Methods and compositions for modified t cells

Abstract

To provide safer methods for modifying T cells, while generating T cells with maximal effector activity and functionality in vivo for T cell based adoptive immunotherapy, and modified T cells.SOLUTION: The disclosure provides a modified T cell and a method for preparing the same. The modified T cell comprises an exogenous nucleic acid encoding a T cell receptor (TCR) having affinity to an antigen on a target cell, and a nucleic acid encoding a bispecific antibody, where (a) the T cell expresses the TCR and the bispecific antibody on the surface of the T cell; (b) the bispecific antibody comprises a bispecific antigen binding domain, where the bispecific antigen binding domain comprises a first and a second single chain variable fragment (scFv) molecules; and (c) the first scFv molecule is specific for at least one antigen on a target cell, and the second scFv molecule is specific for an antigen on an activating T cell.SELECTED DRAWING: None

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 Applications Nos. 62 / 073,144, 62 / 073,343, 62 / 073,467, 62 / 073,540, and 62 / 073,681, all filed October 31, 2014, which are incorporated herein by reference in their entireties.

[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 transfer of multiple genes. However, the main limitation of transient expression of CARs is the suboptimal effector activity and functionality of RNA-transfected T cells. Multiple T cell infusions and / or significant use of low-dose chemotherapy have been used to improve function (Barrett et al., 2013, Hum Gene Ther 24(8):717-27).

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

[0006] Therefore, there is a need for safer methods of modifying T cells while generating T cells with maximal effector activity and functionality in vivo for T cell-based adoptive immunotherapy. [Prior art documents] [Non-patent literature]

[0007] [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

[0008] As described herein, the present invention relates to compositions and methods for modifying T cells.

[0009] In one aspect, the invention includes an engineered T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) comprising affinity for an antigen on a target cell and electroporated RNA encoding a bispecific antibody, wherein the T cell expresses the TCR and bispecific antibody on its surface.

[0010] In another aspect, the invention includes a method for making modified T cells comprising introducing into a T cell a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for an antigen on a target cell and a nucleic acid encoding a bispecific antibody, wherein the electroporated T cell is capable of expressing the TCR and the bispecific antibody.

[0011] In yet another aspect, the invention includes the use of a T cell described herein in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

[0012] In yet another 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 comprising electroporated RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody, wherein the modified T cells express the modified TCR and the bispecific antibody.

[0013] In another aspect, the present invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells that have been electroporated with RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody to prevent or treat an immune response deleterious to the subject.

[0014] 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 a population of modified T cells that have been electroporated with RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody.

[0015] In yet another aspect, the invention includes 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.

[0016] In another aspect, the present invention includes a composition comprising the modified T cells. In another aspect, the present invention includes a pharmaceutical composition comprising the modified T cells of claim 1 and a pharmaceutically acceptable carrier.

[0017] In various embodiments of the above or any other aspects of the present invention depicted herein, the TCR comprises at least one disulfide bond. In one embodiment, the TCR comprises at least one mouse constant region. In another embodiment, the TCR has a higher affinity for a target cell antigen than that of a wild-type TCR. In yet another embodiment, the TCR comprises a TCR alpha chain and a beta chain. In one embodiment, the TCR comprises a costimulatory signaling domain, such as a 4-1BB costimulatory signaling domain, at the C-terminus of at least one of the chains. In one embodiment, the beta chain comprises at least one N-deglycosylation. In one embodiment, the alpha chain comprises at least one N-deglycosylation.

[0018] In another embodiment, the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

[0019] In another embodiment, the bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. In yet another embodiment, the bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules, such that the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on an activated T cell.

[0020] In another embodiment, the activating T cell antigen is selected from the group consisting of CD3, CD4, CD8, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, TCR, PD1 and PD1L.

[0021] In another embodiment, the T cells described herein further comprise an electroporated nucleic acid encoding a costimulatory molecule. In yet 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.

[0022] In another embodiment, the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In yet another embodiment, the methods described herein further comprise electroporating RNA encoding CD3 into the T cells. In one embodiment, the CD3 RNA is co-electroporated with the TCR nucleic acid.

[0023] In another embodiment, the nucleic acid comprises in vitro transcribed RNA or synthetic RNA. In yet another embodiment, the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha chain and a TCR beta chain. In one embodiment, the step of introducing the nucleic acid comprises co-electroporating an RNA encoding the TCR alpha chain and a separate RNA encoding the TCR beta chain.

[0024] In another embodiment, the methods described herein further comprise expanding the T cells. In one embodiment, the expanding comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. In one embodiment, the expanding comprises electroporating the T cells with RNA encoding a chimeric membrane protein, such as a chimeric membrane protein comprising a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB, and culturing the electroporated T cells.

[0025] In another embodiment, the method described herein further comprises cryopreserving the T cells. In one embodiment, the method described herein further comprises thawing the cryopreserved T cells before introducing the nucleic acid into the T cells. In yet another embodiment, the method described herein further comprises cryopreserving the T cells after introducing the TCR nucleic acid. In yet another embodiment, the method described herein further comprises expressing the bispecific antibody as a membrane protein. In yet another embodiment, the method described herein further comprises cryopreserving the bispecific antibody-introduced T cells. In yet another embodiment, the method described herein further comprises inducing lysis of the target cell or tissue. In one embodiment, the induced lysis is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0026] In another embodiment, the condition 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; 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, polychondritis, polyglandular syndrome In another embodiment, the condition is an autoimmune disease, such as an autoimmune disease selected from the group consisting of: rheumatic 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. In yet another embodiment, the condition is a cancer, such as 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.

[0027] In one aspect, the invention includes an engineered T cell comprising a nucleic acid encoding a bispecific antibody comprising dual specificities for an antigen on a target cell and an antigen on a T cell, and a nucleic acid encoding a chimeric ligand engineered activation receptor (CLEAR), wherein the T cell expresses the bispecific antibody and CLEAR.

[0028] In another aspect, the invention includes the use of a T cell described herein in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

[0029] In yet another aspect, the invention includes a method for making an engineered T cell, comprising introducing into a T cell a nucleic acid encoding a bispecific antibody and a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR), wherein the T cell is capable of expressing the bispecific antibody and CLEAR.

[0030] In yet 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 engineered T cells that express a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell.

[0031] In another aspect, the present invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell, to prevent or treat an immune response harmful to the subject.

[0032] 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 a population of engineered T cells comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell.

[0033] In yet another aspect, the invention includes 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.

[0034] In another aspect, the invention includes a composition comprising the modified T cells described herein. In yet another aspect, the invention includes a pharmaceutical composition comprising the modified T cells described herein and a pharmaceutically acceptable carrier.

[0035] In various embodiments of the above or any other aspects of the invention delineated herein, CLEAR comprises an intracellular activation domain and an extracellular domain. In one embodiment, the intracellular activation domain comprises a portion of the intracellular activation domain of CD3 zeta. In one embodiment, the extracellular domain is selected from the group consisting of an antigen-binding domain of an antibody, a ligand-binding domain of a receptor, an antigen, and a ligand. In one embodiment, the extracellular domain is selected from the group consisting of CD27, CD28, CD70, CD80, PD1, and PD-L1. In one embodiment, the extracellular domain is capable of binding to a tumor antigen.

[0036] In another embodiment, CLEAR further comprises a costimulatory domain. In one embodiment, the costimulatory domain is selected from the group consisting of CD4, CD8 and 4-1BB.

[0037] In another embodiment, the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

[0038] In another embodiment, the bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. In yet another embodiment, the bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules, such that the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on a T cell. In yet another embodiment, the bispecific antibody comprises dual specificities for an antigen on a target cell and CLEAR on a T cell.

[0039] In another embodiment, the T cells described herein further comprise a nucleic acid encoding a costimulatory molecule, such as a costimulatory molecule selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In yet another embodiment, the T cells described herein are obtained from the group consisting of peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines.

[0040] In another embodiment, at least one of the nucleic acids is introduced by a method selected from the group consisting of transduction of T cells, transfection of T cells, and electroporation of T cells. In yet another embodiment, at least one of the nucleic acids comprises in vitro transcribed RNA or synthetic RNA.

[0041] In another embodiment, the methods described herein further comprise expanding the T cells. In one embodiment, the expanding comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. In one embodiment, the expanding comprises electroporating the T cells with RNA encoding a chimeric membrane protein, such as a chimeric membrane protein comprising a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB, and culturing the electroporated T cells.

[0042] In another embodiment, the methods described herein further comprise cryopreserving the T cells. In one embodiment, the methods described herein further comprise thawing the cryopreserved T cells before introducing the nucleic acid into the T cells. In yet another embodiment, the methods described herein further comprise cryopreserving the T cells after introducing the CLEAR nucleic acid. In yet another embodiment, the methods described herein further comprise expressing the bispecific antibody as a membrane protein. In yet another embodiment, the methods described herein further comprise cryopreserving the bispecific antibody-introduced T cells. In yet another embodiment, the methods described herein further comprise inducing lysis of the target cell or tissue. In one embodiment, the induced lysis is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0043] In another embodiment, the condition 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; 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, polychondritis, polyglandular syndrome In another embodiment, the condition is an autoimmune disease, such as an autoimmune disease selected from the group consisting of: rheumatic 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. In yet another embodiment, the condition is a cancer, such as 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.

[0044] In one aspect, the invention includes modified T cells that contain a nucleic acid encoding an affinity molecule chimeric receptor that includes a small molecule extracellular domain that has affinity for an antigen on a target cell, wherein the T cells express the affinity molecule chimeric receptor.

[0045] In another aspect, the present invention includes an engineered cell comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain, and wherein the cell expresses the bispecific affinity molecule.

[0046] In yet another aspect, the invention includes a method for generating modified T cells comprising introducing into a population of T cells capable of expressing an affinity molecule chimeric receptor a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell.

[0047] In yet another aspect, the present invention includes a method for making modified cells comprising introducing into a population of cells a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain, and wherein the cells express the bispecific affinity molecule.

[0048] In another aspect, the invention includes the use of a modified T cell described herein or a modified cell described herein in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

[0049] In yet another aspect, the present invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell to prevent or treat an immune response deleterious to the subject.

[0050] In yet another aspect, the present invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

[0051] In 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 a population of engineered cells comprising nucleic acids encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

[0052] In yet another aspect, the invention includes a method of treating a condition in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the T cells described herein or the modified cells described herein.

[0053] 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 modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on the target cell.

[0054] In yet another 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 engineered cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

[0055] In yet another aspect, the invention includes a composition comprising the modified T cells described herein or the modified cells described herein. In yet another aspect, the invention includes a pharmaceutical composition comprising the modified T cells described herein or the modified cells described herein and a pharmaceutically acceptable carrier.

[0056] In various embodiments of the above or any other aspects of the invention delineated herein, the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

[0057] In one embodiment, the small molecule extracellular domain comprises a helical structure lacking disulfide bridges. In another embodiment, the small molecule extracellular domain is less than about 10 kD.

[0058] In another embodiment, the affinity molecule chimeric receptor further comprises an intracellular signaling domain, such as a CD3 signaling domain. In yet another embodiment, the affinity molecule chimeric receptor further comprises a costimulatory signaling domain, such as a 4-1BB costimulatory signaling domain. In yet another embodiment, the affinity molecule chimeric receptor further comprises a transmembrane domain, such as a CD8 transmembrane domain. In yet another embodiment, the affinity molecule chimeric receptor further comprises a TCR variable domain and a TCR constant domain.

[0059] In another embodiment, the T cells described herein further comprise a nucleic acid encoding a costimulatory molecule, such as a co-stimulatory molecule selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In one embodiment, the CD3 comprises at least two different CD3 chains, such as a CD3 zeta chain and a CD3 epsilon chain.

[0060] In another embodiment, the affinity domain capable of binding to a target cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antibody antigen-binding domain. In yet another embodiment, the affinity domain capable of binding to an activating T cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antibody antigen-binding domain.

[0061] In another embodiment, the small molecule antigen-binding domains comprise a helical structure lacking disulfide bridges. In yet another embodiment, the small molecule antigen-binding domains are each less than about 10 kD.

[0062] In another embodiment, the target cell antigen is selected from the group consisting of a tumor-associated antigen (TAA), a bacterial antigen, a parasitic antigen, a viral antigen, and any fragment thereof. In yet another embodiment, the activating T cell antigen is a costimulatory molecule selected from the group consisting of CD3, CD4, CD8, 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, and a CD83-specific binding ligand, and any fragment thereof.

[0063] In another embodiment, the T cells described herein are used in a method of treating an immune response in a subject in need thereof. In yet another embodiment, the cells described herein are used in a method of treating an immune response in a subject in need thereof.

[0064] In another embodiment, the nucleic acid is introduced by a method selected from the group consisting of transducing a population of T cells, transfecting a population of T cells, and electroporating a population of T cells. In yet another embodiment, the introduction of the nucleic acid comprises electroporating RNA encoding the affinity molecule chimeric receptor. In yet another embodiment, the nucleic acid is introduced by a method selected from the group consisting of transducing a population of cells, transfecting a population of cells, and electroporating a population of cells. In yet another embodiment, the introduction of the nucleic acid comprises electroporating RNA encoding the bispecific affinity molecule.

[0065] In another embodiment, the cells described herein are selected from the group consisting of T cells, B cells, natural killer cells, and antigen-presenting cells. In yet 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.

[0066] In another embodiment, the methods described herein further comprise electroporating RNA encoding CD3 into the T cells. In one embodiment, the CD3 RNA is co-electroporated with a nucleic acid encoding an affinity molecule chimeric receptor.

[0067] In another embodiment, the methods described herein further comprise cryopreserving the T cells after introducing the affinity molecule chimeric receptor nucleic acid. In yet another embodiment, the methods described herein further comprise cryopreserving the T cells. In yet another embodiment, the methods described herein further comprise thawing the cryopreserved T cells before introducing the affinity molecule chimeric receptor nucleic acid into the T cells.

[0068] In another embodiment, the methods described herein further comprise expanding the T cells. In one embodiment, the expanding comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. In one embodiment, the expanding comprises electroporating the T cells with RNA encoding a chimeric membrane protein, such as a chimeric membrane protein comprising a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB, and culturing the electroporated T cells.

[0069] In another embodiment, the methods described herein further comprise the step of combining the activated T cells and the target cells with a bispecific affinity molecule.

[0070] In another embodiment, the condition 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; 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, polychondritis, polyglandular syndrome In another embodiment, the condition is an autoimmune disease, such as an autoimmune disease selected from the group consisting of: rheumatic 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. In yet another embodiment, the condition is a cancer, such as 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.

[0071] In another embodiment, the methods described herein further comprise inducing lysis of the target cell or tissue. In one embodiment, the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0072] In one aspect, the invention includes modified T cells comprising electroporated RNA encoding a bispecific T-cell engager (BiTE) molecule, wherein the BiTE molecule comprises a dual specificity for an antigen on a target cell and an antigen on an activated T cell selected from the group consisting of CD3, CD4, CD8, and TCR.

[0073] In another aspect, the invention includes a method for making modified T cells comprising expanding a population of T cells and electroporating the expanded T cells with RNA encoding a bispecific antibody, wherein the electroporated T cells are capable of expressing the bispecific antibody.

[0074] In yet another 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 comprising electroporated RNA encoding a bispecific T cell-enhancing (BiTE) molecule comprising dual specificities for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8 and TCR.

[0075] In yet another aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells to prevent or treat an immune response deleterious to the subject, wherein the modified T cells have been expanded and electroporated with RNA encoding a bispecific T cell-enhancing (BiTE) molecule having dual specificity for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR.

[0076] In another aspect, the invention includes a method of treating a disease or condition associated with immune enhancement in a subject comprising administering to a subject in need thereof a population of modified T cells, wherein the modified T cells have been expanded and electroporated with RNA encoding a bispecific T cell-enhancing (BiTE) molecule having dual specificities for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR.

[0077] In yet another aspect, the invention includes 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.

[0078] In yet another aspect, the invention includes the use of the modified T cell of claim 145 in the manufacture of a medicament for treating an immune response in a subject in need thereof.

[0079] In another aspect, the invention includes a composition comprising the modified T cells described herein. In yet another aspect, the invention includes a pharmaceutical composition comprising the modified T cells described herein and a pharmaceutically acceptable carrier.

[0080] In various embodiments of the above or any other aspects of the invention delineated herein, the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

[0081] In one embodiment, the bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. In another embodiment, the bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules, such that the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on an activated T cell.

[0082] 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.

[0083] In another embodiment, the RNA comprises in vitro transcribed RNA or synthetic RNA.

[0084] In another embodiment, the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. In yet another embodiment, the expanding comprises electroporating the T cells with RNA encoding a chimeric membrane protein, such as a chimeric membrane protein comprising a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB, and culturing the electroporated T cells.

[0085] In another embodiment, the methods described herein further comprise cryopreserving the expanded T cells. In yet another embodiment, the methods described herein further comprise thawing the cryopreserved T cells for electroporation with RNA encoding the bispecific antibody. In yet another embodiment, the methods described herein further comprise expressing the bispecific antibody as a membrane protein. In yet another embodiment, the methods described herein further comprise cryopreserving the bispecific antibody-electroporated T cells.

[0086] In another embodiment, the methods described herein further comprise inducing lysis of the target cells or tissues containing the target cells. In one embodiment, the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0087] In another embodiment, the condition 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; 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, polychondritis, polyglandular syndrome In another embodiment, the condition is an autoimmune disease, such as an autoimmune disease selected from the group consisting of: rheumatic 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. In yet another embodiment, the condition is a cancer, such as 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.

[0088] In one aspect, the invention includes an engineered T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) comprising affinity for a surface antigen on a target cell; and a nucleic acid encoding a costimulatory molecule, wherein the T cell expresses the TCR and the costimulatory molecule.

[0089] In another aspect, the invention includes a method for making a modified T cell comprising introducing into a T cell a nucleic acid encoding a T cell receptor (TCR) that comprises affinity for a surface antigen on a target cell, and introducing into the T cell a nucleic acid encoding a costimulatory molecule, wherein the T cell is capable of expressing the TCR and the costimulatory molecule.

[0090] In yet another aspect, the invention includes the use of the modified T cell of claim 173 in the manufacture of a medicament for treating an immune response in a subject in need thereof.

[0091] In yet 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 modified T cells, wherein the T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) that has affinity for a surface antigen on the target cell.

[0092] In another aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells to prevent or treat an immune response harmful to the subject, wherein the modified T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) with affinity for a surface antigen on a target cell.

[0093] In yet another aspect, the invention includes a method of treating a disease or condition associated with immune enhancement in a subject comprising administering to a subject in need thereof a population of modified T cells, wherein the modified T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) that has affinity for a surface antigen on a target cell.

[0094] In yet another aspect, the invention includes 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.

[0095] In another aspect, the invention includes a composition comprising the modified T cells described herein. In yet another aspect, the invention includes a pharmaceutical composition comprising the modified T cells described herein and a pharmaceutically acceptable carrier.

[0096] In various embodiments of the above or any other aspects of the present invention depicted herein, the TCR comprises at least one disulfide bond. In one embodiment, the TCR comprises at least one mouse constant region. In another embodiment, the TCR has a higher affinity for a target cell antigen than that of a wild-type TCR. In yet another embodiment, the TCR comprises a TCR alpha chain and a beta chain. In one embodiment, the TCR comprises a costimulatory signaling domain, such as a 4-1BB costimulatory signaling domain, at the C-terminus of at least one of the chains. In one embodiment, the beta chain comprises at least one N-deglycosylation. In one embodiment, the alpha chain comprises at least one N-deglycosylation.

[0097] In another embodiment, a nucleic acid encoding a costimulatory molecule is electroporated into T cells. In one embodiment, the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L. In one embodiment, CD3 comprises at least two different CD3 chains. In one embodiment, the different CD3 chains are CD3 zeta chain and epsilon chain.

[0098] In another embodiment, the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

[0099] In another embodiment, at least one of the nucleic acids is introduced by a method selected from the group consisting of transduction of T cells, transfection of T cells, and electroporation of T cells. In yet another embodiment, at least one of the nucleic acids comprises in vitro transcribed RNA or synthetic RNA.

[0100] 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.

[0101] In another embodiment, the methods described herein further comprise expanding the T cells. In one embodiment, the expanding comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. In one embodiment, the expanding comprises electroporating the T cells with RNA encoding a chimeric membrane protein, such as a chimeric membrane protein comprising a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB, and culturing the electroporated T cells.

[0102] In another embodiment, the method described herein further comprises cryopreserving the T cells. In yet another embodiment, the method described herein further comprises thawing the cryopreserved T cells before introducing the TCR-encoding nucleic acid into the T cells. In yet another embodiment, the method described herein further comprises cryopreserving the T cells after introducing the TCR nucleic acid.

[0103] In another embodiment, the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha chain and a TCR beta chain. In one embodiment, the step of introducing the nucleic acid comprises co-electroporating an RNA encoding the TCR alpha chain and a separate RNA encoding the TCR beta chain. In yet another embodiment, the step of introducing the nucleic acid encoding the costimulatory molecule comprises electroporating an RNA encoding CD3 into the T cell. In one embodiment, CD3 RNA is co-electroporated with the TCR nucleic acid.

