Modification of gene expression in CART cells and uses thereof

Abstract

To provide a patient-specific modified T cell intended for treatment of cancer, methods for generating the modified T cell, and pharmaceutical compositions comprising the modified T cell.SOLUTION: The present invention provides a modified T cell comprising: a nucleic acid capable of downregulating expression of an endogenous gene selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, an HLA molecule, CTLA-4, PD1 and FAS; and a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain and an intracellular domain of a co-stimulatory molecule. Also provided are methods for generating the modified T cell. Further provided are pharmaceutical compositions comprising the modified T cell for adoptive therapy and for treating a condition, such as an autoimmune disease.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims 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 herein by reference in its entirety.

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

[0004] Safety concerns associated with integration using lentiviral 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 unwanted 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 allow for the efficient 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 CARs while avoiding the need for combination therapy and additional treatment. Increased RNA during the transfection process adversely affects T cell function, especially anti-tumor activity in vivo (Barrett et al., 2011, Hum Gene Ther 22:1575-1586 (Non-Patent Document 7)). Another construct, in which an anti-CD3 antigen antibody fragment is fused to an anti-tumor antigen antibody fragment, has also been tested in clinical trials for cancer treatment (Bargou et al., 2008, Science 321:974-977 (Non-Patent Document 8); Klinger et al., 2012, Blood 119:6226-6233 (Non-Patent Document 9)). Unfortunately, these constructs have significant limitations in function due to short half-life, poor accessibility to target cell sites, and lack of appropriate long-term signaling function.

[0006] Clinical TCR research has been hampered by low expression levels of transduced TCRs as well as mismatching of α and β chains. The potential for four distinct TCRs to be expressed on the cell surface when T cells transcribe two distinct TCR chains (native α / β, exogenous α / β, and a native / exogenous "mismatched" heterodimer) clearly poses a significant barrier to the use of this approach. Previous studies have clearly demonstrated in preclinical studies that TCR mismatching can lead to harmful self-antigen recognition.

[0007] Although early clinical data on TCR and CAR T cells for the treatment of cancer have shown promising results, risks to patients remain significant, and some patients' T cells lack sufficient potency to be effective treatment, even after forced modification of allogeneic donor-derived T cells via 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, potentially leading to graft-versus-host disease (GVHD). As a result, most current clinical trials using autologous CAR T cell infusions rely on immune tolerance to prevent harmful TCR-mediated recognition of normal tissues after adoptive cell transfer. While this approach has shown initial clinical success, it is limited by the time and expense of producing patient-specific T cell products. Therefore, there is a need for safer methods for engineering T cells that avoid 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 nucleic acids 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 nucleic acids encoding a chimeric antigen receptor (CAR).

[0010] One aspect of the present invention includes modified T cells 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 producing a modified T cell, 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 α chain, a TCR β chain, β-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) comprising an antigen-binding domain and a transmembrane domain.

[0012] In yet another aspect, the present 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 present 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 present 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 harmful to the subject.

