Bispecific chimeric antigen receptors and their therapeutic use

Abstract

To provide immunotherapy that prevents or minimizes the failure of immunotherapy treatment due to the emergence of antigen-loss escape mutations in cancer cells, etc. [Solution] A bispecific chimeric antigen receptor comprising (a) an antigen-specific target-directed region containing an antigen-specific single-chain Fv(scFv) fragment that binds to at least two different antigens, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one costimulatory domain, and (e) an intracellular signaling domain, which is to be co-expressed with a therapeutic regulatory substance such as truncated epidermal growth factor receptor (EGFRt).

Description

[Technical Field]

[0001] This invention relates to a chimeric antigen receptor and genetically modified cells using the same. [Background technology]

[0002] Current immunotherapies are designed to target a single antigen on cancer cells. However, cancer cells are unstable, and some cells may no longer possess the target antigen. These cells, known as antigen loss escape variants, can evade therapeutic destruction, continue to grow, and spread unchecked. Therefore, there is a need in this field for therapies that prevent or minimize treatment failure in cancer and other diseases. [Overview of the project]

[0003] In one embodiment, the present invention provides a bispecific chimeric antigen receptor comprising (a) at least two antigen-specific target-directed regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one costimulatory domain, and (e) an intracellular signaling domain, wherein each antigen-specific target-directed region comprises an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen, and wherein the bispecific chimeric antigen receptor is co-expressed with a therapeutic control agent.

[0004] In one embodiment, the present invention further provides a combination of a bispecific chimeric antigen receptor and a therapeutic control agent, wherein the bispecific chimeric antigen receptor comprises (a) at least two antigen-specific target-directed regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one costimulatory domain, and (e) an intracellular signaling domain, wherein each antigen-specific target-directed region comprises an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen.

[0005] In one embodiment, the present invention further provides a bispecific chimeric antigen receptor comprising (a) at least two antigen-specific target-directed regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one costimulatory domain, and (e) an intracellular signaling domain, wherein each antigen-specific target-directed region comprises an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen, and wherein the bispecific chimeric antigen receptor is co-expressed with a truncated epidermal growth factor receptor (EGFRt).

[0006] In one embodiment, the present invention further provides a bispecific chimeric antigen receptor comprising (a) at least two antigen-specific target-directed regions, (b) a CD8α hinge extracellular spacer domain, (c) a CD8α transmembrane domain, (d) a 4-1BB costimulatory domain, and (vi) a CD3ζ intracellular signaling domain, wherein each antigen-specific target-directed region comprises an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen, wherein the bispecific chimeric antigen receptor is co-expressed with EGFRt, and wherein the bispecific chimeric antigen receptor and EGFRt are linked via a T2A linker.

[0007] In one embodiment, a pharmaceutical composition is also provided comprising the above-described bispecific chimeric antigen receptor, a combination of the bispecific chimeric antigen receptor and a therapeutic control substance, a polypeptide encoding the bispecific chimeric antigen receptor, a vector containing the bispecific chimeric antigen receptor, a virus, and genetically modified cells, a vector containing the combination of the bispecific chimeric antigen receptor and a therapeutic control substance, a virus, and genetically modified cells, or a combination thereof, and a pharmaceutically acceptable carrier. [Invention 1001] a. At least two antigen-specific target-directed regions, b. Extracellular spacer domain, c. Transmembrane domain, d. At least one co-stimulatory domain, and e. Intracellular signaling domains A bispecific chimeric antigen receptor containing and co-expressed with a therapeutic control substance, Each antigen-specific target-directed region contains an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen. Bispecific chimeric antigen receptor. [Invention 1002] The bispecific chimeric antigen receptor of the present invention 1001, wherein the therapeutic control substance comprises one or more of the following: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), select marker, and combinations thereof. [Invention 1003] The bispecific chimeric antigen receptor of the present invention 1002, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof. [Invention 1004] The bispecific chimeric antigen receptor of the present invention 1002, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof. [Invention 1005] The bispecific chimeric antigen receptor of the present invention 1002, wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof. [Invention 1006] The bispecific chimeric antigen receptor of the present invention 1001, wherein the bispecific chimeric antigen receptor and the therapeutic control substance are linked via a cleavable linker. [Invention 1007] The bispecific chimeric antigen receptor of the present invention 1006, wherein the cleavable linker is a self-cleavable linker. [Invention 1008] The bispecific chimeric antigen receptor of the present invention 1007, wherein the cleavable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof. [Invention 1009] The bispecific chimeric antigen receptor of the present invention 1001, wherein the extracellular spacer domain comprises one or more of the following: an antibody Fc fragment, or a functional equivalent, fragment, or derivative thereof; an antibody hinge region, or a functional equivalent, fragment, or derivative thereof; an antibody CH2 region; an antibody CH3 region; an artificial spacer sequence; and combinations thereof. [Invention 1010] The bispecific chimeric antigen receptor of the present invention 1009, wherein the extracellular spacer domain comprises one or more of the following: (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 region of IgG4, (iv) the hinge region of CD8α, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, (vi) the hinge and CH2 region of IgG1, or (vii) any combination thereof. [Invention 1011] The bispecific chimeric antigen receptor of the present invention 1001, wherein the transmembrane domain comprises one or more of the transmembrane region of a type I transmembrane protein, an artificial hydrophobic sequence, and combinations thereof. [Invention 1012] The bispecific chimeric antigen receptor of the present invention 1011, wherein the transmembrane domain comprises one or more of the transmembrane domain of the ζ chain of the T cell receptor complex, CD28, CD8α, and combinations thereof. [Invention 1013] The bispecific chimeric antigen receptor of the present invention 1001, wherein the aforementioned co-stimulatory domain comprises a signaling domain derived from one or more of the following: CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof. [Invention 1014] The bispecific chimeric antigen receptor of the present invention 1001, wherein the intracellular signaling domain comprises one or more signaling domains from among human CD3ζ chain, FcγRIII, FcεRI, the cytoplasmic terminal of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), and combinations thereof. [Invention 1015] The bispecific chimeric antigen receptor of the present invention 1001, wherein each of the at least two antigen-specific target-directing domains targets an antigen independently selected from the group consisting of antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof. [Invention 1016] The aforementioned antigens specific to cancer include 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, and human cell dispersion factor (scatter). The bispecific chimeric antigen receptor of the present invention 1015, comprising one or more of the following: factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and any combination thereof. [Invention 1017] The bispecific chimeric antigen receptor of the present invention 1001, wherein the at least two antigen-specific target-directing regions bind to (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. [Invention 1018] The bispecific chimeric antigen receptor of the present invention 1001, wherein the at least two antigen-specific target-directing regions bind to CD19 and CD20. [Invention 1019] The antigens specific to inflammatory diseases include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, and Lama (Lama A bispecific chimeric antigen receptor according to the present invention 1015, comprising one or more of the following: glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, scleroscin, SOST, TGFβ1, TNF-α, VEGF-A, and combinations thereof. [Invention 1020] The bispecific chimeric antigen receptor of the present invention 1015, wherein the antigen specific to neuropathy comprises one or more of β-amyloid, MABT5102A, and combinations thereof. [Invention 1021] The bispecific chimeric antigen receptor of the present invention 1015, wherein the antigen specific to diabetes includes one or more of L-1β, CD3, and combinations thereof. [Invention 1022] The bispecific chimeric antigen receptor of the present invention 1015, wherein the antigen specific to cardiovascular disease comprises one or more of the following: C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β chain, ITGB2 (CD18), sphingosine-1-phosphate, and combinations thereof. [Invention 1023] The bispecific chimeric antigen receptor of the present invention 1015, wherein the antigen specific to infectious diseases comprises one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, and combinations thereof. [Invention 1024] A combination of the bispecific chimeric antigen receptor of the present invention 1001 and a therapeutic control substance. [Invention 1025] The combination of Invention 1024 wherein the therapeutic control substance comprises one or more of the following: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), select marker, and combinations thereof. [Invention 1026] The combination of the present invention 1025 wherein the EGFRt is bound to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof. [Invention 1027] The combination of the present invention 1025 wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof. [Invention 1028] The combination of the present invention 1025 wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof. [Invention 1029] The combination of the bispecific chimeric antigen receptor and the therapeutic control substance of the present invention 1024, wherein the bispecific chimeric antigen receptor and the therapeutic control substance are linked via a cleavable linker. [Invention 1030] The combination of the invention 1029, wherein the aforementioned severable linker is a self-cutting type severable linker. [Invention 1031] The combination of the invention 1029 wherein the severable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof. [Invention 1032] A polynucleotide encoding the bispecific chimeric antigen receptor of Invention 1001 or a combination of Invention 1024. [Invention 1033] A polypeptide encoded by the polynucleotide of the present invention 1032. [Invention 1034] A vector comprising the polynucleotide of the present invention 1032. [Invention 1035] A virus containing the polynucleotide 1032 of the present invention. [Invention 1036] The virus of the present invention 1035, which is an RNA virus. [Invention 1037] The virus of the present invention 1035, which is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus. [Invention 1038] Genetically modified cells comprising a combination of the polynucleotide of Invention 1032, the chimeric antigen receptor of Invention 1001, or Invention 1024. [Invention 1039] A T lymphocyte (T cell), a genetically modified cell according to Invention 1038. [Invention 1040] Genetically modified cells according to Invention 1039, which are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. [Invention 1041] Genetically modified cells according to Invention 1038, which are natural killer (NK) cells, hematopoietic stem cells (HSCs), embryonic stem cells, or pluripotent stem cells. [Invention 1042] a. One or more of the following: the bispecific chimeric antigen receptor of Invention 1001, the combination of Invention 1024, the polypeptide of Invention 1032, the vector of Invention 1034, the virus of Invention 1035, the genetically modified cell of Invention 1038, and any combination thereof. b. Pharmaceutically acceptable carriers and A pharmaceutical composition containing [the specified substance]. [Invention 1043] The pharmaceutical composition of the present invention 1042, A composition adapted to biochemically interact with a therapeutic control substance for inhibiting the proliferation of cells expressing the therapeutic control substance, and A combination. [Invention 1044] The composition adapted to biochemically interact with the therapeutic control substance is Herceptin, methotrexate, cetuximab, thymidine analog (e.g., ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), 6-thioguanine, synthetic dimerizing agent (dimerizing agent). A combination of the present invention 1043, which is one or more of the following: drug (e.g., AP1903), fludarabine phosphate, linamarin (lin), nucleoside analogs (e.g., BVDU, difluorodeoxycytidine (dFdC), 1-β-D-arabinofuranosylthymine (ala-T)), indole-3-acetic acid (IAA), l-butionine-S,R-sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors, or any combination thereof. [Invention 1045] A method for generating a certain amount of T cells expressing a chimeric antigen receptor, comprising the following steps: (i) A step of transfecting one or more T cells with the vector of the present invention 1034, and (ii) A step of stimulating one or more T cells with cells expressing an antigen targeted by at least two antigen-specific target-directing regions, cells expressing a recombinant antigen targeted by at least two antigen-specific target-directing regions, or cells expressing an antibody against a chimeric antigen receptor, thereby causing the T cells to proliferate to produce a certain amount of T cells. [Invention 1046] A method for treating a disease in a person who requires treatment for the disease, The method is (i) A step of providing a composition of the present invention 1042, and (ii) the step of administering a therapeutically effective amount of the composition to the subject in order to treat the disease. Includes, Each of the at least two antigen-specific targeting regions targets an antigen, and at least one such antigen is associated with the disease. The method. [Invention 1047] a. At least two antigen-specific target-directed regions, b. Extracellular spacer domain, c. Transmembrane domain, d. At least one co-stimulatory domain, and e. Intracellular signaling domains A bispecific chimeric antigen receptor that includes and is co-expressed with truncated epidermal growth factor receptor (EGFRt), Each antigen-specific target-directed region contains an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen. Bispecific chimeric antigen receptor. [Invention 1048] The bispecific chimeric antigen receptor of the present invention 1047, which is co-expressed with a therapeutic control substance comprising one or more of the following: thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), select marker, and combinations thereof. [Invention 1049] The bispecific chimeric antigen receptor of the present invention 1047, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof. [Invention 1050] The bispecific chimeric antigen receptor of the present invention 1048, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof. [Invention 1051] The bispecific chimeric antigen receptor of the present invention 1048, wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof. [Invention 1052] The bispecific chimeric antigen receptor of the present invention 1047, wherein the bispecific chimeric antigen receptor and the therapeutic control substance are linked via a cleavable linker. [Invention 1053] The bispecific chimeric antigen receptor of the present invention 1052, wherein the cleavable linker is a self-cleavable linker. [Invention 1054] The bispecific chimeric antigen receptor of the present invention 1052, wherein the cleavable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof. [Invention 1055] The bispecific chimeric antigen receptor of the present invention 1047, wherein the extracellular spacer domain comprises one or more of the following: an antibody Fc fragment, or a functional equivalent, fragment, or derivative thereof; an antibody hinge region, or a functional equivalent, fragment, or derivative thereof; an antibody CH2 region; an antibody CH3 region; an artificial spacer sequence; and combinations thereof. [Invention 1056] The bispecific chimeric antigen receptor of the present invention 1055, wherein the extracellular spacer domain comprises any one or more of the following: (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 region of IgG4, (iv) the hinge region of CD8α, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, (vi) the hinge and CH2 region of IgG1, and (vii) any combination thereof. [Invention 1057] The bispecific chimeric antigen receptor of the present invention 1047, wherein the transmembrane domain comprises one or more of the transmembrane region of a type I transmembrane protein, an artificial hydrophobic sequence, and combinations thereof. [Invention 1058] The bispecific chimeric antigen receptor of the present invention 1057, wherein the transmembrane domain comprises one or more of the transmembrane domain of the ζ chain of the T cell receptor complex, CD28, CD8α, and combinations thereof. [Invention 1059] The bispecific chimeric antigen receptor of the present invention 1047, wherein the aforementioned co-stimulatory domain comprises a signaling domain derived from one or more of the following: CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof. [Invention 1060] The bispecific chimeric antigen receptor of the present invention 1047, wherein the intracellular signaling domain comprises one or more signaling domains from among human CD3ζ chain, FcγRIII, FcεRI, the cytoplasmic terminal of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), and combinations thereof. [Invention 1061] The bispecific chimeric antigen receptor of the present invention 1047, wherein each of the at least two antigen-specific target-directing domains targets an antigen independently selected from the group consisting of antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof. [Invention 1062] The aforementioned antigens specific to cancer include 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, and fibronectin. Extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human cell dispersion factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH A bispecific chimeric antigen receptor according to the present invention 1061, comprising one or more of the following: 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof. [Invention 1063] The bispecific chimeric antigen receptor of the present invention 1047, wherein the at least two antigen-specific target-directing regions bind to (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. [Invention 1064] The bispecific chimeric antigen receptor of the present invention 1047, wherein at least two antigen-specific target-directing regions bind to CD19 and CD20. [Invention 1065] The aforementioned antigens specific to inflammatory diseases are AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE A bispecific chimeric antigen receptor according to Invention 1061, comprising one or more of the following: Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, llama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, sclerossin, SOST, TGFβ1, TNF-α, VEGF-A, and combinations thereof. [Invention 1066] The bispecific chimeric antigen receptor of the present invention 1061, wherein the antigen specific to neuropathy comprises one or more of β-amyloid, MABT5102A, and combinations thereof. [Invention 1067] The bispecific chimeric antigen receptor of the present invention 1061, wherein the antigen specific to diabetes includes one or more of L-1β, CD3, and combinations thereof. [Invention 1068] The bispecific chimeric antigen receptor of the present invention 1061, wherein the antigen specific to cardiovascular disease comprises one or more of the following: C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β chain, ITGB2 (CD18), sphingosine-1-phosphate, and combinations thereof. [Invention 1069] The bispecific chimeric antigen receptor of the present invention 1061, wherein the antigen specific to an infectious disease comprises one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, and combinations thereof. [Invention 1070] A combination of the bispecific chimeric antigen receptor of the present invention 1047 and EGFRt. [Invention 1071] The combination of the present invention 1070 wherein the EGFRt is bound to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof. [Invention 1072] The combination of the present invention 1070, wherein the bispecific chimeric antigen receptor and the EGFRt are linked via a cleavable linker. [Invention 1073] The combination of the present invention 1072, wherein the aforementioned cuttable linker is a self-cutting type cuttable linker. [Invention 1074] A combination of the present invention 1072, wherein the severable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof. [Invention 1075] A polynucleotide encoding the bispecific chimeric antigen receptor of Invention 1047 or a combination of Invention 1070. [Invention 1076] A polypeptide encoded by the polynucleotide of the present invention 1075. [Invention 1077] A vector comprising the polynucleotide of the present invention 1075. [Invention 1078] A virus containing the polynucleotide of the present invention 1075. [Invention 1079] The virus of this invention 1078, which is an RNA virus. [Invention 1080] The virus of the present invention 1078, which is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus. [Invention 1081] Genetically modified cells comprising a polynucleotide of Invention 1075, a chimeric antigen receptor of Invention 1047, or a combination of Invention 1070. [Invention 1082] A T-lymphocyte (T cell), a genetically modified cell according to the present invention 1081. [Invention 1083] Genetically modified cells according to Invention 1082, which are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. [Invention 1084] Genetically modified cells according to Invention 1081, which are natural killer (NK) cells, hematopoietic stem cells (HSCs), embryonic stem cells, or pluripotent stem cells. [Invention 1085] a. One or more of the following: the bispecific chimeric antigen receptor of Invention 1047, the combination of Invention 1070, the polypeptide of Invention 1076, the vector of Invention 1077, the virus of Invention 1078, the genetically modified cell of Invention 1081, and any combination thereof. b. Pharmaceutically acceptable carriers and A pharmaceutical composition containing [the specified substance]. [Invention 1086] The pharmaceutical composition of the present invention 1085, A composition adapted to biochemically interact with a therapeutic control agent for inhibiting the proliferation of EGFRt-expressing cells. A combination. [Invention 1087] The composition adapted to biochemically interact with the therapeutic control substance is herceptin, methotrexate, cetuximab, thymidine analog (e.g., ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), 6-thioguanine, synthetic dimerizer (e.g., AP1903), phosphate f The combination of the present invention 1086, which is one or more of the following: rudarabine, linamarin (lin), nucleoside analogs (e.g., BVDU, difluorodeoxycytidine (dFdC), 1-β-D-arabinofuranosylthymine (ala-T)), indole-3-acetic acid (IAA), l-butionine-S,R-sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors, or combinations thereof. [Invention 1088] A method for generating a certain amount of T cells expressing a chimeric antigen receptor, comprising the following steps: (i) A step of transfecting one or more T cells with the vector of the present invention 1077, and (ii) A step of stimulating one or more T cells with cells expressing an antigen targeted by at least two antigen-specific target-directing regions, cells expressing a recombinant antigen targeted by at least two antigen-specific target-directing regions, or cells expressing an antibody against a chimeric antigen receptor, thereby causing the T cells to proliferate to produce a certain amount of T cells. [Invention 1089] A method for treating a disease in a person who requires treatment for the disease, The method is (i) A step of providing a composition of the present invention 1085, and (ii) the step of administering a therapeutically effective amount of the composition to the subject in order to treat the disease. Includes, Each of the at least two antigen-specific targeting regions targets an antigen, and at least one such antigen is associated with the disease. The method. [Invention 1090] A bispecific chimeric antigen receptor comprising the sequence described in Figure 4, 9, or 11. [Invention 1091] a. At least two antigen-specific target-directed regions, b. CD8α hinge extracellular spacer domain, c.CD8α transmembrane domain, d.4-1BB co-stimulatory domain, and e.CD3ζ intracellular signaling domain A bispecific chimeric antigen receptor that includes and is co-expressed with truncated epidermal growth factor receptor (EGFRt), Each antigen-specific target-directed region contains an antigen-specific single-chain Fv(scFv) fragment and binds to a different antigen. The bispecific chimeric antigen receptor and EGFRt are linked via a T2A linker. Bispecific chimeric antigen receptor. [Invention 1092] The bispecific chimeric antigen receptor of the present invention 1091, which is co-expressed with a therapeutic control substance comprising one or more of the following: thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), select marker, and combinations thereof. [Invention 1093] The bispecific chimeric antigen receptor of the present invention 1091, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof. [Invention 1094] The bispecific chimeric antigen receptor of the present invention 1092, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated-DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof. [Invention 1095] The bispecific chimeric antigen receptor of the present invention 1092, wherein the CCR comprises one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof. [Invention 1096] The bispecific chimeric antigen receptor of the present invention 1091, wherein each of the at least two antigen-specific target-directing domains targets an antigen independently selected from the group consisting of antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof. [Invention 1097] The aforementioned antigens specific to cancer include 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, and fibronectin. Extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human cell dispersion factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH A bispecific chimeric antigen receptor according to Invention 1096, comprising one or more of the following: 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin, and combinations thereof. [Invention 1098] The bispecific chimeric antigen receptor of the present invention 1096, wherein the at least two antigen-specific target-directing regions bind to (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. [Invention 1099] The bispecific chimeric antigen receptor of the present invention 1096, wherein the at least two antigen-specific target-directing regions bind to CD19 and CD20. [Invention 1100] The aforementioned antigens specific to inflammatory diseases are AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE A bispecific chimeric antigen receptor according to Invention 1096, comprising one or more of the following: Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, llama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, sclerossin, SOST, TGFβ1, TNF-α, VEGF-A, and combinations thereof. [Invention 1101] The bispecific chimeric antigen receptor of the present invention 1096, wherein the antigen specific to neuropathy comprises one or more of β-amyloid, MABT5102A, and combinations thereof. [Invention 1102] The bispecific chimeric antigen receptor of the present invention 1096, wherein the antigen specific to diabetes includes one or more of L-1β, CD3, and combinations thereof. [Invention 1103] The bispecific chimeric antigen receptor of the present invention 1096, wherein the antigen specific to cardiovascular disease comprises one or more of the following: C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β chain, ITGB2 (CD18), sphingosine-1-phosphate, and combinations thereof. [Invention 1104] The bispecific chimeric antigen receptor of the present invention 1096, wherein the antigen specific to an infectious disease comprises one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, and combinations thereof. [Brief explanation of the drawing]

[0008] Exemplary embodiments are illustrated in the reference drawings. The embodiments and drawings disclosed herein are intended to be illustrative, not limiting.

