treatment of cancer shows promising results, the risks to patients are significant, and some patients' T cells are not potent enough to be an effective treatment, even after forced modification of allogeneic donor-derived T cells by TCR or CAR redirection. However, endogenous αβ T cell receptors on infused allogeneic T cells can recognize major and minor histocompatibility antigens in the recipient, leading to graft-versus-host disease (GVHD). As a result, most current clinical trials using infusion of autologous CAR T cells rely on immune tolerance to prevent harmful TCR-mediated recognition of normal tissues after adoptive cell transfer. Although this approach has shown initial clinical success, it is limited by the time and expense of producing patient-specific T cell products. Thus, there is a demand for safer methods to modify T cells without the time and expense of producing patient-specific T cell products. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Louis et al., 2011, Blood 118:6050-6056 [Non-Patent Document 2] Kochenderfer et al., 2010, Blood 116:3875-3886 [Non-Patent Document 3] Porter et al., 2011, N Engl J Med 365:725-733 [Non-Patent Document 4] Zhao, 2006, Mol Ther 13:151-159 [Non-Patent Document 5] Mitchell et al., Smits et al., 2004, Leukemia 18:1898-1902 [Non-Patent Document 6] Barrett et al., 2013, Hum Gene Ther 24(8):717-27 [Non-Patent Document 7] Barrett et al., 2011, Hum Gene Ther 22:1575-1586 [Non-Patent Document 8] Bargou et al., 2008, Science 321:974-977 [Non-Patent Document 9] Klinger et al., 2012, Blood 119:6226-6233 Summary of the Invention

[0009] As described herein, the present invention relates to compositions and methods for generating modified T cells that have a nucleic acid capable of altering gene expression of an endogenous gene selected from the group consisting of a TCR alpha chain, a TCR beta chain, beta-2 microglobulin, an HLA molecule, CTLA-4, PD1, and FAS, and further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0010] One aspect of the invention includes an engineered T cell comprising a nucleic acid capable of downregulating gene expression of an endogenous gene selected from the group consisting of a TCR alpha chain, a TCR beta chain, beta-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 costimulatory molecule.

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

[0012] In yet another aspect, the invention includes 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 cells described herein.

[0013] In yet another aspect, the invention provides 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 cells described herein.

[0014] In another 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 a pharmaceutical composition comprising the modified T cells described herein.

[0015] In yet another aspect, the invention includes 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 described herein to prevent or treat an immune response that is deleterious to the subject.

[0016] In yet another aspect, the invention includes the use of a modified T cell as 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 compositions comprising modified T cells produced according to the methods described herein.

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

[0019] In various embodiments of the above aspects and any other aspects of the invention delineated herein, the nucleic acid capable of downregulating gene expression is 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 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. In another embodiment, the antigen-binding domain of the CAR specifically binds to an antigen on a 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 a modified T cell described herein further comprises electroporating RNA encoding the costimulatory molecule into the T cell. In some embodiments where the costimulatory molecule is CD3, the CD3 comprises at least two different CD3 chains, such as a CD3 zeta chain and a CD3 epsilon chain.

[0022] In another embodiment, 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.

[0023] In yet another embodiment, the method of making a modified T cell as described herein further comprises expanding the T cells. In one embodiment, the 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.

[0024] In yet another embodiment, the method of making a modified T cell as described herein further comprises cryopreserving the T cell. In another embodiment, the method described herein further comprises thawing the cryopreserved T cell prior to introducing the nucleic acid into the T cell.

[0025] In one embodiment, 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 embodiment, the methods described herein further comprise expressing Klf4, Oct3 / 4 and Sox2 in the T cells to induce pluripotency of the T cells.

[0027] In various embodiments of the above aspects and any other aspects of the invention depicted herein, the invention comprises administering modified T cells to a subject. In one embodiment, the subject has a condition such as an autoimmune disease. In some embodiments, the autoimmune disease is 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), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis (CBD), or other conditions. 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 mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, nodular The disease is selected from the group consisting of polyarteritis, 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.

[0028] In another embodiment, the condition is cancer, e.g., a 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 embodiment, the methods described herein further comprise inducing lysis of the target cell or tissue, such as antibody-dependent cell-mediated cytotoxicity (ADCC). [The present invention 1001] 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: [The present invention 1002] The modified T cell of the present invention 1001, wherein the nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA, and CRISPR system. [The present invention 1003] The modified T cell of the present invention 1002, wherein the CRISPR system comprises a pAd5 / F35-CRISPR vector. [The present invention 1004] The modified T cell of the present invention, 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. [The present invention 1005] The modified T cell of the present invention, wherein the antigen-binding domain of the CAR specifically binds to an antigen on a target cell. [The present invention 1006] The modified T cell of the present invention, wherein the intracellular domain of the CAR comprises a dual signaling domain. [The present invention 1007] The modified T cell of the present invention 1001, further comprising an exogenous nucleic acid encoding a costimulatory molecule. [The present invention 1008] 1007. The modified T cell of the present invention, 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 1009] The modified T cell of the present invention, wherein the CD3 comprises at least two different CD3 chains. [The present invention 1010] The modified T cell of the present invention, wherein the different CD3 chains are CD3ζ chain and CD3ε chain. [The present invention 1011] 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: [The present invention 1012] The method of claim 1011, wherein said nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antigenomic RNA, siRNA, shRNA, and CRISPR systems. [The present invention 1013] The method of claim 1012, wherein the CRISPR system comprises a pAd5 / F35-CRISPR vector. [The present invention 1014] The method of the present invention, 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. [The present invention 1015] The method of claim 1011, wherein the antigen-binding domain of the CAR specifically binds to an antigen on a target cell. [The present invention 1016] The method of claim 1011, wherein the intracellular domain of the CAR comprises a dual signaling domain. [The present invention 1017] The method of claim 1011, 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. [The present invention 1018] The method of claim 1011, further comprising expanding the T cells. [The present invention 1019] The method of any one of claims 10 to 18, wherein the step of expanding the T cells comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand. [The present invention 1020] The method of claim 1011, further comprising the step of cryopreserving the T cells. [The present invention 1021] The method of claim 1020, further comprising the step of thawing cryopreserved T cells prior to introducing said nucleic acid into the T cells. [The present invention 1022] The method of claim 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 claim 1011, further comprising the step of electroporating RNA encoding a costimulatory molecule into the T cell. [The present invention 1024] 1025. The method of claim 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 claim 1011, further comprising expressing Klf4, Oct3 / 4 and Sox2 in the T cell to induce pluripotency of the T cell. [The present invention 1026] 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 cells of the present invention. [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 the modified T cells of the present invention. [The present invention 1028] The method of claim 1027, wherein the condition is an autoimmune disease. [The present invention 1029] 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, The method of the present invention 1028, wherein the inflammatory bowel disease 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. [The present invention 1030] The method of claim 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, colon 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. [The present invention 1033] The method of claim 1032, further comprising the step of inducing lysis of the target cell or tissue. [The present invention 1034] The method of the invention 1033, wherein the lysis induced 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 to prevent or treat an immune response that is harmful to the subject. [The present invention 1036] The use of a modified T cell of the present invention in the manufacture of a medicament for the treatment of 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. [The present invention 1038] A pharmaceutical composition comprising a modified T cell produced according to the method of the present invention and a pharma- ceutical acceptable carrier. [Brief description of the drawings]

[0030] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1A]Figure 1, consisting of Figures 1A-1C, is an illustration of CRISPR design and targeting of the TCR αβ-CD3 complex in 293T cells. Figure 1A shows the CRISPR gRNA targeting sites within the genomic locus of the TCR-α and β constant regions. Each exon is indicated by a block. The black blocks represent coding regions. The grey columns represent non-coding regions. Thirteen gRNAs were designed to target exon 1 of the TCR α constant region (TRAC), ten gRNAs were designed to target conserved sequences on exon 1 of the TCR β constant regions 1 (TRBC1) and 2 (TRBC2), and ten gRNAs were designed to target exon 1 of the β-2 microglobin gene. Figure 1B shows a typical gRNA scaffold sequence. gRNA PCR products were generated by overlap PCR and cloned into the MSGV vector with a T7 promoter. FIG. 1C shows Sanger sequencing results showing the presence of multiple peaks in the genomic PCR products of 293T TCR TRAC and TRBC following transfection of CAS9 mRNA and gRNA into cells. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A]Figure 2, consisting of Figures 2A-2E, shows the disruption of TCR αβ-CD3 complex in primary T cells. Figure 2A is a table showing the parameters used for electroporation of CAS9 mRNA and gRNA into primary T cells with BTX830. 360V 1ms in a 2mm cuvette gave the best mean fluorescence intensity (MFI) and efficiency for electroporation of day 3 bead-stimulated primary T cells. Figure 2B is a panel of graphs showing T cells incubated at 32°C 5% CO2 with a much higher MFI than the regular 37°C 5% CO2 condition. Figure 2C is a schematic illustration of the CRISPR system transferred into primary T cells. Electrotransfer of CAS9 mRNA and gRNA into T cells was performed 3 days after bead stimulation of primary T cells. T cells were then cultured with 100 lU / mL IL-2, some cells were cultured at 32°C 5% CO2 for 1 day, followed by an additional 7-9 days. CD3 expression was analyzed by flow cytometry 7-9 days after electroporation. Figure 2D is a panel of graphs showing that targeting efficiency at 37°C is approximately 2.5-fold higher than at 32°C. Figure 2E is a panel of graphs showing downregulation of CD3 6 days after electrotransfer of various amounts and ratios of CAS9 and gRNA targeting TCR β. CD3 expression was analyzed by staining for CD3. Representative flow data at day 6 after electroporation is shown. Quadrants show the percentage of CD3 negative cells in the T cell population. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 3A]Figure 3, consisting of Figures 3A-3D, shows that TCRneg α or β knockouts in T cells can be enriched by depletion of TCRpos T cells. Figure 3A is a panel of graphs showing CD3 expression of TCRneg α or β knockouts in T cells before and after microbead depletion. Flow cytometry illustrates CD3 expression. The numbers in the lower right quadrant represent the percentage of CD3 negative cells in the T cell population. Figure 3B is a panel of sequencing graphs showing that multiple peaks were observed in the genomic PCR products of CD3neg enriched T cells. Figure 3C is a panel of graphs showing CD4 and CD8 T cell repertoire analysis after CD3 microbead enrichment in CRISPR modified α chain, β chain single knockout T cells, and αβ double knockout T cells. The data show that the ratio of CD8 T cell populations was enhanced by CRISPR modification, suggesting that CD8 T cells can be more easily modified than CD4 T cells. FIG. 3D shows the sequencing results of the deletions and insertions introduced into the TCR α and β loci after CRISPR modification. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A]Figure 4, consisting of Figures 4A-4C, shows that multiple electrotransfers of gRNA significantly improved the targeting efficiency of the CRISPR system in primary T cells. Figure 4A is a panel of graphs showing that multiple electroporations of gRNA significantly improved the targeting efficiency. The highest targeting efficiency, close to 80%, was obtained by electroporating T cells up to three times within 24 hours. In early experiments, only 15% TCR targeting efficiency was achieved in T cells. Sustained expression of CAS9 was observed after electrotransfer of CAS9 mRNA into T cells. The reason for the low cleavage efficiency is likely due to rapid degradation of gRNA. A higher CD3 negative population was obtained. Figure 4B is a panel of graphs showing that capping impairs gRNA function, but early introduction of gRNA for the second time resulted in higher efficiency. Figure 4C is a panel of graphs showing that multiple electrotransfer of gRNAs targeting TRAC and TRBC in ND221 resulted in approximately 64.5% and 57.5% cleavage rates, respectively. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A] Figure 5, consisting of Figures 5A and 5B, shows that TCRneg T cells can be expanded under various stimulation conditions. Figure 5A is a panel of graphs showing that TCRneg T cells regained CD3 expression after reintroduction of TCR α and β chains into TCRneg T cells. CD3 and Vb13.1 were detected after electroporation of TCR α and β chains into TCRneg T cells. CD3 expression levels were comparable to TCRpos T cells. Figure 5B is a panel of graphs showing the fold expansion after various conditions used to stimulate TCRneg T cells. PBMC REP resulted in approximately 500-fold expansion, while CD3 / CD28 beads or K562 aAPC restimulation resulted in approximately 25-58-fold expansion. [Figure 5B] See legend to Figure 5A. [Figure 6A]