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

[0019] In various embodiments of the above and any other aspects of the invention delineated herein, the nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA, and a CRISPR system, 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 the modified T cells described herein further comprises electroporating RNA encoding the costimulatory molecule into the T cells. 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 generating modified T cells as described herein further comprises expanding the T cells. In one embodiment, 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 delineated 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 disease-dermatitis herpetiformis, or the like. hepetiformis); chronic fatigue and immune deficiency 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 nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), 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. 1. A modified T cell comprising: [The present invention 1002] 1001. The modified T cell of the present invention, wherein said nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA, and CRISPR systems. [The present invention 1003] 1002. The modified T cell of the present invention, wherein the CRISPR system comprises a pAd5 / F35-CRISPR vector. [The present invention 1004] 1001. 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] 1001. The modified T cell of the present invention, wherein the antigen-binding domain of said CAR specifically binds to an antigen on a target cell. [The present invention 1006] 1001. The modified T cell of the present invention, wherein the intracellular domain of said CAR comprises dual signaling domains. [The present invention 1007] 1001. The modified T cell of the present invention, 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] 1008. 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 1009, wherein the different CD3 chains are a CD3ζ chain and a CD3ε chain. [The present invention 1011] Introducing into the T cells 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) containing an antigen-binding domain and a transmembrane domain into a T cell. 1. A method for producing modified T cells, comprising: [The present invention 1012] 1011. 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] 1012. The method of claim 1012, wherein the CRISPR system comprises a pAd5 / F35-CRISPR vector. [The present invention 1014] The method of claim 1011, 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] 1012. The method of claim 1011, wherein the intracellular domain of said CAR comprises dual signaling domains. [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 claim 1018, wherein the step of expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand. [The present invention 1020] 1011. 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] 1024. 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 hyperactivity 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] 1027. The method of claim 1027, wherein said 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, and celiac disease-dermatitis. hepetiformis); chronic fatigue and immune deficiency 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, 1028. The method of claim 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] 1027. The method of claim 1027, wherein said condition is cancer. [The present invention 1031] 1030. The method of claim 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 claim 1033, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC). [This 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 to prevent or treat an immune response that is harmful to the subject. [The present invention 1036] 1001. 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. [This 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 modified T cells produced according to the method of the present invention and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0030] The following detailed description of 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 represented by a block. The black blocks represent coding regions, and the gray columns represent noncoding regions. Thirteen gRNAs were designed to target exon 1 of the TCR α constant region (TRAC), 10 gRNAs were designed to target conserved sequences in exon 1 of the TCR β constant regions 1 (TRBC1) and 2 (TRBC2), and 10 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 overlapping PCR and cloned into the MSGV vector with a T7 promoter. Figure 1C shows Sanger sequencing results demonstrating the presence of multiple peaks in the genomic PCR products of 293T TCR TRAC and TRBC after 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 disruption of the 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. Using 360V for 1 ms in a 2 mm cuvette, we obtained 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 and 5% CO2, which had a much higher MFI than the normal 37°C and 5% CO2 conditions. Figure 2C is a schematic illustration of the CRISPR system transfected into primary T cells. CAS9 mRNA and gRNA were electrotransferred into T cells three days after bead stimulation. T cells were then cultured with 100 μL / mL IL-2, with some cells cultured at 32°C and 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 was approximately 2.5-fold higher than at 32°C. Figure 2E is a panel of graphs showing CD3 downregulation 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 from day 6 after electroporation are shown. Quadrants indicate 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 knockout T cells can be enriched by depletion of TCRpositive T cells. Figure 3A is a panel of graphs showing CD3 expression in TCRneg α or β knockout 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 multiple peaks 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 may be more easily modified than CD4 T cells. Figure 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, demonstrates that multiple electrotransfers of gRNA significantly improved the targeting efficiency of CRISPR systems in primary T cells. Figure 4A is a panel of graphs demonstrating that multiple electroporations of gRNA significantly improved targeting efficiency. The highest targeting efficiency, approaching 80%, was achieved by electroporating T cells up to three times within a 24-hour period. Initial experiments achieved only a 15% TCR targeting efficiency in T cells. After electrotransfer of CAS9 mRNA into T cells, sustained CAS9 expression was observed. The low cleavage efficiency is likely due to rapid degradation of the gRNA. A higher CD3-negative population was obtained. Figure 4B is a panel of graphs demonstrating that capping impairs gRNA function, but early introduction of the second gRNA resulted in higher efficiency. Figure 4C is a panel of graphs showing that multiple electrotransfers 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, demonstrates 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 bead or K562 aAPC restimulation resulted in approximately 25- to 58-fold expansion. [Figure 5B] See legend to Figure 5A. [Figure 6A] Figure 6A is a panel of graphs showing the phenotypic characteristics of TCR T cells after expansion under various conditions. [Figure 6B] Figure 6B is a panel of graphs showing the phenotypic characteristics of TCR T cells after expansion under various conditions. [Figure 7A] Figure 7, consisting of Figures 7A-7C, shows expanded TCR T cells with potent antitumor activity after in vitro redirection. Figure 7A is a panel of graphs demonstrating that TCR T cells can be redirected by intracellular introduction of anti-NY-ESO 1G4 TCR. Compared with the CAS9 mock (MOCK) group, 1G4 TCR-redirected TCR T cells showed higher levels of Vb13.1 expression, likely due to less mispairing of exogenous and endogenous TCR α and β chains. Figure 7B is a panel of graphs demonstrating that 1G4 TCR-redirected TCR T cells had high degranulation activity when cocultured with tumor (Nalm6-ESO) cell lines. Figure 7C is a panel of graphs demonstrating that 1G4 TCR-redirected TCR 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] Figure 8 is a panel of illustrations showing that committed TCRneg T cells suppress tumor growth in NSG mice after redirection. [Figure 9A]Figure 9, consisting of Figures 9A-9D, shows that disruption of β-2 microglobin resulted in HLA-class I elimination. Figure 9A shows sequencing data of CRISPR capable of disrupting the β-2 microglobin locus in HEK293 cells. Figure 9B is a panel of graphs showing that disruption of β-2 microglobin resulted in a bHLA-class I-negative T cell population. Figure 9C is a panel of graphs showing that cIFNg improved β-2 microglobin targeting efficiency in primary T cells. Figure 9D is a panel of graphs showing that HLA-class I 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 the TCR β constant region (TRBC) and β-2 microglobin. Six days after electroporation, both TCR and β-2 microglobin 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. [Figure 11A]Figure 11, consisting of Figures 11A-11D, shows triple knockout of HLA-class I and TCR α and β chains in primary T cells. Figure 11A is a panel of graphs showing stimulation of CD4 and CD8 T cells with CD3 / CD28 Dynabeads. Three days after stimulation, expanded T cells were electroporated with CAS9 mRNA along with gRNAs targeting the TCR α and 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. Figure 11B is a schematic diagram illustrating the isolation of HLA-class I and TCR α and β chain triple knockout T cells. Figure 11C is a panel of graphs showing electroporation efficiency as assessed 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% of the α and 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] Figure 12, consisting of Figures 12A-12D, shows knockout of FAS in 293T cells. Figure 12A is an image showing Sanger sequencing results of multiple peaks when FAS is knocked out in 293T cells. Figure 12B is a panel of graphs showing FACS data demonstrating that surface expression of FAS protein was disrupted by CRISPR. Figure 12C is a panel of images showing that FAS protein was replaced by GFP after CRISPR-mediated homologous recombination. Figure 12D is a panel of graphs of FACS data showing the percentage of CRISPR-mediated homologous recombination. [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 PD1 was targeted 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, consisting of Figures 15A and 15B, shows knockout of CTLA4 in 293T cells and primary cells, such as CCD1079-SK. Figure 15A is an image showing the results of Sanger sequencing of multiple peaks when CTLA4 is targeted in 293T cells. Figure 15B is an image showing sequence data after limiting dilution and single-cell expansion. Sanger sequencing results identified deletions and insertions at 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 reprogramming process of FASneg T cells into iPSCs. FASneg T cells can be induced to form a typical embryonic stem cell morphology, indicative of a pluripotent state. Figure 17B is a graph showing that FASneg T cells were reprogrammed into iPSCs approximately five times more efficiently than their wild-type counterparts. p53-deficient cell lines have been reported to be easier to reprogram because the apoptotic pathway is blocked. FAS knockout may facilitate the reprogramming process through 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 even after several passages. Figure 18B is a series of graphs showing that CD3neg T cells are reprogrammed approximately five-fold more efficiently than their wild-type counterparts, suggesting that TCR knockout plays a role in the T cell reprogramming process 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, comprising 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, comprising Figures 23A-23D, is a panel of graphs showing that knockdown of the 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 histogram), control siRNA (dotted open histogram), and no siRNA (filled histogram). 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 tumor cell fluorescence after T cell injection into a mouse model. Ten million Nalm6-CBG-ESO-GFP tumor cells, which express 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 various groups as indicated, and tumor cells were detected by fluorescence. [Figure 25] Figure 25 is a panel of images showing the 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 panel of graphs 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 the 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. Figure 29B is a panel of graphs showing that the majority of expanded universal CAR19 T cells retained high levels of CD62L 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 growth kinetics after challenge with Nalm6 tumor cells. [Figure 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 in mice receiving CAR19 T cells. CRISPR-edited TCR single-negative or TCR / HLA-A double-negative universal CAR19 T cells showed the same antitumor activity. [Figure 32A] Figure 32A is a panel of graphs showing that TCR single ablation or dual TCR and HLA-A 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 in 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 anti-tumor activity of CAR19 T cells. FAS-negative CAR19 T cells were generated. FAS ablation was confirmed by flow cytometry analysis. CAR19 gene expression in FAS T cells was comparable to wild-type. Even after a short 4-hour incubation with Nalm6 tumor cells, CD107a expression in FAS 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 CAR cell survival and proliferation 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, FAS 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. [Figure 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 CART 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 of five mice receiving wild-type T cells died of 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 the improved antitumor activity of universal CART cells after blocking the PD1 and Fas pathways with 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. [Figure 42] Figure 42 is a panel of diagrams showing a one-shot system for generating universal CART cells. Because gRNAs are prone to degradation, we developed a simplified one-shot method in which gRNAs are constitutively expressed along with CAR in a single lentiviral vector. [Figure 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. [Figure 44]Figure 44A is a panel of images showing morphological changes during reprogramming of iPSCs from Fas knockout T cells. The formation of typical embryonic stem cell morphology, indicative of FAS T cells, can be induced as a pluripotent state. Figure 44B is a graph showing that FAS T cells were reprogrammed into iPSCs approximately five-fold more efficiently than their wild-type counterparts. p53-deficient cell lines have been reported to be easier to reprogram due to 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 the ES-like morphology of iPSCs from CD3 TCR α- or β-chain knockout T cells under defined reprogramming conditions, which remained consistent 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 that of their wild-type counterparts, suggesting that TCR knockout plays a role in the T cell reprogramming process or affects cell viability after 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 the 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 various 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 for introducing CRISPR / Cas9 into T cells. A schematic of the T7 promoter-driven gRNA 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, then cultured in the presence of IL2 at 32°C for 24 hours before returning to normal culture conditions at 37°C. T cells with disrupted specific genes 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 efficient CRISPR / Cas9-mediated 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 in 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 in CRISPR-transfected T cells using various Cas9:gRNA ratios (top and middle panels) and amounts of total CRISPR RNA (bottom panel). [Figure 51B] Figure 51B is a table showing targeting efficiencies 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. The calculated amount of targeted gene disruption in TRAC and TRBC is shown at the bottom. Arrows indicate the expected bands. [Figure 53A] Figure 53A is an image of indels (in gene disruptions) observed by clonal sequence analysis of PCR amplicons after CRISPR-mediated recombination of the TCR α and β loci. [Figure 53B] Figure 53B is an image of a schematic representation of the human locus encoding the TCR α and β CRISPR gRNA targeting sites within the genomic loci of the TCR α and β 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 / CD3 cells via electrotransfer of 1G4 TCR (alpha and beta) or CAR19 mRNA. [Figure 56] Figure 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 methods is shown. [Figure 58] Figure 58 is a panel of graphs showing relative CD19-CAR expression after electrotransfer of CD19-CAR RNA into Cas9-mock and TCR / CD3 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 a CD107 release assay after incubation with Nalm6 target cells. Representative data from three independent experiments is 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. [Figure 59D] Figure 59D shows images of a group of NOD / scid / γc(- / -) mice (n=12) randomly assigned to three groups after intravenous injection of 1×10 Nalm6 tumor cells. Electroporated CD19-CAR-expressing Cas9 Mock T cells and TCR / CD3 T cells (10×10) were injected intravenously every four 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 one day before the start of T cell administration. Bars, SE=standard error; EP=electroporation; E:T=effector-to-tumor ratio; arrows, time of T cell injection; ns, not significant. ****P<0.0001 by two-way ANOVA with Bonferroni post-hoc test, ns. [Figure 59E] Figure 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] Figure 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 challenging gene-ablated T cells with irradiated allogeneic PBMCs (left panel) or co-culturing allogeneic PBMCs with irradiated and gene-ablated T cells. Individual spots are represented 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 disruption of endogenous TCR by CRISPR / Cas9 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 CD3 T cells electroporated with NY-ESO-1 TCR α RNA (1G4 α, 2 μg), β RNA (1G4 β, 2 μg), or α + β RNA (1G4 α + β, 2 + 2 μg) to disrupt the 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 an HLA-A2 / NY-ESO-1 positive cell line (Nalm6-ESO) or the control cell line Nalm6. [Figure 65B] Figure 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 (TCR T cells) electroporated with two different NY-ESO-1 TCR RNAs (1G4 TCR, 10 ug or 8F TCR, 10 ug) 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 a negative control. *P<0.05, **P<0.01 ****P<0.0001 by two-way ANOVA with 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 and 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 gRNAs targeting the TCR α and TCR β 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 gene 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 is 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 is 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. [Figure 75A]Figure 75A is a graph showing BLI from mice that received a single injection on day 7 using a lentiviral vector to express CD19-CAR and GFP. 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, lentiviral (LV)-transduced T cells (1x107) expressing CD19-CAR were infused by a single injection. T cells expressing LV GFP protein were injected as a control. [Figure 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, not different 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 (2x107) expressing LV-CD19-CAR were infused by a single injection. T cells expressing LV GFP protein served as a control. Imaging began one day before the start of T cell administration. Organs of randomly selected mice from various treatment groups were collected 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] Figure 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 receptors in 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. MD231 cells were transduced with the adenovirus-pAd5F35-CRISPR-PD1 virus. Three days later, genomic DNA was extracted and PCR was performed. Figure 79B shows the sequence of the targeting event in MDA231 cells after adenovirus-CRISPR manipulation. The PD1 PCR product was cloned into the TOPO vector and sequenced. [Figure 80] Figure 80 is a chart showing that reduced gRNA use resulted in increased 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 folds 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 folds (EP#1 and 5). [Figure 82] Figure 82 is a chart showing the 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] Figure 85 is a chart showing T cell numbers (upper chart) and expansion fold (lower chart) following the electroporation and culture procedure. [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 CD19 CAR-transduced and CRISPR-edited T cells (TD / KO). The 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 expanded T cells at day 8. [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 at day 11. [Figure 95] Figure 95 is a panel of graphs showing cytokine production of T cells at 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 INVENTION

[0031] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be 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 a variation 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 a state in which a T cell has been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers, inter alia, to a T cell undergoing cell division.

[0036] As used herein, the term "antibody" 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 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 conformations. The α and β light chains refer to the two major antibody light chain isotypes.

[0040] As used herein, the term "synthetic antibody" refers to 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 that expresses an antibody protein or an amino acid sequence defining that antibody, where the DNA or amino acid sequence is available and well known in the art, and obtained using DNA or amino acid sequence synthesis techniques.

[0041] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production and activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, 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 containing a nucleotide sequence or partial nucleotide sequence encoding 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 may be arranged in various combinations to elicit a 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 produced, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0042] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a 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 to prevent the development of tumors in the first place.

[0043] The term "autoantigen," according to the present invention, refers to 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, and glycoproteins, including cell surface receptors.

