WO2022206975A1 - Cldn18.2抗原结合蛋白及其用途 - Google Patents

Cldn18.2抗原结合蛋白及其用途 Download PDF

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Publication number
WO2022206975A1
WO2022206975A1 PCT/CN2022/084922 CN2022084922W WO2022206975A1 WO 2022206975 A1 WO2022206975 A1 WO 2022206975A1 CN 2022084922 W CN2022084922 W CN 2022084922W WO 2022206975 A1 WO2022206975 A1 WO 2022206975A1
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seq
amino acid
acid sequence
binding protein
sequence shown
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French (fr)
Inventor
杨沙沙
何晓文
李萌
王华菁
杨焕凤
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Oricell Therapeutics Co Ltd
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Oricell Therapeutics Co Ltd
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Priority to CN202280025138.XA priority Critical patent/CN117083299A/zh
Priority to KR1020237035144A priority patent/KR20230166096A/ko
Priority to US18/553,479 priority patent/US20240182562A1/en
Priority to EP22779155.5A priority patent/EP4299593A4/en
Priority to JP2023560013A priority patent/JP2024514246A/ja
Publication of WO2022206975A1 publication Critical patent/WO2022206975A1/zh
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein capable of binding to CLDN18.2.
  • Gastric cancer is the second deadliest malignant tumor in the world. According to the data of the World Health Organization in 2015, 754,000 gastric cancer patients die every year in the world; pancreatic cancer is one of the most malignant tumors, according to the 2016 data of "The Lancet" magazine. , More than 200,000 pancreatic cancer patients die every year worldwide.
  • the standard first-line treatment for advanced or recurrent gastric cancer is chemotherapy.
  • trastuzumab to chemotherapy offers some survival benefit in patients with HER2-positive tumors, but only 15% of all gastric cancers are HER2-positive. The development of safe and effective treatments is imminent.
  • CLDN18.2 (Claudin 18.2) 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, and in solid tumors such as lung cancer, esophageal cancer and ovarian cancer.
  • CLDN18.2 belongs to a family of tight junction proteins that control molecular flow between laminar cells. In tumors, however, the tight junctions are disrupted and the CLDN protein loses its primary role. Because CLDN18.2 is abundant in gastric tumors, the researchers estimate that half of all advanced gastric cancer patients could be candidates for new therapies targeting CLDN18.2 antibodies. Furthermore, this unique target is not present in any healthy tissue other than the stomach wall, thereby minimizing treatment side effects. These characteristics suggest that CLDN18.2 is an ideal target for the development of therapeutic monoclonal antibodies.
  • CLDN18.2 is a membrane protein, it is difficult to obtain high-quality antibodies against native proteins by traditional antibody screening methods. At present, in vivo immunization is mostly used, such as the method of DNA injection, but multiple rounds of immunization in animals are required.
  • CLDN18 has two splice variants, CLDN18.1 and CLDN18.2, which differ only in the N-terminal 69 amino acid sequences, which exist in the first extracellular loop, and the rest of the sequences are identical.
  • CLDN18.1 is expressed in normal lung tissue, so it is necessary to screen out antibodies that can bind to CLDN18.2 while not binding to CLDN18.1.
  • the CLDN18.2 sequence has extremely high homology between human and mouse. Therefore, there is an urgent need to obtain antibodies that specifically bind to CLDN18.2.
  • the present application provides an antigen-binding protein capable of binding CLDN18.2, which exhibits one or more desired functional properties, eg, it can specifically bind to human CLDN18.2 and does not substantially bind to human CLDN18.1, can Has CDC activity and/or has antitumor activity.
  • the present application also provides nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells and methods of making the isolated antigen binding proteins.
  • the isolated antigen binding proteins described herein can be used to prevent and/or treat diseases and/or disorders, such as tumors and/or cancer.
  • the application provides an isolated antigen binding protein comprising HCDR3 comprising the amino acid sequence shown in SEQ ID NO:67.
  • the HCDR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:12, and SEQ ID NO:18.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:68 or SEQ ID NO:11.
  • the HCDR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:17.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:16.
  • the isolated antigen binding protein comprises H-FR1
  • the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1
  • the H-FR1 comprises SEQ ID NO: The amino acid sequence shown in 72.
  • the H-FR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:34, SEQ ID NO:43, and SEQ ID NO:49.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:73 amino acid sequence.
  • the H-FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:35 or SEQ ID NO:44.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO: 80 or SEQ ID The amino acid sequence shown in NO:45.
  • the H-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:36 or SEQ ID NO:42.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the set forth in SEQ ID NO:74 amino acid sequence.
  • the H-FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:46 or SEQ ID NO:37.
  • the isolated antigen binding protein comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:78.
  • the VH of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:15, and SEQ ID NO:19.
  • the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:69.
  • the LCDR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:23, and SEQ ID NO:25.
  • the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:70.
  • the LCDR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:22.
  • the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:71.
  • the LCDR1 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:21.
  • the isolated antigen binding protein comprises L-FR1
  • the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1
  • the L-FR1 comprises SEQ ID NO: The amino acid sequence shown in 75.
  • the L-FR1 of the isolated antigen binding protein comprises the amino acids set forth in any one of SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:50, and SEQ ID NO:53 sequence.
  • the isolated antigen-binding protein comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO:39 amino acid sequence.
  • the isolated antigen binding protein comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO:76 amino acid sequence.
  • the L-FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:40, SEQ ID NO:48 and SEQ ID NO:51.
  • the isolated antigen binding protein comprises L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the set forth in SEQ ID NO:77 amino acid sequence.
  • the L-FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:41, SEQ ID NO:52, and SEQ ID NO:54.
  • the isolated antigen binding protein comprises VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO:79.
  • the VL of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:20, and SEQ ID NO:24.
  • the isolated antigen binding protein comprises any one of the groups of VH and VL selected from the group consisting of:
  • VH comprises the amino acid sequence shown in SEQ ID NO: 1
  • VL comprises the amino acid sequence shown in SEQ ID NO: 5;
  • VH comprises the amino acid sequence shown in SEQ ID NO: 15
  • VL comprises the amino acid sequence shown in SEQ ID NO: 5;
  • VH comprises the amino acid sequence shown in SEQ ID NO:9
  • VL comprises the amino acid sequence shown in SEQ ID NO:13
  • VH comprises the amino acid sequence shown in SEQ ID NO: 19
  • VL comprises the amino acid sequence shown in SEQ ID NO: 20
  • VH comprises the amino acid sequence shown in SEQ ID NO: 19
  • VL comprises the amino acid sequence shown in SEQ ID NO: 24.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  • the antibody heavy chain constant region is derived from a human IgGl heavy chain constant region.
  • the isolated antigen binding protein comprises an antibody light chain constant region.
  • the antibody light chain constant region is derived from a human IgK constant region.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises a Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antibody is selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein does not substantially compete with a reference antibody for binding to CLDN18.2, as verified in FACS, wherein the reference antibody comprises a heavy chain variable region (VH) and a light chain Variable region (VL), the VH of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:55, and the VL of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:56.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the isolated antigen binding protein is capable of specifically binding CLDN18.2 and substantially not binding to CLDN18.1.
  • the CLDN18.2 to which the isolated antigen binding protein is capable of specific binding comprises mouse CLDN18.2, cynomolgus CLDN18.2 and/or human CLDN18.2.
  • the isolated antigen binding protein has CDC activity.
  • the isolated antigen binding protein is capable of inhibiting tumor growth and/or tumor cell proliferation.
  • the present application also provides a chimeric antigen receptor comprising a targeting moiety comprising the antigen binding protein described herein.
  • the chimeric antigen receptor comprises a costimulatory domain comprising a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, Ligands for TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40 and MyD88.
  • a costimulatory domain comprising a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7
  • the costimulatory domain of the chimeric antigen receptor is an intracellular costimulatory signaling region derived from 4-1BB.
  • the costimulatory domain of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:85.
  • the chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10, DAP-12 and at least one ITAM domain.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpes virus
  • the intracellular signaling domain of the chimeric antigen receptor is a CD3 ⁇ -derived signaling domain.
  • the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:86.
  • the chimeric antigen receptor comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • the transmembrane region of the chimeric antigen receptor is a CD8-derived transmembrane region.
  • the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:84.
  • the chimeric antigen receptor includes a hinge region between the targeting moiety and the transmembrane region, the hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of : CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region of the chimeric antigen receptor is a CD8-derived hinge region.
  • the hinge region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:83.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide of the chimeric antigen receptor is derived from the signal peptide of the CD8 protein.
  • the signal peptide of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:88.
  • the chimeric antigen receptor further comprises a low density lipoprotein receptor-related protein or a fragment thereof.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:91.
  • the present application also provides a polypeptide molecule comprising the isolated antigen binding protein or the chimeric antigen receptor.
  • the polypeptide molecule comprises a fusion protein.
  • the present application also provides immunoconjugates comprising the isolated antigen binding protein.
  • the present application also provides isolated one or more nucleic acid molecules encoding the isolated antigen binding protein, the chimeric antigen receptor or the polypeptide molecule.
  • the nucleic acid molecule comprises SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID The nucleotide sequence shown in any one of NO:32 and SEQ ID NO:33.
  • the nucleic acid molecule comprises any group of nucleotide sequences selected from the group consisting of:
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule or the vector.
  • the cells are immune effector cells.
  • the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral Blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • NK cells natural killer cells
  • NKT cells monocytes, dendritic cells
  • monocytes dendritic cells
  • granulocytes lymphocytes
  • leukocytes granulocytes
  • peripheral Blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells are T cells.
  • the cell further comprises and/or expresses a low density lipoprotein receptor-related protein or a fragment thereof.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:91.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the The carrier, and/or the cells, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the antigen binding protein is expressed.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and /or use of the pharmaceutical composition in the preparation of a medicament for preventing, alleviating and/or treating a disease and/or condition.
  • the disease and/or disorder comprises cancer.
  • the cancer comprises solid tumors and/or hematological tumors.
  • the cancer comprises gastric and/or colon cancer.
  • the present application also provides a method for detecting CLDN18.2 in a sample, the method comprising administering the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate , the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the present application also provides a reagent or kit for detecting CLDN18.2 in a sample, comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, and the immunoconjugate , the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and /or use of the pharmaceutical composition in the preparation of a kit for detecting the presence and/or content of CLDN18.2 in a sample.
  • Figure 1 shows the detection of the flow binding activity of the antigen-binding protein of the present application to highly expressed human CLDN18.2 cells;
  • Figure 2 shows the detection of the flow binding activity of the antigen-binding protein of the present application to highly expressed human CLDN18.1 cells
  • Figure 3 shows the detection of the flow binding activity of the antigen-binding protein of the present application to tumor cell lines
  • Figure 4 shows the species cross-binding activity analysis of the antigen-binding proteins of the present application
  • Figures 5a-5b show the detection of the CDC activity of the antigen-binding protein of the present application
  • Figure 6 shows the competition binding activity analysis of the antigen binding protein of the present application
  • Figure 7 shows the body weight change of mice after administration
  • Figure 8 shows the relative change (%) of the body weight of mice after administration
  • Figure 9 shows the tumor volume changes in mice after administration
  • Figure 10 shows the tumor inhibition rate of mice after administration
  • Figure 11 shows the survival curve of mice after administration.
  • Figures 12A-B show the identification of antigen binding activity of CLDN18.2 single chain antibody.
  • Figure 13 shows the identification of the binding activity of CLDN18.2 single chain antibody to non-target cells.
  • Figure 14 shows the antitumor activity of the CLDN18.2 antigen binding protein described in the present application in a mouse model of human gastric cancer.
  • Figure 15A-D shows the expression of CLDN18.2-specific CAR in T cells.
  • Figure 16 shows the in vitro killing activity detection of CAR-T cells.
  • Figure 17A-B shows the detection of factor secretion of CLDN18.2-specific CAR.
  • Figure 18A-D shows the tumor suppressive effect of CLDN18.2-specific CAR-T cells in a mouse colon cancer CDX model.
  • CLDN18.2 or “Claudin18.2” are used interchangeably and generally refer to isoform 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 epitope in common with CLDN18.2.
  • the amino acid sequence of CLDN18.2 (eg, human CLDN18.2) may be known in the art.
  • the human CLDN18.2 nucleotide sequence can be shown under GeneBank Accession No. 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.
  • CLDN18.1 or “Claudin18.1” are used interchangeably and generally refer to isoform 1 of the cell junction claudin Claudin18.
  • the term encompasses "full length", unprocessed CLDN18.1 as well as any form of CLDN18.2 produced by cellular processing.
  • CLDN18.2 may include intact CLDN18.1 and fragments thereof, functional variants, isoforms, species homologues, derivatives, analogs, and analogs having at least one epitope in common with CLDN18.1.
  • the amino acid sequence of CLDN18.1 eg, human CLDN18.1
  • the human CLDN18.1 nucleotide sequence can be shown under GeneBank Accession No.
  • NM_016369.4 the mouse CLDN18.1 nucleotide sequence can be shown under GeneBank Accession No. NM_019815.3.
  • the cynomolgus monkey CLDN18.1 nucleotide sequence can be shown under GeneBank Accession No. XM_005545863.2.
  • isolated generally refers to artificial means obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain 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 isolated of.
  • isolated does not exclude the admixture of artificial or synthetic materials, nor does it exclude the presence of other impurities that do not affect the activity of the material.
  • isolated antigen-binding protein generally refers to a protein with antigen-binding ability obtained by artificial means from the natural state.
  • isolated antigen-binding protein may comprise an antigen-binding moiety and, optionally, a framework or framework portion that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding moiety to the 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; trifunctional antibodies; tetrabodies; Fab, Fab', Fv fragments, F (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.
  • 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 part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • variable generally refers to the fact that certain segments of the variable domains may differ greatly in sequence between antibodies.
  • the variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains are referred to as framework regions (FRs).
  • FRs framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs that form loops connecting, and in some cases forming 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 antigen-binding site of the antibody (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, and encompasses any polypeptide that includes an antigen-binding 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 , mutant 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 should include an 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 antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contains 10 antigen-binding sites, while IgA antibody includes 2-5 that can be combined with J chain to form multivalent Combined basic 4-chain unit.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each L chain is connected to the H chain by one covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • 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 each of the alpha and gamma chains and four CH domains for the mu and epsilon isoforms.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end.
