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WO2022111562A1 - Cellule immunitaire modifiée et son utilisation - Google Patents

Cellule immunitaire modifiée et son utilisation Download PDF

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Publication number
WO2022111562A1
WO2022111562A1 PCT/CN2021/133010 CN2021133010W WO2022111562A1 WO 2022111562 A1 WO2022111562 A1 WO 2022111562A1 CN 2021133010 W CN2021133010 W CN 2021133010W WO 2022111562 A1 WO2022111562 A1 WO 2022111562A1
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seq
modified cell
functionally active
fusion protein
targeting moiety
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Chinese (zh)
Inventor
李本尚
王天怡
万欣宇
杨晓敏
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Sph Biotherapeutics Shanghai Ltd
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Sph Biotherapeutics Shanghai Ltd
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Priority to CN202180079469.7A priority Critical patent/CN116635519A/zh
Publication of WO2022111562A1 publication Critical patent/WO2022111562A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present application relates to the field of biomedicine, in particular to a fusion protein, modified immune cells and applications thereof.
  • Chimeric antigen receptor T cell immunotherapy (Chimeric antigen receptor T-cell immunotherapy, CART) is an effective adoptive cellular immunotherapy developed in recent years. Engineered to make effector T lymphocytes produce targeted cytotoxicity against tumor cells to achieve the purpose of eliminating tumor cells in vivo.
  • T cells The targeting of CART depends on the transformation of T cells through genetic engineering technology, so that they can express specific single-chain antibodies (scFv) that can recognize their own tumor cell-associated antigens (TAA), which are produced by TAA.
  • a specific monoclonal antibody is composed of a light chain variable region (VL) and a heavy chain variable region (VH), with a flexible hinge region between VL and VH.
  • co-stimulatory molecules that can synergistically activate T cells, such as CD28, 4-1BB, and OX4, are simultaneously expressed in T cells.
  • Chain antibodies recognize tumor cells, and on the other hand, through the above-mentioned costimulatory molecules and CD3 ⁇ synergistic activation of T cells to produce targeted cytotoxicity.
  • CART technology has achieved great success in liquid tumors, but there has not been a major change in the application field of solid tumors.
  • the problems faced by CART treatment include: 1) The location of solid tumors is not like leukemia spread throughout the body. CART cells need to reach the foci of solid tumors and infiltrate into the tumor to function. This is the space for CART treatment of solid tumor tissue structure. 2) The inhibitory effect of the immune microenvironment inside the tumor will cause CART cells to fail to function normally, and T cells will be depleted; 3) The internal microenvironment of solid tumors has hypoxia, lack of nutrients, etc., which is not conducive to CART Cells proliferate and produce targeted cytotoxicity. Therefore, it is necessary to develop novel structures to treat tumors.
  • the present application provides a fusion protein, a modified cell comprising and/or expressing the fusion protein, and a modified cell comprising and/or expressing the fusion protein and a multispecific antibody, which have one of the following properties
  • the fusion protein can bind to a first targeting moiety of a multispecific antibody that activates a modified cell comprising the fusion protein, the first targeting moiety of the multispecific antibody;
  • Two targeting moieties can target tumor cells, thereby allowing the modified cells comprising the fusion protein to kill tumors; 2) expanding the scope of cellular immunotherapy; 3) the modified cells comprising the fusion protein attacking tumor cells It has efficacy and uniformity; 4) solves the problem of T cell exhaustion; 5) can treat solid tumors; 6) reduces the risk of cytokine storm; 7) does not cause autoimmune reactions.
  • the application provides a fusion protein comprising: 1) an antigen or a functionally active fragment thereof; 2) a costimulatory domain; and 3) an intracellular signaling domain.
  • the antigen comprises a tumor-associated antigen or a functionally active fragment thereof.
  • the antigen comprises an immune cell activation-associated antigen or a functionally active fragment thereof.
  • the immune cell activation-associated antigen or functionally active fragment thereof comprises an antigen or functionally active fragment thereof selected from the group consisting of CD45, CD56 and CD58.
  • the tumor-associated antigen or functionally active fragment thereof comprises an antigen or functionally active fragment thereof selected from the group consisting of CD19, CD20, CD22, CD33, CD38, CD123, CD7 and BCMA.
  • the tumor-associated antigen or functionally active fragment thereof comprises CD19 protein or functionally active fragment thereof.
  • the tumor-associated antigen or functionally active fragment thereof comprises CD20 protein or functionally active fragment thereof.
  • the tumor-associated antigen or functionally active fragment thereof comprises CD22 protein or functionally active fragment thereof.
  • the tumor-associated antigen or functionally active fragment thereof comprises CD33 protein or functionally active fragment thereof.
  • the tumor-associated antigen or functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, and 10.
  • the costimulatory domain comprises a costimulatory domain derived from a histone selected from the group consisting of 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, Ligands for JAML, CD244, CD100, ICOS, CD83, CD40 and MyD88.
  • a histone selected from the group consisting of 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM
  • the costimulatory domain comprises a costimulatory domain derived from 4-1BB.
  • the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:12.
  • the intracellular signaling domain comprises an intracellular signaling domain derived from a histone selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • a histone selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV),
  • the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:19.
  • the C-terminus of the tumor-associated antigen or functionally active fragment thereof is directly or indirectly linked to the N-terminus of the costimulatory domain.
  • the C-terminus of the co-stimulatory domain is directly or indirectly linked to the N-terminus of the intracellular signaling domain.
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO:20.
  • the application provides one or more isolated nucleic acid molecules encoding the fusion proteins.
  • the isolated nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO:21.
  • the application provides one or more vectors comprising the isolated nucleic acid molecules.
  • the vector comprises a viral vector.
  • the vector comprises a lentiviral vector.
  • the present application provides a modified cell comprising the fusion protein, the nucleic acid molecule and/or the vector.
  • the modified cells include immune cells.
  • the modified cells comprise immune effector cells.
  • the immune effector cells are selected from the group consisting of T cells, macrophages Natural killer cells (NK cells) and NKT cells.
  • the modified cells comprise T cells or macrophages
  • the modified cells comprise NK cells or NKT cells.
  • the modified cells further comprise and/or express one or more multispecific antibodies.
  • the multispecific antibody comprises a first targeting moiety and a second targeting moiety.
  • the first targeting moiety is capable of specifically binding a tumor-associated antigen.
  • the first targeting moiety recognizes the antigen or functionally active fragment thereof in the fusion protein.
  • the tumor-associated antigen targeted by the first targeting moiety is selected from the group consisting of the following histones or functionally active fragments thereof: CD19, CD20, CD22, CD33, CD38, CD123, CD7 and BCMA.
  • the tumor-associated antigen targeted by the second targeting moiety is selected from the following histones or functionally active fragments thereof: GD2, DLL1, DLL3, ROR1, GPC3, HER2, VEGFA, CD1a, CD33, B7H3, CD138, BCMA, EGFRVIII and ERBB2.
  • the first targeting moiety recognizes CD19 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes CD33 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes a CD19 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes a GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes the CD19 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes the B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes CD22 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes CD33 protein or functionally active fragment thereof.
  • the first targeting moiety recognizes a CD22 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes a GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes the CD22 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes the B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes the CD20 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes the CD33 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes a CD20 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes a GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes the CD20 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes the B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes a CD33 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes a GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety recognizes the CD33 protein or a functionally active fragment thereof
  • the second targeting moiety recognizes the B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety comprises a heavy chain variable region VH comprising HCDRl, HCDR2 and HCDR3, the HCDRl comprising SEQ ID NO:23, SEQ ID NO:31, SEQ ID The amino acid sequence shown in any one of NO:39, SEQ ID NO:55, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85 and SEQ ID NO:94.
  • the HCDR2 comprises SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO: :79 and the amino acid sequence shown in any one of SEQ ID NO:95.
  • the HCDR3 comprises SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 86 and SEQ ID NO: 96.
  • the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any of the group consisting of:
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25;
  • HCDR1 SEQ ID NO:31
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33;
  • HCDR1 SEQ ID NO:31
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33;
  • HCDR1 SEQ ID NO:39
  • HCDR2 SEQ ID NO:40
  • HCDR3 SEQ ID NO:41;
  • HCDR1 SEQ ID NO:39
  • HCDR2 SEQ ID NO:40
  • HCDR3 SEQ ID NO:47;
  • HCDR1 SEQ ID NO:39
  • HCDR2 SEQ ID NO:40
  • HCDR3 SEQ ID NO:52;
  • HCDR1 SEQ ID NO:55
  • HCDR2 SEQ ID NO:56
  • HCDR3 SEQ ID NO:57;
  • HCDR1 SEQ ID NO:62
  • HCDR2 SEQ ID NO:63
  • HCDR3 SEQ ID NO:64;
  • HCDR1 SEQ ID NO:70
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:72;
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:80
  • HCDR1 SEQ ID NO:85
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:86;
  • HCDR1 SEQ ID NO: 85
  • HCDR2 SEQ ID NO: 79
  • HCDR3 SEQ ID NO: 86;
  • HCDR1 SEQ ID NO:94
  • HCDR2 SEQ ID NO:95
  • HCDR3 SEQ ID NO:96.
  • the VH comprises SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 84, SEQ ID NO: 90 and SEQ ID NO: 93.
  • the first targeting moiety comprises a light chain variable region VL comprising LCDR1, LCDR2 and LCDR3 comprising SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, and SEQ ID NO: The amino acid sequence shown in any one of 98.
  • the LCDR2 comprises any one of SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:75, and SEQ ID NO:99 amino acid sequence shown.
  • the LCDR3 comprises SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:45, SEQ ID NO:60, SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 83, SEQ ID NO: 89 and SEQ ID NO: 100.
