WO2023134657A1 - Protéine de liaison à l'antigène bispécifique anti-cldn18.2 et 4-1bb et son utilisation - Google Patents

Protéine de liaison à l'antigène bispécifique anti-cldn18.2 et 4-1bb et son utilisation Download PDF

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WO2023134657A1
WO2023134657A1 PCT/CN2023/071504 CN2023071504W WO2023134657A1 WO 2023134657 A1 WO2023134657 A1 WO 2023134657A1 CN 2023071504 W CN2023071504 W CN 2023071504W WO 2023134657 A1 WO2023134657 A1 WO 2023134657A1
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seq
amino acid
acid sequence
sequence shown
antigen binding
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PCT/CN2023/071504
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Chinese (zh)
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杨沙沙
陈晓锐
王华菁
杨焕凤
何晓文
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原启生物科技(上海)有限责任公司
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Publication of WO2023134657A1 publication Critical patent/WO2023134657A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application relates to the field of biomedicine, in particular to an anti-CLDN18.2 bispecific antigen-binding protein and its application.
  • CLDN18.2 (Claudin 18.2) is a family of proteins that form an important part of tight cellular junctions. CLDN18.2 is only expressed in differentiated gastric parietal cells, but not in normal tissues. The latest research shows that CLDN18.2 is overexpressed in more than 77% of gastric cancer patients and more than 80% of pancreatic cancer patients. In addition, it is overexpressed in solid tumors such as lung cancer, esophageal cancer, and ovarian cancer.
  • 4-1BB (CD137, tumor cell necrosis factor receptor superfamily 9), a member of the TNF receptor superfamily (TNFRSF), is a co-stimulatory molecule that activates in immune cells (innate and adaptive immune cells) post expression.
  • 4-1BB plays an important role in regulating the activity of a variety of immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8+ T cells. Many other studies have shown that activation of 4-1BB enhances the immune response and eliminates tumors in mice. Therefore, 4-1BB may be a promising tumor immune target molecule.
  • the present application provides an isolated antigen-binding protein, the antigen-binding protein comprises a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain can specifically bind CLDN18.2.
  • the isolated antigen-binding protein has one of the following properties: 1) can specifically bind CLDN18.2; 2) can specifically bind 4-1BB; 3) can specifically bind human, mouse and CLDN18.2 protein of cynomolgus monkey; 4) can stimulate T cell activation and promote the secretion of IL-2 factor; 5) can inhibit the growth and/or proliferation of tumor cells; 6) have tumor immune memory function.
  • the application provides an isolated antigen-binding protein, which includes: a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain can specifically bind tight junction protein 18.2 (CLDN18.2) , and the first antigen-binding domain comprises at least one CDR in the variable region VH of the antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:100.
  • CLDN18.2 tight junction protein 18.2
  • the first antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97.
  • the first antigen binding domain comprises HCDR3, and the HCDR3 comprises SEQ ID NO:3, SEQ ID NO:57 and SEQ ID NO:71 The amino acid sequence shown in any one.
  • the first antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:98.
  • said first antigen binding domain comprises HCDR2, and said HCDR2 comprises SEQ ID NO:2, SEQ ID NO:56 and SEQ ID NO:70 The amino acid sequence shown in any one.
  • said first antigen binding domain comprises HCDR1, and said HCDR1 comprises SEQ ID NO:99 (X 1 YX 2 X 3 X 4 , wherein X 1 is N or R, X 2 is G, I or V, X 3 is I or M, X 4 is the amino acid sequence shown in H, N or S).
  • the first antigen binding domain comprises HCDR1
  • the HCDR1 comprises SEQ ID NO:1, SEQ ID NO:55 and SEQ ID NO:69
  • said first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO: 99 (X 1 YX 2 X 3 X 4 , Wherein, X1 is N or R, X2 is G, I or V, X3 is I or M, X4 is the amino acid sequence shown in H, N or S), and the HCDR2 comprises SEQ ID NO:98 The amino acid sequence shown, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97.
  • the first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.
  • the first antigen-binding domain comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:43, SEQ ID NO:58 and SEQ ID NO:75.
  • said first antigen binding domain comprises H-FR2
  • said H-FR2 is located between said HCDR1 and said HCDR2
  • said H- FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:44 and SEQ ID NO:59.
  • said first antigen binding domain comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 is located between said HCDR2 and said HCDR3, and said H- FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:45, SEQ ID NO:60 and SEQ ID NO:72.
  • the first antigen-binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and
  • the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:46 and SEQ ID NO:61.
  • the first antigen-binding domain comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:100.
  • said first antigen binding domain comprises a heavy chain variable region VH comprising SEQ ID NO:8, SEQ ID NO:47, SEQ ID NO: The amino acid sequence shown in any one of NO:62, SEQ ID NO:73 and SEQ ID NO:76.
  • the first antigen binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:101.
  • said first antigen binding domain comprises LCDR3, and said LCDR3 comprises SEQ ID NO: 11, SEQ ID NO: 63, SEQ ID NO: 79 and The amino acid sequence shown in any one of SEQ ID NO:86.
  • the first antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 102.
  • the first antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:78.
  • the first antigen binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 103.
  • said first antigen binding domain comprises LCDR1, and said LCDR1 comprises SEQ ID NO:9, SEQ ID NO:48 and SEQ ID NO:77 The amino acid sequence shown in any one.
  • the first antigen binding domain comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 103, and the LCDR2 comprises The amino acid sequence shown in SEQ ID NO:102, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:101.
  • the first antigen-binding domain comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from any one of the following groups:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:77
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:78
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:79.
  • the first antigen-binding domain comprises L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1
  • the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:80 and SEQ ID NO:87.
  • said first antigen binding domain comprises L-FR2
  • said L-FR2 is located between said LCDR1 and said LCDR2
  • said L- FR2 comprises the amino acid sequence shown in SEQ ID NO: 13.
  • said first antigen binding domain comprises L-FR3
  • said L-FR3 is located between said LCDR2 and said LCDR3
  • said L- FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:14, SEQ ID NO:50, SEQ ID NO:65 and SEQ ID NO:81.
  • the first antigen-binding domain comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and
  • the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:82 and SEQ ID NO:88.
  • the first antigen binding domain comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:104.