  • VL corresponds to VH and CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Particular amino acid residues are thought to form the interface between the light and heavy chain variable domains.
  • the VH and VL pair together to form a single antigen binding site.
  • immunoglobulins can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Based on the amino acid sequence of the heavy chain (CH) constant domains, immunoglobulins can be divided into 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 small differences in CH sequence and function, eg, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgK1.
  • CDRs also referred to as “complementarity determining regions” generally refers to regions within the variable domains of antibodies, the sequences of which are highly variable and/or form structurally defined loops.
  • an antibody typically includes 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 also functional and stable 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).
  • FR generally refers to the more highly conserved portions of antibody variable domains, which are referred to as framework regions.
  • the variable domains of native heavy and light chains each comprise four FR regions, namely 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).
  • the VL of the isolated antigen binding proteins 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 the 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 with papain as described above. Papain digestion of the antibody yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • a Fab fragment may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH1) 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 can include all of the light chain, all of the variable regions of the heavy chain, and all or part of the first and second constant regions of the heavy chain.
  • Fab' fragments 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 whole 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 antigens.
  • 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.
  • Heavy chain variable regions and light chain variable regions include, for example, CDRs.
  • Fv fragments include all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.
  • 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 synthetic linkers such as short flexible polypeptide linkers) and are capable of being expressed as single-chain polypeptides, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a scFv may have the VL and VH variable regions described in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may 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 a VH domain or a VL domain, see eg 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 other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • the term "monoclonal antibody” generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are usually highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the 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 antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • Fully human antibody generally refers to an antibody comprising only human immunoglobulin protein sequences.
  • a fully human antibody may contain murine sugar chains if it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells.
  • mouse antibody or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.
  • Fully human antibodies can be generated 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 the use of libraries, are known in the art.
  • the term “directly connected” is opposed to the term “indirectly connected”, which generally refers to a direct connection.
  • the direct connection may be the 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 connected” generally refers to a situation where substances are not directly connected.
  • the indirect linkage may be the case of linkage through a spacer.
  • the C-terminus of L-FR1 and the N-terminus of LCDR1 may be linked directly or indirectly.
  • isolated nucleic acid molecule generally refers to any length of nucleotides in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or synthetically 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.
  • a vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • vectors can include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda phage or M13 Bacteriophages and animal viruses, etc.
  • Animal virus species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses vesicle virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses vesicle virus
  • a vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication site.
  • the carrier may also include components to assist its entry into
  • 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 plasmid or vector, comprising a nucleic acid molecule described herein or a nucleic acid molecule described herein Carrier.
  • a cell can include the progeny of a single cell. Progeny may not necessarily be identical (in morphology or in genome) to the original parent cell due to natural, accidental or intentional mutation.
  • Cells can include cells transfected in vitro with the vectors described herein.
  • the cells can be bacterial cells (eg, E.
  • yeast cells or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells , NSO cells or stem cells (eg ES cells, iPS cells, mesenchymal stem cells) or immune cells (eg T cells, NK cells, NKT cells, macrophages).
  • the cells are mammalian cells.
  • the mammalian cells are HEK293 cells.
  • the term "pharmaceutical composition” generally refers to a composition for the prevention/treatment of a disease or disorder.
  • the pharmaceutical composition may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a carrier described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or suitable preservatives preparation.
  • 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 pharmaceutically acceptable carriers, excipients, or stabilizers that, at the doses and concentrations employed, are effective for the cells or mammals to which they are exposed.
  • 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 “specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be found in a heterogeneous population of molecules, including biomolecules
  • the presence of the target determines the presence of the target.
  • an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets .
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include, but does not require, exclusive binding.
  • the term "reference antibody” generally refers to an antibody that can bind to an antigen (eg, CLDN18.2).
  • an antigen eg, CLDN18.2
  • the antigen binding proteins described herein do not have competitive binding activity compared to the reference antibody.
  • the reference antibody described herein can be zolbetuximab.
  • the term "substantially unbound” generally refers to no binding or binding with very weak binding activity.
  • the very weak binding activity may refer to, for example, in the flow binding activity assay, the average fluorescence intensity value of the antibody that detects binding to CLDN18.1 is at least about 50% lower, about 55% lower than that of the CLDN18.1 antibody positive control %, about 60%, about 70%, about 80%, about 90%, about 91%, about 91%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, About 99%, about 100%.
  • the term "subject” generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • the tumor may be a solid tumor or a hematological tumor.
  • the tumor can be a CLDN18.2 positive tumor.
  • 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 is not limited to, gastric cancer and/or 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.
  • the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, a substitution, deletion or addition of one in the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the CLDN18.2 protein). or multiple amino acid proteins or polypeptides.
  • the functional variant may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the 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 capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • the homologue may be at least about 85% (eg, having an amino acid sequence of at least about 85% with the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the CLDN18.2 protein) 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) homologous proteins or polypeptides.
  • the polypeptide eg, an antibody or fragment thereof that specifically binds the CLDN18.2 protein
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • the same amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • FASTA and BLAST A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, “Rapid and Sensitive Protein Similarity Search", Science, 227: 1435-1441, 1989.
  • a description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215: 403-410 , 1990.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the CDRs of antibodies are part of the variable region. Amino acid residues in this region can make contact with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. in combination. These encoding systems are known in the art, see http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR regions.
  • the CDRs encompass CDR sequences that are divided according to any CDR division; variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR .
  • variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR .
  • homologues thereof are also encompassed, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 92%, amino acid sequences of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the application provides an isolated antigen binding protein, which can comprise HCDR3.
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:67.
  • the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:12.
  • the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:18.
  • the isolated antigen binding protein may comprise HCDR2.
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:68.
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:17.
  • the isolated antigen binding protein may comprise HCDR1.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 16, and the HCDR2 may comprise SEQ ID NO: 68 or SEQ ID The amino acid sequence shown in NO: 11, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 67.
  • the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 16, and the HCDR2 may comprise SEQ ID NO: 16
  • the amino acid sequence shown in any one of NO:3, SEQ ID NO:17 and SEQ ID NO:11, and the HCDR3 may comprise any one of SEQ ID NO:4, SEQ ID NO:12 and SEQ ID NO:18 An amino acid sequence shown.
  • the HCDR1, HCDR2 and HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence selected from any of the following groups:
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 3
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 4 ;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12 ;as well as
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18 .
  • the isolated antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO: The amino acid sequence shown in 72.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:43.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:49.
  • the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO: 73 amino acid sequence.
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:44.
  • the isolated antigen binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO: 80 amino acid sequence.
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:45.
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:42.
  • the isolated antigen binding protein may comprise H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise SEQ ID NO: 74 amino acid sequence.
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 72
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 73
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 73
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 74.
  • the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 34, SEQ ID NO: 43 and SEQ ID NO: 49
  • the H-FR2 may comprise SEQ ID NO: 35 or the amino acid sequence shown in SEQ ID NO:44
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:36 or SEQ ID NO:42
  • the H-FR4 may comprise SEQ ID NO:46 Or the amino acid sequence shown in SEQ ID NO:37.
  • the H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise an amino acid sequence selected from any of the following groups:
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 34
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 35
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 36
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 37;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:43
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:44
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:45
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 46;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 34
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 35
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 42
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 37;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:49
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:35
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:42
  • the amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, HFR2, HFR3 and H-FR4.
  • the HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4 may respectively comprise in sequence: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37; SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO:45 and SEQ ID NO:46; SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:42 and SEQ ID NO:37; or as shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:49,
  • the isolated antigen binding protein may comprise VH.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:78.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:1.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:9.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:15.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:19.
  • the isolated antigen binding protein may comprise LCDR3.
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:69.
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:14.
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the isolated antigen binding protein may comprise LCDR2.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:70.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the isolated antigen binding protein may comprise LCDR1.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:71.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:71
  • the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:70
  • the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:69 .
  • the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:21
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:22
  • the amino acid sequence shown and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 25.
  • the LCDR1, LCDR2 and LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence selected from any of the following groups:
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8 ;
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:14 ;
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 23 ;as well as
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:25 .
  • the isolated antigen binding protein may comprise L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:75.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:47.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:50.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:53.
  • the isolated antigen binding protein may comprise L-FR2, the L-FR2 being located between the LCDR1 and the LCDR2.
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:39.
  • the isolated antigen binding protein may comprise L-FR3, the L-FR3 being located between the LCDR2 and the LCDR3.
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:76.
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:40.
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:48.
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:51.
  • the isolated antigen binding protein may comprise L-FR4, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3.
  • the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:77.
  • the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:41.
  • the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:52.
  • the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4.
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:75
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:39
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:76
  • the amino acid sequence shown and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:77.
  • the L-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:50 and SEQ ID NO:53
  • the L-FR2 can comprise the amino acid sequence shown in SEQ ID NO: 39
  • the L-FR3 can comprise the amino acid sequence shown in any one of SEQ ID NO: 40, SEQ ID NO: 48 and SEQ ID NO: 51 amino acid sequence
  • the L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:41, SEQ ID NO:52 and SEQ ID NO:54.
  • the L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen binding protein may comprise any of the following amino acid sequences:
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:38
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:39
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:40
  • the amino acid sequence shown, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 41;
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 47
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 39
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 48
  • the amino acid sequence shown, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 41;
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:50
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:39
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:51
  • the amino acid sequence shown, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 52;
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 53
  • the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 39
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 40
  • the amino acid sequence shown, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the isolated antigen binding proteins comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4.
  • the LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and L-FR4 may respectively comprise: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:47, SEQ ID NO: 39, SEQ ID NO:48 and SEQ ID NO:41; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:50, SEQ ID NO:39, SEQ ID NO:51 and SEQ ID NO:52; or SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:53, SEQ ID NO:39, SEQ ID NO:40
  • the isolated antigen binding protein comprises VL.
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:79.
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:5.
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:13.
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:20.
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:24.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 16, and the HCDR2 may comprise SEQ ID NO: 68 or SEQ ID
  • the amino acid sequence shown in NO: 11 the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 67
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 71
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 69.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 16, and the HCDR2 may comprise SEQ ID NO: 3, SEQ ID NO: 16
  • the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 12 and SEQ ID NO: 18
  • the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 21
  • the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 22
  • the LCDR3 can The amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 23 and SEQ ID NO: 25 is included.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the isolated antigen binding protein may comprise any of the following amino acid sequences:
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 3
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 4
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8;
  • the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 10
  • the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 11
  • the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 12
  • the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:6,
  • the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO:7
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:14;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6,
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18
  • the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 21
  • the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 22
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 23;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18
  • the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 6
  • the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 7
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 25.
  • the isolated antigen binding protein may comprise VH and VL.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:78
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:79.
  • the VH of the isolated antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 15 and SEQ ID NO: 19, and
  • the VL may comprise the amino acid sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:20, and SEQ ID NO:24.
  • the isolated antigen binding protein may comprise VH and VL selected from any of the following groups:
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 1
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 5;
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 15
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 5;
  • VH may comprise the amino acid sequence shown in SEQ ID NO:9
  • VL may comprise the amino acid sequence shown in SEQ ID NO:13
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 19, and the VL may comprise SEQ ID NO:
  • VH may comprise the amino acid sequence shown in SEQ ID NO: 19
  • VL may comprise SEQ ID NO:
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region.
  • the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgGl heavy chain constant region.
  • the isolated antigen binding protein may comprise an antibody light chain constant region.
  • the antibody light chain constant region may be derived from a human IgK constant region.
  • the isolated antigen-binding proteins described in the present application may comprise heavy chain and/or light chain sequences with one or more conservative sequence modifications.
  • conservative sequence modifications is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody.
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are replacement of amino acid residues with amino acid residues with similar side chains. Sets of amino acid residues with similar side chains are known in the art.
  • amino acid residue groups include those with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains ( For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains ( For example, tyrosine, phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eglycine, as
  • one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described herein may be replaced with other amino acid residues of the same side chain group.
  • Those skilled in the art know that some conservative sequence modifications will not abolish antigen binding, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin.Can.Res.6: 2835-43.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting eg, Western blot
  • flow cytometry eg, FACS
  • immunohistochemistry immunofluorescence, etc.
  • the isolated antigen binding protein is capable of specifically binding the CLDN18.2 antigen.
  • Antigen binding proteins that "specifically bind" to the CLDN18.2 antigen can generally bind CLDN18.2, but not or substantially not bind to other proteins lacking the CLDN18.2 sequence.
  • an antigen binding protein eg, an antibody
  • Whether an antigen binding protein (eg, an antibody) binds the antigen CLDN18.2 can be determined using any assay known in the art.
  • the specific binding activity of the isolated antigen binding protein to CLDN18.2 can be determined using flow cytometry (FACS).
  • the specific binding can be concentration-dependent binding.
  • the mean fluorescence intensity of the CLDN18.2 increased with increasing CLDN18.2 antibody concentration.
  • the antigen binding protein can bind to human CLDN18.2 protein.
  • the antigen binding proteins described herein can also cross-react with murine (eg, mouse) and/or monkey (eg, cynomolgus monkey) CLDN18.2.
  • murine eg, mouse
  • monkey eg, cynomolgus monkey
  • cross-reactivity generally refers to the ability of an antibody to react with homologous proteins from other species.
  • the antigen binding protein is capable of eliciting a CDC effect.
  • the CLDN18.2 antigen binding proteins described herein are able to induce strong CDC effects against SP2/0-human CLDN18.2 cells.
  • the CLDN18.2 antigen binding proteins described herein are able to induce a strong CDC effect against MC38-human CLDN18.2 cells.
  • the induction may be in a dose-dependent manner.
  • the isolated antigen binding protein was verified in FACS to substantially not compete with the reference antibody for binding to CLDN18.2.
  • the reference antibody can comprise a heavy chain variable region (VH) and a light chain variable region (VL), and the reference antibody, for example, its VH can comprise the amino acid sequence shown in SEQ ID NO: 55, and its VL can Contains the amino acid sequence shown in SEQ ID NO:56.