  • the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from any of the group consisting of:
  • LCDR1 SEQ ID NO: 27, LCDR2: SEQ ID NO: 28 and LCDR3: SEQ ID NO: 29;
  • LCDR1 SEQ ID NO:35
  • LCDR2 SEQ ID NO:36
  • LCDR3 SEQ ID NO:37;
  • LCDR1 SEQ ID NO:35
  • LCDR2 SEQ ID NO:36
  • LCDR3 SEQ ID NO:37;
  • LCDR1 SEQ ID NO:43
  • LCDR2 SEQ ID NO:44
  • LCDR3 SEQ ID NO:45;
  • LCDR1 SEQ ID NO: 49
  • LCDR2 SEQ ID NO: 44
  • LCDR3 SEQ ID NO: 50;
  • LCDR1 SEQ ID NO: 49
  • LCDR2 SEQ ID NO: 44
  • LCDR3 SEQ ID NO: 45;
  • LCDR1 SEQ ID NO: 59
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 60;
  • LCDR1 SEQ ID NO:66
  • LCDR2 SEQ ID NO:67
  • LCDR3 SEQ ID NO:68;
  • LCDR1 SEQ ID NO:74
  • LCDR2 SEQ ID NO:75
  • LCDR3 SEQ ID NO:76;
  • LCDR1 SEQ ID NO: 82
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 83;
  • LCDR1 SEQ ID NO: 88
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO: 92
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO:98
  • LCDR2 SEQ ID NO:99
  • LCDR3 SEQ ID NO:100.
  • the VL comprises SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:58, SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 65, SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 87, SEQ ID NO: 91 and SEQ ID NO: 97.
  • the second targeting moiety comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 comprising SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO:85, SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:136, SEQ ID NO: 143 and the amino acid sequence shown in any one of SEQ ID NO: 151.
  • the HCDR2 comprises SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:79, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 118, SEQ ID NO: 127, SEQ ID NO: 137, SEQ ID NO: 144 and SEQ ID NO: 152.
  • the HCDR3 comprises SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:86, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO The amino acid sequence shown in any one of SEQ ID NO: 112, SEQ ID NO: 119, SEQ ID NO: 128, SEQ ID NO: 138, SEQ ID NO: 145 and SEQ ID NO: 153.
  • the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any of the group consisting of:
  • HCDR1 SEQ ID NO:70
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:72;
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:80;
  • HCDR1 SEQ ID NO: 85
  • HCDR2 SEQ ID NO: 79
  • HCDR3 SEQ ID NO: 86;
  • HCDR1 SEQ ID NO:85
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:86;
  • HCDR1 SEQ ID NO:94
  • HCDR2 SEQ ID NO:95
  • HCDR3 SEQ ID NO:96;
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 110
  • HCDR2 SEQ ID NO: 111
  • HCDR3 SEQ ID NO: 112;
  • HCDR1 SEQ ID NO: 117
  • HCDR2 SEQ ID NO: 118
  • HCDR3 SEQ ID NO: 119;
  • HCDR1 SEQ ID NO: 126
  • HCDR2 SEQ ID NO: 127
  • HCDR3 SEQ ID NO: 128;
  • HCDR1 SEQ ID NO: 126
  • HCDR2 SEQ ID NO: 127
  • HCDR3 SEQ ID NO: 128;
  • HCDR1 SEQ ID NO: 136
  • HCDR2 SEQ ID NO: 137
  • HCDR3 SEQ ID NO: 138;
  • HCDR1 SEQ ID NO: 143
  • HCDR2 SEQ ID NO: 144
  • HCDR3 SEQ ID NO: 145;
  • HCDR1 SEQ ID NO: 151
  • HCDR2 SEQ ID NO: 152
  • HCDR3 SEQ ID NO: 153.
  • the VH comprises SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:101, SEQ ID NO :123, SEQ ID NO:109, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:142, and SEQ ID NO:150 amino acid sequence.
  • the second targeting moiety comprises a light chain variable region VL comprising LCDR1, LCDR2 and LCDR3 comprising SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:121, SEQ ID NO:130, SEQ ID NO:140, SEQ ID NO: 147 and the amino acid sequence shown in any one of SEQ ID NO: 155.
  • the LCDR2 comprises SEQ ID NO:75, SEQ ID NO:28, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:131, SEQ ID NO:148 and SEQ ID NO : the amino acid sequence shown in any one of 156.
  • the LCDR3 comprises SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:89, SEQ ID NO:89, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 115, SEQ ID NO: 122, SEQ ID NO: 132, SEQ ID NO: 141, SEQ ID NO: 149 and SEQ ID NO: 157.
  • the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from any of the group consisting of:
  • LCDR1 SEQ ID NO:74
  • LCDR2 SEQ ID NO:75
  • LCDR3 SEQ ID NO:76;
  • LCDR1 SEQ ID NO: 82
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 83;
  • LCDR1 SEQ ID NO: 88
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO: 92
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO: 98
  • LCDR2 SEQ ID NO: 99
  • LCDR3 SEQ ID NO: 100;
  • LCDR1 SEQ ID NO: 106
  • LCDR2 SEQ ID NO: 107
  • LCDR3 SEQ ID NO: 108;
  • LCDR1 SEQ ID NO: 106
  • LCDR2 SEQ ID NO: 107
  • LCDR3 SEQ ID NO: 108;
  • LCDR1 SEQ ID NO: 114
  • LCDR2 SEQ ID NO: 99
  • LCDR3 SEQ ID NO: 115;
  • LCDR1 SEQ ID NO: 121
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 122;
  • LCDR1 SEQ ID NO: 130
  • LCDR2 SEQ ID NO: 131
  • LCDR3 SEQ ID NO: 132;
  • LCDR1 SEQ ID NO: 130
  • LCDR2 SEQ ID NO: 131
  • LCDR3 SEQ ID NO: 132;
  • LCDR1 SEQ ID NO: 140
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 141;
  • LCDR1 SEQ ID NO: 147
  • LCDR2 SEQ ID NO: 148
  • LCDR3 SEQ ID NO: 149;
  • LCDR1 SEQ ID NO: 155
  • LCDR2 SEQ ID NO: 156
  • LCDR3 SEQ ID NO: 157.
  • the VL comprises SEQ ID NO:73, SEQ ID NO:81, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:97, SEQ ID NO:105, SEQ ID NO: :124, SEQ ID NO:113, SEQ ID NO:120, SEQ ID NO:129, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:146, and SEQ ID NO:154 amino acid sequence.
  • the targeting moiety comprises a VHH, scFv, Fab, dAb and/or derived protein structure.
  • the targeting moiety is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the first targeting moiety comprises an scFv.
  • the second targeting moiety comprises an scFv.
  • the first targeting moiety is fused in-frame with the second targeting moiety.
  • the first targeting moiety is linked to the second targeting moiety through a hinge region.
  • the hinge region comprises hinge regions derived from the following histones: CD8, CD28, and 4-1BB.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID NO:17.
  • the modified cell comprises and/or expresses a nucleic acid molecule encoding the fusion protein and/or a nucleic acid molecule encoding the multispecific antibody.
  • nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody are on separate vectors.
  • nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody are on the same vector.
  • the nucleic acid molecule encoding a fusion protein is linked to the nucleic acid molecule encoding a multispecific antibody through a sequence encoding a cleavage peptide.
  • the nucleic acid sequence encoding a cleavage peptide is selected from the group consisting of a P2A sequence, a T2A sequence, an F2A sequence, an E2A sequence, a BmCPV2A sequence, and a BmIFV2A sequence.
  • the cleavage peptide sequence comprises a P2A sequence.
  • the cleavage peptide sequence comprises a T2A sequence.
  • the cleavage peptide sequence in the modified cell comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 13-16.
  • the fusion protein and the multispecific antibody are expressed in different amounts.
  • the present application provides a pharmaceutical composition comprising the modified cells, and/or the one or more multispecific antibodies.
  • the multispecific antibody in the pharmaceutical composition comprises a protein molecule comprising a first targeting moiety and a second targeting moiety prepared and purified using bioengineering techniques.
  • the multispecific antibody is infused into a blood vessel.
  • the present application provides use of the modified cells and/or the one or more multispecific antibodies in the preparation of medicines.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein, the isolated nucleic acid molecule, the carrier, the modified cell, and/or a pharmaceutically acceptable adjuvants and/or excipients.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, It is used to treat tumors.
  • the tumor comprises a hematological tumor.
  • the tumor comprises a solid tumor.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, It is used to treat diseases or disorders associated with the expression of CD33.
  • the disease or disorder associated with expression of CD33 comprises human myeloid leukemia (AML).
  • the present application provides the fusion protein, the isolated nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition for use in A disease or disorder associated with the expression of GD2 is treated.
  • the disease or disorder associated with the expression of GD2 comprises neuroblastoma.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, It is used to treat diseases or disorders associated with the expression of B7H3.
  • the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, It is used to treat diseases or disorders associated with the expression of HER2.
  • the disease or disorder associated with the expression of HER2 comprises breast cancer.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors of any one, the modified cells, and/or the drugs Compositions for the treatment of diseases or disorders associated with the expression of DLL1.
  • the disease or disorder associated with the expression of DLL1 comprises lung cancer.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition related to the expression of CD33.
  • the disease or disorder associated with the expression of CD33 comprises human acute myeloid leukemia (AML).
  • AML human acute myeloid leukemia
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition related to the expression of GD2.
  • the disease or disorder associated with the expression of GD2 comprises neuroblastoma.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition associated with the expression of B7H3.
  • the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
  • the present application provides the fusion protein, the isolated nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition in the preparation of medicine Use of the medicament for the treatment of a disease or condition associated with the expression of HER2.