  • said first antigen binding domain comprises a light chain variable region VL comprising SEQ ID NO: 16, SEQ ID NO: 52, SEQ ID NO: The amino acid sequence shown in any one of NO:66, SEQ ID NO:83 and SEQ ID NO:89.
  • said first antigen binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 Comprising an amino acid sequence selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.
  • the first antigen binding domain comprises VH and VL
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 100
  • the VL comprises SEQ ID NO: 100 Amino acid sequence shown in ID NO:104.
  • the first antigen-binding domain comprises VH and VL, and the VH and VL are selected from any one of the following amino acid sequences:
  • VH comprises the amino acid sequence shown in SEQ ID NO:8
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:16
  • VH comprises the amino acid sequence shown in SEQ ID NO:47
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:52;
  • VH comprises the amino acid sequence shown in SEQ ID NO:62
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:66
  • VH comprises the amino acid sequence shown in SEQ ID NO:73
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:52;
  • VH comprises the amino acid sequence shown in SEQ ID NO:76
  • VL comprises the amino acid sequence shown in SEQ ID NO:83
  • VH comprises the amino acid sequence shown in SEQ ID NO:76
  • VL comprises the amino acid sequence shown in SEQ ID NO:89.
  • said first antigen binding domain comprises a heavy chain constant region.
  • said heavy chain constant region comprises an IgG-derived heavy chain constant region.
  • said heavy chain constant region comprises a heavy chain constant region derived from human IgG.
  • said heavy chain constant region comprises a heavy chain constant region derived from human IgG1.
  • said heavy chain constant region comprises an Fc fragment.
  • said Fc fragment comprises one or more mutations.
  • said Fc fragment comprises one or more mutations of N180A, D239E and L241M.
  • the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 17.
  • said first antigen binding domain comprises a light chain constant region.
  • said light chain constant region comprises a human Ig ⁇ constant region.
  • the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 18.
  • said first antigen binding domain comprises an antibody or antigen binding fragment thereof.
  • said antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab)' 2 , scFv, di-scFv and/or dAb.
  • said second antigen binding domain is capable of specifically binding 4-1BB.
  • the second antigen binding domain comprises at least one CDR in the variable region VH of an antibody heavy chain, the VH comprising SEQ ID NO: 105 amino acid sequence.
  • the second antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
  • the second antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23.
  • the second antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 comprises The amino acid sequence shown in SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
  • the second antigen binding domain comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:91.
  • said second antigen binding domain comprises H-FR2
  • said H-FR2 is located between said HCDR1 and said HCDR2
  • said H- FR2 comprises the amino acid sequence shown in SEQ ID NO:26.
  • said second antigen binding domain comprises H-FR3
  • said H-FR3 is located between said HCDR2 and said HCDR3
  • said H- FR3 comprises the amino acid sequence shown in SEQ ID NO:27.
  • the second antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:28.
  • the second antigen-binding domain comprises a heavy chain variable region VH, and the VH comprises SEQ ID NO:29 or SEQ ID NO:92 amino acid sequence.
  • the second antigen binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the second antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the second antigen binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32.
  • the second antigen binding domain comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 comprises The amino acid sequence shown in SEQ ID NO:33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the second antigen binding domain comprises L-FR1
  • the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:35.
  • the second antigen binding domain comprises L-FR2
  • the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:36.
  • the second antigen binding domain comprises L-FR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:37.
  • the second antigen binding domain comprises L-FR4, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:38.
  • the second antigen binding domain comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:39.
  • the VH and VL are selected from the amino acid sequences of any of the following groups:
  • VH comprises the amino acid sequence shown in SEQ ID NO:29
  • VL comprises the amino acid sequence shown in SEQ ID NO:39
  • VH comprises the amino acid sequence shown in SEQ ID NO:92
  • VL comprises the amino acid sequence shown in SEQ ID NO:39.
  • the VH and VL of said second antigen binding domain are directly or indirectly linked.
  • the VH and VL of said second antigen binding domain are linked by a linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:40.
  • said second antigen binding domain comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment comprises a scFv.
  • the scFv comprises the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:96.
  • said first antigen binding domain and said second antigen binding domain are directly or indirectly linked.
  • said first antigen binding domain and said second antigen binding domain are linked by a linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:21.
  • the scFv of the second antigen-binding domain is directly or indirectly linked to the C-terminus of the Fc fragment of the first antigen-binding domain.
  • the present application also provides an isolated antigen-binding protein, which comprises two first polypeptides and two second polypeptides, and the first polypeptide comprises VH, CH1 in sequence from the N-terminal to the C-terminal , CH2, CH3 and scFv capable of specifically binding to 4-1BB protein, the second polypeptide comprising VL and light chain variable region CL; wherein the VH is paired with the VL and capable of specifically binding to CLDN18.2.
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1
  • the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 56
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57.
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.
  • the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.
  • the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.
  • the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63.
  • the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 78, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79.
  • the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.
  • said CH2 and said CH3 constitute an Fc fragment.
  • said Fc fragment comprises one or more amino acid mutations selected from the group consisting of N180A, D239E and L241M.
  • the scFv comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22
  • the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 23
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24.
  • the scFv comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34.
  • the scFv comprises VH
  • the VH comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:92.
  • the scFv comprises a VL
  • the VL comprises the amino acid sequence shown in SEQ ID NO:39.
  • the present application also provides a polypeptide comprising the antigen-binding protein described in the present application.
  • the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen binding proteins described herein.
  • the present application also provides a vector comprising the nucleic acid molecule described in the present application.
  • the present application also provides a cell comprising the nucleic acid molecule or the vector.
  • the present application also provides a pharmaceutical composition, which comprises the antigen-binding protein described in the present application, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for preparing the antigen-binding protein, which comprises culturing the cell described in the present application under the condition that the isolated antigen-binding protein is expressed.
  • the present application also provides the use of the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of a medicament, the Medicines are used to prevent, alleviate and/or treat diseases and/or conditions.
  • the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of CLDN18.2.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • the tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.
  • the present application also provides the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, which are used for preventing, alleviating and/or or to treat diseases and/or conditions.
  • the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of CLDN18.2.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • the tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.
  • the present application also provides a method for preventing, alleviating and/or treating diseases and/or conditions, the method comprising administering the isolated antigen-binding protein described in the present application to a subject in need, said Polypeptide, said nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition.
  • the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of CLDN18.2.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • the tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.