  • the antigen binding protein is capable of inhibiting tumor growth and/or tumor cell proliferation.
  • the tumor may be a CLDN18.2 expressing tumor, eg, colon cancer.
  • the colon cancer cells may be MC38-human CLDN18.2 cells.
  • the present application also provides a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) may comprise a targeting moiety that binds to the CLDN18.2 protein, for example, that binds to the CLDN18.2 protein
  • the targeting moiety can be an antigen binding protein as described herein.
  • the targeting moiety may be present as an scFv.
  • the scFv can comprise the CDRs, VH and/or VL of the isolated antigen binding proteins described herein.
  • the VH and VL of the scFv can be linked by SEQ ID NO:89.
  • the scFv can comprise the amino acid sequence set forth in SEQ ID NO:90.
  • the CAR includes an extracellular targeting moiety that binds to the CLDN18.2 protein, and may also include other domains.
  • the CAR can include a costimulatory signal region, which can provide a stimulatory signal.
  • the costimulatory signaling region may comprise an intracellular costimulatory signaling region of one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, Ligands for CD244, CD100, ICOS, CD83, CD40 and MyD88.
  • the costimulatory signaling region may be an intracellular costimulatory signaling region derived from 4-1BB.
  • the costimulatory signal region may comprise the amino acid sequence set forth in SEQ ID NO:85.
  • the CAR can comprise an intracellular signaling region, which can comprise a domain with at least one ITAM motif.
  • the intracellular signaling domain can transmit activation signals to the interior of the cell.
  • the intracellular signaling region may comprise an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia Virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and others that contain at least one ITAM domain.
  • EBV Epstein-Barr virus
  • PBj14Nef simian immunodeficiency virus
  • HSKV Kaposi's sarcoma herpes virus
  • the intracellular signaling domain can be a signaling domain derived from CD3 ⁇ .
  • the intracellular signal region may comprise the amino acid sequence set forth in SEQ ID NO:86.
  • the CAR can comprise a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane, which can comprise a hydrophobic alpha helix.
  • the transmembrane domain can be derived from any type I transmembrane protein.
  • the transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.
  • the transmembrane region may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D
  • the transmembrane region can be a CD8-derived transmembrane region.
  • the transmembrane region may comprise the amino acid sequence set forth in SEQ ID NO:84.
  • the CAR can comprise a hinge region, which can be located between the extracellular targeting moiety and the transmembrane domain.
  • the hinge region may comprise the hinge region of one or more proteins selected from the group consisting of CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region can be a CD8-derived hinge region.
  • the hinge region may comprise the amino acid sequence set forth in SEQ ID NO:83.
  • the CAR may further comprise a signal peptide at the N-terminus of the targeting moiety that binds the CLDN18.2 protein.
  • the signal peptide may be a signal peptide derived from the CD8 protein.
  • the signal peptide may comprise the amino acid sequence set forth in SEQ ID NO:88.
  • the CAR may further comprise a low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low density lipoprotein receptor-related protein or fragment thereof can be located at the C-terminus of the CAR.
  • the low-density lipoprotein receptor-related protein or fragments thereof can include low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof may be low-density lipoprotein receptor-related protein 5 and/or 6 or a fragment thereof.
  • the low density lipoprotein receptor-related protein or fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:91.
  • the sequence of the low-density lipoprotein receptor-related protein or its fragment in the CAR can be linked to the C-terminal sequence of the CAR through a self-cleaving peptide (eg, 2A peptides such as T2A, P2A, E2A, etc.) .
  • a self-cleaving peptide eg, 2A peptides such as T2A, P2A, E2A, etc.
  • the low density lipoprotein receptor-related protein or fragment thereof can be linked to the C-terminus of the intracellular signaling region via T2A.
  • the cleavage peptide may comprise the amino acid sequence shown in SEQ ID NO:87.
  • the CAR may sequentially comprise a targeting moiety (eg, the antigen-binding protein) that binds to the CLDN18.2 protein, the hinge region, the transmembrane domain, the costimulatory signaling region and the intracellular signaling region.
  • a targeting moiety eg, the antigen-binding protein
  • the CAR may comprise the scFv, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signaling region derived from 4-1BB, and a CD3 ⁇ derived intracellular signaling region.
  • a vector capable of expressing the CAR or immune effector cells is also included.
  • a targeting moiety eg, the antigen-binding protein
  • encoding the The hinge region encoding the transmembrane domain, encoding the costimulatory signaling region, encoding the intracellular signaling region and encoding the low density lipoprotein receptor-related protein or a nucleic acid molecule fragment thereof.
  • a vector capable of expressing the CAR or immune effector cells is also included.
  • a targeting moiety eg, the antigen-binding protein
  • encoding the Nucleic acids encoding the hinge region, encoding the transmembrane domain, encoding the costimulatory signaling region, encoding the intracellular signaling region, encoding the cleavage peptide, and encoding the low-density lipoprotein receptor-related protein or fragment thereof molecular.
  • Polypeptide molecules Polypeptide molecules, immunoconjugates, nucleic acid molecules, vectors, cells and pharmaceutical compositions
  • the application provides polypeptide molecules, which may comprise the isolated antigen binding proteins described herein or the chimeric antigen receptors described herein.
  • the polypeptide molecule may comprise a fusion protein.
  • the isolated antigen binding proteins described herein can be fused to other functional molecules (eg, antibodies or receptor ligands) to form bispecific or multispecific molecules.
  • the bispecific or multispecific molecule can specifically bind to at least two different binding sites or targeting molecules.
  • the bispecific or multispecific molecules can be prepared by genetic modification, somatic hybridization or chemical methods. See, for example, Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 ( 2000).
  • the present application also provides immunoconjugates, which may comprise the isolated antigen binding proteins described herein.
  • an isolated antigen binding protein or fragment thereof described herein can be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, and the like.
  • the attachment can be through one or more covalent bonds, or non-covalent interactions, and can include chelation.
  • linkers which may be known in the art, can be used to form immunoconjugates.
  • immunoconjugates can be provided in the form of fusion proteins, which can be expressed from polynucleotides encoding the immunoconjugates.
  • the immunoconjugate may also comprise, for example, an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the antibody and therapeutic agent can be cross-linked by a linker that is cleavable, such as a peptidic, disulfide, or hydrazone-like linker.
  • the application provides one or more nucleic acid molecules that can encode the isolated antigen binding proteins described herein or the chimeric antigen receptors described herein.
  • it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • PCR polymerase chain reaction
  • the nucleic acid molecule may comprise SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: The nucleotide sequence shown in any one of SEQ ID NO: 32 and SEQ ID NO: 33.
  • the nucleic acid molecule may comprise any group of nucleotide sequences selected from the group consisting of:
  • the present application provides a vector, which can comprise the nucleic acid molecule described herein.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector can be expressed by transforming, transducing or transfecting a host cell so that the genetic material elements it carries are expressed in the host cell.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the carrier may also include components to assist its entry into cells, such as viral particles, liposomes or protein coats, but not only these materials.
  • the present application provides a cell, which can comprise the isolated antigen-binding protein described in the present application, the chimeric antigen receptor described in the present application, the polypeptide molecule described in the present application, and the nucleic acid described in the present application.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi 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 cells may be immune effector cells.
  • the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes , peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells can be T cells.
  • the cells may contain and/or express the CAR.
  • the cells may comprise and/or express the CAR and the low-density lipoprotein receptor-related protein or fragments thereof.
  • the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the chimeric antigen receptor described in the present application, the polypeptide molecule described in the present application, the The immunoconjugate, the nucleic acid molecule described herein, the vector described herein, and/or the cell 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, typically those active compounds with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as on the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with pharmaceutical administration, and is generally safe and nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intraluminal, 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 various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition can be administered without interruption. The 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 methods for preparing the antigen binding proteins.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application.
  • an animal can be immunized with a linked or naturally occurring CLDN18.2 protein or fragment thereof.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • CLDN18.2 can be used as an immunogen (antigen) for generating non-human antibodies specific for CLDN18.2 and screening the antibodies for biological activity.
  • the priming immunogen can be full-length mature human CLDN18.2, including native homodimers, or peptides containing single/multiple epitopes.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • Chimeric human antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody may be an IgG antibody, and the IgG1 subtype may be used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below.
  • any type of light chain can be used in the compounds and methods of the present application. For example, kappa chains or variants thereof are useful in the compounds and methods of the present application.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, and the carrier , the use of the cells and/or the pharmaceutical composition in the preparation of medicaments for preventing, relieving and/or treating diseases and/or disorders.
  • the present application also provides a method for preventing, alleviating or treating a disease and/or disorder
  • the method may comprise administering the isolated antigen-binding protein described in the present application, the chimeric antigen-binding protein described in the present application to a subject in need thereof
  • the administration can be carried out by different means, eg intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the carrier, the The cells and/or the pharmaceutical composition can be used to prevent, alleviate or treat diseases and/or conditions.
  • the disease and/or disorder may include cancer.
  • the cancer may include solid tumors and/or hematological tumors.
  • the cancer may comprise a CLDN18.2 positive tumor.
  • the cancer may comprise gastric cancer and/or colon cancer.
  • the isolated antigen binding protein can be administered with one or more other antibodies to effectively inhibit tumor growth in a subject.
  • the isolated antigen binding protein can also be administered with a chemotherapeutic agent.
  • the present application also provides a method for detecting CLDN18.2 in a sample.
  • the method comprises administering the isolated antigen binding protein described herein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and /or the pharmaceutical composition.
  • 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 chimeric antigen receptor, the polypeptide molecule, the immune The conjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the carrier, the cell And/or the use of the pharmaceutical composition in the preparation of the reagent or kit for detecting CLDN18.2.
  • the reagents or kits can be used to detect the presence and/or content of CLDN18.2 in a sample.
  • the CLDN18.1 positive control antibody was a commercially available anti-CLDN18 rabbit monoclonal antibody (abcam, Cat#ab203563), which could recognize both CLDN18.1 and CLDN18.2, and the antigen binding site was located in the cell.
  • this antibody is used as CLDN18.1 positive control antibody and named as anti-CLDN18 antibody (Anti-Claudin18 antibody).
  • IMAB362 175D10 clone
  • antibody heavy and light chain such as the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82
  • the constructing plasmids include: pCMV-IMAB362-VL and pCMV-IMAB362-VH.
  • the above two vector plasmids are 3:2 (VL:VH) ratio was co-transfected into Expi293 cells for transient expression, and the cell culture supernatant was harvested 5 days later, purified by Protein A affinity chromatography, and quantified by BCA to obtain a CLDN18.2 positive control antibody, named zolbetuximab.
  • CLDN18 source Genbank accession number Nucleotide sequence hCLDN18.1 NM_016369.4 SEQ ID NO: 61 hCLDN18.2 NM_001002026.3 SEQ ID NO: 62 mouse CLDN18.1 NM_019815.3 SEQ ID NO: 63 mouse CLDN18.2 NM_001194921.1 SEQ ID NO: 64 Cynomolgus monkey CLDN18.1 XM_005545863.2 SEQ ID NO: 65 Cynomolgus monkey CLDN18.2 XM_001114708.4 SEQ ID NO: 66
  • the 96-well plate was Wash once with FACS buffer, then add secondary antibody (goat F(ab')2 anti-human IgG-Fc ( 650) (abcam, Cat#ab98593)); after 30min incubation at 4°C, the 96-well plate was washed twice with FACS buffer, and then the iQue Screener flowmeter (purchased from IntelliCyt) was used to monitor the cell fluorescence and select the highest MFI value. Monoclonal cell expansion and cryopreservation.
  • the cells are divided into two treatment methods: one is to directly resuspend the plate with FACS buffer after cell counting, and the primary antibody is the CLDN18.2 positive antibody zolbetuximab provided in Example 1.
  • Example 1 The other is to fix the membrane after cell counting (Sizhengbo Bio, fixative FXP008 and membrane-breaking agent FXP009), so that the antibody can be combined with the antigenic site located in the cell, the primary antibody is provided in Example 1
  • the commercially available CLDN18 antibody was tested at the same time, and the monoclonal cells that were strongly bound to the commercially available CLDN18 antibody but not bound to zolbetuximab were selected for expansion, culture and cryopreservation.
  • the obtained cell lines with stable and high expression of CLDN18 were marked as: 293T-human CLDN18.1 (293T-human CLDN18.1), 293T-human CLDN18.2 (293T-human CLDN18.2), CHO-human CLDN18.1 (CHO-human CLDN18.1), CHO-human CLDN18.2 (CHO-human CLDN18.2), SP2/0-human CLDN18.1 (SP2/0-human CLDN18.1), SP2/0-human CLDN18.
  • mice A total of 9 Balb/c mice were immunized in this experiment, of which 6 mice were immunized with a combination of cellular antigens and DNA antigens, and the other 3 mice were immunized with whole-cell immunization.
  • the classical mouse immunization schedule was used.
  • mice After twice, collect mouse serum, use 293T-human CLDN18.2 cells to measure the serum titer by FACS, select the mice that meet the titer requirements for the last booster immunization, if the titer does not meet the requirements, it needs to be increased by 1 To 2 times of immunization until the serum titer meets the requirements, 3 days after the last immunization, the spleen tissue of the mice was collected under sterile conditions, ground into a spleen cell suspension, the spleen cells were split into red, and then aliquoted and frozen at -80. °C Refrigerator for use.
  • mice myeloma cells SP2/0 were resuscitated in advance, cultured and passaged in DMEM containing 10% fetal bovine serum at 37°C and 5% CO 2 .
  • PEG 1450 solution Solution
  • high glucose DMEM medium were preheated in a 37°C water bath in advance.
  • the cells are preferably in the logarithmic growth phase. Collect SP2/0 cells by centrifugation at 400g for 5 minutes, resuspend them in an appropriate amount of pre-warmed DMEM medium, and count with trypan blue.
  • the medium is supplemented with 10% FBS, 1% double antibody, and 2% HAT. Supplement and 10% ClonaCell TM -HY Medium C (STEMCELL, CAT#: 03803) in high glucose DMEM medium. The medium was changed the day before the test. The medium after the medium change was high-glucose DMEM medium containing 10% FBS, 1% double antibody, and 1% HT supplement. After 24 hours, the cell culture supernatant of 96-well plate was taken. Perform flow detection.