  • the disease or disorder associated with the expression of HER2 comprises breast cancer.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition associated with the expression of DLL1.
  • the disease or disorder associated with the expression of DLL1 comprises lung cancer.
  • the present application provides a method for preventing and/or treating tumors, comprising administering an effective amount of the fusion protein, the isolated nucleic acid molecule, and the vector to a subject in need thereof , the modified cells, and/or the pharmaceutical composition.
  • the present application provides a method for preventing and/or treating a disease or condition associated with the expression of CD33, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated The nucleic acid molecule, any one of the vector, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of CD33 comprises human acute myeloid leukemia (AML).
  • AML human acute myeloid leukemia
  • the application provides a method for preventing and/or treating a disease or condition associated with the expression of GD2, comprising administering an effective amount of the fusion protein to a subject in need, and the isolated The nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of GD2 comprises neuroblastoma.
  • the application provides a method of preventing and/or treating a disease or condition associated with the expression of B7H3, comprising administering the fusion protein, the isolated nucleic acid molecule to a subject in need thereof , the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
  • the application provides a method for preventing and/or treating a disease or condition associated with the expression of HER2, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated The nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of HER2 comprises breast cancer.
  • the application provides a method of preventing and/or treating a disease or condition associated with the expression of DLL1, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated The nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of DLL1 comprises lung cancer.
  • Figure 1 shows I-1 ⁇ I-4, I-1-1 ⁇ I-1-3, I-2-1 ⁇ I-2-3, I-3-1 ⁇ I-3 described in this application -3. AgCAR structures of I-4-1 to I-4-2.
  • FIG. 2 shows the AgCAR structures of II-1 to II-11 described in this application.
  • Figure 3 shows the targeted cytotoxicity of cells with a genetically engineered vector (AgCAR) structure described in this application.
  • AgCAR genetically engineered vector
  • Figure 4 shows the vector structure diagram of the CD19AgCAR backbone described in this application.
  • Figure 5 shows the vector structure diagram of the CD19AgCAR Double antibody described in this application.
  • Figure 6 shows the vector structure diagram of the CD19AgCAR described in this application.
  • Figure 7 shows the transfection efficiency of human T lymphocytes transfected by lentivirus with CD19AgCAR, lentivirus with CD19AgCAR backbone, and lentivirus with CD19AgCAR Double antibody described in this application.
  • Figures 8A-J show that T cells with CD19AgCAR, T cells with CD19AgCAR backbone, T cells with CD19AgCAR Double antibody, CD19-CART and CD33-CART kill CD33-positive myeloid leukemia cells in vitro as described in the present application The effect of strain HL60.
  • Figure 9A-L shows the killing of GD2-positive neuroblastoma cell line CHP134 by T cells with CD19AgCAR, T cells with CD19AgCAR backbone, T cells with CD19AgCAR Double antibody and GD2-CART described in this application in vitro Effect.
  • Figure 10 shows the residual MV4 in the experiment of co-incubation of T cells with CD22AgCAR, T cells with CD20AgCAR and T cells with CD19AgCAR described in this application with human acute lymphoblastic leukemia cell line MV4;11;11 Flow cytometry results.
  • Figure 11 shows the in vivo effect test of T cells with CD19AgCAR-33 described in this application.
  • Figures 12A-B show the in vivo efficacy assay of T cells with CD19AgCAR-B7H3 as described in the present application.
  • the term "antigen” or “Ag” generally refers to a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immunocompetent cells, or both.
  • the antigens include tumor-associated antigens.
  • tumor-associated antigens can be expressed on the surface of tumor cells.
  • tumor-associated antigens can also be expressed on the surface of other cells (eg, immune cells).
  • tumor-associated antigens can include, but are not limited to, CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA.
  • the antigen includes an immune cell activation-associated antigen.
  • antigens associated with immune cell activation can include, but are not limited to, CD45, CD56, and CD58.
  • any macromolecule including nearly any protein or peptide, can be used as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein.
  • antigen need not be encoded solely by the full-length nucleotide sequence of the gene.
  • an antigen may not be encoded by a "gene”.
  • an antigen can be synthetic or can be derived from a biological sample.
  • biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.
  • the antigen can be from a non-human animal, including but not limited to mammals such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates.
  • single chain antibody or “scFv” generally refers to a molecule of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the heavy and light chain variable regions of the scFv can be from CD19, CD20, CD33, GD2, B7H3, or CD22.
  • CD19 generally refers to an important membrane antigen on human B lymphocytes involved in proliferation and differentiation.
  • the amino acid sequence of human CD19 can be found in UniProt/Swiss-Prot Accession No. P15391.
  • the fusion protein may comprise part or all of the amino acid sequence of human CD19.
  • CD22 generally refers to an important membrane antigen on human B lymphocytes involved in proliferation and differentiation.
  • the amino acid sequence of human CD22 can be found in UniProt/Swiss-Prot Accession No. P20273.
  • the fusion protein may comprise part or all of the amino acid sequence of human CD22.
  • CD20 generally refers to an important membrane antigen on human B lymphocytes involved in proliferation and differentiation.
  • the amino acid sequence of human CD20 can be found in UniProt/Swiss-Prot Accession No. P11836.
  • the fusion protein may comprise part or all of the amino acid sequence of human CD20.
  • CD33 generally refers to myeloid differentiation antigen.
  • the amino acid sequence of human CD33 can be found in UniProt/Swiss-Prot Accession No. P20138.
  • the fusion protein may comprise part or all of the amino acid sequence of human CD33.
  • GD2 generally refers to a ganglioside.
  • GD2 can be expressed on the surface of neuroectodermal tumors, including but not limited to tumors such as neuroblastoma, melanoma, and osteosarcoma.
  • B7H3 generally refers to a transmembrane protein belonging to the B7 family.
  • B7H3 can be expressed on the surface of various tumor cells.
  • the amino acid sequence of human B7H3 can be found in UniProt/Swiss-Prot accession number Q5ZPR3.
  • the fusion protein may comprise part or all of the amino acid sequence of human B7H3.
  • ERBB2 generally refers to one of the members of the epidermal growth factor receptor (EGFR) family.
  • the amino acid sequence of human CD20 can be found in UniProt/Swiss-Prot Accession No. P04626.
  • the term "functionally active fragment” generally refers to an amino acid sequence that has homology to a naturally occurring sequence or is encoded by a homologous nucleotide sequence and is capable of having one or more activities of the naturally occurring sequence.
  • peptide In the context of this application, a variant of any given sequence refers to one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one Sequence of endogenous functions.
  • Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.
  • homology may be equivalent to sequence "identity”.
  • homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • sequence alignments can be performed by various means known to those skilled in the art, eg, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • costimulatory signaling domain generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective lymphocyte responses to antigens.
  • the costimulatory signaling domain may comprise a costimulatory signaling domain derived from a histone selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, Ligands for CD244, CD100, ICOS, CD83, CD40 and MyD88.
  • a costimulatory signaling domain can include a costimulatory signaling domain derived from 4-1BB.
  • intracellular signaling domain generally refers to a domain located inside a cell capable of transducing a signal.
  • the intracellular signaling domain can transmit a signal into a cell.
  • an intracellular signaling domain is any contiguous amino acid sequence used to direct protein targeting.
  • the intracellular signaling domain can comprise an intracellular signaling domain derived from a protein selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, or a combination thereof , bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • the intracellular signaling domain can include an intracellular signaling domain derived from CD3 ⁇ .
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (eg, scFvs), and antibodies with antigens Bound antibody fragments (eg, Fab, Fab' and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • CDR complementarity determining region
  • the heavy chain variable region has 3 CDRs, which are named HCDR1, HCDR2 and HCDR3 for each variable region.
  • the exact boundaries of these CDRs have been defined differently from system to system.
  • the system described by Kabat Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a The residue numbering system is specified, and precise residue boundaries are provided that define the three CDRs. These CDRs may be referred to as Kabat CDRs.
  • the term "cell” generally refers to a plasmid or vector that can or has contained a nucleic acid molecule described herein, or is capable of expressing a chimeric antigen receptor described herein or an antigen binding agent described herein individual cells, cell lines or cell cultures of the protein.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the daughter cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the chimeric antigen receptors or antigen-binding proteins described herein. .
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be immune cells.
  • the immune cells can be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and /or peripheral blood mononuclear cells.
  • the immune cells can be T cells.
  • treating generally refers to: (i) preventing the occurrence of a disease, disorder or condition in a patient who may be susceptible to a disease, disorder and/or condition but has not been diagnosed with the disease; (ii) inhibiting the disease , disease or condition, i.e. arresting its development; and (iii) alleviating the disease, disorder or condition, i.e. causing the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subsided.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (eg, binding to labeling components).
  • amino acid includes natural and/or non-natural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably and generally refer to nucleosides of any length Polymeric forms of acids, such as deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA Transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any sequence
  • a polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation to labeled components.
  • the term "tumor” generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • the tumor may be a solid tumor or a hematological tumor.
  • the solid tumor can include neuroblastoma, Wilms tumor, hepatoblastoma, pancreatoblastoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, pulmonary blastoma, germ cell tumor, lung cancer, liver cancer, Breast cancer, bowel cancer, gastric cancer, gallbladder cancer, pancreatic cancer, prostate cancer, thyroid cancer, central nervous system tumors and other solid tumors.
  • the blood tumor can include acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, mast cell leukemia, plasma cell leukemia, myeloma.
  • multispecific antibody generally refers to antibody molecules that can recognize two or more antigens or epitopes simultaneously.
  • the multispecific antibody can be obtained in eukaryotic expression system or in prokaryotic expression system by chemical coupling method, hybrid-hybridoma method, genetic engineering antibody preparation method, and the like.