[0104] In another embodiment, the methods described herein further comprise inducing lysis of the target cell or tissue. In one embodiment, the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0105] In another embodiment, the condition 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; 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, polychondritis, polyglandular syndrome In another embodiment, the condition is an autoimmune disease, such as an autoimmune disease selected from the group consisting of: rheumatic 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. In yet another embodiment, the condition is a cancer, such as 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. [The present invention 1001] 1. An engineered T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) comprising affinity for an antigen on a target cell, and electroporated RNA encoding a bispecific antibody, wherein the T cell expresses the TCR and bispecific antibody on its surface. [The present invention 1002] 1001. The modified T cell of the present invention, wherein said TCR comprises at least one disulfide bond. [The present invention 1003] 1001. The modified T cell of the present invention, wherein said TCR comprises a TCR alpha chain and a beta chain. [The present invention 1004] 1003. The modified T cell of claim 1003, wherein said TCR comprises a costimulatory signaling domain at the C-terminus of at least one of said chains. [The present invention 1005] 1004. The modified T cell of the present invention, wherein said costimulatory signaling domain is a 4-1BB costimulatory signaling domain. [The present invention 1006] 1003. The modified T cell of the present invention, wherein said beta chain comprises at least one N-deglycosylation. [The present invention 1007] 1003. The modified T cell of the present invention, wherein said alpha chain comprises at least one N-deglycosylation. [The present invention 1008] 1001. The modified T cell of the present invention, wherein said TCR comprises at least one mouse constant region. [The present invention 1009] 1001. The modified T cell of the present invention, wherein said TCR has a higher affinity for a target cell antigen than a wild-type TCR. [The present invention 1010] 1001. The modified T cell of the present invention, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof. [The present invention 1011] 1001. The modified T cell of the present invention, wherein said bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. [The present invention 1012] 1011. The modified T cell of the present invention, wherein said bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules. [The present invention 1013] The modified T cell of the present invention, wherein the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on an activated T cell. [The present invention 1014] 1013. The modified T cell of the present invention, wherein the activating T cell antigen is selected from the group consisting of CD3, CD4, CD8, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, TCR, PD1, and PD1L. [The present invention 1015] 1001. The modified T cell of claim 1001, further comprising an electroporated nucleic acid encoding a costimulatory molecule. [The present invention 1016] 1015. 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 1017] 1. A method for making a modified T cell, comprising introducing into a T cell capable of expressing the TCR and the bispecific antibody a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for an antigen on a target cell and a nucleic acid encoding the bispecific antibody. [The present invention 1018] The method of claim 1017, 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 1019] 1017. The method of claim 1017, wherein said nucleic acid comprises in vitro transcribed RNA or synthetic RNA. [The present invention 1020] The method of claim 1017, further comprising expanding the T cells. [The present invention 1021] The method of claim 1020, wherein the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. [The present invention 1022] The method of claim 1020, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells. [The present invention 1023] 1023. The method of claim 1022, wherein the chimeric membrane protein comprises a single chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB. [The present invention 1024] The method of claim 1017, further comprising the step of cryopreserving the T cells. [The present invention 1025] 1025. The method of claim 1024, further comprising the step of thawing cryopreserved T cells prior to introducing said nucleic acid into the T cells. [The present invention 1026] 1017. The method of claim 1017, wherein the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha chain and a TCR beta chain. [The present invention 1027] 1027. The method of claim 1026, wherein the step of introducing said nucleic acid comprises co-electroporating an RNA encoding a TCR alpha chain and a separate RNA encoding a TCR beta chain. [The present invention 1028] The method of claim 1017, further comprising the step of electroporating the T cells with RNA encoding CD3. [The present invention 1029] The method of claim 1028, wherein the CD3 RNA is co-electroporated with the TCR nucleic acid. [The present invention 1030] The method of claim 1017, further comprising the step of cryopreserving the T cells after introducing the TCR nucleic acid. [The present invention 1031] The method of claim 1017, further comprising the step of expressing the bispecific antibody as a membrane protein. [The present invention 1032] The method of claim 1017, further comprising the step of cryopreserving the bispecific antibody-transduced T cells. [The present invention 1033] 1001. Use of a T cell of the present invention in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof. [The present invention 1034] an effective amount of electroporated modified T cells comprising RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody; to the subject, wherein the modified T cell expresses the modified TCR and the bispecific antibody. [This invention 1035] The method of claim 1034, further comprising the step of inducing lysis of the target cell or tissue. [The present invention 1036] The method of claim 1035, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC). [This invention 1037] 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells that have been electroporated with RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody to prevent or treat an immune response deleterious to the subject. [The present invention 1038] A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells that have been electroporated with RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody. [This invention 1039] 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 1040] 1039. The method of claim 1039, wherein said condition is an autoimmune disease. [The present invention 1041] Autoimmune diseases include acquired immune deficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, inner ear autoimmune disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, and celiac sprue-dermatitis. hepetiformis); Chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, 1040. The method of claim 1040, wherein the inflammatory bowel disease is selected from the group consisting of 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 1042] 1039. The method of claim 1039, wherein said condition is cancer. [This invention 1043] 1042. The method of claim 1042, 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 1044] A composition comprising the modified T cells of the present invention. [This invention 1045] A pharmaceutical composition comprising the modified T cells of the present invention and a pharmaceutically acceptable carrier. [The present invention 1046] 1. An engineered T cell comprising a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and an antigen on a T cell, and a nucleic acid encoding a chimeric ligand engineered activation receptor (CLEAR), wherein the T cell expresses the bispecific antibody and CLEAR. [This invention 1047] 1046. The modified T cell of the present invention, wherein CLEAR comprises an intracellular activation domain and an extracellular domain. [This invention 1048] 1046. The modified T cell of the present invention, wherein the intracellular activation domain comprises a portion of the intracellular activation domain of CD3 zeta. [This invention 1049] 1046. The modified T cell of the present invention, wherein the extracellular domain is selected from the group consisting of an antigen-binding domain of an antibody, a ligand-binding domain of a receptor, an antigen, and a ligand. [The present invention 1050] 1046. The modified T cell of the present invention, wherein the extracellular domain is selected from the group consisting of CD27, CD28, CD70, CD80, PD1 and PD-L1. [This invention 1051] 1046. The modified T cell of the present invention, wherein the extracellular domain is capable of binding to a tumor antigen. [This invention 1052] 1046. The modified T cell of the present invention, wherein CLEAR further comprises a costimulatory domain. [This invention 1053] The modified T cell of the present invention 1052, wherein the costimulatory domain is selected from the group consisting of CD4, CD8 and 4-1BB. [This invention 1054] 1046. The modified T cell of the present invention, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof. [This invention 1055] 1046. The modified T cell of the present invention, wherein said bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. [The present invention 1056] 1055. The modified T cell of the present invention, wherein said bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules. [This invention 1057] 1056. The modified T cell of the present invention, wherein the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on a T cell. [This invention 1058] 1046. The modified T cell of the present invention, wherein the bispecific antibody comprises a dual specificity for an antigen on a target cell and for CLEAR on the T cell. [This invention 1059] The modified T cell of claim 1046, further comprising a nucleic acid encoding a costimulatory molecule. [The present invention 1060] 1058. 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 1061] Use of a T cell of the present invention 1045 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof. [This invention 1062] 1. A method for making an engineered T cell, comprising introducing into a T cell a nucleic acid encoding a bispecific antibody and a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR), wherein the T cell is capable of expressing the bispecific antibody and CLEAR. [The present invention 1063] 1063. The method of claim 1062, wherein at least one of said nucleic acids is introduced by a method selected from the group consisting of transduction of T cells, transfection of T cells, and electroporation of T cells. [This invention 1064] The method of claim 1062, 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. [This invention 1065] 1063. The method of claim 1062, wherein at least one of said nucleic acids comprises in vitro transcribed RNA or synthetic RNA. [The present invention 1066] The method of claim 1062, further comprising expanding the T cells. [This invention 1067] The method of claim 1066, wherein the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. [The present invention 1068] The method of claim 1066, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells. [The present invention 1069] 1068. The method of claim 1068, wherein the chimeric membrane protein comprises a single chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB. [The present invention 1070] The method of claim 1062, further comprising the step of cryopreserving the T cells. [This invention 1071] 107. The method of claim 1070, further comprising the step of thawing cryopreserved T cells prior to introducing said nucleic acid into the T cells. [This invention 1072] The method of claim 1062, further comprising the step of cryopreserving the T cells after introducing the CLEAR nucleic acid. [This invention 1073] 1063. The method of claim 1062, further comprising the step of expressing the bispecific antibody as a membrane protein. [This invention 1074] The method of claim 1062, further comprising the step of cryopreserving the bispecific antibody-transduced T cells. [This invention 1075] 1. An engineered T cell comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on a T cell, wherein the engineered T cell expresses the CLEAR and the bispecific antibody in an effective amount. A method for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising administering to the subject [This invention 1076] The method of claim 1075, further comprising the step of inducing lysis of the target cell or tissue. [This invention 1077] The method of claim 1076, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC). [This invention 1078] 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells, the modified T cells comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell, to prevent or treat an immune response deleterious to the subject. [This invention 1079] 1. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell. [The present invention 1080] 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. [This invention 1081] The method of claim 1080, wherein said condition is an autoimmune disease. [This invention 1082] 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 herpetiformis; Chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, 1081. The method of the present invention, wherein the inflammatory bowel disease is selected from the group consisting of 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. [This invention 1083] The method of claim 1080, wherein said condition is cancer. [This invention 1084] 1083. The method of claim 1083, 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. [This invention 1085] A composition comprising the modified T cells of the present invention. [This invention 1086] A pharmaceutical composition comprising the modified T cells of the present invention 1046 and a pharmaceutically acceptable carrier. [This invention 1087] A modified T cell comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell, wherein the T cell expresses the affinity molecule chimeric receptor. [This invention 1088] The modified T cell of the present invention 1087, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof. [This invention 1089] 1087. The modified T cell of the present invention, wherein the small molecule extracellular domain comprises a helical structure lacking disulfide bridges. [The present invention 1090] The modified T cell of the present invention 1087, wherein the small extracellular domain is less than about 10 kD. [This invention 1091] The modified T cell of the present invention 1087, wherein the affinity molecule chimeric receptor further comprises an intracellular signaling domain. [This invention 1092] 1091. The modified T cell of the present invention, wherein the intracellular signaling domain is a CD3 signaling domain. [This invention 1093] The modified T cell of the present invention 1087, wherein the affinity molecule chimeric receptor further comprises a costimulatory signaling domain. [This invention 1094] The modified T cell of the present invention 1093, wherein the costimulatory signaling domain is a 4-1BB costimulatory signaling domain. [This invention 1095] 1087. The modified T cell of the present invention, wherein the affinity molecule chimeric receptor further comprises a transmembrane domain. [This invention 1096] The modified T cell of the present invention 1095, wherein the transmembrane domain is a CD8 transmembrane domain. [This invention 1097] The modified T cell of the present invention 1087, wherein the affinity molecule chimeric receptor further comprises a TCR variable domain and a TCR constant domain. [This invention 1098] The modified T cell of the present invention 1087, further comprising a nucleic acid encoding a costimulatory molecule. [This invention 1099] 1098. 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 1100] 1099. The modified T cell of the present invention, wherein the CD3 comprises at least two different CD3 chains. [The present invention 1101] 1100. The modified T cell of the present invention, wherein said different CD3 chains are CD3 zeta chain and CD3 epsilon chain. [The present invention 1102] 1. An engineered cell expressing a bispecific affinity molecule, the cell comprising a nucleic acid encoding the bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain. [The present invention 1103] 1102. The modified cell of the present invention, wherein the affinity domain capable of binding to a target cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antigen-binding domain of an antibody. [The present invention 1104] 1102. The modified cell of the present invention, wherein the affinity domain capable of binding to an activating T cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antigen-binding domain of an antibody. [This invention 1105] 1102. The modified cell of the present invention, wherein the small molecule antigen-binding domain comprises a helical structure lacking disulfide bridges. [The present invention 1106] The modified cell of the present invention 1102, wherein each of the small molecule antigen-binding domains is less than about 10 kD. [This invention 1107] 1102. The modified cell of the present invention, wherein the target cell antigen is selected from the group consisting of a tumor-associated antigen (TAA), a bacterial antigen, a parasitic antigen, a viral antigen, and any fragment thereof. [This invention 1108] 1102. The modified cell of the present invention, wherein the activating T cell antigen is a costimulatory molecule selected from the group consisting of CD3, CD4, CD8, 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, and CD83 specific binding ligand, and any fragment thereof. [This invention 1109] The modified cell of the present invention 1087 for use in a method of treating an immune response in a subject in need thereof. [The present invention 1110] The modified cell of the present invention 1102 for use in a method of treating an immune response in a subject in need thereof. [The present invention 1111] The modified cell of the present invention 1102, selected from the group consisting of a T cell, a B cell, a natural killer cell, and an antigen-presenting cell. [The present invention 1112] A method for producing modified T cells, comprising introducing into a population of T cells capable of expressing an affinity molecule chimeric receptor a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell. [The present invention 1113] 1112. The method of claim 1112, wherein said nucleic acid is introduced by a method selected from the group consisting of transduction of a population of T cells, transfection of a population of T cells, and electroporation of a population of T cells. [This invention 1114] 1115. The method of claim 1115, wherein the introducing of the nucleic acid comprises electroporating RNA encoding the affinity molecule chimeric receptor. [This invention 1115] The method of claim 1114, further comprising electroporating the T cells with RNA encoding CD3. [The present invention 1116] 1115. The method of claim 1115, wherein the CD3 RNA is co-electroporated with a nucleic acid encoding an affinity molecule chimeric receptor. [This invention 1117] 1112. The method of claim 1112, 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. [This invention 1118] 1112. The method of claim 1112, further comprising the step of cryopreserving the T cells after introducing the affinity molecule chimeric receptor nucleic acid. [This invention 1119] The method of claim 1112, further comprising expanding the T cells. [The present invention 1120] 1119. The method of claim 1119, wherein the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. [This invention 1121] 1119. The method of claim 1119, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells. [This invention 1122] 1122. The method of claim 1121, wherein the chimeric membrane protein comprises a single chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB. [This invention 1123] 1112. The method of claim 1112, further comprising the step of cryopreserving the T cells. [This invention 1124] The method of claim 1123, further comprising the step of thawing cryopreserved T cells prior to introducing the affinity molecule chimeric receptor nucleic acid into the T cells. [This invention 1125] 1. A method for generating engineered cells that express a bispecific affinity molecule, comprising introducing into a population of cells a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain. [The present invention 1126] 1125. The method of claim 1125, wherein said nucleic acid is introduced by a method selected from the group consisting of transduction of a population of cells, transfection of a population of cells, and electroporation of a population of cells. [This invention 1127] 1126. The method of claim 1125, wherein said population of cells comprises T cells, B cells, natural killer cells, or antigen-presenting cells. [This invention 1128] The method of claim 1125, further comprising the step of combining the activated T cells and the target cells with a bispecific affinity molecule. [This invention 1129] Use of the modified T cell of the invention 1087 or the modified cell of the invention 1102 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof. [The present invention 1130] A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell to prevent or treat an immune response harmful to the subject. [This invention 1131] 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain. [This invention 1132] A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell. [This invention 1133] 1. A method for treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain. [This invention 1134] A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a T cell of the invention 1087 or a modified cell of the invention 1102. [This invention 1135] 1135. The method of claim 1134, wherein said condition is an autoimmune disease. [This invention 1136] 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 herpetiformis; Chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, 1135. The method of the present invention, wherein the inflammatory bowel disease is selected from the group consisting of 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. [This invention 1137] 1135. The method of claim 1134, wherein said condition is cancer. [This invention 1138] 1137. The method of claim 1137, 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. [This invention 1139] 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 comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on the target cell. [This invention 1140] 1. 