[0023] Figure 6A is a panel of graphs showing the phenotypic characteristics of TCRneg T cells after expansion under different conditions. [Figure 6B] Figure 6B is a panel of graphs showing the phenotypic characteristics of TCRneg T cells after expansion under different conditions. [Figure 7A] FIG. 7, consisting of FIG. 7A-7C, shows expanded TCRneg T cells with potent anti-tumor activity after in vitro redirection. FIG. 7A is a panel of graphs showing that TCRneg T cells can be redirected by the introduction of anti-NY-ESO 1G4 TCR into cells. Compared to the CAS9 mock (MOCK) group, TCRneg T cells showed higher levels of Vb13.1 expression when redirected with 1G4 TCR, due to less mispairing of exogenous and endogenous TCR α and β chains. FIG. 7B is a panel of graphs showing that 1G4 TCR-redirected TCRneg T cells had high degranulation activity when co-cultured with tumor (Nalm6-ESO) cell lines. FIG. 7C is a panel of graphs showing that 1G4 TCR-redirected TCRneg T cells had high cytotoxicity against tumor cell lines. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8] FIG. 8 is a panel of illustrations showing that committed TCRneg T cells suppress tumor growth in NSG mice after redirection. [Figure 9A]FIG. 9, consisting of FIGS. 9A-9D, shows that disruption of β-2 microglobin resulted in HLA-class I elimination. FIG. 9A shows sequencing data of CRISPR capable of disrupting the β-2 microglobin locus in HEK293 cells. FIG. 9B is a panel of graphs showing that disruption of β-2 microglobin resulted in a bHLA-class I negative T cell population. FIG. 9C is a panel of graphs showing that cIFNg improved β-2 microglobin targeting efficiency in primary T cells. FIG. 9D is a panel of graphs showing that HLA-class Ineg T cells were enriched by microbead depletion. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10] Figure 10 is a panel of graphs showing simultaneous knockout of HLA-class I and TCR in primary T cells. CD4 and CD8 T cells were stimulated with CD3 / CD28 Dynabeads. Three days after stimulation, expanded T cells were electroporated with CAS9 mRNA along with gRNAs targeting TCR beta constant region (TRBC) and beta-2 microglobin. Six days after electroporation, both TCR and beta-2 microglobin expression were assessed using anti-CD3 monoclonal antibody (mAb) and anti-beta-2 microglobin mAb. Numbers represent the percentage of the population in each quadrant. [Figure 11A]FIG. 11, consisting of FIGS. 11A-11D, shows HLA-class I and TCR α- and β-chain triple knockout in primary T cells. FIG. 11A is a panel of graphs showing CD4 and CD8 T cells stimulated with CD3 / CD28 Dynabeads. Three days after stimulation, expanded T cells were electroporated with CAS9 mRNA along with gRNAs targeting TCR α, β constant regions (TRAC, TRBC) and β-2 microglobin. Six days after electroporation, both TCR and HLA-class I expression were assessed using anti-CD3 monoclonal antibody (mAb) and anti-β-2 microglobin mAb. Numbers represent the percentage of the population in each quadrant. FIG. 11B is a schematic diagram illustrating the isolation of HLA-class I and TCR α- and β-chain triple knockout T cells. FIG. 11C is a panel of graphs showing electroporation efficiency examined by GFP expression. Figure 11D is a panel of graphs showing reintroduction of TCR α and β chains into TCR T cells as measured by flow cytometry. Approximately 64% α and 14% β populations were observed among the total TCR T cells. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 12A] FIG. 12, consisting of FIG. 12A-12D, shows knockout of FAS in 293T cells. FIG. 12A is an image showing Sanger sequencing results of multiple peaks when knocking out FAS in 293T cells. FIG. 12B is a panel of graphs showing FACS data demonstrating that surface expression of FAS protein was disrupted by CRISPR. FIG. 12C is a panel of images showing that FAS protein was replaced by GFP after homologous recombination by CRISPR. FIG. 12D is a panel of graphs showing FACS data showing the percentage of homologous recombination by CRISPR. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13] Figure 13 shows knockout of FAS in primary T cells. FACS data illustrates that surface FAS protein expression was abolished by CRISPR. [Figure 14A] Figure 14, consisting of Figures 14A and 14B, shows knockout of PD1 in 293T cells and primary T cells. Figure 14A is an image showing the results of Sanger sequencing of multiple peaks when targeting PD1 in 293T cells. Figure 14B is a panel of graphs showing FACS data of surface expression of PD1 protein disrupted by CRISPR. [Figure 14B] See legend to Figure 14A. [Figure 15A] Figure 15, which is composed of Figures 15A and 15B, shows the knockout of CTLA4 in 293T cells and primary cells, such as CCD1079-SK. Figure 15A shows the image of the Sanger sequencing results of multiple peaks when CTLA4 is targeted in 293T cells. Figure 15B shows the sequence data after limiting dilution and single cell expansion. The Sanger sequencing results identify deletions and insertions in the CTLA4 genomic locus. [Figure 15B] See legend to Figure 15A. [Figure 16] Figure 16 shows knockout of PPP2r2d in 293T. Sanger sequencing data indicates that PPP2r2d was targeted by CRISPR in 293T cells. [Figure 17]Figure 17, consisting of Figures 17A and 17B, shows the generation of iPSCs from FAS knockout T cells. Figure 17A is a panel of images showing morphological changes during the process of reprogramming FASneg T cells into iPSCs. The formation of typical embryonic stem cell morphology, which indicates FASneg T cells, can be induced as a pluripotent state. Figure 17B is a graph showing that FASneg T cells were reprogrammed into iPSCs about 5 times more efficiently than their wild-type counterparts. It has been reported that p53-deficient cell lines are easier to reprogram because the apoptotic pathway is hindered. FAS knockout can aid the reprogramming process by a similar mechanism. [Figure 18] Figure 18, consisting of Figures 18A and 18B, shows the generation of iPSCs from CD3neg T cells. Figure 18A is a panel of images showing the ES-like morphology formed by CD3neg TCR α or β chain knockout T cells under defined reprogramming conditions. The morphology remains constant after several passages. Figure 18B is a series of graphs showing that reprogramming of CD3neg T cells is about 5-fold more efficient than their wild-type counterparts, suggesting that TCR knockout plays a role in the process of T cell reprogramming or affects cell viability after Sendai virus infection. [Figure 19] FIG. 19 is a graph showing knockdown of the endogenous T cell receptor (TCR) by siRNA and addition of a second disulfide bond and de-N-glycosylation to the β chain. [Figure 20] Figure 20, consisting of Figures 20A and 20B, shows TCR knockout with CAS9 RNA and gRNA. Six days after electroporation, cells were analyzed for TCR expression by assessing CD3. [Figure 21] FIG. 21 is an illustration showing the results of PCR sequencing after CD3 microbead depletion. [Figure 22]Figure 22 is a panel of graphs showing CD3 re-expression 4 hours after NY-ESO-1 TCR RNA electroporation. [Figure 23A] Figure 23, comprised of Figures 23A-23D, is a panel 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 open histograms), control siRNA (dotted open histograms), and T cells without any siRNA (filled histograms). Figure 23B shows transgene (TCR vb13.1) expression in engineered T cells electroporated with wild-type NY-ESO-1 TCR (wt) RNA or TCR (SD) RNA with TCR siRNA, control siRNA, or no siRNA. Figure 23C shows NY-ESO-1 tetramer staining of engineered T cells electroporated with wild-type NY-ESO-1 TCR(wt)RNA or TCR(SD)RNA with 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. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. [Figure 24]Figure 24 is a graph showing the fluorescence of tumor cells after injection of T cells into a mouse model. Ten million Nalm6-CBG-ESO-GFP tumor cells expressing both NY-ESO-1 and GFP (click beetle green) were intravenously injected into NOD / SCID mice. Five days after tumor inoculation, CBR-transduced and RNA-electroporated T cells were injected into different groups as indicated, and tumor cells were detected by fluorescence. [Diagram 25] Figure 25 is a panel of images showing fluorescence of injected tumor and hybrid TCR T cells over time in a mouse model. [Figure 26] Figure 26 is a panel of images showing the generation of universal CAR19 T cells. At the top of the figure is an illustration of the protocol for generating universal CAR19 T cells. The graph on the left shows the percentage of CAR19 positive T cells after lentiviral CAR19 gene transduction. The graph on the right shows the percentage of TCR single negative T cells and TCR / HLA-A double negative T cells before and after sorting. [Figure 27] Figure 27 is a panel of graphs and a table showing fold expansion of CD19 positive cells following stimulation with irradiated CD19-presenting K562 cells. [Figure 28] Figure 28A is a panel of graphs showing endogenous and transgenic gene expression in K562-CD19 expanded cells. Figure 28B is a panel of graphs showing that endogenous TCR expression remained negative in TCR single negative cells, while TCR and HLA-A expression remained negative in TCR / HLA-A double negative T cells after K562-CD19 stimulated expansion. [Figure 29]Figure 29A is a panel of graphs showing that the majority of expanded universal CAR19 T cells were CD45RO positive and expressed moderate levels of CD28 expression. Figure 29B is a panel of graphs showing that the majority of expanded universal CAR19 T cells retained high levels of CD62L expression and low levels of CCR7 expression. [Figure 30A] Figure 30A is a graph showing that CRISPR gene editing did not affect the anti-tumor activity of universal CAR19 T cells in vitro. [Figure 30B] Figure 30B is a panel of graphs showing that TCR single negative and TCR / HLA-A double negative CAR19 T cells exhibited robust lytic capacity when challenged with Nalm6 tumor cells. [Figure 30C] Figure 30C is a panel of graphs showing cytokine secretion as part of the potent anti-tumor activity of these cells. [Figure 30D] Figure 30D is a panel of graphs showing that TCR single ablation or TCR and HLA-A dual ablation in CAR19 T cells exhibited similar proliferation kinetics after challenge with Nalm6 tumor cells. [Diagram 31] Figure 31 is a panel of images showing that CRISPR gene editing did not affect the antitumor activity of universal CAR19 T cells in vivo. Mice receiving unmanipulated T cells and mice injected with lentiviral GFP-transduced wild-type T cells all died within 3 weeks after tumor cell injection. Objective tumor regression was observed for mice receiving CAR19 T cells. CRISPR-edited universal CAR19 T cells with TCR single negative or TCR / HLA-A double negative showed the same antitumor activity. [Figure 32A] Figure 32A is a panel of graphs showing that TCR single or TCR and HLA-A dual ablation in T cells significantly reduced alloreactivity. [Figure 32B]Figure 32B is a panel of graphs showing that exclusion of HLA-A molecules activated NK cells upon long-term co-culture (5 days). [Figure 32C] FIG. 32C is a graph showing that no off-target activity was observed when cells were challenged with allogeneic whole blood PBMCs for 24 hours in an IFNr Eispot assay. [Figure 33-1] Figure 33 is a panel of graphs showing that FAS ablation enhanced the antitumor activity of CAR19 T cells. FAS-negative CAR19 T cells were generated. FAS ablation was confirmed by flow cytometry analysis. CAR19 gene expression of FASneg T cells was comparable to wild type. Even after a short incubation of 4 hours with Nalm6 tumor cells, CD107a expression in FASneg CAR19 T cells was greatly enhanced compared to wild type counterparts. [Figure 33-2] See description of Figure 33-1. [Figure 34A] Figure 34A is a graph showing that FAS ablation in CAR19 T cells enhanced survival and proliferation of CAR T cells under in vitro antigen conditions. FASneg CAR19 T cells expanded faster than wild-type CAR19 T cells when the cells were stimulated with high levels of CD19+ K562 cells. [Figure 34B] Figure 34B is a panel of graphs showing that FASneg CAR19 T cells had reduced levels of apoptosis, as measured by Annexin V staining. [Figure 35A] Figure 35A is a graph showing that FAS ablation in CAR19 T cells enhanced CAR cell function in an animal model. As observed in vitro, FASneg T cells showed enhanced proliferation compared to wild-type T cells. [Figure 35B] Figure 35B is a panel of images showing that the FASneg CAR19 group clearly exhibited superior anti-tumor activity compared to the wild-type group. [Figure 35C] Figure 35C is a graph showing significant differences in bioluminescence data between the FASneg CAR19 and wild-type groups. [Diagram 36] Figure 36 is a panel of graphs showing the generation of PD1-negative PSCA-CAR T cells. PD1 ablation was confirmed by flow cytometry analysis. PD1-negative cells were enriched by microbead depletion. Wild-type or PD1-negative PSCA-CAR T cells were expanded by stimulation with irradiated PSCA antigen-presenting PC3 tumor cells. PSCA-CAR-positive cells were enriched after expansion. [Figure 37] Figure 37 is a panel of graphs showing that PD1 ablation and CD137 expression in PSCA-CAR T cells enhanced CAR T cell activation under in vitro antigen conditions. [Figure 38A] Figure 38A is a panel of images showing PD1 ablation in the in vivo PC3-PSCA-PDL1 NSG model. PSCA-CAR T cells clearly demonstrated enhanced CAR T cell in vivo anti-tumor activity compared to the wild-type group. [Figure 38B] Figure 38B is a graph showing the difference in tumor burden between the PD1 negative and wild type groups. [Figure 39-1] Figure 39 is a panel of histological images showing that TCR or TCR / HLA-I ablated T cells did not cause graft-versus-host disease (GVHD). Mice receiving double or triple knockout CART cells did not develop any signs of GVHD. In contrast, 3 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 39-2] See description of Figure 39-1. [Figure 40A]Figure 40A is a graph showing the survival rate of animals injected with TCR or TCR / HLA-I ablated T cells. Mice were sublethally irradiated and injected. Four out of five mice receiving wild type T cells died due to GVHD during the 60 day study. The PBS group, TCR single ablated and TCR / HLA-I double ablated T cell groups did not show any signs of GVHD. [Figure 40B] Figure 40B is a panel of graphs showing the body weight of mice receiving wild type T cells, PBS, TCR single ablated or TCR / HLA-I double ablated T cells. [Figure 41A] Figure 41A is a panel of images showing improved antitumor activity of universal CART cells after blocking the PD1 and Fas pathways by CRISPR / Cas9. Superior antitumor activity was detected in PD1 knockout universal CD19-CART cells when injected into Nalm6-PDL1-bearing mice. [Figure 41B] Figure 41B is a graph showing quantitative bioluminescence data from mice receiving various CRISPR / Cas9 edited T cells. [Diagram 42] Figure 42 is a panel of diagrams showing a one-shot system for generating universal CART cells. Because gRNA is prone to degradation, we developed a simplified one-shot method to constitutively express gRNA along with CAR in a single lentiviral vector. [Diagram 43] Figure 43 is a panel of graphs showing efficient gene ablation by the one-shot system. Varying amounts of CD3 ablation were observed after electrotransfer of Cas9 mRNA. [Diagram 44]Figure 44A is a panel of images showing morphological changes in the process from reprogramming iPSCs from Fas knockout T cells. The formation of typical embryonic stem cell morphology, indicative of FASneg T cells, can be induced as a pluripotent state. Figure 44B is a graph showing that FASneg T cells were reprogrammed into iPSCs approximately 5-fold more efficiently than their wild-type counterparts. It has been reported that p53-deficient cell lines are easier to reprogram due to the disruption of the apoptotic pathway. FAS knockout may facilitate the reprogramming process using a similar mechanism. [Figure 45A] Figure 45A is a panel of images showing ES-like morphology of iPSCs from CD3neg TCR α-chain or β-chain knockout T cells under defined reprogramming conditions, which remained constant after several passages. [Figure 45B] Figure 45B is a graph showing that the efficiency of reprogramming CD3neg T cells was approximately 5-fold lower than the wild-type counterpart, suggesting that TCR knockout plays a role in the T cell reprogramming process or affects cell viability following Sendai virus infection. [Figure 45C] Figure 45C is a panel of images showing phosphatase staining of CD3neg iPSC cells. [Figure 46] Figure 46 is a panel of graphs showing induction of endogenous pluripotent stem cell genes in various T-iPSC cell lines. [Figure 47] Figure 47A is a panel of images showing immunostaining for expression of Tra-1-60 and SSEA4. Figure 47B is an image showing confirmation of Fas knockout in T-iPSCs by Sanger sequencing. [Figure 48A] Figure 48A is a panel of graphs showing gene ablation in naive T cells by different forms of Cas9. CD3 was knocked out by dCas9 and FokI-Cas9. [Figure 48B]Figure 48B is a panel of graphs showing that two gRNAs were required for dCas9 and FokI-Cas9 gene ablation. [Figure 48C] Figure 48C is an image showing rare off-target events in CRISPR / cas9 genetically modified T cells. [Figure 49] Figure 49 is a panel of images showing the strategy of introducing CRISPR / Cas9 into T cells. A schematic of the gRNA driven by the T7 promoter is shown on the left. A schematic of the generation of gene-edited antigen-specific T cells using the CRISPR system is shown on the right. T cells were electroporated with Cas9 mRNA and gRNA targeting specific genes 3 days after CD3 / CD28 bead stimulation, followed by culture in the presence of IL2 at 32°C for 24 hours, and then returned to normal culture conditions at 37°C. T cells with specific gene disruption were selected on day 8 and redirected with CAR or TCR by lentiviral transduction or mRNA electroporation gene transfer. [Figure 50A] Figure 50A is a panel of graphs showing CRISPR / Cas9-mediated efficient TCR disruption in T cells. CD3 expression of CRISPR / Cas9 edited T cells cultured at 37°C or 32°C. [Figure 50B] Figure 50B is a panel of graphs showing CD3 expression of CRISPR / Cas9 edited T cells cultured after sequential CRISPR RNA electroporation. [Figure 51A] Figure 51A is a panel of graphs showing efficient CRISPR gene disruption in T cells. CD3 expression of CRISPR-transfected T cells using different Cas9:gRNA ratios (top and middle panels) and amounts of total CRISPR RNA (bottom panel). [Figure 51B] FIG. 51B is a table showing targeting efficiency calculated by both flow cytometry and clonal sequencing. [Figure 52]Figure 52 is an image showing the amount of TCR-targeted gene disruption measured by mismatch-selective T7 Surveyor nuclease assay on DNA amplified from cells. Calculated amounts of targeted gene disruption in TRAC and TRBC are shown at the bottom. Arrows indicate expected bands. [Figure 53A] FIG. 53A is an image of indels (in gene disruptions) observed by clonal sequence analysis of PCR amplicons following CRISPR-mediated recombination of the TCR α and β loci. [Figure 53B] Figure 53B is an image of a schematic of the human locus encoding the TCR alpha and beta CRISPR gRNA targeting sites within the genomic loci of the TCR alpha and beta constant regions. Each exon is represented by a block. Arrow: sense strand gRNA targeting site; blue arrow: antisense strand gRNA targeting site. Multiple peaks in the Sanger sequencing results indicate CRISPR-mediated events of NHEJ at the TRAC and TRBC genomic loci. [Figure 54] Figure 54 is a panel of graphs showing CD3 expression in purified TCRneg cells. [Figure 55] Figure 55 is a panel of graphs showing redirection of TCR / CD3neg cells via electrotransfer of 1G4 TCR (alpha and beta) or CAR19 mRNA. [Figure 56] FIG. 56 is a graph showing the expansion of TCR / CD3neg cells after 10 days using various stimulation conditions. [Figure 57] Figure 57 is a panel of graphs showing that CRISPR / Cas9 editing does not impair the anti-tumor efficacy of primary T cells. The phenotype of TCR / CD3 cells after four different expansion procedures is shown. [Figure 58] Figure 58 is a panel of graphs showing relative CD19-CAR expression following electrotransfer of CD19-CAR RNA into Cas9 mock and TCR / CD3neg cells. [Figure 59A] Figure 59A is a panel of graphs showing that no significant functional differences were observed between CD19-CAR redirected Cas9 Mock and TCR / CD3 cells, as confirmed by CD107 release assay after incubation with Nalm6 target cells. Representative data from three independent experiments are shown. Bars, standard error. [Figure 59B] Figure 59B is a panel of graphs showing that no significant functional differences were observed between CD19-CAR redirected Cas9 Mock cells and TCR / CD3 cells, as confirmed by cytotoxicity assays after incubation with Nalm6 target cells. Representative data from three independent experiments are shown. Bars, SE = standard error. [Figure 59C] Figure 59C is a panel of graphs showing that no significant functional differences were observed between CD19-CAR redirected Cas9 Mock and TCR / CD3 cells, as confirmed by IL2 and IFNγ secretion after incubation with Nalm6 target cells. Representative data from three independent experiments are shown. Bars, SE = standard error. [Fig. 59D] Figure 59D shows images of a group of NOD / scid / γc(- / -) mice (n=12) injected (iv) with 1×106 Nalm6 tumor cells, randomized into three groups. Electroporated CD19-CAR expressing Cas9 Mock T cells and TCR / CD3neg T cells (10×106) were injected intravenously every 4 days for a total of three injections (arrows). Mice receiving T cells without RNA electroporation served as controls. Images were obtained from surviving animals as indicated. Imaging began 1 day before the start of T cell administration. Bars, SE=standard error, EP=electroporation; E:T=effector:tumor ratio; arrows, time of T cell injection; ns, not significant. ****P<0.0001 by two-way ANOVA+Bonferroni post-hoc test, ns. [Figure 59E] FIG. 59E is a graph showing the radiance of fluorescent cells. [Figure 60] Figure 60 is a panel of graphs showing double and triple gene ablation by CRISPR / Cas9 to generate universal effector cells. HLA-I disruption by gRNA targeting B2M. [Figure 61] FIG. 61 is a flow diagram of the protocol for generating universal effector cells as described herein. [Figure 62] Figure 62 is a panel of graphs showing that TCR ablation abrogated nonspecific killing activity. 624mel-CBG and PC3-CBG tumor cell lines were incubated with T cells with or without PHA pretreatment at an effector-to-target ratio of 20:1 for 24 hours, and cytotoxicity was calculated based on luciferase assay. Data are mean ± SD; n=3. [Figure 63] Figure 63 is a panel of graphs showing IFNγ Elispot assays to measure TCR and TCR / HLA-disrupted alloreactivity by either loading gene-ablated T cells with irradiated allogeneic PBMC (left panel) or co-culturing allogeneic PBMC with irradiated and gene-ablated T cells. Individual spots are shown on the y-axis as spots generated in the presence of stimulator minus spots generated by effector alone. **P<0.01 by Mann-Whitney test. [Figure 64] Figure 64 is a panel of graphs showing that CRISPR / Cas9 disruption of endogenous TCR improved the function of TCR-redirected T cells. Expression of Vb13.1 and CD3 is shown in T cells transfected with Cas9 mRNA only (Cas9 mock) or in CD3neg T cells electroporated with NY-ESO-1 TCR α RNA (1G4 α, 2ug), β RNA (1G4 β, 2ug) or α+β RNA (1G4 α+β, 2+2ug) to disrupt endogenous TCR α only (α KO), β only (β KO), or both α and β (α+β KO). [Figure 65A] Figure 65A is a panel of graphs showing CD107a upregulation in TCR(1G4)α / β RNA electroporated TCR α or β single knockout or α + β double knockout T cells stimulated with HLA-A2 / NY-ESO-1 positive cell line (Nalm6-ESO) or control cell line Nalm6. [Figure 65B] FIG. 65B is a graph showing the lytic capacity of TCR α or β single knockout or α + β double knockout T cells electroporated with TCR α + β RNA (1G4 TCR) shown in (a) in a luciferase-based CTL assay against Nalm6-ESO. [Figure 66] Figure 66 is a panel of graphs showing Vβ and CD3 expression in TCR α+β double disrupted T cells (TCRneg T cells) electroporated with two different NY-ESO-1 TCR RNAs (1G4 TCR, 10ug or 8F TCR, 10ug) compared to control Cas9 Mock T cells. [Figure 67A] Figure 67A is a panel of graphs showing CD107a upregulation in NY-ESO-1 TCR (1G4 TCR or 8F TCR) RNA electroporated TCR double knockout CD8+ T cells stimulated with HLA-A2 / NY-ESO-1-positive cell lines Nalm6-ESO, 624-mel, or U266. Nalm6 was used as a negative control. [Figure 67B] Figure 67B is a panel of graphs showing cytokine production (IL-2 and TNF-α) of NY-ESO-1 TCR (1G4 TCR or 8F TCR) RNA electroporated TCR double knockout T cells after stimulation with HLA-A2 / NY-ESO-1-positive cell lines Nalm6-ESO or U266; 888mel melanoma cells were used as negative control. *P<0.05, **P<0.01 ****P<0.0001 by two-way ANOVA + Bonferroni post-hoc test. [Figure 68] Figure 68 is a panel of images showing the generation of universal CART cells by combining lentiviral gene transfer with CRISPR / Cas9 electroporation. A flow diagram of the generation of universal CD19-CART cells is shown. T cells were transduced with lentiviral CD19-CAR on day 1 after stimulation, and two days later, T cells were electroporated with Cas9 mRNA and gRNA targeting TCR alpha and beta chains and B2M. The TCR and HLA-I double negative cell population was enriched and then restimulated for expansion. [Figure 69] Figure 69 is a panel of graphs showing CD19-CAR expression in genetically modified lenti-CD19-CAR T cells expanded by CD3 / CD28 bead stimulation after 1G4 TCR electroporation. [Figure 70] Figure 70 is a panel of graphs showing the phenotype of CD19-CAR T cells. [Figure 71] Figure 71 is a graph showing CD107a release in TCR-negative and TCR / HLA-I double-negative CD19-CAR T cells. Representative data from three independent experiments are shown. Bars, SE = standard error. [Figure 72] Figure 72 is a panel of graphs showing cytokine secretion of TCR-negative and TCR / HLA-I double-negative CD19-CAR T cells. Representative data from three independent experiments is shown. Bars, SE = standard error. [Figure 73] Figure 73 is a graph showing the tumor lytic potential of TCR-negative and TCR / HLA-I double-negative CD19-CAR T cells. Representative data from three independent experiments are shown. Bars, SE = standard error. [Figure 74] Figure 74 is a panel of graphs showing CFSE-labeled CD19-CAR T cells and non-transduced T cells incubated with K562 and target K562-CD19 tumor cells at a ratio of 1 to 10 for 72 hours. [Fig. 75A]Figure 75A is a graph showing BLI from mice that received a single injection expressing CD19-CAR and GFP using lentiviral vectors on day 7. ns, no difference by two-way ANOVA+Bonferroni post-hoc test. Tumors were established in NSG mice (n=4 per group) by intravenous injection of 1x106 Nalm6 cells. Starting on day 7, T cells (1x107) expressing CD19-CAR transduced by lentivirus (LV) were injected by a single injection. T cells expressing LV GFP protein were injected as a control. [Fig. 75B] Figure 75B is a graph showing overall survival of mice receiving LV-GFP T cells, LV-CD19-CAR T cells, LV-CD19-CAR-TCR / CD3neg T cells, and LV-CD19-CAR-TCR / HLA-Ineg T cells. ns, no difference by log-rank Mantel-Cox test. [Figure 76] Figure 76 is a panel of images showing that genetically modified CAR T cells maintained antitumor efficacy and did not induce GVHD. Tumors were established in NSG mice (n=4 per group) by intravenous injection of 1x106 Nalm6 cells. Starting on day 7, T cells expressing LV-CD19-CAR (2x107) were injected by a single injection. T cells expressing LV GFP protein were used as a control. Imaging began 1 day before the start of T cell administration. Organs of randomly selected mice from different treatment groups were harvested on day 65 and used for CD3 immunohistochemical staining. [Figure 77] Figure 77 is a series of vector diagrams showing the design of pAd5F35-CRISPR targeting PD1, Fas and the TCR α chain. [Figure 78] FIG. 78 is an illustration showing the design of penton-modified pAd5F35-CRISPR with anti-CD3 ScFv and schematic delivery of pAd5F35-CRISPR for knock-in / out of chimeric antigen receptor into T cells in vitro and in vivo. [Figure 79]Figure 79A is a graph showing Sanger sequencing of PCR products flanking the PD1-gRNA targeting site. Adenovirus-pAd5F35-CRISPR-PD1 virus was transduced into MD231 cells. After 3 days, genomic DNA was extracted and PCR was performed. Figure 79B shows the sequence of the targeting event in MDA231 cells after adenovirus-CRISPR manipulation. PD1 PCR products were cloned into TOPO vector and sequenced. [Figure 80] Figure 80 is a chart showing that reduced gRNA use resulted in improved T cell expansion fold with only a slight decrease in knockout efficiency. [Figure 81] Figure 81 is a chart showing the parameters used to optimize electroporation conditions to obtain high CD3 / B2M knockout efficiency with improved T cell expansion fold. Compared to standard electroporation (EP) conditions in 2 mm cuvettes (EP#10-13) or 4 mm cuvettes. High CD3 / B2M knockout efficiency was observed with improved T cell expansion fold (EP#1 and 5). [Figure 82] FIG. 82 is a chart showing optimization of EP conditions to achieve maximum expansion fold with acceptable knockout efficiency. [Figure 83] Figure 83 is a chart showing further optimization of EP conditions to achieve maximum expansion fold with acceptable knockout efficiency. [Figure 84] Figure 84 is a schematic diagram of the T cell stimulation, lentiviral transduction and CRISPR electroporation procedures. [Figure 85] FIG. 85 is a chart showing T cell numbers (upper chart) and expansion fold (lower chart) following electroporation and culture procedures. [Figure 86]Figure 86 is a panel of graphs showing the mean T cell expansion. Fold expansion (left graph) of T cells transduced with CD19 CAR only (TD only) or T cells transduced with CD19 CAR and CRISPR edited (TD / KO). Fold expansion of T cells at day 10 is shown in the right graph. [Figure 87] Figure 87 is a panel of flow graphs showing CD3 / B2M / CAR expression on day 8 of expanded T cells. [Figure 88] Figure 88 is a panel of graphs showing CD3 / B2M expression after CD3+ T cell depletion. [Figure 89] Figure 89 is a panel of graphs showing CD3 / B2M expression in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells at day 11. Non-transduced ND463 (NOTD) was used as a negative control. [Figure 90] Figure 90 is a panel of graphs showing CD19 CAR expression in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells at day 11. Non-transduced ND463 (NOTD) was used as a negative control. [Figure 91] Figure 91 is a panel of graphs showing CD3 / B2M / CAR expression in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells at day 11. Non-transduced ND463 (NOTD) was used as a negative control. [Figure 92]Figure 92 is a chart summarizing CD3 / B2M / CAR expression in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells. [Figure 93] Figure 93 is a panel of graphs showing CD107a upregulation in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells. [Figure 94] Figure 94 is a panel of graphs showing T cell lytic activity on day 11. [Figure 95] Figure 95 is a panel of graphs showing cytokine production of T cells on day 11. [Figure 96] Figure 96 is a panel of graphs showing T cell expansion. No aberrant T cell proliferation was observed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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 of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0032] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0033] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object 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% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as such variations are appropriate for performing the disclosed methods.