[0009] [Figure 1] A schematic diagram of the chimeric antigen receptor of the present invention according to one embodiment of the present invention is shown. ASTR is the antigen-specific target-directed region, L is the linker, ESD is the extracellular spacer domain, TM is the transmembrane domain, CSD is the costimulatory domain, and ISD is the intracellular signaling domain. [Figure 2] (a) components of an anti-CD19xCD20 CAR according to one embodiment of the present invention, and (b) complete cDNA packaged in an epHIV-7 lentiviral vector transfer plasmid. [Figure 3] The nucleic acid sequence of the bispecific CAR CD19scFv-Gly4Ser1 linker-CD20scFv-IgG4 hinge-CD28tm-41BB-CD3ζ-T2A-EGFRt_epHIV7 according to one embodiment of the present invention is shown. [Figure 4-1]The nucleic acid sequence and amino acid sequence of the bispecific CAR CD19scFv-Gly4Ser1 linker-CD20scFv-IgG4 hinge-CD28tm-41BB-CD3ζ-T2A-EGFRt_epHIV7 according to one embodiment of the present invention are shown. [Figure 4-2] This figure is a continuation of Figure 4-1. [Figure 4-3] This figure is a continuation of Figure 4-2. [Figure 5] The CD19scFv-Gly4Ser1 linker-CD20scFv-IgG4 hinge-CD28tm-CD28gg-CD3ζ transgene construct according to one embodiment of the present invention is shown. [Figure 6] This shows the development of a CγCR platform to support exogenous γc-independent growth according to one embodiment of the present invention. (a) Schematic diagram of wild-type versus chimeric cytokine receptor. The IL-7Rα constitutive cytokine receptor (CγCR7) consists of a human IL-7 cytokine linked to a full-length human IL-7Rα chain via a (G4S)2 linker. The IL-2Rβ constitutive cytokine receptor (CγCR2) is identical to CγCR7 except that the IL-7Rα intracellular signaling domain is replaced with a human IL-2 / IL-15Rβ cytoplasmic domain. (b) Diagram of the expression construct CγCR-T2A-CD19t. [Figure 7] The following is an embodiment of the present invention, namely the nucleic acid sequence and amino acid sequence of a main chain CAR comprising the hinge region of IgG4, the transmembrane domain of CD28, the costimulatory domain of 4-1BB, and the cytoplasmic domain of CD3ζ. [Figure 8] The nucleic acid sequence of GMCSFRss-CD19scFv-Gly4Ser linker-CD20scFv-huIgG hinge / CH2 / CH3-CD28tm / CD28cyto-41BB-CD3ζ is shown according to one embodiment of the present invention. GMCSFRss is a signal sequence derived from GMCSFR. [Figure 9-1]The nucleic acid sequence and amino acid sequence of GMCSFRss-CD19scFv-Gly4Ser linker-CD20scFv-huIgG hinge / CH2 / CH3-CD28tm / CD28cyto-41BB-CD3ζ are shown according to one embodiment of the present invention. GMCSFRss is a signal sequence derived from GMCSFR. [Figure 9-2] This figure is a continuation of Figure 9-1. [Figure 9-3] This figure is a continuation of Figure 9-2. [Figure 9-4] This figure is a continuation of Figure 9-3. [Figure 10] The nucleic acid sequence of GMCSFRss-CD19scFv-Gly4Ser linker-CD20scFv-CD8α hinge-CD8αtm-41BB-CD3ζ-T2A-EGFRt is shown according to one embodiment of the present invention. GMCSFRss is a signal sequence derived from GMCSFR. [Figure 11-1] The nucleic acid sequence and amino acid sequence of GMCSFRss-CD19scFv-Gly4Ser linker-CD20scFv-CD8α hinge-CD8αtm-41BB-CD3ζ-T2A-EGFRt are shown according to one embodiment of the present invention. GMCSFRss is a signal sequence derived from GMCSFR. [Figure 11-2] This figure is a continuation of Figure 11-1. [Figure 11-3] This figure is a continuation of Figure 11-2. [Figure 11-4] This figure is a continuation of Figure 11-3. [Figure 12] The nucleic acid sequence of T2A-EGFRt, according to one embodiment of the present invention, is shown below. [Figure 13-1] The nucleic acid sequence and amino acid sequence of T2A-EGFRt are shown according to one embodiment of the present invention. [Figure 13-2] This figure is a continuation of Figure 13-1. [Modes for carrying out the invention]

[0010] All references cited herein are incorporated herein in their entirety by reference as if they were fully specified. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. Singletonet al., Dictionary of Microbiology and Molecular Biology 3 rd ed., J. Wiley & Sons (New York, NY 2001), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5 th The ed., J. Wiley & Sons (New York, NY 2001), and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide those skilled in the art with many general guidelines for the terminology used in this application.

[0011] Those skilled in the art will understand that there are many methods and materials similar or equivalent to those described in the specification that may be used in practice of the present invention. In fact, the present invention is not limited in any way to the methods and materials described. For the purposes of the present invention, the following terms are defined below.

[0012] The inventions described herein provide chimeric antigen receptors, which are modified receptors that transfer immunospecificity to genetically modified cells. The fact that a single chimeric antigen receptor (CAR) possesses specificity for multiple antigens offers various advantages, most notably a significant reduction in effort compared to producing multiple types of T cell products per patient.

[0013] definition Components of chimeric antigen receptors As used herein, “antigen-specific target-directed region” (ASTR) refers to a region of a CAR that targets a specific antigen. The CAR of the present invention comprises at least two target-directed regions that target at least two different antigens. In one embodiment, the CAR comprises three or more target-directed regions that target at least three or more different antigens. The target-directed regions on the CAR are extracellular. In some embodiments, the antigen-specific target-directed region comprises an antibody or its functional equivalent or a fragment or derivative thereof, and each of the target-directed regions targets a different antigen. The target-directed regions may comprise a full-length heavy chain, a Fab fragment, a single-chain Fv(scFv) fragment, a bivalent single-chain antibody, or a diabody, each of which is specific to the target antigen. However, there are numerous alternatives, each of which can be used in various embodiments of the present invention, such as binding cytokines (leading to recognition by cells having cytokine receptors), aphibodies, ligand-binding domains derived from native receptors, receptor-soluble protein / peptide ligands (e.g., on tumor cells), peptides, and vaccines to stimulate an immune response. In fact, as those skilled in the art will understand, almost any molecule that binds to a given antigen with high affinity can be used as an antigen-specific targeting region.

[0014] As used herein, “chimeric antigen receptor” or “CAR” or “CARs” refers to a modified receptor that imparts antigen specificity to a cell (e.g., a T cell such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. The CARs of the present invention comprise at least two antigen-specific target-directed regions, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. The two or more antigen-specific target-directed regions target at least two different antigens, are arranged in series, and may be separated by a linker sequence. In one embodiment, the extracellular spacer domain is optional. In another embodiment, the CAR is a bispecific CAR. A bispecific CAR is specific to two different antigens.

[0015] As used herein, “co-stimulatory domain” (CSD) refers to a portion of a CAR that enhances the proliferation, survival, and / or development of memory cells. The CAR of the present invention may comprise one or more co-stimulatory domains. Each co-stimulatory domain comprises, for example, one or more co-stimulatory domains from the TNFR superfamily, CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or combinations thereof. Other co-stimulatory domains (e.g., derived from other proteins) may be obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0016] As used herein, “extracellular spacer domain” (ESD) refers to the hydrophilic region between the antigen-specific target-directing region and the transmembrane domain. In some embodiments, the CAR of the present invention includes an extracellular spacer domain. In other embodiments, the CAR of the present invention does not include an extracellular spacer domain. The extracellular spacer domain includes, but is not limited to, an antibody Fc fragment or its fragment or derivative, an antibody hinge region or its fragment or derivative, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, the CD8α hinge, as well as artificial spacers made from polypeptides, which may be as small as, for example, Gly3, or the CH1 and CH3 domains of IgG (e.g., human IgG4). In some embodiments, the extracellular spacer domain is one or more of the following: (i) the hinge, CH2, and CH3 regions of IgG4; (ii) the hinge region of IgG4; (iii) the hinge and CH2 regions of IgG4; (iv) the hinge region of CD8α; (v) the hinge, CH2, and CH3 regions of IgG1; (vi) the hinge region of IgG1; or (vi) the hinge and CH2 regions of IgG1. Other extracellular spacer domains are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0017] As used herein, “intracellular signaling domain” (ISD) or “cytoplasmic domain” refers to a portion of the CAR that transmits effector functional signals, instructing the cell to perform its specialized function. Examples of effector functional signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs (e.g., η chain, FcεR1γ and β chain, MB1(Igα) chain, B29(Igβ) chain, etc.), the human CD3ζ chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transmission such as CD2, CD5, and CD28. Other intracellular signaling domains are apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0018] As used herein, “linker” (L), “linker domain,” or “linker region” refers to an oligo or polypeptide region of about 1 to 100 amino acids in length that links together any of the domains / regions of the CAR of the present invention. The linker may consist of flexible residues such as glycine and serine so that adjacent protein domains can move freely from one another. Longer linkers may be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. The linker may or may not be cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or their functional equivalents, and combinations thereof. In some embodiments, linkers include picornavirus 2A-like linkers, the CHYSEL sequence of porcine tesshouvirus (P2A), Thosea asigna virus (T2A), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence results in a cleavage between 2A glycine and 2B proline, as in Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A)- Pro (2B)Motifs may be included. Other linkers are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0019] As used herein, “transmembrane domain” (TMD) refers to a region of a CAR that crosses the cell membrane. The transmembrane domain of a CAR in this invention is a transmembrane region of a transmembrane protein (e.g., type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains are obvious to those skilled in the art and may be used in connection with alternative embodiments of the invention.

[0020] others As used herein, “antigen-eliminating escape variants” refer to cells that exhibit reduced or absent expression of a target antigen, the antigen being targeted by the CAR of the present invention.

[0021] As used herein, “B-cell related disease” includes B-cell immunodeficiency, autoimmune diseases, and / or excessive / uncontrolled cell proliferation associated with B cells (including lymphoma and / or leukemia). Examples of diseases for which the bispecific CAR of the present invention may be used as a therapeutic agent include, but are not limited to, systemic lupus erythematosus (SLE), diabetes mellitus, rheumatoid arthritis (RA), reactive arthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinemia, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus, unclassifiable immunodeficiency, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and / or IgG subclass deficiency, B-cell lymphomas (Hodgkin lymphoma and / or non-Hodgkin lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinemia and / or hyper-IgM syndrome, and virus-mediated B-cell diseases such as EBV-mediated lymphoproliferative disorders, and chronic infections in which B cells are involved in the pathophysiology.

[0022] “Beneficial outcomes” include, but are not limited to, mitigating or reducing the severity of a disease condition, preventing disease progression, curing a disease condition, preventing the onset of a disease condition, reducing the patient’s chances of developing a disease condition, and extending a patient’s lifespan or expected lifespan.

[0023] "Cancer" and "malignant" typically refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin lymphoma and / or non-Hodgkin lymphoma), brain tumors, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent and androgen-independent prostate cancer.

[0024] As used herein, “co-expression” refers to the simultaneous expression of two or more genes. A gene may be, for example, a nucleic acid encoding a chimeric protein as a single protein or a single polypeptide chain. For example, the CAR of the present invention may be co-expressed with a therapeutic regulatory agent (e.g., truncated epidermal growth factor (EGFRt)), where the CAR is encoded by a first polynucleotide chain and the therapeutic regulatory agent is encoded by a second polynucleotide chain. In one embodiment, the first and second polynucleotide chains are linked by a nucleic acid sequence encoding a cleavable linker. The polynucleotides encoding the CAR and the therapeutic regulatory agent system are linked by an IRES sequence. Alternatively, the CAR and the therapeutic regulatory agent may be encoded by two different polynucleotides, which are not linked via a linker but are instead encoded, for example, by two different vectors. Furthermore, the CAR of the present invention may be co-expressed with a therapeutic regulatory agent and a CCR, a therapeutic regulatory agent and DHFR (e.g., mutant DHFR), or a therapeutic regulatory agent and both a CCR and DHFR (e.g., mutant DHFR). CARs, therapeutic control agents, and CCRs are co-expressed and may be encoded by first, second, and third polynucleotide sequences, respectively, whose first, second, and third polynucleotide sequences are linked via IRES sequences or cleavable linker-coding sequences. Alternatively, these sequences are not linked via linkers but are instead encoded by separate vectors, for example. CARs, therapeutic control agents, and DHFRs (e.g., mutant DHFRs) are co-expressed and may be encoded by first, second, and fourth polynucleotide sequences, whose first, second, and fourth polynucleotide sequences are linked via IRES sequences or cleavable linker-coding sequences. Alternatively, these sequences are not linked via linkers but are instead encoded by separate vectors, for example.CARs, therapeutic control agents, CCRs, and DHFRs (e.g., mutant DHFRs) are co-expressed and may be encoded by first, second, third, and fourth polynucleotide sequences, respectively, which are linked via IRES sequences or cleavable linker-coding sequences. Alternatively, these sequences may not be linked via linkers but instead encoded by separate vectors, for example. When the aforementioned sequences are encoded by separate vectors, these vectors may be transfected simultaneously or sequentially.

[0025] As used herein, “condition,” “disease conditions,” “disease,” and “disease state” include physiological conditions in which affected cells can be targeted by the CAR of the present invention, for example, by expressing antibodies against specific antigens on the affected cells. Examples of antigens that can be targeted include, but are not limited to, antigens expressed on B cells (CD19 and CD20), antigens expressed on carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematological disorders, autoimmune diseases, and / or inflammatory diseases.

[0026] As used herein, “disease targeted by gene-modified cells” encompasses the targeting of any cell involved in any means in any disease by the gene-modified cells of the present invention, regardless of whether the gene-modified cells target affected cells or healthy cells to achieve therapeutically beneficial outcomes. Gene-modified cells include, but are not limited to, gene-modified T cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells, or embryonic stem cells. Gene-modified cells express the CAR of the present invention, which may target any antigen expressed on the surface of target cells. Examples of antigens that may be targeted include, but are not limited to, antigens expressed on B cells, antigens expressed on carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematological disorders, autoimmune diseases, and / or inflammatory diseases. Other antigens that may be targeted are obvious to those skilled in the art and may be targeted by the CAR of the present invention in connection with their alternative embodiments.

[0027] "Effector function" refers to the specialized function of differentiated cells. The effector function of T cells may include, for example, cytolytic activity or helper activity, including cytokine secretion.

[0028] As used herein, "genetically modified cell," "redirected cell," "genetically altered cell," or "modified cell" refers to a cell expressing the CAR of the present invention.

[0029] As used herein, “immune cells” refers to cells of the mammalian immune system, including but not limited to antigen-presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytic cells, plasma cells, and T cells.

[0030] As used herein, “immune response” refers to immunity, including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory responses, acquired (adaptive) immunity, autoimmunity, and / or overactive immunity.

[0031] As used herein, “mammal” refers, without limitation, to any member of the class Mammalia, including humans and non-human primates, such as chimpanzees and other apes and monkey species; domesticated animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and laboratory animals, including rodents such as mice, rats, and guinea pigs. This term does not indicate a specific age or sex. For this reason, adult and neonatal subjects, as well as fetuses, regardless of sex, are intended to be included within the scope of this term.

[0032] As used herein, “polynucleotide” includes, but is not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (nuclear small RNA), snoRNA (nucleolar small RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA.

[0033] As used herein, “naked DNA” refers to CAR-encoding DNA cloned in a suitable expression vector with an orientation appropriate for expression. Viral vectors that may be used include, but are not limited to, SIN lentiviral vectors, retroviral vectors, foamy viral vectors, adeno-associated virus (AAV) vectors, hybrid vectors, and / or plasmid transposons (e.g., the sleeping beauty transposon system) or integrase-based vector systems. Other vectors that may be used in connection with alternative embodiments of the present invention will be obvious to those skilled in the art.

[0034] As used herein, “single-chain variable fragment,” “single-chain antibody variable fragment,” or “scFv” antibody refers to an antibody form that contains variable regions consisting only of the heavy and light chains, linked by a linker peptide.

[0035] As used herein, “target cells” refers to cells involved in a disease that can be targeted by the gene-modified cells of the present invention (including, but not limited to, gene-modified T cells, NK cells, hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells). Other target cells are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0036] The terms “T cell” and “T lymphocyte” are interchangeable and are used synonymously herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, or combinations thereof.

[0037] As used herein, “therapeutic agent” refers to, for example, a drug used to treat, inhibit, prevent, mitigate, reduce the severity of, reduce the likelihood of developing, slow the progression of, and / or cure a disease. Diseases targeted by therapeutic agents include, but are not limited to, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas, as well as antigens expressed on various immune cells, and antigens expressed on cells associated with various hematological disorders, autoimmune diseases, and / or inflammatory diseases.

[0038] As used herein, “therapeutic control agent” refers to an agent that controls cell proliferation, promotes cell selection (e.g., selecting cells expressing the chimeric antigen receptor of the present invention), promotes cell tracking, or a combination thereof. In one embodiment, controlling cell proliferation includes upregulating cell proliferation to promote cell proliferation. In another embodiment, controlling cell proliferation includes downregulating cell proliferation to reduce or inhibit cell proliferation. In some embodiments, an agent that serves as a therapeutic control agent may promote the enrichment of cells expressing a bispecific chimeric antigen receptor that may provide therapeutic benefits.

[0039] As used herein, “transduction” refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0040] As used herein, “transfection” refers to the introduction of foreign nucleic acids into cells using recombinant DNA technology. The term “transformation” means the introduction of a “foreign” (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell, causing the host cell to express the transgene or sequence in order to produce a desired substance, such as a protein or enzyme, encoded by the transgene or sequence. The transgene or sequence may also be called a “cloned” or “foreign” gene or sequence and may include regulatory or regulated sequences, such as initiation, termination, promoter, signaling, secretion, or other sequences, used by the cell’s genetic mechanisms. The gene or sequence may include non-functional sequences or sequences of unknown function. The host cell that receives and expresses the transgene or RNA is “transformed” and is a “transformer” or “clone.” The DNA or RNA introduced into the host cell may originate from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

[0041] As used herein, “treatment” and “to treat” refer to both therapeutic measures and preventive or preventive measures aimed at preventing, slowing (mitigating), preventing a condition, seeking or obtaining beneficial outcomes, or reducing the likelihood of an individual developing the condition, even if the treatment is ultimately unsuccessful. Those in need of treatment include those who already have the condition, as well as those who are susceptible to the condition or whose condition should be prevented.

[0042] As used herein, “tumor” refers to the growth and proliferation of all tumor cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.

[0043] As used herein, “vector,” “cloning vector,” and “expression vector” refer to a medium through which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, and the like.

[0044] Description of the present invention Chimeric antigen receptor While we do not wish to be limited by any assumptions, the chimeric antigen receptors (e.g., bispecific CARs) of the present invention are thought to overcome conventional therapeutic failures due to the growth of antigen-elimination escape variants that may occur during the course of various therapies, for example, when a single antigen is targeted. Accordingly, the present invention relates, in particular, to nucleic acid and amino acid sequences encoding CARs, vectors containing CARs, viruses containing CARs, genetically modified cells (reorientation cells) containing CARs, and methods for producing and using them. In some embodiments, the CAR is a bispecific CAR. In other embodiments, the CAR targets and binds to three or more different antigens.

[0045] In general embodiments, the present invention relates to a CAR (e.g., a bispecific CAR), a nucleic acid sequence encoding a CAR (e.g., a bispecific CAR), a vector containing a nucleic acid encoding a CAR (e.g., a bispecific CAR), a virus containing a nucleic acid sequence encoding a CAR (e.g., a bispecific CAR), a host cell expressing a CAR (e.g., a bispecific CAR) (e.g., a genetically modified cell), a combination of a CAR (e.g., a bispecific CAR) and a therapeutic control substance, and a method for preparing and using a CAR (e.g., a bispecific CAR) as a therapeutic agent.

[0046] The CARs of the present invention target at least two different antigens. The CARs (e.g., bispecific CARs) are co-expressed with therapeutic control agents, such as truncated epidermal growth factor receptor (EGFRt), chimeric cytokine receptor (CCR), and / or dihydroxyfolate receptor (DHFR) (e.g., mutant DHFR). The polynucleotides encoding the CARs and therapeutic control agents can be linked via an IRES sequence or via a polynucleotide sequence encoding a cleavable linker. The CARs of the present invention are constructed such that they are expressed in cells, and the cells can then proliferate in response to the presence of at least one molecule that interacts with at least one antigen-specific target-directed region, such as an antigen.

[0047] In some embodiments, therapeutic control agents for use with the CAR of the present invention include one or more of the following: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), or AP1903 / Fas system. In one embodiment, the CAR of the present invention is linked to EGFRt via a cleavable linker or IRES sequence. In another embodiment, the bispecific CAR is linked to the EGFRt via a cleavable linker or IRES sequence.

[0048] The CAR described herein may be synthesized as a single polypeptide chain and may comprise at least two antigen-specific target-directing regions, an extracellular spacer domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. In this embodiment, the antigen-specific target-directing region is N-terminal, arranged in series, and separated by a linker peptide. The antigen-specific target-directing region is linked to an extracellular spacer domain, which is linked to the transmembrane domain. The transmembrane domain is linked to a costimulatory domain. The costimulatory domain is linked to an intracellular signaling domain at its C-terminus. If more than one costimulatory domain is used, multiple costimulatory domains may be arranged in series with the transmembrane domain at their N-terminus and with the intracellular signaling domain at their C-terminus. The polynucleotides encoding these polypeptides may further comprise an N-terminal signaling sequence that directs the CAR to the cell surface as a type I transmembrane protein. The antigen-specific target-directing region may face extracellularly, and the intracellular signaling domain may be intracytoplasmic.

[0049] Figure 1 shows a schematic diagram of the chimeric antigen receptor of the present invention.

[0050] In one embodiment, the extracellular spacer domain in the CAR is optional. In such a CAR, the antigen-specific target-directing region is located at the N-terminus, arranged in series, and separated by a linker peptide. The antigen-specific target-directing region may be linked to a transmembrane domain. The transmembrane domain may be linked to a co-stimulatory domain. The co-stimulatory domain may be linked to an intracellular signaling domain at the C-terminus. If more than one co-stimulatory domain is used, the multiple co-stimulatory domains may be arranged in series, with a transmembrane domain at their N-terminus and an intracellular signaling domain at their C-terminus. The polynucleotides encoding these polypeptides may further include an N-terminal signaling sequence that directs the CAR to the cell surface as a type I transmembrane protein. The antigen-specific target-directing region may face extracellularly, and the intracellular signaling domain may be intracytoplasmic.