  • the 293T-human CLDN18.2 and CHO-human CLDN18.2 cells constructed in Example 2 were selected to perform the first round of primary flow screening on the parent cloned hybridoma cells obtained after fusion: the cells were digested and counted respectively, and the cells were digested and counted with flow buffer ( PBS containing 0.1% BSA) was resuspended and adjusted to a cell density of 1 ⁇ 10 6 /ml, and 30ul/well was added to a V-bottom 96-well plate; 50ul/well of the hybridoma cell culture supernatant was added to the V that had been plated with cell antigens.
  • flow buffer PBS containing 0.1% BSA
  • a positive control (5ug/ml zolbetuximab antibody, 30ul/well) and a negative control (30ul/well PBS buffer) were set at the same time, incubated at 4°C for 1 hour, washed once with flow buffer, added Corresponding secondary antibody, 30ul/well, incubated at 4°C for 30 minutes, added flow buffer to wash twice, after the cells were loosened, 25ul of FACS buffer was added to each well for on-board detection (iQue Screener flowmeter, IntelliCyt Corporation). The parent clones that showed CLDN18.2 positive in the initial screening were selected and expanded to a 24-well cell culture plate.
  • CHO-human CLDN18.2 Three days later, the cell supernatant was taken for FACS re-screening, and CHO-human CLDN18.2, CHO-human CLDN18.1 and 293T-human CLDN18 were selected. 2.
  • Four kinds of 293T-human CLDN18.1 cells were plated separately, and the CLDN18.2-positive and CLDN18.1-negative hybridoma parent clones were screened out. The positive hybridoma parent clones were monocloned by gradient dilution method, and after two to three rounds of subcloning and FACS screening, hybridoma monoclonal cells secreting CLDN18. 2.
  • CHO-human CLDN18.2 cells produce positive binding, while the binding to 293T-human CLDN18.1 and CHO-human CLDN18.1 cells is negative.
  • the results are shown in Table 2.
  • the subclones with positive binding to CLDN18.2 in the culture supernatant of hybridoma cells include: 3A6, 7E3, 5E6, 14E12 and 17B10, a total of 5 monoclonal cells.
  • the sequences of the heavy chain variable region and light chain variable region of the murine antigen-binding protein of CLDN18.2 were obtained by degenerate primer amplification sequencing.
  • the primers used were referred to PROTOCOL published by Lotta von Boehmer (doi:10.1038/nprot.2016.102), commissioned Primer synthesis was performed by Suzhou Jinweizhi Company.
  • 5 CLDN18.2-positive hybridoma monoclonal cells in Example 3 were collected, and total cell RNA was extracted with QIAGEN RNeasy Plus Mini Kit, 1% agarose gel electrophoresis was used to detect RNA integrity, and NanoDrop nucleic acid was quantified. The analyzer measures the RNA concentration.
  • RNA reverse transcription kit 1 ug of RNA was reverse transcribed into cDNA, and stored at -20°C for later use.
  • 5ul cDNA as the template, configure the PCR reaction system and set the PCR program according to the operation instructions of PCR high-fidelity enzyme (Beijing Quanshijin, AP231), wherein the annealing temperature adopts the method of gradient cooling, and then directly use 1ul of the first round PCR product as
  • the template is subjected to the second round of PCR.
  • the PCR reaction system and procedure are the same as above. All the second round PCR products are loaded and subjected to 1% agarose gel electrophoresis.
  • the specific target band of suitable size is cut out, and the gel is recovered and ligated to the blunt-end cloning vector.
  • (Beijing Quanshi Gold, CB501) transformed Trans1-T1 competent cells, spread on 2YT plate, incubator at 37°C upside down for 12-16 hours, picked up monoclonal colonies with a pipette tip in the ultra-clean workbench, and commissioned Suzhou after the colony activation
  • the VH and VL gene sequences of 5 hybridoma clones were obtained after sequencing, as shown in Table 3.
  • the 5 heavy and light chain variable region sequences obtained by sequencing in Example 4 were used as templates to design primers respectively, and entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to carry out primer synthesis, and high-fidelity enzymes were used to carry out PCR amplification, agarose gel electrophoresis and Gel recovery, the recovered plasmid DNA was homologous recombination (Vazyme, C112) into the eukaryotic expression vector (pCMV-IgG1NDL and pCMV- ⁇ ) with human IgG1 constant region that had been digested and entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. Sequencing was performed to identify the correct positive recombinant vector.
  • the plasmid was extracted, and the heavy and light chains were co-transfected into Expi293 cells. After 5 days, the cell culture supernatant was collected by centrifugation, and purified by ProteinA affinity chromatography to obtain complete human and mouse IgG1 and Ig ⁇ antigen-binding proteins.
  • the five antigen-binding proteins were named c5E6, c7E3, c3A6, c14E12, and c17B10, respectively.
  • the specific binding activity of the above chimeric antibodies to target cells was detected by flow cytometry fluorescence sorting (FACS) using iQue Screener flow meter (purchased from IntelliCyt Company), and PBS containing 0.1% BSA was used as a buffer.
  • FACS flow cytometry fluorescence sorting
  • iQue Screener flow meter purchased from IntelliCyt Company
  • PBS containing 0.1% BSA was used as a buffer.
  • Three kinds of target cells the stably transfected cell line expressing human CLDN18.2, the stably transfected cell line expressing human CLDN18.1, and the tumor cell line, respectively, were tested for binding activity.
  • the cells were 293T-human CLDN18.2, CHO-human CLDN18.2 and SP2/0-human CLDN18.2 cells constructed in Example 2, the cells were digested and counted, and the cells were resuspended in flow buffer and adjusted to 1 ⁇ 10 6 /ml, 30ul/well was added to V-bottom 96-well plate; 30ul/well was added to the primary antibody, the antibody was initially at a concentration of 30ug/ml, and diluted by two or three times with flow buffer to form 7 gradients, each The antibody was set as PBS negative control, and the positive control antibody was zolbetuximab purified in Example 1; incubated at 4°C for 1 hour, washed with flow buffer, added secondary antibody (abcam, Cat#ab98593), 30ul/well, incubated at 4°C 30 minutes; washed twice with flow buffer, the cells were shaken loose, added 25ul/well flow buffer, and waited for the machine. Substitute the original data into GraphPad
  • 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 CLDN18.2 tetrahedral membrane protein, which needs to be analyzed by flow cytometry.
  • the cells were the 293T-human CLDN18.1 and SP2/0-human CLDN18.1 cells constructed in Example 1. After the cells were digested and counted, the membranes were fixed and permeated, and the treated cells were resuspended to 1 with flow buffer.
  • Example 2 Select the tumor cell MC38-human CLDN18.2 constructed according to the method shown in Example 2 and obtained stably and highly expresses human CLDN18.2 as the target cell, and the cells are digested and counted.
  • the cells are resuspended in flow buffer and adjusted to 1 ⁇ 10 6 /ml, 30ul/well was added to V-bottom 96-well plate; 30ul/well was added to primary antibody, the antibody was initially at a concentration of 30ug/ml, and was diluted with flow buffer according to a three-fold gradient to form 7 gradients, each antibody set PBS negative control, the positive control antibody is zolbetuximab purified in Example 1, and the dilution conditions are the same as above; incubate at 4°C for 1 hour, wash with flow buffer, add secondary antibody (abcam, Cat#ab98593), 30ul/well, 4 Incubate at °C for 30 minutes; wash twice with flow buffer, the cells are shaken loose, add 25ul/
  • FACS flow cytometry fluorescence sorting technology
  • 293T-mouse CLDN18.2 and 293T-cynomolgus monkey CLDN18.2 cells were the stably transfected cell lines constructed in Example 2, the cells were digested and counted, and resuspended with flow buffer to a cell density of 1 ⁇ 10 6 /ml, 30ul/well was added to V-bottom 96-well plate; the primary antibody was diluted with flow buffer according to three-fold gradient, the initial concentration was 10ug/ml, forming 6 gradients, each antibody was set as PBS negative control, The positive control antibody is zolbetuximab, and the dilution conditions are the same as above; incubate at 4°C for 1 hour, wash with flow buffer, add secondary antibody (abcam, Cat#ab98593), 30ul/well, incubate at 4°C for 30 minutes; wash with flow buffer for two After the cells were shaken and loosened, 30ul/well flow buffer was added, and the cells were detected on the machine
  • the cytotoxicity detection kit (Promega, Cat#G1780) was used to detect the ability of CLDN18.2 antigen-binding protein to induce CDC effect on SP2/0-human CLDN18.2 cells.
  • the specific process is as follows:
  • Configuration medium (A: DMEM+2%FBS+1% double antibody, B: DMEM+2%FBS+1% double antibody+10% rabbit complement);
  • the target cell SP2/0-human CLDN18.2 was centrifuged at 400g for 5 minutes, and then the cells were resuspended in the above-mentioned medium A to make the target cell density 4 ⁇ 105 cells/ml, and 100ul/well was added to 96-well cells in the culture plate;
  • the antibody was diluted into three different concentrations with the above-mentioned medium B, 10ug/ml, 2ug/ml and 0.4ug/ml respectively, 100ul/well was added to the detection hole, two duplicate wells were set at each concentration point, and zolbetuximab was regarded as positive Control antibody, human IgG-Fc as a negative control antibody;
  • the complement is the rabbit complement Rabbit Complent 3-4 weeks (Cat#31061-3) purchased from PelFreez Bio Company, and the final concentration of the complement used in the experiment is 5%;
  • the c5E6 antigen binding protein was further tested for its ability to induce CDC effect on CHO cells stably overexpressing human CLDN18.2. Briefly, CHO-human CLDN18.2 cells were constructed and obtained by the company's lentiviral transfection system as shown in Example 1. The operation method was the same as that described above. The cells were plated at a density of 1.2 ⁇ 10 4 cells per well, and the rabbit complement was finally used.
  • the concentration is 5%, and the antigen-binding protein is diluted in a five-fold ratio, namely 10ug/ml, 2ug/ml, 0.4ug/ml, 0.08ug/ml, 0.016ug/ml, 0.0032ug/ml, 0.64ng/ml, each Two duplicate wells were set for the concentration, zolbetuximab was used as positive control antibody, and human IgG-Fc was used as isotype control antibody. The absorbance value after co-incubation for 4 h was detected by a microplate reader, the percentage of target cell lysis was calculated, and GraphPad prism 8 was used to analyze and process the data.
  • the c5E6 antigen-binding protein can induce a strong CDC effect on CHO-human CLDN18.2 cells in a dose-dependent manner, and exhibits higher CDC activity than zolbetuximab.
  • C5E6 and zolbetuximab were labeled with biotin (EZ-Link Sulfo-NHS-LC-Biotin, Thermo, A39257), each labeled 500ug, and the biotinylated antigen-binding protein molecules were labeled with a molecular interaction analyzer OctetRED384 (sartorius) to determine the labeling effect , Select SA sensor immobilized antibody, the antibody concentration is 100nM, labelled for 180s, the labelled biotinylated antigen-binding protein is named as biotinylated-c5E6 (Biotinylated-c5E6), biotinylated-c7E3 (Biotinylated-c7E3), biotin Biotinylated-c17B10, Biotinylated-zolbetuximab (Biotinylated-zolbetuximab).
  • biotinylated-c5E6 Biotin
  • the antigen binding proteins (c5E6, c7E3, c17B10) in Example 5, the positive control antibody zolbetuximab and the isotype control antibody human IgG-Fc were selected to test their antitumor activity in C57BL/6 mice inoculated with mouse colon cancer cells .
  • the above six antigen-binding proteins were purified and obtained, and the endotoxin of the antigen-binding protein was controlled below 4EU/mg.
  • the specific implementation is as follows:
  • mice 115 female C57BL/6 mice were subcutaneously inoculated with MC38-human CLDN18.2 cells in the right flank, and the inoculated cell amount was 1.1 ⁇ 10 6 .
  • the tumor size was 31.33-116.31 mm 3 (average tumor size was 71.59 mm).
  • 80 mice (mm 3 ) were divided into 10 groups according to tumor volume, with 8 mice in each group, and the day of random grouping of mice was defined as Day0. After grouping, the mice were administered by tail vein, once a week, for four consecutive weeks, and divided into two groups of 30 mg/kg and 7.5 mg/kg.
  • lentivirus infection and flow sorting were used to obtain cells stably expressing human CLDN18.2, namely SP2/0-hCLDN18.2 cells.
  • the immunogen is SP2/0-hCLDN18.2 cells, using mouse hybridoma technology, flow screening and subcloning to obtain CLDN18.2 positive monoclonal cells, and in vitro culture to obtain IgG class CLDN18.2 murine monoclonal antibody.
  • Degenerate primer amplification method was used to sequence the heavy chain variable region ( VH ) and light chain variable region ( VL ) sequences .
  • Cells SP2/0-hCLDN18.1 stably expressing human CLDN18.1 were obtained by simultaneous lentivirus infection and flow sorting.
  • VH contains the amino acid sequence shown in SEQ ID NO: 92
  • VL contains the amino acid sequence shown in SEQ ID NO: 93
  • flow cytometry SP2/0-hCLDN18.2 cells were counted, cells were resuspended in flow buffer and adjusted to 1 ⁇ 10 6 /ml, 30ul/well was added to V-bottom 96-well plate; 30ul/well was added with primary antibody, and the primary antibody was 80ug/ Starting from the working concentration of ml, it was diluted by 4-fold gradient with flow buffer to form 7 gradients.
  • Each antibody was set as a PBS negative control; incubated at 4°C for 1 hour, washed once with flow buffer, and added secondary antibody: goat F (ab')2 anti-human IgG-Fc ( 650) (abcam, Cat#ab98593), 30ul/well, incubated at 4°C for 30 minutes; washed twice with flow buffer, the cells were shaken loose, added 25ul/well flow buffer, and waited for the machine. At the same time, SP2/0-hCLDN18.1 cells were counted and plated. The working concentration of the primary antibody started from 10ug/ml and was diluted 3-fold to form a total of 5 concentration gradients.