  • the multispecific antibody can be a bispecific antibody.
  • the term "subject” generally refers to any organism, including but not limited to mammals such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates.
  • a subject may be a human, pet or livestock.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides a fusion protein comprising: 1) an antigen or a functionally active fragment thereof; 2) a costimulatory domain; and 3) an intracellular signaling domain.
  • the fusion protein can include antigens or functionally active fragments thereof.
  • the antigen or functionally active fragment thereof may include an immune cell activation-associated antigen or functionally active fragment thereof.
  • the immune cell activation-related antigen or functionally active fragment thereof may include an antigen or functionally active fragment thereof selected from the group consisting of CD45, CD56 and CD58.
  • the antigens or functionally active fragments thereof may include tumor-associated antigens.
  • the tumor-associated antigen or functionally active fragment thereof may comprise an antigen or functionally active fragment thereof selected from the group consisting of CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA and B7H3.
  • the tumor-associated antigen may be an antigen associated with B lymphocyte development.
  • the tumor-associated antigen or a functionally active fragment thereof may include CD19 protein or a functionally active fragment thereof.
  • the tumor-associated antigen or a functionally active fragment thereof may include CD20 protein or a functionally active fragment thereof.
  • the tumor-associated antigen or a functionally active fragment thereof may include CD22 protein or a functionally active fragment thereof.
  • the tumor-associated antigen or a functionally active fragment thereof may include CD33 protein or a functionally active fragment thereof.
  • the tumor-associated antigen or a functionally active fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8 and 10, and the tumor-associated antigen or functionally active fragment thereof encodes the amino acid sequence.
  • the nucleic acid sequence may comprise the sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the costimulatory domain may comprise a costimulatory domain derived from a histone selected from the group consisting of: 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3 , SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML , CD244, CD100, ICOS, ligands for CD83, CD40 and MyD88.
  • a histone selected from the group consisting of: 4-1BB, CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3 , SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BT
  • the costimulatory domain may comprise a costimulatory domain derived from 4-1BB.
  • the costimulatory domain may comprise the amino acid sequence set forth in SEQ ID NO:12.
  • the nucleic acid sequence encoding the costimulatory domain may comprise the nucleic acid sequence shown in SEQ ID NO: 11.
  • the intracellular signaling domain may comprise an intracellular signaling domain derived from a histone selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa , bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • the intracellular signaling domain can include an intracellular signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence set forth in SEQ ID NO:19.
  • the nucleic acid sequence encoding the intracellular signaling domain may comprise the nucleic acid sequence shown in SEQ ID NO:18.
  • the C-terminus of the tumor-associated antigen or its functionally active fragment may be directly or indirectly linked to the N-terminus of the costimulatory domain.
  • the C-terminus of the co-stimulatory domain may be directly or indirectly linked to the N-terminus of the intracellular signaling domain.
  • the fusion protein from the N-terminus to the C-terminus, may comprise an antigen or a functionally active fragment thereof, a costimulatory domain and an intracellular signaling domain.
  • the antigen or functionally active fragment thereof, the costimulatory domain and the intracellular signaling domain may be fused in-frame (as shown in Figure 1).
  • the antigen or its functionally active fragment can be CD19 protein or its functionally active fragment, and its amino acid sequence can comprise the sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the amino acid sequence shown in SEQ ID NO:2 %, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • Its nucleic acid sequence may comprise the sequence shown in SEQ ID NO: 1.
  • the nucleic acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the nucleic acid sequence set forth in SEQ ID NO: 1 %, 95%, 96%, 97%, 98%, 99% or higher) sequence homology.
  • the antigen or its functionally active fragment can be CD20 protein or its functionally active fragment, and its amino acid sequence can comprise the sequence shown in SEQ ID NO:4.
  • the amino acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the amino acid sequence shown in SEQ ID NO:4 %, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • Its nucleic acid sequence may comprise the sequence shown in SEQ ID NO:3.
  • the nucleic acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the nucleic acid sequence shown in SEQ ID NO:3 %, 95%, 96%, 97%, 98%, 99% or higher) sequence homology.
  • the antigen or its functionally active fragment can be CD22 protein or its functionally active fragment, and its amino acid sequence can comprise the sequence shown in SEQ ID NO:6.
  • the amino acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the amino acid sequence shown in SEQ ID NO:6 %, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • Its nucleic acid sequence may comprise the sequence shown in SEQ ID NO:5.
  • the nucleic acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the nucleic acid sequence set forth in SEQ ID NO:5 %, 95%, 96%, 97%, 98%, 99% or higher) sequence homology.
  • the antigen or its functionally active fragment can be CD33 protein or its functionally active fragment, and its amino acid sequence can comprise the sequence shown in SEQ ID NO:8.
  • the amino acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the amino acid sequence set forth in SEQ ID NO:8 %, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • Its nucleic acid sequence may comprise the sequence shown in SEQ ID NO:9.
  • the nucleic acid sequence of the antigen or functionally active fragment thereof may comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the nucleic acid sequence set forth in SEQ ID NO:9 %, 95%, 96%, 97%, 98%, 99% or higher) sequence homology.
  • the costimulatory domain may comprise a costimulatory domain derived from 4-1BB, which may comprise the amino acid sequence shown in SEQ ID NO:12.
  • the costimulatory domain can comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequence homology.
  • the intracellular signaling domain may include an intracellular signaling domain derived from CD3 ⁇ , which may comprise the amino acid sequence shown in SEQ ID NO:19.
  • the amino acid sequence of the intracellular signaling domain can comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%) of the amino acid sequence set forth in SEQ ID NO: 19 %, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the fusion protein may comprise CD19 protein or its functionally active fragment, 4-1BB and CD3 ⁇ from the N-terminus to the C-terminus.
  • the fusion protein may comprise CD22 protein or its functionally active fragment, 4-1BB and CD3 ⁇ from N-terminus to C-terminus.
  • the fusion protein may comprise CD20 protein or its functionally active fragment, 4-1BB and CD3 ⁇ , from the N-terminus to the C-terminus.
  • the fusion protein may comprise CD33 protein or its functionally active fragment, 4-1BB and CD3 ⁇ from the N-terminus to the C-terminus.
  • the application provides one or more multispecific antibodies, which may include a first targeting moiety and a second targeting moiety, the first targeting moiety recognizing the antigen or functionally active fragment thereof in the fusion protein .
  • the first targeting moiety may bind to the antigen or functionally active fragment thereof in the fusion protein.
  • the first targeting moiety can specifically bind to a tumor-associated antigen in the fusion protein.
  • the tumor-associated antigen targeted by the first targeting moiety may be selected from the following histones or functionally active fragments thereof: CD19, CD20, CD22, CD33, CD38, CD123, CD7, BCMA and B7H3.
  • the second targeting moiety may target a tumor-associated antigen.
  • the tumor-associated antigen targeted by the second targeting moiety may be selected from the following histones or functionally active fragments thereof: GD2, DLL1, DLL3, ROR1, GPC3, HER2, VEGFA, CD1a, CD33, B7H3, CD138, BCMA, EGFRVIII.
  • the targeting moiety may include, but is not limited to, VHH, scFv, Fv fragments, Fab, Fab' fragments, F(ab')2 fragments, dAbs and/or derived protein structures.
  • the targeting moiety may include, but is not limited to, recombinant antibodies, monoclonal antibodies, fully human antibodies, murine antibodies, humanized antibodies, and chimeric antibodies.
  • the first targeting moiety may comprise a scFv.
  • the second targeting moiety may comprise a scFv (as shown in Figure 1).
  • the first targeting moiety can recognize CD19 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize CD33 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD19 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD19 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD22 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize CD33 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD22 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD22 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD20 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize CD33 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD20 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD20 protein or its functionally active fragment
  • the second targeting moiety can recognize B7H3 protein or its functionally active fragment
  • the first targeting moiety can recognize CD33 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize GD2 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD33 protein or a functionally active fragment thereof
  • the second targeting moiety can recognize B7H3 protein or a functionally active fragment thereof.
  • the first targeting moiety can recognize CD19 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise any one of SEQ ID NO: 22 and SEQ ID NO: 30
  • the amino acid sequence shown in the item can comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:30 , 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the first targeting moiety can recognize CD22 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise any one of SEQ ID NO:54 and SEQ ID NO:61
  • the amino acid sequence shown in the item can comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO:61 , 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the first targeting moiety can recognize the CD20 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID
  • the first targeting moiety can comprise at least 80% (eg, at least 85%, 90%, 91%) of the amino acid sequence set forth in SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:51 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the first targeting moiety can recognize CD33 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID The amino acid sequence shown in any one of NO:84, SEQ ID NO:90 and SEQ ID NO:93.
  • the first targeting moiety can comprise at least 80% of the amino acid sequence set forth in SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO:93 (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequences of sequence homology.
  • the second targeting moiety can recognize the CD33 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID The amino acid sequence shown in any one of NO:84, SEQ ID NO:90 and SEQ ID NO:93.
  • the first targeting moiety can comprise at least 80% of the amino acid sequence set forth in SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90 and SEQ ID NO:93 (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequences of sequence homology.
  • the second targeting moiety can recognize the GD2 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise SEQ ID NO: 101, SEQ ID NO: 123, SEQ ID The amino acid sequence shown in any one of NO: 109 and SEQ ID NO: 116.