[0051] Antigen-specific target-directed region of chimeric antigen receptors The CAR of the present invention may target several (two or more, three or more, etc.) different antigens. In one embodiment, the CAR is a bispecific CAR and targets two different antigens. As described above, the antigen-specific targeting regions of the CAR may be arranged in series and separated by a linker peptide. The antigen targeted by the CAR may be an antigen on a single affected cell (such as a cancerous B cell) or an antigen expressed on separate cells, each contributing to the disease. The antigen targeted by the CAR is an antigen that is directly or indirectly involved in the disease.

[0052] In a bispecific CAR, at least two different antigen-specific antibodies or their fragments or derivatives can be cloned within the antigen-specific target-directed region. The antibodies may be specific to any, but at least two distinct, optimal antigens. Antigen-specific antibodies may be Fab fragments of the antibody or single-chain variable fragments (scFv) of the antibody.

[0053] For example, Figure 2 shows one embodiment of the present invention depicting CD19 and CD20-specific CARs. Using methods well known to those skilled in the art, multiple scFvs specific to at least two different antigens are cloned upstream (i.e., to the N-terminus) of the IgG4-CD28-ζ domain on cells that can be targeted by the genetically modified cells described below, insofar as the target antigens are expressed. Such techniques are fully described in the literature. (Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989), Current Protocols in Molecular Biology.Volumes I-III [Ausubel, RM, ed. (1994)], Cell Biology: A Laboratory Handbook. Volumes I-III [JECelis, ed. (1994))], Current Protocols in Immunology. Volumes I-III [Coligan, JE, ed. (1994)], Oligonucleotide Synthesis. (MJ Gait ed. 1984), Nucleic Acid Hybridization [BD Hames & S. J. Higgins eds. (1985)], Transcription And Translation [BD Hames & S. J. Higgins, eds. (1984)], Animal Cell Culture [RIFreshney, ed. (1986)], Immobilized Cells And Enzymes [IRL Press, (1986)], Practical Guide To Molecular Cloning B. Perbal (1984), Current Proptocols in Immunology (JEColigan, AMKruisbeek, DH Margulies, EMS Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and research books in academic journals such as Advances in Immunology.

[0054] In one embodiment, each antigen-specific targeting region is V sufficient alone to confer antigen specificity (a "single domain antibody"), and includes a full-length IgG heavy chain (specific for the target antigen) having V H , CH1, hinge, and CH2 and CH3 (Fc) Ig domains. The full-length IgG heavy chain can be linked via an appropriate transmembrane domain to a co-stimulatory domain and an intracellular signaling domain. Both V H and V H are required to generate a fully active antigen-specific targeting region, and both the CAR containing V L and the full-length lambda light chain (IgL) are introduced into the cell to generate an active antigen-specific targeting region. In one embodiment, the extracellular spacer domain can be linked between the antigen-specific binding domain and the transmembrane domain. The cell includes, but is not limited to, T-lymphocytes (T cells), natural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells that can bring about a result related to treatment.

[0055] In another embodiment, each antigen-specific targeting region of the CAR includes at least two single-chain antibody variable fragments (scFvs) that are each specific for a different target antigen. The scFv has the C-terminus of one variable domain (V H or V L ) linked via a polypeptide linker to the other (V L or V H ​​The linker is attached to the N-terminus of ), and scFv has developed without significantly disrupting antigen binding or binding specificity. (Chaudhary et al., A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin. 1990 Proc. Natl. Acad. Sci., 87:9491; Bedzyk et al. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody. 1990 J. Biol. Chem., 265:18615). The linker is V H The N-terminus of V L At the C-terminus, or V H The C-terminus of V L It binds to the N-terminus. These scFvs lack the constant region (Fc) found in the heavy and light chains of innate antibodies. ScFvs specific to at least two different antigens are arranged in series and linked to the costimulatory domain and intracellular signaling domain via transmembrane domains. In one embodiment, an extracellular spacer domain may be linked between the antigen-specific binding region and the transmembrane domain.

[0056] In another embodiment, each scFv fragment may be fused to all or part of the constant domain of the heavy chain. The resulting antigen-specific target-directed region, specific to at least two different antigens, is bound to the costimulatory domain and the intracellular signaling domain via the transmembrane domain. In one embodiment, the extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

[0057] In further embodiments, each antigen-specific target-directed region of the CAR contains a divalent (or bivalent) single-chain variable fragment (di-scFv, bi-scFv). In a CAR containing di-scFv, two scFvs specific to each antigen result in two Vs that make up a series scFv. H and two V L They are linked together by generating a single peptide chain containing the region. (Xiong, Cheng-Yi; Natarajan, A; Shi, XB; Denardo, GL; Denardo, SJ (2006). "Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine ​​location on PEGylation and tumor binding." Protein Engineering Design and Selection 19(8):359-367; Kufer, Peter; Lutterbuse, Ralf; Baeuerle, Patrick A. (2004). "A revival of bispecific antibodies." Trends in Biotechnology 22(5):238-244). A CAR containing at least two antigen-specific targeting regions will express two scFv specific to each of the two antigens. The resulting antigen-specific targeting regions, specific to at least two different antigens, are bound to the costimulatory domain and the intracellular signaling domain via the transmembrane domain. In one embodiment, the extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

[0058] In further embodiments, each antigen-specific target-directed region of the CAR includes a diabody. In the diabody, the scFv is constructed using a linker peptide that is too short for the two variable regions to fold together, thereby dimerizing the scFv. Even shorter linkers (one or two amino acids) result in the formation of a trimer, a so-called triabody or tribody. Tetrabodies may also be used.

[0059] To construct the CAR of the present invention, two or more individual antigen-specific target-directed regions are linked to each other either covalently or noncovalently on a single protein molecule. Oligo or polypeptide linkers, Fc hinges, or membrane hinge regions may be used to link these domains to each other. The CAR of the present invention may comprise two or more different antigen-specific target-directed regions linked together in different combinations. For example, two or more antigen-specific target-directed regions containing immunoglobulin sequences (e.g., scFv and / or single-domain antibodies) may be linked to each other.

[0060] Targets of the antigen-specific target-directed region of chimeric antigen receptors In some embodiments, the antigen-specific target-directing region of a CAR (e.g., a bispecific CAR) targets antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, or combinations thereof. Examples of antigens that can be targeted by the CARs of the present invention (e.g., bispecific CARs) include, but are not limited to, antigens expressed on B cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematological disorders, autoimmune diseases, and / or inflammatory diseases. A CAR of the present invention, specific to at least two different target antigens, may be able to redirect the effector function of the expressing cell to either of the two target antigens. This feature of the construct can overcome, for example, the problem of antigen-loss escape variants when targeting genetically unstable B-cell malignancies using single antigen specificity.

[0061] Cancer-specific antigens that can be targeted by the CAR of the present invention (e.g., bispecific CAR) include 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TN FRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human cell dispersion factor (scatter The present invention includes, but is not limited to, one or more of the following: factor receptor kinases, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin. Other cancer-specific antigens are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. Examples of CARs targeting the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR, and DHFR (e.g., mutant DHFR).

[0062] In some embodiments, the bispecific chimeric antigen receptor targets and binds to at least two different antigens. Examples of pairs of at least two antigens bound by the bispecific CAR of the present invention include, but are not limited to, CD19 and CD20, CD19 and CD22, CD20 and L1-CAM, L1-CAM and GD2, EGFR and L1-CAM, EGFR and C-MET, EGFR and HER2, C-MET and HER2, and EGFR and ROR1. Other pairs of cancer-specific antigens will be obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. In yet another embodiment, the bispecific chimeric antigen receptor targets CD19 and CD20. Examples of CARs targeting the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR and DHFR (e.g., mutant DHFR).

[0063] Antigens specific to inflammatory diseases that can be targeted by the CAR of the present invention include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, and llama (Lama) The following are examples of CARs that target the above antigens, but are not limited to: glama), LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, scleroscin, SOST, TGFβ1, TNF-α, or VEGF-A. Other antigens specific to inflammatory diseases are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. Examples of CARs that target the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR and DHFR (e.g., mutant DHFR).

[0064] Antigens specific to neurological disorders that can be targeted by the CARs of the present invention include, but are not limited to, one or more of β-amyloid or MABT5102A. Other antigens specific to neurological disorders are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. Examples of CARs targeting the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR and DHFR (e.g., mutant DHFR).

[0065] Antigens specific to diabetes that can be targeted by the CARs of the present invention include, but are not limited to, one or more of L-1β or CD3. Other antigens specific to diabetes or other metabolic disorders are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. Examples of CARs targeting the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR and DHFR (e.g., mutant DHFR).

[0066] Antigens specific to cardiovascular disease that can be targeted by the CARs of the present invention include, but are not limited to, one or more of C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β-chain, ITGB2 (CD18), and sphingosine-1-phosphate. Other antigens specific to cardiovascular disease are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. Examples of CARs targeting the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR, and DHFR (e.g., mutant DHFR).

[0067] Antigens specific to infectious diseases that can be targeted by the CAR of the present invention include, but are not limited to, one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF-α. Other antigens specific to infectious diseases are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. Examples of CARs targeting the above antigens include, but are not limited to, bispecific CARs, bispecific CARs co-expressed with EGFRt, bispecific CARs co-expressed with EGFRt and CCR, bispecific CARs co-expressed with EGFRt and DHFR (e.g., mutant DHFR), or bispecific CARs co-expressed with EGFRt, CDR, and DHFR (e.g., mutant DHFR).

[0068] Further examples of target antigens include, but are not limited to, surface proteins found on cancer cells in a specific or amplified manner (e.g., IL-14 receptor, CD19, CD20, and CD40 in B-cell lymphoma; Lewis Y and CEA antigens in various carcinomas; Tag72 antigen in breast and colorectal cancer; EGF-R in lung cancer; folate-binding protein and HER-2 protein, which are often amplified in human breast and ovarian cancer), or viral proteins (e.g., gp120 and gp41 envelope proteins of HIV; envelope proteins derived from hepatitis B and C viruses; glycoprotein B and other envelope glycoproteins of human cytomegalovirus; envelope proteins derived from oncoviruses such as Kaposi's sarcoma-associated herpesvirus). Other potential targets of the CAR of the present invention include CD4, when the ligand is HIV gp120 envelope glycoprotein, as well as other viral receptors, such as ICAM, the receptor for human rhinovirus, and poliovirus-associated receptor molecules.

[0069] Further targets of the CAR of the present invention include antigens involved in B-cell-related diseases. Even further targets of the CAR of the present invention will be obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0070] Co-stimulatory domain of chimeric antigen receptor The CAR of the present invention also includes a co-stimulatory domain. This domain can enhance cell proliferation, cell survival, and the development of memory cells. The CAR of the present invention may include one or more co-stimulatory domains. Each co-stimulatory domain includes, for example, one or more co-stimulatory domains from the TNFR superfamily, CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or combinations thereof. Co-stimulatory domains derived from other proteins may also be used in conjunction with the CAR of the present invention. Further co-stimulatory domains will be obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention. If the CAR includes more than one co-stimulatory domain, these domains may be arranged in series and optionally separated by a linker.

[0071] Extracellular spacer domain of chimeric antigen receptor The CAR of the present invention may further include an extracellular spacer domain. In some embodiments, this domain facilitates proper protein folding. The extracellular spacer domain includes an antigen-specific target-directing region and a hydrophilic region attached to the transmembrane domain. The extracellular spacer domain may include, but is not limited to, an antibody Fc fragment or its fragment or derivative, an antibody hinge region or its fragment or derivative, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, the CD8α hinge, artificial spacers made from polypeptides such as Gly3, or the CH1 and CH3 domains of IgG (such as human IgG4). Specifically, the extracellular spacer domain may be (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 regions of IgG4, (iv) the hinge region of CD8α, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vi) the hinge and CH2 regions of IgG1, or a combination thereof. Further extracellular spacer domains are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0072] Transmembrane domain of chimeric antigen receptor The CAR of the present invention may also include a transmembrane domain. The transmembrane domain may include a transmembrane sequence derived from any protein having a transmembrane domain, including any type I, type II, or type III transmembrane protein. The transmembrane domain of the CAR of the present invention may also include an artificial hydrophobic sequence. The transmembrane domain of the CAR of the present invention may be selected to not dimerize. Further transmembrane domains are obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0073] Intracellular signaling domain of chimeric antigen receptors The CAR of the present invention may also include an intracellular signaling domain. This domain may be cytoplasmic and may transmit effector functional signals, instructing cells to perform their specialized functions. Examples of intracellular signaling domains include, but are not limited to, the ζ chain of a T cell receptor or any of its homologs (e.g., η chain, FcεR1γ and β chain, MB1(Igα) chain, B29(Igβ) chain, etc.), CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transmission such as CD2, CD5, and CD28. Specifically, the intracellular signaling domain may be the human CD3ζ chain, FcγRIII, FcεRI, the cytoplasmic end of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), or a combination thereof. Further intracellular signaling domains will be obvious to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0074] Linkers in chimeric antigen receptors In some embodiments, two or more components of the CAR of the present invention are separated by one or more linkers. For example, in a CAR comprising at least two antigen-specific target-directed regions, a first target-directed region on the CAR may be separated from a second target-directed region on the CAR via a linker. Furthermore, the CAR may be linked to a therapeutic control substance via a linker. The linker is an oligo or polypeptide region about 1 to 100 amino acids long that links any of the domains / regions of the CAR of the present invention together. In some embodiments, the linker may be, for example, 5 to 12 amino acids long, 5 to 15 amino acids long, or 5 to 20 amino acids long. The linker may consist of flexible residues such as glycine and serine so that adjacent protein domains can move freely from one another. For example, longer linkers, longer than 100 amino acids, may be used in connection with alternative embodiments of the present invention, for example, to ensure that two adjacent domains do not sterically interfere with each other. Examples of linkers that may be used in the present invention include, but are not limited to, 2A linkers (e.g., T2A), 2A-like linkers, or their functional equivalents.

[0075] therapeutic controlled substance Therapeutic control agents control cell proliferation, promote cell selection (e.g., selecting cells expressing the chimeric antigen receptor of the present invention), or perform a combination thereof. In one embodiment, controlling cell proliferation includes upregulating cell proliferation to promote it. In another embodiment, controlling cell proliferation includes downregulating cell proliferation to decrease or inhibit it. In some embodiments, an active agent serving as a therapeutic control agent may promote the enrichment of cells expressing a bispecific chimeric antigen receptor that may provide therapeutic benefits. In some embodiments, an active agent serving as a therapeutic control agent may biochemically interact with additional compositions to control the function of that therapeutic control agent. For example, EGFRt (a therapeutic control agent) may biochemically interact with cetuximab to control the function of EGFRt in selection, tracking, cell ablation, or a combination thereof.

[0076] Examples of therapeutic control agents include, but are not limited to, one or more of the following: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimeras, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptors (CCRs), selection markers, and combinations thereof. In some embodiments, therapeutic control agents are co-expressed with bispecific chimeric antigen receptors.

[0077] Examples of drugs that control the function of therapeutic control agents include Herceptin, methotrexate, cetuximab, thymidine analogs (e.g., ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), 6-thioguanine, and synthetic dimerizing agents. Examples of drugs include, but are not limited to, one or more of the following: (e.g., AP1903), fludarabine phosphate, linamarin (lin), nucleoside analogs (e.g., BVDU, difluorodeoxycytidine (dFdC), 1-β-D-arabinofuranosylthymine (ala-T)), indole-3-acetic acid (IAA), l-butionine-S,R-sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors, or combinations thereof. These drugs may be administered before, during, or after the use of the therapeutic control agent.

[0078] As described above, the CAR of the present invention can be synthesized as a single polypeptide chain. If the CAR is a bispecific CAR, the polynucleotide sequence encoding the CAR may be arranged, for example, in the N-terminus to C-terminus direction as follows: N-terminal signal sequence - antigen-specific target-directed region 1 - linker - antigen-specific target-directed region 2 - extracellular spacer domain - transmembrane domain - costimulatory domain - intracellular signaling domain. In one embodiment, such a CAR may contain two or more costimulatory domains.

[0079] Alternatively, the polynucleotide sequence encoding the CAR may be arranged in the following configuration from N-terminus to C-terminus: N-terminal signal sequence - antigen-specific target-directed region 1 - linker - antigen-specific target-directed region 2 - transmembrane domain - co-stimulatory domain - intracellular signaling domain. In one embodiment, such a CAR may contain two or more co-stimulatory domains.

[0080] If a CAR contains more than two antigen-specific target-directed regions, the polynucleotide sequence encoding the CAR may be arranged in the following order from the N-terminus to the C-terminus: N-terminal signal sequence - antigen-specific target-directed region 1 - linker - antigen-specific target-directed region 2 - linker - (antigen-specific target-directed region) n -Transmembrane domain-costimulatory domain-intracellular signaling domain. Such a CAR may further include an extracellular spacer domain. Each antigen-specific target-directed region may be separated by a linker. In one embodiment, such a CAR may include two or more costimulatory domains.

[0081] The present invention provides a nucleic acid sequence of an example CAR backbone comprising an extracellular spacer domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. Specifically, an example CAR backbone may comprise IgG4 hinge-CD28™-41BB-CD3ζ in the N-terminus-C-terminus direction, where the extracellular spacer domain is the IgG4 hinge region, the transmembrane domain is a CD28-derived transmembrane region, the costimulatory domain is derived from 4-1BB, and the intracellular signaling domain is derived from the CD3ζ chain (Figure 7). At least two antigen-specific target-directed regions may be inserted into the N-terminus relative to the IgG4 hinge.

[0082] The present invention provides nucleic acid sequences of exemplary embodiments in which the CAR is specific to CD19 and CD20. In one embodiment, a sequence encoding a bispecific anti-CD19xCD20 CAR is shown in Figures 3, 8, or 10. In another embodiment, a sequence encoding a bispecific anti-CD19xCD20 CAR is shown in Figures 4, 9, or 11. In this exemplary embodiment, the bispecific CAR comprises scFvs specific to CD19 and CD20, each scFv being separated by a linker and bound to an extracellular spacer domain which is bound to a co-stimulatory domain and an intracellular signaling domain via a transmembrane domain. While the example CAR shows one set of scFv sequences, any scFvs specific to CD19 and CD20 may be used. In certain embodiments, the CD19 and CD20-specific bispecific CAR is CD19scFv-Gly4Ser linker-CD20scFv-IgG4-hinge-CD28tm-41BB(cyto)-ζ(cyto), encoded by the sequence shown in Figures 3 and 4. This bispecific CAR comprises a CD19 and CD20-specific single-chain Fv fragment linked by a Gly4Ser linker, an IgG4 hinge extracellular spacer domain, a CD28 transmembrane domain, a 41BB costimulatory domain, and a CD3ζ chain-derived cytoplasmic domain.

[0083] In another embodiment, a CD19 and CD20-specific bispecific CAR comprises CD19scFv-Gly4ser linker-CD20scFv-hulgG4-hingeCH2CH3-CD28tm / cyto-41BB-ζ (Figures 9-10). This bispecific CAR includes CD19 and CD20-specific single-chain Fv fragments linked by a Gly4Ser linker, a human IgG4 hinge, CH2 and CH3 extracellular spacer domains, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a cytoplasmic domain derived from the CD3ζ chain.

[0084] In a further embodiment, a CD19 and CD20-specific bispecific CAR is CD19-Gly4ser linker-CD20scFv-CD8α hinge-CD8α™-41BB costimulator-ζcyto (Figures 11-12). This bispecific CAR comprises a CD19 and CD20-specific single-chain Fv fragment linked by a Gly4Ser linker, a CD8α hinge extracellular spacer domain, a CD8α transmembrane domain, a 41BB costimulatory domain, and a CD3ζ chain-derived cytoplasmic domain.

[0085] Truncate epidermal growth factor receptor (EGFRt) Human epidermal growth factor receptor (huEGFR) (EGFR in humans; ErbB-1, HER1) is a receptor tyrosine kinase of the ErbB family of growth factor receptors that is not expressed by hematopoietic and lymphocyte-generating cells. Ligand (EGF, TGF-α) binding occurs within the N-terminal extracellular domains I and II of EGFR as a result of the translocation of the receptor tyrosine kinase inactive monomer to the active homodimer.

[0086] The extracellular domain III of EGFR contains antibody (e.g., cetuximab (Erbitux), IgG1 chimeric antibody) binding sites. Human EGFR may retain intact antibody binding sites (e.g., cetuximab binding sites) within extracellular domains III, IV, or combinations thereof, but may be unable to bind to ligands (EGF, TGF-α) due to the removal of domains I and II, as well as the loss of signaling activity at its cytoplasmic end (Wang et al., A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells Blood 118(5)1255-1263).

[0087] The truncated EGFRt polypeptides described herein have at least three uses for genetic modification in cell-based therapies: ex vivo cell purification, in vivo cell tracking, and cell ablation. In one embodiment, EGFRt for use with the CAR of the present invention as a therapeutic control agent is conjugated to one or more of the following: EGFR-specific siRNA, EGFR-targeting small molecules, anti-EGFR antibodies, or a combination thereof. In another embodiment, EGFRt comprises the sequence shown in Figure 12 or 13, or a sequence that is approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95% homologous to the sequence shown in Figure 12 or 13.

[0088] In one embodiment of the present invention, huEGFRt may be co-expressed with the CAR of the present invention to control cells expressing the CAR (e.g., in vivo, in vitro, or ex vivo) by purifying cells expressing the CAR (e.g., ex vivo cell purification), tracking cells expressing the CAR (e.g., in vitro or in vivo cell tracking), or, if necessary, inducing cell ablation. In one embodiment, the CAR is a bispecific CAR.

[0089] Chimeric cytokine receptor (CCR) Based on the limitations of using exogenous γc cytokines in adoptive immunotherapy, the present invention provides T cells possessing an endogenous γc cytokine signaling mechanism. The usefulness of forced-constitutive chimeric cytokine receptor IL-2 / IL-15Rβ (CγCR2) and IL-7Rα (CγCR7) receptor signaling was compared. As described below, chimeric cytokine receptors have the ability to improve the survival, retention, and in vivo engraftment of cytotoxic T cells (CTLs).