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

[0036] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or may be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as 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 refers to the antigen-determining variable region of the 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 all antibody molecules in their naturally occurring conformations.

[0039] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. The α and β light chains refer to the two major antibody light chain isotypes.

[0040] The term "synthetic antibody" as used herein means an antibody made using recombinant DNA technology, such as, for example, the bacteriophage expressed antibodies described herein. The term should also be taken to mean an antibody made by synthesis of an antibody-encoding DNA molecule expressing an antibody protein or an amino acid sequence defining that antibody, the DNA or amino acid sequence being obtained using synthetic techniques of DNA or amino acid sequences that are available and well known in the art.

[0041] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production or activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely 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 the desired immune response. Furthermore, those skilled 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] The term "anti-tumor effect" as used herein refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present invention in preventing the development of tumors in the first place.

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

[0044] The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune disease is the result of an inappropriate and excessive response to an autoantigen. 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 mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, among others.

[0045] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into that individual.

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

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

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

[0049] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial T cell receptor that is engineered to be expressed on immune effector cells and 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, CARs are expressed with specificity for, for example, a tumor-associated antigen. CARs can also include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen binding region. In some aspects, CARs include 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, CARs can target cancer by redirecting the specificity of T cells expressing the CAR specific for a tumor-associated antigen.

[0050] The term "cleavage" refers to the cleavage of covalent bonds, 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 breaks are possible. Double-strand breaks can occur as a result of two different single-strand break events. DNA cleavage can result in the generation of either blunt ends or staggered ends. In some embodiments, fusion polypeptides can be used to target cleaved double-stranded DNA.

[0051] As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or change the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody of the present 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 have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for antigen binding ability using the functional assays described herein.

[0052] "Costimulatory ligand," as that term is used herein, includes 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 signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by binding of, e.g., 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 beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, among others, antibodies that specifically bind to costimulatory molecules present on T cells such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

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

[0054] As used herein, a "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 up- or down-regulation of key molecules.

[0055] The term "CRISPR / CAS," "clustered regularly interspaced short palindromic repeats system," or "CRISPR" refers to DNA loci that contain short repeats of a base sequence. Each repeat is followed by a short segment of spacer DNA from previous 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 adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage.

[0056] In type II CRISPR / Cas systems, short segments of exogenous DNA, termed "spacers," are integrated 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 Cas9 proteins 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 guide Cas9 to cleave a sequence of interest, a crRNA-tracrRNA fusion transcript, hereafter referred to as "guide RNA" or "gRNA", can be designed from a human U6 polymerase III promoter. CRISPR / CAS-mediated genome editing and regulation has highlighted its transformative potential for basic science, cell engineering, and therapeutics.

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

[0059] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

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

[0061] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable 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, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence 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, the nucleotide sequence of which is identical to the mRNA sequence and which is usually shown in a 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 a protein or other product of that gene or cDNA.

[0063] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue or system.

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

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

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

[0067] "Expression vector" refers to a vector that contains a recombinant polynucleotide that contains an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements 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) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) that incorporate recombinant polynucleotides.

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

[0069] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody comprises 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 those of a human immunoglobulin sequence. A humanized antibody also optimally comprises at least a portion of an 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, where the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody.

[0071] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, such as between two polypeptide molecules, particularly between two amino acid molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The degree or identity of two amino acid sequences having 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 direct function of the number of positions that are matched or identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer 10 amino acids long) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0072] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but may act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering 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 the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0074] As used herein, "induced pluripotent stem cell" or "iPS cell" refers to pluripotent stem cells generated from adult cells such as T cells. Expression of reprogramming factors such as Klf4, Oct3 / 4 and Sox2 in adult cells converts the cells into pluripotent cells that can proliferate and differentiate 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 communicate the utility of the compositions and methods of the invention. The instructional materials of the kits of the invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the invention, or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the instructional materials and the compounds are used conjointly by the recipient.

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

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

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

[0079] "Lentivirus" as used herein refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors.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] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of a nucleic acid.

[0081] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of a response in a subject, compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses disrupting 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 versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding an RNA or protein can also include introns to the extent that a nucleotide sequence encoding a protein, depending on the type, may contain introns.

[0084] The term "operably linked" refers to a functional link 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 operably linked to a second nucleic acid sequence if the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0085] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in cells from a disease area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0086] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0087] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides, which 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 means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., as well as synthetic means.

[0088] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds 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 may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, e.g., commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

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

[0090] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for 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 an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that causes 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 in a cell 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 in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

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

[0094] "Sendai virus" refers to a genus in the family Paramyxoviridae. Sendai virus is a negative single-stranded RNA virus that does not integrate into the host genome or alter the genetic information of the host cell. Sendai virus has a remarkably broad host range and is not pathogenic to humans. Sendai virus is used as a recombinant viral vector and is capable of transient but strong gene expression.

[0095] A "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0096] "Single chain antibody" refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region through an engineered amino acid stretch.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 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0097] The term "specifically binds" as used herein with respect to an antibody 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-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may bind to different allelic forms of that antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" may be used in relation to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure rather than the entire protein. If an antibody is specific for epitope "A", then 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 involving labeled "A" and that antibody.

[0098] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or rearrangement of cytoskeletal structures.

[0099] "Stimulatory molecule," as that term is used herein, 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" refers to 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, thereby mediating 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 peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0101] The term "subject" is intended to include organisms in which an immune response can be elicited (e.g., mammals). As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include farm animals and pets, such as, for example, ovine, bovine, porcine, canine, feline 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. A substantially purified cell also refers to a cell that is separated from other cell types that it is normally associated with in its natural state. In some instances, a population of substantially purified cells refers to a homogenous cell population. In other instances, the term simply refers to a cell that is separated from the cells that it is normally associated with in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0103] "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 the presentation of antigen. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (a) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γ / δ) chains. TCRs 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: a variable domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0105] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0106] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced 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 cell includes the primary subject cell and its progeny.

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

[0108] As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and 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 to the inside of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated 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 transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0110] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a 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. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0111] explanation Universal T cells to avoid graft-versus-host disease (GVHD) are highly desirable in clinical situations. However, the use of allogeneic T cells carries risks due to rejection by the host's immune system through the recognition of HLA-A molecules. Targeting strategies for manipulating multiple genes are complex, and efforts have only achieved low efficiency in T cells, and have failed to simultaneously prevent GVHD and host-versus-graft reactions.

[0112] The FAS receptor / FAS ligand (FAS / FASL) apoptotic signaling pathway negatively regulates T cell function. PD1 and CTLA4 are two major inhibitory signaling pathways in T cells. Enhanced antitumor immunity resulting from antibody-mediated blockade of CTLA-4, PD-1 or PD-L1 suggests that inhibiting these pathways may improve the efficiency of immunotherapy. The present invention includes the generation of modified T cells with ablation of TCR α and β chains, β-2 microglobulin, HLA molecules, CTLA-4, PD-1, and / or FAS as a means to generate modified T cells with reduced immunogenicity.