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

[0045] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later 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 the rapid and uncontrollable proliferation of abnormal cells.Cancer cells can spread locally or to other parts of the body through the 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] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor engineered to be expressed on immune effector cells and specifically bind 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 containing an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen-binding region. In some aspects, CARs comprise a fusion of a single-chain variable fragment (scFv)-derived monoclonal antibody fused to the CD3ζ transmembrane and intracellular domains. The specificity of the CAR design can be derived from the receptor's ligand (e.g., a peptide). In some embodiments, CARs can target cancer by redirecting the specificity of T cells expressing the CAR to tumor-associated antigens.

[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 modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies 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 antibody 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., an aAPC, a dendritic cell, a 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, for example, by binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind B7-H3. Costimulatory ligands also include 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, among others.

[0053] A "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates 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-regulation 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 due to previous exposure to a virus. Bacteria and archaea have developed an adaptive immune defense called the CRISPR-CRISPR-associated (Cas) system, which uses short RNAs to direct the degradation of foreign nucleic acids. In bacteria, the CRISPR system confers adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage.

[0056] In the type II CRISPR / Cas system, 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 the Cas protein to sequence-specific cleavage and silencing of pathogenic DNA. Recent studies have shown that target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region.

[0057] To 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 the 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 unless the disease is 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 without the disorder. If left untreated, a disorder does not necessarily cause a further decline in the animal's health.

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

[0061] "Effective amount" or "therapeutically effective amount," used interchangeably herein, 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, having either 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, which is the nucleotide sequence identical to the mRNA sequence and 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 the 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] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, ex vivo expanded T cells are increased in number relative to the number originally present in the culture. In another embodiment, ex vivo expanded T cells are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells 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] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a 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) incorporating a recombinant polynucleotide.

[0068] As used herein, "homology" 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 molecules is occupied by the same monomer subunit; for example, if a position in each of 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 matching or homologous positions; for example, if half of the positions in 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 matching 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 instances, 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 will comprise 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] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide 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 alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or identical, the two amino acid sequences are 90% identical.

[0072] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this protein class are IgA, IgG, IgM, IgD, and IgE. IgA is the predominant antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in most subjects during the primary immune response. 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 function 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, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen.

[0074] As used herein, "induced pluripotent stem cells" or "iPS cells" refer 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 capable of proliferation and differentiation into multiple cell types.

[0075] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to 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 material and the compounds be used conjointly by the recipient.

[0076] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide 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 " used herein refers to a genus of Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cell, so they are one of the most efficient methods of gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant level of gene transfer in vivo.

[0080] As used herein, the term "modified" 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 nucleic acids.

[0081] As used herein, the term "modulate" 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 subject that has not received the treatment. 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 nucleotide sequences that encode proteins, depending on their form, may contain introns.

[0084] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when 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 refer to an abnormal level of expression of the tumor antigen in cells from a diseased 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, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide 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 cellular genomes using conventional cloning techniques and PCR™, 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 comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing 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. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0089] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a 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 Paramyxoviridae family. 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 via 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] As used herein, the term "specifically binds" with respect to antibodies refers to 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 also bind to that antigen from one or more species. However, such cross-species reactivity does not, in and of itself, change the antibody's classification as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in and of itself, change the antibody's classification as specific. In some cases, the terms "specific binding" or "specifically binds" are used in reference 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 specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction 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., a 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.), is capable of specifically binding 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, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0101] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0102] As used herein, "substantially purified" cells are cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their 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 cells that have been separated from the cells with which they are normally associated in their 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 nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0104] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. 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, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in α / β and γ / δ forms, which 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 containing a TCR (e.g., including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells).

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

[0106] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is 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 phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct position 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 into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids 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. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 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 prevent graft-versus-host disease (GVHD) are highly desirable in clinical settings. However, the use of allogeneic T cells carries the risk of rejection by the host's immune system through 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 not been able to simultaneously prevent GVHD and host-versus-graft reactions.

[0112] The FAS receptor / FAS ligand (FAS / FASL) apoptosis signaling pathway negatively regulates T cell function. PD1 and CTLA4 are two major inhibitory signaling pathways in T cells. The enhanced antitumor immunity resulting from antibody-mediated blockade of CTLA-4, PD-1, or PD-L1 suggests that inhibiting these pathways may improve the efficacy of immunotherapy. The present invention includes the generation of modified T cells in which TCR α and β chains, β-2 microglobulin, HLA molecules, CTLA-4, PD-1, and / or FAS have been deleted 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 a modified T cell receptor or a chimeric antigen receptor. In some embodiments, the present invention includes methods for generating modified T cells. Such modified T cells can be placed into a therapeutic composition and administered to a patient 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 chains 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, T cells with downregulated gene expression have reduced immunogenicity in an allogeneic environment. In another embodiment, T cells with reduced immunogenicity express a 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 into T cells a nucleic acid capable of downregulating endogenous gene expression, wherein the gene is selected from the group consisting of TCR α chain, TCR β chain, β-2 microglobulin, HLA molecule, CTLA-4, PD1, and FAS. Downregulating the expression of endogenous genes involved in generating an immune response against the cells, such as TCR α chain, TCR β chain, β-2 microglobulin, or HLA molecule, reduces immune-mediated rejection of the modified T cells. For example, downregulating the expression of endogenous TCR, MHC, or β-2 microglobulin genes eliminates surface presentation of alloantigens on the T cells that could otherwise cause rejection by the host immune system. Additionally, 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 cells when exposed to an immunosuppressive microenvironment.

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

[0117] The nucleic acid that can regulate endogenous gene expression can down-regulate endogenous gene expression.In one embodiment, the nucleic acid that can down-regulate endogenous gene expression is selected from the group consisting of antisense RNA, antigomer RNA, siRNA, shRNA and CRISPR system.Endogenous gene expression can be down-regulated, 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 (e.g., 293T cells), primary cells, and CAR T cells. The CRISPR / CAS system can simultaneously target multiple genomic loci by coexpressing a single CAS9 protein with two or more gRNAs, making this system uniquely suited for editing multiple genes or synergistic activation of target genes.

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

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

[0121] In one embodiment, inducing 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, which 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-strand break.The sequence of the guide nucleic acid sequence can be located within the locus of the gene.In one embodiment, the length of 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 or more.

[0123] The guide nucleic acid sequence may be specific for any sequence, such as a gene thought 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 an α, β, γ, and / or δ chain), β-2 microglobulin, FAS, PD1, a major histocompatibility complex protein (such as an HLA class I molecule and / or an HLA class II molecule), CTLA-4, or any combination thereof.

[0124] The guide nucleic acid sequence includes an RNA sequence, a DNA sequence, a combination thereof (RNA-DNA 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. Genetic engineering of 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 present invention also includes modified T cells having downregulated gene expression as described herein and an exogenous T cell receptor (TCR). In one aspect, the present 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) that comprises 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 a TCR alpha chain, a 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 modified T cells 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 populations 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 antigen presentation. 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 a TCR heterodimer, which is involved in ligand recognition. TCRs exist in α / β and γ / δ forms, which are structurally similar but have different anatomical locations and functions. In one embodiment, a TCR comprises a TCR α chain and a TCR β chain, and thus, the nucleic acid encoding a TCR comprises a nucleic acid encoding a TCR α chain and a TCR β chain. In another embodiment, the TCR α chain or the 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 contains at least one mouse constant region. The constant domain is located proximal to the cell membrane, followed by a transmembrane domain and a short cytoplasmic tail. In one embodiment, the modified TCR contains a cytoplasmic domain that includes a costimulatory signaling domain, such as a 4-1BB costimulatory signaling domain. The variable domain contributes to determining the specific antigen and MHC molecule to which the TCR has binding specificity. 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. TCR sequence diversity 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, while 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 TCRs, high-affinity TCRs, and chimeric TCRs. When a TCR is modified, it may have a higher affinity for a target cell surface antigen than a 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 a modified chain, such as a modified α chain or β chain. Such modifications may include, but are not limited to, N-deglycosylation, altered domains (such as variable regions engineered to target specific antigens or increase affinity), the addition of one or more disulfide bonds, whole or fragmented chains from different species, and any combination thereof.

[0134] In one embodiment, the TCR has specificity for a target cell antigen. The target cell surface antigen can include any type of ligand that defines the surface of a target cell. For example, the target cell surface antigen can be selected to recognize a ligand that serves as a cell surface marker on target cells 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 includes 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 the 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 TCRs include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In one embodiment, the target antigen includes either a tumor-associated antigen (TAA) or a viral antigen, or any fragment 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) comprising 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 a TCR alpha chain, a 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 receptor (CAR) The present invention also includes modified T cells that have downregulated gene expression as described herein and have a CAR. Thus, the present invention encompasses modified T cells that comprise 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 present invention includes a method of making a modified T cell, comprising introducing into a T cell a nucleic acid capable of downregulating 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 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 whose downregulation is 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 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 affinity for a surface antigen on a target cell as described elsewhere herein. The invention also includes populations of cells comprising the modified T cells described herein.

[0141] One or more domains or fragments of a CAR can be human. In one embodiment, the present invention comprises 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 that express the gene using standard techniques, by deriving the gene from a vector known to contain it, or by directly isolating it from cells and tissues that contain it. 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 depends on the type and number of antigens present on the surface of the target cell. For example, the antigen-binding domain may be selected to recognize an antigen that acts as a cell surface marker on 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 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 (CD2 subset 1, also referred to as 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 S (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; 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 (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (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 β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulator 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 moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular 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 breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian 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 anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0148] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as 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 Regarding 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 the binding of this domain to the transmembrane domain of the same or different surface membrane protein, so as to minimize the interaction with other members of 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 chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some cases, various human hinges, including human Ig (immunoglobulin) hinges, may also be used.