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 cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain. [This invention 1141] The method of any of claims 1139 or 1140, further comprising the step of inducing lysis of the target cell or tissue. [This invention 1142] The method of any of claims 1139 or 1140, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC). [This invention 1143] A composition comprising the modified T cell of the present invention 1087 or the modified cell of the present invention 1102. [This invention 1144] A pharmaceutical composition comprising the modified T cell of the present invention 1087 or the modified cell of the present invention 1102 and a pharmaceutically acceptable carrier. [Invention 1145] 1. A modified T cell comprising electroporated RNA encoding a bispecific T-cell engager (BiTE) molecule, wherein the BiTE molecule comprises a dual specificity for an antigen on a target cell and an antigen on an activated T cell selected from the group consisting of CD3, CD4, CD8, and TCR. [Invention 1146] The modified T cell of the present invention 1145, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof. [This invention 1147] 1145. The modified T cell of the present invention, wherein said bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. [This invention 1148] 1147. The modified T cell of claim 1147, wherein said bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules. [This invention 1149] The modified T cell of the present invention 1148, wherein the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on an activated T cell. [This invention 1150] Expanding the population of T cells; and Electroporating the expanded T cells with RNA encoding the bispecific antibody wherein electroporated T cells are capable of expressing the bispecific antibody. [This invention 1151] 1150. The method of claim 1150, 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. [This invention 1152] 1150. The method of claim 1150, wherein said RNA comprises in vitro transcribed RNA or synthetic RNA. [This invention 1153] The method of claim 1450, wherein the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. [This invention 1154] The method of claim 1150, wherein the expansion comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells. [This invention 1155] 1154. The method of claim 1154, wherein the chimeric membrane protein comprises a single chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB. [Invention 1156] The method of claim 1150, further comprising the step of cryopreserving the expanded T cells. [This invention 1157] 1156. The method of claim 1156, further comprising the step of thawing cryopreserved T cells for electroporation with RNA encoding the bispecific antibody. [This invention 1158] The method of claim 1150, further comprising the step of expressing the bispecific antibody as a membrane protein. [This invention 1159] 1150. The method of claim 1150, further comprising the step of cryopreserving the bispecific antibody-electroporated T cells. [The present invention 1160] 1. 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 comprising electroporated RNA encoding a bispecific T cell-enhancing (BiTE) molecule comprising dual specificities for an antigen on the target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8 and TCR. [This invention 1161] The method of claim 1160, further comprising the step of inducing lysis of the target cells or tissue containing the target cells. [This invention 1162] The method of claim 1160, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC). [This invention 1163] 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells to prevent or treat an immune response deleterious to the subject, wherein the modified T cells have been expanded and electroporated with RNA encoding a bispecific T cell inducer (BiTE) molecule having dual specificity for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR. [This invention 1164] 1. A method of treating a disease or condition associated with immune enhancement in a subject comprising administering to a subject in need thereof a population of modified T cells, wherein the modified T cells have been expanded and electroporated with RNA encoding a bispecific T cell-enhancing (BiTE) molecule having dual specificities for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR. [Invention 1165] A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising modified T cells of the present invention. [Invention 1166] The method of claim 1165, wherein the immune response is an autoimmune disease. [This invention 1167] 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 herpetiformis; Chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, 1166. The method of the present invention, wherein the inflammatory bowel disease is selected from the group consisting of 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. [Invention 1168] 1165. The method of claim 1165, wherein said condition is cancer. [This invention 1169] 1168. The method of claim 1168, 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. [This invention 1170] Use of a modified T cell of the present invention 1145 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof. [This invention 1171] A composition comprising the modified T cells of the present invention. [This invention 1172] A pharmaceutical composition comprising the modified T cells of the present invention 1145 and a pharmaceutically acceptable carrier. [This invention 1173] An engineered T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) having affinity for a surface antigen on a target cell; and a nucleic acid encoding a costimulatory molecule, wherein the T cell expresses the TCR and the costimulatory molecule. [This invention 1174] 1173. The modified T cell of the present invention, wherein said TCR comprises at least one disulfide bond. [This invention 1175] 1173. The modified T cell of the present invention, wherein said TCR comprises a TCR alpha chain and a beta chain. [Invention 1176] 1175. The modified T cell of the present invention, wherein said TCR comprises a costimulatory signaling domain at the C-terminus of at least one of said chains. [This invention 1177] 1176. The modified T cell of claim 1176, wherein the costimulatory signaling domain is a 4-1BB costimulatory signaling domain. [This invention 1178] 1175. The modified T cell of the present invention, wherein said beta chain comprises at least one N-deglycosylation. [This invention 1179] 1175. The modified T cell of the present invention, wherein said alpha chain comprises at least one N-deglycosylation. [This invention 1180] 1173. The modified T cell of the present invention, wherein said TCR comprises at least one mouse constant region. [This invention 1181] The modified T cell of the present invention 1173, wherein a nucleic acid encoding a costimulatory molecule is electroporated into the T cell. [This invention 1182] 1181. 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. [This invention 1183] 1182. The modified T cell of the present invention, wherein the CD3 comprises at least two different CD3 chains. [This invention 1184] The modified T cell of the present invention 1183, wherein said different CD3 chains are CD3 zeta chain and CD3 epsilon chain. [This invention 1185] The modified T cell of the present invention 1173, wherein the TCR has a higher affinity for a target cell antigen than a wild-type TCR. [Invention 1186] The modified T cell of the present invention 1173, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof. [This invention 1187] introducing into the T cell a nucleic acid encoding a T cell receptor (TCR) that has affinity for a surface antigen on a target cell; and introducing nucleic acids encoding costimulatory molecules into T cells wherein said T cell is capable of expressing said TCR and a costimulatory molecule. [This invention 1188] 1187. The method of claim 1187, wherein at least one of said nucleic acids is introduced by a method selected from the group consisting of transduction of T cells, transfection of T cells, and electroporation of T cells. [This invention 1189] 1187. The method of claim 1187, 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. [This invention 1190] 1187. The method of claim 1187, wherein at least one of said nucleic acids comprises in vitro transcribed RNA or synthetic RNA. [This invention 1191] The method of claim 1187, further comprising expanding the T cells. [This invention 1192] 1191. The method of claim 1191, wherein the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand. [This invention 1193] 1191. The method of claim 1191, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells. [This invention 1194] 1193. The method of claim 1193, wherein the chimeric membrane protein comprises a single chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB. [This invention 1195] 1187. The method of claim 1187, further comprising the step of cryopreserving the T cells. [Invention 1196] 1195. The method of claim 1195, further comprising the step of thawing cryopreserved T cells prior to introducing the nucleic acid encoding said TCR into the T cells. [This invention 1197] 1187. The method of claim 1187, wherein the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha chain and a TCR beta chain. [This invention 1198] 1198. The method of claim 1198, wherein the step of introducing said nucleic acid comprises co-electroporating an RNA encoding a TCR alpha chain and a separate RNA encoding a TCR beta chain. [This invention 1199] 1187. The method of claim 1187, wherein the step of introducing a nucleic acid encoding a costimulatory molecule comprises electroporating RNA encoding CD3 into the T cell. [The present invention 1200] The method of claim 1199, wherein the CD3 RNA is co-electroporated with the TCR nucleic acid. [The present invention 1201] 1187. The method of claim 1187, further comprising the step of cryopreserving the T cells after introducing the TCR nucleic acid. [This invention 1202] Use of a modified T cell of the present invention 1173 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof. [This invention 1203] 1. 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, wherein the T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) having affinity for a surface antigen on the target cell. [The present invention 1204] The method of claim 1203, further comprising the step of inducing lysis of the target cell or tissue. [This invention 1205] The method of claim 1204, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC). [The present invention 1206] 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells to prevent or treat an immune response deleterious to the subject, wherein the modified T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) having affinity for a surface antigen on a target cell. [This invention 1207] 1. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells, wherein the modified T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) having affinity for a surface antigen on a target cell. [This invention 1208] A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising modified T cells of the present invention. [This invention 1209] The method of claim 1208, wherein the immune response is an autoimmune disease. [The present invention 1210] 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 herpetiformis; Chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, 1209. The method of claim 1209, wherein the inflammatory bowel disease is selected from the group consisting of 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 1211] 1208. The method of claim 1208, wherein said condition is cancer. [The present invention 1212] 12. The method of claim 12, 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. [This invention 1213] A composition comprising the modified T cells of the present invention. [This invention 1214] A pharmaceutical composition comprising the modified T cells of the present invention 1173 and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0106] 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 1] Figure 1 is a panel of graphs showing transgene expression in T cells co-electroporated with TCR and BiTE. T cells were co-electroporated with CD19.CD3 (top panel) or 4D5.CD3 (ErbB2) (middle panel) BiTE, with or without CD3 zeta and epsilon. 18 hours after electroporation, T cells were stained for TCR vb13.1 and mIgG Fab (or Her2-Fc). The bottom panel shows TCR (vb13.1) expression 3 days after electroporation. [Figure 2] Figure 2 is a panel of graphs showing that CD107a was upregulated in tumor-stimulated T cells. T cells were co-electroporated with the indicated RNAs and stimulated with tumor cell lines single- or double-positive for CD19 and NY-ESO-1 (ESO). CD107a upregulation was assessed 4 hours later. [Figure 3] Figure 3 is a panel of graphs showing that CD107a was upregulated in tumor-stimulated T cells. T cells were co-electroporated with the indicated RNAs and stimulated with tumor cell lines single- or double-positive for CD19 and NY-ESO-1 (ESO). CD107a upregulation was assessed 4 hours later. [Figure 4]FIG. 4 is a graph showing IFN-γ production in RNA-electroporated T cells stimulated with tumor cell lines. [Figure 5] FIG. 5 is a graph showing IL-2 production in RNA-electroporated T cells stimulated with tumor cell lines. [Figure 6] FIG. 6 is a panel of graphs showing upregulation of CD107a in T cells expressing NY-ESO-1 TCR (1G4) and mesothelin BiTE (ss1.CD3) stimulated with tumor cells. [Figure 7] Figure 7 is a panel of images showing tumor growth in mice intravenously injected with Naml6-ESO-CBG cells (2 x 10 cells). Five days after tumor cell injection, mice were treated with RNA-electroporated T cells as indicated (5 mice per group). Tumor growth was visualized by bioluminescence imaging. [Figure 8] FIG. 8 is a graph showing T cells expressing bispecific RNA against target tumor-associated antigens have enhanced T cell function. [Figure 9] FIG. 9 is a panel of graphs showing that TCRs on T cells recognized both cognate and MHC / peptide antigens and surface tumor antigens without HLA restriction by combining with bispecific antibodies on the T cells. [Figure 10] FIG. 10, comprising FIGS. 10A and 10B, shows a list of bispecific antibody RNA and TCR RNA constructs that were electroporated into T cells. [Figure 11] FIG. 11 is a panel of graphs showing that T cells recognized both the cognate antigen (HLA-A2 / NY-ESO-1) and CD19 or Her2 with the modified NY-ESO-1 TCR and bispecific antibody. [Figure 12] FIG. 12 shows enhanced chimeric ligand-modified activating receptor (CLEAR) binding between a T cell and a target tumor cell, and a bispecific antibody on another T cell binding to the same target tumor cell. [Figure 13]FIG. 13, comprising FIGS. 13A and 13B, is a list of CLEAR and bispecific antibody constructs made in the present invention. [Figure 14] Figure 14 is a panel of graphs showing CD27 expression in T cells or K562 cells 18 hours after electroporation with CD27-BBZ or CD27-Z CLEAR RNA (top panel), and CD70 expression in cell lines (bottom panel). [Figure 15] FIG. 15 is a panel of graphs showing upregulation of CD107a in CD27 CLEAR RNA electroporated T cells stimulated with CD70-positive tumor cell lines. [Figure 16] Figure 16 is a panel of graphs showing PD1 expression in PD1 CLEAR RNA electroporated T cells (top panel) and upregulation of CD107a in the T cells after stimulation with PD-L1-positive tumor cells, Nalm6-PD-L1. [Figure 17] Figure 17 is a graph showing cytokine production in PD1 CLEAR RNA electroporated T cells stimulated with PD-L1 positive tumor cells Nalm6-PD-L1. [Figure 18] Figure 18 is a panel of graphs showing transgene expression in T cells co-electroporated with PD1-Z and aPD-ameso bispecific antibody RNA. [Figure 19] Figure 19 is a panel of graphs showing upregulation of CD107a in PD1-Z and aPD-ameso bispecific antibody RNA co-electroporated T cells stimulated with MESO-positive tumor cells (K562-meso, SK-OV3, and PC3), or MESO / PD-L1 double-positive tumor cells (PC3-PDL1). [Figure 20] Figure 20 is a panel of graphs showing cytokine production of PD1-Z and aPD-ameso bispecific antibody RNA co-electroporated T cells stimulated with MESO-positive tumor cells (K562-meso and SK-OV3). [Figure 21] Figure 21 is a panel of graphs showing transgene expression in T cells co-electroporated with CD27-Z and aCD27-aErbB2 bispecific antibody RNA (top panel) or CD19 bispecific antibody RNA (bottom panel). [Figure 22] FIG. 22 is a panel of graphs showing upregulation of CD107a in CD27-Z and aCD27-aErbB2 bispecific antibody RNA electroporated T cells stimulated with tumor cell lines expressing CD70 and / or ErbB2. [Figure 23] FIG. 23 is a panel of graphs showing upregulation of CD107a in CD27-Z and aCD27-aCD19 bispecific antibody RNA-electroporated T cells stimulated with tumor cell lines expressing CD70 and / or CD19. [Figure 24] Figure 24 is a panel of graphs showing T cells electroporated with PD1-Z and anti-PD1 / anti-ErbB2 bispecific antibody RNA, as indicated. Eighteen hours later, electroporated T cells were stained for anti-PD1 and anti-mIgG Fab. [Figure 25] Figure 25 is a panel of graphs showing tumor cell lines (top panel) and K562 cells electroporated with ErbB2 or PD-L1 RNA (bottom panel, 18 hours after electroporation). Cells were stained for PD-L1 and ErbB2 expression. [Figure 26] Figure 26 is a panel of graphs showing electroporated T cells stimulated with tumor lines for 4 hours. Upregulation of CD107a was detected by flow cytometry. [Figure 27] Figure 27 is a panel of graphs showing electroporated T cells stimulated with tumor lines for 4 hours. Upregulation of CD107a was detected by flow cytometry. [Figure 28] FIG. 28 is an image showing an illustration of affinity antibody mimic constructs. [Figure 29] Figure 29 is a panel of graphs showing the expression of affinity antibody mimic redirected T cells (ART) by staining with anti-His antibody. T cells were electroporated with 10 micrograms of RNA encoding ART against EGFR (955.BBZ, 1853.BBZ, or 1970.BBZ) or ErbB2 (342.BBZ, 432-15.BBZ, 342-14.BBZ, or 342-4.BBZ) and cultured overnight in R10. One hundred microliters of electroporated T cells were stained with anti-His tag antibody for flow cytometric detection of ART expression (lower panel). Compared to non-electroporated T cells (no EP), T cells electroporated with all ART RNAs were shown to stain positively. [Figure 30] Figure 30 is a panel of graphs showing the specific CD107a upregulation of EGFR and ErbB2 ARTs. Electroporated T cells shown in Figure 29 were stimulated with four tumor cell lines expressing EGFR and / or ErbB2, as indicated under each tumor's name. After 4 hours of incubation, CD107a upregulation was measured by staining the cells with CD107a-PE, CD3-APC, and CD8-FITC. As shown in Figure 29, all EGFR ARTs strongly reacted only with EGFR-positive tumor lines (SK-OV3, MDA231, and MDA468), but not with the EGFR-negative tumor MCF7. Meanwhile, all ErbB2 ARTs strongly reacted with ErbB2-positive tumor lines (SK-OV3, MDA231, and MCF7), but not with the ErbB2-negative tumor MDA468. 4D5.BBZ and 2224.BBZ were CARs against ErbB2 and EGFR, respectively. [Figure 31]Figure 31 is a graph showing the specific IFN-γ production of EGFR and ErbB2 ARTs. Electroporated T cells, as shown in Figure 29, were stimulated with four tumor cell lines expressing EGFR and / or ErbB2, as indicated under each tumor's name. After overnight incubation, IFN-γ production was measured by ELISA using the supernatants. As shown in this figure, IFN-γ was detectable at different levels for all EGFR ARTs stimulated with EGFR-positive tumor lines (SK-OV3, MDA231, and MDA468), but not for the EGFR-negative tumor MCF7. Meanwhile, all ErbB2 ARTs strongly reacted with ErbB2-positive tumor lines (SK-OV3, MDA231, and MCF7), but not with the ErbB2-negative tumor MDA468. 4D5.BBZ and 2224.BBZ were CARs against ErbB2 and EGFR, respectively. [Figure 32] Figure 32 is a graph showing the specific IL-2 production of EGFR and ErbB2 ARTs. The electroporated T cells shown in Figure 29 were stimulated with four tumor cell lines expressing EGFR and / or ErbB2, as indicated under each tumor name. After overnight incubation, IL-2 production was measured by ELISA using the supernatants. As shown in this figure, IL-2 was detectable at different levels for all EGFR ARTs stimulated with EGFR-positive tumor lines (SK-OV3, MDA231, and MDA468) but not for the EGFR-negative tumor MCF7. Meanwhile, all ErbB2 ARTs strongly reacted with ErbB2-positive tumor lines (SK-OV3, MDA231, and MCF7) but not with the ErbB2-negative tumor MDA468. 4D5.BBZ and 2224.BBZ were CARs against ErbB2 and EGFR, respectively. [Figure 33]Figure 33 is a graph showing the specific lytic activity of EGFR and ErbB2 ARTs. In separate experiments, the killing ability of two EGFR ARTs and two ErbB2 ARTs was tested against the tumor cell line SK-OV3, which is positive for both EGFR and ErbB2, compared with their associated CARs. As shown in this figure, ART T cells killed tumor cells as effectively as the associated CAR T cells. [Figure 34A] Figure 34A is a panel of images showing affinity antibody mimic modified TCRs. Figure 34A is a schematic diagram of affinity antibody mimic modified TCRs (Affi-TCRs) by adding affinity antibody mimic and His tag sequences to the N' of either the alpha or beta chain of the TCR. [Figure 34B] Figure 34B is a panel of images showing affinity antibody mimic-modified TCRs. Figure 34B is a panel of graphs showing Vb13.1 TCR and His-tag detection in affinity antibody mimic-redirected TCR (Affi-TCR) RNA-electroporated T cells. T cells were co-electroporated with NY-ESO-1 (1G4) TCR alpha (a) and beta (b), or their ErbB2 affinity antibody mimic (342, 342.15, 342, or 342.4) variants. After overnight incubation, vb13.1 and His-tag were detected by flow cytometry. [Figure 34C] Figure 34C is a panel of images showing affinity antibody mimic modified TCRs. Figure 34C shows the sequence of ErbB2 affinity antibody mimics. [Figure 35]Figure 35 is a panel of graphs showing CD107a upregulation in Affi-TCR RNA-electroporated T cells. T cells were co-electroporated with TCR alpha (a) and beta (b), or their ErbB2-affinity antibody mimic (342, 342.15, 342, or 342.4) variants, as shown in Figure 34B, and stimulated with tumor lines Nalm-6-ESO (NY-eso-1+, ErbB2-), A549-ESO (NY-eso-1+, ErbB2+), SK-OV3 (NY-eso-1-, ErbB2+), A549 (NY-eso-1-, ErbB2+), or Nalm6 (NY-eso-1-, ErbB2-) for CD107a assay. The results show that T cells bearing ErbB2 Affi-TCR were able to specifically recognize Ny-ESO-1 and ErbB2-positive tumors. [Figure 36A] Figure 36A is a panel of images showing affinity antibody mimic modified CD3 epsilon.Figure 36A is a schematic diagram of affinity antibody mimic modified CD3 epsilon by adding an affinity antibody mimic and a G4S linker to the N' of CD3 epsilon. [Figure 36B] Figure 36B is a panel of images showing affinity antibody mimic modified CD3 epsilon. Figure 36B is a table showing electroporation of T cells. T cells were co-electroporated with NY-ESO-1 (1G4) TCR alpha (a) and beta (b) or together with ErbB2 affinity antibody mimic (342, 342.15, 342, or 342.4) modified CD3 epsilon (e). [Figure 36C] Figure 36C is a panel of images showing affinity antibody mimic modified CD3 epsilon. Figure 36C is a panel of graphs showing maintenance of TCR expression and dual targeting by co-delivery of affinity antibody mimic modified CD3 epsilon. After overnight culture of the T cells in Figure 36B, vb13.1 expression was detected by flow cytometry. [Figure 37]Figure 37 is a panel of graphs showing CD107a upregulation in Affi-TCR RNA-electroporated T cells. T cells were co-electroporated with NY-ESO-1 (1G4) TCR alpha (a) and beta (b) or with ErbB2 affinity antibody mimic (342, 342.15, 342, or 342.4)-modified CD3 epsilon (e) as shown in Figure 36B and stimulated with tumor lines Nalm-6-ESO (NY-eso-1+, ErbB2-), Nalm6 (NY-eso-1-, ErbB2-), SK-OV3 (NY-eso-1-, ErbB2+), or MDA231 (NY-eso-1-, ErbB2+) for CD107a assay. [Figure 38A]Figure 38, including Figures 38A-38C, demonstrates that bispecific antibodies can be secreted by electroporating bispecific antibody-encoding RNA (Bis-RNA) into T cells. T cells were electroporated with RNA encoding a CD19 CAR (CAR RNA), blinatumomab Bis-RNA (Bis-RNA), or GFP, or co-electroporated with both CD19 CAR and blinatumomab Bis-RNA (CAR RNA / Bis-RNA). 