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

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

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

[0116] In one aspect, the nucleic acid capable of downregulating endogenous gene expression is introduced into the T cell, such as by electroporation, transfection, or lentiviral or other viral transduction. In another aspect, the present invention includes modified T cells comprising electroporated nucleic acid capable of downregulating endogenous gene expression. In yet another aspect, the modified T cell comprises electroporated nucleic acid capable of downregulating endogenous TCR gene expression. In another aspect, a composition comprising modified T cells is produced according to the methods described herein. In yet another aspect, the present invention includes pharmaceutical compositions 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 pharma- ceutically acceptable carrier.

[0117] The nucleic acid that can regulate endogenous gene expression can downregulate endogenous gene expression.In one embodiment, the nucleic acid that can downregulate endogenous gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA and CRISPR system.Endogenous gene expression can be downregulated, knocked down, reduced and / or inhibited, for example, by antisense RNA, antigomer RNA, siRNA, shRNA, CRISPR system, etc.

[0118] CRISPR / Cas The CRISPR / Cas system is a facile 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 simultaneously target multiple genomic loci by co-expressing a single CAS9 protein with two or more gRNAs, making this system uniquely suitable for multigene editing or synergistic activation of target genes.

[0119] CRISPRi, an example of a CRISPR / Cas system used to inhibit gene expression, is described in US Patent Application Publication No. 2014 / 0068797. CRISPRi induces permanent gene disruption utilizing RNA-guided Cas9 endonuclease to introduce DNA double-strand breaks that 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 silences the expression of targeted genes.

[0120] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell to form a complex that allows the Cas endonuclease to introduce a double-stranded break at the target gene. In one embodiment, the CRISPR system comprises an expression vector, such as, but not limited to, the pAd5F35-CRISPR vector. In one embodiment, the modified T cell is generated by introducing a Cas expression vector and a guide nucleic acid sequence specific for a gene into a T cell. 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, other nucleases known in the art, and any combination thereof.

[0121] In one embodiment, the induction of the Cas expression vector comprises exposing the T cell to an agent that activates an inducible promoter in the Cas expression vector. In such an embodiment, the Cas expression vector comprises an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., 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 induction of the inducible promoter. This results in expression of the Cas expression vector.

[0122] The guide nucleic acid sequence is specific to a gene and targets the gene for Cas endonuclease-induced double-stranded break.The sequence of the guide nucleic acid sequence can be within the locus of the 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 believed to reduce immunogenicity or reduce susceptibility to an immunosuppressive microenvironment. In one embodiment, the gene may include a sequence specific for a T cell receptor (TCR) chain (such as alpha, beta, gamma, and / or delta chain), beta-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 complex sequence), or a sequence having synthetic nucleotides. The guide nucleic acid sequence can 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 antigen-specific TCRs has therapeutic potential in the treatment of cancer and certain chronic viral infections. Engineering T cells with specific TCRs has the advantage of redirecting T cells against intracellular antigens. Given that most oncogenic proteins are intracellular, the development of a panel of TCRs specific for oncogenic driver proteins is highly attractive.

[0126] The invention also includes modified T cells having downregulated gene expression as described herein and an exogenous T cell receptor (TCR). In one aspect, the invention includes a method for making a modified T cell comprising introducing into a T cell a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for a surface antigen on a target cell, and a nucleic acid capable of regulating endogenous gene expression selected from the group consisting of TCR alpha chain, TCR beta chain, beta-2 microglobulin, PD1 and FAS, wherein the T cell is capable of expressing the modified TCR.

[0127] In another aspect, the invention includes a modified T cell comprising a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for a surface antigen on an exogenous target cell, and a nucleic acid capable of downregulating endogenous gene expression selected from the group consisting of TCR alpha chain, TCR beta chain, beta-2 microglobulin, PD1 and FAS, wherein the T cell expresses the modified TCR and endogenous gene expression is downregulated in the T cell. The 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 involved in the activation of T cells in response to the presentation of antigen. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex, inducing a series of intracellular signaling cascades.

[0129] TCRs are generally composed of six different membrane-bound chains that form TCR heterodimers that are 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, TCRs comprise TCR α chains and TCR β chains, and thus the nucleic acid encoding a TCR comprises the nucleic acid encoding a TCR α chain and TCR β chain. In another embodiment, the TCR α chain or TCR β chain or both chains comprise at least one N-deglycosylation.

[0130] Each chain is composed of two extracellular domains, a variable domain and a constant domain. In one embodiment, the TCR comprises at least one mouse 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 comprises a costimulatory signaling domain, such as a 4-1BB costimulatory signaling domain. The variable domain contributes to the determination of the specific antigen and MHC molecule that the TCR has binding specificity for. The specificity of a T cell for a unique antigen-MHC complex then resides in the specific TCR expressed by the T cell.

[0131] Each of the constant and variable domains may contain intrachain disulfide bonds. In one embodiment, the TCR contains 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 generated through somatic rearrangement of linked variable (V), diversity (D), joining (J) and constant region genes.

[0132] Functional α and γ chain polypeptides are formed by rearranged VJC regions, whereas β and δ chains consist of VDJC regions. The extracellular constant domain contains a membrane proximal region and an immunoglobulin region.

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

[0134] In one embodiment, the TCR comprises specificity for a target cell antigen. The target cell surface antigen may comprise any type of ligand that defines the surface of a target cell. For example, the target cell surface antigen may be selected to recognize a ligand that serves as a cell surface marker on target cells that is 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 embodiment, the target cell surface antigen comprises any tumor-associated antigen (TAA) and viral antigen, disease cell-associated antigen, or any fragment thereof.

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

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

[0137] Techniques for engineering and expression of T cell receptors include, but are not limited to, the generation of TCR heterodimers containing native disulfide bridges connecting 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 receptors (CARs) The present invention also includes modified T cells having downregulated gene expression as described herein and a CAR. Thus, the present invention encompasses modified T cells comprising a CAR or a nucleic acid encoding a CAR, where the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0139] In one aspect, the invention includes a method of making a modified T cell comprising introducing into a T cell a nucleic acid capable of downregulating endogenous gene expression selected from the group consisting of TCR alpha chain, TCR beta chain, beta-2 microglobulin, an HLA molecule, CTLA-4, PD1 and FAS, and introducing into a T cell a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the antigen-binding domain, transmembrane domain and intracellular domain of a costimulatory molecule.

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

[0141] One or more domains of CAR, or fragments of domains, can be human. In one embodiment, the present invention includes a fully human CAR. The nucleic acid sequence encoding the desired domain can be obtained by recombinant methods known in the art, such as by screening a library from cells expressing the gene, by deriving the gene from a vector known to contain it, or by directly isolating it from cells and tissues that contain it, using standard techniques. Alternatively, the gene of interest can be produced 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 incorporated by reference herein in their entireties.

[0143] Antigen-binding domain In one embodiment, the CAR comprises an antigen binding domain that binds to an antigen on a 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 will depend 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 the target cell associated with a particular disease state.

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

[0146] The antigen-binding domain can comprise any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and fragments thereof. Thus, in one embodiment, 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, CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to 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 (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); Louis Y antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific fetal 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; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100);Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; 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 beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulatory receptor β (SEQ ID NO: 1); stimulatory 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 portion of globoH glycoceramide (GloboH);mammary differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cell receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3);pannexin 3 (PANX3);G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K9 (LY6K);olfactory receptor 51E2 (OR51E2);TCR gamma-alternative 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; prostein; 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) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; 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 myelocytoma 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 anchoring 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); legumain; human papilloma virus E6 (HPV E6); human papilloma virus 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 the one in which the CAR will ultimately be used. For example, for human use, it may be 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] It is also beneficial that the antigen binding domain is operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, both of which are described elsewhere herein, for expression in a cell. In one embodiment, the nucleic acid encoding the antigen binding domain is operably linked to the nucleic acid encoding the transmembrane domain and the nucleic acid encoding the intracellular domain.

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

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

[0152] In one embodiment, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0153] Intracellular domain The intracellular domain, or alternatively, the cytoplasmic domain, of CAR is responsible for the activation of the cell in which the CAR is expressed. The term "intracellular domain" is therefore meant to include any portion of the intracellular domain that is sufficient to transmit an activation signal. In one embodiment, the intracellular domain includes a domain that is responsible for effector function. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

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

[0155] Examples of intracellular domains for use in the present invention include the cytoplasmic portion of T cell receptor (TCR) and any co-stimulatory molecule that act in concert to initiate signal transduction after antigen receptor engagement, as well as any derivative or mutant of these elements and any synthetic sequence that has the same functional ability.In one embodiment, the intracellular domain of CAR comprises a dual signal transduction domain.The dual signal transduction domain can comprise any fragment or domain from any of the molecules described herein.

[0156] Examples of intracellular domains include TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (Fcε R1b), 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, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SL AMF7, 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, ITG B2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CR TAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BL These include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AME (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 capability, any synthetic sequence of a costimulatory molecule, and any combination thereof.

[0157] In one embodiment, the intracellular domain of the CAR comprises any portion of a costimulatory molecule, e.g., at least one signaling domain from CD3, CD27, CD28, ICOS, 4-1BB, PD-1, the T cell receptor (TCR), any derivative or variant thereof having the same functional capability, 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 a 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, and most preferably 25-50 amino acids. In another embodiment, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular domain of the CAR. An example of a linker includes a glycine-serine doublet.

[0159] Human antibodies When using the antigen-binding domain of bispecific antibody or CAR, it may be preferable to use human antibody or its fragment.For therapeutic treatment of human subjects, fully human antibody is particularly desirable.Human antibody can be produced by various methods known in the art, including phage display method using antibody library derived from human immunoglobulin sequence, together with the improvement of these methods.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] Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, such as the whole or a portion of a polypeptide of the invention. Antibodies directed against a selected target can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, such techniques can be used to produce therapeutically useful IgG, IgA, IgM and IgE antibodies, including but not limited to IgG1 (gamma 1) and IgG3. For a review 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, as well as protocols for producing such antibodies, see, e.g., 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 incorporated herein by reference in its entirety. Additionally, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be contracted to obtain human antibodies directed against a selected antigen using technology similar to that described above. For specific discussion of the transfer of human germ-line immunoglobulin gene arrays into germ-line mutant mice, which would result in the production of human antibodies upon antigen challenge, see, e.g., 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 immunoglobulin variable (V) domain gene repertoires of unimmunized donors. According to this approach, antibody V domain genes are cloned in frame into major or minor coat protein genes of filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats; for reviews, 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.Repertoires of V genes can be constructed from unimmunized human donors and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the procedures 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, each of which is incorporated herein by reference in its entirety.

[0162] Human antibodies can also be produced by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety.) Human antibodies can also be produced in vitro using hybridoma techniques such as, but not limited to, those described by Roder et al. (Methods Enzymol, 121:140-167 (1986)).

[0163] Humanized antibodies Alternatively, in some embodiments, non-human antibodies can be humanized, in which certain sequences or regions of the antibody are modified to increase similarity to antibodies naturally produced in humans. For example, in the present invention, the antibody or fragment thereof can comprise 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. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated 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 by reference in its entirety), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332).No. 6,407,213; U.S. Pat. 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 JS, 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. Often, framework residues in the framework regions will be replaced with the corresponding residue from the CDR donor antibody to alter, and preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of the CDRs and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, each of which is incorporated herein by reference in its entirety).

[0165] A humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. A humanized antibody thus comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Antibody humanization is well known in the art and essentially involves substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR grafting (EP 239,400; PCT Publication No. WO 2004 / 0231112). 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, the contents of which are incorporated herein by reference in their entireties. In such humanized chimeric antibodies, substantially less than an intact human variable domain is substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also 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), the contents of which are incorporated herein by reference in their entireties.

[0166] The selection of both light and heavy human variable domains used in making humanized antibodies aims to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of rodent antibodies is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of rodent is then 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 incorporated herein by reference in their entirety). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. 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 incorporated herein by reference in their entireties).

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

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

[0169] Other molecules The present invention also includes modified T cells described herein that include a costimulatory molecule or a nucleic acid encoding a costimulatory molecule. In one embodiment, the modified T cells of the present invention further include 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, transfecting, or electroporating the T cells. In another embodiment, the costimulatory molecule is selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In another embodiment, the costimulatory molecule includes CD3 and includes at least two different CD3 chains, such as CD3 zeta chain and CD3 epsilon chain.

[0170] In another embodiment, the modified T cell further comprises Klf4, Oct3 / 4 and / or Sox2, or a nucleic acid encoding Klf4, Oct3 / 4 and / or Sox2 to induce pluripotency of the T cell. The T cell 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 embodiment, a viral vector encoding Klf4, Oct3 / 4 and Sox2 is introduced into the T cell to induce pluripotency. In another embodiment, a Sendai virus vector is introduced into the T cell to induce pluripotency, where the Sendai virus vector encodes Klf4, Oct3 / 4 and Sox2.

[0171] Nucleic acid introduction Methods for introducing nucleic acid 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 biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0172] Biological methods for introducing polynucleotide of interest into host cell 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, e.g., human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus, etc.See, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362.

[0173] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0174] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl 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; dimyristyl 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. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vehicles formed by the formation of sealed 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 aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have a different structure in solution than the normal vesicle structure are also encompassed. For example, lipids may adopt 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 acid into host cells or to otherwise expose cells to the inhibitor of the present invention, various assay methods can be carried out to confirm the presence of nucleic acid in host cells.Such assay methods include "molecular biological" 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 specific peptides by immunological means (ELISA and Western blot) or by the assay methods described herein to identify agents that fall within the scope of the present invention.

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

[0177] Furthermore, the nucleic acid can be introduced by any means, such as transduction of the expanded T cells, transfection of the expanded T cells, and electroporation of the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid to be introduced into the T cells may 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 polymerase chain reaction (PCR) generated template. The 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 sequence or any other suitable DNA source. The desired template for in vitro transcription is a chimeric membrane protein. As an example, the template encodes an antibody, an antibody fragment or a portion of an antibody. As another example, the template includes an extracellular domain, including a single chain variable domain of an antibody, such as anti-CD3, and an intracellular domain of a costimulatory molecule. In one embodiment, the RNA chimeric membrane protein template encodes a chimeric membrane protein comprising an extracellular domain comprising an antigen-binding domain derived from an antibody against a costimulatory molecule, and an intracellular domain derived from a portion of the intracellular domain of CD28 and 4-1BB.

[0179] PCR can be used to generate templates for in vitro mRNA transcription, which are then introduced into cells. Methods for carrying out PCR are well known in the art. Primers for use in PCR are designed to have a region that is substantially complementary to the region of DNA used as a 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 with 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 the portion of a gene that is normally transcribed in cells (open reading frame), including the 5' and 3' UTR. Primers can also be designed to amplify a portion of a gene that codes for a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or a portion of the 5' and 3' UTR. Primers useful for PCR are made by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position 5' of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that are substantially complementary to a double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position 3' of the DNA sequence to be amplified relative to the coding strand.

[0180] Chemical structures with 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 0-3000 nucleotides in length. The length 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 technique, one skilled in the art can vary the length 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, the UTR sequence that is not endogenous to the gene of interest can be added by incorporating the UTR sequence into the forward and reverse primers, or by any other modification of the template. The use of the UTR sequence that is not endogenous to the gene of interest can be useful for changing the stability and / or translation efficiency of RNA. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce the stability of mRNA. Therefore, the 3' UTR can be selected or designed to increase the stability of transcribed RNA based on the characteristics of UTRs that are well known in the art.

[0182] In one embodiment, the 5' UTR can include the Kozak sequence of the endogenous gene. Alternatively, if 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 increase the translation efficiency of some RNA transcripts, but does not seem to be required for all RNAs to allow efficient translation. The need for the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of mRNA.

[0183] To allow synthesis of RNA from a DNA template without the need for gene cloning, a transcription promoter should be added upstream of the sequence to be transcribed relative to the DNA template. If 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 open reading frame to be transcribed. In one embodiment, 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 T7, T3 and SP6 promoters are known in the art.

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

[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 traditional method for the incorporation of polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, since plasmid DNA templates obtained from bacterial cells are often highly compromised with deletions and other abnormalities. This makes cloning procedures not only laborious and time-consuming, but also often unreliable. That is why a method that allows the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0187] The polyA / T segment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail, such as a 100T tail (size can be 50-5000 T), or 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 the RNA and reduces RNA degradation. In general, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100-5000 adenosines.

[0188] The poly(A) tail of the RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as Escherichia coli (E. coli) polyA 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 the RNA by about 2-fold. Furthermore, 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 poly(A) polymerase. The ATP analogs can further increase the stability of the RNA.

[0189] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and 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] The RNA produced by the method disclosed herein can also include 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 with mRNA and facilitates the initiation of translation.Can include any solute suitable for cell electroporation, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

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

[0192] The disclosed methods can be applied to modulating T cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infectious diseases, and autoimmune diseases, including assessing the ability of engineered T cells to kill targeted cancer cells.