  • Figure 1A shows a schematic diagram of the construction of the isolated antigen-binding protein described in this application
  • Figure 1B shows the SEC-HPLC purity test results of the purified antigen-binding protein.
  • Figure 2A shows the binding activity of the antigen-binding protein to 4-1BB-NF ⁇ B-293T cells
  • Figure 2B shows the binding activity of the antigen-binding protein to CHO-hCLDN18.2 cells.
  • Figure 3 shows the flow cytometric verification of the activity assay of the double-head simultaneous binding of the antigen-binding protein.
  • Figure 4A shows the flow cytometric binding activity of the antigen-binding protein and 293T-mouse CLDN18.2 cells
  • Figure 4B shows the flow cytometric binding activity of the antigen-binding protein and 293T-cynomolgus monkey CLDN18.2 cells.
  • Figure 5 shows a flow cytometric competition binding experiment of the antigen binding proteins described in this application.
  • Figure 6 shows the results of luciferase activity analysis experiments.
  • Figure 7A shows the secretion level of IL-2 under the condition of PBMC activated by SEA superantigen
  • Figure 7B shows the level of IL-2 secretion under the condition of activation of PBMC by anti-human CD3 antibody.
  • Figure 8 shows the detection of tumor inhibitory activity of antigen-binding proteins in a mouse colon cancer tumor model.
  • Figure 9 shows the statistics of tumor volume in mice after administration.
  • Figure 10 shows the detection of the flow cytometry binding activity between the targeting part of CLDN18.2 described in the present application and the cells with high expression of human CLDN18.2.
  • Figure 11 shows the detection of the flow cytometric binding activity between the targeting part of CLDN18.2 described in the present application and high-expressing human CLDN18.1 cells.
  • Figure 12 shows the detection of the flow cytometry binding activity of the targeting moiety of CLDN18.2 described in the present application to tumor cell lines.
  • CLDN18.2 or “Claudin18.2” are used interchangeably and generally refer to subtype 2 of the cell junction claudin Claudin18.
  • the term encompasses "full length", unprocessed CLDN18.2 as well as any form of CLDN18.2 produced by cellular processing.
  • CLDN18.2 may include intact CLDN18.2 and fragments thereof, functional variants, isoforms, species homologues, derivatives, analogs and analogs having at least one common epitope with CLDN18.2.
  • the amino acid sequence of CLDN18.2 (eg, human CLDN18.2) is known in the art.
  • the human CLDN18.2 nucleotide sequence can be shown under GeneBank accession number NM_001002026.3.
  • the mouse CLDN18.2 nucleotide sequence can be shown under GeneBank Accession No. NM_001194921.1.
  • the cynomolgus monkey CLDN18.2 nucleotide sequence can be shown under GeneBank accession number XM_001114708.4.
  • 4-1BB also known as 4-1BB or TNFRS9
  • 4-1BB also known as 4-1BB or TNFRS9
  • TNFRS9 tumor necrosis factor receptor superfamily
  • CD137 agonistic monoclonal antibodies increase the expression of co-stimulatory molecules in many models, and significantly enhance the response of cytolytic T lymphocytes, exerting anti-tumor effects.
  • the anti-tumor effect of CD137-targeted therapy can be confirmed by in vivo anti-tumor efficacy studies in mice using agonistic anti-mouse CD137 monoclonal antibodies.
  • CD137 has emerged as a potent activator of immune cells and an important candidate antigen for the treatment of various diseases. (See Vinay, Dass S., and Byoung S. Kwon.”4-1BB(CD137), an inducible costimulatory receptor, as a specific target for cancer therapy.”BMB reports 47.3(2014):122.)
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • isolated antigen-binding protein generally refers to a protein with antigen-binding ability obtained from a natural state through artificial means.
  • isolated antigen binding protein may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives.
  • Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. See eg Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).
  • antigen binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; triabodies; tetrabodies; (ab') 2 , F(ab) 2 , scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgGl antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.
  • the isolated antigen binding protein may comprise more than one antigen binding domain.
  • the antigen binding domains may target different antigens.
  • the antigen binding domains may target different epitopes of the same antigen.
  • the isolated antigen binding protein can comprise a first antigen binding domain and a second antigen binding domain.
  • the first antigen binding domain can target CLDN18.2 protein, for example, the second antigen binding domain can target 4-1BB protein.
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.
  • variable generally means that some segments of the variable domain may have large differences in sequence between antibodies.
  • the variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains are called the framework regions (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of the ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • the term “antibody” also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind CLDN18.2). Typically, such fragments will include the antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 antigen-binding sites that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.
  • the gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl and IgKl.
  • CDR also referred to as “complementarity determining region” generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structurally defined loop.
  • antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • variable domains of native heavy and light chains each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3, and L-FR4).
  • VL of an isolated antigen binding protein described herein can include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the VH of the isolated antigen binding proteins described herein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, CLDN18.2).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region ( CH 1 ) of the H chain ( VH ).
  • Fab' fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 generally refers to antibody fragments produced by pepsin digestion of intact antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions.
  • the heavy and light chain variable regions include, for example, CDRs.
  • an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable
  • the regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv can have the VL and VH variable regions described in any order (eg, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include a VL-linker-VH Or VH-linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • monoclonal antibody generally refers to a preparation of antibody molecules of single molecular composition.
  • Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants.
  • monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins.
  • the modifier "monoclonal” denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody”), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more effective in a human individual than the parental (e.g., mouse-derived) antibody. Less likely to trigger an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen.
  • a humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigen specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin.
  • CDR region residues in a human immunoglobulin can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region.
  • donor antibody such as mouse, rat
  • rabbit or non-human primate residue substitution in the CDR region such as mouse, rat
  • FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues.
  • humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.
  • Fully human antibody generally refers to an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a “mouse antibody” or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as using libraries, are known in the art.
  • the term “directly connected” is opposite to the term “indirectly connected”, and the term “directly connected” generally refers to a direct connection.
  • the direct connection may be a case where substances are directly connected without a spacer.
  • the spacer may be a linker.
  • the linker can be a peptide linker.
  • the term “indirectly linked” generally refers to the situation where substances are not directly linked.
  • the indirect connection may be through a spacer.
  • the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 Phages and animal viruses, etc.
  • Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, and papillomaviruses.
  • vascular viruses such as SV40.
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes or protein coats, but not only.
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule as described herein or a nucleic acid molecule as described herein. Carrier.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • Cells may include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (e.g., E.