18 hours after electroporation, T cells were stained with goat anti-mouse IgG Fab (mIgG Fab) to detect the CD19 CAR expressed on the T cells or the bispecific antibody tethered to the T cell surface via binding to CD3 (Figure 38A, top panel shows mIgG Fab staining and GFP expression of electroporated T cells). Immediately after electroporation, aliquots of T cells carrying CAR RNA or Bis-RNA were mixed with an equal number of GFP RNA T cells and co-cultured for 18 hours. mIgG Fab and GFP were then assessed (Figure 38A, middle panel). 18 hours after electroporation, aliquots of T cells carrying CAR RNA or Bis-RNA were mixed with an equal number of GFP RNA T cells and stained for mIgG Fab and GFP (Figure 38A, lower panel). 18 hours after electroporation, T cells electroporated with different RNAs, or T cells electroporated with CAR RNA or Bis-RNA, were mixed with GFP RNA-electroporated T cells for 18 hours (GFP T, 18h) or 0 hours (GFP T, 0h), and then stimulated with CD19-expressing cell lines (Nalm6, K562-CD19, and Raji) or CD19-negative K562 cells. The mixture was then assessed for CD107a staining (Figure 38B).Eighteen hours after electroporation, CD19 CAR RNA (CAR RNA) or Bis-RNA electroporated T cells were either alone or mixed with an equal number of GFP RNA electroporated T cells (GFP-T) and analyzed for lytic activity after a 4-hour flow-based cytotoxic T lymphocyte assay at an effector:target ratio of 5:1 (Figure 38C). [Figure 38B] See legend to Figure 38A. [Figure 38C] See legend to Figure 38A. [Figure 39A] Figure 39, including Figures 39A-39D, highlights the increased T cell activation and tumor-killing capacity of Bis-RNA-electroporated T cells. T cells were electroporated with different amounts (µg RNA / 0.1 ml T cells) of blinatumomab Bis-RNA (Bis-RNA) or CD19 CAR RNA (CAR RNA), as indicated. Eighteen hours after electroporation, T cells bearing Bis-RNA or CAR RNA were stimulated with a CD19-positive cell line with or without an equal number of GFP RNA-electroporated T cells (GFP-T) and assessed for CD107a expression (Figure 39A). Eighteen hours after electroporation of the above T cells, electroporated T cells were subjected to intracellular staining for IFN-γ and granzyme B (Figure 39B). IFN-γ ELISA was performed after overnight stimulation of T cells with CD19-positive (Nalm6, K562-CD19, and Raji) or CD19-negative (K562) cell lines (Figure 39C). T cells were electroporated with either blinatumomab Bis-RNA (Bis-RNA) or CD19bbz CAR RNA (CAR RNA at RNA doses of 1, 5, or 10 μg / 0.1 ml T cells), or co-electroporated with 5 μg each of Bis-RNA and CAR RNA (Bis-RNA + CAR RNA). 18 hours after electroporation, lytic activity was assessed by a 4-hour flow-based cytotoxic T lymphocyte assay at the indicated effector:target ratios (Figure 39D). [Figure 39B] See legend to Figure 39A. [Figure 39C] See legend to Figure 39A. [Figure 39D] See legend to Figure 39A. [Figure 40A] Figure 40, comprising Figures 40A-40D, demonstrates the long-term tumor reactivity of Bis-RNA electroporated T cells and the sensitivity of CAR RNA and Bis-RNA T cells. T cells were electroporated with different amounts of blinatumomab Bis-RNA at 1, 5, and 10 μg / 0.1 ml T cells and compared with T cells electroporated with CD19BBZ CAR RNA at a 10 μg RNA dose. CD107a staining was performed on different days after electroporation, and the results were plotted as dot plots of CD107a / CD8 expression on days 3, 8, and 12 (Figure 40A), mean fluorescence intensity (MFI) values ​​(Figure 40B, upper panel), or percentage of CD107a-expressing cells (Figure 40B, lower panel) by days after electroporation. T cells electroporated with 5 or 10 μg of CD19BBZ (19BBZ) or blinatumomab (Blina) RNA were stimulated with K562 cells, and the T cells were electroporated with decreasing amounts of CD19 mRNA. The cells were co-cultured for 18 hours, and the supernatants were subjected to IFN-γ ELISA (Figure 40C). T cells electroporated with 10 μg of 4D5BBZ or 4D5-CD3 RNA were stimulated with K562 cells, and the T cells were electroporated with decreasing amounts of ErbB2 (Her2) mRNA. The cells were co-cultured for 18 hours, and the supernatants were subjected to IFN-γ ELISA (Figure 40D). [Figure 40B] See legend to Figure 40A. [Figure 40C] See legend to Figure 40A. [Figure 40D] See legend to Figure 40A. [Figure 41A]Figure 41, comprising Figures 41A-41D, highlights that Bis-RNA electroporated T cells became less dependent on costimulation and exhibited enhanced division and proliferation. CFSE-labeled resting CD4+ T cells were electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with either irradiated K562-CD19 cells (mixed with an equal number of irradiated K562 cells as a control for K562-CD86 cells) or irradiated K562-CD19 cells (mixed with an equal number of irradiated K562-CD86 cells). CFSE staining on day 6 (Figure 41A) and T cell counts at different days after stimulation (Figure 41B) were monitored. CFSE-labeled CD45RO+ (memory cells) or CD45RO- (naive cells) resting CD4 T cells were electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with either irradiated K562-CD19 cells (mixed with an equal number of irradiated K562 cells as a control for K562-CD86 cells) or irradiated K562-CD19 cells (mixed with an equal number of irradiated K562-CD86 cells). CFSE staining on day 6 (Figure 41C) and T cell counts at different days after stimulation (Figure 41D) were monitored. [Figure 41B] See legend to Figure 41A. [Figure 41C] See legend to Figure 41A. [Figure 41D] See legend to Figure 41A. [Figure 42A]Figure 42, including Figures 42A-42E, shows the enhanced antitumor activity of Bis-RNA T cells in a Nalm-6 xenograft model. Leukemia was established after intravenous injection of Nalm-6 cells (1 x 106 cells, i.v.) into NOD / scid / yc(- / -) (NSG) mice (n = 5). Seven days after tumor cell injection, mice were randomized and treated with T cells electroporated with either blinatumomab Bis-RNA or CD19BBZ RNA. Mice treated with anti-mesothelin RNA CAR T cells (ss1BBZ) served as controls. T cells were injected intravenously as a single injection of 25e6 (1X) or weekly for 3 weeks (3X) at a dose of 20e6 for the first injection and 5e6 for the second and third injections. Animals were imaged at the indicated time points after injection (Figure 42A). BLI data from this experiment are plotted with total photon flux—SE—shown on the y-axis; 1e5 p / sec / cm2 / sr corresponds to mice without luciferase-containing cells (Figure 42B). The leukemia-established or leukemia-free NSG mice described above were injected with 20e6 T cells electroporated with either CD19BBZ or Bis-RNA, or with control ss1BBZ RNA. Bone marrow and splenocytes were harvested from two mice in each group on days 1, 3, and 7 after T cell injection. T cells were purified from each mouse by depleting mouse cells from pooled bone marrow and splenocytes. T cell function was assessed by CD137 expression (* indicates p<0.05) (Figure 42C), cytokine production (by ELISA) (Figure 42D), and CD107a expression (Figure 42E) after stimulation with the CD19-positive cell line K562-CD19 for 18 hours (CD137 and IFN-γ secretion) and 4 hours (CD107a), respectively. [Figure 42B] See legend to Figure 42A. [Figure 42C] See legend to Figure 42A. [Figure 42D] See legend to Figure 42A. [Figure 42E] See legend to Figure 42A. [Figure 43A]Figure 43, comprising Figures 43A-43E, shows different Bis-RNAs directed against different tumor antigens. T cells were electroporated with one of seven different Bis-RNA constructs encoding fully human CD19-CD3 using single-chain variable fragments (scFvs) derived from the fully human anti-CD19 (21D4) antibody and the fully human anti-CD3 scFv (28F11) antibody, respectively. After stimulation of the electroporated T cells with the CD19-positive cell line K562-CD19 for 4 hours, upregulation of CD107a was monitored by flow cytometry (Figure 43A). After stimulation of the electroporated T cells with the CD19-positive cell lines Nalm6, K562-CD19, and Raji for 18 hours, IFN-γ production was detected by ELISA. CD19BBZ CAR RNA (CAR RNA), blinatumomab Bis-RNA (Blina-Bis-RNA), and no electroporation (no EP) were used as controls (Figure 43B). Figure 43C shows detection of scFvs on T cells electroporated with Bis-RNA or CAR RNA against mesothelin, cMet, PSCA, and GD2 using an anti-mouse IgG Fab antibody (for scFvs against mesothelin and GD2) or an anti-human IgG Fab (for cMet and PSCA). T cells electroporated with Bis-RNA or CAR RNA against mesothelin, cMet, PSCA, and GD2 (CAR) were stimulated for 4 hours with cell lines expressing mesothelin (K562-meso, SK-OV3), PSCA (K562-PSCA), cMet (SK-OV3), or GD2 (LY5Y), and CD107a upregulation was monitored by flow cytometry (Figure 4D). NSG mice (n=6) were intravenously injected with 1e6 Nalm6-CBG and 7 days later were injected with 5e6 T cells lentivirally transduced with CD19BBZ or D4F11, or 20e6 T cells electroporated with CD19BBZ or D4F11 RNA. Mice treated with T cells electroporated with ss1BBZ CAR RNA served as controls.On days 16 and 20 after tumor injection, mice injected with RNA-electroporated T cells were intraperitoneally administered Cytoxan (40 mg / kg, i.p.). The next day, the mice were injected with 5e6 T cells electroporated with RNA as described. Bioluminescence was measured one day before T cell injection and two days after each subsequent injection as described for Figure 42 (Figure 43E). [Figure 43B] See legend to Figure 43A. [Figure 43C] See legend to Figure 43A. [Figure 43D] See legend to Figure 43A. [Figure 43E] See legend to Figure 43A. [Figure 44A] Figure 44, including Figures 44A-44D, shows that Bis-RNA-electroporated T cells were more resistant to programmed cell death 1 (PD1)- and regulatory T cell (Treg)-induced suppression. T cells were co-electroporated with different amounts of CD19BBZ RNA or CD19-CD3 Bis-RNA and 5 μg or 10 μg of PD1 RNA and stimulated with the CD19-positive cell lines K652-CD19 or Raji. CD107a expression was measured by flow cytometry (Figure 44A), and IFN-γ production was detected by ELISA (Figure 44B). Tregs purified from fresh resting CD4+ T cells were added to resting CD4+ T cells electroporated with CFSE-labeled blinatumomab Bis-RNA or CD19BBZ RNA (19BBZ) at different T effector:Treg ratios and stimulated with K562-CD19 cells. Six days after stimulation, cells were stained with anti-CD3, and CFSE staining was monitored by flow cytometry (Figures 44C-D). [Figure 44B] See legend to Figure 44A. [Figure 44C] See legend to Figure 44A. [Figure 44D] See legend to Figure 44A. [Figure 45]Figure 45, comprising Figures 45A-45B, shows the expression of T cells pulsed with supernatant from Bis-RNA-electroporated T cells. T cells were electroporated with CD19BBZ RNA (CAR RNA) or Bis-RNA for blinatumomab (Blina-Bis-RNA) or D4F11 RNA (D4F11-Bis-RNA). 18 hours after electroporation, supernatant was collected from Bis-RNA-electroporated T cells, and diluted supernatant (indicated as 10X or 100X) was added to non-electroporated T cells cocultured with CD19-positive cell lines, Nalm6, K562-CD19, or Raji. T cells electroporated with CAR RNA or Bis-RNA served as controls. Upregulation of CD107a was monitored after 4 hours of stimulation. [Figure 46] Figure 46 is a graph showing that Bis-RNA electroporated T cells have significantly enhanced lytic activity. T cells were electroporated with either blinatumomab Bis-RNA (Bis-RNA) or CD19bbz CAR RNA at RNA doses of 1, 5, or 10 μg per 0.1 ml of T cells. After 18 hours, lytic activity was measured using a 4-hour flow-based cytotoxic T lymphocyte assay at a T effector:target ratio of 30:1. [Figure 47] Figure 47 is a panel of graphs showing CD107a upregulation in Bis-RNA electroporated T cells. T cells were electroporated with 10 μg of either 4D5BBZ or 4D5-CD3 RNA and stimulated by co-culturing them for 4 hours with K562 cell lines electroporated with decreasing amounts of ErbB2 (Her2) mRNA, as indicated. CD107a upregulation was detected by flow cytometry. [Figure 48A]Figure 48A shows Bis-RNA electroporated T cells. Figure 48A is a panel of graphs showing CFSE labeling of resting CD4+ T cells electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with either irradiated K562-CD19 cells (mixed with the same number of irradiated K562 cells as a control for K562-CD86 cells) (W / O CD86) or irradiated K562-CD19 cells (mixed with the same number of irradiated K562-CD86 cells) (W / CD86). CFSE dilution rates on day 6 are shown. The left panel shows the mean fluorescence intensity (MFI) for CFSE labeling (higher MFI indicates less T cell division and proliferation). The right panel shows the relative CFSE dilution percentage of T cells electroporated with 1 μg of Bis-RNA. CD3 / CD28 bead-stimulated T cells and T cells without electroporation were used as positive and negative controls, respectively. [Figure 48B] Figure 48B shows Bis-RNA electroporated T cells. Figure 48B is a graph showing CFSE labeling of resting CD4+ T cells electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with either irradiated K562-CD19 cells (mixed with the same number of irradiated K562 cells as a control for K562-CD86 cells) (K562-CD19 / K562) or irradiated K562-CD19 cells (mixed with the same number of irradiated K562-CD86 cells) (K562-CD19 / K562-CD86). The relative CFSE dilution starting on day 3 is shown (T cells on day 6 electroporated with 1 μg Bis-RNA and stimulated with K562-CD19 / K562 cells were set as 50%). [Figure 48C]Figure 48C is a panel of graphs showing CFSE labeling of CD45RO+ (memory cells) or CD45RO- (naive cells) resting CD4 T cells electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with either irradiated K562-CD19 cells (mixed with an equal number of irradiated K562 cells as a control for K562-CD86 cells) (K562-CD19 / K562) or irradiated K562-CD19 cells (mixed with an equal number of irradiated K562-CD86 cells) (K562-CD19 / K562-CD86). CFSE dilution starting on day 3 was expressed as MFI of CFSE or relative CFSE dilution (CD45RO+ T cells on day 6 electroporated with 1 μg of Bis-RNA and stimulated with K562-CD19 / K562 cells were set as 50%). [Figure 48D] Figure 48D shows Bis-RNA electroporated T cells. Figure 48D is a panel of graphs showing CFSE labeling of resting CD4+ T cells electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with either irradiated K562-CD19 cells (mixed with the same number of irradiated K562 cells as a control for K562-CD86 cells) (K19 / K562) or irradiated K562-CD19 cells (mixed with the same number of irradiated K562-CD86 cells) (K19 / K86). Eight days after stimulation, supernatants from the cultures were subjected to ELISA for IFN-γ and IL-2 detection. [Figure 48E] Figure 48E shows Bis-RNA electroporated T cells. Figure 48E is a graph showing CFSE labeling of CD45RO+ (memory cells) or CD45RO- (naive cells) resting CD4 T cells electroporated with different RNA doses of blinatumomab Bis-RNA or CD19BBZ RNA (CAR RNA) and stimulated with Raji (CD19+) or K562 (Cd19-) cells for 18 hours. IL-2 secretion was assayed by ELISA. [Figure 48F] Figure 48F shows Bis-RNA electroporated T cells. Figure 48F is a graph showing CFSE-labeled T cells electroporated with 1 or 5 μg of CD19BBZ (19BBZ), CD19-28Z (19-28Z), or Blinatumomab (Blina) RNA and stimulated with irradiated K562, a 1:1 mixture of K562 and K562-CD19, or a 1:1 mixture of K562-CD19 and K562-CD86. After 6 days, T cells were subjected to flow cytometry analysis for CFSE dilution. [Figure 48G] Figure 48G is a graph showing the overall viability of 5 x 10 T cells electroporated with 5 μg of CD19BBZ (19BBZ) or CD19-28Z (19-28Z) or blinatumomab (Blina) RNA at day 6 and stimulated with a 1:1 mixture of K562 and K562-CD19 cells or a 1:1 mixture of K562-CD19 and K562-CD86 cells. [Figure 48H] Figure 48H is a graph showing Bis-RNA electroporated T cells. T cells were electroporated with 5 μg of CD19BBZ (19BBZ) or CD19-28Z (19-28Z) or blinatumomab (Blina) RNA and analyzed in a flow-based 4-hour cytotoxic T lymphocyte assay. [Figure 49A]Figure 49A shows the expression levels of T cells electroporated with various Bis-RNA constructs. Figure 49A is a graph showing T cells electroporated with one of seven different Bis-RNA constructs encoding fully human CD19-CD3 using scFvs derived from a fully human anti-CD19 antibody (21D4) and a fully human anti-CD3 scFv antibody (28F11). The electroporated T cells were stimulated with the CD19-positive cell lines Nalm6, K562-CD19, and Raji for 18 hours, after which IL-2 production was detected by ELISA. CD19BBZ CAR RNA (CAR RNA), blinatumomab Bis-RNA (Blina-Bis-RNA), and no electroporation (no EP) were used as controls. [Figure 49B] Figure 49B shows the expression levels of T cells electroporated with various Bis-RNA constructs. Figure 49B is a graph showing T cells electroporated with CD19BBZ RNA (CAR RNA), blinatumomab Bis-RNA (Blina-Bis-RNA), or fully human CD19-CD3 Bis-RNA (D4F11). Four hours after electroporation, T cells were stimulated with K562-CD19 for 18 hours, and the supernatants were subjected to Luminex for detection of multiple cytokines / chemokines. Data were normalized by setting CAR RNA T cells to 100%. [Figure 49C] Figure 49C shows the expression levels of T cells electroporated with various Bis-RNA constructs. Figure 49C is a graph showing T cells electroporated with 1 μg or 10 μg of RNA doses of either CD19BBZ, blinatumomab Bis-RNA, D4BBZ, or D4F11 Bis-RNA. The cells were stimulated with the CD19-positive cell line K562-CD19 or Raji, and cytokine production (IFN-γ and IL-2) was detected by ELISA. [Figure 49D]Figure 49D shows the expression level of T cells electroporated with various Bis-RNA constructs. Figure 49D is a graph showing T cells electroporated with 1, 5 or 10 μg of RNA dose of either CD19BBZ, Blinatumomab Bis-RNA, D4BBZ or D4F11 Bis-RNA. The cells were stimulated with CD19-positive cell lines K562-CD19, Raji or Nalm6, and as a control, with CD19-negative cell line K562-meso. CD107a expression was detected by flow cytometry. [Figure 49E] Figure 49E shows the levels of expression in T cells electroporated with various constructs of Bis-RNA. Figure 49E is a graph showing CD107a upregulation in T cells electroporated with RNA encoding EGFRviii CAR (MR1-BBZ or 139-BBZ) or EGFRviii Bis-RNA (MR1-Bis-RNA or 139-Bis-RNA) and stimulated with EGFRviii RNA-electroporated K562 cells or K562 cells. [Figure 49F] Figure 49F shows the expression levels of T cells electroporated with various Bis-RNA constructs. Figure 49F is a graph showing CD107a upregulation in T cells electroporated with RNA encoding CD19 CAR (CD19BBZ) or ErBB2 CAR (4D5-BBZ) or ErBB2 Bis-RNA (4D5-OKT3). The upper panel shows staining of T cells containing ErBB2 fusion protein (Her2-Fc) 18 hours after electroporation. The lower panel shows CD107a staining of T cells stimulated with either an ErBB2-positive / CD19-negative tumor cell line (SK-OV3) or a CD19-positive / ErBB2-negative tumor cell line (Nalm6). [Figure 49G] Figure 49G is a panel of graphs showing the expression of ErBB2 fusion protein (Her2-Fc), which shows the levels of expression in T cells electroporated with various constructs of Bis-RNA. [Figure 49H]Figure 49H is a panel of graphs showing CD107a expression in CD8+ cells. [Figure 50] Figure 50 is a graph showing T cells co-electroporated with different amounts of CD19BBZ RNA or CD19-CD3 Bis-RNA and 5 μg or 10 μg of PD1 RNA. The cells were stimulated with the CD19-positive cell line Raji, and IFN-γ secretion was detected by ELISA. Data are plotted as the percentage of cytokine production by T cells relative to cells without PD1 co-transfection. [Figure 51A] Figure 51A is a panel of graphs characterizing co-electroporated T cells. Figure 51B is a panel of flow graphs showing the increased T cell activation and tumor killing capacity of Bis-RNA electroporated T cells. T cells were electroporated with different amounts (μg RNA / 0.1 ml T cells) of either blinatumomab Bis-RNA (Bis-RNA) or CD19 CAR RNA (CAR RNA), as indicated. Eighteen hours after electroporation, T cells bearing Bis-RNA or CAR RNA were stimulated with a CD19-positive cell line with or without the addition of an equal number of GFP RNA electroporated T cells (GFP-T) and assessed for CD107a expression. [Figure 51B] Figure 51B is a panel of graphs characterizing co-electroporated T cells. Figure 51B is a panel of graphs showing expression of IFN-γ and granzyme B after Bis-RNA electroporation. 18 hours after electroporation of the T cells, electroporated T cells were subjected to intracellular staining for IFN-γ and granzyme B (Figure 2B). [Figure 51C]Figure 51C is a panel of graphs characterizing co-electroporated T cells. Figure 51D is a graph showing ELISA detection of IFN-γ. ELISA for IFN-γ was performed after overnight stimulation of T cells with CD19-positive (Nalm6, K562-CD19, and Raji) or CD19-negative (K562) cell lines. [Figure 51D] Figure 51D is a panel of graphs characterizing co-electroporated T cells. Figure 51D is a graph showing T cell specificity after electroporation and expression of Bis-RNA. T cells were electroporated with either blinatumomab Bis-RNA (Bis-RNA) or CD19bbz CAR RNA (CAR RNA at RNA doses of 1, 5, or 10 μg / 0.1 ml T cells), or co-electroporated with 5 μg each of Bis-RNA and CAR RNA (Bis-RNA + CAR RNA). 