  • the first targeting moiety can comprise at least 80% (eg, at least 85%) the amino acid sequence set forth in SEQ ID NO: 101, SEQ ID NO: 123, SEQ ID NO: 109, SEQ ID NO: 116 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the second targeting moiety can recognize B7H3 protein or a functionally active fragment thereof, which comprises a heavy chain variable region VH, and the VH can comprise any one of SEQ ID NO: 125, SEQ ID NO: 133
  • the first targeting moiety can comprise at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence set forth in SEQ ID NO: 125, SEQ ID NO: 133 , 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the first targeting moiety may comprise a heavy chain variable region VH
  • the heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3
  • the HCDR1 may comprise SEQ ID NO: 23, SEQ ID NO :31, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85, and SEQ ID NO:94 amino acid sequence.
  • the HCDR1 may comprise a combination of SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:55, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78,
  • the amino acid sequence set forth in any one of SEQ ID NO:85 and SEQ ID NO:94 has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%) %, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the HCDR2 may comprise SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:40, SEQ ID NO:56.
  • the HCDR2 may comprise a combination of SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79 and
  • the amino acid sequence set forth in any one of SEQ ID NO:95 has at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) %, 99% or higher) amino acid sequences of sequence homology.
  • the HCDR3 may comprise SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO: 64.
  • the HCDR3 may comprise a combination of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:52,
  • the amino acid sequences set forth in SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:86, and SEQ ID NO:96 have at least 80% (eg, at least 85%) , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the first targeting moiety may comprise a heavy chain variable region VH, which may include HCDR1, HCDR2 and HCDR3.
  • VH heavy chain variable region
  • the HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences selected from any one of the group, or the HCDR1, HCDR2, and HCDR3 may comprise at least 80% ( For example, amino acid sequences of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology:
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25;
  • HCDR1 SEQ ID NO:31
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33;
  • HCDR1 SEQ ID NO:31
  • HCDR2 SEQ ID NO:32
  • HCDR3 SEQ ID NO:33;
  • HCDR1 SEQ ID NO:39
  • HCDR2 SEQ ID NO:40
  • HCDR3 SEQ ID NO:41;
  • HCDR1 SEQ ID NO:39
  • HCDR2 SEQ ID NO:40
  • HCDR3 SEQ ID NO:47;
  • HCDR1 SEQ ID NO:39
  • HCDR2 SEQ ID NO:40
  • HCDR3 SEQ ID NO:52;
  • HCDR1 SEQ ID NO:55
  • HCDR2 SEQ ID NO:56
  • HCDR3 SEQ ID NO:57;
  • HCDR1 SEQ ID NO:62
  • HCDR2 SEQ ID NO:63
  • HCDR3 SEQ ID NO:64;
  • HCDR1 SEQ ID NO:70
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:72;
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:80
  • HCDR1 SEQ ID NO:85
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:86;
  • HCDR1 SEQ ID NO: 85
  • HCDR2 SEQ ID NO: 79
  • HCDR3 SEQ ID NO: 86;
  • HCDR1 SEQ ID NO:94
  • HCDR2 SEQ ID NO:95
  • HCDR3 SEQ ID NO:96.
  • the first targeting moiety may comprise a light chain variable region VL
  • the heavy chain variable region may comprise LCDR1, LCDR2 and LCDR3
  • the LCDR1 may comprise SEQ ID NO: 27, SEQ ID NO :35, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92 and the amino acid sequence shown in any one of SEQ ID NO:98.
  • the LCDR1 may comprise a combination of SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:66, SEQ ID NO:74,
  • the amino acid sequence set forth in any one of SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, and SEQ ID NO:98 has at least 80% (eg, at least 85%, 90%, 91%, 92%) %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the LCDR2 may comprise SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:28, SEQ ID NO:67, SEQ ID NO:75 and SEQ ID NO: The amino acid sequence shown in any one of 99.
  • the LCDR2 can be included in the combination with SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:28, SEQ ID NO:67, SEQ ID NO:75, and SEQ ID NO:99
  • the amino acid sequence shown in any one has at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) Amino acid sequences with high) sequence homology.
  • the LCDR3 may comprise SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:45, SEQ ID NO:60, SEQ ID NO: 68.
  • the LCDR3 may comprise a combination of SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:45, SEQ ID NO:60, SEQ ID NO:68,
  • the amino acid sequences set forth in SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:89, and SEQ ID NO:100 have at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%) , 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the light chain variable region VL may be included, and the heavy chain variable region may include LCDR1, LCDR2 and LCDR3.
  • the LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences selected from any one of the group, or the LCDR1, LCDR2, and LCDR3 may comprise at least 80% ( For example, amino acid sequences of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology:
  • LCDR1 SEQ ID NO: 27, LCDR2: SEQ ID NO: 28 and LCDR3: SEQ ID NO: 29;
  • LCDR1 SEQ ID NO:35
  • LCDR2 SEQ ID NO:36
  • LCDR3 SEQ ID NO:37;
  • LCDR1 SEQ ID NO:35
  • LCDR2 SEQ ID NO:36
  • LCDR3 SEQ ID NO:37;
  • LCDR1 SEQ ID NO:43
  • LCDR2 SEQ ID NO:44
  • LCDR3 SEQ ID NO:45;
  • LCDR1 SEQ ID NO: 49
  • LCDR2 SEQ ID NO: 44
  • LCDR3 SEQ ID NO: 50;
  • LCDR1 SEQ ID NO: 49
  • LCDR2 SEQ ID NO: 44
  • LCDR3 SEQ ID NO: 45;
  • LCDR1 SEQ ID NO: 59
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 60;
  • LCDR1 SEQ ID NO:66
  • LCDR2 SEQ ID NO:67
  • LCDR3 SEQ ID NO:68;
  • LCDR1 SEQ ID NO:74
  • LCDR2 SEQ ID NO:75
  • LCDR3 SEQ ID NO:76;
  • LCDR1 SEQ ID NO: 82
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 83;
  • LCDR1 SEQ ID NO: 88
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO: 92
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO:98
  • LCDR2 SEQ ID NO:99
  • LCDR3 SEQ ID NO:100.
  • the second targeting moiety may comprise a heavy chain variable region VH
  • the heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3
  • the HCDR1 may comprise SEQ ID NO: 70, SEQ ID NO :78, SEQ ID NO:85, SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:102, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:126
  • the HCDR1 can comprise a combination of SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:85, SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:102, Amino acid sequence shown in any one of SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:126, SEQ ID NO:136, SEQ ID NO:143 and SEQ ID NO:151 Amino acids with at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology sequence.
  • the HCDR2 may comprise SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO: 118.
  • the HCDR2 may comprise a combination with SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:79, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:118,
  • the amino acid sequence set forth in any one of SEQ ID NO: 127, SEQ ID NO: 127, SEQ ID NO: 137, SEQ ID NO: 144, and SEQ ID NO: 152 has at least 80% (e.g., at least 85%, 90%) %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the HCDR3 may comprise SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:86, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO: 112.
  • the HCDR3 can comprise a combination of SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:86, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO:104, SEQ ID NO:104, SEQ ID NO:104, The amino acid sequences set forth in SEQ ID NO: 112, SEQ ID NO: 119, SEQ ID NO: 128, SEQ ID NO: 128, SEQ ID NO: 138, SEQ ID NO: 145, and SEQ ID NO: 153 have at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequences of sequence homology.
  • variable region VH of the heavy chain can be included, and the variable region of the heavy chain can include HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences selected from any one of the group, or the HCDR1, HCDR2, and HCDR3 may comprise at least 80% ( For example, amino acid sequences of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology:
  • HCDR1 SEQ ID NO:70
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:72;
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:80;
  • HCDR1 SEQ ID NO: 85
  • HCDR2 SEQ ID NO: 79
  • HCDR3 SEQ ID NO: 86;
  • HCDR1 SEQ ID NO:85
  • HCDR2 SEQ ID NO:79
  • HCDR3 SEQ ID NO:86;
  • HCDR1 SEQ ID NO:94
  • HCDR2 SEQ ID NO:95
  • HCDR3 SEQ ID NO:96;
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 110
  • HCDR2 SEQ ID NO: 111
  • HCDR3 SEQ ID NO: 112;
  • HCDR1 SEQ ID NO: 117
  • HCDR2 SEQ ID NO: 118
  • HCDR3 SEQ ID NO: 119;
  • HCDR1 SEQ ID NO: 126
  • HCDR2 SEQ ID NO: 127
  • HCDR3 SEQ ID NO: 128;
  • HCDR1 SEQ ID NO: 126
  • HCDR2 SEQ ID NO: 127
  • HCDR3 SEQ ID NO: 128;
  • HCDR1 SEQ ID NO: 136
  • HCDR2 SEQ ID NO: 137
  • HCDR3 SEQ ID NO: 138;
  • HCDR1 SEQ ID NO: 143
  • HCDR2 SEQ ID NO: 144
  • HCDR3 SEQ ID NO: 145;
  • HCDR1 SEQ ID NO: 151
  • HCDR2 SEQ ID NO: 152
  • HCDR3 SEQ ID NO: 153.
  • the second targeting moiety may comprise a light chain variable region VL
  • the heavy chain variable region may comprise LCDR1, LCDR2 and LCDR3
  • the LCDR1 may comprise SEQ ID NO: 74, SEQ ID NO :82, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114, SEQ ID NO:121, SEQ ID NO:130, SEQ ID NO:140 , SEQ ID NO: 147 and the amino acid sequence shown in any one of SEQ ID NO: 155.
  • the LCDR1 may comprise a combination of SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:88, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:114,
  • the amino acid sequence set forth in any one of SEQ ID NO: 121, SEQ ID NO: 130, SEQ ID NO: 140, SEQ ID NO: 147, and SEQ ID NO: 155 has at least 80% (eg, at least 85%, 90%) %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequences of sequence homology.
  • the LCDR2 may comprise SEQ ID NO:75, SEQ ID NO:28, SEQ ID NO:28, SEQ ID NO:28, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO: 99.