  • the cells are mammalian cells. In certain embodiments, the mammalian cells are HEK293 cells.
  • the term "pharmaceutical composition” generally refers to a composition for preventing/treating a disease or condition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally includes a pharmaceutically acceptable carrier, excipient or stabilizer that is inert to the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous.
  • Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • the term "specifically binds" or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets .
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • a tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • a tumor may be a solid tumor or a non-solid tumor.
  • the tumor may be a CLDN18.2 positive tumor.
  • a tumor may be a disease and/or disorder related to abnormal expression of CLDN18.2.
  • cancer generally refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Cancer in this application includes but not limited to gastric cancer, colon cancer and the like.
  • tumor and cancer are used interchangeably herein, eg, both terms encompass solid tumors and liquid tumors, eg, diffuse or circulating tumors.
  • cancer or “tumor” can include premalignant as well as malignant cancers and tumors.
  • protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, passed through in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to CLDN18.2 and/or 4-1BB protein).
  • the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion.
  • Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the homologue may have at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or polypeptides.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • Perfectage of sequence homology can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity.
  • Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA FASTA and BLAST.
  • a description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J.
  • 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%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. These coding systems are known in the art, see http://www.bioinf.org.uk/abs/index.html#kabatnum for details. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the CDR may be divided according to the Kabat method.
  • the isolated antigen-binding protein may comprise a first antigen-binding domain capable of specifically binding to CLDN18.2 or a functionally active fragment thereof.
  • the first antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof or a variant thereof.
  • the antibody may be selected from the following groups: monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.
  • the antigen-binding fragment can be selected from the following group: Fab, Fab', Fv fragment, F(ab)' 2 , scFv, di-scFv and/or dAb.
  • the first antigen-binding domain may comprise at least one CDR in the variable region VH of the antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:100.
  • the first antigen-binding domain may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:97.
  • the first antigen binding domain may comprise HCDR3, and the HCDR3 may comprise SEQ ID NO:3, SEQ ID NO:57 and SEQ ID NO:71 The amino acid sequence shown in any one.
  • the first antigen-binding domain may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:98.
  • the first antigen binding domain may comprise HCDR2, and the HCDR2 may comprise SEQ ID NO:2, SEQ ID NO:56 and SEQ ID NO:70 The amino acid sequence shown in any one.
  • the first antigen binding domain may comprise HCDR1, and the HCDR1 may comprise SEQ ID NO:99 (X 1 YX 2 X 3 X 4 , wherein, X 1 is N or R, X 2 is G, I or V, X 3 is I or M, X 4 is the amino acid sequence shown in H, N or S).
  • the first antigen binding domain may comprise HCDR1, and the HCDR1 may comprise SEQ ID NO:1, SEQ ID NO:55 and SEQ ID NO:69 The amino acid sequence shown in any one.
  • the first antigen binding domain may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO: 99 (X 1 YX 2 X 3 X 4 , Wherein, X1 is N or R, X2 is G, I or V, X3 is I or M, X4 is the amino acid sequence shown in H, N or S), and the HCDR2 may comprise SEQ ID NO:98
  • the amino acid sequence shown in SEQ ID NO:97, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:97.
  • the first antigen-binding domain may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise any amino acid sequence selected from the following group:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.
  • the first antigen-binding domain may comprise H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the The H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:43, SEQ ID NO:58 and SEQ ID NO:75.
  • the first antigen binding domain may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2
  • the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:44 and SEQ ID NO:59 may be included.
  • the first antigen binding domain may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3
  • the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:45, SEQ ID NO:60 and SEQ ID NO:72 may be included.
  • the first antigen-binding domain may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the
  • the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:46 and SEQ ID NO:61.
  • the first antigen-binding domain may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:100.
  • the first antigen-binding domain may comprise a heavy chain variable region VH, and the VH may comprise SEQ ID NO:8, SEQ ID NO:47, SEQ ID The amino acid sequence shown in any one of NO:62, SEQ ID NO:73 and SEQ ID NO:76.
  • the first antigen-binding domain may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:101.
  • the first antigen binding domain may comprise LCDR3, and the LCDR3 may comprise SEQ ID NO: 11, SEQ ID NO: 63, SEQ ID NO: 79 and The amino acid sequence shown in any one of SEQ ID NO:86.
  • the first antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 102.
  • the first antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:78.
  • the first antigen-binding domain may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:103.
  • the first antigen binding domain may comprise LCDR1, and the LCDR1 may comprise SEQ ID NO:9, SEQ ID NO:48 and SEQ ID NO:77.
  • the first antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 103, and the LCDR2 may comprise Comprising the amino acid sequence shown in SEQ ID NO: 102, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 101.
  • the first antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise any group of amino acid sequences selected from the following group:
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:77
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:78
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:79.
  • the first antigen-binding domain may comprise L-FR1
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1
  • the The L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:80 and SEQ ID NO:87.
  • the first antigen binding domain may comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 Can comprise the aminoacid sequence shown in SEQ ID NO:13.
  • the first antigen binding domain may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3
  • the amino acid sequence shown in any one of SEQ ID NO:14, SEQ ID NO:50, SEQ ID NO:65 and SEQ ID NO:81 may be included.
  • the first antigen-binding domain may comprise L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the The L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:82 and SEQ ID NO:88.
  • the first antigen-binding domain may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:104.
  • the first antigen binding domain may comprise a light chain variable region VL, and the VL may comprise SEQ ID NO: 16, SEQ ID NO: 52, SEQ ID The amino acid sequence shown in any one of NO:66, SEQ ID NO:83 and SEQ ID NO:89.
  • the first antigen binding domain may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may be Comprising an amino acid sequence selected from any one of the following groups:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.
  • the first antigen-binding domain may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 100
  • the VL may comprise Amino acid sequence shown in SEQ ID NO: 104.
  • the first antigen-binding domain may comprise VH and VL, and the VH and VL may be selected from any one of the following amino acid sequences:
  • VH comprises the amino acid sequence shown in SEQ ID NO:8
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:16
  • VH comprises the amino acid sequence shown in SEQ ID NO:47
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:52;
  • VH comprises the amino acid sequence shown in SEQ ID NO:62
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:66
  • VH comprises the amino acid sequence shown in SEQ ID NO:73
  • VL shown comprises the amino acid sequence shown in SEQ ID NO:52;
  • VH comprises the amino acid sequence shown in SEQ ID NO:76
  • VL comprises the amino acid sequence shown in SEQ ID NO:83
  • VH comprises the amino acid sequence shown in SEQ ID NO:76
  • VL comprises the amino acid sequence shown in SEQ ID NO:89.