18 hours after electroporation, lytic activity was assessed by a 4-hour flow-based cytotoxic T lymphocyte assay at the indicated effector:target ratios. [Figure 52] FIG. 52 is a table listing the soluble fusion protein RNAs that were electroporated into T cells. [Figure 53] Figure 53 shows the construction of a bispecific antibody using anti-PD-L1 scFv and anti-CD28 scFv. [Figure 54A] Figure 54A is a panel of graphs showing cytokine production in electroporated T cells. Figure 54A is a graph showing IL-2 production by T cells electroporated with different RNAs and activated by incubation with tumor cells. [Figure 54B] Figure 54B is a panel of graphs showing cytokine production in electroporated T cells. Figure 54B is a graph showing IFN-γ production by T cells electroporated with different RNAs and activated by incubation with tumor cells. [Figure 55]FIG. 55 shows the construction of a bispecific antibody using anti-TGFb receptor II scFv and anti-CD28 scFv. [Figure 56A] Figure 56A is a panel of graphs showing the reactivity of electroporated T cells. Figure 56B is a graph showing that T cells electroporated with 4D5-CD3 Bis-RNA responded to tumor cells overexpressing ErbB2. T cells electroporated with ErbB2 CAR or Bis-RNA were stimulated with tumor lines expressing high levels of ErbB2, SK-OV3 and N87, or low levels, MFC-7, MDA-231, PC3, and A549. CD107a assays showed that T cells expressing 4D5-6.CD3 were only reactive to ErbB2-overexpressing tumor cells. [Figure 56B] Figure 56B is a panel of graphs showing the reactivity of electroporated T cells. Figure 56B is a panel of graphs showing the results of a repeat of the experiment in Figure 56A. [Figure 57] Figure 57 is a panel of graphs showing the lytic activity of T cells electroporated with 4D5-CD3 Bis-RNA against ErbB2-overexpressing tumor cells. T cells electroporated with ErbB2 CAR or Bis-RNA were tested for their lytic activity against ErbB2-overexpressing tumor cells SK-OV3-CBG or ErbB2-low-expressing tumor cells mel624 (624-CBG). Luciferase-based CTL assays showed that T cells expressing 4D5-6.CD3 were reactive only against ErbB2-overexpressing tumor cells SK-OV3, as were the affinity-tuned ErbB2 CARs, 4D5-5.BBZ and 4D5-3.BBZ. [Figure 58]Figure 58 is a panel of graphs showing that T cells transduced with 4D5-CD3 Bis-RNA via lentivirus are reactive to ErbB2-overexpressing tumor cells. ErbB2 CAR or Bis-RNA-transduced T cells were stimulated with tumor lines expressing high levels of ErbB2, SK-OV3 and N87, or low levels, MDA-231, PC3, and A549. CD107a assays showed that 4D5-CD3-expressing T cells were reactive only to ErbB2-overexpressing tumor cells. [Figure 59] Figure 59 is a panel of graphs showing that T cells transduced with 4D5-CD3 Bis-RNA lentivirally are reactive to ErbB2-overexpressing tumor cells. T cells transduced with ErbB2 CAR or Bis-RNA as indicated were stimulated with tumor lines expressing high levels of ErbB2, SK-OV3 and N87, or low levels, MDA-231, PC3, and A549. Cytokine production, assayed by ELISA, indicates that T4D5-CD3-expressing T cells are reactive only to ErbB2-overexpressing tumor cells. [Figure 60A] Figure 60A is a panel of images showing the regression of advanced angiogenic tumors in mice treated with T cells. Figure 60B is a panel of images showing that affinity-tuned ErbB2 BiTE cells increase the therapeutic index and induce regression of advanced angiogenic tumors in mice. T cells engineered with ErbB2 CARs or BiTEs of different affinities by lentiviral transduction were tested in dual-tumor-implanted NSG mice. On day 0, mice (n=4-5) were implanted with PC3-CBG tumor cells (1 x 10 cells / mouse, subcutaneous) in the right flank. On day 5, the same mice were implanted with SK-OV3-CBG tumor cells (5 x 10 cells / mouse, subcutaneous) in the left flank. On day 23 after PC3 tumor inoculation, mice were treated with T cells (intravenously). T cells were administered as a single injection of 1 x 10 cells / mouse. Mice treated with untransduced T cells (no TD) served as controls. Animals were imaged at the indicated time points after PC3 tumor inoculation. [Figure 60B]Figure 60B is a panel of images showing the regression of progressive angiogenic tumors in mice treated with T cells. Figure 60B is a graph showing SK-OV3 tumor size in a dual tumor-implanted NSG mouse model. Tumor size was measured, and tumor volume was calculated and plotted. [Figure 60C] Figure 60C is a panel of images showing the regression of progressive angiogenic tumors in mice treated with T cells. Figure 60D is a graph showing PC3 tumor size in a dual tumor-implanted NSG mouse model. Tumor size was measured, and tumor volume was calculated and plotted. [Figure 61] Figure 61, comprising Figures 61A-61B, is a panel of images showing the PD1-CD28 switch receptor. Figure 61A is a diagram of a construct for co-expressing the PD1-CD28 switch receptor and the affinity-regulated T4D5-6.CD3 BiTE. Figure 61B is a panel of graphs showing detection of the PD1-CD28 switch receptor in T cells lentivirally transduced with PD1-CD28 and T4D5-CD3 co-expression vectors. [Figure 62A]Figure 62, comprising Figures 62A-62M, is a panel of images. Figure 62A is a graph showing the increased lytic activity of T cells co-expressing both the PD1-CD28 switch receptor and the T4D5-6.CD3 affinity-regulated BiTE. Figures 62B-62G are a panel of images showing Bis-RNA electroporated T cells generated by the rapid expansion protocol (REP), which further improved in vivo anti-leukemia activity. The phenotype of T cells expanded by REP or anti-CD3 / anti-CD28 beads (beads) was evaluated (Figure 62B). REP T cells or anti-CD3 / anti-CD28 beaded T cells were electroporated with different amounts of CAR RNA or Bis-RNA and stimulated with different cell lines for 18 hours. CD137 upregulation was analyzed by flow cytometry (gating on CD3+ T cells) (Figure 62C). Lytic activity was measured in REP T cells (Figure 62D, left panel) or anti-CD3 / anti-CD28 beaded T cells (Figure 62D, right panel) electroporated with different amounts of CAR RNA or blinatumomab Bis-RNA. NSG mice were intravenously injected with 1 × 10 Nalm6-CBG and treated 5 days later with 30 × 10 CAR RNA or blinatumomab Bis-RNA (Blina) T cells for the first treatment, followed by 5 × 10 twice-weekly treatments for 3 weeks, starting 8 days after Nalm6-CBG injection. Bioluminescence imaging (BLI) was performed at the indicated time points (Figure 62E), and BLI and viability results are plotted in Figure 62F and Figure 62G, respectively. Figures 62H-62I are panels of images showing the generation of K562-based artificial antigen-presenting cells (aAPCs) expressing membrane-bound OKT3. Lentiviral vectors (pLENS) expressing chimeric proteins of membrane forms of OKT3 with either a CD8 hinge and transmembrane (OKT3.8) or a CD8 hinge and CD28 transmembrane (OKT3.8.28) are shown in Figure 62H.K562-based aAPCs, K562-CD86-CD137L (KT) or K562-CD137L (2D11) cell lines, were transduced with lentivirus OKT3.8 or OKT3.8.28, and membrane-bound OKT3 expression was detected using an antibody against mouse IgG Fab (Figure 62I). Figure 62J is a panel of images showing the characterization of membrane-bound OKT3-transduced K562 aAPC clones. The clones were selected by limiting dilution based on the expression of membrane-bound OKT3 from OKT3.8.28-transduced KT aAPCs. Figures 62K-62M are panels of graphs showing REP using K562-based artificial aAPCs. Figure 62K is a graph showing that OKT3-loaded K562-CD86-CD137L (KT) or K562-CD137L (2D11), or KT expressing membrane-bound OKT3 (KT.OKT), were irradiated and cultured for 1 day (D1) or 2 days (D2) before being used to stimulate T cells at a T cell:aAPC ratio of 1:250. Figure 62L is a panel of graphs showing expanded T cells independently expanded in REP and stained for CD62L and CD28. Figure 62M is a graph showing different B cell lines in the REP experiment compared to KT cells. [Figure 62B] See legend to Figure 62A. [Figure 62C] See legend to Figure 62A. [Figure 62D] See legend to Figure 62A. [Figure 62E] See legend to Figure 62A. [Figure 62F] See legend to Figure 62A. [Figure 62G] See legend to Figure 62A. [Figure 62H] See legend to Figure 62A. [Figure 62I] See legend to Figure 62A. [Figure 62J] See legend to Figure 62A. [Figure 62K] See legend to Figure 62A. [Figure 62L] See legend to Figure 62A. [Figure 62M] See legend to Figure 62A. [Figure 63] Figure 63 is a graph showing NSG mice with leukemia established by intravenous injection of 1 x 10 Naml6-CBG cells. Seven days later, the mice were treated with 20 x 10 RNA-electroporated T cells. On days 13 and 27, the mice were treated with 60 mg / kg of Cytoxan intraperitoneally 24 hours before injection of 5 x 10 T cells electroporated with RNA. Animals were imaged at the indicated time points after injection, with total photon flux shown on the Y axis (n = 5). [Figure 64] Figure 64 is a graph showing NSG mice with leukemia established by intravenous injection of 1 x 10 Naml6-CBG cells. Seven days later, the mice were treated with 20 x 10 RNA-electroporated T cells. On days 13 and 27, the mice were treated with 60 mg / kg of Cytoxan intraperitoneally 24 hours before injection of 5 x 10 T cells electroporated with RNA. Animals were imaged at the indicated time points after injection, with total photon flux shown on the Y axis (n = 5). [Figure 65] Figure 65 is a graph showing that TCR RNA electroporated T cells controlled tumor growth better than lenti-transduced T cells. 2.5x10 A549-ESO / A2 tumor cells were infused intravenously on day 0. Treatment began on day 5. Lenti-T cells were administered at a single dose of 10x10 on day 5, and RNA electroporated T cells were infused twice weekly starting on day 5 in four doses of 30x10, 10x10, 10x10, and 10x10. [Figure 66] Figure 66 is a panel of images showing that TCR RNA electroporated T cells controlled tumor cell growth. [Figure 67] Figure 67 is a panel of images showing that lenti-transduced T cells did not control tumor growth as effectively as TCR RNA electroporated T cells. [Figure 68] Figure 68 is a graph showing that lenti-transduced T cells controlled tumor growth more effectively than lenti-transduced T cells in a tumor model. [Figure 69] Figure 69 is a panel of images showing diagrams of CD3 constructs and graphs showing TCR and CD3 expression in T cells electroporated with TCR and CD3 RNA. [Figure 70] Figure 70 is a graph showing the expression levels of TCR (vb13.1), CD3 and TCR (vb13.1) / CD3 detected in electroporated T cells. [Figure 71] Figure 71 is a panel of graphs showing expression of TCR RNA electroporated into T cells. [Figure 72] Figure 72 is a table showing lentiviral vector titers for 1G4 wild type (1G4wt) and high affinity (1G4.LY95a) NY-ESO-1 TCR. [Figure 73] Figure 73 is a table showing the titers of different lentiviral vectors for 1G4 wild type (1G4wt) and high affinity (1G4.LY95a) NY-ESO-1 TCR. [Figure 74] Figure 74 is a table showing the efficiency of viral infection of T cells. [Figure 75] Figure 75 is a panel of graphs showing transgene expression in T cells co-electroporated with TCR and CD3. [Figure 76] Figure 76 is a panel of graphs showing transgene expression in T cells electroporated with TCR and CD3 RNA. [Figure 77] Figure 77 is a panel of graphs showing transgene expression in T cells co-electroporated with TCR and CD3 or T cells electroporated with TCR and stimulated with CD3 beads. [Figure 78] Figure 78 is a panel of flow diagrams showing T cells electroporated with TCR RNA (y-axis) with or without CD3 and incubated with Naml6 tumor cells (x-axis). [Figure 79]Figure 79 is a panel of flow diagrams showing T cells electroporated with TCR RNA (y-axis) with or without CD3 and incubated with Naml6 tumor cells expressing OKT (x-axis). [Figure 80] Figure 80 is a panel of flow diagrams showing T cells electroporated with TCR RNA (y-axis) with or without CD3 and incubated with Naml6 tumor cells expressing ND340 (x-axis). [Figure 81] Figure 81 is a panel of flow graphs showing TCR and CD3 expression in T cells electroporated with either full units (4 subunits) or fewer (3 subunits, 2 subunits, or 1 subunit) of CD3 delta, gamma, epsilon, and zeta, along with NY-ESO-1 TCR (1G4) alpha and beta chain RNA. CD3 and TCR (vb13.1) were detected by flow cytometry 18 hours after electroporation. [Figure 82] Figure 82 is a bar graph showing the mean fluorescence intensity (MFI) of CD3 and TCR expression after electroporation of CD3 RNA into the 293 cell line. [Figure 83] Figure 83 is a panel of flow graphs showing CD107a upregulation in TCR RNA and CD3 RNA co-electroporated T cells stimulated with tumor cells. [Figure 84] Figure 84 is a panel of graphs showing IFN-γ expression in RNA-electroporated T cells incubated with different tumor cell lines. T cells were electroporated with NY-ESO-1 TCR alpha and beta RNA along with different combinations of CD3 RNA. The cells were co-cultured with NY-ESO-1 / HLA-A2-positive Naml6-ESO or 624mel tumor cells. IFN-γ production levels were measured 18 hours later. [Figure 85]Figure 85 is a graph showing IFN-γ expression of different RNA-electroporated T cells incubated with tumor cell lines. [Figure 86] Figure 86 is a panel of graphs showing IL-2 expression in RNA-electroporated T cells incubated with different tumor cell lines. T cells were electroporated with NY-ESO-1 TCR alpha and beta RNA along with different combinations of CD3 RNA. The cells were cultured with NY-ESO-1 / HLA-A2-positive Naml6-ESO or 624mel tumor cells. IL-2 production levels were measured 18 hours later. [Figure 87] Figure 87 is a panel of graphs showing TCR expression by T cells electroporated with TCR or CD3 RNA constructs containing 4-1BB. T cells were co-electroporated with 1G4 TCR alpha (or alpha.BB) and beta (alpha / beta or alpha.BB / beta), or alpha / beta with CD3 zeta or epsilon (zeta, zeta.BB, epsilon, or epsilon.BB), with or without 4-1BB. After 18 hours, CD3 and vb13.1 were measured by flow cytometry. [Figure 88]Figure 88 is a panel of graphs showing that RNA-electroporated T cells incubated with different tumor cell lines were effective against multiple tumor cell lines. Providing costimulatory signals to the C-terminus of the TCR or CD3 maintained T cell function, potentially providing a direct costimulatory signal to T cells. T cells were co-electroporated with 1G4 TCR alpha (or alpha.BB) and beta (alpha / beta or alpha.BB / beta), or alpha / beta with CD3 zeta or epsilon (zeta, zeta.BB, epsilon, or epsilon.BB), with or without 4-1BB. Eighteen hours later, T cells were stimulated with the tumor cell lines Naml6-ESO (HLA-A2+ / NY-ESO-1+), A549ENA (HLA-A2+ / NY-ESO-1+), 624mel (HLA-A2+ / NY-ESO-1+), 526mel (HLA-A2+ / NY-ESO-1+), or 888mel (HLA-A2+ / NY-ESO-1+), and CD107a production was measured. [Figure 89] Figure 89 is a panel of flow diagrams showing Naml6 tumor cells (x-axis) and T cells (y-axis) electroporated with RNA encoding different TCR chains with or without CD3 chains. [Figure 90] Figure 90 is a panel of flow diagrams showing 624 tumor cells (x-axis) and T cells (y-axis) electroporated with RNA encoding different TCR chains with or without CD3 chains. [Figure 91] Figure 91 is a panel of flow diagrams showing 526 tumor cells (x-axis) and T cells (y-axis) electroporated with RNA encoding different TCR chains with or without CD3 chains. [Figure 92] Figure 92 is a panel of flow diagrams showing 888 tumor cells (x-axis) and T cells (y-axis) electroporated with RNA encoding different TCR chains with or without CD3 chains. [Figure 93]Figure 93, comprising Figures 93A-93D, is a panel of graphs showing the achieved maximal transgene expression (Figure 93A) and function of electroporated T cells with a TCR having a disulfide bond and a beta chain having both a disulfide bond and N-deglycosylation. N-deglycosylation of the TCR alpha chain impaired the function of electroporated T cells (Figures 93A-93D). [Figure 94] Figure 94 is a graph showing expression of 10x106 Naml6-CBG-ESO-GFP (click beetle green) cells expressing both NY-ESO-1 and GFP after intravenous injection into NOD / SCID mice. Five days after tumor inoculation, cells were first transduced with CBR (click beetle red) and then RNA-electroporated T cells were intravenously injected as indicated (n=5). wt1G4: wild-type 1G4 TCR, m1G4: second disulfide bond alpha / second disulfide bond alpha and N-deglycosylated beta, CD19: CD19BBZ CAR, meso: ss1BBZ CAR, and saline. [Figure 95] Figure 95 is a panel of images showing that N-deglycosylation in the TCR alpha chain impaired the function of RNA-electroporated T cells. [Figure 96] Figure 96 is a panel of images showing transgene expression and functionality of T cells electroporated with RNA encoding a hybrid TCR containing a mouse constant region. [Figure 97] Figure 97 is a panel of images showing the fluorescence of tumor cells and hybrid TCR T cells injected into a mouse model over time. [Figure 98] Figure 98, comprising Figures 98A-98D, is a panel of images showing that the addition of disulfide bonds to the alpha and beta chains of a TCR, or N-deglycosylation of the beta chain, enhanced transgene expression and function of electroporated T cells compared to the addition of disulfide bonds to the TCR or hybrid TCR. [Figure 99]Figure 99 is a panel of images showing TCR expression by T cells electroporated with TCR or CD3 containing 4-1BB. T cells were co-electroporated with 1G4 TCR alpha (or alpha.BB) and beta (a / b or a.BB / b), or a / b with CD3 zeta or epsilon (z, z.BB, e, or e.BB), with or without 4-1BB. After 18 hours, CD3 and vb13.1 were measured by flow cytometry. [Figure 100] Figure 100 is a panel of images showing T cells co-electroporated with 1G4 TCR alpha (or alpha.BB) and beta (a / b or a.BB / b), or a / b with CD3 zeta or epsilon (z, z.BB, e, or e.BB), with or without 4-1BB. Eighteen hours later, T cells were stimulated with tumor lines Nalm6-ESO (HLA-A2+ / NY-ESO-1+), A549-ESO (HLA-A2+ / NY-ESO-1+), 624mel (HLA-A2+ / NY-ESO-1+), 526mel (HLA-A2+ / NY-ESO-1-), or 888mel (HLA-A2- / NY-ESO-1-) and assessed in a CD107a assay. [Figure 101] Figure 101 is a panel of images showing T cells co-electroporated with 1G4 TCR alpha and beta, or a / b bearing CD3 epsilon (E) and / or zeta (Z), with or without CD27 (or CD28). Eighteen hours later, T cells were stimulated with tumor lines Nalm6-ESO (HLA-A2 / NY-ESO-1), 624mel (HLA-A2 / NY-ESO-1), U266 (HLA-A2 / NY-ESO-1), or 888mel (HLA-A2 / NY-ESO-1) and assessed in a CD107a assay. [Figure 102]Figure 102 is a panel of images showing detection of PD1 and vb13.1 in T cells lentivirally transduced with PD1 constructs and 1G4 TCR with or without the PD1-CD28 switch receptor and CD27. [Figure 103] Figure 103 is a graph showing that 5E6 A549-ESO (HLA-A2 and NY-ESO-1 transduced A549) was injected subcutaneously on day 0. 1 x 10 transduced T cells were injected on day 14 and tumor size was measured. Preliminary results showed that TCR alone (1G4) had no effect, but CD27 costimulatory signal (1G4.CD27), or PD1-CD28 switch receptor (PD1-CD28.1G4), or both CD27 costimulatory signal and PD1-CD28 switch receptor (PD1-CD28.1G4.CD27) delayed tumor growth. [Figure 104] Figure 104 is a schematic diagram of an engineered TCR capable of non-MHC restricted tumor antigen recognition along with functional cognate antigen recognition. [Figure 105] Figure 105 is a panel of images showing transgene expression in co-electroporated T cells. A seven-amino acid influenza (HA1) peptide sequence was added to the N'-terminus of either the TCR alpha chain or the CD3 zeta or epsilon chain to generate HA1.alpha (HA1.a), HA1.zeta (HA1.z), and HA1.epsilon (HA1.e). T cells were co-electroporated with RNA encoding a bispecific antibody against influenza HA1 (17-9 or 26-9) and a tumor antigen (CD19 or Her2 (4D5)). 18 hours after electroporation, transgenic TCR (vb13.1) expression was examined by flow cytometry. [Figure 106] Figure 106 is a panel of images showing that T cells with modified NY-ESO-1 TCR recognized the cognate antigen (HLA-A2 / NY-ESO-1) along with CD19 or Her2. [Figure 107]Figure 107 is a graph showing IFN-γ secretion of T cells transfected with modified TCR together with bis-RNA. [Figure 108] Figure 108 is a graph showing IL-2 secretion of T cells transfected with modified TCR together with bis-RNA. [Figure 109] Figure 109 is a panel of images showing the anti-tumor activity of T cells with modified TCR and bis-RNA administered to tumor-bearing mice. Mice were intravenously injected with Nalm6-ESO and treated with T cells electroporated with RNA as indicated. [Figure 110] Figure 110, comprising Figures 110A-110B, is a panel of images showing T cells with modified TCRs. Figure 110A is a schematic diagram of small molecule modified TCRs (Affi-TCRs) by adding a small molecule (AFFIBODY®) and a His-tag sequence to the N'-terminus of either the alpha or beta chain of the TCR. Figure 110B is a panel of images showing that T cells were co-electroporated with NY-ESO-1 (1G4) TCR alpha (a) and beta (b), or their ErbB2 small molecule (342, 342.15, 342, or 342.4) variants. After overnight incubation, vb13.1 and the His-tag were detected by flow cytometry. [Figure 111] Figure 111 is a panel of images showing CD107 upregulation in Affi-TCR RNA electroporated T cells. [Figure 112]Figure 112, comprising Figures 112A-112C, is a panel of images showing TCR expression and dual targeting by co-delivery of small molecule-modified CD3 epsilon. Figure 112A is a schematic diagram of small molecule-modified CD3 epsilon by adding a small molecule and a G4S linker to the N'-terminus of the CD3 epsilon chain. Figure 112B is a table showing T cells co-electroporated with NY-ESO-1 (1G4) TCR alpha (a) and beta (b) or together with ErbB2 small molecule (342, 342.15, 342, or 342.4)-modified CD3 epsilon (e). Figure 112C is a panel of images showing vb13.1 expression detected by flow cytometry after overnight incubation. [Figure 113] Figure 113 is a panel of images showing CD107a upregulation in Affi-TCR RNA electroporated T cells. T cells were co-electroporated with NY-ESO-1 (1G4) TCR alpha (a) and beta (b) or together with ErbB2 small molecule (342, 342.15, 342, or 342.4) modified CD3 epsilon (e) as shown in Figure 101 and stimulated with tumor cells Nalm-6-ESO (NY-eso-1+, ErbB2-), Nalm6 (NY-eso-1-, ErbB2-), SK-OV3 (NY-eso-1-, ErbB2+), or MDA231 (NY-eso-1-, ErbB2+) and assessed for CD107a expression. DETAILED DESCRIPTION OF THE INVENTION