[0193] This method also provides the ability to control expression levels over a wide range, for example by varying the amount of promoter or input RNA, allowing expression levels to be individually regulated. Furthermore, the technique of PCR-based mRNA production greatly facilitates 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. RNA transgenes can be delivered to lymphocytes as minimal expression cassettes without the need for any additional viral sequences and expressed therein after simple in vitro cell activation. Under these conditions, integration of transgenes into host cell genome is unlikely to occur. Due to the efficiency of RNA transfection and its ability to uniformly modify the entire lymphocyte population, cell cloning is not required.

[0195] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been tested sequentially in various animal models: 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, which are utilized in a standardized manner as templates for in vitro transcription and genetically engineered to produce stabilized RNA transcripts. Currently, protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest flanked on either the 3' and / or 5' sides by untranslated regions (UTRs), 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). The polyadenylation cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced in cells from such constructs.

[0197] RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. In addition, RNA only needs access to the cytoplasm, not the nucleus, and therefore typical transfection methods provide extremely high transfection rates. Furthermore, plasmid-based approaches require that the promoter driving the expression of the gene of interest is active in the cells under study.

[0198] In another aspect, RNA construct is delivered to cell by electroporation.See, for example, the formulation and methodology of electroporation of nucleic acid construct into mammalian cell as taught in US2004 / 0014645, US2005 / 0052630A1, US2005 / 0070841A1, US2004 / 0059285A1, US2004 / 0092907A1.Various parameters including electric field strength required for electroporation of any known cell type are generally known in related research literature and many patents and applications in the art.See, for example, US6,678,556, US7,171,264, and US7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation can also be used for in vitro cell transfection, as described, for example, in U.S. Pat. No. 20070128708A1. Electroporation can also be used to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, to cells using any of the many available devices and electroporation systems known to those skilled in the art represents an exciting new means to deliver RNA of interest to target cells.

[0199] In one embodiment, the method includes electroporating RNA encoding the TCR alpha and beta chains. The TCR alpha and beta chains can be encoded on the same or separate RNAs. When alpha and beta are encoded by separate RNAs, the RNAs can be co-electroporated.

[0200] In another embodiment, the method may further comprise electroporating a nucleic acid encoding a costimulatory molecule. The costimulatory molecule nucleic acid may 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 a 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 embodiments, any number of T cell lines available in the art can be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis or leukapheresis transfusion. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium, such as phosphate buffered saline (PBS) or wash solution, which may be calcium-free and magnesium-free, or may be lacking many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in medium.

[0202] In another embodiment, 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 either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

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

[0204] Enrichment of T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers specific to negatively selected cells.Preferred methods are cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on 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 isolation of desired cell populations 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 beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of cells and beads. 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 further embodiments, more than 100 million cells / ml is used. In further embodiments, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used. Using a high concentration can result in increased cell yield, cell activation, and cell expansion.

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

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

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

[0209] The expression of the chimeric membrane protein allows interaction with other cells in the population, such as cells expressing CD3, which stimulates and activates the expansion of the electroporated T cells. Without wishing to be bound by any particular theory, it is believed that cells expressing CD3 come into contact with 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 the expansion of the electroporated T cells.

[0210] In one embodiment, the T cells are expanded prior to downregulation of the endogenous gene. In another embodiment, the 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. A costimulatory molecule includes any molecule that costimulates T cells, such as, but not limited to, CD3, CD28, or a combination thereof. In one embodiment, the extracellular domain may comprise an antigen-binding domain derived from anti-CD3, anti-CD28, or a combination thereof. In another embodiment, the extracellular domain comprises a single chain variable fragment (scFv) against CD3.

[0212] In another embodiment, the extracellular domain may comprise 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. In some cases, it is beneficial for the extracellular domain to be derived from the same species as the one in which the chimeric membrane protein will ultimately be used. For example, for human use purposes, it may be beneficial for the extracellular domain of the chimeric membrane protein to comprise a human antibody or a fragment thereof. Thus, in one embodiment, the extracellular domain portion comprises a human antibody, or a fragment thereof, as described elsewhere herein. Or, in some embodiments, the extracellular domain portion comprises a non-human antibody that has been humanized, as described elsewhere herein.

[0213] Intracellular domain The intracellular or cytoplasmic domain comprises 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 antigens.

[0214] The cytoplasmic domain or intracellular signaling domain of the chimeric membrane protein is responsible for activating at least one of the effector functions of the T cell. 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 truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits an effector function signal. The intracellular signaling domain therefore includes any truncated portion of the intracellular signaling domain that is sufficient to transmit an effector function signal.

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

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

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

[0218] T cell expansion As demonstrated by the data disclosed herein, the step of expanding T cells by the methods disclosed herein can result in an increase of 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 integers 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 the culture device in cell medium for a period of time or until the cells reach confluence or high cell density for optimal passaging, followed by passage to another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or more before the cells are passaged to another culture device. More preferably, the level of confluence is 90% or more. The period of time may be any time suitable for culturing cells in vitro. The replacement of the T cell medium may be performed at any time during the culture of the T cells. Preferably, the T cell medium is replaced about every 2-3 days. The T cells are then harvested from the culture device, whereupon the T cells can be used immediately or cryopreserved for storage for later use. In one embodiment, the invention includes a step of cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing the nucleic acid into the T cells.

[0220] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the invention further includes cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding the 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 in addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors such as those described in U.S. Patent No. 5,199,942, or other factors such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding T cells includes culturing T cells with factors selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0222] The culturing step described herein (following contact with an agent described herein or electroporation) may be very short, e.g., 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 culturing step described further herein (contact with an agent described herein) may be longer, e.g., 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 taken from an organism and grown under controlled conditions. Primary cell culture is the culture of cells, tissues, or organs taken directly from an organism and prior to the first subculture. Cells are expanded in culture when placed in a growth medium under conditions that promote cell growth and / or division, resulting in a larger population of cells. When cells are expanded in culture, the rate of cell growth is typically measured by the amount of time required for the cells to double, also known as doubling time.

[0224] Each time 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 times they have been passaged. For example, a cultured cell population that has been passaged 10 times may be referred to as a P10 culture. A primary culture, i.e., the first culture after cell isolation from 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 may be many population doublings during a passaging; therefore, the number of population doublings of a culture is greater than the number of passages. The expansion of cells during the period between passaging (i.e., the number of population doublings) depends on many factors, including, but not limited to, seeding density, substrate, medium, and time between passaging.

[0225] In one embodiment, the cells may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of time units therebetween. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) that may contain 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 additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media can include 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, either serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, and / or cytokines in amounts 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 infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0226] The medium used to culture the T cells may contain an agent capable of costimulating the 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 about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x, or more. In one embodiment, T cells are expanded within the range of about 20x to about 50x or more by culturing the electroporated population.

[0227] In one embodiment, the method includes introducing into the expanded T cells a nucleic acid encoding a T cell receptor (TCR) that has affinity for a surface antigen on a target cell, and electroporating into the T cells an RNA encoding a costimulatory molecule, wherein the electroporated T cells are capable of expressing the TCR and the costimulatory molecule.

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

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

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

[0231] In one aspect, the present 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 embodiment, the T cells are modified as described elsewhere herein. The modified T cells can be administered to induce lysis of a target cell or tissue, such as where the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0232] In another aspect, the invention includes 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 described herein to prevent or treat an immune response deleterious to the subject.

[0233] In yet another embodiment, a method of treating a disease or condition associated with immune enhancement in a subject comprises 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 generated as described herein can be homogenous and retain T cell functions. Furthermore, the modified T cells can be administered to animals, preferably mammals, and more preferably humans, to suppress immune responses, such as those common to autoimmune diseases, such as diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhanced allograft tolerance induction, and graft rejection. Furthermore, the cells of the present invention can be used to treat any condition in which a weakened or otherwise inhibited immune response, particularly a cell-mediated immune response, is desired to treat or alleviate the disease. In one aspect, the present invention includes treating a condition, such as an autoimmune disease, in a subject, comprising 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 immune deficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune disease of the inner ear (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-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, These include, but are not limited to, 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, and Wegener's granulomatosis.

[0236] The T cell 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 and ventilator-induced lung injury.

[0237] In another embodiment, 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 dosages and routes and for times determined in appropriate preclinical and clinical experiments and tests. The cell composition can be administered multiple times at dosages within these ranges. The administration of the cells of the present invention can be combined with other methods useful for treating the desired disease or condition as determined by one of skill in the art.

[0239] The cells of the invention that are administered can be autologous, allogeneic, or xenogeneic with respect to the subject undergoing treatment.

[0240] Administration of the cells of the invention may be performed in any convenient manner known to those of skill in the art. The cells of the invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In another example, the cells of the invention are directly injected into a site of inflammation in a subject, a site of local disease 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. The present invention utilizes such matrices in a novel context, acting as artificial lymphoid organs to support, maintain or regulate the immune system, typically by regulating T cells. Thus, the present invention can utilize matrix compositions and formulations that have demonstrated utility in tissue engineering. Thus, the types of matrices that can be used in the compositions, devices and methods of the present invention are virtually limitless and can include both biological and synthetic matrices. In one particular example, 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 thus these patents are incorporated herein by reference in their entirety. The matrix includes characteristics generally associated with being biocompatible when administered to a mammalian host. The matrix may be formed from natural and / or synthetic materials. The matrix may be non-biodegradable if it is desired to leave a permanent or removable structure in the animal's body, such as an implant; or it may be biodegradable. The matrix may take the form of a sponge, an implant, a tube, a telfa pad, a fiber, a hollow fiber, a lyophilized component, a gel, a powder, a porous composition, or a nanoparticle. Furthermore, the matrix may be designed to allow sustained release of seeded cells or produced cytokines or other active agents. In certain embodiments, the matrix of the present invention may be described as a semi-solid scaffold that is flexible and stretchable and permeable to substances such as inorganic salts, aqueous liquids, and dissolved gaseous agents, including oxygen.

[0242] A matrix is ​​used herein as an example of a biocompatible material, however, the invention is not limited to matrices and any reference to the term matrix should be read to include devices and other materials that retain or allow cells to traverse, are biocompatible, and allow macromolecules to traverse directly through the material, either being a semipermeable membrane in itself or being used in conjunction with a particular semipermeable material.

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

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

[0245] When an "immunologically effective amount," "anti-immune response effective amount," "immune response inhibiting effective amount," 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). 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6It can be generally stated that the T cell composition can be administered at a dose of 10000 cells / kg body weight, including all integer values ​​within these ranges. The T cell composition can also be administered multiple times at these doses. The cells can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the medical arts by monitoring the patient for symptoms of disease and adjusting the treatment accordingly.

[0246] In certain embodiments, it may be desirable to administer activated T cells to a subject, then draw blood again (or perform apheresis), activate the T cells therefrom according to the present invention, and reinfuse these activated and expanded T cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, T cells can be activated from a blood draw of 10 ml to 400 ml. In certain embodiments, 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 wishing to be bound by theory, this multiple blood draw / multiple reinfusion protocol can be used to single out certain populations of T cells.

[0247] In certain embodiments of the invention, cells expanded and modified using the methods described herein, or other methods known in the art whereby T cells are expanded to therapeutic levels, are administered to a patient along with (e.g., prior to, concurrently with, or following) a number of relevant treatments, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be combined with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (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 composition of the present invention is administered to a patient together with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell depletion therapy with chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B cell depletion therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation.In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present 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 exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to art-accepted practices. Doses of CAMPATH, for example, typically range from 1 to about 100 mg for adult patients and are usually administered daily for 1 to 30 days. A preferred daily dose is 1 to 10 mg / day, although in some cases higher doses of up to 40 mg / day may be used (as described in U.S. Pat. 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 provided to provide those of skill in the art with a complete disclosure and description of the methods of making and using the cells, the expansion and culture methods, and the treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0250] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the scope of those skilled in the art. Such techniques are well explained 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 therefore may be considered in making and practicing the present invention. Techniques that are particularly useful for certain embodiments are discussed in the following sections. EXAMPLES

[0251] Experimental Examples The present invention will be described in further 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. Therefore, the present invention should not be construed as being limited to the following examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0252] Without further description, it is believed that one of ordinary skill in the art can, using the above description and the following examples, make and utilize the compounds of the present invention, and practice the methods claimed. Accordingly, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[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 stimulatory antibodies (Life Technologies, Grand Island, NY, catalogue) as described (Human gene therapy 2011, 22(12):1575-1586). On day 10, T cells were cultured at 1 × 10 in a solution of 90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO). 8 Cells / vial were stored frozen.

[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. 624mel melanoma line was purchased from the Surgery Branch (NCI / NIH). All cell lines were cultured as indicated and routinely tested negative for mycoplasma contamination.

[0256] Preparation of TCR constructs for mRNA electroporation and lentiviral transduction: Based on the sequencing information obtained from related publications, 1G4 NY-ESO-1 TCRs with various mutations (1G4 and 8F) and CARs (PSCA or CD19) were synthesized and / or amplified by PCR (The Journal of experimental medicine 2005, 201(8):1243-1255; J Immunol 2008, 180(9):6116-6131) and subcloned into pGEM.64A RNA-based vector or pTRPE lentiviral vector.

[0257] Preparation of human primary T cells: Primary human CD4 and CD8 T cells were isolated from healthy volunteer donors after leukapheresis with 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] CRISPR design and construction: Cas9 DNA was synthesized by PCR and subsequently inserted into the PGEM vector. gRNAs were selected with an NGG PAM site from GN19 and some with an NGG PAM site from N20. All gRNAs contained complementary sequences consisting of mismatches of more than 13 base pairs, and potential 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 microglobin. gRNAs were designed to target either sequences within exon 1 of the TCR α constant region, a consensus sequence common to exon 1 of both TCR β constant regions 1 and 2, β-2 microglobulin or PD1. The gRNA coding sequence was assembled using overlap PCR and cloned into the MSGV vector containing a T7 promoter. These plasmids were linearized with EcoRI. gRNAs were in vitro transcribed. Cas9 mRNA was in vitro transcribed using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, Carlsbad, CA). mRNA was stored at -80°C in nuclease-free vials for single use. The gRNA targeting sequence used for animal testing is as follows: TIFF0007657474000001.tif34128

[0259] Flow cytometry: The following monoclonal antibodies and reagents with the indicated specificities were used with appropriate isotype controls: APC-conjugated anti-CD3 (555335), FITC-anti-CD8 (555366), PE-anti-CD8 (555635), FITC-anti-CD27 (555440), PE-anti-CD107 (555801), PE-anti-β-2 microglobin (551337), FITC-anti-HLA (555552) from BD Biosciences (San Jose, CA); FITC-anti-CD45RO (304204), APC-anti-CD62L (304814), APC-anti-CCR7 (353214) from Biolegend; and PE-anti-Vb13.1 (IM2021U) from Beckman Coulter (Pasadena, CA). Data were acquired by FACS Accuri (BD Biosciences, San Jose, CA) using CellQuest version 3.3 (BD ​​Biosciences, San Jose, CA) and analyzed by FCS Express version 3.00 (De Novo Software, Los Angeles, CA) or FlowJo version 7.6.1 (Tree Star, Inc. Ashland, OR).

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

[0261] CD3 neg Generation of T cells: DNA supercoiled plasmids were linearized by SpeI and EcoRI, respectively. gRNAs were in vitro transcribed by 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 mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, AM1345, Carlsbad, CA). T cells were stimulated with CD3 / CD28 Dynabeads for 3 days prior to electroporation. Ten million primary T cells were debeaded and then electrotransferred with 20 μg Cas9, 10 μg gRNA seeds into cells with BTX830 at 360V, 1 ms parameters, followed by a second and / or third electrotransfer of 10 μg gRNA. Furthermore, T cells were washed three times with OPTI-MEM and then cultured in OPTI-MEM (Invitrogen) at 1–3 × 10 cells. 8 The cells were resuspended at a final concentration of 50 ...

[0262] After electroporation, cells were immediately placed into 2 mL of prewarmed medium and cultured at 37° C., 5% CO2 or cultured at 32° C., 5% CO2 for 1 day and then returned to 37° C., 5% CO2.

[0263] TCR α and β double disruption, or TRAC, TRBC and B2M triple disruption: To generate TCR α and β double knockout T cells, Cas9 mRNA was co-electroporated with two different gRNAs targeting the TCR α chain (TRAC) and TCR β chain (TRBC). TCR α and β double knockout T cells could be purified in two steps: 1) electroporation of 1G4 TCR α chain RNA followed by depletion of TCR positive cells and α chain single knockout cells with anti-CD3 microbeads, and 2) electroporation of TCR β chain RNA followed by depletion of TCR β chain single knockout cells with anti-CD3 microbeads. 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 next day the TCR-negative cell population was sorted to obtain universal T cells. On day 18, the universal T cells were electroporated with TCR or CAR RNA to generate universal effector cells. Expression of TCR and HLA-I molecules was confirmed at each stage.