[0090] Therefore, in one embodiment of the present invention, the CAR of the present invention can be co-expressed with CCR. For example, the bispecific CAR can be co-expressed with EGFRt and CCR. Alternatively, the bispecific CAR can be co-expressed with CCR. Examples of chimeric cytokine receptors include, but are not limited to, IL-7 cytokine-linker-IL7Rα, IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, and IL-7 cytokine-linker-IL2Rβ.

[0091] The CCR, which includes the IL-7 cytokine-linker-IL7Rα, contains an N-terminal signal sequence bound to the N-terminus of the IL-7 cytokine, and the IL-7 cytokine is linked via the linker to the extracellular domain, transmembrane domain, and cytoplasmic domain of IL-7Rα (the α-chain of the IL-7 receptor).

[0092] The CCR, which includes the IL-7 cytokine, a linker, the extracellular domain of IL-7Rα, the transmembrane domain of IL-7Rα, and the cytoplasmic domain of IL-2Rβ, contains an N-terminal signal sequence bound to the N-terminus of the IL-7 cytokine, and the IL-7 cytokine is linked via the linker to the extracellular and transmembrane domains of IL-7Rα, as well as the cytoplasmic domain of IL-2Rβ (the β-chain of the IL-2 receptor).

[0093] The CCR, which includes IL-7 cytokine-linker-IL2Rβ, contains an N-terminal signaling sequence bound to the N-terminus of the IL-7 cytokine, and the IL-7 cytokine is linked to the extracellular domain, transmembrane domain, and cytoplasmic domain of IL-2Rβ via the linker.

[0094] Dihydroxyfolate receptor (DHFR) Genetic modification of T cells to co-express therapeutic transgenes and drug resistance transgenes that confer resistance to lymphotoxic drugs provides an opportunity to select therapeutic cells both in vivo and ex vivo. A mutated human enzyme transgene, dihydrofolate reductase double mutant (DHFR), is one such example. FS L22F, F31S) confers resistance to methotrexate (MTX) to modified T cells and enables the selection of cells that co-express CD19-specific chimeric antigen receptors (CD19CARs) that specifically target B-cell tumor cells.

[0095] In one embodiment, the CAR of the present invention (e.g., a bispecific CAR) may be co-expressed with DHFR (e.g., mutant DHFR). In a further embodiment, the bispecific CAR may be co-expressed with EGFRt, CCR, and DHFR (including mutant DHFR). Alternatively, the bispecific CAR may be co-expressed with EGFRt and DHFR (including mutant DHFR).

[0096] Other select markers that may be used with the CAR of the present invention include, but are not limited to, methylated DNA-protein-cysteine ​​methyltransferase (MDMT), inosine monophosphate dehydrogenase II (IMDHP2), or combinations thereof. MDMT may be used when cells are made resistant to chemotherapy and therefore a synergistic effect between chemotherapy and T-cell therapy is desired.

[0097] Vectors encoding the CAR of the present invention are also provided herein. Vectors encoding the CAR also encode EGFRt. In some embodiments, vectors encoding the CAR and EGFRt also encode CCR or DHFR (e.g., mutant DHFR). In other embodiments, vectors encoding the CAR and EGFRt also encode CCD and DHFR (e.g., mutant DHFR). In some specific embodiments, the vector may encode bispecific CAR and EGFRt, bispecific CAR and EGFRt and CCR, bispecific CAR and EGFRt and DHFR (e.g., mutant DHFR), or bispecific CAR and EGFRt and CCR and DHFR (e.g., mutant DHFR). Vectors that can be used to express the CAR of the present invention include, but are not limited to, lentiviral vectors, γ retroviral vectors, foamy virus vectors, AAV vectors, adenovirus vectors, modified hybrid viruses, and naked DNA (including, but not limited to, transposon-mediated vectors such as Sleeping Beauty and Piggybak, and integrases such as Phi31).

[0098] In an embodiment representing an example of the present invention, the CD19 and CD20-specific bispecific CAR disclosed herein is expressed by a lentiviral vector, as illustrated in Figure 5.

[0099] Genetically modified cells of the present invention The present invention also provides genetically modified cells that contain and stably express the CAR of the present invention. The CAR expressed by the genetically modified cells may include at least two antigen-specific targeting regions, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. The polynucleotide sequence encoding the CAR may also include an N-terminal signaling sequence. In one embodiment, the CAR is a bispecific CAR. Each of the at least two antigen-specific targeting regions, an extracellular spacer domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain is described above. The antigen-specific targeting domain is capable of specifically binding to antigens that are not normally bound by T cell receptors in an MHC-unrestricted manner.

[0100] In one embodiment, genetically modified cells expressing the CAR of the present invention (e.g., a bispecific CAR) co-express EGFRt. In a further embodiment, genetically modified cells expressing the CAR (e.g., a bispecific CAR) co-express EGFRt and CCR. In a further embodiment, genetically modified cells expressing the CAR (e.g., a bispecific CAR) co-express EGFRt and DHFR (e.g., mutant DHFR). In another embodiment, genetically modified cells expressing the CAR (e.g., a bispecific CAR) co-express EGFRt, CCR, and DHFR (e.g., mutant DHFR).

[0101] Genetically modified cells express CARs having at least two antigen-specific target-directed regions that are specific to at least two different target antigens. In one embodiment, the antigen-specific target-directed regions include target-specific antibodies or their functional equivalents, fragments, or derivatives. The antigen-specific antibodies may be Fab fragments of antibodies or single-chain variable fragments (scFv) of antibodies.

[0102] Genetically modified cells containing and expressing the CAR of the present invention can produce therapeutic results, including T lymphocytes (T cells) and naive T cells (TN ), memory T cells (for example, central memory T cells (T CM ), effector memory cells (T EM This includes, but is not limited to, natural killer cells, hematopoietic stem cells and / or pluripotent embryonic / inducible stem cells. In one embodiment, the genetically modified cells are autologous cells. For example, individual T cells of the present invention may be CD4+ / CD8-, CD4- / CD8+, CD4- / CD8-, or CD4+ / CD8+. The T cells may be a mixed group of CD4+ / CD8- and CD4- / CD8+ cells, or a group of single clones. When co-cultured in vitro with cells expressing a target antigen (e.g., CD20+ and / or CD19+ tumor cells), the CD8 T cells of the present invention may produce IL-2, IFNγ, TNFα, and other T cell effector cytokines. + When T cells are co-cultured in vitro with antigen-specific target cells, they can lyse those target cells. In some embodiments, T cells are CD45RA + CD62L + Naive cells, CD45RO + CD62L + Central memory cells, CD62L - This could be one or more effector memory cells, or a combination thereof. (Berger et al., Adoptive transfer of virus-specific and tumor-specific T cell immunity. Curr Opin Immunol 2009 21(2)224-232).

[0103] Genetically modified cells can be produced by stably transfecting cells with DNA encoding the CAR of the present invention. The DNA encoding the CAR of the present invention (e.g., a bispecific CAR) may also encode EGFRt, CCR, and / or DHFR (e.g., a mutant DHFR). In one embodiment, a first polynucleotide encoding the CAR (e.g., a bispecific CAR) is linked to a second polynucleotide encoding EGFRt via an IRES sequence or a polynucleotide encoding a cleavable linker. In another embodiment, the first polynucleotide encoding the CAR (e.g., a bispecific CAR) is linked to a second polynucleotide encoding EGFRt via an IRES sequence or a polynucleotide encoding a cleavable linker, and the first or second polynucleotide is similarly linked to a third polynucleotide encoding CCR or DHFR (e.g., a mutant DHFR) via an IRES sequence or a polynucleotide encoding a cleavable linker. In further embodiments, the first polynucleotide encodes a CAR (e.g., a bispecific CAR) and is linked to a second polynucleotide encoding EGFRt via an IRES sequence or a polynucleotide encoding a cleavable linker, and the first and second polynucleotides are linked to a third polynucleotide encoding a CCR and a fourth polynucleotide encoding a DHFR (e.g., a mutant DHFR) via an IRES sequence or a polynucleotide encoding a cleavable linker. Viral vectors are commonly used to deliver heterologous genes to cells (e.g., T cells). Examples of viral vectors that may be used to generate genetically modified cells include, but are not limited to, SIN lentiviral vectors, retroviral vectors, foamy viral vectors, adeno-associated virus (AAV) vectors, and / or plasmid transposons (e.g., the sleeping beauty transposon system).

[0104] Various methods generate stable transfectants expressing the CAR of the present invention. In one embodiment, a method for stably transfecting and reorienting cells is by electroporation using naked DNA. By using naked DNA, the time required to generate reorienting cells can be significantly reduced. Further methods for genetically modifying cells using the naked DNA encoding the CAR of the present invention include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer, and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection and / or magnetofection using gene guns). Transfected cells showing the presence of a single integrated non-rearrangement vector and CAR expression can be expanded ex vivo. In one embodiment, cells selected for ex vivo expansion are CD8 + It exhibits the ability to specifically recognize and lyse antigen-specific target cells.

[0105] Viral transduction methods may also be used to generate reoriented cells expressing the CAR of the present invention. Cell types that may be used to generate genetically modified cells expressing the bispecific CAR of the present invention include, but are not limited to, T lymphocytes (T cells), natural killer cells, hematopoietic stem cells, and / or pluripotent embryonic / inducible stem cells that can yield therapeutic outcomes.

[0106] Stimulation of T cells with an antigen under appropriate conditions results in cell proliferation (expansion) and / or IL-2 production. Cells containing the CAR of the present invention expand in number in response to the binding of one or more antigens to the antigen-specific target-directing region of the CAR. The present invention also provides a method for producing and expanding cells that express a CAR. The method comprises transfecting or transducing cells with a vector that expresses a CAR, and stimulating the cells with cells that express a target antigen, recombinant target antigen, or antibody against a receptor, the cells proliferating to produce and expand T cells. In one embodiment, the cells may be one or more of T lymphocytes (T cells), natural killer (NK) cells, hematopoietic stem cells (HSCs), or pluripotent embryonic / inducible stem cells that can produce therapeutic outcomes.

[0107] In one exemplary embodiment, the genetically modified cells of the present invention express a bispecific CAR specific to the CD19 and CD20 antigens. In a further embodiment, the genetically modified T cells express the bispecific CAR CDl9scFv-Gly4ser-linker-CD20scFv-hulgG4-hinge-CD28-41BB(cyto)-ζ(cyto), or CDl9scFv-Gly4ser-linker-CD20scFv-hulgG4-hingeCH2CH3-CD28tm / cyto-ζ, or CD19-Gly4ser-linker-CD20scFv-CD8α-hinge-CD8αTM-41BB-costimulator-ζcyto.

[0108] In one exemplary embodiment, the present invention provides a method for producing and expanding T cells expressing CD19-specific and CD20-specific CARs. The method involves transducing a vector expressing CD19 and CD20 bispecific CARs into CD3xCD28 bead-stimulated purified central memory T cells (such as peripheral blood-derived T cells) using a lentivirus, growing the T cells in the presence of rhuIL-2 and / or IL-15, and transforming the T cells into CD19-specific T cells. + and CD20 +The process involves restimulating cells with antibodies against recombinant CD19 and CD20, or their receptors, to proliferate T cells in order to produce and expand CD19-specific and CD20-specific T cells.

[0109] Therapeutic method of the present invention The CAR of the present invention can be used to overcome therapeutic failures resulting from antigen elimination escape variants, reduce resistance to existing therapies, and / or treat diseases associated with antigens targeted by this CAR.

[0110] Accordingly, the present invention also provides a method for treating a disease in a subject requiring treatment of a disease associated with an antigen targeted by the CAR of the present invention. The method comprises providing a composition comprising the CAR of the present invention and administering an effective amount of the composition to treat a disease associated with the antigen in a subject.

[0111] The present invention also provides a method for overcoming therapeutic failure in disease conditions in subjects (e.g., B-cell diseases) where it is necessary to overcome therapeutic failure resulting from antigen-eliminating escape variants. The method comprises providing a composition comprising the CAR of the present invention and administering an effective amount of the composition to treat an antigen-associated disease in a subject.

[0112] In some embodiments, the composition comprises polynucleotides encoding CARs, proteins containing CARs, or gene-modified cells containing CARs. In another embodiment, the gene-modified cells of the composition are T lymphocytes (T cells), naive T cells (T cells), which can produce therapeutic outcomes. N ), memory T cells (for example, central memory T cells (T CM ), effector memory cells (T EMThe cells are natural killer (NK) cells, hematopoietic stem cells (HSCs), or pluripotent embryonic / inducible stem cells, which express the CAR of the present invention. The compositions of the present invention may be administered alone or in combination with existing therapies. When other therapies are used in combination, the compositions of the present invention may be administered simultaneously with or sequentially with the other existing therapies.

[0113] Pharmaceutical composition In various embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of the CAR of the present invention (e.g., a bispecific CAR). The CAR of the present invention in the composition may be one or more of the following: a polynucleotide encoding the CAR, a protein containing the CAR, or a genetically modified cell containing the CAR. The composition may further comprise a polynucleotide encoding EGFRt, CCR, and / or DHFR (e.g., mutant DHFR), a protein containing EGFRt, CCR, and / or DHFR co-expressed with the CAR, or a genetically modified cell expressing the CAR and co-expressing EGFRt, CCR, and / or DHFR. "pharmaceutically acceptable excipient" generally means an excipient that is safe, non-toxic, and useful in the preparation of a desirable pharmaceutical composition, and includes excipients acceptable for veterinary use as well as for pharmaceutical use in humans. Such excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gas.

[0114] In various embodiments, pharmaceutical compositions according to the present invention may be formulated for delivery via any route of administration. "Route of administration" means any route of administration known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsule, and / or injection.

[0115] Pharmaceutical compositions according to the present invention may also contain any pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium that is involved in carrying or transporting a compound of interest from one tissue, organ, or body part to another. For example, a carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulation material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other components of the formulation. It must also be suitable for use in contact with any tissue or organ it may come into contact with in that it must not pose a risk of toxicity, hypersensitivity, allergic reaction, immunogenicity, or any other complication that would unduly outweigh its therapeutic benefits.

[0116] Pharmaceutical compositions according to the present invention may also be encapsulated in capsules, tablets, or prepared as emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition or to facilitate the preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, acacia, agar, or gelatin. Carriers may also include sustained-release materials such as glyceryl monostearate or glyceryl distearate, either alone or with wax.

[0117] Pharmaceutical preparations are prepared, as necessary, according to conventional pharmaceutical techniques involving grinding, mixing, granulation, and pressurization for tablet formation, or grinding, mixing, and filling for rigid gelatin capsule formation. When liquid carriers are used, preparations are in the form of syrups, elixirs, emulsions, or aqueous or non-aqueous suspensions. Such liquid formulations may be administered directly orally or filled into flexible gelatin capsules.

[0118] Pharmaceutical compositions according to the present invention can be delivered in a therapeutically effective dose. The precise therapeutically effective dose is the amount of composition that yields the most effective result in terms of therapeutic efficacy in a given subject. This amount varies depending on a variety of factors, including, but not limited to, the properties of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, disease type and stage, overall health status, responsiveness to a given dose, and type of pharmacotherapy), the properties of pharmaceutically acceptable carriers in the formulation, and the route of administration. Technicians in the clinical and pharmacological fields can determine the therapeutically effective dose through conventional experimental methods, for example, by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000). [Examples]

[0119] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. With respect to the extent to which specific materials are mentioned, this is merely illustrative and not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without exercising originality and without departing from the scope of the invention.

[0120] Example 1 Figure 1 is a schematic diagram of the bispecific chimeric antigen receptor of the present invention. In an example embodiment of the present invention, Figure 2 shows the components of a bispecific anti-CD19x anti-CD20 bispecific CAR. Figure 2 also shows a schematic diagram of the complete cDNA packaged in an epHIV-7 lentiviral vector transfer plasmid. Figures 3 and 4 show the nucleic acid sequence and amino acid sequence of an example bispecific CAR, namely GMCSFss-CD19scFv-Gly4Ser1 linker-CD20scFv-IgG4 hinge-CD28tm-41BBζ-T2A-EGFRt_epHIV7.

[0121] Example 2 Figure 5 is a schematic diagram showing an example of a CAR vector construct of the present invention, namely the CD19scFv-CD20scFv-IgG4-CD28tm-CD28 costimulatory-CD3ζ transgene construct. The CD19scFv-CD20scFv-IgG4-CD28tmCD28 costimulatory-CD3ζ transgene was constructed using a one-step isothermal DNA assembly method previously described by Gibson et al. (Enzymatic assembly of DNA molecules upto several hindred kilobases. Nature Methods. 2009;6:343-345). The V of the CD19 scFv construct L and V H The domain was sequenced by polymerase chain reaction (PCR) from a previously described CD19CAR-CD28-ζ transgene. (Schmitz N, Dreger P, Glass B, Sureda A. Allogeneic transplantation in lymphoma: current status. Haematologica. 2007;92(11):1533-1548). Using a previously described CD20R-CD28-ζ transgene, the V of CD20 scFv was sequenced by spliced-overlap polymerase chain reaction. H and V LThe domain was constructed (Michael Jensen et al., CD20 is a molecular target for scFvFc:zeta receptor redirected T-cells:implications for cellular immunotherapy of CD20). + malignancy. Biology of Blood and Marrow Transplant. 1998;4:75-83). V of CD19 scFv and CD20 scFv. H and V L The domain was ligated to an 18-residue linker peptide as previously described. The IgG4-CD28tm-CD28 costimulatory domain was sequenced using the CD19R-CD28-CD3ζ transgene by PCR. The CD3ζ-T2A-EGFRt_epHIV7 lentiviral destination vector was prepared by restriction digestion of the CD19R-CD28 portion of the previously described CD19R-CD28-ζ-T2A-EGFRt_epHIV7 plasmid (Seitaro Terakura et al., Generation of CD19-CAR modified CD8+ T-cells derived from virus-specific central memory T-cells. Blood. Oct. 26, 2011) using NheI and RsrII. Using restricted digested ζ-epHIV7 destination vectors, and primers for each containing a 30bp duplication at the 5' end, CD19scFv, CD20scFv, and IgG4-CD28tm-CD28 costimulated-CD3ζ constructs were constructed by one-step isothermal Gibson DNA assembly.

[0122] (Table 1) Regulatory elements in bispecific CAR epHIV-7 transfer plasmids TIFF2026088275000002.tif143135

[0123] Example 3 HEK293T cells were transfected with either the anti-CD19xCD20CAR-T2A-EGFRt epHIV-7 transfer plasmid or the anti-CD20xCD19CAR-T2A-EGFRt epHIV-7 transfer plasmid. The transfected cells were stained with biotinylated anti-Fc antibody and streptavidin PE (SA-PE), and then subjected to flow cytometry analysis to detect the expression of the two CARs. Both the anti-CD19xCD20 CAR and the anti-CD20xCD19 CAR were expressed on the transfected HEK293T cells.

[0124] The epHIV-7 transfer plasmid co-expressed the two bispecific CARs described above and EGFRt. EGFRt co-expression was detected on similarly transfected cells using a combination of biotinylated anti-EGFR antibody / SA-PE staining and flow cytometry analysis.

[0125] Example 4 Primary human peripheral blood T cells were activated with OKT3 and then lentivirally transduced using monospecific anti-CD19 CAR, monospecific anti-CD20 CAR, or bispecific anti-CD19xCD20 CAR-T2A-EGFRt epHIV7 lentiviral vectors. The epHIV7 lentiviral vectors also encoded EGFRt along with monospecific anti-CD19 CAR, monospecific anti-CD20 CAR, or bispecific anti-CD19xCD20. Thus, cells expressing a CAR co-expressed EGFRt. Transfected cells were stained with biotinylated anti-EGFR antibody and SA-PE, and then subjected to flow cytometry analysis to detect EGFRt expression and co-expression of monospecific or bispecific CARs. Of the cells transfected with a single-specific anti-CD19 CAR, 51% expressed EGFRt; of the cells transfected with a single-specific anti-CD20 CAR, 38.5% expressed EGFRt; and of the cells transfected with a bispecific anti-CD19xCD20 CAR, 63.8% expressed EGFRt.

[0126] T cell receptor (TCR) complexes in transfected cells were also detected in the same transfected cells using FITC-conjugated anti-TCRα and anti-TCRβ antibody staining and flow cytometry analysis.

[0127] Example 5 H9 cells were genetically modified to express CD19, CD20, or both CD19 and CD20. The cells were stained with anti-CD19 and anti-CD20 antibodies and then subjected to flow cytometry analysis to detect CD19 and CD20 expression. Cytometry analysis was performed to detect CD19 + CD20 - CD19 - CD20 + , and CD19 + CD20 +The desired expression profile of H9 cells was confirmed, i.e., the genetically modified H9 cells expressed CD19 or CD20, or both CD19 and CD20, thereby stimulating cancer target cells including antigen-negative antigen-loss escape variants. As described later, these cell lines were then used as target cells to stimulate CAR-expressing T cell lines and acted as effector cells to kill the target cells.

[0128] Similarly, the endogenous levels of CD19 and CD20 expression in the SUP-B15 and DHL-6 cell lines were analyzed using anti-CD19 APC and anti-CD20 PE staining and flow cytometry analysis. The SUP-B15 cell line expressed high levels of CD19 and low levels of CD20 (therefore, CD19 + CD20 - ), and the DHL-16 cell line expressed high levels of CD20 and low levels of CD19 (therefore, CD19 - CD20 + ).

[0129] Example 6 The lysis of target cells by effector cells was measured using a 4-hour chromium release assay. The effector cells were primary human T cells transduced with lentivirus to express a single-specific anti-CD19 CAR, a single-specific anti-CD20 CAR, or a bispecific anti-CD19xCD20 CAR. The bispecific anti-CD19xCD20 CAR effector T cells were CD19 + CD20 - H9 cells, CD19 - CD20 + H9 cells, CD19 + CD20 + H9 cells, and CD19 + CD20 - , CD19 - CD20 + , and CD19 + CD20 +All of the target cells were effectively lysed. At effector-to-target ratios of 1:1, 3:1, 10:1, and 30:1, approximately 25%, 45%, 50%, and 60% of the target cells were lysed, respectively.

[0130] In contrast, the single-specificity CAR-expressing T cell line was unable to lyse antigen-negative antigen-loss escape variants that escaped from the single-specificity CAR effector cells. The anti-CD19 CAR effector T cells were unable to lyse CD19 - CD20 + targets, and the anti-CD20 CAR effector T cells were unable to lyse CD19 + CD20 - targets.