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] As used herein, "activation" refers to a state in which a T cell is sufficiently stimulated 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.

[0112] The phrase "affinity molecule chimeric receptor" refers to a recombinant receptor comprising an affinity molecule, such as a small molecule, antibody mimic, Affibody™, or any fragment thereof, that binds to a target protein or peptide with high affinity. In some embodiments, the affinity molecule chimeric receptor comprises a transmembrane domain and an intracellular domain. In some other embodiments, the affinity molecule chimeric receptor comprises a T cell receptor domain, such as a constant domain and a variable domain.

[0113] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin obtained from a natural or recombinant source, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Tetramers may occur naturally or may be reconstituted from single-chain antibodies or antibody fragments. Antibodies also include dimers that may occur naturally or may be constructed from single-chain antibodies or antibody fragments. Antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab')2, as well as single-chain antibodies (scFv), humanized antibodies, and human 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).

[0114] The term "antibody fragment" refers to a region of an intact antibody, specifically 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, single-domain antibodies such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38), which are composed of either a VL or VH domain that exhibits sufficient affinity for a target, and multispecific antibodies formed from antibody fragments. Antibody fragments also include human or humanized antibodies, or fragments of such human or humanized antibodies.

[0115] 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.

[0116] 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.

[0117] As used herein, a "bispecific antibody" refers to an antibody having binding specificities for at least two different antigen epitopes. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitopes are derived from two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using coexpression of two immunoglobulin heavy chain / light chain pairs. See, e.g., Milstein et al. (1983) Nature 305:537-39. Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan et al. (1985) Science 229:81. Bispecific antibodies include bispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-48, Gruber et al. (1994) J. Immunol. 152:5368.

[0118] 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.

[0119] 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 produced 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 obtained from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0125] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0126] The phrase "bispecific affinity molecule" refers to a molecule that contains two different binding specificities and is therefore capable of simultaneously binding to two targets, molecules, or antigens. A bispecific affinity molecule comprises an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell. In some embodiments, one or more affinity domains are small molecule antigen-binding domains and may comprise a small molecule, an antibody mimetic, an Affibody™, or any fragment thereof.

[0127] As used herein, "bispecific" refers to a molecule that has binding specificity for at least two different binding epitopes. In one embodiment, the epitopes are derived from the same binding partner. In another embodiment, the epitopes are derived from two different binding partners. Molecules with bispecificity for different epitopes can include bispecific antibodies.

[0128] 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 spread 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, thyroid cancer, etc.

[0129] 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 may 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 CD3 zeta 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.

[0130] As used herein, the phrase "chimeric ligand-modified activating receptor" or "CLEAR" refers to an engineered receptor that contains at least an extracellular domain for ligand recognition and an intracellular domain for activation signaling within a T cell. CLEAR is engineered to contain an extracellular domain that binds to either a tumor or viral antigen or other molecule, while the intracellular domain provides an activation signal within the T cell after binding of a ligand or antigen to the receptor's extracellular domain.

[0131] The term "chimeric membrane protein" refers to an engineered membrane protein having an extracellular domain and an intracellular domain derived from or capable of activating one or more signaling molecules and / or receptor molecules. For example, the chimeric membrane protein described herein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB.

[0132] 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.

[0133] "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.

[0134] 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.

[0135] 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.

[0136] The term "derived from" refers to made, synthesized, or obtained from a particular source, such that the derived material is related to the source. The derived material need not be identical to the particular source. In one embodiment, the antigen is derived from a protein. In another embodiment, the single-chain variable fragment is derived from a monoclonal antibody.

[0137] 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.

[0138] "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.

[0139] "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.

[0140] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue, or system.

[0141] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0142] 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).

[0143] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0144] "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., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide.

[0145] "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.

[0146] "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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] The phrases "immunologically effective amount," "anti-immune response effective amount," "immune response inhibiting effective amount," or "therapeutic amount" refer to the amount of a composition of the present invention administered to a subject, as determined by a physician, optionally in consultation with a scientist, taking into account individual differences in the subject's (patient's) age, weight, immune response, type of disease / condition, and health status, so as to obtain the desired result in the subject.

[0151] 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.

[0152] "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.

[0153] " 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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. Nucleotide sequences that encode RNA and proteins can contain introns.

[0159] 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.

[0160] 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.

[0161] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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. An example of a "cell surface receptor" is human FSHR.

[0170] As used herein, "similarity" 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 similar at that position. The similarity between two sequences is a linear function of the number of matching or similar positions; for example, if half of the positions in two sequences (e.g., 5 positions in a polymer 10 subunits long) are similar, the two sequences are 50% similar; if 90% of the positions (e.g., 9 out of 10) are similar or identical, the two sequences are 90% similar.

[0171] "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.

[0172] The term "small molecule" refers to a peptide having about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids that has the ability to bind to a target, e.g., a molecule or antigen. Small molecules have low molar masses, e.g., less than about 12 kD, 11 kD, 10 kD, 9 kD, 8 kD, 7 kD, 6 kD, 5 kD, or any molar mass therebetween or less. In some embodiments, a small molecule is a small molecule extracellular domain of an affinity molecule chimeric receptor. In some embodiments, a small molecule is a small molecule binding domain of a bispecific affinity molecule. Small molecules can be characterized by their ability to bind to a target and their structure. In some embodiments, a small molecule contains at least one helix, e.g., an alpha helix, or two helices, three helices, or more. Small molecules are also chemically inert and can withstand high temperatures, such as 85°C or higher.

[0173] 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.

[0174] 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-beta and / or rearrangement of cytoskeletal structures.

[0175] "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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a region of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0180] 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 alpha / beta and gamma / delta 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 gamma delta T cells).

[0181] 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.

[0182] 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.

[0183] "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.

[0184] 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.

[0185] 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, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0186] 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.

[0187] explanation The present invention includes methods and compositions for generating engineered T cells capable of expressing bispecific antibodies. In some embodiments, the present invention includes methods for generating engineered T cells. Other embodiments include engineered T cells or populations of engineered T cells. The bispecific antibody comprises dual specificities for an antigen on a target cell and an antigen on an activated T cell, such as CD3, CD4, CD8, and TCR.

[0188] T cell receptor The present invention includes T cells having an exogenous T cell receptor (TCR). In one aspect, the present invention includes a method for generating modified T cells, comprising expanding a population of T cells and introducing into the expanded T cells a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for an antigen on a target cell. In this embodiment, the T cells are capable of expressing the modified TCR.

[0189] In another aspect, the invention includes a method for making modified T cells, comprising expanding a population of T cells and introducing into the expanded T cells a nucleic acid encoding a modified T cell receptor (TCR) that comprises affinity for a surface antigen on a target cell. In this embodiment, the T cells are capable of expressing the modified TCR.

[0190] 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.

[0191] TCR is generally composed of six different membrane-bound chains that form a TCR heterodimer that is involved in ligand recognition.TCR exists in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions.In one embodiment, TCR comprises a TCR alpha chain and a beta chain, and thus the nucleic acid encoding TCR comprises the nucleic acid encoding the TCR alpha chain and the TCR beta chain.In another embodiment, the alpha chain or the beta chain, or both, comprise at least one N-deglycosylation.

[0192] 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 costimulatory signaling domain is 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.

[0193] 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.

[0194] Functional alpha and gamma chain polypeptides are formed by rearranged VJC regions, while beta and delta chains consist of VDJC regions. The extracellular constant domain includes a membrane proximal region and an immunoglobulin region.

[0195] 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 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 alpha chain or beta 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.

[0196] Examples of target cell-associated antigens are described elsewhere herein, all of which may be targeted by the TCRs of the present invention.

[0197] In one aspect, the invention includes a population of modified T cells comprising electroporated RNA encoding a modified T cell receptor (TCR) comprising affinity for an antigen on a target cell, the population of T cells being expanded prior to electroporation with the TCR RNA.

[0198] In another aspect, the invention includes a modified T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) having affinity for an antigen on a target cell and an electroporated nucleic acid encoding a costimulatory molecule, wherein the T cell expresses the TCR and the costimulatory molecule. The costimulatory molecule may be selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L.

[0199] In another aspect, the invention includes a modified T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) comprising affinity for a surface antigen on a target cell; and an electroporated nucleic acid encoding a costimulatory molecule, wherein the T cell expresses the TCR and the costimulatory molecule.

[0200] In yet another aspect, the invention includes modified T cells comprising electroporated RNA encoding a modified T cell receptor (TCR), wherein the modified TCR comprises affinity for an antigen on a target cell, and the T cells are expanded prior to electroporation with the TCR RNA.

[0201] In yet another aspect, the invention includes modified T cells comprising electroporated RNA encoding a modified T cell receptor (TCR), wherein the modified TCR comprises affinity for a surface antigen on a target cell, and the T cells are expanded prior to electroporation with the TCR RNA.

[0202] In one embodiment, the invention involves introducing into expanded T cells a nucleic acid encoding a modified T cell receptor (TCR) that comprises affinity for an antigen on a target cell. In this embodiment, the T cells are capable of expressing the modified TCR.

[0203] Techniques for engineering and expression of T cell receptors include, but are not limited to, the production of TCR heterodimers containing native disulfide bridges linking the individual subunits (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).

[0204] In one embodiment, the TCR has specificity for a target cell antigen. The target cell antigen can include any type of protein associated with the target cell. For example, the target cell antigen can be selected to recognize a specific disease state of the target cell. 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 antigen includes any tumor-associated antigen (TAA) and viral antigen, or any fragment thereof.

[0205] Target cell antigens can include any protein that can be processed and presented by major histocompatibility complex.For example, target antigens can be associated with certain disease states.Therefore, examples of cell markers that can act as targets for TCR include those associated with virus, bacterial and parasitic infection, autoimmune disease and cancer cells.In one embodiment, target antigens include tumor-associated antigens (TAA) and viral antigens, or any fragments thereof.

[0206] bispecific antibody The present invention includes bispecific antibodies. Bispecific antibodies have two different binding specificities and therefore bind to two different antigens. In one embodiment, the bispecific antibody comprises a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen.

[0207] In another embodiment, the bispecific antibody comprises an antigen-binding domain comprising a first and a second single-chain variable fragment (scFv) molecule. In such an embodiment, the first and second scFvs bind to an antigen on a target cell and an antigen on an activated T cell. In another embodiment, the first scFv molecule is specific for at least one antigen on a target cell, and the second scFv molecule is specific for an antigen on an activated T cell. For example, the activating T cell antigen can bind to CD3, CD4, CD8, or TCR. In these examples, the bispecific antibody recognizes a T cell antigen and is referred to as a bispecific T cell engager (BiTE).

[0208] In another embodiment, the first and second scFvs bind to an antigen on a target cell and an antigen on a T cell. For example, the T cell antigen can include CLEAR, CD3, CD4, CD8, or TCR. In these examples, the bispecific antibody recognizes a T cell antigen, such as CD3, CD4, CD8, or TCR, and is referred to as a bispecific T cell derivative (BiTE). In another embodiment, the bispecific antibody comprises a dual specificity for an antigen on a target cell and CLEAR on a T cell.

[0209] However, the present invention is not limited by the use of any particular bispecific antibody. Rather, any bispecific antibody or BiTE can be used. Bispecific antibody or BiTE molecules can also be expressed as membrane proteins with specificity for at least one target cell-associated antigen. Examples of target cell-associated antigens are described elsewhere herein, all of which can be targeted by the bispecific antibodies of the present invention. Techniques for producing human and humanized antibodies are also described elsewhere herein. In one embodiment, the bispecific antibody or BiTE molecule comprises a bispecific antigen-binding domain. In this embodiment, the bispecific antigen-binding domain includes a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof.

[0210] In one aspect, the invention includes a population of modified T cells that contain nucleic acid, such as RNA, encoding a bispecific antibody that contains a dual specificity for an antigen on a target cell and an antigen on a T cell. The population of T cells is expanded prior to introduction of the nucleic acid.

[0211] In one aspect, the invention includes a population of modified T cells comprising electroporated mRNA encoding a bispecific antibody comprising a dual specificity for an antigen on a target cell and an antigen on activated T cells, the population of T cells having been expanded prior to BiTE electroporation.

[0212] In another aspect, the invention includes modified T cells comprising electroporated mRNA encoding a bispecific T cell engager (BiTE) molecule. In this embodiment, the BiTE molecule comprises dual specificities for an antigen on a target cell and an antigen on an activated T cell selected from the group consisting of CD3, CD4, CD8, and TCR.

[0213] In yet another aspect, the invention includes a population of modified T cells comprising nucleic acid, such as RNA, encoding a bispecific antibody comprising dual specificity for an antigen on a target cell and an antigen on a T cell. The population of T cells can be expanded prior to introduction of the nucleic acid.

[0214] In yet another aspect, the present invention includes modified T cells comprising electroporated RNA encoding a bispecific antibody. The bispecific antibody comprises dual specificities for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR. This embodiment also includes that the T cells are expanded prior to electroporation with the bispecific antibody mRNA.

[0215] In yet another aspect, the invention includes modified T cells comprising nucleic acid, such as RNA, encoding a bispecific antibody. The bispecific antibody comprises two specificities for an antigen on a target cell and an antigen on the T cell, such as CLEAR. This embodiment also includes expanding the T cells prior to electroporation with the bispecific antibody RNA.

[0216] In one embodiment, the invention involves electroporating expanded T cells with mRNA encoding a bispecific antibody, such as a BiTE molecule. In another embodiment, the invention involves introducing nucleic acids encoding a bispecific antibody, such as a BiTE molecule, into expanded T cells. In such embodiments, the T cells are capable of expressing the bispecific antibody. Techniques for engineering and expressing bispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and the "knob-in-hole" technique (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); by cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science 229:81 (1985)); by using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); by using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); as well as by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).Engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576A1). Bispecific antibodies can be constructed by linking two different antibodies or portions thereof. For example, bispecific antibodies can include Fab, F(ab')2, Fab', scFv, and sdAb derived from two different antibodies.

[0217] In one embodiment, the bispecific antibody and / or BiTE molecule comprises a bispecific antigen-binding domain comprising a first and a second single-chain variable fragment (scFv) molecule. In such an embodiment, the bispecific antigen-binding domain can comprise dual specificities for an antigen on a target cell and an antigen on a T cell, such that the first scFv molecule is specific for at least one antigen on the target cell and the second scFv molecule is specific for at least one antigen on the T cell. In another embodiment, the bispecific antigen-binding domain can comprise dual specificities for an antigen on a target cell and an antigen on an activated T cell, such that the first scFv molecule is specific for at least one antigen on the target cell and the second scFv molecule is specific for at least one antigen on the activated T cell. In another embodiment, the bispecific antibody is expressed as a membrane protein.

[0218] In one embodiment, the bispecific antibody comprises specificity for a target cell antigen. The target cell antigen may comprise the same target cell antigen as that bound by the T cell receptor, or may comprise a different target cell antigen. The target cell antigen may comprise any type of ligand that defines the target cell. For example, the target cell antigen may be selected to recognize a ligand that acts as a cellular marker on target cells associated with a particular disease state. Thus, examples of cellular markers that can act as ligands for the antigen moiety domain in a BiTE molecule include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0219] In one embodiment, the target cell antigen comprises any tumor-associated antigen (TAA) and viral antigen, or any fragment thereof. In this embodiment, the bispecific antibody and / or BiTE molecule comprises an antibody, such as a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof, that specifically binds to the target cell antigen. Examples of activating T cell antigens can include tumor antigens, viral antigens, and fragments thereof. Examples of target cell antigens can include tumor antigens, viral antigens, and fragments thereof.

[0220] In one embodiment, the bispecific antibody and / or BiTE molecule contains specificity for at least one antigen on activated T cells. Activating T cell antigens include antigens found on the surface of T cells that can activate other cells. Activating T cell antigens can include costimulatory molecules. Costimulatory molecules are cell surface molecules that are neither antigen receptors nor their ligands, but are required for efficient lymphocyte responses to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83-specific binding ligands. Other costimulatory elements are also within the scope of the present invention.

[0221] In one embodiment, the activating T cell antigen is CD3, CD4, CD8, T cell receptor (TCR), or any fragment thereof. In this embodiment, the bispecific antibody and / or BiTE molecule comprises an antibody, such as a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof, that specifically binds to the activating T cell antigen. Examples of activating T cell antigens may include anti-CD3, anti-CD4, anti-CD8, anti-TCR, and fragments thereof.

[0222] In another embodiment, the bispecific antibody and / or BiTE molecule comprises specificity for at least one antigen on a T cell. T cell antigens include antigens found on the surface of T cells, such as CLEAR. The T cell antigen may comprise a costimulatory signal or domain. The costimulatory signal or domain is derived from a cell surface molecule that is not an antigen receptor or its ligand, but is required for an efficient lymphocyte response to the antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83-specific binding ligands. Other costimulatory elements are also within the scope of the present invention. In one embodiment, the bispecific antibody comprises dual specificity for an antigen on a target cell and CLEAR on a T cell.

[0223] In another embodiment, the T cell antigen is CD3, CD4, CD8, T cell receptor (TCR), or any fragment thereof. In this embodiment, the bispecific antibody and / or BiTE molecule comprises an antibody, such as a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof, that specifically binds to the T cell antigen. Examples of activating T cell antigens can include anti-CD3, anti-CD4, anti-CD8, anti-TCR, and fragments thereof.

[0224] Affinity molecule chimeric receptor The present invention includes engineered T cells having chimeric affinity molecule receptors. In one aspect, the present invention includes a method for generating engineered T cells, comprising introducing into a population of T cells capable of expressing the chimeric affinity molecule receptor a nucleic acid encoding a chimeric affinity molecule receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell.

[0225] Affinity molecule chimeric receptors generally consist of a small molecule extracellular domain for antigen recognition, such as an extracellular domain with affinity for an antigen on a target cell. In some embodiments, the small molecule extracellular domain is derived from an antibody mimic. The small molecule extracellular domain of a bispecific affinity molecule is generally a peptide having about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. The molar mass of the small molecule extracellular domain may be less than that of a single-domain antibody, such as less than about 10 kD. In one embodiment, the affinity molecule chimeric receptor comprises a small molecule extracellular domain that is less than about 10 kD. The molar mass of the small molecule extracellular domain may be less than about 12 kD, 11 kD, 10 kD, 9 kD, 8 kD, 7 kD, 6 kD, 5 kD, or any molar mass therebetween. The small molecule extracellular domain may also comprise a helical structure lacking disulfide bridges. Some small molecule extracellular domains contain at least one alpha helix, two alpha helices, three alpha helices, or more. Small molecule extracellular domains may also be chemically inert and able to withstand high temperatures, such as about 85° C. or higher.

[0226] In one embodiment, the affinity molecule chimeric receptor further comprises an intracellular signaling domain, such as a CD3 signaling domain; a transmembrane domain, such as a CD8 transmembrane domain; and a costimulatory domain, such as a 4-1BB costimulatory domain.

[0227] The affinity molecule chimeric receptor may also be based on a T cell receptor (TCR). In this embodiment, the affinity molecule chimeric receptor comprises a small extracellular domain having affinity for an antigen on a target cell, a TCR variable domain, and a TCR constant domain. The TCR variable domain may be derived from a chimera of the alpha chain or beta chain, or both. Similarly, the TCR constant domain may be derived from a chimera of the alpha chain or beta chain, or both. 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.

[0228] Examples of target cell-associated antigens are described elsewhere herein, all of which may be targeted by the affinity molecule chimeric receptors of the present invention.

[0229] In one aspect, the invention includes a population of modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain that has affinity for an antigen on a target cell, wherein the population of T cells expresses the affinity molecule chimeric receptor.

[0230] In another aspect, the invention includes modified T cells that contain a nucleic acid encoding an affinity molecule chimeric receptor that includes a small molecule extracellular domain that has affinity for an antigen on a target cell, wherein the T cells express the affinity molecule chimeric receptor.

[0231] In one embodiment, the small molecule extracellular domain comprises specificity for a target cell antigen. The target cell antigen can include any type of protein associated with the target cell. For example, the target cell antigen can be selected to recognize a specific disease state of the target cell. Thus, examples of cell surface markers can act to bind to small molecule extracellular domains for antigens such as those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells. In one embodiment, the target cell antigen includes any tumor-associated antigen (TAA), bacterial antigen, parasitic antigen, viral antigen, or any fragment thereof.

[0232] Target cell antigens can include any protein that can be processed and presented by major histocompatibility complex.For example, target antigens can be associated with certain disease states.Therefore, examples of cell markers that can act as targets for TCR include those associated with virus, bacterial and parasitic infection, autoimmune disease and cancer cells.In one embodiment, target antigens include tumor-associated antigens (TAA) and viral antigens, or any fragments thereof.

[0233] Bispecific affinity molecules The present invention also includes bispecific affinity molecules. Bispecific affinity molecules contain two different binding specificities and can therefore bind to two targets, molecules, or antigens. In one aspect, the present invention includes engineered cells containing nucleic acids encoding bispecific affinity molecules containing an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell. In this embodiment, at least one affinity domain contains a small molecule antigen-binding domain, and the cells express the bispecific affinity molecule. In another embodiment, activated T cells and target cells bind to the bispecific affinity molecule.

[0234] Either affinity domain of the bispecific affinity molecule may comprise a small molecule antigen-binding domain that may have affinity for a target cell antigen or an activated T cell antigen. In one embodiment, the affinity domain capable of binding to a target cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antibody antigen-binding domain. In another embodiment, the affinity domain capable of binding to an activated T cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antibody antigen-binding domain. In yet another embodiment, the bispecific affinity molecule comprises a small molecule antigen-binding domain with affinity for a target cell antigen and a small molecule antigen-binding domain with affinity for an activated T cell antigen. In one embodiment, the bispecific affinity molecule comprises a small molecule antigen-binding domain with affinity for a target cell antigen and an antibody antigen-binding domain with affinity for an activated T cell antigen. In another embodiment, the bispecific affinity molecule comprises an antibody antigen-binding domain with affinity for a target cell antigen and a small molecule antigen-binding domain with affinity for an activated T cell antigen.