[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 of CAR, or in other words, the cytoplasmic domain, 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 responsible for effector function. The term "effector function" refers to the specialized function of a cell. For example, the effector function of T cells may be cytolytic activity or helper activity, including the 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 cellular 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 costimulatory molecule that act cooperatively to initiate signal transduction after antigen receptor engagement, and any derivative or variant of these elements and any synthetic sequence that have 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 derived 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, a ligand that specifically binds to CD83, CD5, 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 Included are 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 derivative, variant or fragment 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, a T cell receptor (TCR), any derivative or variant thereof, any synthetic sequence thereof, and any combination thereof, that has the same functional capability.

[0158] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of a CAR, or between the intracellular and transmembrane domains of a 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 and intracellular domains of a 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 the 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, and the improved method of these methods.Also see U.S. Patent No. 4,444,887 and 4,716,111; and PCT Publication No. 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 are capable of expressing 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, constant, 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 nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. These modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of a polypeptide of the present invention. Antibodies directed against the selected target can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such techniques, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies, including, but not limited to, IgG1 (γ1) and IgG3. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (Int. Rev. Immunol, 13:65-93 (1995)).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, 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 technique, 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 a review, 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.V gene repertoires can be constructed from unimmunized human donors to isolate antibodies against a diverse array of antigens (including self-antigens) essentially according to the techniques 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 specific 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 produced 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 herein 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. In many cases, framework residues in the framework regions will be substituted 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, such as by modeling the interactions between 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, often referred to as "import" residues, are typically taken from an "import" variable domain. Thus, a humanized antibody comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region of human origin. 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 2014 / 023106). 91 / 09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; and 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 has been 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. Antibody humanization 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. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entireties.

[0166] The selection of human variable domains, both light and heavy, used to create humanized antibodies aims to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then adopted 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 particular light or heavy chain subgroup. 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 the target antigen 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 using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are commonly 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 for analysis of the possible role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve desired antibody characteristics, 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 for 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 comprise a costimulatory molecule or a nucleic acid encoding a costimulatory molecule. In one embodiment, the modified T cells of the present invention further comprise an exogenous nucleic acid encoding a costimulatory molecule such that the modified T cells express the costimulatory molecule. The nucleic acid can be introduced into the T cells by transducing, 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 comprises CD3 and at least two different CD3 chains, such as a CD3ζ chain and a CD3ε chain.

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

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

[0172] The biological method for introducing the polynucleotide of interest into host cell includes the use of DNA and RNA vector.Virus vector, and especially retrovirus vector, has become the most widely used method for inserting genes into mammalian, for example, human cells.Other virus 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., 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 approximately -20°C. Chloroform evaporates more readily than methanol, so it is used as the sole solvent. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of sealed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures 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 forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures in solution that differ from the typical 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 otherwise expose cells to the inhibitors of the present invention, various assays can be performed to confirm the presence of the nucleic acid in host cells. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0176] In one embodiment, a nucleic acid encoding a T cell receptor (TCR) that has 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 as or a separate 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 or sequentially with the nucleic acid that can downregulate endogenous TCR gene expression. In one embodiment, the nucleic acid encoding the TCR is introduced before the nucleic acid that can downregulate endogenous TCR gene expression.

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

[0178] RNA In one embodiment, the nucleic acid introduced into T cells 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. DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR 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. For example, the template encodes an antibody, an antibody fragment, or a portion of an antibody. For 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 portions of the intracellular domains 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 performing 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 PCR template. 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 in which 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 in PCR. Primers can be designed to be substantially complementary to any part 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 encoding a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or a portion of the 5' and 3' UTR. Primers useful for PCR are prepared 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 the 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 the 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 capable of promoting 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 to 3,000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various 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] 5' and 3' UTR can be the naturally occurring endogenous 5' and 3' UTR of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into forward primers and reverse primers, or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for changing RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in 3' UTR sequences can reduce mRNA stability. Therefore, 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 contain 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 a 5' UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but it does not appear to be required for all RNAs to enable efficient translation. The need for a Kozak sequence for many mRNAs is known in the art. In another embodiment, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of mRNA.

[0183] To enable RNA synthesis 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. When a sequence that functions as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the transcribed open reading frame. 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. Consensus nucleotide sequences for 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. In circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells. Transcription of linearized plasmid DNA at the end of the 3' UTR results in mRNA of normal size, which 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 incorporating a polyA / T stretch into a DNA template is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, because plasmid DNA templates obtained from bacterial cells are often highly damaged with deletions and other abnormalities. This makes the cloning procedure not only tedious and time-consuming, but also often unreliable. Therefore, a method that allows for the construction of DNA templates with a polyA / T 3' stretch without cloning is highly desirable.

[0187] The poly(A) / T segment of the transcription DNA template can be generated during PCR by using a reverse primer containing a poly(T) tail, such as a 100T tail (which can range in size from 50 to 5000T), 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 RNA and reduces RNA degradation. Generally, 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 to 5000 adenosines.

[0188] The poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as Escherichia coli (E. coli) poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the RNA translation efficiency by approximately two-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. ATP analogs can further increase RNA stability.

[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 contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates translation initiation. Any solute suitable for cell electroporation can be included, including factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[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 genetically engineered T cells to kill target 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 adjusted. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0194] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and vector-free. 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 the transgene into the 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 using in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been sequentially tested 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 and are genetically engineered to serve as templates for in vitro transcription in a standardized manner, resulting in the production of stabilized RNA transcripts. Currently, protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter that enables RNA transcription, followed by a gene of interest flanked on either the 3' and / or 5' ends 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 therefore corresponds to the subsequent poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, 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 intracellularly 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. Furthermore, RNA only needs to access the cytoplasm, not the nucleus, and therefore typical transfection methods can achieve extremely high transfection rates. Furthermore, plasmid-based approaches require that the promoter driving the expression of the gene of interest be active in the cells under study.

[0198] In another aspect, RNA constructs are delivered to cells by electroporation.For example, refer to the formulation and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US Patent No. 2004 / 0014645, US Patent No. 2005 / 0052630A1, US Patent No. 2005 / 0070841A1, US Patent No. 2004 / 0059285A1, US Patent No. 2004 / 0092907A1.Various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in relevant research literature and many patents and applications in the art.For example, refer to US Patent No. 6,678,556, US Patent No. 7,171,264 and US Patent No. 7,173,116. 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 to transfect cells in vitro, 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 for delivering RNA of interest to target cells.

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

[0200] In another embodiment, the method can further comprise electroporating a nucleic acid encoding a costimulatory molecule. The costimulatory molecule nucleic acid can be co-electroporated with the TCR RNA.

[0201] Source of T cells Prior to expansion, a source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is human. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain 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. Apheresis products typically contain 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 a wash solution that may lack calcium and magnesium, or may lack 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 cords. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0203] The cord blood mononuclear cells isolated in this manner 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 achieved using isolated antibodies, antibody-containing biological samples such as ascites, antibodies bound to physical supports, and cell-bound antibodies.

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

[0205] For isolation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the 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 a further embodiment, more than 100 million cells / ml is used. In a further embodiment, 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 a further embodiment, 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 depletion 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 from 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, as well as instantaneous, uncontrolled freezing at -20°C or in liquid nitrogen, can be used.

[0207] In one embodiment, the population of T cells is contained within cells such as peripheral blood mononuclear cells, 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 bearing 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, wherein the electroporated T cells in the population are expanded at least 10-fold. The chimeric membrane proteins of the present invention comprise an extracellular domain and an intracellular domain. The extracellular domain comprises a target-specific binding element, such as an antibody. In one embodiment, the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain derived from portions of the intracellular domains of CD28 and 4-1BB.

[0209] Expression of the chimeric membrane protein allows interaction with other cells in the population, such as cells expressing CD3, thereby stimulating and activating the expansion of the electroporated T cells. Without wishing to be bound by any particular theory, it is believed that cells expressing CD3 contact and bind to the chimeric membrane protein expressed on the surface of the electroporated T cells. At least one T cell expressing the chimeric membrane protein interacts with another cell expressing CD3. This interaction stimulates 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. Costimulatory molecules include any molecule that costimulates T cells, such as, but not limited to, CD3, CD28, or a combination thereof. In one embodiment, the extracellular domain can 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 can comprise any portion of an antibody that binds to an antigen, including, but not limited to, the antigen-binding domain of a synthetic antibody, a human antibody, a humanized antibody, a single-domain antibody, a single-chain variable fragment, 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, 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. Alternatively, in some embodiments, the extracellular domain portion comprises a humanized non-human antibody as described elsewhere herein.

[0213] Intracellular domain The intracellular or cytoplasmic domain contains 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 lymphocyte response to an antigen.

[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. Typically, the entire intracellular signaling domain can be used, although 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 the effector function signal. The intracellular signaling domain, therefore, includes any truncated portion of the intracellular signaling domain sufficient to transmit the 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 molecule, derivatives or variants of these sequences that have the same functional capability, any synthetic sequence, 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 A spacer domain, such as an oligopeptide or polypeptide, which functions to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain in the polypeptide chain, may be incorporated between the extracellular domain and the transmembrane domain of the chimeric membrane protein, or between the cytoplasmic domain and the transmembrane domain of the chimeric membrane protein. The spacer domain is composed of up to 300 amino acids, preferably 10 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, for example, a CD28 transmembrane domain and a CD8-α hinge domain.

[0218] T cell expansion As demonstrated by the data disclosed herein, expanding T cells by the methods disclosed herein can result in an increase of 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, and any and all whole and partial integers therebetween. In one embodiment, T cells are expanded in the range of about 20x to about 50x.