  • the secondary antibody was goat F(ab')2 anti-human IgG.
  • FACS flow cytometry fluorescence sorting technology
  • HFF human foreskin fibroblasts
  • HaCaT human immortalized epidermal cells
  • LO2 human normal hepatocytes
  • MSC mesenchymal stem cells
  • KERA human normal epidermal cells
  • A549 human breast cancer cells
  • MCF-7 human skin fibroblasts
  • BJ human skin fibroblasts
  • the secondary antibody was goat F(ab')2 anti-human IgG-Fc ( 650) (abcam, Cat#ab98593), centrifuged after incubation, resuspended with flow buffer and put on the machine, and the results were substituted into GraphPad 8.0 software for graphing. The results are shown in Figure 13.
  • the 5E6-scFv single-chain antibody does not have non-specific binding to various non-target cells in human tissues. Compared with the Standard antibody, its specificity to tumor targets is preliminarily verified, that is, there is no "off-target effect" phenomenon.
  • the human CLDN18.2 antigen binding protein (c5E6), the positive control antibody zolbetuximab and the isotype control antibody human IgG-Fc were selected to test their antitumor effects in female nude mice inoculated with human gastric cancer cells (NUGC-4). active.
  • NUGC-4 human gastric cancer cells
  • mice 28 female nude mice were subcutaneously inoculated with NUGC-4 cells, and the amount of inoculated cells was 3 ⁇ 10 6 .
  • 20 mice with an average tumor size of 80 mm 3 were selected and divided into 4 groups according to the tumor volume, with 5 mice in each group.
  • Day0 the day of random grouping of mice was defined as Day0.
  • mice The body weight changes of the mice were shown in Figure 14A; during the observation period, the tumor volume change curve of the mice was shown in Figure 14B, G3: zolbetuximab, 10 mg/kg group and G4:c5E6, 10mg/kg group of mice showed a certain anti-tumor activity relative to the G2 isotype control group, and there were statistically significant differences (p ⁇ 0.05); no anti-tumor activity between G3 and G4 Significant differences, with similar levels of tumor inhibition.
  • Example 14 CLDN18.2-specific CAR is stably expressed in T cells
  • the CLDN18.2-specific CAR structure in this example is composed of human CD8 signal peptide (SEQ ID NO: 88), anti-human CLDN18.2 single-chain antibody (SEQ ID NO: 90), human CD8 Hinge region (SEQ ID NO:83), human CD8 transmembrane region (SEQ ID NO:84), human 4-1BB intracellular costimulatory domain (SEQ ID NO:85), human CD3 ⁇ intracellular activation domain (SEQ ID NO:85) NO: 86) and the added Ori new element (SEQ ID NO: 91) together.
  • SEQ ID NO: 88 human CD8 signal peptide
  • SEQ ID NO: 90 anti-human CLDN18.2 single-chain antibody
  • SEQ ID NO:83 human CD8 Hinge region
  • human CD8 transmembrane region SEQ ID NO:84
  • human 4-1BB intracellular costimulatory domain SEQ ID NO:85
  • human CD3 ⁇ intracellular activation domain SEQ ID NO:85
  • NO: 86
  • CAR-T cells cultured for 9-14 days detect the positive rate of cells: the virus used to infect cells carries Myc-tag, so after the virus infects cells, the positive rate of Myc is detected by flow cytometry to obtain the positive rate of CAR expression,
  • the direct-labeled detection antibody used was Myc-Tag (9B11) Mouse mAb (Alexa 488Conjugate) (Cell Signaling, 2279S).
  • Example 15 CLDN18.2-specific CAR can specifically kill target cells in vitro
  • CHO-hCLDN18.2 cells stably and highly expressing human CLDN18.2 and CHO-hCLDN18.1 cells stably and highly expressing human CLDN18.1 were obtained by lentivirus infection and flow sorting.
  • a cytotoxicity detection kit Promega, Cat#G1780 was further used to evaluate the specific killing ability of CAR-T cells in vitro by LDH method. The steps are as follows:
  • the CAR-T and Mock T cells routinely cultured for 9 days in the above Example 14 were resuspended with blank X-VIVO medium after centrifugation, so that the cell density was 1 ⁇ 10 5 /ml; the target cells were CHO, CHO-hCLDN18.2 and CHO-hCLDN18.1, the three target cells were digested and counted, and then resuspended in blank X-VIVO medium to make the cell density 5 ⁇ 10 5 /ml; then According to the volume system of 100ul target cell suspension + 100ul CAR-T/Mock T cell suspension per well, it was mixed into a sterile v-bottom 96-well plate. Control wells were set according to the kit requirements.
  • Human IFN- ⁇ ELISA kit (R&D, DY285B) and Human IL-2 ELISA kit (R&D, DY202) were used to analyze the secretion of IFN- ⁇ and IL-2 by CAR-T in the process of killing target cells quantity.
  • target cells CHO-hCLDN18.2
  • control cells CHO-hCLDN18.1
  • CLDN18.1 highly expressing CLDN18.1
  • negative cells were divided according to the cell amount of 1 ⁇ 10 4 cells per well.
  • the human CLDN18.2 gene was first introduced into MC38 cells by lentivirus, and then the mouse colon cancer cells with high expression of human CLDN18.2, namely MC38-hCLDN18.2 cells, were sorted by flow sorting. .
  • MC38-hCLDN18.2 cells were inoculated at a dose of 1.5 ⁇ 10 6 per mouse. Eight days after the mice received tumors, mice with a tumor size of 41.68-120.6 mm 3 (average tumor size of 80.01 mm 3 ) were randomly divided into 3 groups.
  • mice in each group 8 mice in each group, the CAR-T (or Mock T) cells that have been cultured for 10 days were reinfused into the tail vein, and the CAR-T reinfusion dose in the 5E6-CART and Standard-CART groups was 3 ⁇ 10 6 cells/mouse , the infusion dose of the blank control Mock T cell group was 1 ⁇ 107 cells/mouse.
  • the tumor size and body weight of the mice were measured 3 times a week for 4 weeks, as shown in Figure 18A. After infusion of CAR-T (or Mock T) cells into the tail vein of mice, mice in the 5E6-CART group lost more than 10% of their body weight on D13, and the relative change in the body weight of a single mouse on D15 was -34.51%.
  • mice in the 5E6-CART group showed intolerance problems and obvious side effects in this tumor model, which were manifested as weight loss, erect back hair, curled motion and stomach bleeding, etc.; Mock T group The mice showed tolerance, and the weight of the mice was normal.

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Abstract

提供了一种分离的抗原结合蛋白，其能够结合CLDN18.2。还提供了包含所述分离的抗原结合蛋白的嵌合抗原受体，以及所述抗原结合蛋白和所述嵌合抗原受体的制备方法和应用。

Description

CLDN18.2抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种能够结合CLDN18.2的抗原结合蛋白。
背景技术
胃癌是全球第二致死率的恶性肿瘤，根据世界卫生组织2015年数据，全球每年有75.4万例胃癌患者死亡；胰腺癌是恶性程度最高的肿瘤之一，根据《柳叶刀》杂志2016数据显示，全球每年有超过20万胰腺癌患者死亡。晚期或复发性胃癌的标准一线治疗是化疗。曲妥珠单抗加入化疗为HER2-阳性肿瘤患者提供一定的生存获益，但是所有胃癌中只有15％为HER2-阳性。研发安全、有效的治疗手段迫在眉睫。
CLDN18.2(Claudin 18.2)只表达于分化型胃壁细胞，在正常组织中无表达。最新研究表明，CLDN18.2在77％以上胃癌患者以及80％以上胰腺癌患者中都过表达，此外在肺癌，食管癌以及卵巢癌等实体瘤中过表达。CLDN18.2属于紧密连接蛋白质家族，可以控制层细胞之间的分子流动。然而，在肿瘤中，紧密连接被破坏，CLDN蛋白质失去了其主要作用。因为CLDN18.2大量存在于胃部肿瘤中，研究者估计所有晚期胃癌患者的一半可以作为CLDN18.2抗体靶点新疗法的候选人。此外，这一独特靶点不存在于除了胃壁的任何健康组织，从而最大程度地减少治疗副作用。这些特点表明CLDN18.2是一个理想的治疗性单克隆抗体开发的靶点。
由于CLDN18.2是膜蛋白，采用传统抗体筛选方式很难获得抗天然蛋白的高质量抗体。目前大多采用体内免疫，例如注射DNA的方法，但需要在动物上进行多轮免疫。CLDN18有两种剪切变体CLDN18.1和CLDN18.2，这两种变体仅在N端69个氨基酸序列有不同，存在于第一个胞外环中，其余序列均相同。而CLDN18.1在正常肺组织中有表达，因此需筛选出能与CLDN18.2结合同时不与CLDN18.1结合的抗体。另一方面，CLDN18.2序列在人与鼠中同源性极高。因此，亟需获得特异性结合CLDN18.2抗体。
发明内容
本申请提供了一种能够结合CLDN18.2的抗原结合蛋白，其表现出一种或多种期望的功能特性，例如其可以特异性结合人CLDN18.2且基本上不结合人CLDN18.1、能够具有CDC活性和/或具有抗肿瘤活性。本申请还提供编码所述分离的抗原结合蛋白的核酸分子、表达载 体、宿主细胞和制备所述分离的抗原结合蛋白的方法。本申请所述分离的抗原结合蛋白可以用于预防和/或治疗疾病和/或病症，如肿瘤和/或癌症。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的HCDR3包含SEQ ID NO:4、SEQ ID NO:12和SEQ ID NO:18中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:68或SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的HCDR2包含SEQ ID NO:3或SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:2、SEQ ID NO:10和SEQ ID NO:16中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的H-FR1包含SEQ ID NO:34、SEQ ID NO:43和SEQ ID NO:49中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的H-FR2包含SEQ ID NO:35或SEQ ID NO:44所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:80或SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的H-FR3包含SEQ ID NO:36或SEQ ID NO:42所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:74所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的H-FR4包含SEQ ID NO:46或SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:78所 示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的VH包含SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:15和SEQ ID NO:19中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR3，且所述LCDR3包含SEQ ID NO:69所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的LCDR3包含SEQ ID NO:8、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:25中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR2，且所述LCDR2包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的LCDR2包含SEQ ID NO:7或SEQ ID NO:22所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，且所述LCDR1包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的LCDR1包含SEQ ID NO:6或SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:75所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的L-FR1包含SEQ ID NO:38、SEQ ID NO:47、SEQ ID NO:50和SEQ ID NO:53中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:39所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:76所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的L-FR3包含SEQ ID NO:40、SEQ ID NO:48和SEQ ID NO:51中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:77所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的L-FR4包含SEQ ID NO:41、SEQ ID NO:52和SEQ ID NO:54中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VL，且所述VL包含SEQ ID NO:79 所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的VL包含SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:20和SEQ ID NO:24中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含选自下述的任一组VH和VL：
1)所述VH包含SEQ ID NO:1所示的氨基酸序列，且所述VL包含SEQ ID NO:5所示的氨基酸序列；
2)所述VH包含SEQ ID NO:15所示的氨基酸序列，且所述VL包含SEQ ID NO:5所示的氨基酸序列；
3)所述VH包含SEQ ID NO:9所示的氨基酸序列，且所述VL包含SEQ ID NO:13所示的氨基酸序列；
4)所述VH包含SEQ ID NO:19所示的氨基酸序列，且所述VL包含SEQ ID NO:20所示的氨基酸序列；以及
5)所述VH包含SEQ ID NO:19所示的氨基酸序列，且所述VL包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链恒定区。
在某些实施方式中，所述抗体重链恒定区源自人IgG重链恒定区。
在某些实施方式中，所述抗体重链恒定区源自人IgG1重链恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体轻链恒定区。