[0235] In some embodiments, the small molecule antigen-binding domain of the bispecific affinity molecule comprises an antibody mimic or a fragment thereof. The small molecule antigen-binding domain of the bispecific affinity molecule is generally a peptide having about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. The molar mass of the small molecule may be less than that of a single-domain antibody, such as less than about 10 kD. In one embodiment, the bispecific affinity molecule comprises a small molecule antigen-binding domain that is less than about 10 kD.

[0236] When the affinity domain is a small molecule antigen-binding domain, the small molecule may have a molar mass less than or equal to about 12 kD, 11 kD, 10 kD, 9 kD, 8 kD, 7 kD, 6 kD, 5 kD, or any molar mass therebetween. The molar mass of a small molecule antigen-binding domain with affinity for a target cell antigen may be greater or less than the molar mass of a small molecule antigen-binding domain with affinity for an activating T cell antigen. The small molecule antigen-binding domain may also comprise a helical structure lacking disulfide bridges. Some small molecule antigen-binding domains contain at least one alpha helix, two alpha helices, three alpha helices, or more. The small molecule antigen-binding domain may also be chemically inert and able to withstand high temperatures, such as about 85°C or higher.

[0237] In another embodiment, the nucleic acid encoding the bispecific affinity molecule may further comprise a linker or spacer between the affinity domains. As used herein, the term "linker" or "spacer" generally refers to any oligopeptide or polypeptide that functions to link one affinity domain to another, either a small molecule antigen-binding domain to another small molecule antigen-binding domain, or a small molecule antigen-binding domain to an antibody antigen-binding domain. The spacer or linker may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0238] The present invention is not limited by the use of only a particular affinity domain. Rather, any affinity domain can be used. Bispecific affinity molecules can also be expressed as membrane proteins with specificity for at least one target cell antigen. Examples of target cell antigens are described elsewhere herein, all of which can be targeted by the bispecific affinity molecules of the present invention.

[0239] In one embodiment, the bispecific affinity molecule comprises an antibody antigen-binding domain. Techniques for producing human and humanized antibodies or fragments thereof are described elsewhere herein. In this embodiment, the antibody antigen-binding domain comprises a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof.

[0240] In one aspect, the present invention includes a population of engineered cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain, and the population of cells expresses the bispecific affinity molecule. The population of engineered cells includes lymphocytes, such as T cells, B cells, or natural killer cells; antigen-presenting cells, or non-lymphocytes. In one embodiment, the population of cells includes T cells, B cells, natural killer cells, or antigen-presenting cells.

[0241] In another aspect, the present invention includes an engineered cell comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain, and the cell expresses the bispecific affinity molecule. The engineered cell can be selected from a lymphocyte, such as a T cell, a B cell, or a natural killer cell; an antigen-presenting cell; or a non-lymphocyte. In one embodiment, the cell is a T cell, a B cell, a natural killer cell, or an antigen-presenting cell.

[0242] In yet another aspect, the present invention includes a method of making modified cells, comprising introducing into a population of cells a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell. In this embodiment, at least one affinity domain comprises a small molecule antigen-binding domain, and the cells express the bispecific affinity molecule. In one embodiment, the population of cells comprises T cells, B cells, natural killer cells, or antigen-presenting cells. Techniques for engineering and expressing bispecific affinity molecules include, but are not limited to, engineering Affibody™ molecules (see Lofblom et al., FEBS Letters 584: 2670 (2010); Feldwisch et al., J Mol Biol, 398(2): 232 (2010); U.S. Patent Nos. 8,501,909, 8,426,557, 8,247,375, and 7,993,650; and U.S. Patent Application Publication No. 2014 / 0295521), and "knob-in-hole" technology (see, e.g., U.S. Patent No. 5,731,168).Multispecific antibodies can also be made by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); by cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science 229:81 (1985)); by using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); by using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994); as well as by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991). Engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies," are also included herein (see, for example, US 2006 / 0025576A1). Bispecific antibodies can be constructed by linking two different antibodies or portions thereof. For example, one of the affinity domains of the bispecific affinity molecule can comprise Fab, F(ab')2, Fab', scFv, or sdAb derived from an antibody.

[0243] In one embodiment, the bispecific affinity molecule comprises an affinity domain that can bind to a target cell antigen.The target cell antigen can comprise any type of ligand or antigen that defines the target cell.For example, the target cell antigen can be selected to recognize a ligand that acts as a cell marker on the target cell and is associated with a specific disease state.Therefore, examples of target cell antigens include those associated with virus, bacterial and parasitic infections, pathological conditions, autoimmune diseases and cancer cells.

[0244] In one embodiment, the target cell antigen includes any tumor-associated antigen (TAA), bacterial antigen, parasitic antigen, viral antigen, and any fragment thereof. When the affinity domain is an antigen-binding domain of an antibody, the antibody antigen-binding domain can include antibodies, such as synthetic antibodies, human antibodies, humanized antibodies, single-chain variable fragments, single-domain antibodies, antigen-binding fragments thereof, and any combination thereof, that specifically bind to the target cell antigen.

[0245] In another embodiment, the bispecific affinity molecule comprises an affinity domain capable of binding to an activating T cell antigen. Activating T cell antigens include antigens found on the surface of T cells that can activate other cells. Activating T cell antigens may include costimulatory molecules. Costimulatory molecules are cell surface molecules that are neither antigen receptors nor their ligands, but are required for efficient lymphocyte responses to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83-specific binding ligands. Other costimulatory elements are also within the scope of the present invention.

[0246] In yet another embodiment, the activating T cell antigen is CD3, CD4, CD8, T cell receptor (TCR), or any fragment thereof. In this embodiment, the bispecific antibody and / or BiTE molecule comprises an antibody, such as a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof, that specifically binds to the activating T cell antigen. Examples of activating T cell antigens may include anti-CD3, anti-CD4, anti-CD8, anti-TCR, and fragments thereof.

[0247] Chimeric Ligand-Modified Activated Receptors The present invention includes T cells having a chimeric ligand-modified activating receptor. In one aspect, the invention includes a method for generating modified T cells, comprising expanding a population of T cells and introducing a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR). In this embodiment, the T cells are capable of expressing CLEAR.

[0248] CLEAR generally consists of an extracellular domain for ligand recognition and an intracellular domain for activation signal transduction. In one embodiment, CLEAR comprises an intracellular activation domain and an extracellular domain.

[0249] The extracellular domain can be engineered for recognition by antibodies, receptors, ligands, or other binding molecules. In one embodiment, the extracellular domain specifically binds to either a tumor antigen or molecule that is not normally expressed by healthy cells or tissues. Examples of tumor antigens or abnormal molecules include, but are not limited to, viral, bacterial, and parasitic antigens, tumor-associated antigens (TAA), or any fragments thereof. In one embodiment, the extracellular domain is selected from an antigen-binding domain of an antibody, a ligand-binding domain of a receptor, an antigen, or a ligand. In another embodiment, the extracellular domain is selected from CD27, CD28, CD70, CD80, PD1, or PD-L1. In yet another embodiment, the extracellular domain can bind to a tumor antigen.

[0250] The intracellular domain can be engineered to deliver an activation signal within the T cell. Examples of intracellular domains include, but are not limited to, the intracellular domain from CD3, CD3 zeta with or without a costimulatory signal, CD28, CD4, CD8, 4-1BB, the TCR alpha chain and / or beta chain, or a fragment of any of these intracellular domains. In one embodiment, the intracellular activation domain comprises the intracellular activation domain of CD3 zeta. In another embodiment, the activation signal delivered within the T cell can be a positive signal that induces proliferation, cytokine production, lytic activity, and other activities of the T cell. In yet another embodiment, the activation signal delivered within the T cell can be a negative signal that shuts down activities within the T cell, such as inhibiting proliferation, inducing senescence and / or anergy, inhibiting cytokine production, and other activities of the T cell.

[0251] In another embodiment, CLEAR comprises a costimulatory domain. Costimulatory domains derived from molecules including, but not limited to, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 specific binding ligand. Other costimulatory elements are within the scope of the present invention.

[0252] costimulatory molecules In one embodiment, the modified T cells of the present invention further comprise a 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 yet another embodiment, the costimulatory molecule is CD3, and CD3 comprises at least two different CD3 chains, such as the CD3 zeta chain and the CD3 epsilon chain. In an exemplary embodiment, RNA encoding a costimulatory molecule, such as CD3, is electroporated into the T cell or cells. In another embodiment, a nucleic acid encoding a costimulatory molecule, such as CD3 RNA, is co-electroporated with another nucleic acid, such as a nucleic acid or RNA encoding an affinity molecule chimeric receptor.

[0253] In another embodiment, the TCR is modified to include a costimulatory domain selected from at least one domain from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.

[0254] 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)).

[0255] 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.

[0256] 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).

[0257] 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.

[0258] 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.

[0259] In one embodiment, a nucleic acid encoding a T cell receptor (TCR) that contains affinity for an antigen on a target cell is introduced into a T cell. The nucleic acid can be introduced by any means, such as transduction of expanded T cells, transfection of expanded T cells, and electroporation of expanded T cells.

[0260] In another embodiment, the nucleic acid encoding the affinity molecule chimeric receptor or bispecific affinity molecule is introduced by a method selected from the group consisting of transduction of a cell population, transfection of a cell population, and electroporation of a cell population.

[0261] In another embodiment, a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell is placed into the cell population by a method selected from the group consisting of transduction of the cell population, transfection of the cell population, and electroporation of the cell population.

[0262] In yet another embodiment, nucleic acids encoding bispecific antibodies, such as BiTE molecules, are introduced into T cells. 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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)).

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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)).

[0275] 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.

[0276] 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.

[0277] 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 chimeric receptor mRNAs with different structures and their domain combinations. For example, varying the intracellular effector / costimulatory domains on multiple chimeric receptors in the same cell allows the determination of receptor combination structures that evaluate the highest level of cytotoxicity against multiple antigen targets and the lowest cytotoxicity against normal cells.

[0278] 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.

[0279] 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.

[0280] Several IVT vectors are known in the literature and are used in a standardized manner as templates for in vitro transcription, genetically engineered to produce stabilized RNA transcripts. Currently, protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter 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.

[0281] 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.

[0282] 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.

[0283] In some embodiments, RNA encoding a TCR is electroporated into cells. In one embodiment, the RNA encoding a TCR is in vitro transcribed RNA. In some embodiments, mRNA encoding a bispecific antibody is electroporated into cells. In another embodiment, the mRNA encoding a bispecific antibody is in vitro transcribed mRNA.

[0284] In some embodiments, RNA encoding the bispecific antibody is electroporated into cells. In one embodiment, the RNA encoding the bispecific antibody is in vitro transcribed RNA.

[0285] In some embodiments, RNA encoding the affinity molecule chimeric receptor or bispecific affinity molecule is electroporated into the cell. In one embodiment, the RNA encoding the affinity molecule chimeric receptor or bispecific affinity molecule is in vitro transcribed RNA.

[0286] In one embodiment, the method includes electroporating RNAs encoding the TCR alpha and beta chains. The TCR alpha and beta chains can be encoded on the same or separate RNAs, such as by co-electroporating an RNA encoding the TCR alpha chain and a separate RNA encoding the TCR beta chain. When the alpha and beta are encoded by separate RNAs, the RNAs can be co-electroporated.

[0287] In some embodiments, the method further comprises electroporating a nucleic acid encoding the bispecific antibody or BiTE molecule. The bispecific antibody nucleic acid may be co-electroporated with the TCR RNA.

[0288] 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.

[0289] In one embodiment, the method includes electroporating an RNA encoding the affinity molecule chimeric receptor. In another embodiment, the method further includes electroporating an RNA encoding a costimulatory molecule, such as CD3. The affinity molecule chimeric receptor and the costimulatory molecule can be encoded on the same RNA or separate RNAs. When the affinity molecule chimeric receptor and the costimulatory molecule are encoded by separate RNAs, the RNAs can be co-electroporated.

[0290] In another embodiment, the method comprises electroporating a nucleic acid encoding the bispecific affinity molecule. A costimulatory molecule nucleic acid can also be co-electroporated with the bispecific affinity molecule nucleic acid.

[0291] Chimeric membrane proteins In one embodiment, the modified T cells are expanded before introducing the nucleic acid. In another embodiment, the modified T cells are expanded before electroporating with RNA encoding a TCR, bispecific antibody, or BiTE molecule. Expanding the T cells can include electroporating T cells with RNA encoding a chimeric membrane protein and culturing the electroporated T cells. The chimeric membrane proteins of the present invention comprise an extracellular and an intracellular domain. The extracellular domain comprises a target-specific binding element, such as an antibody. In one embodiment, the extracellular domain of the chimeric membrane protein targets a molecule on T cells, including, but not limited to, TCR, CD3, CD28, etc.

[0292] Extracellular domain The present invention includes an extracellular domain comprising an antigen-binding domain comprising an antibody or fragment thereof directed against a molecule on a T cell. This molecule can include any molecule that costimulates T cells, such as, but not limited to, TCR, CD3, CD28, or a combination thereof. In one embodiment, the extracellular domain can comprise an antigen-binding domain comprising the CD3-binding domain of an anti-CD3 antibody, an anti-TCR antibody, an anti-CD28 antibody, or a combination thereof.

[0293] In another embodiment, the extracellular domain can comprise any fragment of an antibody that binds to an antigen, including, but not limited to, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, a single-chain variable fragment, and the antigen-binding domain of a fragment thereof. In some cases, it is beneficial for the extracellular domain to be derived from the same species as the chimeric membrane protein will ultimately be used in. For example, when used in humans, 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 comprises a human antibody or a fragment thereof.

[0294] In one embodiment, the antibody is a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, a single chain variable fragment, and antigen-binding fragments thereof.

[0295] 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 that is neither an antigen receptor nor its ligand, but is required for an efficient lymphocyte response to an antigen.

[0296] The cytoplasmic domain or intracellular signaling domain of the chimeric membrane protein is involved in activating at least one effector function of a T cell. The term "effector function" refers to a specialized function of a cell. For example, a T cell effector function can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and causes the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be utilized, although in many cases, it is not necessary to use the entire chain. As long as 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. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0297] Non-limiting examples of intracellular signaling domains for use in chimeric membrane proteins include any fragment of the intracellular domain of CD28, 4-1BB, T cell receptor (TCR), costimulatory molecules, any derivative or variant of these sequences, any synthetic sequence having the same functional capability, and any combination thereof.

[0298] Other domains of chimeric membrane proteins A spacer domain may be incorporated between the extracellular and transmembrane domains of a chimeric membrane protein, or between the cytoplasmic and transmembrane domains of a chimeric membrane protein. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular domain or the cytoplasmic domain in a polypeptide chain. A spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0299] 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 mRNA encoding the chimeric membrane protein further comprises a transmembrane domain and a hinge domain, such as a CD28 transmembrane domain and a CD8 alpha hinge domain.

[0300] Human antibodies For in vivo use of antibodies in humans, it may be preferable to use human antibodies. Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be produced by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications 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. Human antibodies can also be antibodies whose heavy and light chains are encoded by nucleotide sequences derived from one or more sources of human DNA.

[0301] Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, 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, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to 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 invention. Antibodies directed against the target of choice 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 (gamma 1) 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 and 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.) may be engaged to provide human antibodies against a selected antigen using technology similar to that described above. For specific discussions regarding the transfer of human germ-line immunoglobulin gene arrays in germ-line mutant mice that 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).

[0302] 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)). Using phage display technology (McCafferty et al., Nature, 348:552-553 (1990)), human antibodies and antibody fragments can be produced in vitro from immunoglobulin variable (V) domain gene repertoires derived from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a 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 an 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, e.g., 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 wide variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of unimmunized mice.Repertoires of V genes from unimmunized human donors can be constructed, and antibodies to a wide variety of antigens (including self-antigens) can be isolated essentially according to the techniques described by 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.

[0303] 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)).

[0304] humanized antibodies Alternatively, in some embodiments, non-human antibodies are humanized, in which particular sequences or regions of the antibody are modified to increase their similarity to antibodies naturally produced in humans, hi one embodiment, the antigen-binding domain is humanized.

[0305] A "humanized" antibody retains the same antigen specificity as the original antibody. However, using certain methods of humanization, the binding affinity and / or specificity of the antibody to the human CD3 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.

[0306] A humanized antibody has one or more amino acid residues introduced into it from a source that is not human. These non-human amino acid residues are often referred to as "import" residues, which 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 can be performed by CDR grafting, essentially replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; (See, e.g., US Pat. No. 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 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.

[0307] The selection of human variable domains, both light and heavy, used to create humanized antibodies is intended 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 accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entireties).

[0308] 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 the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] In another embodiment, the T cells are isolated from cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, the methods described herein further comprise isolating the population of T cells from peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, or T cell lines.

[0317] T cell expansion In one embodiment, expanding the T cells further comprises culturing the electroporated T cells. In another embodiment, the source of the electroporated and expanded T cells is peripheral blood mononuclear cells.

[0318] 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 electroporated T cells, comprising culturing an electroporated population, wherein the electroporated T cells within the population expand at least 10-fold. Expression of the chimeric membrane protein allows interactions with other cells within the population to stimulate and activate the expansion of the electroporated T cells. In one embodiment, at least one cell in the cell population expresses CD3. Without being bound by any particular theory, a cell expressing CD3 may contact and bind to the chimeric membrane protein expressed on the surface of the electroporated cell. At least one cell expressing the chimeric membrane protein may interact with another cell expressing CD3. This interaction may stimulate the expansion of the electroporated T cells.

[0319] Alternatively, cells can be expanded ex vivo using the methods described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 can be an alternative to or in addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors, such as those described in U.S. Patent No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-2, IL-3, and c-kit ligand, in the case of rapid expansion protocols (REP), such as those described in Dudley et al., J. Immunol., 26(4):332-342, 2003. In one embodiment, expanding T cells includes culturing T cells with factors selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand.

[0320] As demonstrated by the data disclosed herein, expansion of electroporated T cells by the methods disclosed herein can be by approximately 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 or partial integers therebetween. In one embodiment, T cells are expanded within the range of about 20x to about 50x.

[0321] After culturing, the T cells can be incubated in the cell culture medium in the culture device for a set period of time, or until the cells reach confluence or a high cell density for optimal passaging before passing the cells to another culture device. The culture device can be any culture device commonly used for culturing cells in vitro. The set period of time can be any time suitable for culturing cells in vitro. The T cell medium can be replaced at any time during the culture of the T cells. Preferably, the T cell medium is replaced approximately every 2-3 days. The T cells are then harvested from the culture device, and the T cells can be used immediately or cryopreserved and stored for later use. In one embodiment, the present invention includes cryopreserving the expanded T cells. The cryopreserved, expanded T cells are then thawed prior to electroporation with RNA. In another embodiment, the cryopreserved T cells are thawed prior to introducing a nucleic acid into the T cells.

[0322] In one aspect, the method of expanding T cells can further include electroporation prior to isolating the T cells followed by culturing. In another embodiment, the invention further includes cryopreserving the expanded T cells. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding a bispecific antibody or BiTE molecule. In yet another embodiment, the cryopreserved T cells are thawed for transfection with an affinity molecule chimeric receptor or bispecific affinity molecule nucleic acid. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding a TCR.

[0323] The culturing steps described herein (contacting with an agent described herein) 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 steps described further herein (contacting 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.

[0324] 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.

[0325] 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.

[0326] 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).

[0327] 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.

[0328] 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, where...

Claims

1. 1. An engineered T cell comprising an exogenous nucleic acid encoding a T cell receptor (TCR) comprising affinity for an antigen on a target cell, and electroporated RNA encoding a bispecific antibody, wherein the T cell expresses the TCR and bispecific antibody on its surface.

2. The modified T cell of claim 1, wherein the TCR comprises at least one disulfide bond.

3. The modified T cell of claim 1 , wherein the TCR comprises a TCR alpha chain and a beta chain.

4. The modified T cell of claim 3, wherein the TCR comprises a costimulatory signaling domain at the C-terminus of at least one of the chains.

5. The modified T cell of claim 4, wherein the costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

6. The modified T cell of claim 3, wherein the beta chain comprises at least one N-deglycosylation.

7. The modified T cell of claim 3, wherein the alpha chain comprises at least one N-deglycosylation.

8. The modified T cell of claim 1, wherein the TCR comprises at least one mouse constant region.

9. The modified T cell of claim 1, wherein the TCR has a higher affinity for a target cell antigen than a wild-type TCR.

10. 2. The modified T cell of claim 1, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

11. 2. The engineered T cell of claim 1, wherein the bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof.

12. 12. The modified T cell of claim 11, wherein the bispecific antigen-binding domain comprises a first and a second single-chain variable fragment (scFv) molecule.

13. The modified T cell of claim 12, wherein the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on an activated T cell.

14. 14. The modified T cell of claim 13, wherein the activating T cell antigen is selected from the group consisting of CD3, CD4, CD8, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, TCR, PD1 and PD1L.