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

[0265] CD3neg T cell enrichment: Cells washed with AutoMACS buffer were incubated with CD3 microbeads (Miltenyi Biotec, 130-050-101, Auburn, CA) for 30 min at 4°C. After washing twice, cells were passed through an LD column (MiltenyiBiotec, 130-042-901, Auburn, CA) and the flow-through was collected for further use. CD3 neg CD3 expression in T cells was restored by co-electroporation of 1G4TCR α and β mRNA, and cells were expanded by a single Rapid Expansion Protocol (REP), CD3 / CD28 Dynabeads or K562-based aAPCs.

[0266] CD3 neg T cell generation and expansion: CD3 neg CD3 expression was restored in T cells by electrotransfer of exogenous 1G4 TCR α- and β-chain in vitro transcribed mRNA (5 μg for each chain). These cells were expanded using a single Rapid Expansion Protocol (REP). PBMCs from three different donors: ND052 105×10 6 pcs, ND405 83×10 6 pcs, ND410 136×10 6 Each was irradiated and then mixed to give a total of 324 × 10 6 PBMCs were obtained. The PBMCs were resuspended to a final volume of 90 ml, then R10 was added to 300 ml, mixed and split into two T150 ml flasks. OKT was added to a final concentration of 30 ng / ml. On day 2, IL-2 was added at 50 CU / ml. From day 5, cells were counted and nutrients were added every 2 days, and when T cells appeared to have entered a resting state as judged by both slowed growth kinetics and cell size, they were used for functional assays or cryopreserved.

[0267] Sanger sequencing: The levels of genomic disruption of the TCR alpha chain (TRAC), TCR beta chain 1 (TRBC1) and TCR beta chain 2 (TRBC2) in T cells were determined by Surveyor Nuclease assay (Transgenomics, Omaha, NE). Target disruption rates were quantified by densitometry. PCR primers used for amplification of the target loci were: TIFF0007657474000002.tif27140

[0268] The PCR products were purified and ligated into the TOPO cloning vector (Invitrogen) and subsequently transformed into E. coli. Single clones were picked and sequenced to count indels.

[0269] Preparation of siRNA and CRISPRi for electroporation: RNA duplexes targeting the TCR constant region for either TIFF0007657474000003.tif34164 were designed using the Custom RNAi Design Tool (Integrated DNA Technologies, Coralville, IA), and siRNAs were synthesized (Integrated DNA Technologies, Coralville, IA). siRNAs for both TCR α and β were mixed and electroporated into stimulated T cells 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). CD3 / CD28 bead-stimulated T cells were electroporated with IVT RNA using BTX EM830 (Harvard Apparatus BTX) as previously described (Cancer research 2010, 70(22):9053-9061). Briefly, T cells were washed three times and electroporated with IVT RNA in OPTI-MEM (Invitrogen) at a final concentration of 1–3 × 10 8 Cells were resuspended at 1000 cells / ml. 0.1 ml of cells were then mixed with 10 μg of IVT RNA (or as indicated) and electroporated in a 2 mm cuvette.

[0271] ELISA Assay: Target cells, different tumor cell lines expressing CD19, were washed and cultured at 1 × 10 6 Effector T cells were washed and suspended at 1 × 10 cells / ml. 100 μl of each target cell type was added in duplicate to a 96-well round-bottom plate (Corning). 6 After resuspension at 100 cells / ml, 100 μl of T cells were combined with target cells in the indicated wells. Also, wells containing only T cells were included as controls. The plates were incubated at 37°C for 18-20 hours. After incubation, the supernatants were collected and subjected to ELISA assay (eBioscience).

[0272] CD107a staining: Assay 96-well plates were cultured in 160 μl complete RPMI medium at an effector:T cell ratio of 1:1 (1 × 10 5 1x10 effectors per 5Cells were seeded with 100 μl of phycoerythrin-labeled anti-CD107a antibody (BD Biosciences, 555801) and the plates were incubated at 37° C. for 1 h, after which Golgi Stop (2 μl Golgi Stop in 3 ml RPMI medium, 20 μl / well; BD Biosciences, 51-2092KZ) was added and the plates were incubated for another 2.5 h. 5 μl FITC-anti-CD8 and 5 μl APC-anti-CD3 were then added and incubated at 37° C. for 30 min. After incubation, 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 in R10 medium at 1 × 10 5 Cells / ml were resuspended and 100 μl of CBG-labeled cells were incubated with different ratios of T cells (e.g., 30:1, 15:1, etc.) at 37° C. overnight. 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. 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 studies: The study was performed as previously described with some modifications (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, 6–10 week-old NOD / SCID γ (NSG) mice were inoculated with 1 × 10 6 PC3-CBG tumor cells were subcutaneously injected into the left flank of the same mice on day 5, followed by SK-OV3-CBG tumor cells (5 × 10 6 The mice were treated with T cells via the tail vein 23 days after inoculation of the PC3-CBG tumor, and both tumors grew to a volume of approximately 200 mm. 3 Lentiviral transduced T cells were 1 × 10 7 Cells / mouse (10M) or 3 x 10 6 Briefly, for the Nalm6 tumor model, 6- to 10-week-old NOD / SCID γ (NSG) mice were administered 1 × 10 6 Click beetle green protein (CBG)-transduced Nalm6 (Nalm6-CBG) cells were injected via the tail vein on day 0. T cell administration began on day 7 after tumor inoculation. For the PC3-PDL1 solid tumor model, 1 × 10 6 PSCA, PD-L1 and CBG transduced PC3 (PC3-PSCA-PDL1-CBG) tumor cells were injected subcutaneously into the right flank on day 0. Mice were administered T cells via the tail vein on day 22 after PC3-PDL1-CBG tumor inoculation, resulting in tumors growing to approximately 200 mm 3 The volume of T cells was 2 × 10 6 The animals were dosed at 100 mg / mouse (2M). Animals were randomized and divided into groups based on baseline tumor size. All animals were included in the study and blinded tumor assessments were performed for all animal studies performed.

[0275] Procedure for T cell stimulation, lentiviral transduction and CRISPR electroporation: Figure 84 shows the procedures used to perform T cell stimulation, lentiviral transduction and CRISPR electroporation. On day 0, T cells were obtained from three donors (100 x 10 6 Cells were stimulated with anti-CD3 / anti-CD28 beads at a T cell:bead ratio of 1:3. The cell concentration was 0.5 × 10 6 The cells were adjusted to 100 mL / flask with 50 mL (25 × 10 6 100 × 100 T cells were reserved as unmodified T cells (Group 9). On day 3, the beads were removed and the cells were washed twice in Opti-MEM medium, and the transduced T cells from each donor were incubated in CART / Mock EP (10 mL, 50 × 100 6 cells / mL) and CART / CRISPR (10 mL, 50 × 10 6 The cells were then electroporated with CAS9 RNA (1st EP) using 120 μg CAS9 RNA / 400 μL T cells at 500 V / 1 ms. After electroporation, cells in groups 1, 3, 5 and 7 were then split by culturing half the T cells in fresh medium and half in culture conditioned medium. On day 4, cells were washed twice and plated at 50 × 10 cells / mL in Opti-MEM. 6 20 μg TRBC4 and B2M gRNA were electroporated into 400 μL of T cells. After electroporation, cells were diluted to 1 × 10 in half fresh medium and half conditioned medium. 6 On days 5 and 7, cells were split and resuspended in half fresh medium and half conditioned medium. On day 8, CD3+ cells were removed from groups 2, 4, and 6 by low-density columns. CD3- T cells were cultured at 0.5–1 × 10 cells / mL in half fresh medium and half conditioned medium. 6On day 11, T cells were harvested and cultured to expand the cells at 25×10 cells / mL from three donors. 5 One was shipped for karyotype analysis, and the remaining cells were aliquoted and frozen.

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

[0277] Example 1: CRISPR-based disruption of the TCR-CD3 complex on T cells Thirteen gRNAs targeting the constant region of the TCR α chain, ten gRNAs targeting the constant region of the TCR β chain, and ten gRNAs targeting the β-2 microglobin gene (Figures 1A-1C and 9A-9D) were developed and tested in 293T cells. Primary human T cells were grown ex vivo for 3 days with anti-CD3 / anti-CD28 Dynabeads for 3 days. Because transient expression of CRISPR is sufficient to mediate gene knockout, we developed a "hit-and-run" delivery strategy to transiently express CRISPR by utilizing electrotransfer of in vitro transcribed RNA encoding CAS9 and gRNA (Figure 2C).

[0278] To measure TCR expression, we used a mAb specific for CD3, which is present on the cell surface only when TCR antibodies are expressed. Six days after electrotransfer, flow cytometry analysis revealed that CRISPR targeting TRBCs eliminated CD3 expression on primary T cells in donor ND147 at a level of 13.7 (Figure 2D). The efficiency of TCR knockout correlated with the amount of electrotransferred mRNA (Figure 2D). Although RNA electrotransfer in primary T cells was well tolerated, some reduction in cell viability was observed, which correlated with increasing amounts of introduced RNA. It has been reported that gene disruption mediated by ZFNs and TALENs is more efficient when cells are transiently exposed to mild hypothermia. The same phenomenon was observed with this CRISPR system.

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

[0280] Two and three rounds of gRNA electrotransfer significantly improved the level of efficiency in eliminating CD3 expression on primary T cells. Targeting of TRAC reached a level of 77% after three electrotransfers of gRNA (Figure 4A). Targeting of TRAC or TRBC was accompanied by some decrease in viability, reaching levels of 64.5% or 57.5%, respectively, after the second electrotransfer of gRNA (Figure 4C).

[0281] To confirm that electroporated T cells were genetically modified at the intended gRNA target site (TCR α or β locus), Sanger sequencing was performed using specific oligonucleotide primers flanking the target site within TRAC, TRBC1 or TRBC2. Multiple peaks in the designated PCR products originating from the target site were only present after CRISPR electrotransfer, and the rate of disruption 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 resulted in a permanent disruption of αβ TCR expression, as assessed by Sanger sequencing and confirmed by flow cytometric analysis of CD3.

[0282] Example 2: Enrichment of TCR αβ-negative T cells A rapid and robust method is available to isolate sources of TCR-disrupted populations for future clinical applications. To begin to address this issue, we isolated TCR / CD3 neg The population was enriched for CD3 neg The CD3 population was enriched by >99% (Figure 3A). neg The population could not be enriched from non-transfected control cells. Successive depletion steps resulted in >99% enrichment, with no balance shift toward CD4 or CD8 T cell subsets (Figure 3C). Sequencing results also demonstrated that deletions and insertions were introduced into the TCR α and β loci after CRISPR modification (Figure 3D).

[0283] Example 3: HLA-class I by CRISPR neg Generation of T cells To examine the ability of CRISPR to knock out HLA-class I expression from allogeneic T cells, we designed a gRNA targeting β-2 microglobin. The β-2 microglobin locus could be engineered by CRISPR in 293 T cells (Figure 9A). Evidence showed that disruption of β-2 microglobin abolished HLA-class I expression on the T cell surface (Figure 9B).

[0284] IFN-γ enhanced the targeting efficiency of β-2 microglobin in T cells by approximately 10-fold (Figure 9C). Multiple electrotransfers of β-2 microglobin gRNA generated a population that was 66% β-2 microglobin negative (Figure 11A).

[0285] Future clinical applications of allogeneic transplantation will require a rapid and robust method to isolate HLA-class I null populations. To begin to address this problem, we labeled cells with PE-anti-β-2 microglobulin antibodies and isolated HLA-class I null populations by negative selection using clinically approved paramagnetic anti-PE microbeads and depletion columns. neg The population was enriched for HLA-class I by a single depletion step. neg The population was enriched by >99%. HLA-class I neg The population could not be enriched from non-transfected control cells. Enriched HLA-class I population via flow cytometry neg Analysis of the HLA-class I repertoire in T cells demonstrated the exclusion of HLA-class I expression from the cell surface (Fig. 9D).

[0286] Example 4: CD3 neg T cells can be expanded by a variety of methods CD3 neg T cells regained CD3 expression after electrotransfer of exogenous 1G4-TCR α- and β-chain in vitro transcribed mRNA (5 μg each). These cells were (1) expanded by a single Rapid Expansion Protocol (REP) and subsequently examined for activity and specificity. PBMCs were obtained from three different donors: ND052 105×10 6 pcs, ND405 83×10 6 pcs, ND410 136×10 6 The cells were irradiated and then mixed to give a total of 324 × 10 6 PBMCs were obtained. 2 × 10 6 10 cells were electrotransferred with RNA. CD3 negT were resuspended to a final volume of 90 ml and R10 medium was added to a total volume of 300 ml. Cells were split into two T150 ml flasks. OKT was added to a final concentration of 30 ng / ml. On day 2, IL-2 was added at 50 CU / ml. From day 5, cells were counted and nutrients were added every 2 days, and when T cells appeared to have entered a quiescent state as judged by both slowed growth kinetics and cell size, they were used for functional assays or cryopreserved.

[0287] After a single REP, CD3 neg T cells were expanded until their numbers increased 500-fold. These cells were then expanded by (2) stimulation with anti-CD3 / anti-CD28 coated magnetic beads at a cell-to-bead ratio of 1:3.

[0288] After a single REP, CD3 neg T cells were expanded until their numbers increased 500-fold. These cells were then cultured at a concentration of 1 × 10 6 The cells were expanded by co-cultivation with an equal mixture of 1000 cells / ml.

[0289] After a single REP, CD3 neg T cells were expanded until their numbers increased 500-fold. These cells were then cultured at a concentration of 1 × 10 6 The cells were expanded by co-cultivation with an equal mixture of 100 ng / ml of erythrocytes and 30 ng / ml of OKT.

[0290] After a single REP, CD3 neg T cells were expanded until their numbers increased 500-fold. These cells were then cultured at a concentration of 1 × 10 6 The cells were expanded by co-cultivation with an equal mixture of 1 mg / ml NY-ESO peptide and 1 mg / ml NY-ESO peptide.

[0291] Example 5: TCR by electrotransfer of TCR neg T cell redirection TCR neg To study T cell function, these cells were redirected by TCR electrotransfer. By introducing the TCR α and TCR β chains, these cells expressed high levels of TCR. Expression of Vb13.1 was significantly higher in the electrotransferred TCR compared to the CAS9 mock control. neg The expression of 107a was much higher in T cells (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 increased degranulation activity (Figure 7B). Killing assays also showed strong toxicity against this cell line (Figure 7C). This indicates that these cells are potentially safer than conventional clinical trials using CAR and TCR expressing T cells, as they do not appear to induce GVHD and are less mismatch toxic than TCR treated normal T cells.

[0292] Some 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 eliminating T cells expressing unwanted αβ TCR also include the incomplete knockout of endogenous TCR for treating GVHD and inhibiting endogenous TCR from adversely affecting CAR function (e.g., through competition with transcription factors).For this reason, a genetic approach is designed to use designer ZFN to permanently destroy α and β constant region sequences in T cells, thereby eliminating TCR expression.

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

[0294] Different from ZFN and TALEN, the CRISPR / Cas system has recently emerged as a potential facile and efficient alternative to ZFN and TALEN for inducing targeted genetic changes. Recent studies have shown that target recognition by 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 retargeted to cleave virtually any DNA sequence by redesigning the crRNA. The data disclosed herein demonstrate the feasibility of CRISPR / CAS 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. Multiple genes in T cells can be simultaneously disrupted by administering different gRNAs together with CAS9.

[0295] Example 6: Triple knockout of HLA CLASS I and TCR α, β chains by CRISPR To work towards an "off-the-shelf" allogeneic T cell therapy for malignancies and infectious diseases, cell therapy by infusion of T cells has been designed to reconstitute immunity against pathogens and malignancies. The time required to ex vivo produce sufficient numbers of T cells with desired characteristics is often not compatible with the time frame for patients. Moreover, autologous T cells from patients with advanced disease may be functionally impaired and tolerant to the desired antigen.

[0296] To address this, allogeneic T cells can be infused into patients to avoid immune-mediated rejection caused by host T cells that recognize different major or minor histocompatibility antigens on the infused cells. A rapid and robust method can be created to extend the application of T cell therapy and to isolate sources of TCR and HLA-class I-destroyed populations for future allogeneic transplantation.

[0297] ZFN and TALEN comprise zinc finger DNA binding domain designed to bind specific DNA sequence fused with the cleavage domain of Fokl endonuclease.When multiple genes need to be manipulated, the design and construction of ZFN and TALEN is very complicated and time-consuming, because genes must be targeted individually.By using the CRISPR system described herein, it is possible to obtain the efficiency and shortened time course of gene disruption.