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

[0220] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises 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 involves 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 the T cells involves culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0222] The 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 cell population. 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 the doubling time.

[0224] Each round of subculture is referred to as a passage. When cells are subcultured, they are said to have been passaged. A particular cell population or cell line may 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 passages (i.e., the number of population doublings) depends on many factors, including, but not limited to, the seeding density, the substrate, the medium, and the time between passages.

[0225] In one embodiment, cells can be cultured for a few hours (about 3 hours) to about 14 days, or any integer value in between. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) that can 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 may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, or Optimizer supplemented with amino acids, sodium pyruvate, and vitamins, and may be 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 that can costimulate the T cells, for example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can be expanded by 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 comprises introducing into expanded T cells a nucleic acid encoding a T cell receptor (TCR) that comprises affinity for a surface antigen on a target cell, and electroporating into the T cells 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 include co-electroporation with RNA encoding the costimulatory molecule. In such an embodiment, the expanded T cells are further electroporated or co-electroporated with RNA encoding CD3. CD3 includes at least two different CD3 chains, such as CD3ζ chain and CD3ε 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 a nucleic acid encoding an agent into the expanded T cell population, such as by transducing, transfecting, or electroporating the expanded T cells with a nucleic acid encoding a TCR, where the agent further stimulates the T cells. The agent can stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function. In one embodiment, the agent nucleic acid is co-electroporated with a chimeric membrane protein RNA. In another embodiment, the agent nucleic acid, such as a TCR RNA, is electroporated after culturing the electroporated population. In a further embodiment, the agent nucleic acid, such as a TCR RNA, is electroporated into 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 pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition containing 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 the 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 harmful 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 engineered T cells generated as described herein can be homogeneous and retain T cell function. Furthermore, the engineered T cells can be administered to animals, preferably mammals, and more preferably humans, to suppress immune responses, such as those common in 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 desirable 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 engineered 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 inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac disease-dermatitis herpetiformis; Chronic fatigue and immune deficiency 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, 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 a dosage, route, and time determined in appropriate preclinical and clinical experiments and tests. The cell composition can be administered multiple times at dosages within these ranges. Administration of the cells of the present invention can be combined with other methods useful for treating the desired disease or condition, as determined by those skilled 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 present invention can be carried out in any convenient manner known to those skilled in the art. The cells of the present invention can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In another example, the cells of the present invention are injected directly into a site of inflammation in a subject, a site of local disease in a subject, a lymph node, an organ, a tumor, or the like.

[0241] The cells described herein can also be administered using a number of matrices. The present invention utilizes such matrices in a novel context: as artificial lymphoid organs, acting to support, maintain, or regulate the immune system, typically through modulation of 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 specific example, compositions and devices described in 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 these patents are incorporated herein by reference in their entireties. 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, such as an implant, if desired to leave a permanent or removable structure in the animal's body; or it may be biodegradable. The matrix may take the form of a sponge, implant, tube, telfa pad, fiber, hollow fiber, lyophilized component, gel, powder, porous composition, or 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 gases, including oxygen.

[0242] A matrix is ​​used herein as an example of a biocompatible material, however, the invention is not limited to matrices and all references 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 as a semipermeable membrane or in conjunction with a particular semipermeable material.

[0243] Pharmaceutical Compositions The pharmaceutical composition of the present invention can comprise the modified T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid such as glycine; antioxidant; chelating agent such as EDTA or glutathione; adjuvant (e.g., aluminum hydroxide); and preservative.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 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). Pharmaceutical compositions comprising the modified T cells described herein can be administered in doses up to 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6It can be generally stated that T cell compositions can be administered at a dose of 1000 cells / kg body weight, including all integer values ​​within these ranges. The T cell compositions can also be administered multiple times at these doses. The cells can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0246] In certain embodiments, it may be desirable to administer activated T cells to a subject, subsequently 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 being bound by theory, this multiple blood draw / multiple reinfusion protocol can be used to select for specific 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 for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) 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 used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibody or other antibody therapy, cytoxin, fludaribine, 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 signaling (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 a further embodiment, 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 using 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 peripheral blood stem cell transplantation after standard treatment with high-dose chemotherapy.In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the 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. CAMPATH dosages, for example, generally 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 this invention are not limited to the particular formulations described in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of the cell methods, expansion and culture methods, and therapeutic 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 skill of those in the art. Such techniques are fully 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); and "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 particularly useful for certain embodiments are discussed in the following sections. [Example]

[0251] Experimental Examples The present invention is described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the present invention should not be construed as 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 foregoing description and the following examples, make and utilize the compounds of the present invention and practice the methods claimed. The following examples, therefore, 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, catalog) 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. The 624mel melanoma line was purchased from the Surgery Branch (NCI / NIH). All cell lines were cultured as directed and routinely tested negative for mycoplasma contamination.

[0256] Preparation of TCR constructs for mRNA electroporation and lentiviral transduction: Based on sequencing information obtained from relevant 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 vectors or pTRPE lentiviral vectors.

[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 using an NGG PAM site from GN19, and some were selected using an NGG PAM site from N20. All gRNAs contained complementary sequences consisting of mismatches greater than 13 base pairs, eliminating potential off-target mRNA sites (Table 1). gRNAs were designed as shown in Figure 1A and synthesized by overlapping PCR. All gRNA PCR products were ligated into the MSGV vector. In vitro transcribed CAS9 and gRNAs targeted the TCR α and β chains and β-2 microglobulin constant regions. gRNAs were designed to target either a sequence within exon 1 of the TCR α constant region, a consensus sequence shared by exons 1 of both TCR β constant regions 1 and 2, β-2 microglobulin, or PD1. The gRNA-encoding sequences were assembled using overlapping 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 the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies, Carlsbad, CA). The mRNA was stored at -80°C in nuclease-free vials for single use. The gRNA targeting sequence used for animal testing is as follows: TIFF0007792114000001.tif34128

[0259] Flow cytometry: The following monoclonal antibodies and reagents with the indicated specificities were used, along 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 microglobulin (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 (San Diego, CA); 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 nutrient supplemented every two days. 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.

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

[0262] After electroporation, cells were immediately placed in 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, we co-electroporated Cas9 mRNA 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] Generating 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, T cells were electroporated with Cas9 and gRNAs targeting TCR α and β chains, B2M, and PD1. Six days after CRISPR delivery, CD3-, HLA-I-, and PD1-negative T cells 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 minutes at 4°C. After two washes, the cells were passed through an LD column (Miltenyi Biotec, 130-042-901, Auburn, CA), and the flow-through fraction was collected for further use. neg CD3 expression on 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 of 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 were irradiated and then mixed together to give a total of 324 × 10 6 PBMCs were obtained. The PBMCs were resuspended to a final volume of 90 ml, followed by the addition of R10 to 300 ml, mixed, and divided 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 two days, and once 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. The PCR primers used for amplification of the target loci were: TIFF0007792114000002.tif27140

[0268] The PCR product was 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 of either TIFF0007792114000003.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 diluted in OPTI-MEM (Invitrogen) at a final concentration of 1–3 × 10. 8 Cells were resuspended at 0.1 ml 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 plated at 1 × 10 in R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum; Invitrogen). 6 Effector T cells were washed and suspended at 1 x 10 cells / ml. 100 μl of each target cell type was added to a 96-well round-bottom plate (Corning) in duplicate. 6 After resuspension at 100 cells / ml, 100 μl of T cells were combined with target cells in the indicated wells. Control wells containing T cells alone were also included. The plates were incubated at 37°C for 18–20 hours. After incubation, supernatants were collected and subjected to ELISA assays (eBioscience).

[0272] CD107a staining: In a 96-well plate, plate effector cells:T cells at a 1:1 ratio (1 x 10) in 160 μl of complete RPMI medium. 5 1×10 effectors 5Cells were plated with 1000 ng / well of ...

[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 The cells were resuspended at 100 μl per ml, 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 percent 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- to 10-week-old NOD / SCIDγ (NSG) mice were inoculated with 1 × 10 IgG into the right flank. 6 PC3-CBG tumor cells were subcutaneously injected into the left flank of the same mice on day 5, and 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 Lentivirally 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 / SCIDγ (NSG) mice were administered 1 × 10 cells / mouse (3M). 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 cells were injected into 6- to 10-week-old NOD / SCIDγ (NSG) mice. 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, allowing tumors to grow to approximately 200 mm 3 The volume of T cells was 2 × 10 6 The dose was 2M / mouse. Animals were randomized and divided into groups based on baseline tumor size. All animals were included in the study, and blinded tumor assessment was performed for all animal experiments performed.

[0275] T cell stimulation, lentiviral transduction and CRISPR electroporation procedures: 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 / donor). 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 cells / ml, 100 mL / flask. On day 1, stimulated T cells were transduced with CD19 CAR lentivirus at a multiplicity of infection (MOI) of 2. 50 mL (25 x 10 6 ) 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 added to CART / Mock EP (10 mL, 50 × 10 6 cells / mL) and CART / CRISPR (10 mL, 50 × 10 6 The cells were then electroporated with CAS9 RNA (first EP) using 120 μg of CAS9 RNA / 400 μL of T cells at 500 V / 1 ms. After electroporation, cells in groups 1, 3, 5, and 7 were subsequently split by culturing half of the T cells in fresh medium and half in conditioned medium. On day 4, the cells were washed twice and resuspended in Opti-MEM at 50 × 10 6 T cells were resuspended at 1 x 10 cells / mL. 20 μg TRBC4 and B2M gRNA were electroporated into 400 μL of T cells. After electroporation, cells were resuspended at 1 x 10 cells / mL 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 column depletion. CD3- T cells were resuspended 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 to 25 x 10 cells / mL. 5 One was shipped for karyotyping, 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, 10 gRNAs targeting the constant region of the TCR β chain, and 10 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. Because transient expression of CRISPR is sufficient to mediate gene knockout, we developed a "hit-and-run" delivery strategy to transiently express CRISPR by electrotransferring 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. Gene disruption mediated by ZFNs and TALENs has been reported to be 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 sample, 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. TRAC targeting reached 77% after three rounds of gRNA electrotransfer (Figure 4A). Targeting of TRAC or TRBC was accompanied by a slight 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 product originating from the target site were present only 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 persistent disruption of αβ TCR expression, as assessed by Sanger sequencing and confirmed by flow cytometry analysis of CD3.