15. The modified T cell of claim 1, further comprising an electroporated nucleic acid encoding a costimulatory molecule.

16. 16. The modified T cell of claim 15, wherein the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.

17. 1. A method for making a modified T cell, comprising introducing into a T cell capable of expressing the TCR and the bispecific antibody a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for an antigen on a target cell and a nucleic acid encoding the bispecific antibody.

18. 18. The method of claim 17, 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.

19. 18. The method of claim 17, wherein the nucleic acid comprises in vitro transcribed RNA or synthetic RNA.

20. 20. The method of claim 17, further comprising expanding the T cells.

21. 21. The method of claim 20, wherein the expanding step comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-2, IL-3, and c-kit ligand.

22. 21. The method of claim 20, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells.

23. The method of claim 22, wherein the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB.

24. 18. The method of claim 17, further comprising cryopreserving the T cells.

25. 25. The method of claim 24, further comprising thawing cryopreserved T cells prior to introducing said nucleic acid into said T cells.

26. 18. The method of claim 17, wherein the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha chain and a TCR beta chain.

27. 27. The method of claim 26, wherein the step of introducing nucleic acids comprises co-electroporating an RNA encoding a TCR alpha chain and a separate RNA encoding a TCR beta chain.

28. 18. The method of claim 17, further comprising electroporating the T cells with RNA encoding CD3.

29. 29. The method of claim 28, wherein the CD3 RNA is co-electroporated with the TCR nucleic acid.

30. 18. The method of claim 17, further comprising cryopreserving the T cells after introducing the TCR nucleic acid.

31. 18. The method of claim 17, further comprising expressing the bispecific antibody as a membrane protein.

32. 18. The method of claim 17, further comprising cryopreserving the bispecific antibody-transduced T cells.

33. 10. Use of the T cells of claim 1 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

34. an effective amount of electroporated modified T cells comprising RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody; to the subject, wherein the modified T cell expresses the modified TCR and the bispecific antibody.

35. 35. The method of claim 34, further comprising inducing lysis of the target cell or tissue.

36. 36. The method of claim 35, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

37. 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells that have been electroporated with RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody to prevent or treat an immune response deleterious to the subject.

38. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells that have been electroporated with RNA encoding a modified T cell receptor (TCR) and RNA encoding a bispecific antibody.

39. 10. 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 claim 1.

40. 40. The method of claim 39, wherein the condition is an autoimmune disease.

41. Autoimmune diseases include acquired immune deficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, inner ear autoimmune disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, and celiac sprue-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, polychondritis, 41. The method of claim 40, wherein the disease is selected from the group consisting of 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.

42. 40. The method of claim 39, wherein the condition is cancer.

43. 43. The method of claim 42, 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.

44. A composition comprising the modified T cells of claim 1.

45. 10. A pharmaceutical composition comprising the modified T cells of claim 1 and a pharmaceutically acceptable carrier.

46. 1. An engineered T cell comprising a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and an antigen on a T cell, and a nucleic acid encoding a chimeric ligand engineered activation receptor (CLEAR), wherein the T cell expresses the bispecific antibody and CLEAR.

47. The modified T cell of claim 46, wherein CLEAR comprises an intracellular activation domain and an extracellular domain.

48. The modified T cell of claim 46, wherein the intracellular activation domain comprises a portion of the intracellular activation domain of CD3 zeta.

49. 47. The modified T cell of claim 46, wherein the extracellular domain is selected from the group consisting of an antigen-binding domain of an antibody, a ligand-binding domain of a receptor, an antigen, and a ligand.

50. 47. The modified T cell of claim 46, wherein the extracellular domain is selected from the group consisting of CD27, CD28, CD70, CD80, PD1 and PD-L1.

51. 47. The modified T cell of claim 46, wherein the extracellular domain is capable of binding to a tumor antigen.

52. The modified T cell of claim 46, wherein CLEAR further comprises a costimulatory domain.

53. 53. The modified T cell of claim 52, wherein the costimulatory domain is selected from the group consisting of CD4, CD8 and 4-1BB.

54. 47. The modified T cell of claim 46, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

55. 47. The modified T cell of claim 46, wherein the bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof.

56. 56. The modified T cell of claim 55, wherein the bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules.

57. 57. The modified T cell of claim 56, wherein the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on a T cell.

58. 47. The modified T cell of claim 46, wherein the bispecific antibody comprises a bispecific for an antigen on a target cell and for CLEAR on the T cell.

59. 47. The modified T cell of claim 46, further comprising a nucleic acid encoding a costimulatory molecule.

60. 59. The modified T cell of claim 58, wherein the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.

61. 46. ​​Use of the T cell of claim 45 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

62. 1. A method for making an engineered T cell, comprising introducing into a T cell a nucleic acid encoding a bispecific antibody and a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR), wherein the T cell is capable of expressing the bispecific antibody and CLEAR.

63. 63. The method of claim 62, wherein at least one of the nucleic acids is introduced by a method selected from the group consisting of transduction of T cells, transfection of T cells, and electroporation of T cells.

64. 63. The method of claim 62, 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.

65. 63. The method of claim 62, wherein at least one of said nucleic acids comprises in vitro transcribed or synthetic RNA.

66. 63. The method of claim 62, further comprising expanding the T cells.

67. 67. The method of claim 66, wherein the expanding step comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-2, IL-3 and c-kit ligand.

68. 67. The method of claim 66, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells.

69. The method of claim 68, wherein the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB.

70. 63. The method of claim 62, further comprising cryopreserving the T cells.

71. 71. The method of claim 70, further comprising thawing cryopreserved T cells prior to introducing said nucleic acid into said T cells.

72. The method of claim 62, further comprising cryopreserving the T cells after introducing the CLEAR nucleic acid.

73. 63. The method of claim 62, further comprising expressing the bispecific antibody as a membrane protein.

74. 63. The method of claim 62, further comprising cryopreserving the bispecific antibody-transduced T cells.

75. 1. An engineered T cell comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on a T cell, wherein the engineered T cell expresses the CLEAR and the bispecific antibody in an effective amount. A method for stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising administering to the subject

76. 76. The method of claim 75, further comprising inducing lysis of the target cell or tissue.

77. 77. The method of claim 76, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

78. 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells, the modified T cells comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell, to prevent or treat an immune response deleterious to the subject.

79. 1. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding a chimeric ligand-modified activating receptor (CLEAR) and a nucleic acid encoding a bispecific antibody having dual specificity for an antigen on a target cell and for CLEAR on the T cell.

80. 47. A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cell of claim 46.

81. 81. The method of claim 80, wherein the condition is an autoimmune disease.

82. 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 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, polychondritis, 82. The method of claim 81, wherein the disease is selected from the group consisting of 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.

83. 81. The method of claim 80, wherein the condition is cancer.

84. 84. The method of claim 83, 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.

85. 47. A composition comprising the modified T cell of claim 46.

86. 47. A pharmaceutical composition comprising the modified T cell of claim 46 and a pharmaceutically acceptable carrier.

87. A modified T cell comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell, wherein the T cell expresses the affinity molecule chimeric receptor.

88. 88. The modified T cell of claim 87, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

89. The modified T cell of claim 87, wherein the small molecule extracellular domain comprises a helical structure lacking disulfide bridges.

90. 88. The modified T cell of claim 87, wherein the small extracellular domain is less than about 10 kD.

91. The modified T cell of claim 87, wherein the affinity molecule chimeric receptor further comprises an intracellular signaling domain.

92. 92. The modified T cell of claim 91, wherein the intracellular signaling domain is a CD3 signaling domain.

93. The modified T cell of claim 87, wherein the affinity molecule chimeric receptor further comprises a costimulatory signaling domain.

94. The modified T cell of claim 93, wherein said costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

95. The modified T cell of claim 87, wherein the affinity molecule chimeric receptor further comprises a transmembrane domain.

96. 96. The modified T cell of claim 95, wherein the transmembrane domain is a CD8 transmembrane domain.

97. The modified T cell of claim 87, wherein the affinity molecule chimeric receptor further comprises a TCR variable domain and a TCR constant domain.

98. 88. The modified T cell of claim 87, further comprising a nucleic acid encoding a costimulatory molecule.

99. 99. The modified T cell of claim 98, wherein the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.

100. The modified T cell of claim 99, wherein the CD3 comprises at least two different CD3 chains.

101. The modified T cell of claim 100, wherein the different CD3 chains are CD3 zeta chain and CD3 epsilon chain.

102. 1. An engineered cell expressing a bispecific affinity molecule, the cell comprising a nucleic acid encoding the bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

103. The modified cell of claim 102, wherein the affinity domain capable of binding to a target cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antibody antigen-binding domain.

104. The modified cell of claim 102, wherein the affinity domain capable of binding to an activating T cell antigen is selected from the group consisting of a small molecule antigen-binding domain and an antigen-binding domain of an antibody.

105. The modified cell of claim 102, wherein the small molecule antigen-binding domain comprises a helical structure lacking disulfide bridges.

106. The modified cell of claim 102, wherein each of said small molecule antigen-binding domains is less than about 10 kD.

107. The modified cell of claim 102, wherein the target cell antigen is selected from the group consisting of a tumor-associated antigen (TAA), a bacterial antigen, a parasitic antigen, a viral antigen, and any fragment thereof.

108. The modified cell of claim 102, wherein the activating T cell antigen is a costimulatory molecule selected from the group consisting of CD3, CD4, CD8, 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, and CD83 specific binding ligand, and any fragment thereof.

109. 88. The modified cell of claim 87 for use in a method of treating an immune response in a subject in need thereof.

110. 103. The modified cell of claim 102 for use in a method of treating an immune response in a subject in need thereof.

111. 103. The modified cell of claim 102, wherein the modified cell is selected from the group consisting of a T cell, a B cell, a natural killer cell, and an antigen-presenting cell.

112. A method for producing modified T cells, comprising introducing into a population of T cells capable of expressing an affinity molecule chimeric receptor a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell.

113. 113. The method of claim 112, wherein said nucleic acid is introduced by a method selected from the group consisting of transduction of a population of T cells, transfection of a population of T cells, and electroporation of a population of T cells.

114. 116. The method of claim 115, wherein the introducing of the nucleic acid comprises electroporating RNA encoding the affinity molecule chimeric receptor.

115. The method of claim 114, further comprising electroporating the T cell with RNA encoding CD3.

116. The method of claim 115, wherein the CD3 RNA is co-electroporated with a nucleic acid encoding an affinity molecule chimeric receptor.

117. 113. The method of claim 112, 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.

118. 113. The method of claim 112, further comprising cryopreserving the T cells after introducing the affinity molecule chimeric receptor nucleic acid.

119. 113. The method of claim 112, further comprising expanding the T cells.

120. 120. The method of claim 119, wherein the expanding step comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-2, IL-3 and c-kit ligand.

121. 120. The method of claim 119, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells.

122. The method of claim 121, wherein the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB.

123. 113. The method of claim 112, further comprising cryopreserving the T cells.

124. 124. The method of claim 123, further comprising thawing cryopreserved T cells prior to introducing the affinity molecule chimeric receptor nucleic acid into the T cells.

125. 1. A method for generating engineered cells that express a bispecific affinity molecule, comprising introducing into a population of cells a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

126. 126. The method of claim 125, wherein said nucleic acid is introduced by a method selected from the group consisting of transduction of a population of cells, transfection of a population of cells, and electroporation of a population of cells.

127. 126. The method of claim 125, wherein the population of cells comprises T cells, B cells, natural killer cells, or antigen-presenting cells.

128. 126. The method of claim 125, further comprising combining the activated T cells and the target cells with a bispecific affinity molecule.

129. 104. Use of the modified T cell of claim 87 or the modified cell of claim 102 in the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

130. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell to prevent or treat an immune response harmful to the subject.

131. 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

132. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on a target cell.

133. 1. A method for treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

134. 104. A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the T cell of claim 87 or the modified cell of claim 102.

135. 135. The method of claim 134, wherein the condition is an autoimmune disease.

136. 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 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, polychondritis, polyarteritis nodosa ...

136. The method of claim 135, wherein the disease is selected from the group consisting of glandular 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.

137. 135. The method of claim 134, wherein the condition is cancer.

138. 138. The method of claim 137, 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.

139. 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 comprising a nucleic acid encoding an affinity molecule chimeric receptor comprising a small molecule extracellular domain having affinity for an antigen on the target cell.

140. 1. 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 cells comprising a nucleic acid encoding a bispecific affinity molecule comprising an affinity domain capable of binding to an antigen on a target cell and an affinity domain capable of binding to an antigen on an activated T cell, wherein at least one affinity domain comprises a small molecule antigen-binding domain.

141. 141. The method of any one of claims 139 or 140, further comprising inducing lysis of the target cell or tissue.

142. The method of any one of claims 139 or 140, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

143. 104. A composition comprising the modified T cell of claim 87 or the modified cell of claim 102.

144. 104. A pharmaceutical composition comprising the modified T cell of claim 87 or the modified cell of claim 102 and a pharmaceutically acceptable carrier.

145. 1. A modified T cell comprising electroporated RNA encoding a bispecific T-cell engager (BiTE) molecule, wherein the BiTE molecule comprises a dual specificity for an antigen on a target cell and an antigen on an activated T cell selected from the group consisting of CD3, CD4, CD8, and TCR.

146. The modified T cell of claim 145, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

147. 146. The modified T cell of claim 145, wherein said bispecific antibody comprises a bispecific antigen-binding domain selected from the group consisting of a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof.

148. The modified T cell of claim 147, wherein said bispecific antigen-binding domain comprises first and second single-chain variable fragment (scFv) molecules.

149. The modified T cell of claim 148, wherein the first scFv molecule is specific for at least one antigen on a target cell and the second scFv molecule is specific for an antigen on an activated T cell.

150. Expanding the population of T cells; and Electroporating the expanded T cells with RNA encoding the bispecific antibody wherein electroporated T cells are capable of expressing the bispecific antibody.

151. 151. The method of claim 150, 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.

152. 151. The method of claim 150, wherein said RNA comprises in vitro transcribed RNA or synthetic RNA.

153. The method of claim 450, wherein the expanding step comprises culturing the T cells with an agent selected from the group consisting of flt3-L, IL-1, IL-2, IL-3 and c-kit ligand.

154. The method of claim 150, wherein the expansion comprises electroporating the T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells.

155. The method of claim 154, wherein the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB.

156. The method of claim 150, further comprising cryopreserving the expanded T cells.

157. The method of claim 156, further comprising thawing the cryopreserved T cells for electroporation with RNA encoding the bispecific antibody.

158. 151. The method of claim 150, further comprising expressing the bispecific antibody as a membrane protein.

159. The method of claim 150, further comprising cryopreserving the bispecific antibody-electroporated T cells.

160. 1. 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 comprising electroporated RNA encoding a bispecific T cell-enhancing (BiTE) molecule comprising dual specificities for an antigen on the target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8 and TCR.

161. 161. The method of claim 160, further comprising inducing lysis of the target cell or tissue containing the target cell.

162. 161. The method of claim 160, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

163. 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells to prevent or treat an immune response deleterious to the subject, wherein the modified T cells have been expanded and electroporated with RNA encoding a bispecific T cell inducer (BiTE) molecule having dual specificity for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR.

164. 1. A method of treating a disease or condition associated with immune enhancement in a subject comprising administering to a subject in need thereof a population of modified T cells, wherein the modified T cells have been expanded and electroporated with RNA encoding a bispecific T cell-enhancing (BiTE) molecule having dual specificities for an antigen on a target cell and an antigen on activated T cells selected from the group consisting of CD3, CD4, CD8, and TCR.

165. 146. A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cell of claim 145.

166. The method of claim 165, wherein the immune response is an autoimmune disease.

167. 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 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, polychondritis, polyarteritis nodosa ...

167. The method of claim 166, wherein the disease is selected from the group consisting of glandular 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.

168. 166. The method of claim 165, wherein the condition is cancer.

169. 169. The method of claim 168, 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.

170. 146. Use of the modified T cell of claim 145 in the manufacture of a medicament for treating an immune response in a subject in need thereof.

171. 146. A composition comprising the modified T cell of claim 145.

172. 146. A pharmaceutical composition comprising the modified T cell of claim 145 and a pharmaceutically acceptable carrier.

173. 1. A modified T cell comprising: an exogenous nucleic acid encoding a T cell receptor (TCR) having affinity for a surface antigen on a target cell; and a nucleic acid encoding a costimulatory molecule, wherein the T cell expresses the TCR and the costimulatory molecule.

174. The modified T cell of claim 173, wherein said TCR comprises at least one disulfide bond.

175. The modified T cell of claim 173, wherein said TCR comprises a TCR alpha chain and a beta chain.

176. The modified T cell of claim 175, wherein said TCR comprises a costimulatory signaling domain at the C-terminus of at least one of said chains.

177. The modified T cell of claim 176, wherein said costimulatory signaling domain is a 4-1BB costimulatory signaling domain.

178. The modified T cell of claim 175, wherein said beta chain comprises at least one N-deglycosylation.

179. The modified T cell of claim 175, wherein said alpha chain comprises at least one N-deglycosylation.

180. The modified T cell of claim 173, wherein said TCR comprises at least one mouse constant region.

181. The modified T cell of claim 173, wherein the nucleic acid encoding the costimulatory molecule is electroporated into the T cell.

182. The modified T cell of claim 181, wherein the costimulatory molecule is selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.

183. The modified T cell of claim 182, wherein the CD3 comprises at least two different CD3 chains.

184. The modified T cell of claim 183, wherein said different CD3 chains are CD3 zeta chain and CD3 epsilon chain.

185. The modified T cell of claim 173, wherein said TCR has a higher affinity for a target cell antigen than a wild-type TCR.

186. The modified T cell of claim 173, wherein the target cell antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, and any fragment thereof.

187. introducing into the T cell a nucleic acid encoding a T cell receptor (TCR) that has affinity for a surface antigen on a target cell; and introducing nucleic acids encoding costimulatory molecules into T cells wherein said T cell is capable of expressing said TCR and a costimulatory molecule.

188. The method of claim 187, wherein at least one of said nucleic acids is introduced by a method selected from the group consisting of transduction of T cells, transfection of T cells, and electroporation of T cells.

189. The method of claim 187, 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.

190. 188. The method of claim 187, wherein at least one of said nucleic acids comprises in vitro transcribed or synthetic RNA.

191. The method of claim 187, further comprising expanding the T cells.

192. 192. The method of claim 191, wherein the expanding step comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-2, IL-3 and c-kit ligand.

193. 192. The method of claim 191, wherein the expanding step comprises electroporating T cells with RNA encoding the chimeric membrane protein and culturing the electroporated T cells.

194. The method of claim 193, wherein the chimeric membrane protein comprises a single-chain variable fragment (scFv) against CD3 and an intracellular domain comprising fragments of the intracellular domains of CD28 and 4-1BB.

195. The method of claim 187, further comprising cryopreserving the T cells.

196. The method of claim 195, further comprising thawing cryopreserved T cells prior to introducing said TCR-encoding nucleic acid into said T cells.

197. The method of claim 187, wherein the nucleic acid encoding the TCR comprises a nucleic acid encoding a TCR alpha chain and a TCR beta chain.

198. 200. The method of claim 198, wherein said introducing nucleic acid step comprises co-electroporating an RNA encoding a TCR alpha chain and a separate RNA encoding a TCR beta chain.

199. The method of claim 187, wherein the step of introducing a nucleic acid encoding a costimulatory molecule comprises electroporating RNA encoding CD3 into the T cell.

200. 200. The method of claim 199, wherein the CD3 RNA is co-electroporated with the TCR nucleic acid.

201. The method of claim 187, further comprising cryopreserving the T cells after introducing the TCR nucleic acid.

202. 174. Use of the modified T cell of claim 173 in the manufacture of a medicament for treating an immune response in a subject in need thereof.

203. 1. 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, wherein the T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) having affinity for a surface antigen on the target cell.

204. The method of claim 203, further comprising inducing lysis of the target cell or tissue.

205. The method of claim 204, wherein the lysis induced is antibody-dependent cell-mediated cytotoxicity (ADCC).

206. 1. A method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified T cells to prevent or treat an immune response deleterious to the subject, wherein the modified T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) having affinity for a surface antigen on a target cell.

207. 1. A method of treating a disease or condition associated with immune enhancement in a subject, comprising administering to a subject in need thereof a population of modified T cells, wherein the modified T cells have been expanded and electroporated with RNA encoding a modified T cell receptor (TCR) having affinity for a surface antigen on a target cell.

208. 174. A method of treating a condition in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified T cell of claim 173.

209. The method of claim 208, wherein the immune response is an autoimmune disease.

210. 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 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, polychondritis, polyarteritis nodosa ...

209. The method of claim 209, wherein the disease is selected from the group consisting of 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.

211. 209. The method of claim 208, wherein the condition is cancer.

212. 212. The method of claim 211, 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.

213. 174. A composition comprising the modified T cell of claim 173.

214. 174. A pharmaceutical composition comprising the modified T cell of claim 173 and a pharmaceutically acceptable carrier.

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