  • the first antigen-binding domain may comprise a heavy chain constant region.
  • the heavy chain constant region may comprise an IgG-derived heavy chain constant region.
  • the heavy chain constant region may comprise a heavy chain constant region derived from human IgG.
  • the heavy chain constant region may comprise a heavy chain constant region derived from human IgG1.
  • the heavy chain constant region may comprise an Fc fragment.
  • the Fc fragment in the isolated antigen-binding protein, may contain one or more mutations.
  • the Fc fragment in the isolated antigen-binding protein, may contain one or more mutations among N180A, D239E and L241M.
  • the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the first antigen binding domain may comprise a light chain constant region.
  • the light chain constant region may comprise a human Ig ⁇ constant region.
  • the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the first antigen-binding domain may comprise a monoclonal antibody capable of binding CLDN18.2.
  • the isolated antigen-binding protein may comprise a second antigen-binding domain capable of specifically binding to the 4-1BB protein or a functionally active fragment thereof.
  • the second antigen binding domain of the isolated antigen binding protein may comprise scFv.
  • the second antigen-binding domain in the isolated antigen-binding protein, may comprise at least one CDR in the variable region VH of the heavy chain of an antibody, and the VH may comprise SEQ ID NO: 105. amino acid sequence.
  • the second antigen-binding domain comprises HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the second antigen-binding domain comprises HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the second antigen-binding domain comprises HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the second antigen-binding domain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 may comprise Comprising the amino acid sequence shown in SEQ ID NO:23, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:24.
  • the second antigen-binding domain may comprise H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:91.
  • the second antigen binding domain may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 Can comprise the aminoacid sequence shown in SEQ ID NO:26.
  • the second antigen binding domain may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 Can comprise the aminoacid sequence shown in SEQ ID NO:27.
  • the second antigen-binding domain may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the The H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the second antigen-binding domain may comprise a heavy chain variable region VH, and the VH may comprise SEQ ID NO:29 or SEQ ID NO:92 amino acid sequence.
  • the second antigen-binding domain may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the second antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the second antigen-binding domain may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:32.
  • the second antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 may comprise Comprising the amino acid sequence shown in SEQ ID NO:33, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:34.
  • the second antigen-binding domain may comprise L-FR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the second antigen-binding domain may comprise L-FR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the second antigen-binding domain in the isolated antigen-binding protein, may comprise L-FR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the second antigen-binding domain may comprise L-FR4, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the second antigen-binding domain may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:39.
  • the VH and VL may be selected from the amino acid sequences of any of the following groups:
  • VH comprises the amino acid sequence shown in SEQ ID NO:29
  • VL comprises the amino acid sequence shown in SEQ ID NO:39
  • VH comprises the amino acid sequence shown in SEQ ID NO:92
  • VL comprises the amino acid sequence shown in SEQ ID NO:39.
  • the VH and VL of the second antigen-binding domain may be directly or indirectly linked.
  • the VH and VL of the second antigen-binding domain may be linked by a linker.
  • the linker may comprise the amino acid sequence shown in SEQ ID NO:40.
  • the antigen-binding fragment may comprise scFv.
  • the scFv may comprise the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:96.
  • the second antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof.
  • the framework region may be selected from the group consisting of human consensus framework sequences and human germline sequences.
  • the second antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof or a variant thereof.
  • the antibody may be selected from the following groups: monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.
  • the antigen-binding fragment can be selected from the following group: Fab, Fab', Fv fragment, F(ab)' 2 , scFv, di-scFv and/or dAb.
  • the second antigen binding domain of the isolated antigen binding protein can be a scFv.
  • the present application provides an antigen-binding protein, which includes a first antigen-binding domain and a second antigen-binding domain.
  • the first antigen binding domain can specifically bind CLDN18.2 protein.
  • the second antigen binding domain can specifically bind a 4-1BB protein.
  • the first antigen binding domain can be a monoclonal antibody.
  • the second antigen binding domain may be a scFv.
  • the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked.
  • the first antigen-binding domain may be connected to the second antigen-binding domain through a linker.
  • the linker can be a peptide linker.
  • the linker can include the amino acid sequence shown in SEQ ID NO: 21.
  • the scFv of the second antigen-binding domain can be connected to the C-terminus of the Fc fragment of the first antigen-binding domain through a linker.
  • the VH of the second antigen-binding domain can be connected to the C-terminus of the Fc fragment of the first antigen-binding domain through a linker.
  • the VL of the second antigen-binding domain can be connected to the C-terminus of the Fc fragment of the first antigen-binding domain through a linker.
  • the application provides an isolated antigen binding protein, which may comprise two first polypeptides and two second polypeptides.
  • the first polypeptide may comprise a heavy chain of an antigen-binding protein capable of specifically binding CLDN18.2 protein and a scFv capable of specifically binding 4-1BB protein.
  • the second polypeptide may comprise a light chain of an antigen binding protein capable of specifically binding a CLDN18.2 protein.
  • the first polypeptide sequentially comprises VH, CH1, CH2, CH3 and scFv capable of specifically binding to 4-1BB protein from N-terminus to C-terminus
  • the second polypeptide comprises VL and light chain variable region CL ; wherein said VH is paired with said VL and is capable of specifically binding to CLDN18.2.
  • the VH may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.
  • the VL may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.
  • the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 11.
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79.
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.
  • said CH2 and said CH3 may constitute an Fc fragment.
  • the Fc fragment may comprise one or more amino acid mutations selected from the group consisting of N180A, D239E and L241M.
  • the scFv capable of specifically binding to 4-1BB protein may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
  • the scFv capable of specifically binding to 4-1BB protein may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the scFv capable of specifically binding 4-1BB protein may comprise a VH.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:92.
  • the scFv capable of specifically binding 4-1BB protein may comprise a VL.
  • the VL comprises the amino acid sequence shown in SEQ ID NO:39.
  • the scFv capable of specifically binding to 4-1BB protein may comprise VH and VL.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:29
  • the VL may comprise the amino acid sequence set forth in SEQ ID NO:39.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:92
  • the VL may comprise the amino acid sequence set forth in SEQ ID NO:39.