[0131] Example 7 The bispecific CAR-expressing CD4-enriched T cells were activated against cytokine secretion (interferon γ (IFN-g, IFN-γ)) in response to stimulation by CD19 + CD20 - H9 cells, CD19 - CD20 + H9 cells, CD19 + CD20 + H9 cells, and SUP-B15 cells, including CD19 + CD20 - , CD19 - CD20 + , and CD19 + CD20 + target cells. The amount of IFN-γ was measured by cytokine bead array of the culture supernatants of T cells and target cells 24 hours after co-culture. The activated bispecific CAR-expressing CD4-enriched T cells secreted at least 2500 pg / ml INF-g in response to stimulation by all types of target cells. In contrast, the single-specificity CAR-expressing T cell line was not activated for cytokine INF-g secretion in response to stimulation by antigen-negative antigen-loss escape variants that escaped from the single-specificity CAR effector cells. The CD19 CAR T cells were unable to lyse CD19 - CD20 +CD20 CAR T cells, which are unable to secrete IGN-γ when co-cultured with target cells, are CD19 + CD20 - IGN-γ could not be secreted during co-culture with target cells. In vitro stimulation assay TIFF2026088275000003.tif62128

[0132] Example 8 The following examples illustrate a CD19-specific chimeric antigen receptor linked to a truncated epidermal growth factor receptor (EGFRt) via a T2A sequence. EGFRt can be linked to and co-expressed with other chimeric antigen receptors, such as bispecific chimeric antigen receptors.

[0133] The applicant demonstrated the usefulness of such truncated EGFR (huEGFRt) expressed by transduced T cells for immunomagnetic purification, cell tracking by flow cytometry and immunohistochemistry, and in vivo cell ablation after systemic cetuximab administration, using biotinylated cetuximab. In this exemplary embodiment, domains I and II of EGFRt were removed, while domains III and IV were retained.

[0134] The CD19CAR-T2A-EGFRt-epHIV7 lentiviral construct is shown as (1) V of CD19-specific FMC63 monoclonal antibody (mAb) H and V L Gene fragment, IgG4 hinge-C H2 -C H3A chimeric antigen receptor (CAR) sequence consisting of the transmembrane and cytoplasmic signaling domains of the costimulatory molecule CD28, and the cytoplasmic domain of the CD3ζ chain (Kowolik CK.et al., CD28 costimuation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 2006, 66(22):10995-11004); (2) a self-cleaving T2A sequence (Szymczak AL.et al., Correction of multi-gene deficiency in vivo using a "self-cleaving" 2A peptide-based retroviral vector. Nat Biotechnol2004;22(5)589-594); and (3) a truncated EGFR sequence.

[0135] huEGFRt after lentiviral transduction + Immunomagnetic enrichment of human T cells huEGFRt + Biotinylated cetuximab was used for either immunomagnetic selection or FACS sorting of the cells. The applicants used biotinylated cetuximab in combination with commercially available anti-biotin microbeads for the immunomagnetic selection of human T cells transduced with a self-inactivating lentivirus that directs the co-expression of CD19CAR and huEGFRt.

[0136] PBMC or purified central memory (CD45RO + CD62L + T CM ) or effector memory (CD45RO + CD62L + T EMA subset of T cells was stimulated with anti-CD3 / anti-CD28 beads and then transduced with a lentiviral vector to generate a panel of primary human T cell lines; 2.6%–40% of these expressed huEGFRt and CAR. Unselected cells were labeled with biotinylated cetuximab and anti-biotin microbeads and then isolated to consistently obtain a selected cell population in which 90% expressed huEGFRt and CAR.

[0137] Unselected T cells and selected fractions were stained with biotinylated cetuximab and either PE-conjugated streptavidin or PE-conjugated antibiotin Ab, and then subjected to flow cytometry analysis. CD19CAR + EGFRt + Cell selection was performed 3 days after transduction of OKT3 blast cells (concentrated from 38% to 98%), or using the transduction effector memory CD62LCD45RO. + After one cycle of rapid expansion of derived cells (enriched from 20% to 96%), after three cycles of rapid expansion of transduced CMVpp65-specific TCM-derived cells (enriched from 12% to 91%), or transduced CD8 + The procedure was performed either after two cycles of rapid expansion of TCM-derived cells (enriched from 3% to 97%). CD19CAR + EGFRt + IMPDH2dm + Cell selection was performed after one cycle of rapid expansion of transduced TCM-derived cells (enriched from 25% to 92%).

[0138] The CD19CAR-T2A-EGFRt-IMPDH2dm construct contained in the lentiviral vector includes a codon-optimized sequence portion of a CD19-specific CD28-costimulated CAR (CD19CAR), followed by a self-cleavable T2A, and the selection markers huEGFRt and IMPDH2dm (double mutants of the inosine monophosphate dehydrogenase 2 gene that enable cell survival upon addition of mycophenolate 27), along with an elongation factor 1 promoter sequence (EF-1p), a GM-CSF receptor α-chain signaling sequence (GMCSFRss), and a 3-nucleotide stop codon.

[0139] In cultures of unselected transduced T cells used to expand OKT3-mediated activity, huEGFRt is used before immunomagnetic selection. - Cellular huEGFRt + A proliferative advantage over the cells was observed. However, after immunomagnetic selection, the level of huEGFRt expression and the frequency of expressing cells expanded the OKT3 base. 14 EGFRt remained stable for three consecutive 14-day cycles. + Cell magnification is huEGFRt in unselected cultures. + It was significantly enhanced compared to that obtained from cells.

[0140] These data suggest that huEGFRt can serve as a unique cell surface marker for transduced human T cells, enabling subsequent cetuximab-based immunomagnetic purification of a stable huEGFRt-expressing cell population that also expresses CAR.

[0141] Adoptive transfer using flow cytometry and immunohistochemistry huEGFRt + T cell tracking To test the usefulness of huEGFRt for tracking the engraftment of adoptive T cells, the applicants used CD19CAR + EGFRt + NOD / Scid IL-2RγC transplanted with human T cells null Blood and bone marrow specimens were collected from mice.

[0142] First, unfixed peripheral blood and bone marrow mononuclear cell samples were stained with biotinylated cetuximab and PE-bound streptavidin, and then subjected to flow cytometry analysis. Human CD45 + The level of T cell engraftment (20%–25%) was similar in animals administered either EGFRt-negative or EGFRt-positive T cells, but double staining for human CD45 and EGFR was different for huEGFRt. + This made it possible to isolate human T cells (that is, those expressing the transgene) from their huEGFRt-negative counterparts.

[0143] Next, the applicants used an EGFR-specific diagnostic kit to test standard paraffin-embedded fixed tissue specimens for huEGFRt + We attempted to determine whether it was suitable for detecting T cell infiltration. The applicants performed immunohistochemical analysis of paraffin-embedded femurs from transplanted mice and detected huEGFRt in the bone marrow. + Cells were detected. These data support the usefulness of huEGFRt as a tracking marker for quantifying the frequency and tissue distribution of adoptive T cells.

[0144] Cetuximab, which binds to huEGFRt, sensitizes human T cells to ADCC. A useful feature of cell surface selection / tracking markers is their ability to serve as targets for in vivo cell ablation. The applicants have shown that cetuximab bound to huEGFRt on T cells can be used in vitro to target huEGFRt. + The degree to which T cells activate ADCC, and the effect of cetuximab administration on adoptive transfer in NOD / scid mice huEGFRt + We evaluated whether it could attenuate T cell engraftment.

[0145] As target cells 51 Cr-labeled huEGFRt +T cells and human GM-CSF-activated fresh PBMCs were co-cultured as effectors. Subsequently, cetuximab was added to huEGFRt + T cells were specifically sensitized to ADCC cell lysis induced by an effector. huEGFRt + T cell lysis was measured by a 4-hour chromium release assay, and the results showed that cetuximab addition significantly increased lysis from less than 5% to approximately 50%, 45%, 40%, and 15% at effector-to-target (effector:target) ratios of 50:1, 25:1, 5:1, and 1:1, respectively.

[0146] In contrast, in this assay, the addition of CD20-specific mAb rituxan was effective against huEGFRt + It had no effect on inducing ADCC in T cells.

[0147] The applicants then further modified huEGFRt to secrete autocrine IL-2 and express a firefly luciferase biophotonic reporter. + We induced T cells in CTLL-2 mice and used these ffLuc + huEGFRt + CTLL-2 cells were adopted into NOD / scid mice via intravenous injection, and these mice were subsequently administered cetuximab or rituximab. In vivo engraftment of the transferred CTLL-2 cells was significantly inhibited in mice administered Erbitux (1 mg intraperitoneally daily), as measured by in vivo biophotonic imaging (97%, P<0.05). ffLuc + huEGFRt + Cetuximab-mediated elimination of CTLL-2 cells occurred within 4–6 days. These data were obtained from huEGFRt. + We support the use of cetuximab as a therapeutic control agent in patients who have received T-cell therapy.

[0148] Example 9 This example describes T cells having an endogenous γc cytokine signaling mechanism, and shows that the chimeric cytokine receptors (CCRs) IL-2 / IL-15Rβ (CγCR2) and IL-7Rα (CγCR7), which are chimeric cytokine receptors, have the ability to improve the survival, persistence, and in vivo engraftment of cytotoxic T cells (CTLs). The truncated CD19 antigen (CD19t) was linked to the CγCR via a T2A linker so as to show the expression of CγCR on the cell surface. The chimeric cytokine receptors described herein can be linked to the chimeric antigen receptors of the present invention, such as the bispecific CARs described herein.

[0149] To generate an intracellular, ligand-independent γc cytokine platform, the applicants generated a chimeric γc cytokine receptor (CγCR) consisting of the IL-7 cytokine linked to the extracellular domain of IL-7Rα by 10 amino acids. To generate a CγCR that provides IL-7R signaling, the IL-7 cytokine was linked to the full-length IL-7Rα chain (CγCR7). The CγCR that provides IL-2 / IL-15Rβ signaling was generated by linking the IL-7 cytokine to the extracellular and transmembrane domains of IL-7Rα fused to the cytoplasmic domain of IL-2 / IL-15Rβ (CγCR2). These single-chain chimeric receptors are expected to require the endogenous γc chain for signaling.

[0150] The construct was then generated such that the CγCR transgene was followed by a self-cleavable T2A sequence and a cytoplasmic truncated CD19 antigen (CD19t). CγCR and CD19t are expressed as single transcripts and, post-translation, are cleaved at the C-terminus of the T2A self-cleaving peptide to yield two separate type 1 membrane proteins, CγCR(T2A) and CD19t. Based on the expression of the two proteins from a single transcript, the ratio of CγCR(T2A) to CD19t expression is 1:1, and therefore, cell surface CD19t is an indicator of CγCR cell surface expression. Lentiviral transduction and the expression of these constructs can then be measured by surface CD19t expression, as observed in both Jarcutt and NK-92 cell lines.

[0151] A third CγCR (CγCR7t) was also created, possessing an IL-7 cytokine linked to a truncated IL-7Rα lacking 1 to 126 amino acids from the extracellular domain of IL-7Rα. A molecular model of CγCR7t dimerization with the endogenous γc chain is essential for signal transduction. The deletion of 1 to 126 amino acids from the extracellular domain of IL-7Rα renders CγCR7t non-functional.

[0152] Truncate-type CγCR7 expression does not functionally signal or support cytokine-independent cell growth. Flow cytometry shows that lentitransduction jarcuts (95% CD19t) + CγCR7t + ) and teff cell lines (97% CD19t + CγCR7t + CD19t was detected on the cell surface. Western blot analysis of STAT5 phosphorylation in CγCR7t-expressing Jurcut cell lines did not detect a significant increase in phosphorylated STAT5 compared to non-transduced control Jurcut cell lines. When positive control OKT3-stimulated PBMCs were cultured in 50 U / ml IL-2 and 10 ng / ml IL-15, K562 showed increased activation of phosphorylated STAT5. Therefore, the expansion and viability of CγCR7t-transduced CTLs cultured for 20 days were still cytokine-dependent.

[0153] Functional CγCRs such as CγCR2 and CγCR7 react to CD8 in the absence of exogenous cytokines. + To determine whether they could support the growth of human primary T cells, the inventors measured the expansion of CTLs expressing each CγCR. Human primary T cells expressing CγCR7t could not expand in the absence of exogenous cytokines. Both CγCR2 and CγCR7 maintained viability by CD8 in a similar manner to that of parental cells cultured in 5 U / ml and 0.5 U / ml IL-2, respectively. + It was possible to support the survival and proliferation of T cells. CγCR2 + vs CγCR7 + Increased overall cell expansion measured for CTLs correlates with increased expression of Ki67, a nuclear antigen protein present in the G1, S, G2, and M phases of the cell cycle (i.e., 26 MFIs for CγCR7 vs. 52 MFIs for CγCR2). Higher expression of Bcl-2, a major anti-apoptotic protein induced in response to IL-2 and IL-7 signaling, correlates with increased expression of CγCR7 + vs CγCR2 + Observed in CTLs, this supports the ability of CγCR7 to maintain the survival of human primary T cells. Combined, these data support cytokine-independent T cell viability and expansion, but CγCR7 is the effector CD8 + It is suggested that CγCR2 provides a proliferative advantage while maintaining the survival of CTLs.

[0154] CD8 expressing CγCR + T cells exhibit cytokine-independent engraftment in vivo. Research conducted by the inventor's laboratory and others has been conducted on NOD / Scid IL-2RγC null This study demonstrates that the engraftment of human CTLs in mice depends on the exogenous administration of human IL-15 or IL-2. To overcome this dependence, we investigated the possibility of CγCR expression in CTLs using parental effector T cells and CγCR7. + CTL and CγCR2 +CTLs in immunodeficiency NOD / Scid IL-2RγC in the absence of exogenous cytokine administration null The T cell was injected into the tail vein of mice. At least four mice per group were collected on days 8, 17, 24, and 48, and overall engraftment was compared by analyzing T cell levels in the blood and bone marrow.

[0155] In the blood, CγCR2 + CTL stands for CγCR7 + Compared to CTLs and parental cells, it exhibited very significant (P<0.007) exogenous cytokine-independent engraftment. In the bone marrow, CγCR7 + CTL (P<0.03) and CγCR2 + Both CTLs (P<0.0005) exhibited significantly exogenous cytokine-independent engraftment compared to parental cells. CγCR2 + CTL stands for CγCR7 + It showed higher engraftment compared to CTL. This is because CγCR7 + CTL and CγCR2 + Both CTLs can support exogenous cytokine-independent engraftment, but the overall percentage of cells differs. Blood compared to bone marrow. + This supported a higher engraftment rate of CTLs. Bone marrow showed long-term CγCR7 + This supported the engraftment of CTLs. Importantly, engraftment was not indefinite, as cells were no longer present in the blood and bone marrow in either group by day 48.

[0156] Intracellular γc cytokine signaling can replace the need for exogenous cytokine administration to support adoptive CTLs. Providing an intracellular cytokine receptor can overcome a major limitation of adoptive immunotherapy (long-term persistence of adoptive CTLs). This eliminates the need for exogenous cytokine administration and reduces toxicity and bystander effects on endogenous cell types.

[0157] Example 10 This example demonstrates that CD19 chimeric antigen receptors linked to EGFRt and DHFR can be controlled by methotrexate. Using the method described herein, the dihydroxyfolate receptor described herein can be linked to the bispecific chimeric antigen receptor of the present invention.

[0158] The applicants have developed a human selectable transgene that utilizes a human dihydrofolate reductase (hDHFR) variant that can enable T cell selection using the less toxic, pharmaceutically available drug methotrexate (MTX). MTX exerts its antiproliferative effect through competitive inhibition of DHFR, a key enzyme essential for the novel synthesis of thymidylate nucleotides.

[0159] In this embodiment, the applicants have found that primary human T cells co-expressing a CD19-specific chimeric antigen receptor (CD19CAR for targeting CD19-expressing tumors) have DHFR FS The potential for (hDHFR L22F / F31S variant)-mediated in vitro selection was evaluated. In this strategy, the inventors found that exposure of a mixed group of transduced T cells to the lymphocytotoxic drug MTX eliminated non-transduced T cells and DHFR FS This should lead to a selective expansion of / CD19CAR T cells, and overall, it was hypothesized that co-expressing T cells would enhance the antitumor effect of the T cell population. Here, the applicants considered the DHFR of gene-modified T cells. FS Mediating selection forces CD19CAR therapeutic transgene expression and, in the presence of clinically achievable concentrations of MTX (e.g., 0.1 μM MTX), CAR + This demonstrates that it has become possible to induce stable embedded systems.

[0160] hDHFR for potential therapeutic utility FS To provide a selective approach, the applicants have combined a truncated human EGFR polypeptide as a CD19-specific chimeric antigen receptor (CD19CAR) and a tracking marker (huEGFRt), each separated by a ribosome-skipped T2A sequence, with hDHFR FSWe designed a lentiviral vector that co-expresses [the specified gene].

[0161] First, in CTLL2 T cells, this CD19CAR-huEGFRt-hDHFR FS Lentiviral vectors were transduced, and their resistance to MTX was evaluated. Ten days after lentitransduction, 7-8% of cells were positive for CD19CAR and huEGFRt expression.

[0162] In the absence of MTX, non-transduced and transduced CTLL2 cells expanded at equal rates (21-fold and 27-fold, respectively). After 8 days of incubation with MTX (0-0.1 μM), transduced cells showed a 7-fold expansion with 80% survival, while exposure of non-transduced CTLL2 cells to MTX at concentrations of 0.05 μM or higher strongly inhibited the expansion and viability of non-transduced CTLL2 cells.

[0163] Evaluation of huEGFRt expression levels in transduced CTLL2 cells after 8 days in cultures at different concentrations of MTX indicates transgene expression of huEGFRt. + We further revealed the remarkable MTX-mediated enrichment of cells (49%, 93%, 98.5%, and 99% with 0.01, 0.025, 0.05, and 0.1 μM MTX, respectively).

[0164] To further characterize the highest dose of MTX that may be resistant to selected CTLL2 cells, transduced CTLL2 cells cultured with 0.1 μM MTX for 8 days were replaced with a wide range of MTX concentrations (up to 0.75 μM). These transduced and pre-MTX-selected cells were able to expand 90–100 times at MTX concentrations up to 0.25 μM, which was equivalent to the expansion of untransduced control CTLL2 in the absence of MTX.

[0165] The applicants found that primary human T cells had the same CD19CAR-huEGFRt-hDHFR FS A lentiviral vector was transduced. Purified CD62L + CD45RO+ T cells were used as the starting group based on their potential for survival after adoptive transfer. Ten days after transduction, these T cells were cultured in different concentrations of MTX, and cell number and viability were evaluated over time. After 10 days, transduced and non-transduced T cells expanded similarly (80-fold) in the absence of MTX. Furthermore, even at 0.1 μM of MTX, transduced T cells maintained 63% viability, while non-transduced primary human T cells, initiated at a low MTX concentration of about 0.025 μM, showed strong inhibition of both viability and expansion.

[0166] Flow cytometry evaluation of transdextrin T cells after 10 days in cultures at different concentrations of MTX revealed significant MTX-mediated enrichment of transgene-expressing cells (e.g., 0.025 μM MTX resulted in approximately 54% CD19CAR). + and 79% EGFRt + Concentrated; 0.05 μM MTX contains approximately 76% CD19CAR + and 89% EGFRt + (Concentrated).

[0167] A comparison of CD19CAR and EGFRt expression in cultures at day 6 versus day 10 is a time-dependent comparison of MTX / DHFR. FS The stable progression of mediating selection was revealed (Day 0: 18% CD19CAR) + , 28% EGFRt + Day 6: 48% CD19CAR + 71% EGFRt + Day 10: 70% CD19CAR + , 88% EGFRt + ).

[0168] All references cited herein are incorporated herein in their entirety by reference as if they were fully specified. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the invention pertains. Singletonet al., Dictionary of Microbiology and Molecular Biology 3 rd ed., J. Wiley & Sons (New York, NY 2001), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5 th The ed., J. Wiley & Sons (New York, NY 2001), and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide those skilled in the art with many general guidelines for the terminology used in this application.

[0169] Those skilled in the art will understand that there are many methods and materials that may be used in practice of the present invention that are similar or equivalent to those described herein. In fact, the present invention is not limited in any way to the methods and materials described herein. For the purposes of the present invention, the following terms are defined below.

[0170] While these descriptions directly illustrate the embodiments described above, those skilled in the art should understand that they may also consider modifications and / or variations of the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are also intended to be included therein. Unless otherwise specifically mentioned, the words and phrases in this specification and the claims are intended to be used in their ordinary and idiomatic sense to those skilled in the art of this application.

[0171] The above description of various embodiments of the invention, known to the applicant at the time of filing the patent application, is proposed and intended for illustrative and explanatory purposes. This description is not intended to be comprehensive or to limit the invention to the exact form disclosed, and many modifications and variations are possible in terms of the above teachings. The embodiments described are useful for explaining the principles of the invention and its practical applications, and for enabling those skilled in the art to utilize the invention in various embodiments and with various modifications suitable for the specific use to be intended. Accordingly, the invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention.

[0172] While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that modifications and alterations can be made based on the teachings herein without departing from the present invention and its broader embodiments. In general, it will be understood to those skilled in the art that the terms used herein are generally intended to be “not limited” terms (for example, the term “contains” should be interpreted as “contains but not limited to,” the term “has” should be interpreted as “has at least,” and the term “contains” should be interpreted as “contains but not limited to,” etc.).