  • the LCDR2 may comprise a combination of SEQ ID NO:75, SEQ ID NO:28, SEQ ID NO:28, SEQ ID NO:28, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:107, The amino acid sequence shown in any one of SEQ ID NO:99, SEQ ID NO:28, SEQ ID NO:131, SEQ ID NO:131, SEQ ID NO:28, SEQ ID NO:148 and SEQ ID NO:156 Amino acids with at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology sequence.
  • the LCDR3 may comprise SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:89, SEQ ID NO:89, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO: 108, SEQ ID NO: 115, SEQ ID NO: 122, SEQ ID NO: 132, SEQ ID NO: 132, SEQ ID NO: 141, SEQ ID NO: 149 and SEQ ID NO: 157 shown in any one of amino acid sequence.
  • the LCDR3 may comprise a combination of SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:89, SEQ ID NO:89, SEQ ID NO:100, SEQ ID NO:108, SEQ ID NO:108, SEQ ID NO:108,
  • the amino acid sequences set forth in SEQ ID NO: 115, SEQ ID NO: 122, SEQ ID NO: 132, SEQ ID NO: 132, SEQ ID NO: 141, SEQ ID NO: 149, and SEQ ID NO: 157 have at least 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequences of sequence homology.
  • the light chain variable region VL may be included, and the heavy chain variable region may include LCDR1, LCDR2 and LCDR3.
  • the LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences selected from any one of the group, or the LCDR1, LCDR2, and LCDR3 may comprise at least 80% ( For example, amino acid sequences of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology:
  • LCDR1 SEQ ID NO:74
  • LCDR2 SEQ ID NO:75
  • LCDR3 SEQ ID NO:76;
  • LCDR1 SEQ ID NO: 82
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 83;
  • LCDR1 SEQ ID NO: 88
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO: 92
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 89;
  • LCDR1 SEQ ID NO: 98
  • LCDR2 SEQ ID NO: 99
  • LCDR3 SEQ ID NO: 100;
  • LCDR1 SEQ ID NO: 106
  • LCDR2 SEQ ID NO: 107
  • LCDR3 SEQ ID NO: 108;
  • LCDR1 SEQ ID NO: 106
  • LCDR2 SEQ ID NO: 107
  • LCDR3 SEQ ID NO: 108;
  • LCDR1 SEQ ID NO: 114
  • LCDR2 SEQ ID NO: 99
  • LCDR3 SEQ ID NO: 115;
  • LCDR1 SEQ ID NO: 121
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 122;
  • LCDR1 SEQ ID NO: 130
  • LCDR2 SEQ ID NO: 131
  • LCDR3 SEQ ID NO: 132;
  • LCDR1 SEQ ID NO: 130
  • LCDR2 SEQ ID NO: 131
  • LCDR3 SEQ ID NO: 132;
  • LCDR1 SEQ ID NO: 140
  • LCDR2 SEQ ID NO: 28
  • LCDR3 SEQ ID NO: 141;
  • LCDR1 SEQ ID NO: 147
  • LCDR2 SEQ ID NO: 148
  • LCDR3 SEQ ID NO: 149;
  • LCDR1 SEQ ID NO: 155
  • LCDR2 SEQ ID NO: 156
  • LCDR3 SEQ ID NO: 157.
  • the first targeting moiety may be fused in-frame with the second targeting moiety.
  • the first targeting moiety may be linked to the second targeting moiety through a hinge region.
  • the hinge region may comprise hinge regions derived from the following histones: CD8, CD28 and 4-1BB.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 17.
  • the application also provides isolated one or more nucleic acid molecules that encode the fusion proteins described herein.
  • each of the one or more nucleic acid molecules may encode the entire fusion protein or a portion thereof (eg, HCDR1-3, one or more of the heavy chain variable regions) kind).
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
  • nucleic acid encoding the fusion protein can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the nucleic acid molecule of the fusion protein described in the present application .
  • the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more of the nucleic acid molecules may be included in each vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc.
  • a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, the promoters for SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's A portion may be fused to a portion of the gene promoter for other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector can be an expression vector.
  • the vector can be a viral vector.
  • the viral vector can be administered directly to the patient (in vivo) or can be administered in an indirect form, eg, by treating cells with the virus in vitro, and then administering the treated cells to the patient (ex vivo).
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise long terminal repeats 5'LTR and truncated 3'LTRs, RREs, rev response elements (cPPT), central termination sequences (CTS) and/or post-translational regulatory elements (WPRE).
  • the vectors described herein can be introduced into cells.
  • the vectors described herein can be lentiviral vectors.
  • the application provides one or more modified cells that can comprise and/or express the fusion protein, one or more (eg, 2 or more) of the nucleic acid molecules, and /or one or more (eg, 2 or more) of said carriers.
  • the cells may also contain and/or express the multispecific antibodies described herein.
  • the cells can include immune cells.
  • the cells can include immune effector cells.
  • the cells can include T cells, macrophages Natural killer cells (NK cells) and/or NKT cells.
  • NK cells Natural killer cells
  • the cells can include T cells.
  • the T cells may include thymocytes, naive T cells, immature T cells, mature T cells, resting T cells, or activated T cells.
  • the T cells may be helper T cells (Th), such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • the T cells may be CD4+ helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; CD8+ T cells), tumor infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+/CD8 + T cells, CD4-/CD8- T cells or any other T cell subtype.
  • the modified T cells are human T cells.
  • a source of cells can be obtained from a subject, eg, a patient, by various non-limiting methods.
  • T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available and known to those of skill in the art can be used.
  • the cells can be derived from a healthy donor, from a patient diagnosed with cancer, or obtained from a patient diagnosed with an infection.
  • the cells are part of a mixed population of cells presenting different phenotypic properties.
  • the method of treatment using the modified cells can be referred to as antigen-coupled antibody redirected T-cell immunotherapy (Antigen Coupling Antibody Redirect T-cell Immunotherapy, AgCAR- T).
  • the cells can include NK cells.
  • the NK cells can include CD56bright and CD56dim.
  • the NK cells can include NK1 and NK2.
  • the NK cells can include A-NK and NA-NK.
  • the method of treatment using the modified cells can be referred to as antigen-coupled antibody redirected NK cell immunotherapy (Antigen Coupling Antibody Redirect NK-cell Immunotherapy, AgCAR- NK).
  • the cells can include NKT cells.
  • the NKT cells can include type 1 NKT cells, type 2 NKT cells, and NKT-like cells.
  • the method of treatment using the modified cells can be referred to as antigen-coupled antibody redirected NKT cell immunotherapy (Antigen Coupling Antibody Redirect NKT-cell Immunotherapy, AgCAR- NKT).
  • the cells can include macrophages
  • a macrophage is a species that can engulf and digest cellular debris, microorganisms, cancer cells, and all other substances that lack the surface markers expressed on the surface of normal cells, a process called phagocytosis. Macrophages are present in almost all tissues and search for possible pathogens through the movement of amoeba. In addition to their important role in nonspecific innate immune responses, they can also help initiate adaptive immunity by recruiting other immune cell types, such as lymphocytes.
  • modified cells when used cells, using the modified cell therapy method can be referred to as antigen-conjugated antibody redirection Cellular Immunotherapy (Antigen Coupling Antibody Redirect Immunotherapy, ).
  • nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody may be located on different vectors.
  • the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding the multispecific antibody may be located on the same vector.
  • the fusion protein-encoding nucleic acid molecule can be linked to the multispecific antibody-encoding nucleic acid molecule through a sequence encoding a cleavage peptide.
  • the nucleic acid sequence encoding the cleavage peptide may be selected from the group consisting of P2A sequence, T2A sequence, F2A sequence, E2A sequence, BmCPV2A sequence and BmIFV2A sequence.
  • the nucleic acid sequence encoding the cleavage peptide may be a P2A sequence.
  • the nucleic acid sequence encoding the cleavage peptide may comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 13-16.
  • the expression level of the fusion protein and the multispecific antibody may be different.
  • the expression level of the fusion protein may differ from the expression level of the multispecific antibody by at least 1%.
  • the expression level of the fusion protein may differ from the expression level of the multispecific antibody by 1%, 5%, 6%, 7%, 10%, 15%, 20%, 25%, 30%, 40% , 50%, 60%, 70%, 80% or 90%.
  • the vector may comprise, in order, a nucleic acid sequence encoding an antigen or a functionally active fragment thereof, a nucleic acid sequence encoding a costimulatory domain, a nucleic acid sequence encoding a costimulatory domain, The nucleic acid sequence of the intracellular signaling domain, the cleavage peptide sequence, the nucleic acid sequence encoding the first targeting moiety and the nucleic acid sequence encoding the second targeting moiety (as shown in Figure 2).
  • the modified cell may express the fusion protein and the multispecific antibody, wherein the fusion protein is located on the surface of the modified cell, and the multispecific antibody is secreted to the modified cell Outside of the modified cell, the fusion protein can bind to and activate the modified cell with the first targeting moiety of the multispecific antibody, which can target the corresponding Tumor cells that exert cytotoxicity to kill tumors (eg, acute myeloid leukemia, neuroblastoma, or solid tumors), as shown in Figure 3.
  • tumors eg, acute myeloid leukemia, neuroblastoma, or solid tumors
  • compositions uses and methods
  • the application provides a pharmaceutical composition that may comprise the modified cells, and/or the one or more multispecific antibodies.
  • the multispecific antibody may comprise a purified protein molecule comprising a first targeting moiety and a second targeting moiety prepared using bioengineering techniques.
  • multispecific antibodies can be injected into the human body.
  • the multispecific antibody can be infused into the body via a blood vessel.