  • the first polypeptide of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:42.
  • the second polypeptide of the isolated antigen binding protein may comprise any one of SEQ ID NO:20, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:85 and SEQ ID NO:90. The amino acid sequence shown.
  • the two first polypeptides of the isolated antigen binding protein may comprise the same amino acid sequence.
  • the two second polypeptides of the isolated antigen binding protein may comprise the same amino acid sequence.
  • Polypeptide molecules Polypeptide molecules, nucleic acid molecules, vectors, cells and pharmaceutical compositions
  • polypeptide molecules which may comprise an isolated antigen binding protein described herein.
  • the application provides one or more nucleic acid molecules that encode the isolated antigen binding proteins described herein.
  • it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.
  • PCR polymerase chain reaction
  • the present application provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • 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 permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled 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 vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
  • the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application.
  • each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like.
  • the cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells.
  • the vectors described herein can be introduced into the host cells by methods known in the art.
  • the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application and /or the cells described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutical compositions may also contain more than one active compound, generally those with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, generally safe, nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application provides a method for preparing the antigen-binding protein.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.
  • the present application also provides the described isolated antigen-binding protein, the described polypeptide molecule, the described nucleic acid molecule, the described carrier, the described cell and/or the described pharmaceutical composition
  • the purposes in, described medicine is used for preventing, alleviating and/or treating disease and/or disease.
  • the present application also provides a method for preventing, alleviating or treating diseases and/or conditions, the method may include administering the isolated antigen-binding protein, the polypeptide molecule described in the present application to a subject in need , the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition.
  • the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition described in the present application can be used to prevent , Alleviate or treat diseases and/or conditions.
  • the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of CLDN18.2.
  • the disease and/or condition may include a tumor.
  • the tumor may comprise solid tumors and/or non-solid tumors.
  • the tumor may comprise a CLDN18.2 positive tumor.
  • the tumor may comprise gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer, and/or colon cancer.
  • the present application also provides a method for detecting CLDN18.2 in a sample.
  • the method comprises administering the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition described herein.
  • the method may be an ex vivo and/or in vitro method.
  • the present application also provides a reagent or kit for detecting CLDN18.2 in a sample, which may comprise the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell And/or the pharmaceutical composition.
  • the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition are used in the preparation of the detection CLDN18.2 Uses in reagents or kits.
  • the reagent or kit can be used to detect the presence and/or content of CLDN18.2 in a sample.
  • the humanized Claudin18.2 monoclonal antibody 5E6 independently developed by the company (VH amino acid sequence shown in SEQ ID NO: 8, VL amino acid sequence shown in SEQ ID NO: 16) and fully human 4-1BB monoclonal antibody YN006 ( The VH amino acid sequence is shown in SEQ ID NO:29, and the VL amino acid sequence is shown in SEQ ID NO:39), and the combined construction forms an anti-CLDN18.2/4-1BB bispecific antibody.
  • the double antibody adopts the human IgG1-Fc backbone with amino acid mutations, the anti-CLDN18.2 part of the complete IgG form is linked to the N-terminal of the Fc segment, and the anti-4-1BB part of the single-chain scFv form is linked to the C-terminal of the Fc segment ;
  • the amino acid mutation in the human IgG1-Fc framework region can silence the ADCC and other effector functions of the double antibody.
  • the amino acid mutation is specifically: N180A/D239E/L241M, and the molecular structure is shown in Figure 1A.
  • the double antibody was purified by Protein A one-step affinity chromatography in the 293F transient expression system, the expression level of the antibody could reach about 130 mg/L, and the SEC-HPLC purity was 96%.
  • the double antibody was named OriAb-362. ( Figure 1B)
  • the antigen-binding activity of the OriAb-362 double antibody was verified by flow cytometry, and the specific method was as follows: target cells overexpressing human 4-1BB (41BB-NFkB-293T) or overexpressing human CLDN18. 2 target cells (hCLDN18.2-CHO); add serially diluted primary antibodies (ie OriAb-362 double antibody and 4-1BB parent monoclonal antibody YN006; OriAb-362 double antibody and CLDN18.2 parent monoclonal antibody 5E6), a The initial working concentration of the antibody was 100nM, and it was serially diluted 3 times to form a total of 11 concentration gradients; after incubation at 4°C for 1 hour, the plate was washed by centrifugation, and then the corresponding secondary antibody (Goat pAb to human IgG-Dylight 650, abcam, Cat#ab98593), incubated at 4°C for 30 min, centrifuged to wash the plate, and then used an iQue Screener flow cyto
  • the method of flow cytometry was used to verify the simultaneous binding activity of both ends of the OriAb-362 double antibody.
  • the method was as follows: select CHO-hCLDN18.2 cells overexpressing human CLDN18.2 to plate, and then add serially diluted OriAb-362 double antibody, antibody from 25nM At the beginning of the working concentration, two-fold gradient dilution was performed to form a total of 9 concentration gradients.
  • monoclonal antibody 5E6 was set as a negative control, and the antibody concentration was the same as that of Ori-Ab362 double antibody; an equal volume of fixed concentration (1.5ug/ml working concentration) of biotinylated 4-1BB protein (h4-1BB-Biotin), incubated at 4°C for 1 hour, centrifuged to wash the plate, added secondary antibody (BD phamingen APC Streptavidin, Cat#554067), incubated at 4°C for 30 minutes The plate was washed by centrifugation, and then an iQue Screener flow cytometer (purchased from IntelliCyt) was used to detect the fluorescent signal on the machine. The results are shown in Figure 3.
  • the OriAb-362 double antibody can simultaneously bind to CHO-CLDN18.2 cells and biotinylated 4-1BB protein, that is, the OriAb-362 double antibody has two simultaneous binding activities.
  • the OriAb-362 double antibody is basically the same as the parental monoclonal antibody 5E6, and the EC50 value is basically the same; the results are shown in Figure 4B.
  • the binding activity to 293T-macacaCLDN18.2 cells In terms of binding activity, the OriAb-362 double antibody is slightly weaker than the parental monoclonal antibody 5E6, showing a relationship of 1.5 times the EC50 value.
  • the OriAb-362 double antibody basically retains the binding activity of the parent monoclonal antibody 5E6 to the mouse/cynomolgus monkey CLDN18.2 antigen, and can simultaneously bind to human, mouse, and cynomolgus monkey CLDN18.2, and has cross-species binding activity.