在某些实施方式中，所述抗体轻链恒定区源自人Igκ恒定区。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述抗体选自下组中的一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，在FACS中验证，所述分离的抗原结合蛋白基本上不与参比抗体竞争结合CLDN18.2，其中所述参比抗体包含重链可变区(VH)和轻链可变区(VL)，所述参比抗体的VH包含SEQ ID NO:55所示的氨基酸序列，所述参比抗体的VL包含SEQ ID NO:56所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合CLDN18.2，且基本上不结合CLDN18.1。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合的CLDN18.2包含小鼠CLDN18.2、食蟹猴CLDN18.2和/或人CLDN18.2。
在某些实施方式中，所述分离的抗原结合蛋白具有CDC活性。
在某些实施方式中，所述分离的抗原结合蛋白能够抑制肿瘤生长和/或肿瘤细胞的增殖。
另一方面，本申请还提供了嵌合抗原受体，其包含靶向部分，所述靶向部分包含本申请所述的抗原结合蛋白。
在某些实施方式中，所述嵌合抗原受体包含共刺激域，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述嵌合抗原受体的共刺激域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述嵌合抗原受体的共刺激域包含SEQ ID NO:85所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号传导域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述嵌合抗原受体的所述胞内信号传导域为源自CD3ζ的信号传导域。
在某些实施方式中，所述嵌合抗原受体的所述胞内信号传导域包含SEQ ID NO:86所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体包含跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述嵌合抗原受体的所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述嵌合抗原受体的所述跨膜区包含SEQ ID NO:84所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体在靶向部分和跨膜区之间包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述嵌合抗原受体的所述铰链区为源自CD8的铰链区。
在某些实施方式中，所述嵌合抗原受体的所述铰链区包含SEQ ID NO:83所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含信号肽。
在某些实施方式中，所述嵌合抗原受体所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述嵌合抗原受体的所述信号肽包含SEQ ID NO:88所示的氨基酸序列。
在某些实施方式中，所述嵌合抗原受体还包含低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:91所示的氨基酸序列。
另一方面，本申请还提供了多肽分子，其包含所述分离的抗原结合蛋白或所述嵌合抗原受体。
在某些实施方式中，所述多肽分子包含融合蛋白。
另一方面，本申请还提供了免疫缀合物，其包含所述分离的抗原结合蛋白。
另一方面，本申请还提供了分离的一种或多种核酸分子，其编码所述分离的抗原结合蛋白、所述嵌合抗原受体或所述的多肽分子。
在某些实施方式中，所述核酸分子包含SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33中任一项所示的核苷酸序列。
在某些实施方式中，所述核酸分子包含选自下述的任一组核苷酸序列：
1)SEQ ID NO:26所示的核苷酸序列，以及SEQ ID NO:27所示的核苷酸序列；
2)SEQ ID NO:30所示的核苷酸序列，以及SEQ ID NO:27所示的核苷酸序列；
3)SEQ ID NO:28所示的核苷酸序列，以及SEQ ID NO:29所示的核苷酸序列；
4)SEQ ID NO:31所示的核苷酸序列，以及SEQ ID NO:32所示的核苷酸序列；以及
5)SEQ ID NO:31所示的核苷酸序列，以及SEQ ID NO:33所示的核苷酸序列。
另一方面，本申请还提供了载体，其包含所述核酸分子。
另一方面，本申请还提供了细胞，其包含所述分离的抗原结合蛋白、所述嵌合抗原受体、所述多肽分子、所述核酸分子或所述载体。
在某些实施方式中，所述细胞为免疫效应细胞。
在某些实施方式中，所述细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
在某些实施方式中，所述细胞为T细胞。
在某些实施方式中，所述细胞还包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
在某些实施方式中，所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:91所示的氨基酸序列。
另一方面，本申请还提供了药物组合物，其包含所述分离的抗原结合蛋白、所述嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、和/或所述细胞，以及任选地药学上可接受的载剂。
另一方面，本申请还提供了制备所述分离的抗原结合蛋白的方法，所述方法包括在使得所述抗原结合蛋白表达的条件下，培养所述细胞。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括癌症。
在某些实施方式中，所述癌症包括实体瘤和/或血液瘤。
在某些实施方式中，所述癌症包含胃癌和/或结肠癌。
另一方面，本申请还提供了检测样品中CLDN18.2的方法，所述方法包括施用所述分离 的抗原结合蛋白、所述嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
另一方面，本申请还提供了检测样品中CLDN18.2的试剂或试剂盒，其包含所述分离的抗原结合蛋白、所述嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中CLDN18.2的存在和/或含量。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请的抗原结合蛋白与高表达人CLDN18.2细胞的流式结合活性检测；
图2显示的是本申请的抗原结合蛋白与高表达人CLDN18.1细胞的流式结合活性检测；
图3显示的是本申请的抗原结合蛋白与肿瘤细胞系的流式结合活性检测；
图4显示的是本申请的抗原结合蛋白的种属交叉结合活性分析；
图5a-5b显示的是本申请的抗原结合蛋白的CDC活性检测；
图6显示的是本申请的抗原结合蛋白的竞争结合活性分析；
图7显示的是给药后小鼠的体重变化；
图8显示的是给药后小鼠的体重相对变化(％)；
图9显示的是给药后小鼠的肿瘤体积变化；
图10显示的是给药后小鼠的肿瘤抑制率；
图11显示的是给药后小鼠的生存曲线。
图12A-B显示的是CLDN18.2单链抗体的抗原结合活性鉴定。
图13显示的是CLDN18.2单链抗体与非靶细胞的结合活性鉴定。
图14显示的是本申请所述的CLDN18.2抗原结合蛋白的在人胃癌小鼠模型中的抗肿瘤活性。
图15A-D显示的是CLDN18.2特异性CAR在T细胞中的表达情况。
图16显示的是CAR-T细胞的体外杀伤活性检测。
图17A-B显示的是CLDN18.2特异性CAR的因子分泌情况检测。
图18A-D显示的是CLDN18.2特异性CAR-T细胞在小鼠结肠癌CDX模型中的抑瘤作用。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“CLDN18.2”、或“Claudin18.2”可以互换使用，通常指细胞连接密蛋白Claudin18的亚型2。所述术语涵盖“全长”、未加工的CLDN18.2以及由细胞加工所产生的任何形式的CLDN18.2。CLDN18.2可以包括完整的CLDN18.2及其片段，其功能性变体、同工型、物种同源物、衍生物、类似物以及具有至少一个与CLDN18.2共同表位的类似物。CLDN18.2(例如人CLDN18.2)的氨基酸序列可以是本领域已知的。例如，人CLDN18.2核苷酸序列可以显示于GeneBank登录号NM_001002026.3下。例如，小鼠CLDN18.2核苷酸序列可以显示于GeneBank登录号NM_001194921.1下。例如，食蟹猴CLDN18.2核苷酸序列可以显示于GeneBank登录号XM_001114708.4下。
在本申请中，术语“CLDN18.1”、或“Claudin18.1”可以互换使用，通常指细胞连接密蛋白Claudin18的亚型1。所述术语涵盖“全长”、未加工的CLDN18.1以及由细胞加工所产生的任何形式的CLDN18.2。CLDN18.2可以包括完整的CLDN18.1及其片段，其功能性变体、同工型、物种同源物、衍生物、类似物以及具有至少一个与CLDN18.1共同表位的类似物。CLDN18.1(例如人CLDN18.1)的氨基酸序列可以是本领域已知的。例如，人CLDN18.1核苷酸序列可以显示于GeneBank登录号NM_016369.4下。例如，小鼠CLDN18.1核苷酸序列可以显示于GeneBank登录号NM_019815.3下。例如，食蟹猴CLDN18.1核苷酸序列可以显示于GeneBank登录号XM_005545863.2下。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境 下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的抗原结合蛋白”通常指从天然状态下经人工手段获得的具有抗原结合能力的蛋白。该“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的CDR或CDR衍生物的备选蛋白框架区或人工框架区。此类框架包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的框架区以及包含例如生物相容性聚合物的完全合成的框架区。参见例如Korndorfer等,2003,Proteins:Structure,Function,andBioinformatics,53(1):121-129(2003)；Roque等,Biotechnol.Prog.20:639-654(2004)。抗原结合蛋白的实例包括但不限于：人抗体、人源化抗体；嵌合抗体；重组抗体；单链抗体；双功能抗体；三功能抗体；四功能抗体；Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv，dAb，IgD抗体；IgE抗体；IgM抗体；IgG1抗体；IgG2抗体；IgG3抗体；或IgG4抗体以及其片段。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH”和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“可变”通常指在抗体之间可变结构域的某些区段在序列上可能存在较大差异。可变结构域介导抗原结合并决定特定抗体对其特定抗原的特异性。然而，可变性并非在整个可变结构域范围内均匀分布。它通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成(参见Kabat et al,Sequences of Immunological Interest,Fifth Edition,National Institute of Health,Bethesda,Md.(1991))。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆 的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体”中，为了本发明的目的，术语“抗体”也包括抗体片段，比如Fab、F(ab') 2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合CLDN18.2)的其它抗体片段。通常，这样的片段应当包括抗原结合结构域。基本的4链抗体单元是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般为约150,000道尔顿。每个L链通过一个共价二硫键与H链连接，而两个H链通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还具有规则间隔的链内二硫化桥键。每个H链在N末端具有可变结构域(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域。VL与VH对应，且CL与重链的第一恒定结构域(CH1)相对应。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL配对一起形成单个抗原结合位点。对于不同类别抗体的结构和性质，参见例如Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw(eds),Appleton&Lange,Norwalk,Conn.,1994，第71页和第6章。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。基于CH序列和功能方面的相对小的差异，将γ和α类进一步分成亚类，例如，人表达下述亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgK1。
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如,Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。
在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3，和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3，和L-FR4)。例如，本申请所述 的分离的抗原结合蛋白的VL可以包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。本申请所述的分离的抗原结合蛋白的VH可以包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在本申请中，术语“抗原结合片段”通常指具有特异结合抗原(例如，CLDN18.2)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2，scFv，di-scFv和/或dAb。
在本申请中，术语“Fab”通常指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(V H)的第一恒定区(C H1)组成。
在本申请中，术语“Fab′片段”通常指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。
在本申请中，术语“F(ab')2”通常指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。
在本申请中，术语“Fv片段”通常指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。
在本申请中，术语“scFv”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。
在本申请中，术语“单克隆抗体”通常指单分子组成的抗体分子制备物。单克隆抗体通 常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
术语“全人源抗体”通常指仅包含人类免疫球蛋白蛋白质序列的抗体。如果其是在小鼠中、在小鼠细胞中或在衍生自小鼠细胞的杂交瘤中生产，那么全人源抗体可能含有鼠糖链。类似地，“小鼠抗体”或“大鼠抗体”分别指仅包含小鼠或大鼠免疫球蛋白序列的抗体。可通过噬菌体展示或其它分子生物学方法，在人体内、在具有人类免疫球蛋白种系序列的转基因动物体内生成全人源抗体。可用于制造抗体的示例性技术在美国专利：6,150,584、6,458,592、6,420,140中描述。其它技术，如使用文库，是本领域已知的。
在本申请中，术语“直接相连”与术语“间接相连”相对，术语“直接相连”通常是指直接连接。例如，所述直接相连可以为物质间没有间隔子而直接相连的情况。所述间隔子可以是连接子。例如，所述连接子可以为肽连接子。术语“间接相连”通常是指物质间不直接相连的情况。例如，所述间接相连可以为通过间隔子而连接的情况。例如，在本申请所述的分 离的抗原结合蛋白中，所述L-FR1的C末端与所述LCDR1的N末端可以直接或间接相连。
在本申请中，术语“分离的核酸分子”通常指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类可以包括逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“细胞”通常指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本发明所述的核酸分子或本发明所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或干细胞(例如ES细胞、iPS细胞、间充质干细胞)或免疫细胞(例如T细胞、NK细胞、NKT细胞、巨噬细胞)。在某些实施方案中，细胞为哺乳动物细胞。在某些实施方案中，哺乳动物细胞为HEK293细胞。
在本申请中，术语“药物组合物”通常指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载剂、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的 载体可包括例如缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，例如钠；和/或非离子表面活性剂。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“参比抗体”通常是指可以与抗原(例如，CLDN18.2)结合的抗体。在某些情形中，本申请所述抗原结合蛋白与参比抗体相比，不具有竞争结合活性。在某些情形中，本申请所述参比抗体可以为zolbetuximab。
在本申请中，术语“基本上不结合”通常指不结合或以很弱的结合活性结合。其中，很弱的结合活性可以指例如在流式结合活性检测中，检测到与CLDN18.1结合的抗体的平均荧光强度值与CLDN18.1抗体阳性对照相比，至少低约50％、约55％、约60％、约70％、约80％、约90％、约91％、约91％、约93％、约94％、约95％、约96％、约97％、约98％、约99％、约100％。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“肿瘤”通常指由异常细胞生长形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤。例如，在本申请中，肿瘤可以是CLDN18.2阳性的肿瘤。
术语“癌症”通常指以异常细胞快速且失控生长为特征的疾病。癌细胞可以局部地或通过血流和淋巴系统扩散到身体其他部分。本申请中的癌症包括但不限于胃癌和/或结肠癌等。术语“肿瘤”和“癌症”在本文中互换地使用，例如，两种术语涵盖实体瘤和液体肿瘤，例如，弥散型或循环型肿瘤。如本文所用，术语“癌症”或“肿瘤”可以包括恶变前以及恶性癌症和肿瘤。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合CLDN18.2蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸 的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合CLDN18.2蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。
一方面，本申请提供一种分离的抗原结合蛋白，其可以包含HCDR3。在本申请中，所述HCDR3可以包含SEQ ID NO:67所示的氨基酸序列。例如，所述HCDR3可以包含SEQ ID NO:4所示的氨基酸序列。例如，所述HCDR3可以包含SEQ ID NO:12所示的氨基酸序列。例如，所述HCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR2。在本申请中，所述HCDR2可以包含SEQ ID NO:68所示的氨基酸序列。在本申请中，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列。例如，所述HCDR2可以包含SEQ ID NO:3所示的氨基酸序列。例如，所述HCDR2可以包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1。在本申请中，所述HCDR1可以包含SEQ ID NO:2所示的氨基酸序列。在本申请中，所述HCDR1可以包含SEQ ID NO:10所示的氨基酸序列。在本申请中，所述HCDR1可以包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3。在本申请中，所述HCDR1可以包含SEQ ID NO:2、SEQ ID NO:10和SEQ ID NO:16中任一项所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:68或SEQ ID NO:11所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:67所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:2、SEQ ID NO:10和SEQ ID NO:16中任一项所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:3、SEQ  ID NO:17和SEQ ID NO:11中任一项所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:4、SEQ ID NO:12和SEQ ID NO:18中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的HCDR1，HCDR2和HCDR3可以包含选自下述任一组的氨基酸序列：
1)所述HCDR1可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:3所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:4所示的氨基酸序列；
2)所述HCDR1可以包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:12所示的氨基酸序列；以及