  • the modified cells can be injected into the human body.
  • the modified cell and the multispecific antibody can be co-administered.
  • the modified cell and the multispecific antibody can be administered separately.
  • the modified cells can be administered before the multispecific antibody.
  • the multispecific antibody can be administered before the modified cells.
  • the modified cell and the multispecific antibody can be administered in the same manner, eg, the modified cell and the multispecific antibody can be administered in a different manner.
  • the modified cells and the multispecific antibody can be placed in the same container.
  • the modified cells and the multispecific antibody can be placed in separate containers.
  • the multispecific antibody may comprise a protein molecule comprising a first targeting moiety and a second targeting moiety prepared and purified using bioengineering techniques, and the multispecific antibody may be imported into Intravascular and function with the fusion proteins and/or the modified cells described herein.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein, one or more (eg, 2 or more) of the isolated nucleic acid molecules, one or more (eg, 2 or more) the carrier, the modified cell, and/or a pharmaceutically acceptable adjuvant and/or excipient.
  • the present application provides the use of the modified cells and/or the one or more multispecific antibodies in the preparation of medicines.
  • the present application provides the fusion protein, the isolated nucleic acid molecule, the vector, the modified cell, and/or the pharmaceutical composition, which can be used to treat tumors .
  • the tumor can comprise a hematological tumor.
  • the tumor can comprise a solid tumor.
  • the present application provides a described fusion protein, described isolated nucleic acid molecule, described carrier, described modified cell, and/or described pharmaceutical composition, which can be used for A disease or disorder associated with the expression of CD33 is treated.
  • the disease or disorder associated with the expression of CD33 can include human myeloid leukemia.
  • the disease or disorder associated with the expression of CD33 can include human acute myeloid leukemia (AML).
  • AML human acute myeloid leukemia
  • the present application provides the fusion protein, the isolated nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition for use in A disease or disorder associated with the expression of GD2 is treated.
  • the disease or disorder associated with the expression of GD2 comprises neuroblastoma.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, It is used to treat diseases or disorders associated with the expression of B7H3.
  • the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, It is used to treat diseases or disorders associated with the expression of HER2.
  • the disease or disorder associated with the expression of HER2 comprises breast cancer.
  • the present application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions, which For the treatment of diseases or disorders associated with the expression of DLL1.
  • the disease or disorder associated with the expression of DLL1 comprises lung cancer.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition related to the expression of CD33.
  • the disease or disorder associated with the expression of CD33 comprises human myeloid leukemia.
  • the disease or disorder associated with the expression of CD33 comprises human acute myeloid leukemia (AML).
  • AML human acute myeloid leukemia
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition related to the expression of GD2.
  • the disease or disorder associated with the expression of GD2 comprises neuroblastoma.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition associated with the expression of B7H3.
  • the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
  • the present application provides the fusion protein, the isolated nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition in the preparation of medicine Use of the medicament for treating a disease or condition associated with the expression of HER2.
  • the disease or disorder associated with the expression of HER2 comprises breast cancer.
  • the application provides one or more of the fusion proteins, the isolated nucleic acid molecules, the vectors, the modified cells, and/or the pharmaceutical compositions in Use in the preparation of a medicament for treating a disease or condition associated with the expression of DLL1.
  • the disease or disorder associated with the expression of DLL1 comprises lung cancer.
  • the present application provides a method for preventing and/or treating tumors, comprising administering an effective amount of the fusion protein, the isolated nucleic acid molecule, and the vector to a subject in need thereof , the modified cells, and/or the pharmaceutical composition.
  • the present application provides a method for preventing and/or treating a disease or condition associated with the expression of CD33, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated The nucleic acid molecule, any one of the vector, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of CD33 comprises human myeloid leukemia.
  • the disease or disorder associated with the expression of CD33 comprises human acute myeloid leukemia (AML).
  • AML human acute myeloid leukemia
  • the application provides a method for preventing and/or treating a disease or condition associated with the expression of GD2, comprising administering an effective amount of the fusion protein to a subject in need, and the isolated The nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of GD2 comprises neuroblastoma.
  • the application provides a method of preventing and/or treating a disease or condition associated with the expression of B7H3, comprising administering the fusion protein, the isolated nucleic acid molecule to a subject in need thereof , the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with expression of B7H3 comprises neuroblastoma.
  • the application provides a method for preventing and/or treating a disease or condition associated with the expression of HER2, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated The nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of HER2 comprises breast cancer.
  • the application provides a method of preventing and/or treating a disease or condition associated with the expression of DLL1, comprising administering to a subject in need thereof an effective amount of the fusion protein, the isolated The nucleic acid molecule, the carrier, the modified cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with the expression of DLL1 comprises lung cancer.
  • the RRE sequence is inserted upstream of the multiple cloning site of the expression vector pWPXL-X, the WPRE sequence and cPPT sequence are inserted downstream of the multiple cloning site, and the EF1A promoter is inserted into the RRE between sequences and multiple cloning sites.
  • the main components of the genetic engineering vector are as follows, and the corresponding sequences are synthesized by the method of gene synthesis:
  • I-1 CD19 antigen sequence-costimulatory molecule 4-1BB-intracellular signaling domain CD3 ⁇ ;
  • I-2 CD22 antigen sequence-costimulatory molecule 4-1BB-intracellular signaling domain CD3 ⁇ ;
  • I-3 CD20 antigen sequence-costimulatory molecule 4-1BB-intracellular signaling domain CD3 ⁇ ;
  • I-4 CD33 antigen sequence - costimulatory molecule 4-1BB - intracellular signaling domain CD3 ⁇ .
  • the amino acid sequence of CD19 antigen is shown in SEQ ID NO:2
  • the amino acid sequence of CD22 antigen is shown in SEQ ID NO:6
  • the amino acid sequence of CD20 antigen is shown in SEQ ID NO:4
  • the amino acid sequence of CD33 antigen is shown in SEQ ID NO: 8
  • the amino acid sequence of the costimulatory molecule 4-1BB is shown in SEQ ID NO: 12
  • the intracellular signaling domain CD3 ⁇ is shown in SEQ ID NO: 19.
  • CD19 scFv for CD19, CD20, CD22, CD33
  • VH is shown as SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:30, VL is shown as SEQ ID NO:26, SEQ ID NO: 34, shown in SEQ ID NO: 34
  • CD20 scFv VH is shown in SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 51, VL is shown in SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 53
  • CD22 scFv VH is shown in SEQ ID NO: 54, SEQ ID NO: 61, VL is shown in SEQ ID NO: 58, SEQ ID NO: 65
  • CD33 scFv VH is shown in SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:90, SEQ ID NO:93, VL as SEQ ID NO:73, SEQ ID NO:81, SEQ
  • I-1-1 CD19 scFv-CD33 scFv;
  • I-1-2 CD19 scFv-GD2 scFv;
  • I-1-3 CD19 scFv-B7H3 scFv;
  • I-2-1 CD22 scFv-CD33 scFv;
  • I-2-2 CD22 scFv-GD2 scFv;
  • I-2-3 CD22 scFv-B7H3 scFv;
  • I-3-1 CD20 scFv-CD33 scFv;
  • I-3-3 CD20 scFv-B7H3 scFv;
  • I-4-1 CD33 scFv-GD2 scFv;
  • I-4-2 CD33 scFv-B7H3 scFv;
  • I-1 ⁇ I-4 and I-1-1 ⁇ I-1-3, I-2-1 ⁇ I-2-3, I-3-1 ⁇ I-3-3 , I-4-1 to I-4-2 can be expressed on different expression vectors respectively.
  • I-1 can be connected to I-1-1, I-1-2, and I-1-3 through the cleavage peptide sequence (SEQ ID NO: 13), respectively, and I-2 can be cleaved through
  • the peptide sequences are connected to I-2-1, I-2-2, and I-2-3, respectively, and I-3 can be connected to I-3-1, I-3-2, I-3- 3 is connected, and I-4 can be connected with I-4-1 and I-4-2 by cutting the peptide sequence respectively, and the corresponding sequence can be synthesized by the method of gene synthesis to obtain the following structure:
  • the genetic engineering vector CD19AgCAR backbone (I-1, as shown in Figure 4) is obtained.
  • the expression vector is digested with the I-1-1 structural restriction enzyme.
  • the genetic engineering vector CD19AgCAR Double antibody (I-1-1, the secondary antibody is CD33 scFv, as shown in Figure 5) is obtained.
  • the genetic engineering vector CD19AgCAR is obtained. (II-1, as shown in Figure 6).
  • the genetic engineering vector CD19AgCAR backbone (I-1, as shown in Figure 4) is obtained after the expression vector is connected with the I-1 structural restriction enzyme, and the genetic engineering vector is obtained after the expression vector is connected with the I-1-2 structural restriction enzyme
  • the vector CD19AgCAR Double antibody (I-1-2, the secondary antibody is GD2 scFv)
  • the genetic engineering vector CD19AgCAR (II-2) is obtained after the expression vector is ligated with II-2 structural restriction enzyme.
  • the genetic engineering vector CD19AgCAR backbone, CD19AgCAR Double antibody, CD19AgCAR and viral packaging plasmid were transfected according to the molar ratio of 1:1:1 respectively, with a total mass of 6ug/10cm Dish, and the lentivirus was transfected into 293T cells for 72h and 96h after collection and culture. Supernatant and virus concentration.
  • protein tags (tags such as myc, flag, HA, etc.) are added to the 5' end of the scFv fragment of the primary antibody or secondary antibody, and the corresponding fluorescently labeled antibody can be used.
  • the transfection efficiency was detected by flow cytometry or real-time quantitative PCR, and the virus-transfected T cells were named AgCAR-T cells.