  • the antibodies were named Biotin-OriAb362, Biotin-5E6, Biotin-IMAB362.
  • OriAb362 double antibody, 5E6 monoclonal antibody and IMAB362 monoclonal antibody may have different antigen-binding epitopes, and the OriAb-362 double antibody is more competitive than IMAB362 in terms of antigen binding activity, and the OriAb-362 double antibody completely retains the parental monoclonal antibody
  • the antigen-binding epitope of 5E6 has the same competitive binding activity.
  • this example uses the luciferase experimental method to detect: 1)
  • the effector cell 41BB-NFkB-293T is a stably integrated NFkB luciferase reporter
  • 2) CHO-hCLDN18.2 is a lentivirus-transfected high-expression human CLDN18.2 cell
  • NUGC-4 is a natural low-expression human CLDN18.2 cell
  • CHO-hCLDN18.2 and NUGC-4 were selected as target cells with different expression levels of CLDN18.2 in this example, and CHO-hCL
  • OriAb-362 double antibody can stimulate target cells to activate 4-1BB luciferase signaling, which is dependent on the expression of CLDN18.2, and its activation intensity is positively correlated with the expression abundance of human CLDN18.2 in target cells (Fig. 6A, the target cells with high expression of human CLDN18.2, the activation fold is about 5 times; Fig. 6B, the target cells with low expression of human CLDN18.2, the activation fold is about 2 times), and it is antibody concentration dependent, while Neither the parental mAb nor its combination could activate 4-1BB luciferase signaling.
  • Example 7 OriAb-362 double antibody can stimulate T cell activation and promote the secretion of IL-2 factor
  • Superantigens such as SEA can cross-link TCR and MHC class II molecules to activate CD4+ T cells.
  • SEA superantigen was used to continuously stimulate human PBMC, and the factor release experiment was used to verify the activity of OriAb-362 double antibody in promoting the immune response of human PBMC.
  • "X-VIVO+5%FBS+5ug/ml SEA" culture medium was resuspended, the cell density was 1.25*10 6 /well, 100ul cell suspension/well, spread evenly in a V-bottom sterile 96-well plate; Positive target cells are CHO-hCLDN18.2 cells with high expression of human CLDN18.2 and human gastric cancer cell NUGC-4 with natural low expression of human CLDN18.2.
  • Negative target cells are CHO and CHO-hCLDN18.1 cells.
  • Solid-phase binding of high-density anti-CD3 antibody can cause cross-linking of TCR-CD3 complex, directly generate activation signal, and stimulate T cell activation.
  • the solid-phase-bound anti-human CD3 antibody is used to continuously stimulate human PBMC, and the factor release experiment is used to verify the activity of the OriAb-362 double antibody in promoting the immune response of human PBMC, as follows: Coating with a sterile 96-well plate that can absorb proteins Anti-human CD3 antibody, diluted with sterile PBS to a concentration of 1 ⁇ g/mL, 100mL per well, coated overnight at 4°C; washing CD3 protein: suck up the PBS in the 96-well plate with an aspirator the next day, and use X-VIVO+ Wash the plate three times with 5% FBS medium; then add PBMC, target cells and antibodies in sequence, the antibody concentration and cell amount are the same as above, and the resuspension buffer is X-VIVO+5% FBS; IL
  • human 4-1BB transgenic C57BL/6 mice Biocytogen, 110004, B-h4-1BB mice
  • tumors were inoculated subcutaneously Mouse colon cancer cells overexpressing human CLDN18.2, that is, MC38-hCLDN18.2 cells (1.5*10 6 /mouse), were grouped when the average tumor volume of the mice was 80 mm 3 , and the grouping information was shown in Table 1 below.
  • mice The body weight and tumor volume of the mice were continuously observed until D30, and the body weight of the mice was normal throughout the observation period, as shown in Figure 8A; the tumor inhibition of the mice is shown in Figure 8B, compared to Group1: PBS group and Group2: hIgG-Fc group, Group3/Group4 /Group5/Group6 all had significant tumor inhibitory activity (p ⁇ 0.0001), among which Group3: 5E6 monoclonal antibody group had no mice that completely eliminated tumors (0/6), and the tumor volume inhibition rate TGI TV (%) was 39.22% , Group4: 4 mice in the YN006 monoclonal antibody group completely eliminated tumors (4/6), TGI TV (%) was 102.62%, Group5: 6 mice in the OriAb-362 double antibody group completely eliminated tumors (6/6 ), TGI TV (%) was 102.81%, and in Group6: 5E6+YN006 combined administration group, 5 mice completely eliminated tumors (5/6), and TGI TV (%) was 98.62%.
  • Table 2 The specific
  • mice Administration route & cycle Group1 PBS / 6 IP, Biw*3 weeks Group2 hIgG-Fc 10 6 IP, Biw*3 weeks Group3 5E6 10 6 IP, Biw*3 weeks Group4 YN006 10 6 IP, Biw*3 weeks Group5 OriAb-362 13.3 6 IP, Biw*3 weeks Group6 5E6+YN006 10+10 6 IP, Biw*3 weeks
  • mice TGI TV (%) value 30 days after 1st administration G1, PBS, 10ml/kg 0/6 / G2, hIgG-Fc, 10mg/kg 0/6 13.89% G3, 5E6, 10mg/kg 0/6 39.22% G4, YN006, 10mg/kg 4/6 102.62% G5, OriAb362, 13.3mg/kg 6/6 102.81% G6, 5E6+YN006, 10+10mg/kg 5/6 98.62%
  • this example reduced the dosage of the OriAb-362 double antibody and designed the following experimental scheme: the mice were human 4-1BB transgenic C57BL/6 mice (Biocytogen, 110004, B-h4-1BB mice), subcutaneously transplanted mouse colon cancer cells overexpressing human CLDN18.2, that is, MC38-hCLDN18.2 cells (1.5*10 6 /only), and treated mice Grouping was performed when the average tumor volume was 80 mm 3 , and the grouping information was shown in Table 3.
  • mice On the day of grouping, intraperitoneal administration (BIW*3; twice a week for three weeks) was performed and recorded as D1, and the mice were measured three times a week Body weight and tumor volume, when the mouse tumor volume is greater than 3000mm 3 Euthanize the mouse. Analyze the tumor inhibition data of mice on Day 31 after 1st administration, the tumor elimination status of mice in each group is shown in Table 4: G2: OriAb-362, 13.3mg/kg high-dose group and G3: OriAb-362, 3.325mg/kg All the mice in the kg low-dose group eliminated tumors (6/6), and 3 mice in the G4:5E6+YN006 combined administration group eliminated tumors (3/6).