3)所述HCDR1可以包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:72所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:34所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:43所示的氨基酸序列。例如，所述H-FR1可以包含SEQ ID NO:49所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:73所示的氨基酸序列。例如，所述H-FR2可以包含SEQ ID NO:35所示的氨基酸序列。例如，所述H-FR2可以包含SEQ ID NO:44所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:80所示的氨基酸序列。在本申请中，所述H-FR3可以包含SEQ ID NO:45所示的氨基酸序列。例如，所述H-FR3可以包含SEQ ID NO:36所示的氨基酸序列。例如，所述H-FR3可以包含SEQ ID NO:42所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4可以包含SEQ ID NO:74所示的氨基酸序列。例如，所述H-FR4可以包含SEQ ID NO:46所示的氨基酸序列。例如，所述H-FR4可以包含SEQ  ID NO:37所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4。在本申请中，所述H-FR1可以包含SEQ ID NO:72所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:73所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:80或SEQ ID NO:45所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:74所示的氨基酸序列。
在本申请中，所述H-FR1可以包含SEQ ID NO:34、SEQ ID NO:43和SEQ ID NO:49中任一项所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:35或SEQ ID NO:44所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:36或SEQ ID NO:42所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:46或SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的H-FR1，H-FR2，H-FR3和H-FR4可以包含选自下述任一组的氨基酸序列：
1)所述H-FR1可以包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:36所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:37所示的氨基酸序列；
2)所述H-FR1可以包含SEQ ID NO:43所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:44所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:45所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:46所示的氨基酸序列；
3)所述H-FR1可以包含SEQ ID NO:34所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:42所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:37所示的氨基酸序列；以及
4)所述H-FR1可以包含SEQ ID NO:49所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:35所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:42所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，H-FR1，HFR2，HFR3和H-FR4。例如，所述HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和H-FR4可分别依次包含：SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37；SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46；SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:42和SEQ ID NO:37；或SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:49、SEQ ID NO: 35、SEQ ID NO:42和SEQ ID NO:37所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VH。在本申请中，所述VH可以包含SEQ ID NO:78所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:9所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:15所示的氨基酸序列。例如，所述VH可以包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3。在本申请中，所述LCDR3可以包含SEQ ID NO:69所示的氨基酸序列。例如，所述LCDR3可以包含SEQ ID NO:8所示的氨基酸序列。例如，所述LCDR3可以包含SEQ ID NO:14所示的氨基酸序列。例如，所述LCDR3可以包含SEQ ID NO:23所示的氨基酸序列。例如，所述LCDR3可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR2。在本申请中，所述LCDR2可以包含SEQ ID NO:70所示的氨基酸序列。例如，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列。例如，所述LCDR2可以包含SEQ ID NO:22所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1。在本申请中，所述LCDR1可以包含SEQ ID NO:71所示的氨基酸序列。例如，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列。例如，所述LCDR1可以包含SEQ ID NO:21所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，LCDR2和LCDR3。例如，所述LCDR1可以包含SEQ ID NO:71所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:70所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:69所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的LCDR1可以包含SEQ ID NO:6或SEQ ID NO:21所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7或SEQ ID NO:22所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:8、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:25中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的LCDR1，LCDR2和LCDR3可包含选自下述任一组的氨基酸序列：
1)所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:8所示的氨基酸序列；
2)所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ  ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:14所示的氨基酸序列；
3)所述LCDR1可以包含SEQ ID NO:21所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:22所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:23所示的氨基酸序列；以及
4)所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连。在本申请中，所述L-FR1可以包含SEQ ID NO:75所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:38所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:47所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:50所示的氨基酸序列。例如，所述L-FR1可以包含SEQ ID NO:53所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间。在本申请中，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间。在本申请中，所述L-FR3可以包含SEQ ID NO:76所示的氨基酸序列。例如，所述L-FR3可以包含SEQ ID NO:40所示的氨基酸序列。例如，所述L-FR3可以包含SEQ ID NO:48所示的氨基酸序列。例如，所述L-FR3可以包含SEQ ID NO:51所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连。在本申请中，所述L-FR4可以包含SEQ ID NO:77所示的氨基酸序列。例如，所述L-FR4可以包含SEQ ID NO:41所示的氨基酸序列。例如，所述L-FR4可以包含SEQ ID NO:52所示的氨基酸序列。例如，所述L-FR4可以包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，L-FR2，L-FR3和L-FR4。例如，所述L-FR1可以包含SEQ ID NO:75所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:76所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:77所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的L-FR1可以包含SEQ ID NO:38、SEQ ID NO: 47、SEQ ID NO:50和SEQ ID NO:53中任一项所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:40、SEQ ID NO:48和SEQ ID NO:51中任一项所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:41、SEQ ID NO:52和SEQ ID NO:54中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的L-FR1，L-FR2，L-FR3和L-FR4可包含下述任一组氨基酸序列：
1)所述L-FR1可以包含SEQ ID NO:38所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:40所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:41所示的氨基酸序列；
2)所述L-FR1可以包含SEQ ID NO:47所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:48所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:41所示的氨基酸序列；
3)所述L-FR1可以包含SEQ ID NO:50所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:51所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:52所示的氨基酸序列；以及
4)所述L-FR1可以包含SEQ ID NO:53所示的氨基酸序列，所述L-FR2可以包含SEQ ID NO:39所示的氨基酸序列，所述L-FR3可以包含SEQ ID NO:40所示的氨基酸序列，且所述L-FR4可以包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包含LCDR1，LCDR2，LCDR3，L-FR1，L-FR2，L-FR3和L-FR4。例如，所述LCDR1，LCDR2，LCDR3，L-FR1，L-FR2，L-FR3和L-FR4可分别依次包含：SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41；SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:14、SEQ ID NO:47、SEQ ID NO:39、SEQ ID NO:48和SEQ ID NO:41；SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:50、SEQ ID NO:39、SEQ ID NO:51和SEQ ID NO:52；或SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:25、SEQ ID NO:53、SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包含VL。在本申请中，所述VL可以包含SEQ ID NO:79所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:5所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:13所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:20所示的氨基酸序列。例如，所述VL可以包含SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3。在本申请中，所述HCDR1可以包含SEQ ID NO:2、SEQ ID NO:10和SEQ ID NO:16中任一项所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:68或SEQ ID NO:11所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:67所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:71所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:70所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:69所示的氨基酸序列。
在本申请中，所述HCDR1可以包含SEQ ID NO:2、SEQ ID NO:10和SEQ ID NO:16中任一项所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:3、SEQ ID NO:17和SEQ ID NO:11中任一项所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:4、SEQ ID NO:12和SEQ ID NO:18中任一项所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:6或SEQ ID NO:21所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7或SEQ ID NO:22所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:8、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:25中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的HCDR1，HCDR2，HCDR3，LCDR1，LCDR2和LCDR3可包含下述任一组氨基酸序列：
1)所述HCDR1可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:4所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:8所示的氨基酸序列；
2)所述HCDR1可以包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:12所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:14所示的氨基酸序列；
3)所述HCDR1可以包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:17所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:18所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:8所示的氨基酸序列；
4)所述HCDR1可以包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:18所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:21所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:22所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:23所示的氨基酸序列；
5)所述HCDR1可以包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:17所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:18所示的氨基酸序列，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列，所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可以包含SEQ ID NO:25所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含VH和VL。在本申请中，所述VH可以包含SEQ ID NO:78所示的氨基酸序列，且所述VL可以包含SEQ ID NO:79所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白的VH可以包含SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:15和SEQ ID NO:19中任一项所示的氨基酸序列，且所述VL可以包含SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:20和SEQ ID NO:24中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含选自下述任一组VH和VL：
1)所述VH可包含SEQ ID NO:1所示的氨基酸序列，且所述VL可包含SEQ ID NO:5所示的氨基酸序列；
2)所述VH可包含SEQ ID NO:15所示的氨基酸序列，且所述VL可包含SEQ ID NO:5所示的氨基酸序列；
3)所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:13所示的氨基酸序列；
4)所述VH可包含SEQ ID NO:19所示的氨基酸序列，且所述VL可包含SEQ ID NO:
20所示的氨基酸序列；以及
5)所述VH可包含SEQ ID NO:19所示的氨基酸序列，且所述VL可包含SEQ ID NO:
24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区。所述抗体重链恒定区可以源自人IgG重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG1重链恒定区。
在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区。所述抗体轻链恒定区可以源自人Igκ恒定区。
此外，需要说明的是，本申请所述分离的抗原结合蛋白可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述分离的抗原结合蛋白中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。在某些实施方式中，本申请所述分离的抗原结合蛋白的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。本领域内的技术人员知道，一些保守序列修改不会使抗原结合性消失，具体可以参见，例如，Brummell et al.,(1993)Biochem 32:1180-8；de Wildt et al.,(1997)Prot.Eng.10:835-41；Komissarov et al.,(1997)J.Biol.Chem.272:26864-26870；Hall et al.,(1992)J.Immunol.149:1605-12；Kelley and O'Connell(1993)Biochem.32:6862-35；Adib-Conquy et al.,(1998)Int.Immunol.10:341-6 and Beers et al.,(2000)Clin.Can.Res.6:2835-43。