[0282] Example 2: Enrichment of TCR αβ-negative T cells For future clinical applications, a rapid and robust method for isolating sources of TCR-disrupted populations is available. To begin addressing this issue, we have identified TCR / CD3 T cells by negative selection using clinically approved paramagnetic beads and depletion columns. 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 untransfected control cells. Successive depletion steps resulted in >99% enrichment, with no shift in balance toward CD4 or CD8 T cell subsets (Figure 3C). Sequencing results also demonstrated that CRISPR modification introduced deletions and insertions into the TCR α and β loci (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 was successfully manipulated 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 increased the targeting efficiency of β-2 microglobulin in T cells by approximately 10-fold (Figure 9C). Multiple electrotransfers of β-2 microglobulin gRNA resulted in a population that was 66% β-2 microglobulin negative (Figure 11A).

[0285] For future clinical applications of allogeneic transplantation, a rapid and robust method for isolating HLA-class I null populations will be necessary. To begin addressing this issue, 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 more than 99%. HLA-class I neg The population could not be enriched from non-transfected control cells. Enriched HLA-class I populations 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 expanded by a single Rapid Expansion Protocol (REP) and subsequently tested 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 2 x 10 PBMCs were obtained. 6 Electrotransfer of CD3 cells with RNA was performed. negT cells 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 added every two days until T cells appeared to have entered a resting state, as judged by both slowed growth kinetics and cell size, at which point 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 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 1000 cells / ml and 30 ng / ml 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 TCR electrotransfer neg T cell redirection TCR neg To investigate 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 exhibited high levels of 107a, indicating increased degranulation activity (Figure 7B). Killing assays also demonstrated potent cytotoxicity against this cell line (Figure 7C). This indicates that these cells are potentially safer than previous clinical trials using CAR and TCR-expressing T cells, as they do not appear to induce GVHD and are less susceptible to mismatch cytotoxicity than TCR-treated conventional 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 the adverse effect of endogenous TCR on CAR function (for example, through competition with transcription factors). Therefore, a genetic approach has been designed that uses designer ZFN to permanently disrupt the 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] Unlike ZFNs and TALENs, the CRISPR / CAS system has recently emerged as a potentially facile and efficient alternative to ZFNs and TALENs for inducing targeted genetic changes. Recent studies have shown that target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region. Therefore, the CRISPR / CAS system can 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 coexpressing a single CAS9 protein with two or more gRNAs, making this system uniquely suited for multiplexed gene editing or synergistic activation of target genes. Simultaneous disruption of multiple genes in T cells can be achieved by administering various gRNAs together with CAS9.

[0295] Example 6: CRISPR-mediated triple knockout of HLA CLASS I and TCR α, β chains To work toward "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 the desired characteristics is often incompatible with the time frame for patients. Furthermore, autologous T cells from patients with advanced disease may be functionally impaired and may be tolerant to the desired antigen.

[0296] To address this, patients can be infused with allogeneic T cells to circumvent 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 broaden the application of T cell therapy and to isolate sources of TCR- and HLA-class I-disrupted populations for future allogeneic transplantation.

[0297] ZFN and TALEN comprise zinc finger DNA binding domain designed to bind with 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 each gene must be targeted individually.By using the CRISPR system described herein, it is possible to obtain gene disruption efficiency and shorten the time course.

[0298] To address this issue, we electrotransferred CAS9 with three different gRNAs targeting TRAC, TRBC, and β-2 microglobulin. Cells were labeled with PE-anti-β-2 microglobulin antibody and then negatively selected for HLA-class I expression using clinically approved paramagnetic anti-PE microbeads and a depletion column. 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 again enriched using microbeads. Two days later, TCR electrotransfer was performed on these triple knockout cells. The day after electrotransformation, the cells were stimulated with CD3 / CD28 Dynabeads. The next day, the 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) apoptosis signaling pathway has been extensively studied and characterized in T cells. PD1 and CTLA4 are two major inhibitory signaling pathways in T cells and 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, which have demonstrated enhanced antitumor immunity after antibody-mediated blockade of CTLA-4, PD-1, or PD-L1. Similar antibodies have been developed for human use, and early clinical data have shown promising results. Ppp2r2d knockdown can inhibit T cell apoptosis, enhance T cell proliferation, and enhance 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 for FAS, PD1, CTLA4, and PPP2r2d were modified by CRISPR. FAS was further replaced with GFP by CRISPR-induced homologous recombination. FACS data showed that 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 to reprogram primary and T cells. There are many methods for generating iPSCs, including virus-mediated gene transduction and chemical induction. While lentiviral and retroviral vectors require integration into host chromosomes to express reprogramming genes, 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 reprogramming efficiency is relatively low. Similarly, mRNA-based reprogramming is complex 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, and it 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 day 3 post-stimulation, the beads were removed, and the cells were resuspended in 1 mL of prewarmed complete T cell medium. Subsequently, they were incubated with a calculated volume of CytoTune Sendai virus (Life Technologies, Carlsbad, CA) containing a polycistronic vector for the expression of hKlf4, hOct3 / 4, and hSox2 in the cells. The treated T cells were seeded into 24-well plates and centrifuged at 2250 rpm for 90 minutes 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 two days. Half of the medium was changed daily. On day 3 post-infection, the cells were transferred to MEF feeder plates and cultured in T cell medium without cytokines. Four days after infection, the cells were cultured in standard hES medium. The medium was changed daily. ES-like colonies were observed approximately on day 7. 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 start of the reprogramming process. T cells underwent dramatic morphological changes during the reprogramming process 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 clear borders. A typical embryonic stem cell morphology was observed, which is consistent with FAS neg , CD3 negThese results indicate that TCR α-chain and TCR 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 efficiently than their wild-type counterparts (Figure 17B). neg The efficiency of T cell reprogramming was approximately five times higher than that of wild-type counterparts (Figure 18B). p53-deficient cell lines have been reported to be easier to reprogram due to disruption of the apoptotic pathway. FAS knockout also induces increased resistance to apoptosis. Loss of TCR expression renders 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 by 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). The endogenous T cell receptor (TCR) was 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, comprising 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 results showing multiple peaks in enriched CD3-negative T cells following electroporation of either CAS9 mRNA and gRNA to knockdown 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 undergone knockdown of endogenous TCR β (TRB-7) re-expressed CD3 4 hours after electroporation of NY-ESO-1 TCR α and β (1G4LY95 TCR) RNA. Normal T cells (ND424 beads) served as a control, which were nearly 100% CD3 positive with 5.25% endogenous TCR vb13.1 expression.

[0312] Figure 23, comprising 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 histogram), control siRNA (dotted open histogram), and no siRNA (filled histogram). 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.

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

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

[0315] Example 10: Universal CAR19 T cells 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 the cells were CAR19-positive as detected by flow cytometry. Because transient expression of CRISPR is sufficient to mediate gene knockout, we developed a "hit-and-run" delivery strategy to transiently express CRISPR by electrotransferring in vitro transcribed RNAs of CAS9 and gRNAs targeting the TCR α chain, TCR β chain constant region, and β-2 microglobulin gene on day 3. Following 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 the CRISPR construct (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 over 100 (Figure 27).

[0317] After expansion, cells remained TCR single-negative or TCR / HLA-A double-negative, resulting in enrichment of the CAR19-positive population. 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 (Fig. 29A) and retained high levels of CD62L expression (Fig. 29B), intermediate levels of CD28 expression (Fig. 29A), and low levels of CCR7 expression (Fig. 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 a minor effect on CAR19 expression and antitumor activity (Figures 30B and 30C). TCR single-negative and TCR / HLA-A double-negative CAR19 T cells exhibited robust lytic activity 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 growth kinetics after challenge with CD19-expressing cells (Figure 30D).

[0320] To examine the antitumor activity of CRISPR / CAS9-edited CAR19 T cells, we injected TCR single-negative and TCR and HLA-A double-negative CAR19 T cells 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 of tumor cell injection. Objective tumor regression was observed in mice receiving CAR19 T cells (Figure 6). CRISPR / CAS9 did not affect the in vivo tumor-killing activity of CAR19 T cells, confirming the benefits 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 the cells were challenged with HLA-mismatched tumor cell lines (Figure 32A). Elimination of HLA-A molecules resulted in activation of NK cells after prolonged coculture (5 days). No off-target activity was observed in IFNr Elispot assays after 24 hours of challenge with allogeneic whole blood PBMCs. The lack of off-target activity suggests that T cells may play a key role in acute immune responses following 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 can serve as a source of universal effector donor cells.

[0322] CAS9 and various gRNAs targeting FAS were electrotransferred into T cells. FASneg cells were sorted and subsequently transduced with lentiviral CAR19. Flow cytometry and Sanger sequencing data demonstrated 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 significantly enhanced compared to wild-type counterparts, even within 4 hours of coculture.