[0173] Sequence information SEQUENCE LISTING <110> Seattle Children's Hospital d / b / a Seattle Children's Xare Institute <120> Bispecific Chimeric Antigen Receptors and Therapeutic Uses Thereof <150> US 61 / 598,216 <151> 2012-02-13 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 3273 <212> DNA <213> Artificial Sequence <220> <223> GMCSFRss-CD19scFv-Gly4Ser1linker-CD20scFv-IgG4Hinge-CD28tm-41BB-C D3zeta-T2A-EGFRt_epHIV7 <400> 1 atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60 atccccatga cccagaccac ctccagcctg agcgccagcc tgggcgaccg ggtgaccatc 120 agctgccggg ccagccagga catcagcaag tacctgaact ggtatcagca gaagcccgac 180 ggcaccgtca agctgctgat ctaccacacc agccggctgc acagcggcgt gcccagccgg 240 tttagcggca gcggctccgg caccgactac agcctgacca tctccaacct ggaacaggaa 300 gatatcgcca cctacttttg ccagcagggc aacacactgc cctacacctt tggcggcgga 360 acaaagctgg aaatcaccgg cagcacctcc ggcagcggca agcctggcag cggcgagggc 420 agcaccaagg gcgaggtgaa gctgcaggaa agcggccctg gcctggtggc ccccagccag 480 agcctgagcg tgacctgcac cgtgagcggc gtgagcctgc ccgactacgg cgtgagctgg 540 atccggcagc cccccaggaa gggcctggaa tggctgggcg tgatctgggg cagcgagacc 600 acctactaca acagcgccct gaagagccgg ctgaccatca tcaaggacaa cagcaagagc 660 caggtgttcc tgaagatgaa cagcctgcag accgacgaca ccgccatcta ctactgcgcc 720 aagcactact actacggcgg cagctacgcc atggactact ggggccaggg caccagcgtg 780 accgtgagca gcggaggtgg tggatccgag gtgcagctgc agcagtctgg ggctgagctg 840 gtgaagcctg gggcctcagt gaagatgtcc tgcaaggctt ctggctacac atttaccagt 900 tacaatatgc actgggtaaa gcagacacct ggacagggcc tggaatggat tggagctatt 960 tatccaggaa atggtgatac ttcctacaat cagaagttca aaggcaaggc cacattgact 1020 gcagacaaat cctccagcac agcctacatg cagctcagca gcctgacatc tgaggactct 1080 gcggactatt actgtgcaag atctaattat tacggtagta gctactggtt cttcgatgtc 1140 tggggcgcag ggaccacggt caccgtctcc tcaggcagta ctagcggtgg tggctccggg 1200 ggcggttccg gtgggggcgg cagcagcgac attgtgctga cccaatctcc agctatcctg 1260 tctgcatctc caggggagaa ggtcacaatg acttgcaggg ccagctcaag tgtaaattac 1320 atggactggt accagaagaa gccaggatcc tcccccaaac cctggattta tgccacatcc 1380 aacctggctt ctggagtccc tgctcgcttc agtggcagtg ggtctgggac ctcttactct 1440 ctcacaatca gcagagtgga ggctgaagat gctgccactt attactgcca gcagtggagt 1500 tttaatccac ccacgttcgg agggggacc aagctggaaa taaagagag caagtacgga 1560 ccgccctgcc ccccttgccc tatgttctgg gtgctggtgg tggtcggagg cgtgctggcc 1620 tgctacagcc tgctggtcac cgtggccttt atcatctttt gggtgaaacg gggcagaaag 1680 1740 gatggctgta gctgccgatt tccagaagaa gaaggag gatgtgaact gcgggtgaag 1800 ttcagcagaa gcgccgacgc ccctgcctac cagcagggcc agaatcagct gtacaacgag 1860 1920 gagatgggcg gcaagcctcg gcggagaac cccagaggaag gcctgtataa cgaactgcag 1980 aaagacaaga tggccgaggc ctacagcgag atcggcatga agggcgagcg gaggcggggc 2040 aagggccacg acggcctgta tcagggcctg tccaccgcca ccaaggatac ctacgacgcc 2100 ctgcacatgc aggccctgcc cccaaggctc gagggcggcg gagagggcag aggaagtctt 2160 ctaacatgcg gtgacgtgga ggagaatccc ggccctagga tgcttctcct ggtgacaagc 2220 cttctgctct gtgagttacc acacccagca ttcctcctga tcccacgcaa agtgtgtaac 2280 ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 2340 ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 2400 gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 2460 aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac ggacctccat 2520 gccttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt 2580 gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 2640 ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 2700 aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc 2760 tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 2820 gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag 2880 tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 2940 cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac 3000 tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga 3060 gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac 3120 ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg 3180 aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 3240 gtggtggccc tggggatcgg cctcttcatg tga 3273 <210> 2 <211> 3273 <212> DNA <213> Artificial Sequence <220> <223> GMCSFRss-CD19scFv-Gly4Ser1linker-CD20scFv-IgG4Hinge-CD28tm-41BB-C D3zeta-T2A-EGFRt_epHIV7 <220> <221> CDS <222> (1)..(3273) <400> 2 atg ctg ctg ctg gtg acc agc ctg ctg ctg tgc gag ctg ccc cac ccc 48 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 gcc ttt ctg ctg atc ccc atg acc cag acc acc tcc agc ctg agc gcc 96 Ala Phe Leu Leu Ile Pro Met Thr Gln Thr Thr Ser Ser Leu Ser Ala 20 25 30 agc ctg ggc gac cgg gtg acc atc agc tgc cgg gcc agc cag gac atc 144 Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile 35 40 45 agc aag tac ctg aac tgg tat cag cag aag ccc gac ggc acc gtc aag 192 Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 50 55 60 ctg ctg atc tac cac acc agc cgg ctg cac agc ggc gtg ccc agc cgg 240 Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg 65 70 75 80 ttt agc ggc agc ggc tcc ggc acc gac tac agc ctg acc atc tcc aac 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn 85 90 95 ctg gaa cag gaa gat atc gcc acc tac ttt tgc cag cag ggc aac aca 336 Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr 100 105 110 ctg ccc tac acc ttt ggc ggc gga aca aag ctg gaa atc acc ggc agc 384 Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser 115 120 125 acc tcc ggc agc ggc aag cct ggc agc ggc gag ggc agc acc aag ggc 432 Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly 130 135 140 gag gtg aag ctg cag gaa agc ggc cct ggc ctg gtg gcc ccc agc cag 480 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 145 150 155 160 agc ctg agc gtg acc tgc acc gtg agc ggc gtg agc ctg ccc gac tac 528 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 165 170 175 ggc gtg agc tgg atc cgg cag ccc ccc agg aag ggc ctg gaa tgg ctg 576 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 180 185 190 ggc gtg atc tgg ggc agc gag acc acc tac tac aac agc gcc ctg aag 624 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 195 200 205 agc cgg ctg acc atc atc aag gac aac agc aag agc cag gtg ttc ctg 672 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 210 215 220 aag atg aac agc ctg cag acc gac gac acc gcc atc tac tac tgc gcc 720 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 225 230 235 240 aag cac tac tac tac ggc ggc agc tac gcc atg gac tac tgg ggc cag 768 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 245 250 255 ggc acc agc gtg acc gtg agc agc gga ggt ggt gga tcc gag gtg cag 816 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Gln 260 265 270 ctg cag cag tct ggg gct gag ctg gtg aag cct ggg gcc tca gtg aag 864 Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys 275 280 285 atg tcc tgc aag gct tct ggc tac aca ttt acc agt tac aat atg cac 912 Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His 290 295 300 tgg gta aag cag aca cct gga cag ggc ctg gaa tgg att gga gct att 960 Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile 305 310 315 320 tat cca gga aat ggt gat act tcc tac aat cag aag ttc aaa ggc aag 1008 Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys 325 330 335 gcc aca ttg act gca gac aaa tcc tcc agc aca gcc tac atg cag ctc 1056 Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu 340 345 350 agc agc ctg aca tct gag gac tct gcg gac tat tac tgt gca aga tct 1104 Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser 355 360 365 aat tat tac ggt agt agc tac tgg ttc ttc gat gtc tgg ggc gca ggg 1152 Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly 370 375 380 acc acg gtc acc gtc tcc tca ggc agt act agc ggt ggt ggc tcc ggg 1200 Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly 385 390 395 400 ggc ggt tcc ggt ggg ggc ggc agc agc gac att gtg ctg acc caa tct 1248 Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu Thr Gln Ser 405 410 415 cca gct atc ctg tct gca tct cca ggg gag aag gtc aca atg act tgc 1296 Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 420 425 430 agg gcc agc tca agt gta aat tac atg gac tgg tac cag aag aag cca 1344 Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln Lys Lys Pro 435 440 445 gga tcc tcc ccc aaa ccc tgg att tat gcc aca tcc aac ctg gct tct 1392 Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser 450 455 460 gga gtc cct gct cgc ttc agt ggc agt ggg tct ggg acc tct tac tct 1440 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 465 470 475 480 ctc aca atc agc aga gtg gag gct gaa gat gct gcc act tat tac tgc 1488 Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 485 490 495 cag cag tgg agt ttt aat cca ccc acg ttc gga ggg ggg acc aag ctg 1536 Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 500 505 510 gaa ata aaa gag agc aag tac gga ccg ccc tgc ccc cct tgc cct atg 1584 Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met 515 520 525 ttc tgg gtg ctg gtg gtg gtc gga ggc gtg ctg gcc tgc tac agc ctg 1632 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 530 535 540 ctg gtc acc gtg gcc ttc atc atc ttt tgg gtg aaa cgg ggc aga aag 1680 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys 545 550 555 560 aaa ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa act 1728 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 565 570 575 act caa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa 1776 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 580 585 590 gga gga tgt gaa ctg cgg gtg aag ttc agc aga agc gcc gac gcc cct 1824 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 595 600 605 gcc tac cag cag ggc cag aat cag ctg tac aac gag ctg aac ctg ggc 1872 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 610 615 620 aga agg gaa gag tac gac gtc ctg gat aag cgg aga ggc cgg gac cct 1920 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 625 630 635 640 gag atg ggc ggc aag cct cgg cgg aag aac ccc cag gaa ggc ctg tat 1968 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 645 650 655 aac gaa ctg cag aaa gac aag atg gcc gag gcc tac agc gag atc ggc 2016 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670 atg aag ggc gag cgg agg cgg ggc aag ggc cac gac ggc ctg tat cag 2064 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 675 680 685 ggc ctg tcc acc gcc acc aag gat acc tac gac gcc ctg cac atg cag 2112 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 690 695 700 gcc ctg ccc cca agg ctc gag ggc ggc gga gag ggc aga gga agt ctt 2160 Ala Leu Pro Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu 705 710 715 720 cta aca tgc ggt gac gtg gag gag aat ccc ggc cct agg atg ctt ctc 2208 Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu 725 730 735 ctg gtg aca agc ctt ctg ctc tgt gag tta cca cac cca gca ttc ctc 2256 Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu 740 745 750 ctg atc cca cgc aaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa 2304 Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys 755 760 765 gac tca ctc tcc ata aat gct acg aat att aaa cac ttc aaa aac tgc 2352 Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys 770 775 780 acc tcc atc agt ggc gat ctc cac atc ctg ccg gtg gca ttt agg ggt 2400 Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly 785 790 795 800 gac tcc ttc aca cat act cct cct ctg gat cca cag gaa ctg gat att 2448 Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile 805 810 815 ctg aaa acc gta aag gaa atc aca ggg ttt ttg ctg att cag gct tgg 2496 Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp 820 825 830 cct gaa aac agg acg gac ctc cat gcc ttt gag aac cta gaa atc ata 2544 Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile 835 840 845 cgc ggc agg acc aag caa cat ggt cag ttt tct ctt gca gtc gtc agc 2592 Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser 850 855 860 ctg aac ata aca tcc ttg gga tta cgc tcc ctc aag gag ata agt gat 2640 Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp 865 870 875 880 gga gat gtg ata att tca gga aac aaa aat ttg tgc tat gca aat aca 2688 Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr 885 890 895 ata aac tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att 2736 Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile 900 905 910 ata agc aac aga ggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc 2784 Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys 915 920 925 cat gcc ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac 2832 His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp 930 935 940 tgc gtc tct tgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag 2880 Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys 945 950 955 960 tgc aac ctt ctg gag ggt gag cca agg gag ttt gtg gag aac tct gag 2928 Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu 965 970 975 tgc ata cag tgc cac cca gag tgc ctg cct cag gcc atg aac atc acc 2976 Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr 980 985 990 tgc aca gga cgg gga cca gac aac tgt atc cag tgt gcc cac tac att 3024 Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile 995 1000 1005 gac ggc ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga 3069 Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly 1010 1015 1020 gaa aac aac acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg 3114 Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 1025 1030 1035 tgc cac ctg tgc cat cca aac tgc acc tac gga tgc act ggg cca 3159 Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro 1040 1045 1050 ggt ctt gaa ggc tgt cca acg aat ggg cct aag atc ccg tcc atc 3204 Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile 1055 1060 1065 gcc act ggg atg gtg ggg gcc ctc ctc ttg ctg ctg gtg gtg gcc 3249 Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala 1070 1075 1080 ctg ggg atc ggc ctc ttc atg tga 3273 Leu Gly Ile Gly Leu Phe Met 1085 1090 <210> 3 <211> 1090 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Thr Gln Thr Thr Ser Ser Leu Ser Ala 20 25 30 Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile 35 40 45 Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 50 55 60 Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn 85 90 95 Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr 100 105 110 Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser 115 120 125 Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly 130 135 140 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 145 150 155 160 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 165 170 175 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 180 185 190 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 195 200 205 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 210 215 220 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 225 230 235 240 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 245 250 255 Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val Gln 260 265 270 Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys 275 280 285 Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His 290 295 300 Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile 305 310 315 320 Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys 325 330 335 Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu 340 345 350 Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser 355 360 365 Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly 370 375 380 Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly 385 390 395 400 Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu Thr Gln Ser 405 410 415 Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys 420 425 430 Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln Lys Lys Pro 435 440 445 Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser 450 455 460 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 465 470 475 480 Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 485 490 495 Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 500 505 510 Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met 515 520 525 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 530 535 540 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys 545 550 555 560 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 565 570 575 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 580 585 590 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 595 600 605 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 610 615 620 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 625 630 635 640 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 645 650 655 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 675 680 685 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 690 695 700 Ala Leu Pro Pro Arg Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu 705 710 715 720 Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu 725 730 735 Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu 740 745 750 Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys 755 760 765 Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys 770 775 780 Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly 785 790 795 800 Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile 805 810 815 Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp 820 825 830 Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile 835 840 845 Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser 850 855 860 Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp 865 870 875 880 Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr 885 890 895 Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile 900 905 910 Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys 915 920 925 His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp 930 935 940 Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys 945 950 955 960 Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu 965 970 975 Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr 980 985 990 Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile 995 1000 1005 Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly 1010 1015 1020 Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 1025 1030 1035 Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro 1040 1045 1050 Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile 1055 1060 1065 Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala 1070 1075 1080 Leu Gly Ile Gly Leu Phe Met 1085 1090 <210> 4 <211> 582 <212> DNA <213> Artificial Sequence <220> <223> IgG4hinge-CD28tm-41BB-CD3Zeta <400> 4 gagagcaagt acggaccgcc ctgcccccct tgccctatgt tctgggtgct ggtggtggtc ggaggcgtgc tggcctgcta cagcctgctg gtcaccgtgg ccttcatcat cttttgggtg 120 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggagatgg ctgtagctgc cgatttccag aagaagaga aggagatgt gaactgcggg tgaagttcag cagaagcgcc gacgcccctg cctaccagca gggccagaat cagctgtaca acgagctga cctgggcaga aggagagt acgacgtcct ggataagcgg agaggccggg accctgagat gggcggcaag cctcggcgga agaaccccca ggaaggcctg 420 father tgcagaaaga caagatggcc gaggcctaca gcgagatcgg catgaagggc gagcggaggc ggggcaaggg ccacgacggc ctgtatcagg gcctgtccac cgccaccag 540 gatacctacg acgccctgca catgcaggcc ctgccccc gg <210> 5 <211> 582 <212> DNA <213> Artificial Sequence <220> <223> IgG4hinge-CD28tm-41BB-CD3Zeta <220> <221> CDS <222> (1)..(582) <400> 5 gag agc aag tac gga ccg ccc tgc ccc cct tgc cct atg ttc tgg gtg 48 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val 1 5 10 15 ctg gtg gtg gtc gga ggc gtg ctg gcc tgc tac agc ctg ctg gtc acc 96 Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr 20 25 30 gtg gcc ttc atc atc ttt tgg gtg aaa cgg ggc aga aag aaa ctc ctg 144 Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu 35 40 45 tat ata ttc aaa caa cca ttt atg aga cca gta caa act act caa gag 192 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 50 55 60 gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa gga gga tgt 240 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 65 70 75 80 gaa ctg cgg gtg aag ttc agc aga agc gcc gac gcc cct gcc tac cag 288 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 85 90 95 cag ggc cag aat cag ctg tac aac gag ctg aac ctg ggc aga agg gaa 336 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 100 105 110 gag tac gac gtc ctg gat aag cgg aga ggc cgg gac cct gag atg ggc 384 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 115 120 125 ggc aag cct cgg cgg aag aac ccc cag gaa ggc ctg tat aac gaa ctg 432 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 130 135 140 cag aaa gac aag atg gcc gag gcc tac agc gag atc ggc atg aag ggc 480 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 145 150 155 160 gag cgg agg cgg ggc aag ggc cac gac ggc ctg tat cag ggc ctg tcc 528 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 165 170 175 acc gcc acc aag gat acc tac gac gcc ctg cac atg cag gcc ctg ccc 576 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 180 185 190 cca agg 582 Pro Arg <210> 6 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val 1 5 10 15 Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr 20 25 30 Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu 35 40 45 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 50 55 60 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 65 70 75 80 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 85 90 95 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 100 105 110 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 115 120 125 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 130 135 140 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 145 150 155 160 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 165 170 175 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 180 185 190 Pro Arg <210> 7 <211> 2910 <212> DNA <213> Artificial Sequence <220> <223> GMCSFRss-CD19scFv-Gly4serlinker-CD20scFv-huIgG4hinge / CH2 / CH3-CD28 tm / CD28cyto-41BB-CD3Zeta <400> 7 atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60 atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120 gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180 aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240 cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300 gaacgaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360 ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420 ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480 cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540 gtgagctgga tccggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600 agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660 agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720 tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780 accagcgtga ccgtgagcag cggaggtggt ggatccgagg tgcagctgca gcagtctggg 840 gctgagctgg tgaagcctgg ggcctcagtg aagatgtcct gcaaggcttc tggctacaca 900 tttaccagtt acaatatgca ctgggtaaag cagacacctg gacagggcct ggaatggatt 960 ggagctattt atccaggaaa tggtgatact tcctacaatc agaagttcaa aggcaaggcc 1020 acattgactg cagacaaatc ctccagcaca gcctacatgc agctcagcag cctgacatct 1080 gaggactctg cggactatta ctgtgcaaga tctaattatt acggtagtag ctactggttc 1140 ttcgatgtct ggggcgcagg gaccacggtc accgtctcct caggcagtac tagcggtggt 1200 ggctccgggg gcggttccgg tgggggcggc agcagcgaca ttgtgctgac ccaatctcca 1260 gctatcctgt ctgcatctcc aggggagaag gtcacaatga cttgcagggc cagctcaagt 1320 gtaaattaca tggactggta ccagaagaag ccaggatcct cccccaaacc ctggatttat 1380 gccacatcca acctggcttc tggagtccct gctcgcttca gtggcagtgg gtctgggacc 1440 tcttactctc tcaaatcag cagagtggag gctgaagaatg ctgccactta ttactgccag 1500 cagtggagtt ttaatccacc cacgttcgga ggggggacca agctggaaat aaaagagagc 1560 aagtacggac cgccctgccc cccttgccct gccccccgagt tcctgggcgg acccagcgtg 1620 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccggacccc cgaggtgacc 1680 tgcgtggtgg tggacgtgag ccaggaagat cccgaggtcc agttcaattg gtacgtggac 1740 ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagttcaa cagcacctac 1800 cgggtggtgt ctgtgctgac cgtgctgcac caggactggc tgaacggcaa agaatacaag 1860 tgcaaggtgt ccaacaaggg cctgcccagc agcatcgaaa agaccatcag caaggccaag 1920 ggccagcctc gccagcccca ggtgtacacc ctgcctccct cccaggaga gatgaccaag 1980 aaccaggtgt ccctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 2040 tgggagagca acggccagcc tgagaacaac tacaagacca cccctcccgt gctggacagc 2100 gacggcagct tcttcctgta cagccggctg accgtggaca agagccggtg gcaggaaggc 2160 aacgtcttta gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 2220 ctgagcctgt ccctgggcaa gatgttctgg gtgctggtgg tggtgggcgg ggtgctggcc 2280 tgctacagcc tgctggtgac agtggccttt atcatctttt gggtgcggag caagcggagc 2340 agaggcggcc acagcgacta catgaacatg acccccagac ggcctggccc cacccggaag 2400 cactaccagc cctacgcccc acccagggac tttgccgcct agaagcaa acggggcaga 2460 aagaaactcc tgtatatatt caaacaacca tttatgagac cagtaccaaac tactcaagag 2520 gaagatggct gtagctgccg atttccagaa gaagaagaag gagatgtga actgcgggtg 2580 aagttcagca gaagcgccga cgcccctgcc taccagcagg gccagaatca gctgtacaac 2640 gagctgaacc tgggcagaag ggaagagtac gacgtcctgg ataagcggag aggccgggac 2700 cctgagatgg gcggcaagcc tcggcggaag aacccccagg aaggcctgta taacgaactg 2760 cagaaagaca agatggccga ggcctacagc gagatcggca tgaagggcga gcggaggcgg 2820 ggcaagggcc acgacggcct gtatcagggc ctgtccaccg ccaccaagga tacctacgac 2880 gccctgcaca tgcaggccct gcccccaagg 2910 <210> 8 <211> 2910 <212> DNA <213> Artificial Sequence <220> <223> GMCSFR-ssCD19scFv-Gly4serlinker-CD20scFv-huIgG4hinge / CH2 / CH3-CD28 tm / CD28cyto-41BB-CD3Zeta <220> <221> CDS <222> (1)..(2910) <400> 8 atg ctg ctg ctg gtg acc agc ctg ctg ctg tgc gag ctg ccc cac ccc 48 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 gcc ttt ctg ctg atc ccc gac atc cag atg acc cag acc acc tcc agc 96 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 ctg agc gcc agc ctg ggc gac cgg gtg acc atc agc tgc cgg gcc agc 144 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 cag gac atc agc aag tac ctg aac tgg tat cag cag aag ccc gac ggc 192 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 acc gtc aag ctg ctg atc tac cac acc agc cgg ctg cac agc ggc gtg 240 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 ccc agc cgg ttt agc ggc agc ggc tcc ggc acc gac tac agc ctg acc 288 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 atc tcc aac ctg gaa cag gaa gat atc gcc acc tac ttt tgc cag cag 336 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 ggc aac aca ctg ccc tac acc ttt ggc ggc gga aca aag ctg gaa atc 384 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 acc ggc agc acc tcc ggc agc ggc aag cct ggc agc ggc gag ggc agc 432 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 acc aag ggc gag gtg aag ctg cag gaa agc ggc cct ggc ctg gtg gcc 480 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 ccc agc cag agc ctg agc gtg acc tgc acc gtg agc ggc gtg agc ctg 528 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 ccc gac tac ggc gtg agc tgg atc cgg cag ccc ccc agg aag ggc ctg 576 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 gaa tgg ctg ggc gtg atc tgg ggc agc gag acc acc tac tac aac agc 624 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 gcc ctg aag agc cgg ctg acc atc atc aag gac aac agc aag agc cag 672 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 gtg ttc ctg aag atg aac agc ctg cag acc gac gac acc gcc atc tac 720 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 tac tgc gcc aag cac tac tac tac ggc ggc agc tac gcc atg gac tac 768 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 tgg ggc cag ggc acc agc gtg acc gtg agc agc gga ggt ggt gga tcc 816 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser 260 265 270 gag gtg cag ctg cag cag tct ggg gct gag ctg gtg aag cct ggg gcc 864 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 275 280 285 tca gtg aag atg tcc tgc aag gct tct ggc tac aca ttt acc agt tac 912 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 290 295 300 aat atg cac tgg gta aag cag aca cct gga cag ggc ctg gaa tgg att 960 Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 305 310 315 320 gga gct att tat cca gga aat ggt gat act tcc tac aat cag aag ttc 1008 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 325 330 335 aaa ggc aag gcc aca ttg act gca gac aaa tcc tcc agc aca gcc tac 1056 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 340 345 350 atg cag ctc agc agc ctg aca tct gag gac tct gcg gac tat tac tgt 1104 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys 355 360 365 gca aga tct aat tat tac ggt agt agc tac tgg ttc ttc gat gtc tgg 1152 Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp 370 375 380 ggc gca ggg acc acg gtc acc gtc tcc tca ggc agt act agc ggt ggt 1200 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly 385 390 395 400 ggc tcc ggg ggc ggt tcc ggt ggg ggc ggc agc agc gac att gtg ctg 1248 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu 405 410 415 acc caa tct cca gct atc ctg tct gca tct cca ggg gag aag gtc aca 1296 Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr 420 425 430 atg act tgc agg gcc agc tca agt gta aat tac atg gac tgg tac cag 1344 Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln 435 440 445 aag aag cca gga tcc tcc ccc aaa ccc tgg att tat gcc aca tcc aac 1392 Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn 450 455 460 ctg gct tct gga gtc cct gct cgc ttc agt ggc agt ggg tct ggg acc 1440 Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 465 470 475 480 tct tac tct ctc aca atc agc aga gtg gag gct gaa gat gct gcc act 1488 Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr 485 490 495 tat tac tgc cag cag tgg agt ttt aat cca ccc acg ttc gga ggg ggg 1536 Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly 500 505 510 acc aag ctg gaa ata aaa gag agc aag tac gga ccg ccc tgc ccc cct 1584 Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 515 520 525 tgc cct gcc ccc gag ttc ctg ggc gga ccc agc gtg ttc ctg ttc ccc 1632 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 530 535 540 ccc aag ccc aag gac acc ctg atg atc agc cgg acc ccc gag gtg acc 1680 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 545 550 555 560 tgc gtg gtg gtg gac gtg agc cag gaa gat ccc gag gtc cag ttc aat 1728 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 565 570 575 tgg tac gtg gac ggc gtg gaa gtg cac aac gcc aag acc aag ccc aga 1776 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 580 585 590 gag gaa cag ttc aac agc acc tac cgg gtg gtg tct gtg ctg acc gtg 1824 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 595 600 605 ctg cac cag gac tgg ctg aac ggc aaa gaa tac aag tgc aag gtg tcc 1872 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 610 615 620 aac aag ggc ctg ccc agc agc atc gaa aag acc atc agc aag gcc aag 1920 