[0298] To address this issue, we electrotransferred CAS9 with three different gRNAs targeting TRAC, TRBC, and β-2 microglobin. Cells were labeled with PE-anti-β-2 microglobin antibody and depleted by negative selection using clinically approved paramagnetic anti-PE microbeads and depletion columns to selectively express HLA-class I. neg The population was enriched for HLA-class I by a single depletion step. negThe population was enriched by >99% (Figure 9D). The cells were then reintroduced with the TCR α chain to express 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 with microbeads. Two days later, TCR electrotransfer was performed on these triple knockout cells. The day after electrotransformation, cells were stimulated with CD3 / CD28 Dynabeads. The next day, cells were then subjected to lentiviral delivery of antigen-specific TCR and culture expansion.

[0299] Example 7: CRISPR-mediated knockout of FAS, PD1, CTLA4, and PPP2R2D The FAS receptor / FAS ligand (FAS / FASL) apoptotic signaling pathway has been extensively studied and characterized in T cells. PD1 and CTLA4 are the two major inhibitory signaling pathways in T cells that have also been studied in detail. Direct evidence of the potential therapeutic impact of targeting these pathways has been provided in preclinical mouse tumor model studies that demonstrate enhanced antitumor immunity following antibody-mediated blockade of CTLA-4, PD-1, or PD-L1. Similar antibodies have been developed for human use, and early clinical data show promising results. Ppp2r2d knockdown can inhibit T cell apoptosis and enhance T cell proliferation as well as cytokine production. Ppp2r2d is a potential target for improving human T cell function.

[0300] To address this issue, we electrotransferred CAS9 and three different gRNAs targeting FAS, PD1, CTLA4, and PPP2r2d into T cells. 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 CRISPR-induced homologous recombination. FACS data showed that the surface expression of FAS and PD1 was lost.

[0301] Example 8: Generation of IPS cells by genetically modified primary cells and T cells Progress in adoptive T cell therapy for cancer and infectious diseases has been hindered by the lack of readily available, antigen-specific human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes. To address this issue, we disrupted the expression of FAS, PD1, CTLA4, and PPP2r2d in primary cells and T cells.

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

[0303] Unlike these methods, Sendai virus does not integrate into the host genome or alter the genetic information of the host cell. Sendai virus also has reprogramming capabilities comparable to lentivirus- and retrovirus-based gene transduction.

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

[0305] The day after transduction, 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 every day. On day 3 post-infection, the cells were transferred to MEF feeder plates and cultured in T cell medium without added cytokines. Four days after infection, the cells were cultured in standard hES medium. The medium was changed every day. At approximately day 7, ES-like colonies were observed. From day 15, the cells were cultured in conditioned hES medium, and culture was continued for an additional 10 days. Colonies were picked approximately 25-30 days after transduction.

[0306] At approximately day 4, cell clusters formed on the feeder cells, indicating the beginning of the reprogramming process. T cells underwent dramatic morphological changes during reprogramming to iPSCs. At approximately day 12, large cell clusters with loose borders began to appear. At approximately day 18, T cells transformed into typical ES-like colonies with well-defined borders. A typical embryonic stem cell morphology was observed, which is consistent with the FAS neg , CD3 negThese results indicate that TCR α- and β-chain knockout T cells were induced to a pluripotent state under defined reprogramming conditions (Figures 17A and 18A).

[0307] FAS neg T cells were easier to reprogram from iPSCs, approximately five times more efficient than their wild-type counterparts (Figure 17B). neg The efficiency of T cell reprogramming was about 5-fold higher than that of wild-type counterparts (Figure 18B). p53-deficient cell lines have been reported to be easier to reprogram due to the disruption of the apoptotic pathway. FAS knockout also induces more resistance to apoptosis. Loss of TCR expression makes T cells less healthy, 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 N-deglycosylation in the β-chain (S / SD). Endogenous T cell receptors (TCRs) were knocked down with siRNA and T cells were electroporated with RNA. 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, consisting of Figures 20A and 20B, shows TCR alpha knockdown by co-electroporation of CAS9 RNA and gRNA. Six days after electroporation, cells were analyzed for TCR expression by assessing CD3.

[0310] Figure 21 shows Sanger sequencing. The results show multiple peaks in CD3-enriched T cells upon electroporation of either CAS9 mRNA and gRNA to knock down TCR α (TRAC-5) or TCR β (TRBC-7).

[0311] Figure 22 is a panel of graphs showing that CD3 negative T cells that had endogenous TCR β (TRB-7) knockdown 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 were nearly 100% CD3 positive with endogenous TCR vb13.1 expression at 5.25%.

[0312] Figure 23, consisting of Figures 23A-23D, is a panel of graphs showing that knockdown of endogenous TCR enhanced both transgene expression and function of TCR RNA electroporated T cells. Figure 23A shows TCR expression in T cells electroporated with TCR siRNA (solid open histograms), control siRNA (dotted open histograms), and T cells without any siRNA (filled histograms). Figure 23B shows transgene (TCR vb13.1) expression in engineered T cells electroporated with wild-type NY-ESO-1 TCR(wt)RNA or TCR(SD)RNA with TCR siRNA, control siRNA, or no siRNA. Figure 23C shows NY-ESO-1 tetramer staining of engineered T cells electroporated with wild-type NY-ESO-1 TCR(wt)RNA or TCR(SD)RNA with TCR siRNA, control siRNA, or no siRNA. FIG. 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. Ten million Nalm6-CBG-ESO-GFP tumor cells expressing both NY-ESO-1 and GFP (click beetle green protein) were injected intravenously into NOD / SCID mice. Five days after tumor inoculation, CBR (click beetle red) transduced and RNA electroporated T cells were injected into different 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 different time points.

[0315] Example 10: Universal CAR19 T cells generated by combining lentiviral transduction and disruption of the TCR-CD3 complex on T cells using CRISPR As shown in Figure 26, primary T cells were stimulated with anti-CD3 / anti-CD28 beads on day 0 and subsequently transduced with lenti-CAR19. More than 70% of cells were CAR19 positive as detected by flow cytometry. Since transient expression of CRISPR is sufficient to mediate gene knockout, we developed a "hit-and-run" delivery strategy to transiently express CRISPR by utilizing electrotransfer of in vitro transcribed RNA of CAS9 and gRNA targeting the TCR α chain, TCR β chain constant region and β-2 microglobulin gene on day 3. After electrotransfer, 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. CD3 was chosen because it is present on the cell surface only when the TCR is expressed. Primary T cells were electroporated with CRISPR constructs (Figure 26). TCR single negative and TCR / HLA-A double negative cells were expanded by exposure to CD19-presenting K562 cells, with an expansion fold of >100 (Figure 27).

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

[0318] The majority of expanded universal T cells were CD45RO positive (Figure 29A) and retained high levels of CD62L expression (Figure 29B), intermediate levels of CD28 expression (Figure 29A) and low levels of CCR7 expression (Figure 29B).

[0319] CRISPR gene editing did not affect the antitumor activity of universal CAR19 T cells in vitro (Figure 30A). TCR or TCR / HLA-A ablation had only minor effects on CAR19 expression and antitumor activity (Figures 30B and 30C). TCR single-negative and TCR / HLA-A double-negative CAR19 T showed robust lytic ability when challenged with Nalm6 tumor cells (Figure 30B). CD107a release and cytokine secretion also indicated potent antitumor activity in universal cells (Figure 30C). TCR single-ablated or TCR and HLA-A double-ablated CAR19 T cells exhibited similar proliferation kinetics after challenge with CD19-expressing cells (Figure 30D).

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

[0321] Complete ablation of TCR α and β chains and HLA-A molecules on T cells completely abolished nonspecific killing when 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 challenge of these cells with allogeneic whole blood PBMCs in IFNr Elispot assays. The lack of off-target activity suggests that T cells may play a major role in acute immune responses after encounter with allogeneic cells. All these results suggest that CRISPR / CAS9-edited TCR α and β chains and HLA-A molecules (triple negative) T cells may serve as a source of universal effector donor cells.

[0322] CAS9 and various gRNAs targeting FAS were electrotransferred into T cells. FASneg cells were selected 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 comparable to wild type. Even after short-term incubation with Nalm6 tumor cells, CD107a expression in FASneg CAR19 T cells was greatly enhanced compared to wild type counterparts, even within 4 hours of co-culture.

[0323] Several reports have shown that even weak antigen stimulation induces FAS activation to promote 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, suggesting that under high-level antigen conditions, FAS / FASL induced apoptosis rather than activation (Figure 34A). FASneg CAR19 T cells further showed reduced levels of apoptosis as measured by Annexin V staining (Figure 34B).

[0324] As observed in vitro, FASneg T cells showed enhanced proliferation compared to wild-type T cells. Similar proliferation results were observed when True Count assay of CAR19 T cells was performed after injecting the cells into Nalm6-bearing mice. The FASneg CAR19 group showed better antitumor activity when 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 FAS ablation in CAR T cells enhanced their antitumor activity.

[0325] CAS9 and various gRNAs targeting PD1 were electrotransferred into T cells 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 wild-type and PD1-negative groups. After incubation with PC3-PSCA-PDL1 tumor cells, PD1 expression was rapidly upregulated on the surface of wild-type PSCA-CAR T cells, whereas very low levels of PD1 expression were detected on PD1-negative PSCA-CAR T cells (Figure 37). PD1-negative PSCA-CAR T cells also showed greatly enhanced and sustained high-level 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 the in vivo PC3-PSCA-PDL1 NSG model, significantly enhanced 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 investigate the effect of CRISPR-engineered universal CART cells on graft-versus-host disease (GVHD), high doses of T cells were administered to NSG mice bearing Nalm6 leukemia. Mice administered double or triple knockout CART cells did not show any signs of developing GVHD. 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 injected intravenously after sublethally irradiating mice. Clinical GVHD was monitored 2-3 times per week. Four of five mice receiving wild-type T cells died during the 60-day study, whereas mice receiving PBS, TCR single-ablated, and TCR / HLA-I double-ablated T cells did not show any signs of GVHD. Mice receiving wild-type T cells lost weight; however, mice receiving PBS, TCR single-ablated, and TCR / HLA-I double-ablated T cells gained some weight over the study 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. Mice receiving PD1 / CD3 / HLA-I triple knockout cells showed better antitumor activity compared to 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 method to enhance the treatment of universal CART cells with CRISPR / Cas9.

[0330] Since gRNA is prone to degradation, we developed a simplified one-shot method to generate universal CART cells. gRNA was constitutively expressed along with CAR in a single lentiviral vector. Naive T cells were transduced with lentivirus encoding gRNA and CAR one day after stimulation with CD3 / CD28 Dynabeads. Cells were electroporated with Cas9 mRNA on day 3 (Figure 42). This system allows the manipulation of several genes with one 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 to the extent of 90% in each of the different Cas9 mRNA groups (Figure 43).

[0331] Progress in adoptive T cell therapy for cancer and infectious diseases has been hindered by the lack of readily available, antigen-specific human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes. To address this issue, we disrupted the expression of FAS, PD1, CTLA4, and PPP2r2d in primary cells and T cells.

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

[0333] In contrast, Sendai viruses do not integrate into the host genome or alter the genetic information of the host cell, and they also have reprogramming capabilities comparable to lentivirus- and retrovirus-based gene transduction.

[0334] One hundred thousand wild-type, FASneg, CD3neg TCR α-chain and TCR β-chain knockout T cells were seeded into each well in a 24-well plate. 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 prewarmed T cell complete medium, followed by incubation with a calculated volume of CytoTune Sendai virus (Life Technologies, Carlsbad, CA), which contains a polycistronic vector for expression of hKlf4, hOct3 / 4 and hSox2 in the cells. The treated T cells were seeded into a 24-well plate and centrifuged at 2250 rpm for 90 min at room temperature. 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] The day after transduction, 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 every day. On day 3 post-infection, the cells were transferred to MEF feeder plates and cultured in T cell medium without added cytokines. Four days after infection, the cells were cultured in standard hES medium. The medium was changed every day. At approximately day 7, ES-like colonies were observed. From day 15, the cells were cultured in conditioned hES medium, and 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 the feeder cells, indicating the beginning of the reprogramming process. T cells underwent dramatic morphological changes during reprogramming to iPSCs (Figure 44A). At approximately day 12, large cell clusters with loose borders began to appear. At approximately day 18, T cells transformed into typical ES-like colonies with clear borders. FASneg T cells were reprogrammed to iPSCs approximately 5-fold more efficiently than their wild-type counterparts (Figure 44B). It has been reported that p53-deficient cell lines are easier to reprogram because the apoptotic pathway is blocked. FAS knockout can aid the reprogramming process by a similar mechanism.

[0337] An ES-like morphology was observed for iPSCs reprogrammed from CD3neg TCR α or β chain knockout T cells (Figure 45A). This morphology remained constant after several passages. The efficiency of reprogramming CD3neg T cells was about 5-fold lower than that of 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 panel of images showing phosphatase staining of CD3neg iPSC cells.

[0338] Typical embryonic stem cell morphology was observed, indicating that FASneg, CD3neg TCR α- and β-chain knockout T cells were induced to a pluripotent state under defined reprogramming conditions. Although loss of TCR expression renders T cells less healthy, the data presented here indicate that apoptosis plays an important role in the reprogramming process.

[0339] 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). Fas knockout in T-iPSCs was confirmed by Sanger sequencing (Figure 47B).

[0340] dCas9 and FokI-Cas9 have been reported to have relatively weak off-target activity. T cells were evaluated for whether they could be edited by modified versions of 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 knockout events more precise and specific.

[0341] To investigate off-target events of CRISPR / CAS9 in T cells, we performed surveyor assays at off-target sites. No obvious cleavage was observed at the genomic locus for the genes examined (Figure 48C).

[0342] Example 11: Multiplex genome editing CART cells were generated by using the CRISPR / Cas9 system to simultaneously disrupt multiple genomic loci. The CART cells are deficient in the expression of endogenous TCR and HLA class I (HLA-I) molecules for use as allogeneic universal CART cells. Genes for 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 with mRNA encoding Cas9. Universal TCR or CART cells were generated by combining lentiviral (LV) delivery of CAR with CRISPR RNA electroporation to simultaneously disrupt endogenous TCR and B2M genes. Furthermore, disruption of endogenous PD1 also enhanced the efficacy of CAR therapy in solid tumor models.

[0343] Multiplexed delivery of gRNAs results in highly efficient disruption of multiple genes in primary human T cells without compromising effector function Efficient multiplex genome editing is required to generate universal T cells with defects in TCR, HLA and other genes. To achieve efficient gene disruption in T cells, we optimized CRISPR / gRNA RNA electroporation. First, we performed co-electroporation of Cas9 and gRNA with RNA generated using an in vitro transcription system (Figure 49, left), and then developed a "hit-and-run" delivery strategy to transiently deliver Cas9 mRNA and gRNA into T cells by electroporation (Figure 49, right).

[0344] Early experiments targeting the TCR α constant region (TRAC) or β constant region (TRBC) using a single electroporation technique resulted in 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 was increased 4-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 gene disruption efficiency correlated with the amount of electrotransferred mRNA (Figure 51A).

[0345] gRNA is subject to rapid degradation compared to mRNA, which may limit its targeting efficiency. For this reason, 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, with 82.4% of cells expressing CD3+ after the third gRNA electroporation. neg (Figure 50B). Clonal sequencing showed that the genome targeting efficiency reached 89.4% after the third gRNA electroporation (Figure 51B). Surveyor assay confirmed that the cleavage rates at the TRAC and TRBC genomic loci were 81.7% and 49.3%, respectively, after the third gRNA electroporation (Figure 52). Multiple peaks in the Sanger sequencing data flanking the TRAC and TRBC target sites confirmed that the genome reading frame had shifted downstream of the target sites (Figure 53A). The occurrence of insertions or deletions (indels) caused by CRISPR / Cas9-mediated NHEJ was confirmed by clonal sequencing (Figure 53B). TCR was amplified by TCR / CD3 by a single-step CD3 negative selection. neg The population was enriched by more than 99% (99.70±0.20%) (FIG. 54).

[0346] TCR / CD3 neg To develop a method to expand T cells,neg 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). Following T cell stimulation / expansion methods, the following were compared: (1) 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 expressing CD19 (K562-CD19). After a single stimulation for 10 days, fold expansion 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 investigate whether CRISPR / Cas9 gene editing affects T cell phenotype and function, we investigated TCR / CD3 neg When the phenotype of the T cells was examined, all expanded cells remained CD3 negative and most retained high levels of CD27 (79.8%-93.4%), consistent with the phenotype of central memory cells (Figure 57). neg T cells were electroporated a second time with CD19 CAR mRNA to examine their antitumor activity. neg Surface CAR expression on T cells was comparable 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 T cells were comparable to those of wild-type control cells. CD19 CAR TCR / CD3 neg T cells were injected into Nalm6-bearing NSG mice to examine their in vivo antitumor activity. Tumor regression was evident, with efficacy comparable to that of CART19 wild-type counterparts (Figures 59D and 59E). These results indicate that CRISPR / Cas9 editing of endogenous TCR does not adversely affect the function of primary T cells for adoptive immunotherapy.