[0323] Several reports have shown that even weak antigen stimulation induces FAS activation and promotes T cell proliferation (Rethi, et al., Blood, vol. 112(4):1195-1204, 2008). Interestingly, FASneg CAR19 T cells expanded much faster than wild-type CAR19 T cells when stimulated with high levels of CD19+ K562 cells. This suggests that under high-level antigen conditions, FAS / FASL induced apoptosis rather than activation (Figure 34A). FASneg CAR19 T cells also 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 assays of CAR19 T cells were performed after cell injection into Nalm6-bearing mice. The FASneg CAR19 group exhibited superior antitumor activity compared to the wild-type group (Figure 35B). This difference is illustrated in Figure 35C, which shows bioluminescence data between these two groups. These data indicate that FAS ablation in CAR19 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 the 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 significantly enhanced and sustained high expression of CD137, a marker of T cell activation (Figure 37), indicating that the PD1 / PDL1 inhibitory signaling pathway was blocked.

[0326] When tested in an 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 examine 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 showed no signs of developing GVHD. In contrast, three of four mice in the wild-type CD19 CART group developed GVHD by day 65, as confirmed by histological examination of various organs (Figure 39).

[0328] In another experiment, cells were resuspended in FBS and intravenously infused into mice after sublethally irradiating them. Clinical GVHD was monitored two to three times weekly. 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 dual-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 dual-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 superior antitumor activity compared with 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 for enhancing universal CART cell treatment with CRISPR / Cas9.

[0330] Because gRNAs are susceptible to degradation, we developed a simplified one-shot method for generating universal CART cells. The gRNA was constitutively expressed along with the CAR in a single lentiviral vector. Naive T cells were transduced with lentivirus encoding the gRNA and CAR one day after stimulation with CD3 / CD28 Dynabeads. On day 3, cells were electroporated with Cas9 mRNA (Figure 42). This system allows for the manipulation of several genes with a single vector (Figure 42). CD3 expression was confirmed by flow cytometry on day 6. T cells treated with this one-shot system showed consistent gene ablation, with rates of up to 90% across the various 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 to reprogram primary and T cells. There are many methods for generating iPSCs, including viral-mediated gene transduction and chemical induction. Lentiviral and retroviral vectors require integration into host chromosomes to express reprogramming genes. DNA-based vectors, such as adenoviral, adeno-associated viral, 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 efficiencies. Similarly, mRNA-based reprogramming is complex and has been shown to have very low efficiency.

[0333] In contrast, Sendai virus does not integrate into the host genome or alter the genetic information of the host cell, and it also has 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 of a 24-well plate. The cells were stimulated with CD3 / CD28 beads. On day 3 post-stimulation, the beads were removed, and the cells were resuspended in 1 mL of prewarmed complete T cell medium. They were then incubated with a calculated volume of CytoTune Sendai virus (Life Technologies, Carlsbad, CA) containing a polycistronic vector for the expression of hKlf4, hOct3 / 4, and hSox2 in the cells. The treated T cells were seeded into a 24-well plate and centrifuged at 2250 rpm for 90 minutes 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 with 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 two days. Half of the medium was changed daily. On day 3 post-infection, the cells were transferred to MEF feeder plates and cultured in T cell medium without cytokines. Four days after infection, the cells were cultured in standard hES medium. The medium was changed daily. ES-like colonies were observed approximately on day 7. 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 five-fold more efficiently than their wild-type counterparts (Figure 44B). p53-deficient cell lines have been reported to be easier to reprogram due to disruption of the apoptotic pathway. FAS knockout can facilitate the reprogramming process through a similar mechanism.

[0337] We observed ES-like morphology of iPSCs reprogrammed from CD3neg TCR α- or β-chain knockout T cells (Figure 45A). This morphology remained constant after several passages. The efficiency of CD3neg T cell reprogramming was approximately 5-fold lower than that of wild-type counterparts (Figure 45B), suggesting that TCR knockout plays a role in the T cell reprogramming process 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 their ability to be edited by modified versions of the CRISPR / dCAS9 and CRISPR / FokI-CAS9 systems (Figure 48A). Flow cytometry data demonstrated that primary T cells were edited by both CRISPR / dCAS9 and CRISPR / FokI-CAS9 (Figure 48B). The CRISPR / dCAS9 gene knockout system demonstrated 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. These CART cells are deficient in the expression of endogenous TCR and HLA class I (HLA-I) molecules for use as allogeneic universal CART cells. The T cell receptor (TCR) α chain, TCR β chain, and β-2 microglobulin (B2M) genes 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 CARs with CRISPR RNA electroporation to simultaneously disrupt the endogenous TCR and B2M genes. Furthermore, disruption of endogenous PD1 enhanced the efficacy of CAR therapy in solid tumor models.

[0343] Multiplexed delivery of gRNAs leads to highly efficient disruption of multiple genes in human primary T cells without compromising effector function Efficient multiplex genome editing is required to generate universal T cells deficient in TCR, HLA, and other genes. To achieve efficient gene disruption in T cells, 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). Then, we 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 resulted in 1%–3% CD3-negative (CD3 neg) T cells were obtained (Figure 50A, upper graph). To determine whether transient exposure to mild hypothermia enabled more efficient gene disruption, cell editing was performed at 37°C or 32°C. CRISPR-mediated disruption of TRAC and TRB increased fourfold 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 maximal disruption efficiency was 1:1 to 2:1, and gene disruption efficiency correlated with the amount of electrotransferred mRNA (Figure 51A).

[0345] gRNA is prone to rapid degradation compared to mRNA, which may limit targeting efficiency. Therefore, multiple sequential electroporations of gRNA were investigated 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 transfected by a single-step CD3 negative selection to identify TCR / CD3 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, TCR / CD3neg T cells were co-electroporated with HLA-A2-restricted 1G4 NY-ESO-1 TCR (α + β) RNA to restore CD3 expression (Figure 55, left panel). Following T cell stimulation / expansion methods, the following were compared: (1) Rapid T Cell Expansion Protocol (REP) using PBMCs as feeder cells, (2) anti-CD3 / CD28 Dynabeads (beads), or (3) OKT3 loaded with K562-based artificial antigen-presenting cells (K562 aAPC) expressing CD28 and 4-1BB ligands. TCR / CD3 neg T cells were also electroporated with CD19 CAR RNA (Figure 55, right panel) and subsequently stimulated with irradiated K562 aAPCs 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 aAPCs, and K562-CD19, respectively (Figure 56).

[0347] To investigate whether CRISPR / Cas9 gene editing affects T cell phenotype and function, we investigated TCR / CD3 T cells expanded by various methods. neg When the T cell phenotype was examined, all expanded cells remained CD3 negative, and most (79.8%-93.4%) retained high levels of CD27, 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 TCRs 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 the TCR α and β chains is necessary to prevent TCR mispairing-associated toxicity in adoptive immunotherapy of TCR-redirected T cells, and B2M is essential for the assembly and expression of the HLA-I complex. In light of this, we developed a triple disruption of the TCR α and β chains and B2M to generate universal T cells. We first investigated whether disrupting 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 and β chains 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 chain and B2M triple knockout T cells abolished nonspecific killing of HLA-mismatched tumor cell lines (Figure 62). When these cells were challenged with allogeneic whole-blood irradiated PBMCs in an IFNγ Elispot assay, no response was observed (Figure 63, left panel). Ablation of HLA-I molecules also significantly reduced alloreactivity, as confirmed by co-culture of allogeneic PBMCs with irradiated B2M-depleted cells (Figure 63, right panel). These results suggest that triple-negative T cells, lacking TCR α and β chains and B2M, may serve as a universal source of T cells for adoptive immunotherapy, resisting rejection by the host immune system while simultaneously being unable to cause graft-versus-host disease.

[0350] Enhanced antitumor activity of TCR-redirected endogenous TCR-ablated T cells T cells in which the TCR α and β chains were disrupted by CRISPR / Cas9 showed increased transgenic TCR expression on the cell surface after redirection with NY-ESO-1 TCR (1G4). ​​Transgenic TCR expression was 67.6%, 78.8%, or 94.3% in TCR α or β single knockouts or α / β double knockouts, respectively, compared with 46.8% in wild-type T cells. Enhanced transgenic TCR expression led to enhanced T cell function, as evidenced by increased antigen-specific CD107a expression (Figure 65A) and enhanced cytotoxicity (Figure 65B), particularly 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 67A and 67B). These results strongly suggest that the endogenous TCR differentially influences the expression and function of the transgenic TCR.

[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). Upon cell expansion, 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 cells exhibited 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 a disrupted endogenous TCR (LV-CD19 CAR TCR) were injected into NSG mice bearing disseminated Nalm6 leukemia. neg ) or TCR and HLA-I co-disrupted CART cells (LV-CD19 CAR TCR / HLA-I neg) showed tumor regression to the same extent as mice treated with 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 (100 / 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 T cells 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, three of four mice in the wild-type CD19 CART (LV-CD19 CAR) group developed GVHD by day 65, as 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 simultaneously eliminating alloreactivity.

[0355] Adenoviral CRISPR delivery into primary T cells The CRISPR / Cas9 system is rapidly being utilized for gene regulation and editing in model organisms and cell lines. Viral vectors are 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 approximately 71% (Figures 79A and 79B). Adenoviruses provide a useful platform for introducing CRISPR into human T cells, even in their quiescent state. This approach will be useful for exploring the potential of CRISPR for gene regulation and editing in numerous experimental contexts.