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 625 630 635 640 ggc cag cct cgc gag ccc cag gtg tac acc ctg cct ccc tcc cag gaa 1968 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 645 650 655 gag atg acc aag aac cag gtg tcc ctg acc tgc ctg gtg aag ggc ttc 2016 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 660 665 670 tac ccc agc gac atc gcc gtg gag tgg gag agc aac ggc cag cct gag 2064 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 675 680 685 aac aac tac aag acc acc cct ccc gtg ctg gac agc gac ggc agc ttc 2112 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 690 695 700 ttc ctg tac agc cgg ctg acc gtg gac aag agc cgg tgg cag gaa ggc 2160 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 705 710 715 720 aac gtc ttt agc tgc agc gtg atg cac gag gcc ctg cac aac cac tac 2208 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 725 730 735 acc cag aag agc ctg agc ctg tcc ctg ggc aag atg ttc tgg gtg ctg 2256 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu 740 745 750 gtg gtg gtg ggc ggg gtg ctg gcc tgc tac agc ctg ctg gtg aca gtg 2304 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 755,760,765 gcc ttc atc atc ttt tgg gtg cgg agc aag cgg agc aga ggc ggc cac 2352 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His 770,775,780 agc gac tac atg aac atg acc ccc aga cgg cct ggc ccc acc cgg aag 2400 Ser Asp Tyr Met Asn Met Thr Pro Arg Pro Gly Pro Thr Arg Lys 785,790,795,800 cac tac cag ccc tac gcc cca ccc agg gac tt gcc gcc tac aga agc 2448 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 805 810 815 aaa cgg ggc aga aag aaa ctc ctg tat ata ttc aaa caa cca ttt atg Lys Arg Gly Arg Lys Leu Leu Tyr Ile Phe Phe Lys Gln Pro Phe Met 820 825 830 aga cca gta caa act act caa gag ga gat ggc tgt agc tgc cga ttt 2544 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 835 840 845 cca gaa gaa gaa gaa gga gga tgt gaa ctg cgg gtg aag ttc agc aga 2592 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 850 855 860 agc gcc gac gcc cct gcc tac cag cag ggc cag aat cag ctg tac aac 2640 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 865 870 875 880 gag ctg aac ctg ggc aga agg gaa gag tac gac gtc ctg gat aag cgg 2688 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 885 890 895 aga ggc cgg gac cct gag atg ggc ggc aag cct cgg cgg aag aac ccc 2736 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 900 905 910 cag gaa ggc ctg tat aac gaa ctg cag aaa gac aag atg gcc gag gcc 2784 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 915 920 925 tac agc gag atc ggc atg aag ggc gag cgg agg cgg ggc aag ggc cac 2832 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 930 935 940 gac ggc ctg tat cag ggc ctg tcc acc gcc acc aag gat acc tac gac 2880 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 945 950 955 960 gcc ctg cac atg cag gcc ctg ccc cca agg 2910 Ala Leu His Met Gln Ala Leu Pro Pro Arg 965 970 <210> 9 <211> 970 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser 260 265 270 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 275 280 285 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 290 295 300 Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 305 310 315 320 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 325 330 335 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 340 345 350 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys 355 360 365 Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp 370 375 380 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly 385 390 395 400 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu 405 410 415 Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr 420 425 430 Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln 435 440 445 Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn 450 455 460 Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 465 470 475 480 Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr 485 490 495 Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly 500 505 510 Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 515 520 525 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 530 535 540 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 545 550 555 560 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 565 570 575 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 580 585 590 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 595 600 605 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 610 615 620 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 625 630 635 640 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 645 650 655 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 660 665 670 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 675 680 685 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 690 695 700 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 705 710 715 720 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 725 730 735 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu 740 745 750 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 755 760 765 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His 770 775 780 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 785 790 795 800 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 805 810 815 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 820 825 830 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 835 840 845 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 850 855 860 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 865 870 875 880 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 885 890 895 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 900 905 910 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 915 920 925 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 930 935 940 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 945 950 955 960 Ala Leu His Met Gln Ala Leu Pro Pro Arg 965 970 <210> 10 <211> 3402 <212> DNA <213> Artificial Sequence <220> <223> GMCSFRss-CD19scFv-Gly4serlinker-CD20scFv-CD8alphaHinge-CD8alphaTM -41BB-CD3Zeta-T2A-EGFRt <400> 10 atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60 atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120 gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180 aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240 cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300 gaacgaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360 ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420 ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480 cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540 gtgagctgga tccggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600 agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660 agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720 tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780 accagcgtga ccgtgagcag cggaggtggt ggatccgagg tgcagctgca gcagtctggg 840 gctgagctgg tgaagcctgg ggcctcagtg aagatgtcct gcaaggcttc tggctacaca 900 tttaccagtt acaatatgca ctgggtaaag cagacacctg gacagggcct ggaatggatt 960 ggagctattt atccaggaaa tggtgatact tcctacaatc agaagttcaa aggcaaggcc 1020 acattgactg cagacaaatc ctccagcaca gcctacatgc agctcagcag cctgacatct 1080 gaggactctg cggactatta ctgtgcaaga tctaattatt acggtagtag ctactggttc 1140 ttcgatgtct ggggcgcagg gaccacggtc accgtctcct caggcagtac tagcggtggt 1200 ggctccgggg gcggttccgg tgggggcggc agcagcgaca ttgtgctgac ccaatctcca 1260 gctatcctgt ctgcatctcc aggggagaag gtcacaatga cttgcagggc cagctcaagt 1320 gtaaattaca tggactggta ccagaagaag ccaggatcct cccccaaacc ctggatttat 1380 gccacatcca acctggcttc tggagtccct gctcgcttca gtggcagtgg gtctgggacc 1440 tcttactctc tcacaatcag cagagtggag gctgaagatg ctgccactta ttactgccag 1500 cagtggagtt ttaatccacc cacgttcgga ggggggacca agctggaaat aaaagagagc 1560 aagtacggac cgccctgccc cccttgccct aagcctacca ccacccctgc ccctagacct 1620 ccaacacccg ccccaacaat cgccagccag cctctgtctc tgaggcccga ggcttgtaga 1680 ccagctgctg gcggagccgt gcacaccaga ggactggatt tcgcctgcga catctacatc 1740 tgggcccctc tggccggcac atgtggcgtg ctgctgctga gcctcgtgat caccaagcgg 1800 ggcagaaaga aactgctgta catctttaag cagcccttca tgcggcccgt gcagaccacc 1860 caggaagagg acggctgctc ctgcagattc cccgaggaag aagaaggcgg ctgcgagctg 1920 agagtgaagt tcagcagatc cgccgacgcc cctgcctacc agcagggaca gaaccagctg 1980 tacaacgagc tgaacctggg cagacgggaa gagtacgacg tgctggacaa gcggagaggc 2040 cgggaccctg agatgggcgg aaagcccaga agaaagaacc cccaggaagg cctgtataac 2100 gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgg 2160 agaagaggca agggccacga tggcctgtac cagggcctga gcaccgccac caaggacacc 2220 tatgacgccc tgcacatgca ggccctgcct ccaagactcg agggcggcgg agagggcaga 2280 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctaggat gcttctcctg 2340 gtgacaagcc ttctgctctg tgagttacca cacccagcat tcctcctgat cccacgcaaa 2400 gtgtgtaacg gaataggtat tggtgaattt aaagactcac tctccataaa tgctacgaat 2460 attaaacact tcaaaaactg cacctccatc agtggcgatc tccacatcct gccggtggca 2520 tttaggggtg actccttcac acatactcct cctctggatc cacaggaact ggatattctg 2580 aaaaccgtaa aggaaatcac agggtttttg ctgattcagg cttggcctga aaacaggacg 2640 gacctccatg cctttgagaa cctagaaatc atacgcggca ggaccaagca acatggtcag 2700 tttctcttg cagtcgtcag cctgaacata acatccttgg gattacgctc cctcaaggag 2760 ataagtgatg gagatgtgat aatttcagga aaaaaatt tgtgctatgc aaatacaata 2820 aactggaaaa aactgtttgg gacctccggt cagaaaacca aaattataag caacagaggt 2880 gaaaacagct gcaaggccac aggccaggtc tgccatgcct tgtgctcccc cgagggctgc 2940 tggggcccgg agcccaggga ctgcgtctct tgccggaatg tcagccgagg cagggaatgc 3000 gtggacaagt gcaaccttct ggagggtgag ccaagggagt ttgtggagaa ctctgagtgc 3060 atacagtgcc acccagagtg cctgcctcag gccatgaaca tcacctgcac aggacgggga 3120 ccagacaact gtatccagtg tgcccactac attgacggcc cccactgcgt caagacctgc 3180 ccggcaggag tcatgggaga aaacaacacc ctggtctgga agtacgcaga cgccggccat 3240 gtgtgccacc tgtgccatcc aaactgcacc tacggatgca ctgggccagg tcttgaaggc 3300 tgtccaacga atgggcctaa gatcccgtcc atcgccactg ggatggtggg ggccctcctc 3360 ttgctgctgg tggtggccct ggggatcggc ctcttcatgt ga 3402 <210> 11 <211> 3402 <212> DNA <213> Artificial Sequence <220> <223> GMCSFRss-CD19scFv-Gly4serlinker-CD20scFv-CD8alphaHinge-CD8alphaTM -41BB-CD3Zeta-T2A-EGFRt <220> <221> CDS <222> (1)..(3402) <400> 11 atg ctg ctg ctg gtg acc agc ctg ctg ctg tgc gag ctg ccc cac ccc 48 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 gcc ttt ctg ctg atc ccc gac atc cag atg acc cag acc acc tcc agc 96 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 ctg agc gcc agc ctg ggc gac cgg gtg acc atc agc tgc cgg gcc agc 144 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 cag gac atc agc aag tac ctg aac tgg tat cag cag aag ccc gac ggc 192 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 acc gtc aag ctg ctg atc tac cac acc agc cgg ctg cac agc ggc gtg 240 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 ccc agc cgg ttt agc ggc agc ggc tcc ggc acc gac tac agc ctg acc 288 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 atc tcc aac ctg gaa cag gaa gat atc gcc acc tac ttt tgc cag cag 336 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 ggc aac aca ctg ccc tac acc ttt ggc ggc gga aca aag ctg gaa atc 384 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 acc ggc agc acc tcc ggc agc ggc aag cct ggc agc ggc gag ggc agc 432 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 acc aag ggc gag gtg aag ctg cag gaa agc ggc cct ggc ctg gtg gcc 480 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 ccc agc cag agc ctg agc gtg acc tgc acc gtg agc ggc gtg agc ctg 528 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 ccc gac tac ggc gtg agc tgg atc cgg cag ccc ccc agg aag ggc ctg 576 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 gaa tgg ctg ggc gtg atc tgg ggc agc gag acc acc tac tac aac agc 624 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 gcc ctg aag agc cgg ctg acc atc atc aag gac aac agc aag agc cag 672 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 gtg ttc ctg aag atg aac agc ctg cag acc gac gac acc gcc atc tac 720 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 tac tgc gcc aag cac tac tac tac ggc ggc agc tac gcc atg gac tac 768 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 tgg ggc cag ggc acc agc gtg acc gtg agc agc gga ggt ggt gga tcc 816 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser 260 265 270 gag gtg cag ctg cag cag tct ggg gct gag ctg gtg aag cct ggg gcc 864 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 275 280 285 tca gtg aag atg tcc tgc aag gct tct ggc tac aca ttt acc agt tac 912 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 290 295 300 aat atg cac tgg gta aag cag aca cct gga cag ggc ctg gaa tgg att 960 Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 305 310 315 320 gga gct att tat cca gga aat ggt gat act tcc tac aat cag aag ttc 1008 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 325 330 335 aaa ggc aag gcc aca ttg act gca gac aaa tcc tcc agc aca gcc tac 1056 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 340 345 350 atg cag ctc agc agc ctg aca tct gag gac tct gcg gac tat tac tgt 1104 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys 355 360 365 gca aga tct aat tat tac ggt agt agc tac tgg ttc ttc gat gtc tgg 1152 Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp 370 375 380 ggc gca ggg acc acg gtc acc gtc tcc tca ggc agt act agc ggt ggt 1200 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly 385 390 395 400 ggc tcc ggg ggc ggt tcc ggt ggg ggc ggc agc agc gac att gtg ctg 1248 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu 405 410 415 acc caa tct cca gct atc ctg tct gca tct cca ggg gag aag gtc aca 1296 Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr 420 425 430 atg act tgc agg gcc agc tca agt gta aat tac atg gac tgg tac cag 1344 Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln 435 440 445 aag aag cca gga tcc tcc ccc aaa ccc tgg att tat gcc aca tcc aac 1392 Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn 450 455 460 ctg gct tct gga gtc cct gct cgc ttc agt ggc agt ggg tct ggg acc 1440 Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 465 470 475 480 tct tac tct ctc aca atc agc aga gtg gag gct gaa gat gct gcc act 1488 Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr 485 490 495 tat tac tgc cag cag tgg agt ttt aat cca ccc acg ttc gga ggg ggg 1536 Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly 500 505 510 acc aag ctg gaa ata aaa gag agc aag tac gga ccg ccc tgc ccc cct 1584 Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 515 520 525 tgc cct aag cct acc acc acc cct gcc cct aga cct cca aca ccc gcc 1632 Cys Pro Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 530 535 540 cca aca atc gcc agc cag cct ctg tct ctg agg ccc gag gct tgt aga 1680 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 545 550 555 560 cca gct gct ggc gga gcc gtg cac acc aga gga ctg gat ttc gcc tgc 1728 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 565 570 575 gac atc tac atc tgg gcc cct ctg gcc ggc aca tgt ggc gtg ctg ctg 1776 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 580 585 590 ctg agc ctc gtg atc acc aag cgg ggc aga aag aaa ctg ctg tac atc 1824 Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 595 600 605 ttt aag cag ccc ttc atg cgg ccc gtg cag acc acc cag gaa gag gac 1872 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 610 615 620 ggc tgc tcc tgc aga ttc ccc gag gaa gaa gaa ggc ggc tgc gag ctg 1920 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 625 630 635 640 aga gtg aag ttc agc aga tcc gcc gac gcc cct gcc tac cag cag gga 1968 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 645 650 655 cag aac cag ctg tac aac gag ctg aac ctg ggc aga cgg gaa gag tac 2016 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 660 665 670 gac gtg ctg gac aag cgg aga ggc cgg gac cct gag atg ggc gga aag 2064 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 675 680 685 ccc aga aga aag aac ccc cag gaa ggc ctg tat aac gaa ctg cag aaa 2112 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 690 695 700 gac aag atg gcc gag gcc tac agc gag atc gga atg aag ggc gag cgg 2160 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 705 710 715 720 aga aga ggc aag ggc cac gat ggc ctg tac cag ggc ctg agc acc gcc 2208 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 725 730 735 acc aag gac acc tat gac gcc ctg cac atg cag gcc ctg cct cca aga 2256 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 740 745 750 ctc gag ggc ggc gga gag ggc aga gga agt ctt cta aca tgc ggt gac 2304 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 755 760 765 gtg gag gag aat ccc ggc cct agg atg ctt ctc ctg gtg aca agc ctt 2352 Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu 770 775 780 ctg ctc tgt gag tta cca cac cca gca ttc ctc ctg atc cca cgc aaa 2400 Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys 785 790 795 800 gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata 2448 Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile 805 810 815 aat gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc 2496 Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly 820 825 830 gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat 2544 Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His 835 840 845 act cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag 2592 Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys 850 855 860 gaa atc ​​aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg 2640 Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr 865 870 875 880 gac ctc cat gcc ttt gag aac cta gaa atc ​​ata cgc ggc agg acc aag 2688 Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys 885 890 895 caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc 2736 Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser 900 905 910 ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att 2784 Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile 915 920 925 tca gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa 2832 Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys 930 935 940 ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt 2880 Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly 945 950 955 960 gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc 2928 Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser 965 970 975 ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg 2976 Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg 980 985 990 aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag 3024 Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu 995 1000 1005 ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc 3069 Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys 1010 1015 1020 cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga 3114 His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 1025 1030 1035 cgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac ggc 3159 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 1040 1045 1050 ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac 3204 Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn 1055 1060 1065 aac acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac 3249 Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His 1070 1075 1080 ctg tgc cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt 3294 Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu 1085 1090 1095 gaa ggc tgt cca acg aat ggg cct aag atc ccg tcc atc gcc act 3339 Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr 1100 1105 1110 ggg atg gtg ggg gcc ctc ctc ttg ctg ctg gtg gtg gcc ctg ggg 3384 Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly 1115 1120 1125 atc ggc ctc ttc atg tga 3402 Ile Gly Leu Phe Met 1130 <210> 12 <211> 1133 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30 Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45 Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60 Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95 Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110 Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140 Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160 Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205 Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220 Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240 Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser 260 265 270 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 275 280 285 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 290 295 300 Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 305 310 315 320 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 325 330 335 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 340 345 350 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys 355 360 365 Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp 370 375 380 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly 385 390 395 400 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Ile Val Leu 405 410 415 Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr 420 425 430 Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met Asp Trp Tyr Gln 435 440 445 Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn 450 455 460 Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 465 470 475 480 Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr 485 490 495 Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gly Gly 500 505 510 Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 515 520 525 Cys Pro Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 530 535 540 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 545 550 555 560 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 565 570 575 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 580 585 590 Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 595 600 605 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 610 615 620 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 625 630 635 640 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 645 650 655 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 660 665 670 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 675 680 685 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 690 695 700 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 705 710 715 720 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 725 730 735 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 740 745 750 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 755 760 765 Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu 770 775 780 Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys 785 790 795 800 Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile 805 810 815 Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly 820 825 830 Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His 835 840 845 Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys 850 855 860 Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr 865 870 875 880 Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys 885 890 895 Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser 900 905 910 Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile 915 920 925 Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys 930 935 940 Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly 945 950 955 960 Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser 965 970 975 Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg 980 985 990 Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu 995 1000 1005 Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys 1010 1015 1020 His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 1025 1030 1035 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 1040 1045 1050 Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn 1055 1060 1065 Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His 1070 1075 1080 Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu 1085 1090 1095 Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr 1100 1105 1110 Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly 1115 1120 1125 Ile Gly Leu Phe Met 1130 <210> 13 <211> 1146 <212> DNA <213> Artificial Sequence <220> <223> T2A-EGFRt <400> 13 ctcgagggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 60 cccggcccta ggatgcttct cctggtgaca agccttctgc tctgtgagtt accacaccca 120 gcattcctcc tgatcccacg caaagtgtgt aacggaatag gtattggtga atttaaagac 180 tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc catcagtggc 240 gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatac tcctcctctg 300 gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt tttgctgatt 360 caggcttggc ctgaaaacag gacggacctc catgcctttg agaacctaga aatcatacgc 420 ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa cataacatcc 480 ttgggattac gctccctcaa ggagataagt gatggagatg tgataatttc aggaaacaaa 540 aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc cggtcagaaa 600 accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca ggtctgccat 660 gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt ctcttgccgg 720 aatgtcagcc gaggcaggga atgcgtggac aagtgcaacc ttctggaggg tgagccaagg 780 gagtttgtgg agaactctga gtgcatacag tgccacccag agtgcctgcc tcaggccatg 840 aacatcacct gcacaggacg gggaccagac aactgtatcc agtgtgccca ctacattgac 900 ggcccccact gcgtcaagac ctgcccggca ggagtcatgg gagaaaacaa caccctggtc 960 tggaagtacg cagacgccgg ccatgtgtgc cacctgtgcc atccaaactg cacctacgga 1020 tgcactgggc caggtcttga aggctgtcca acgaatgggc ctaagatccc gtccatcgcc 1080 actgggatgg tgggggccct cctcttgctg ctggtggtgg ccctggggat cggcctcttc 1140 atgtga 1146 <210> 14 <211> 1146 <212> DNA <213> Artificial Sequence <220> <223> T2A-EGFRt <220> <221> CDS <222> (1)..(1146) <400> 14 ctc gag ggc ggc gga gag ggc aga gga agt ctt cta aca tgc ggt gac 48 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 gtg gag gag aat ccc ggc cct agg atg ctt ctc ctg gtg aca agc ctt 96 Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu 20 25 30 ctg ctc tgt gag tta cca cac cca gca ttc ctc ctg atc cca cgc aaa 144 Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys 35 40 45 gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata 192 Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile 50 55 60 aat gct acg aat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc 240 Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly 65 70 75 80 gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat 288 Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His 85 90 95 act cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag 336 Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys 100 105 110 gaa atc ​​aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg 384 Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr 115 120 125 gac ctc cat gcc ttt gag aac cta gaa atc ​​ata cgc ggc agg acc aag 432 Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys 130 135 140 caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc 480 Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser 145 150 155 160 ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att 528 Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile 165 170 175 tca gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa 576 Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys 180 185 190 ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt 624 Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly 195 200 205 gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc 672 Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser 210 215 220 ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg 720 Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg 225 230 235 240 aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag 768 Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu 245 250 255 ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac 816 Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His 260 265 270 cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga 864 Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly 275 280 285 cca gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc 912 Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys 290 295 300 gtc aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc 960 Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val 305 310 315 320 tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac 1008 Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn 325 330 335 tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat 1056 Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn 340 345 350 ggg cct aag atc ccg tcc atc gcc act ggg atg gtg ggg gcc ctc ctc 1104 Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu 355 360 365 ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg tga 1146 Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 370 375 380 <210> 15 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 15 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu 20 25 30 Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys 35 40 45 Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile 50 55 60 Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly 65 70 75 80 Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His 85 90 95 Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys 100 105 110 Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr 115 120 125 Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys 130 135 140 Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser 145 150 155 160 Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile 165 170 175 Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys 180 185 190 Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly 195 200 205 Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser 210 215 220 Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg 225 230 235 240 Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu 245 250 255 Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His 260 265 270 Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly 275 280 285 Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys 290 295 300 Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val 305 310 315 320 Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn 325 330 335 Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn 340 345 350 Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu 355 360 365 Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 370 375 380