[0348] Reduced alloreactivity of TCR α, β and B2M triple-disrupted T cells Disruption of both TCR α and β chains is necessary to prevent TCR mismatch-associated toxicities in adoptive immunotherapy of TCR-redirected T cells, and B2M is essential for the assembly and expression of HLA-I complexes. In light of this, we developed a triple disruption of TCR α and β chains and B2M to generate universal T cells. We first investigated whether disruption of B2M could eliminate HLA-I expression on T cells. T cells were electroporated with Cas9 / gRNA RNA targeting B2M. This resulted in a 79.9% B2M and HLA-I double negative population. HLA-I neg The population could be further enriched by negative selection (Figure 60).

[0349] To generate triple knockout T cells lacking TCR α, β and B2M, we co-electroporated Cas9 mRNA with three different gRNAs targeting TRAC, TRBC and B2M. The resulting CD3 and HLA-I double negative cell population was 65.4% (Figure 61). After enrichment of double and triple knockout cells, TCR α and β and B2M triple knockout T cells abolished non-specific killing of HLA-mismatched tumor cell lines (Figure 62). No response was observed when these cells were challenged with allogeneic whole blood irradiated PBMC in an IFNγ Elispot assay (Figure 63, left panel). Ablation of HLA-I molecules also significantly reduced alloreactivity, which was confirmed by co-culture of allogeneic PBMC with irradiated B2M-disrupted cells (Figure 63, right panel). These results suggest that triple-negative T cells, which lack TCR α and β chains and B2M, may serve as a universal source of T cells for adoptive immunotherapy, resisting rejection by the host immune system while at the same time being unable to cause graft-versus-host disease.

[0350] Enhanced antitumor activity of endogenous TCR-redirected T cells T cells with CRISPR / Cas9 disruption of the TCR α and β chains had 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% for TCR α or β single knockouts or α / β double knockouts, respectively, compared to 46.8% for wild-type T cells. 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), especially for α / β double knockout T cells.

[0351] In another experiment, α / β double knockout T cells were transfected with a different NY-ESO-1 TCR (8F). In contrast to the 1G4 TCR, the 8F TCR was significantly higher than the transgenic TCR expression (Figure 66; TCR / CD3 neg 60.1% vs. 44.7% for wild type T cells (Cas9 Mock T cells) (with approximately 5% endogenous TCR Vβ8 background)) and showed highly significant enhancement in both function (CD107a expression in FIG. 67A and cytokine production in FIG. 67B). These results strongly indicate that the endogenous TCR has a differential impact on transgenic TCR expression and function.

[0352] Universal CART cells retain antitumor efficacy and do not cause GVHD By combining LV transduction of CD19 CAR with RNA electroporation of Cas9 / gRNA, universal CD19 CART cells were generated (Figure 68). When the cells were expanded, the remaining CD3 neg The cells had high levels of CD19 CAR expression (Figure 69). The majority of expanded T cells were CD45RO positive and retained high levels of CD62L expression and moderate levels of CD28 expression, consistent with central memory cell status (Figure 70). Expanded TCR / HLA-I double negative CD19 CART demonstrated robust in vitro antitumor activity, including CD107a release (Figure 71), cytokine secretion (Figure 72), lytic capacity (Figure 73), and proliferation (Figure 74), which were as potent as wild-type CD19 CART cells.

[0353] T cells were infused into NSG mice bearing disseminated Nalm6 leukemia. CART cells with ablated endogenous TCR (LV-CD19 CAR TCR neg ) or TCR and HLA-I co-disrupted CAR TCR / HLA-I (LV-CD19 CAR TCR / HLA-I negMice receiving LV-CD19 CAR showed tumor regression to the same extent as mice receiving wild-type CD19 CART cells (LV-CD19 CAR) (Figures 75A and 75B), suggesting that neither TCR disruption alone nor TCR and B2M disruption affects the anti-tumor activity of CART cells.

[0354] To examine the effect of engineered T cells on GVHD, a high dose of T cells (20 × 10 6 LV-CD19 CAR TCR / CD3 cells / mouse) were administered to NSG mice bearing Nalm6 leukemia. As shown in Figure 76, 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 Mice treated with LV-CD19 CAR 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, neither TCR disruption alone nor TCR and HLA-I disruption affects the in vivo antitumor activity of CART cells while abrogating alloreactivity.

[0355] Adenoviral CRISPR delivery into primary T cells The CRISPR / Cas9 system is rapidly being exploited for gene regulation and editing purposes in model organisms and cell lines. Viral vectors may be particularly suitable for extending the applicability of CRISPR to other cell types, including dividing 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 of tumor cells (Figure 78) resulted in high rates of targeted mutagenesis, up to about 71% (Figures 79A and 79B). Adenoviruses provide a useful platform for introducing CRISPR into human T cells, even when they are in a quiescent state. This approach may be useful for exploring the gene regulation and editing potential of CRISPR in numerous experimental contexts.

[0356] Optimization of electroporation CD3 and B2M knockout efficiency as well as T cell expansion were evaluated after electroporation (EP) of Cas9 and gRNA in 4mm and 2mm cuvettes. Standard EP conditions using 2mm cuvettes (360v / 1ms, 1st EP-20μg Cas9 RNA+ 10μg gRNA / 100μl T cells, 2nd EP-5μg gRNA / 100μl T cells) showed the highest CD3 and B2M knockout rates of 81.8% and 88.6%, respectively, at approximately 2.7-fold T cell expansion (EP#1), compared to an expansion fold of approximately 18.8 for control EP T cells (EP#12). Decreasing the gRNA dose (EP#2-5) dramatically increased T cell expansion but had only a minor effect on CD3 and B2M knockout efficiency. See Figure 80. Standard EP conditions using 4 mm cuvettes significantly reduced the knockout efficiency of CD3 and B2M (EP#8), suggesting that EP conditions (voltage and / or pulse width) need to be further optimized for use with 4 mm cuvettes.

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

[0358] To further optimize the EP conditions with the aim of achieving maximum T cell expansion with CD3 / B2M knockout efficiency of over 60%, various EP conditions and RNA amounts were examined. The results showed improved expansion with relatively high CD3 / B2M knockout efficiency (63.5% for CD3 and 84.8% for B2M) for EP#4, where EP#1 is (400v / 2ms / 120μg CAS9 RNA) and EP#2 is (500v / 1ms / 20μg gRNA). See Figure 82.

[0359] Further experiments were carried out to optimize EP conditions. As a result, compared to the most favorable conditions examined (EP#1 in Figure 82), 500v / 1ms / 120μg CAS9 RNA (EP#1) and 500v / 1ms / 20μg gRNA (EP#2) resulted in increased CD3 / B2M knockout efficiency and T cell expansion (EP#3). See Figure 83.

[0360] Large-scale electroporation and expansion An experiment was performed to determine whether large-scale electroporation could provide high knockout and expansion efficiency. On day 0, T cells (100 × 10 6 0.5 x 10 pieces / donor 6 On day 1, stimulated T cells were transduced with CD19 CAR lentivirus. 50 mL (25 × 10 6 ) T cells were reserved as unmodified T cells (Group 9). On day 3, the beads were removed and the transduced T cells from each donor were resuspended in CART / Mock EP (10 mL, 5 × 106 pcs) and CART / CRISPR (10mL, 50×10 6 The cells were then electroporated with CAS9 RNA (first EP) and divided into groups 1, 3, 5, and 7. On the fourth day, gRNA was electroporated into T cells, and the cells were divided into 1 × 10 6 On days 5 and 7, cells were split. On day 8, CD3+ cells were removed from groups 2, 4, and 6. On day 11, T cells were harvested and 25×10 cells from three donors were cultured. 5 One was sent for karyotype analysis.

[0361] Table 1: Experimental groups TIFF0007657474000004.tif61128

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

[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 three donor populations at day 8. On day 11, T cells were subjected to FACS staining to detect CD3, B2M and CAR expression. Non-transduced ND463 (NOTD) was used as a negative control. Figure 89 shows CD3 and B2M expression in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells. Figure 90 shows CAR expression in CD19 CAR TD / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells. Figure 91 shows CD3 / B2M / CAR expression in CD19 CAR TD (transduced) / CRISPR electroporated CD3 depleted T cells; CD19 CAR TD / CRISPR electroporated T cells; and CD19 CAR TD T cells at day 11. Figure 92 summarizes CD3 / B2M / CAR expression in different T cell populations.

[0364] On day 11, the various T cell populations were stimulated with CD19 positive cell lines Raji or Nalm6 as indicated in 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 T cells was examined using a luminescent cytotoxic lymphocyte (CTL) assay after co-culture of T cells with CD19 positive target cells, Nalm6-CBG, as indicated in Figure 94. Also on day 11, the cytokine production of T cells was analyzed by stimulating T cell populations with Nalm6 target cells. See Figure 95.

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

[0367] As one of the most attractive applications of the CRISPR / Cas9 system, multiplex genome editing holds great promise for advancing T cell-based adoptive immunotherapy. However, the low targeting efficiency of DNA transfection has limited the use of multiplex genome engineering in primary T cells. We developed a “hit-and-run” delivery strategy to introduce CRISPR into T cells via co-electroporation of Cas9 mRNA and gRNA. By combining up to three rounds of gRNA electroporation with transient exposure to mild hypothermia, targeting efficiency above 80% at the protein level was consistently achieved for single gene disruptions. More promisingly, triple gene disruptions of TRAC, TRBC and B2M yielded approximately 65% ​​double-negative CD3 and HLA-I without any purification or selection. These results also demonstrated that enrichment of gene-disrupted T cells to >99% purity could be easily achieved using clinically approved paramagnetic beads, and purified T cells could be expanded up to 500-fold in 10 days. The expanded T cells maintained their gene-disrupted phenotype and exhibited characteristics consistent with central memory T cells. Disrupted T cells did not cause GVHD, suggesting that they could be used as allogeneic CAR T cells. Importantly, gene-edited T cells showed antitumor activity both in vitro and in various tumor mouse models, and they were as effective as or better than non-gene-edited T cells. Therefore, it is believed that the process described herein for making synthetic cells can be easily converted into current GMP-compliant manufacturing procedures.

[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 to eliminate endogenous TCR α- and β-chain expression to avoid GVHD. Due to the complexity of targeting strategies to manipulate multiple genes in T cells by zinc finger nucleases (ZFNs) and TAL effector nucleases TALENs, previous studies have not been able to simultaneously prevent GVHD and host-versus-graft reactions in preclinical animal models. NK cell activation can also be interrupted by CRISPR / Cas9 or by removal of stimulatory NK ligands by expression of non-classical HLA class I molecules such as HLA-E, which may protect universal T cells from NK cell-mediated rejection.

[0369] In summary, we were able to efficiently generate clinical-scale universal CART cells with potent antitumor activity and reduced alloreactivity using multiplex CRISPR technology. This approach can be integrated into current GMP-compliant manufacturing procedures and is likely to be translated into the clinic, given the successful translation of ZFN-based transfer therapy for HIV / AIDS. Universal CAR T cells and TCR T cells may represent an alternative to autologous T cells. In fact, checkpoint-disabled universal CAR T cells and TCR T cells may be more effective and versatile than current autologous T cell CART therapy for cancer and infectious diseases.

[0370] Other Aspects The recitation of a list of elements in a definition of a variable herein includes definition of that variable as a single element or as a combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as a single embodiment or in combination with other embodiments or portions thereof.

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

[0372] Sequence 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 sequencing <400> 1 tgtgctagac atgaggtcta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequencing <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. (i) CRISPR-mediated insertions or deletions at the endogenous TCR α chain (TRAC) and endogenous TCR β chain (TRBC) loci, resulting in downregulated gene expression of the endogenous TRAC and TRBC genes. (ii) CRISPR-mediated insertion or deletion at the endogenous beta 2-microglobulin (B2M) locus, which results in downregulated gene expression of the B2M gene; and (iii) a nucleic acid encoding a chimeric antigen receptor (CAR) having affinity for a tumor-associated antigen (TAA) on a target cell; A CRISPR-modified T cell, comprising: a CRISPR system that mediates insertion or deletion in an endogenous TRAC gene, an endogenous TRBC gene, or an endogenous B2M gene, and a CRISPR-associated (Cas) nuclease; (a) a first guide RNA comprising a nucleic acid sequence capable of targeting the sequence of SEQ ID NO: 15 in the coding sequence of the TRAC gene; (b) a second guide RNA comprising a nucleic acid sequence capable of targeting the sequence of SEQ ID NO: 16 in the coding sequence of the TRBC gene; or (c) a third guide RNA comprising a nucleic acid sequence capable of targeting the sequence of SEQ ID NO: 44 in the coding sequence of the B2M gene; and CRISPR modified T cells.

2. The CRISPR modified T cell of claim 1, wherein the CRISPR modified T cell further comprises a CRISPR-mediated insertion or deletion at a locus that causes downregulated gene expression of an HLA molecule, and the HLA molecule is not a class I HLA molecule.

3. The antigen-binding domain of the CAR is (a) 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; and (b) specifically binds to an antigen on a target cell; The CRISPR modified T cell of claim 1.

4. The CRISPR modified T cell of claim 3, wherein the antigen comprises CD19, prostate specific membrane antigen (PSMA), or prostate stem cell antigen (PSCA).

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

6. The intracellular domain is (a) a costimulatory signaling domain and an intracellular signaling domain, and / or (b) one or more costimulatory domains of a protein selected from the group consisting of CD3, CD83, CD86, CD27, CD28, 4-1BB (CD137), CD127, 4-1BBL, CD134, PD-1, PDL1, CD7, LIGHT, DAP10, DAP12, CD2, ICAM-1, LFA-1, lymphocyte-specific protein tyrosine kinase (LCK), TNFR2, CD30, CD40, ICOS (CD278), NKG2C, and B7-H3; and / or (c) an intracellular domain selected from the group consisting of the cytoplasmic signaling domains of TCR, CD3 zeta chain (CD3ζ), common FcRγ, FcγRIIa, FcεRI, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, and CD79b The CRISPR modified T cell of claim 1 .

7. (a) a TRAC gene comprising any one of the nucleic acid sequences of SEQ ID NOs: 20 to 28, and / or (b) a TRBC gene comprising any one of the nucleic acid sequences of SEQ ID NOs: 30 to 36 and 38 to 43; The CRISPR modified T cell of claim 1 .

8. A pharmaceutical composition for treating a disease or condition in a subject comprising the CRISPR modified T cell of any one of claims 1 to 7, wherein the disease or condition is (i) is associated with immune enhancement; or (ii) 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-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, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polyarteritis nodosa an autoimmune disease selected from the group consisting of myeloarthritis, 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; or (iii) 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; Pharmaceutical compositions.

9. 10. A pharmaceutical composition for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising the CRISPR modified T cell of any one of claims 1 to 7, and inducing lysis of the target cell or tissue.

10. 10. The pharmaceutical composition of claim 9, wherein the induced lysis is antibody-dependent cell-mediated cytotoxicity (ADCC).

11. 10. A pharmaceutical composition for preventing or treating a harmful immune response in a subject in adoptive cell transfer therapy, comprising the CRISPR modified T cell of any one of claims 1 to 7.

12. 13. Use of the CRISPR modified T cell of any one of claims 1 to 7 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

13. A composition comprising the CRISPR modified T cell of any one of claims 1 to 7.

14. 8. A pharmaceutical composition comprising the CRISPR modified T cell of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

15. An in vitro method for generating a CRISPR modified T cell according to any one of claims 1 to 7, comprising: (a) a nucleic acid encoding the CAR, and (b) one or more nucleic acids encoding a CRISPR system that mediates an insertion or deletion in an endogenous TRAC gene, an endogenous TRBC gene, or an endogenous B2M gene. in vitro,

16. 16. The in vitro method of claim 15, wherein the CRISPR system comprises a pAd5 / F35 CRISPR vector.

17. 16. The in vitro method of claim 15, 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. 16. The in vitro method of claim 15, further comprising expanding the T cells, thereby obtaining expanded T cells.

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

20. 16. The in vitro method of claim 15, further comprising, prior to the step of introducing at least one of the nucleic acids described in (a) and (b) of claim 15 into the T cells, cryopreserving the T cells to obtain cryopreserved cells, and thawing the cryopreserved cells.

21. 21. The in vitro method of claim 20, wherein the step of introducing at least one of the nucleic acids described in (a) and (b) of claim 15 is at least one selected from the group consisting of transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells.

22. The in vitro method of claim 15, further comprising electroporating RNA encoding the costimulatory molecule into the T cell.

23. 23. The in vitro method of claim 22, wherein the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PDL1.

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