  • 4.1 Collect 0.5 ⁇ 2ml/Kg of peripheral blood, obtain CD3 positive T cells or CD56 to obtain NK cells, or TCR ⁇ / ⁇ separation to obtain ⁇ / ⁇ T cells, and add CD3/CD28 immunomagnetic beads to stimulate activation and virus After transfection, continue to give CD3/CD28 immunomagnetic beads, interleukin-7 and interleukin-15 for in vitro expansion. Generally, it can expand more than 100 times in 7 to 8 days. After reaching a suitable number of cells, it will be washed and returned to the human body. Adjusted according to the body load, the number of back-infused human AgCAR-T cells ranges from 2 ⁇ 10 5 /Kg to 2 ⁇ 10 7 /Kg.
  • the virus prepared by the genetic engineering vector generally has a transfection efficiency of more than 20% after in vitro transfection, and most of the viruses are between 40% and 70%, as shown in FIG. 7 .
  • FIG. 7 the transfection efficiency of human T lymphocytes after the virus with CD19AgCAR, CD19AgCAR backbone and CD19AgCAR Double antibody was transfected in turn.
  • the virus with CD19AgCAR (II-1), CD19AgCAR backbone (I-1) and CD19AgCAR Double antibody (I-1-1) was transfected into human T lymphocytes and incubated with human myeloid leukemia cell line HL60.
  • CD19-CART cells and CD33-CART cells were co-incubated with human myeloid leukemia cell line HL60, respectively.
  • flow cytometry detection of residual HL60 cells in the co-incubation system was performed at 0 h and 24 h of co-incubation.
  • a and F are the results of co-incubating T cells with CD19AgCAR (II-1) and co-incubated with HL60 cells for 0h and 24h, respectively;
  • B and G are the results of CD19AgCAR backbone (I- 1) The results of co-incubation with HL60 cells for 0h and 24h after lentivirus-transfected T cells;
  • C and H were incubated with HL60 cells after lentivirus-transfected T cells with CD19AgCAR Double antibody (I-1-1), respectively The results of 0h and 24h;
  • D and I are the results of co-incubating CD19-CART cells with HL60 cells for 0h and 24h, respectively;
  • E and J are the results of co-incubating CD33-CART cells with HL60 cells for 0h and 24h, respectively.
  • the lentivirus with CD19AgCAR (II-1) can completely kill the target cell CD33-positive HL60 cells 24h after transfection of T cells, resulting in the same cytotoxic effect as CD33-CART cells, which is more efficient than CD33-CART cells.
  • the cytotoxic effects of lentiviral-transfected T cells with CD19AgCAR backbone (I-1), lentiviral-transfected T cells with CD19AgCAR Double antibody (I-1-1), and CD19-CART cells on target cells were significantly enhanced .
  • Human T lymphocytes were transfected with virus with CD19AgCAR(II-2), CD19AgCAR backbone(I-1) and CD19AgCAR Double antibody(I-1-2) and incubated with human neuroblastoma cell line CHP134 , and GD2-CART cells were co-incubated with human neuroblastoma cell line CHP134, and the residual CHP134 cells in the co-incubation system were detected by flow cytometry at 0h, 24h and 48h of co-incubation.
  • A, B, and C are the results of co-incubation with CHP134 cells for 0h, 24h, and 48h after lentivirus-transfected T cells with CD19AgCAR (II-2); D, E, and F are in turn The results of co-incubation with CHP134 cells for 0h, 24h and 48h after lentivirus transfected with CD19AgCAR backbone (I-1); G, H, and I are the lentiviruses with CD19AgCAR Double antibody (I-1-2).
  • Human T lymphocytes were transfected with the virus with CD22AgCAR(II-4), CD20AgCAR(II-7) and CD19AgCAR(II-2) and co-incubated with human acute lymphoblastic leukemia cell line MV4;11 (MV4; 11 cell lines express CD33 and CD123, but not GD2), and the residual MV4 in the co-incubation system was detected at 0h, 24h and 48h; 11 cells were detected by flow cytometry.
  • A, B, C are the results of co-incubating T cells with CD22AgCAR (II-4) after transfection with MV4; 11 cells are incubated for 0h, 24h and 48h; D, E, F are in turn Indicates the results of co-incubating T cells with CD20AgCAR (II-7) and MV4 after transfection with MV4; 11 cells were incubated for 0h, 24h and 48h; G, H, and I were lentiviruses with CD19AgCAR (II-2) in turn Results of co-incubation with MV4;11 cells for 0h, 24h and 48h after transfection of T cells.
  • the lentivirus with CD22AgCAR (II-4) and CD20AgCAR (II-7) can completely kill the target cells MV4; 11 cells 24h and 48h after transfection of T cells (CD123 positive cells were kill), resulting in better cytotoxicity than CD19AgCAR(II-2) cells.
  • the leukemia cells of this patient specifically expressed CD33.
  • the patient's own T lymphocytes transfected with CD19AgCAR-33 (II-1) virus were prepared into 5*10 6 /kg CD19AgCAR-33 T cells and injected into the patient's blood vessels via peripheral intravenous injection (day 0).
  • the patient's bone marrow minimal residual disease (MRD) was detected as 43.8% before treatment (illustration shown in Figure 11A), and on day 14 after CD19AgCAR-33T cell infusion, the patient's MRD decreased to ⁇ 0.1% (illustration shown in Figure 11B ). ), and achieved remission at the molecular level, suggesting that CD19AgCAR-33T cells have clinical therapeutic value.
  • the patient's tumor cells specifically expressed B7H3.
  • the patient's own T lymphocytes transfected with CD19AgCAR-B7H3 (II-3) virus were prepared into 6.3*10 6 /kg CD19AgCAR-B7H3 T cells and injected into the patient's blood vessels via peripheral intravenous injection (day 0).
  • 5% of neuroblastoma cells were morphologically visible in the bone marrow of the patient.
  • the patient's tumor cells specifically expressed GD2.
  • the patient's own T lymphocytes transfected with CD20AgCAR-GD2 (II-8) virus were prepared into 4.8*10 6 /kg CD20AgCAR-GD2 T cells and injected into the patient's blood vessels via peripheral intravenous injection (day 0). Morphologically, 2% of neuroblastoma cells were seen in the bone marrow of the patient before treatment. On day 30 after CD20AgCAR-GD2T cell infusion, there were no morphologically visible tumor cells in the patient's bone marrow. The patient's bone marrow was recently followed up to day 103. still in remission. The above results suggest that CD20AgCAR-GD2T cells have clinical therapeutic value.
  • the virus with CD19AgCAR-ROR1 was transfected into human T lymphocytes, and incubated with human neuroblastoma cell line SK-N-SH (expressing ROR1 protein target), and the CD19-ROR1 obtained by protein expression
  • the dual-target antibody supernatant (illustrated in Figure 12A) was added to the above co-incubated culture system at different doses, and flow cytometry of residual SK-N-SH cells in the co-incubated system was performed at 0 h, 24 h and 48 h of co-incubation Technical inspection.

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Abstract

La présente invention concerne une protéine de fusion, et en particulier une cellule immunitaire modifiée contenant la protéine de fusion et son utilisation. La protéine de fusion contient un antigène ou un fragment fonctionnellement actif de celui-ci, un domaine co-stimulateur et un domaine de signalisation intracellulaire. La cellule modifiée contenant la protéine de fusion se lie à un anticorps multispécifique et a un bon effet de destruction de cellules tumorales, et est nommée anticorps de couplage à l'antigène redirigeant l'immunothérapie par lymphocytes T (AgCAR-T).
PCT/CN2021/133010 2020-11-26 2021-11-25 Cellule immunitaire modifiée et son utilisation Ceased WO2022111562A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014011988A2 (fr) * 2012-07-13 2014-01-16 The Trustees Of The University Of Pennsylvania Renforcement de l'activité des lymphocytes t car grâce à la co-introduction d'un anticorps bispécifique
CN105392888A (zh) * 2013-03-16 2016-03-09 诺华股份有限公司 使用人源化抗cd19嵌合抗原受体治疗癌症
CN105829349A (zh) * 2013-10-15 2016-08-03 加州生物医学研究所 肽嵌合抗原受体t细胞开关和其用途
CN107106670A (zh) * 2014-10-31 2017-08-29 宾夕法尼亚大学董事会 用于修饰的t细胞的方法和组合物
US20180118808A1 (en) * 2015-03-26 2018-05-03 The California Institute For Biomedical Research SWITCHABLE NON-scFv CHIMERIC RECEPTORS, SWITCHES, AND USES THEREOF
CN109071663A (zh) * 2016-02-05 2018-12-21 奥里尼斯生物科学公司 双特异性信号传导剂及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014011988A2 (fr) * 2012-07-13 2014-01-16 The Trustees Of The University Of Pennsylvania Renforcement de l'activité des lymphocytes t car grâce à la co-introduction d'un anticorps bispécifique
CN105392888A (zh) * 2013-03-16 2016-03-09 诺华股份有限公司 使用人源化抗cd19嵌合抗原受体治疗癌症
CN105829349A (zh) * 2013-10-15 2016-08-03 加州生物医学研究所 肽嵌合抗原受体t细胞开关和其用途
CN107106670A (zh) * 2014-10-31 2017-08-29 宾夕法尼亚大学董事会 用于修饰的t细胞的方法和组合物
US20180118808A1 (en) * 2015-03-26 2018-05-03 The California Institute For Biomedical Research SWITCHABLE NON-scFv CHIMERIC RECEPTORS, SWITCHES, AND USES THEREOF
CN109071663A (zh) * 2016-02-05 2018-12-21 奥里尼斯生物科学公司 双特异性信号传导剂及其用途

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