Claims

1. a. At least two antigen-specific target-directed regions, b. Extracellular spacer domain, c. Transmembrane domain, d. At least one co-stimulatory domain, and e. Intracellular signal transduction domains A bispecific chimeric antigen receptor containing and co-expressed with a therapeutic control substance, Each antigen-specific target-directed region contains an antigen-specific single-chain Fv (scFv) fragment and binds to a different antigen. Bispecific chimeric antigen receptor.

2. The bispecific chimeric antigen receptor according to claim 1, wherein the therapeutic control substance comprises one or more of the following: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), selection marker, and combinations thereof.

3. The bispecific chimeric antigen receptor according to claim 2, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof.

4. The bispecific chimeric antigen receptor according to claim 2, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof.

5. The bispecific chimeric antigen receptor according to claim 2, wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof.

6. The bispecific chimeric antigen receptor according to claim 1, wherein the bispecific chimeric antigen receptor and the therapeutic control substance are linked via a cleavable linker.

7. The bispecific chimeric antigen receptor according to claim 6, wherein the cleavable linker is a self-cleavable linker.

8. The bispecific chimeric antigen receptor according to claim 7, wherein the cleavable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof.

9. The bispecific chimeric antigen receptor according to claim 1, wherein the extracellular spacer domain comprises one or more of the following: an antibody Fc fragment, or a functional equivalent, fragment, or derivative thereof; an antibody hinge region, or a functional equivalent, fragment, or derivative thereof; an antibody CH2 region; an antibody CH3 region; an artificial spacer sequence; and combinations thereof.

10. The bispecific chimeric antigen receptor according to claim 9, wherein the extracellular spacer domain comprises any one or more of the following: (i) the hinge, CH2, and CH3 regions of IgG4; (ii) the hinge region of IgG4; (iii) the hinge and CH2 region of IgG4; (iv) the hinge region of CD8α; (v) the hinge, CH2, and CH3 regions of IgG1; (vi) the hinge region of IgG1; (vii) the hinge and CH2 region of IgG1; or (vii) any combination thereof.

11. The bispecific chimeric antigen receptor according to claim 1, wherein the transmembrane domain comprises one or more of the transmembrane region of a type I transmembrane protein, an artificial hydrophobic sequence, and combinations thereof.

12. The bispecific chimeric antigen receptor according to claim 11, wherein the transmembrane domain comprises one or more of the transmembrane domain of the ζ chain of the T cell receptor complex, CD28, CD8α, and combinations thereof.

13. The bispecific chimeric antigen receptor according to claim 1, wherein the co-stimulatory domain comprises a signaling domain derived from one or more of the following: CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof.

14. The bispecific chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain comprises one or more signaling domains from among human CD3ζ chain, FcγRIII, FcεRI, the cytoplasmic terminal of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), and combinations thereof.

15. The bispecific chimeric antigen receptor according to claim 1, wherein each of the at least two antigen-specific target-directing domains targets an antigen independently selected from the group consisting of antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof.

16. The cancer-specific antigens mentioned above are 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, Fibronectin extradomain-B, Folate receptor 1, GD2, GD3 ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human cell dispersion factor (scatter) Factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH The bispecific chimeric antigen receptor according to claim 15, comprising one or more of the following: 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGF-1, VEGF-2, vimentin, and combinations thereof.

17. The bispecific chimeric antigen receptor according to claim 1, wherein the at least two antigen-specific target-directing regions bind to (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viiii) C-MET and HER2, or (ix) EGFR and ROR1.

18. The bispecific chimeric antigen receptor according to claim 1, wherein the at least two antigen-specific target-directing regions bind to CD19 and CD20.

19. The antigens specific to inflammatory diseases include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, and Lama (Lama) The bispecific chimeric antigen receptor according to claim 15, comprising one or more of the following: glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, scleroscin, SOST, TGFβ1, TNF-α, VEGF-A, and combinations thereof.

20. The bispecific chimeric antigen receptor according to claim 15, wherein the antigen specific to neurological disorders comprises one or more of β-amyloid, MABT5102A, and combinations thereof.

21. The bispecific chimeric antigen receptor according to claim 15, wherein the antigen specific to diabetes comprises one or more of L-1β, CD3, and combinations thereof.

22. The bispecific chimeric antigen receptor according to claim 15, wherein the antigen specific to cardiovascular disease comprises one or more of the following: C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β chain, ITGB2 (CD18), sphingosine-1-phosphate, and combinations thereof.

23. The bispecific chimeric antigen receptor according to claim 15, wherein the antigen specific to an infectious disease comprises one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, and combinations thereof.

24. A combination of the bispecific chimeric antigen receptor described in claim 1 and a therapeutic control substance.

25. The combination according to claim 24, wherein the therapeutic control substance comprises one or more of the following: truncated epidermal growth factor receptor (EGFRt), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), selection marker, and combinations thereof.

26. The combination according to claim 25, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof.

27. The combination according to claim 25, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof.

28. The combination according to claim 25, wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof.

29. The combination according to claim 24, wherein the bispecific chimeric antigen receptor and the therapeutic control substance are linked via a cleavable linker.

30. The combination according to claim 29, wherein the cuttable linker is a self-cutting type cuttable linker.

31. The combination according to claim 29, wherein the severable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof.

32. A polynucleotide encoding the bispecific chimeric antigen receptor described in claim 1 or the combination described in claim 24.

33. A polypeptide encoded by the polynucleotide described in claim 32.

34. A vector comprising the polynucleotide described in claim 32.

35. A virus comprising the polynucleotide described in claim 32.

36. The virus according to claim 35, which is an RNA virus.

37. The virus according to claim 35, which is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus.

38. Genetically modified cells comprising the polynucleotide described in claim 32, the chimeric antigen receptor described in claim 1, or the combination described in claim 24.

39. A genetically modified cell according to claim 38, which is a T lymphocyte (T cell).

40. The genetically modified cell according to claim 39, which is a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof.

41. The genetically modified cell according to claim 38, which is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.

42. a. Any one or more of the bispecific chimeric antigen receptor according to claim 1, the combination according to claim 24, the polypeptide according to claim 32, the vector according to claim 34, the virus according to claim 35, the genetically modified cell according to claim 38, and any combination thereof, b. Pharmaceutically acceptable carriers and A pharmaceutical composition containing [the specified substance].

43. The pharmaceutical composition according to claim 42, A composition adapted to biochemically interact with a therapeutic control substance for inhibiting the proliferation of cells expressing the therapeutic control substance, and A combination.

44. The composition adapted to biochemically interact with the therapeutic control substance is Herceptin, methotrexate, cetuximab, thymidine analog (e.g., ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), 6-thioguanine, synthetic dimerizing agent (dimerizing agent) The combination according to claim 43, which is one or more of the following: drug (e.g., AP1903), fludarabine phosphate, linamarin (lin), nucleoside analogs (e.g., BVDU, difluorodeoxycytidine (dFdC), 1-β-D-arabinofuranosylthymine (Ala-T)), indole-3-acetic acid (IAA), l-butionine-S,R-sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors, or any combination thereof.

45. A method for generating a certain amount of T cells expressing a chimeric antigen receptor, comprising the following steps: (i) the step of transfecting one or more T cells with the vector described in claim 34, and (ii) A step of stimulating one or more T cells with cells expressing an antigen targeted by at least two antigen-specific target-directing regions, cells expressing a recombinant antigen targeted by at least two antigen-specific target-directing regions, or cells expressing an antibody against a chimeric antigen receptor, such that the T cells proliferate to produce a certain amount of T cells.

46. A method for treating a disease in a person who requires treatment for the disease, The method is (i) A step of providing the composition according to claim 42, and (ii) the step of administering a therapeutically effective amount of the composition to a subject in order to treat the disease. Includes, At least two antigen-specific targeting regions each target an antigen, and at least one such antigen is associated with the disease. The method.

47. a. At least two antigen-specific target-directed regions, b. Extracellular spacer domain, c. Transmembrane domain, d. At least one co-stimulatory domain, and e. Intracellular signal transduction domains A bispecific chimeric antigen receptor that includes and is co-expressed with truncated epidermal growth factor receptor (EGFRt), Each antigen-specific target-directed region contains an antigen-specific single-chain Fv (scFv) fragment and binds to a different antigen. Bispecific chimeric antigen receptor.

48. The bispecific chimeric antigen receptor according to claim 47, which is co-expressed with a therapeutic control substance comprising one or more of the following: thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), select marker, and combinations thereof.

49. The bispecific chimeric antigen receptor according to claim 47, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof.

50. The bispecific chimeric antigen receptor according to claim 48, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated-DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof.

51. The bispecific chimeric antigen receptor according to claim 48, wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof.

52. The bispecific chimeric antigen receptor according to claim 47, wherein the bispecific chimeric antigen receptor and the therapeutic control substance are linked via a cleavable linker.

53. The bispecific chimeric antigen receptor according to claim 52, wherein the cleavable linker is a self-cleavable linker.

54. The bispecific chimeric antigen receptor according to claim 52, wherein the cleavable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof.

55. The bispecific chimeric antigen receptor according to claim 47, wherein the extracellular spacer domain comprises one or more of the following: an antibody Fc fragment, or a functional equivalent, fragment, or derivative thereof; an antibody hinge region, or a functional equivalent, fragment, or derivative thereof; an antibody CH2 region; an antibody CH3 region; an artificial spacer sequence; and combinations thereof.

56. The bispecific chimeric antigen receptor according to claim 55, wherein the extracellular spacer domain comprises any one or more of the following: (i) the hinge, CH2, and CH3 regions of IgG4; (ii) the hinge region of IgG4; (iii) the hinge and CH2 region of IgG4; (iv) the hinge region of CD8α; (v) the hinge, CH2, and CH3 regions of IgG1; (vi) the hinge region of IgG1; (vi) the hinge and CH2 region of IgG1; and (vii) any one or more combinations thereof.

57. The bispecific chimeric antigen receptor according to claim 47, wherein the transmembrane domain comprises one or more of the transmembrane region of a type I transmembrane protein, an artificial hydrophobic sequence, and combinations thereof.

58. The bispecific chimeric antigen receptor according to claim 57, wherein the transmembrane domain comprises one or more of the transmembrane domain of the zeta chain of the T cell receptor complex, CD28, CD8α, and combinations thereof.

59. The bispecific chimeric antigen receptor according to claim 47, wherein the co-stimulatory domain comprises a signaling domain derived from one or more of the following: CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof.

60. The bispecific chimeric antigen receptor according to claim 47, wherein the intracellular signaling domain comprises one or more signaling domains from among human CD3ζ chain, FcγRIII, FcεRI, the cytoplasmic terminal of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), and combinations thereof.

61. The bispecific chimeric antigen receptor according to claim 47, wherein each of the at least two antigen-specific target-directing domains targets an antigen independently selected from the group consisting of antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof.

62. The cancer-specific antigens are 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, and fibronectin. Extra Domain-B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Cell Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, Insulin-like Growth Factor I Receptor, Integrin α5β1, Integrin αvβ3, MORAb-009, MS4A1, MUC1, Mucin CanAg, N-Glycolylneuraminic Acid, NPC-1C, PDGF-Rα, PDL192, Phosphatidylserine, Prostate Cancer Cells, RANKL, RON, ROR1, SCH The bispecific chimeric antigen receptor according to claim 61, comprising one or more of the following: 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGF-1, VEGF-2, vimentin, and combinations thereof.

63. The bispecific chimeric antigen receptor according to claim 47, wherein the at least two antigen-specific target-directing regions bind to (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viiii) C-MET and HER2, or (ix) EGFR and ROR1.

64. The bispecific chimeric antigen receptor according to claim 47, wherein the at least two antigen-specific target-directing regions bind to CD19 and CD20.

65. The antigens specific to inflammatory diseases are AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE The bispecific chimeric antigen receptor according to claim 61, comprising one or more of the following: Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, llama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, sclerossin, SOST, TGFβ1, TNF-α, VEGF-A, and combinations thereof.

66. The bispecific chimeric antigen receptor according to claim 61, wherein the antigen specific to neurological disorders comprises one or more of β-amyloid, MABT5102A, and combinations thereof.

67. The bispecific chimeric antigen receptor according to claim 61, wherein the antigen specific to diabetes includes one or more of L-1β, CD3, and combinations thereof.

68. The bispecific chimeric antigen receptor according to claim 61, wherein the antigen specific to cardiovascular disease comprises one or more of the following: C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β chain, ITGB2 (CD18), sphingosine-1-phosphate, and combinations thereof.

69. The bispecific chimeric antigen receptor according to claim 61, wherein the antigen specific to an infectious disease comprises one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, and combinations thereof.

70. A combination of the bispecific chimeric antigen receptor according to claim 47 and EGFRt.

71. The combination according to claim 70, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof.

72. The combination according to claim 70, wherein the bispecific chimeric antigen receptor and the EGFRt are linked via a cleavable linker.

73. The combination according to claim 72, wherein the cuttable linker is a self-cutting type cuttable linker.

74. The combination according to claim 72, wherein the severable linker is one or more of a 2A linker, a 2A-like linker, or a functional equivalent thereof.

75. A polynucleotide encoding the bispecific chimeric antigen receptor described in claim 47 or the combination described in claim 70.

76. A polypeptide encoded by the polynucleotide described in claim 75.

77. A vector comprising the polynucleotide described in claim 75.

78. A virus comprising the polynucleotide described in claim 75.

79. The virus according to claim 78, which is an RNA virus.

80. The virus according to claim 78, which is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus.

81. Genetically modified cells comprising the polynucleotide described in claim 75, the chimeric antigen receptor described in claim 47, or the combination described in claim 70.

82. A genetically modified cell according to claim 81, which is a T lymphocyte (T cell).

83. The genetically modified cell according to claim 82, which is a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof.

84. The genetically modified cell according to claim 81, which is a natural killer (NK) cell, a hematopoietic stem cell (HSC), an embryonic stem cell, or a pluripotent stem cell.

85. a. Any one or more of the bispecific chimeric antigen receptor according to claim 47, the combination according to claim 70, the polypeptide according to claim 76, the vector according to claim 77, the virus according to claim 78, the genetically modified cell according to claim 81, and any combination thereof, b. Pharmaceutically acceptable carriers and A pharmaceutical composition containing [the specified substance].

86. The pharmaceutical composition according to claim 85, A composition adapted to biochemically interact with a therapeutic control substance for inhibiting the proliferation of cells expressing EGFRt. A combination.

87. The composition adapted to biochemically interact with the therapeutic control substance is herceptin, methotrexate, cetuximab, thymidine analog (e.g., ganciclovir), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-flurocytosine (5-FC), 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), 6-thioguanine, synthetic dimerizer (e.g., AP1903), flu phosphate The combination according to claim 86, which is one or more of the following: darabine, linamarin (lin), nucleoside analogs (e.g., BVDU, difluorodeoxycytidine (dFdC), 1-β-D-arabinofuranosylthymine (Ala-T)), indole-3-acetic acid (IAA), l-butionine-S,R-sulfoximine (BSO), rituximab (RTX), doxycycline, tyrosine kinase inhibitors, or combinations thereof.

88. A method for generating a certain amount of T cells expressing a chimeric antigen receptor, comprising the following steps: (i) the step of transfecting one or more T cells with the vector described in claim 77, and (ii) A step of stimulating one or more T cells with cells expressing an antigen targeted by at least two antigen-specific target-directing regions, cells expressing a recombinant antigen targeted by at least two antigen-specific target-directing regions, or cells expressing an antibody against a chimeric antigen receptor, such that the T cells proliferate to produce a certain amount of T cells.

89. A method for treating a disease in a person who requires treatment for the disease, The method is (i) A step of providing the composition according to claim 85, and (ii) the step of administering a therapeutically effective amount of the composition to a subject in order to treat the disease. Includes, At least two antigen-specific targeting regions each target an antigen, and at least one such antigen is associated with the disease. The method.

90. A bispecific chimeric antigen receptor comprising the sequence described in Figure 4, 9, or 11.

91. a. At least two antigen-specific target-directed regions, b. CD8α hinge extracellular spacer domain, c. CD8α transmembrane domain, d. 4-1BB costimulatory domain, and e. CD3ζ intracellular signal transduction domain A bispecific chimeric antigen receptor that includes and is co-expressed with truncated epidermal growth factor receptor (EGFRt), Each antigen-specific target-directed region contains an antigen-specific single-chain Fv (scFv) fragment and binds to a different antigen. The bispecific chimeric antigen receptor and EGFRt are linked via a T2A linker. Bispecific chimeric antigen receptor.

92. The bispecific chimeric antigen receptor according to claim 91, which is co-expressed with a therapeutic control substance comprising one or more of the following: thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), select marker, and combinations thereof.

93. The bispecific chimeric antigen receptor according to claim 91, wherein the EGFRt binds to one or more of the following: EGFR-specific siRNA, small molecules, anti-EGFR antibodies or their fragments, or combinations thereof.

94. The bispecific chimeric antigen receptor according to claim 92, wherein the selection marker comprises one or more of the following: dihydroxyfolate receptor (DHFR), mutant DHFR, methylated-DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), and combinations thereof.

95. The bispecific chimeric antigen receptor according to claim 92, wherein the CCR comprises any one or more of the following: (i) IL-7 cytokine-linker-IL7Rα, (ii) IL-7 cytokine-linker-extracellular domain of IL-7Rα-transmembrane domain of IL-7Rα-cytoplasmic domain of IL-2Rβ, (iii) IL-7 cytokine-linker-IL2Rβ, and (iv) any combination thereof.

96. The bispecific chimeric antigen receptor according to claim 91, wherein each of the at least two antigen-specific target-directing domains targets an antigen independently selected from the group consisting of antigens specific to cancer, inflammatory diseases, neuropathy, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof.

97. The cancer-specific antigens are 4-1BB, 5T4, adenocarcinoma antigen, α-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, and fibronectin. Extra Domain-B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Cell Dispersion Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, Insulin-like Growth Factor I Receptor, Integrin α5β1, Integrin αvβ3, MORAb-009, MS4A1, MUC1, Mucin CanAg, N-Glycolylneuraminic Acid, NPC-1C, PDGF-Rα, PDL192, Phosphatidylserine, Prostate Cancer Cells, RANKL, RON, ROR1, SCH The bispecific chimeric antigen receptor according to claim 96, comprising one or more of the following: 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGF-1, VEGF-2, vimentin, and combinations thereof.

98. The bispecific chimeric antigen receptor according to claim 96, wherein the at least two antigen-specific target-directing regions bind to (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viiii) C-MET and HER2, or (ix) EGFR and ROR1.

99. The bispecific chimeric antigen receptor according to claim 96, wherein the at least two antigen-specific target-directing regions bind to CD19 and CD20.

100. The antigens specific to inflammatory diseases are AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidine), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE The bispecific chimeric antigen receptor according to claim 96, comprising one or more of the following: Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, llama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAbβ7, sclerossin, SOST, TGFβ1, TNF-α, VEGF-A, and combinations thereof.

101. The bispecific chimeric antigen receptor according to claim 96, wherein the antigen specific to neurological disorders comprises one or more of β-amyloid, MABT5102A, and combinations thereof.

102. The bispecific chimeric antigen receptor according to claim 96, wherein the antigen specific to diabetes includes one or more of L-1β, CD3, and combinations thereof.

103. The bispecific chimeric antigen receptor according to claim 96, wherein the antigen specific to cardiovascular disease comprises one or more of the following: C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, β chain, ITGB2 (CD18), sphingosine-1-phosphate, and combinations thereof.

104. The bispecific chimeric antigen receptor according to claim 96, wherein the antigen specific to an infectious disease comprises one or more of the following: anthrax toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, and combinations thereof.

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