[0356] Optimizing electroporation CD3 and B2M knockout efficiency and T cell expansion were evaluated after electroporation (EP) of Cas9 and gRNA in 4 mm and 2 mm cuvettes. Standard EP conditions using 2 mm cuvettes (360v / 1ms, 1st EP: 20 μg Cas9 RNA + 10 μg gRNA / 100 μl T cells, 2nd EP: 5 μg gRNA / 100 μl T cells) demonstrated 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 with 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 little effect on CD3 and B2M knockout efficiency. See Figure 80. Under standard EP conditions using a 4 mm cuvette, the knockout efficiency of CD3 and B2M was significantly reduced (EP#8), suggesting that the 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 folds (EP#1 and 5). See Figure 81.

[0358] To further optimize the EP conditions with the goal of achieving maximum T cell expansion with a CD3 / B2M knockout efficiency of over 60%, various EP conditions and RNA amounts were investigated. 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 was (400v / 2ms / 120μg CAS9 RNA) and EP#2 was (500v / 1ms / 20μg gRNA). See Figure 82.

[0359] Further experiments were carried out to optimize the 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 conducted to determine whether large-scale electroporation could result in high knockout and expansion efficiencies. On day 0, T cells (100 × 10) obtained from three donors were electroporated using anti-CD3 / anti-CD28 beads. 6 0.5 x 10 cells / donor 6 On day 1, stimulated T cells were transduced with CD19 CAR lentivirus. 50 mL (25 x 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 added to CART / Mock EP (10 mL, 5 × 106 ) and CART / CRISPR (10 mL, 50 × 10 6 On day 4, T cells were electroporated with gRNA and divided into 1 × 10 cells. 6 Cells were cultured at 25 x 10 cells / mL. 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 x 10 cells from three donors were cultured at 25 x 10 cells / mL. 5 One was sent for karyotype analysis.

[0361] (Table 1) Experimental groups TIFF0007792114000004.tif61128

[0362] T cell numbers (top chart in Figure 85) and expansion folds (bottom chart in Figure 85) were assessed after the electroporation and culture procedures. The expansion folds of T cells transduced with CD19 CAR only (TD only) or CD19 CAR-transduced and CRISPR-edited T cells (TD / KO) are shown in the left graph in Figure 86, and the T cell expansion folds at day 10 are shown in the right graph in Figure 86. By optimizing the 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 groups on 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, various T cell populations were stimulated with the 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, 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 manipulation 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 gRNA electroporations with transient exposure to mild hypothermia, targeting efficiencies exceeding 80% at the protein level were consistently achieved for single gene disruptions. Even more promising, triple gene disruption of TRAC, TRBC, and B2M yielded approximately 65% ​​double-negative CD3 and HLA-I expression without any purification or selection. These results also demonstrated that enrichment of gene-disrupted T cells to greater than 99% purity can be easily achieved using clinically approved paramagnetic beads, and purified T cells 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. The disrupted T cells did not cause GVHD, suggesting that they could be used as allogeneic CAR T cells. Importantly, the gene-edited T cells exhibited anti-tumor activity both in vitro and in various tumor mouse models, and their efficacy was comparable to or even superior to that of non-gene-edited T cells. Therefore, it is believed that the process described herein for producing synthetic cells can be easily adapted to 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 with ZFNs or TALENs to eliminate expression of endogenous TCR α and β chains to avoid GVHD. Due to the complexity of targeting strategies using zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) to manipulate multiple genes in T cells, 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 disrupted by CRISPR / Cas9 or by removing stimulatory NK ligands through the expression of non-classical HLA class I molecules, such as HLA-E, which may protect general T cells from NK cell-mediated rejection.

[0369] In summary, we have efficiently generated clinical-scale universal CART cells with potent antitumor activity and reduced alloreactivity using multiplexed CRISPR technology. This approach can be incorporated into current GMP-compliant manufacturing procedures and is likely to be clinically applicable, given the successful translation of ZFN-based transfer therapy for HIV / AIDS. Universal CAR T cells and TCR T cells may offer an alternative to autologous T cells. Indeed, checkpoint-disabled universal CAR T cells and TCR T cells may be more effective and versatile than current autologous T-cell CART therapies for cancer and infectious diseases.

[0370] Other Aspects The recitation of a list of elements in a definition of a variable herein includes definitions 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. While the present invention has been disclosed with reference to specific embodiments, it will be 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.

Claims

1. (a) a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of a first costimulatory molecule; (b) one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating an endogenous beta-2 microglobulin gene; (c) one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating an endogenous TCR α chain gene or an endogenous TCR β chain gene; and (d) one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating the endogenous Fas gene.

1. A modified T cell comprising: the modified T cells do not express Fas, beta-2 microglobulin, a TCR alpha chain, or a TCR beta chain; the expansion of the modified T cells is faster than the expansion of wild-type CAR T cells; and the modified T cells exhibit superior anti-tumor activity when compared to wild-type CAR T cells; Modified T cells.

2. The modified T cell of claim 1, wherein the CRISPR / Cas system comprises a pAd5 / F35-CRISPR vector.

3. 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; or the antigen-binding domain of the CAR specifically binds to an antigen on a target cell; or the antigen-binding domain of the CAR comprises a single-chain variable fragment that specifically binds to CD19 or PSMA on a target cell; The modified T cell of claim 1.

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

5. The CRISPR / Cas system (a) SEQ ID NO: 5 in the coding sequence of the Fas gene; and (b) SEQ ID NO: 15 in the coding sequence of the TCR α gene; and / or (c) SEQ ID NO: 16 in the coding sequence of the TCR β gene; and (d) SEQ ID NO: 44 in the coding sequence of the B2M gene The modified T cell of claim 1 , comprising a nucleic acid sequence capable of targeting a sequence of

6. The modified T cell of claim 1 , wherein the nucleic acid capable of downregulating Fas gene expression comprises the sequence CCATGCTGGGCATCTGGACCCTC.

7. The modified T cells (a) a TCR α gene comprising the nucleic acid sequence of any one of SEQ ID NOs: 20-28; and / or (b) a TCR β gene comprising any one of the nucleic acid sequences of SEQ ID NOs: 30-36 and 38-43; The modified T cell of any one of claims 1 to 6, comprising:

8. 8. The modified T cell of claim 7, wherein the modified T cell comprises one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating FAS, CD3, and B2M gene expression.

9. (a) (i) one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating an endogenous beta-2 microglobulin (B2M) gene; (ii) one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating an endogenous TCR α chain gene or an endogenous TCR β chain gene; and (iii) one or more nucleic acids encoding a CRISPR / Cas system capable of downregulating the endogenous Fas gene; into T cells ex vivo; (b) isolating T cells that have reduced or eliminated expression of the endogenous FAS gene, the endogenous B2M gene, the endogenous TCR α chain gene, and the endogenous TCR β chain gene; (c) introducing into the T cell a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of a first costimulatory molecule.

1. An ex vivo method for generating modified T cells, comprising: the modified T cells do not express Fas, beta-2 microglobulin, TCR alpha chain, and TCR beta chain; method.

10. 10. The method of claim 9, wherein the CRISPR / Cas system comprises a pAd5 / F35-CRISPR vector.

11. 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 antigen-binding domain of the CAR specifically binds to an antigen on a target cell, and / or wherein the intracellular domain of the CAR comprises dual signaling domains.

10. The method of claim 9.

12. The method of any one of claims 9 to 11, wherein the intracellular domain of the CAR comprises dual signaling domains.

13. The method of any one of claims 9 to 12, 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.

14. The method of any one of claims 9 to 13, further comprising expanding the T cells, thereby obtaining expanded T cells.

15. 15. The method of claim 14, wherein said expanding step comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

16. 16. The method of any one of claims 9 to 15, further comprising the steps of cryopreserving the T cells to obtain cryopreserved cells, and thawing the cryopreserved T cells prior to introducing at least one of the nucleic acids into the T cells.

17. 17. The method of any one of claims 14 to 16, wherein the step of introducing at least one of the nucleic acids 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.

18. The CRISPR / Cas system (a) SEQ ID NO: 5 in the coding sequence of the Fas gene; and (b) SEQ ID NO: 15 in the coding sequence of the TCR α gene; and / or (c) SEQ ID NO: 16 in the coding sequence of the TCR β gene; and (d) SEQ ID NO: 44 in the coding sequence of the B2M gene The method of claim 9, comprising a nucleic acid sequence capable of targeting a sequence of

19. The modified T cells (a) a TCR α gene comprising the nucleic acid sequence of any one of SEQ ID NOs: 20-28; and / or (b) a TCR β gene comprising any one of the nucleic acid sequences of SEQ ID NOs: 30-36 and 38-43; 10. The method of claim 9, comprising:

20. The method of any one of claims 9 to 19, further comprising expressing Klf4, Oct3 / 4 and Sox2 in the T cells to induce pluripotency of the T cells.

21. A pharmaceutical composition for treating a disease or condition in a subject, comprising the modified T cell of any one of claims 1 to 8, wherein the disease or condition is (i) is associated with immune enhancement, or (ii) Acquired immune deficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac disease-dermatitis hepetiformis), chronic fatigue and immune deficiency 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 ... an autoimmune disease selected from the group consisting of myelopathic chondritis, 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.

22. 10. A pharmaceutical composition for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising the modified T cell of any one of claims 1 to 8, wherein the pharmaceutical composition induces lysis of the target cell or tissue.

23. 23. The pharmaceutical composition of claim 22, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

24. A pharmaceutical composition for preventing or treating an immune reaction that is harmful to a subject in adoptive cell transfer therapy, comprising the modified T cells of any one of claims 1 to 8.

25. 10. Use of the modified T cell of any one of claims 1 to 8 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

26. A composition comprising modified T cells produced according to the method of any one of claims 9 to 20.

27. A pharmaceutical composition comprising modified T cells produced according to the method of any one of claims 9 to 20 and a pharmaceutically acceptable carrier.

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