  • mice whose tumors had been eliminated continued to be observed until Day40, and then each mouse was inoculated subcutaneously on the contralateral side.
  • the tumor cells and doses used for inoculation were the same as above (MC38-hCLDN18.2 cells, 1.5*10 6 /mouse)
  • 6 blank mice of the same strain without any experiment were selected as the Naive control group and subcutaneously received tumor treatment at the same time. After receiving the tumor, it was observed for 20 days, and the tumor volume of the mice was measured three times a week. The tumor growth of the mice is shown in Figure 9.
  • the OriAb-362 double antibody can induce complete tumor elimination in all the mice enrolled (6/6), and re-inoculate tumors on the contralateral side of the tumor-eliminated mice, The mice still maintain the state of tumor elimination, no new tumor formation, showing good tumor immune memory function, and have long-term protective immune memory.
  • FACS flow cytometry fluorescence sorting technique
  • iQue Screener flow meter purchased from IntelliCyt Company
  • PBS containing 0.1% BSA as buffer to detect the specific binding activity of the above-mentioned chimeric antibody to target cells
  • select Three kinds of target cells a stably transfected cell line expressing human CLDN18.2, a stably transfected cell line expressing human CLDN18.1, and a tumor cell line were tested for their binding activity.
  • the cell lines that obtained stable and high expression of CLDN18 were constructed and marked as 293T-human CLDN18.2, CHO-human CLDN18.2 and SP2/0-human CLDN18.2 cells respectively, the cells were digested and counted, and the cells were resuspended and adjusted in flow buffer To 1 ⁇ 10 6 /ml, add 30ul/well into a V-bottom 96-well plate; add 30ul/well of the primary antibody, starting at a concentration of 30ug/ml, dilute with flow buffer in a two- or three-fold gradient to form a 7
  • Each antibody was set as a PBS negative control, and the positive control antibody was zolbetuximab purified in Example 1; incubated at 4°C for 1 hour, washed once with flow buffer, and added secondary antibody (abcam, Cat#ab98593), 30ul/ Well, incubate at 4°C for 30 minutes; wash the flow buffer twice, shake the cells loose, add 25ul/well flow buffer, and wait
  • the positive control antibody is a commercially available anti-CLDN18 antibody (Anti-Claudin18 antibody) (abcam, Cat#ab203563), whose antigen-binding site is located in the intracellular part of the CLDN18.2 tetrachorotin protein, and flow cytometric intracellular staining analysis is required.
  • the cells are constructed 293T-human CLDN18.1 and SP2/0-human CLDN18.1 cells.
  • the cells After the cells are digested and counted, they are fixed and ruptured, and the treated cells are resuspended to 1 ⁇ 10 6 in flow buffer /ml, 30ul/well was added to a V-bottom 96-well plate; 30ul/well was added to the primary antibody, the antibody was initially diluted at a concentration of 30ug/ml, and was diluted with flow buffer in a three-fold ratio to form 7 gradients.
  • PBS negative control, positive control antibody dilution conditions are the same as above; incubate at 4°C for 1 hour, wash with flow buffer, add secondary antibody (abcam, Cat#ab98593 and Cat#ab150079), 30ul/well, incubate at 4°C for 30 minutes; Wash twice with flow buffer, shake the cells loose, add 25ul/well flow buffer, and wait for the machine. Substituting the original data into GraphPad8.0 software for drawing and calculation, the results are shown in Figure 11.

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Abstract

L'invention concerne une protéine de liaison à l'antigène isolée, son procédé de préparation et son utilisation. La protéine de liaison à l'antigène comprend : un premier domaine de liaison à l'antigène et un second domaine de liaison à l'antigène, le premier domaine de liaison à l'antigène pouvant se lier spécifiquement à la claudine 18.2 (CLDN18.2), et le premier domaine de liaison à l'antigène contenant au moins une CDR dans une séquence d'acides aminés telle que représentée dans SEQ ID NO : 100.
PCT/CN2023/071504 2022-01-11 2023-01-10 Protéine de liaison à l'antigène bispécifique anti-cldn18.2 et 4-1bb et son utilisation WO2023134657A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180086828A1 (en) * 2015-04-15 2018-03-29 Patricius Hendrikus Corne Van Berkel Site-specific antibody-drug conjugates
CN111788228A (zh) * 2018-05-18 2020-10-16 礼新医药科技(上海)有限公司 抗密蛋白18.2抗体及其用途
CN113121698A (zh) * 2018-04-09 2021-07-16 原启生物科技(上海)有限责任公司 抗pd-l1抗体及其用途
CN113166265A (zh) * 2019-08-12 2021-07-23 天境生物科技(上海)有限公司 抗紧密连接蛋白18.2和抗4-1bb双特异性抗体及其用途
WO2021239026A1 (fr) * 2020-05-29 2021-12-02 杭州邦顺制药有限公司 Anticorps dirigé contre la claudine 18.2 et utilisation associée
WO2022206975A1 (fr) * 2021-04-02 2022-10-06 原启生物科技(上海)有限责任公司 Protéine de liaison à l'antigène cldn18.2 et son utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180086828A1 (en) * 2015-04-15 2018-03-29 Patricius Hendrikus Corne Van Berkel Site-specific antibody-drug conjugates
CN113121698A (zh) * 2018-04-09 2021-07-16 原启生物科技(上海)有限责任公司 抗pd-l1抗体及其用途
CN111788228A (zh) * 2018-05-18 2020-10-16 礼新医药科技(上海)有限公司 抗密蛋白18.2抗体及其用途
CN113166265A (zh) * 2019-08-12 2021-07-23 天境生物科技(上海)有限公司 抗紧密连接蛋白18.2和抗4-1bb双特异性抗体及其用途
WO2021239026A1 (fr) * 2020-05-29 2021-12-02 杭州邦顺制药有限公司 Anticorps dirigé contre la claudine 18.2 et utilisation associée
WO2022206975A1 (fr) * 2021-04-02 2022-10-06 原启生物科技(上海)有限责任公司 Protéine de liaison à l'antigène cldn18.2 et son utilisation

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