可以通过本领域已知的各种测定鉴别、筛选或表征本申请所述的CLDN18.2抗原结合蛋白的物理/化学特性和/或生物活性。
在某些实施方式中，例如可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白或融合蛋白的抗原结合活性。
在本申请中，所述分离的抗原结合蛋白能够特异性结合CLDN18.2抗原。“特异性结合”CLDN18.2抗原的抗原结合蛋白通常可以结合CLDN18.2，但不结合或基本上不结合缺乏CLDN18.2序列的其它蛋白。抗原结合蛋白(例如，抗体)是否结合抗原CLDN18.2可使用本领域中已知的任何测定法确定。例如，可以使用流式细胞荧光分选技术(FACS)测定所述分离的抗原结合蛋白与CLDN18.2的特异性结合活性。在某些实施方式中，所述特异性结合可以为浓度依赖性结合。例如，在FACS结合活性实验中，所述CLDN18.2的平均荧光强度随 着CLDN18.2抗体浓度的增加而增强。
在本申请中，所述抗原结合蛋白可以结合人CLDN18.2蛋白。在某些情形中，本申请所述的抗原结合蛋白还可以与鼠(例如，小鼠)和/或猴(例如，食蟹猴)的CLDN18.2交叉反应。例如，通过FACS所检测的。在本申请中，“交叉反应”通常指抗体与来自其他物种的同源蛋白反应的能力。
在本申请中，所述抗原结合蛋白能够引发CDC效应。例如，本申请所述CLDN18.2抗原结合蛋白针对SP2/0-人CLDN18.2细胞能够诱导强CDC效应。例如，本申请所述CLDN18.2抗原结合蛋白针对MC38-人CLDN18.2细胞能够诱导强CDC效应。例如，诱导方式可以是以剂量依赖方式。
在本申请中，所述分离的抗原结合蛋白在FACS中验证，其基本上不与参比抗体竞争结合CLDN18.2。所述参比抗体可以包含重链可变区(VH)和轻链可变区(VL)，所述参比抗体例如，其VH可以包含SEQ ID NO:55所示的氨基酸序列，其VL可以包含SEQ ID NO:56所示的氨基酸序列。
在本申请中，所述抗原结合蛋白能够抑制肿瘤生长和/或肿瘤细胞的增殖。所述肿瘤可以是表达CLDN18.2的肿瘤，例如，结肠癌。所述结肠癌的细胞可以是MC38-人CLDN18.2细胞。
嵌合抗原受体
另一方面，本申请还提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体(CAR)可以包含结合CLDN18.2蛋白的靶向部分，例如，结合CLDN18.2蛋白的靶向部分可以是本申请所述的抗原结合蛋白。例如，所述靶向部分可以以scFv形式存在。例如，所述scFv可以包含本申请所述的分离的抗原结合蛋白的CDR、VH和/或VL。例如，所述scFv的VH和VL可以通过SEQ ID NO:89相连。例如，所述scFv可以包含SEQ ID NO:90所示的氨基酸序列。
在本申请中，所述CAR包含胞外的结合CLDN18.2蛋白的靶向部分外，还可包含其他结构域。
在本申请中，所述CAR可以包括共刺激信号区域，其可提供刺激信号。例如，所述共刺激信号区域可以包括选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
例如，所述共刺激信号区域可以为源自4-1BB的胞内共刺激信号区域。例如，所述共刺 激信号区域可以包含SEQ ID NO:85所示的氨基酸序列。
在某些情形中，所述CAR可包含胞内信号区域，其可以包含至少有一个ITAM基序的结构域。所述胞内信号传导结构域可以将激活信号传输到细胞内部。例如，所述胞内信号区域可以包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和其他至少包含一个ITAM的结构域。
例如，所述胞内信号区域可以为源自CD3ζ的信号传导结构域。例如，所述胞内信号区域可以包含SEQ ID NO:86所示的氨基酸序列。
在某些情形中，所述CAR可包含跨膜域，所述跨膜域是细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。所述跨膜域可以源自任意的I型跨膜蛋白。跨膜域可以是预测为形成疏水螺旋的合成序列。例如，所述跨膜区可以包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
例如，所述跨膜区可以为源自CD8的跨膜区。例如，所述跨膜区可以包含SEQ ID NO:84所示的氨基酸序列。
在某些情形中，所述CAR可包含铰链区，所述铰链区可以位于所述胞外的靶向部分和所述跨膜域之间。例如，所述铰链区可以包含选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
例如，所述铰链区可以为源自CD8的铰链区。例如，所述铰链区可以包含SEQ ID NO:83所示的氨基酸序列。
在本申请中，所述CAR在所述结合CLDN18.2蛋白的靶向部分的N端还可包含信号肽。例如，所述信号肽可以为源自CD8蛋白的信号肽。例如，所述信号肽可以包含SEQ ID NO:88所示的氨基酸序列。
在本申请中，所述CAR还可包含低密度脂蛋白受体相关蛋白或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述CAR的C端。例如，所述低密度脂蛋白受体 相关蛋白或其片段可以包括低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以是低密度脂蛋白受体相关蛋白5和/或6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包括SEQ ID NO:91所示的氨基酸序列。
在本申请中，所述CAR中的所述低密度脂蛋白受体相关蛋白或其片段的序列可通过自剪切肽(例如，T2A、P2A、E2A等2A肽)连接至CAR的C端序列。例如，所述低密度脂蛋白受体相关蛋白或其片段可以通过T2A与胞内信号区域的C端连接。例如，所述剪切肽可以包含SEQ ID NO:87所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含结合CLDN18.2蛋白的靶向部分(例如，所述抗原结合蛋白)、所述铰链区、所述跨膜域、所述共刺激信号区域和所述胞内信号区域。例如，自N端至C端，所述CAR可依次包含所述scFv，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，和源自CD3ζ的胞内信号区域。
在本申请中，还包含能够表达所述CAR或免疫效应细胞的载体，在所述载体中，可依次包含编码结合CLDN18.2蛋白的靶向部分(例如，所述抗原结合蛋白)、编码所述铰链区、编码所述跨膜域、编码所述共刺激信号区域、编码所述胞内信号区域和编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子。
在本申请中，还包含能够表达所述CAR或免疫效应细胞的载体，在所述载体中，可依次包含编码结合CLDN18.2蛋白的靶向部分(例如，所述抗原结合蛋白)、编码所述铰链区、编码所述跨膜域、编码所述共刺激信号区域、编码所述胞内信号区域、编码所述剪切肽和编码所述低密度脂蛋白受体相关蛋白或其片段的核酸分子。
多肽分子、免疫缀合物、核酸分子、载体、细胞和药物组合物
另一方面，本申请提供了多肽分子，其可以包含本申请所述的分离的抗原结合蛋白或本申请所述的嵌合抗原受体。
在本申请中，所述多肽分子可以包含融合蛋白。例如，本申请所述分离的抗原结合蛋白可以与其他功能分子(例如抗体或受体配体)融合形成双特异性分子或多特异性分子。所述双特异性分子或多特异性分子可以特异性结合至少两个不同结合位点或靶向分子。所述双特异性分子或多特异性分子可以通过基因改造、体细胞杂交或化学法进行制备。具体可以参见，例如Kufer et al,cited supra；Cao and Suresh,Bioconjugate Chemistry,9(6),635-644(1998)；和van Spriel et al.,Immunology Today,21(8),391-397(2000)。
另一方面，本申请还提供了免疫缀合物，其可以包含本申请所述的分离的抗原结合蛋白。
在本申请中，可以将本申请所述的分离的抗原结合蛋白或其片段与另一试剂，如化学治 疗剂、毒素、免疫治疗剂、成像探针、分光镜探针等的连接。该连接可以通过一个或多个共价键，或非共价相互作用，并且可以包括螯合作用。可以使用多种接头(所述接头可以为本领域所知)以形成免疫缀合物。此外，可以以融合蛋白质的形式提供免疫缀合物，其可以由编码免疫缀合物的多核苷酸表达。所述免疫缀合物还可以包含例如抗体-药物缀合物(ADC)。在ADC中，抗体和治疗剂可以通过接头交联，该接头可切割，例如肽类接头、二硫类接头或腙类接头。
另一方面，本申请提供了一种或多种核酸分子，其可以编码本申请所述的分离的抗原结合蛋白或本申请所述的嵌合抗原受体。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。
在本申请中，所述核酸分子可以包含SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33中任一项所示的核苷酸序列。
在本申请中，所述核酸分子可以包含选自下述的任一组核苷酸序列：
1)SEQ ID NO:26所示的核苷酸序列，以及SEQ ID NO:27所示的核苷酸序列；
2)SEQ ID NO:30所示的核苷酸序列，以及SEQ ID NO:27所示的核苷酸序列；
3)SEQ ID NO:28所示的核苷酸序列，以及SEQ ID NO:29所示的核苷酸序列；
4)SEQ ID NO:31所示的核苷酸序列，以及SEQ ID NO:32所示的核苷酸序列；以及
5)SEQ ID NO:31所示的核苷酸序列，以及SEQ ID NO:33所示的核苷酸序列。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请提供了一种细胞，其可以包含本申请所述分离的抗原结合蛋白、本申请所述的嵌合抗原受体、本申请所述的多肽分子、本申请所述的核酸分子或本申请所述的载 体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中。
在某些实施方案中，所述的细胞可以是免疫效应细胞。在某些实施方案中，所述的细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
在某些实施方案中，所述的细胞可以是T细胞。
在本申请中，所述细胞可以包含和/或表达所述CAR。在本申请中，所述细胞可以包含和/或表达所述CAR和所述低密度脂蛋白受体相关蛋白或其片段。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的嵌合抗原受体、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)佐剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方案中，所述药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量可以取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
在某些实施方案中，所述药学上可接受的载剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方案中，所述药物组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者 体内的治疗剂，如WO2015/036583所述。
制备方法
另一方面，本申请提供了制备所述的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白。例如，可以用连接或天然存在的CLDN18.2蛋白或其片段，免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的CLDN18.2都可以作为免疫原(抗原)，用于产生对CLDN18.2特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人CLDN18.2，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。
嵌合人源抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体可以是IgG抗体，可以使用IgG1亚型。可以通过用下文实施例中描述的生物学测定筛选抗体实现必需恒定结构域序列的优化，以产生所需生物学活性。同样，任一类轻链都可以在本申请的化合物和方法中使用。例如，κ链或其变体在本申请的化合物和方法中是可以用的。
方法和用途
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的嵌合抗原受体、所述的多肽分子、所述的免疫缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防、缓解或治疗疾病和/或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述分离的抗原结合蛋白、所述的嵌合抗原受体、所述的多肽分子、所述的免疫缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请所述分离的抗原结合蛋白、所述的嵌合抗原受体、所述的多肽分子、所述的免疫缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其可以用于预防、缓解或治疗疾病和/或病症。
在本申请中，所述疾病和/或病症可以包括癌症。
在本申请中，所述癌症可以包括实体瘤和/或血液瘤。
在本申请中，所述癌症可以包括CLDN18.2阳性的肿瘤。
在本申请中，所述癌症可以包含胃癌和/或结肠癌。
在本申请中，所述分离的抗原结合蛋白可以与一种或多种其他抗体一起施用，以便有效抑制受试者中的肿瘤生长。所述分离的抗原结合蛋白还可以与化疗剂一起施用。
另一方面，本申请还提供了检测样品中CLDN18.2的方法。所述方法包括施用本申请所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。所述方法可以是离体和/或体外方法。
另一方面，本申请还提供了检测样品中CLDN18.2的试剂或试剂盒，其可以包含所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的嵌合抗原受体、所述多肽分子、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备所述检测CLDN18.2的试剂或试剂盒中的用途。
在本申请中，所述试剂或试剂盒可以用于检测样品中CLDN18.2的存在和/或含量。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 CLDN18.1和CLDN18.2阳性对照抗体的制备
CLDN18.1阳性对照抗体选择市售的抗CLDN18的兔单克隆抗体(abcam,Cat#ab203563)，该抗体可以同时识别CLDN18.1和CLDN18.2，且抗原结合位点位于胞内。在本申请中，将此抗体作为CLDN18.1阳性对照抗体，并命名为抗CLDN18抗体(Anti-Claudin18 antibody)。
根据专利申请CN103509114A提供的175D10克隆(IMAB362)抗体重轻链全长氨基酸序列(如SEQ ID NO:81和SEQ ID NO:82所示的氨基酸序列)，委托苏州金唯智生物科技有限公司进行常规基因合成并克隆至本公司提供的真核表达载体pCMV-k、pCMV-IgG1(NDL)中，构建质粒包括：pCMV-IMAB362-VL和pCMV-IMAB362-VH，将以上两种载体质粒按3:2(VL:VH)的比例共转染Expi293细胞瞬时表达，5天后收获细胞培养上清，经Protein A亲和层析纯化，BCA定量即可获得CLDN18.2阳性对照抗体，命名为zolbetuximab。
实施例2 CLDN18.1和CLDN18.2高表达稳定细胞株的构建
根据表1给出的人CLDN18.1、人CLDN18.2、小鼠CLDN18.1、小鼠CLDN18.2、食蟹猴CLDN18.1、食蟹猴CLDN18.2的核苷酸序列，委托苏州金唯智生物科技有限公司进行常规基因合成并克隆至本公司提供的慢病毒表达载体pHAGE-full EF1a-IzsGreen中，将得到的含质粒的穿刺菌经活化后进行质粒中提，然后利用本公司的慢病毒包装和感染体系分别对293T、CHO、SP2/0细胞进行慢病毒感染，感染后获得的细胞经FACS检测感染效率后，梯度稀释法进行单克隆化，再经过FACS筛选验证，获得稳定高表达CLDN18.1和CLDN18.2的单克隆细胞株。
表1.CLDN18.1和CLDN18.2的核苷酸序列
CLDN18来源 Genbank登录号 核苷酸序列
人CLDN18.1 NM_016369.4 SEQ ID NO:61
人CLDN18.2 NM_001002026.3 SEQ ID NO:62
小鼠CLDN18.1 NM_019815.3 SEQ ID NO:63
小鼠CLDN18.2 NM_001194921.1 SEQ ID NO:64
食蟹猴CLDN18.1 XM_005545863.2 SEQ ID NO:65
食蟹猴CLDN18.2 XM_001114708.4 SEQ ID NO:66
对于稳定高表达CLDN18.2细胞株的FACS验证，取30000个转染细胞铺到V形96孔板中，然后加入阳性抗体zolbetuximab并设PBS阴性对照孔，4℃孵育1小时后，96孔板用FACS缓冲液洗涤1遍，再加入二抗(山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2022084922-appb-000001
650)(abcam,Cat#ab98593))；4℃孵育30min后，96孔板用FACS缓冲液洗涤2遍，之后使用iQue Screener流式仪(购自IntelliCyt公司)监测细胞荧光度，挑选MFI值最高的单克隆细胞扩大培养和冻存。
对于稳定高表达CLDN18.1细胞株的FACS验证，细胞分为两种处理方式：一种是细胞计数后直接用FACS缓冲液重悬铺板，一抗为实施例1提供的CLDN18.2阳性抗体zolbetuximab；另一种是细胞计数后进行固定破膜处理(四正柏生物，固定液FXP008和破膜剂FXP009)，以使抗体可以和位于胞内的抗原位点结合，一抗为实施例1提供的市售CLDN18抗体；同时上机检测，挑选与市售CLDN18抗体强结合，与zolbetuximab不结合的单克隆细胞扩大培养和冻存。
构建获得的稳定高表达CLDN18的细胞株分别标记为：293T-人CLDN18.1(293T-human  CLDN18.1)、293T-人CLDN18.2(293T-human CLDN18.2)、CHO-人CLDN18.1(CHO-human CLDN18.1)、CHO-人CLDN18.2(CHO-human CLDN18.2)、SP2/0-人CLDN18.1(SP2/0-human CLDN18.1)、SP2/0-人CLDN18.2(SP2/0-human CLDN18.2)、293T-鼠CLDN18.1(293T-mouseCLDN18.1)、293T-鼠CLDN18.2(293T-mouseCLDN18.2)、293T-食蟹猴CLDN18.1(293T-macacaCLDN18.1)、293T-食蟹猴CLDN18.2(293T-macacaCLDN18.2)，均为单克隆稳定细胞株。
实施例3 CLDN18.2鼠源抗原结合蛋白的生成
1.小鼠免疫
采用人CLDN18.2第一个胞外段全长核苷酸序列，委托苏州金唯智生物科技有限公司进行常规基因合成并克隆至本公司提供的真核表达载体中，构建质粒为pCMV-CLDN18.2-D1，将得到的穿刺菌液进行质粒中提，获得DNA免疫原；细胞免疫原为实施例2中构建获得的SP2/0-人CLDN18.2细胞。本实验共对9只Balb/c小鼠进行免疫，其中6只小鼠为细胞抗原和DNA抗原组合免疫方式，另外3只小鼠为全细胞免疫方式，采用经典的小鼠免疫时间表，免疫两次后，采集小鼠血清，用293T-人CLDN18.2细胞通过FACS对血清滴度进行测定，选择符合滴度要求的小鼠进行最后一次强化免疫，若滴度不符合要求，需要增加1到2次免疫直至血清滴度符合要求，最后一次免疫3天后，在无菌条件下收集小鼠脾脏组织，并研磨成脾细胞悬液，脾细胞裂红处理，然后分装冻存于-80℃冰箱待用。
2.杂交瘤细胞融合
提前复苏小鼠骨髓瘤细胞SP2/0，用含10％胎牛血清的DMEM培养基于37℃，5％CO 2条件下培养、传代。融合当天，提前37℃水浴预热PEG 1450溶液(Solution)(Sigma Aldrich，Cat#25322-68-3)和高糖DMEM培养基。镜检观察SP2/0细胞状态，细胞最好处于对数生长期，400g，5分钟离心收集SP2/0细胞，用适量的预热DMEM培养基重悬，台盼蓝计数，要求细胞活率在90％以上，然后放入37℃水浴锅中待用。37℃水浴复苏小鼠脾细胞，用20ml预热DMEM洗涤1次，400g，5分钟，弃上清，加入25mL预热DMEM重悬细胞，稀释一定比例后用血球计数板计数。按细胞数比例(脾细胞：SP2/0＝4:1)混合，400g，5分钟，离心，弃上清。缓慢加入1mL预热的PEG1450溶液，进行细胞融合，按照每块96孔板为1×10 7个脾细胞的总量进行铺板，培养基为含10％FBS、1％双抗、2％HAT补充剂(Supplement)和10％ClonaCell TM-HY Medium C(STEMCELL,CAT#:03803)的高糖DMEM培养基。检测前一天进行换液处理，换液后的培养基为含10％FBS、1％双抗、1％HT补充剂(Supplement)的高糖DMEM培养基，24h后取96孔板细胞培养上清进行流式检测。
3.杂交瘤细胞的流式筛选
选择实施例2中构建的293T-人CLDN18.2和CHO-人CLDN18.2细胞对融合后获得的母克隆杂交瘤细胞进行第一轮流式初筛：细胞分别消化计数，用流式缓冲液(含0.1％BSA的PBS)重悬调整至细胞密度为1×10 6/ml，30ul/孔加入V底96孔板；取50ul/孔的杂交瘤细胞培养上清加入已经铺好细胞抗原的V底96孔板中，同时设阳性对照(5ug/ml的zolbetuximab抗体，30ul/孔)和阴性对照(30ul/孔的PBS缓冲液)，4℃共孵育1小时，流式缓冲液清洗一次，加入相对应的二抗，30ul/孔，4℃共孵育30分钟，加入流式缓冲液清洗两次，细胞震松散后每孔加入25ul的FACS缓冲液，进行上机检测(iQue Screener流式仪，IntelliCyt公司)。挑选初筛显示CLDN18.2阳性的母克隆扩大培养至24孔细胞培养板，三天后取细胞上清进行FACS复筛，选择CHO-人CLDN18.2、CHO-人CLDN18.1和293T-人CLDN18.2、293T-人CLDN18.1四种细胞分别铺板，从中筛选出CLDN18.2阳性且CLDN18.1阴性的杂交瘤母克隆。利用梯度稀释法对阳性的杂交瘤母克隆进行单克隆化，经过两到三轮的亚克隆和FACS筛选，获得分泌CLDN18.2特异性抗体的杂交瘤单克隆细胞，能与293T-人CLDN18.2、CHO-人CLDN18.2细胞产生阳性结合，而与293T-人CLDN18.1、CHO-人CLDN18.1细胞结合为阴性。
结果如表2所示，杂交瘤细胞培养上清与CLDN18.2结合阳性的亚克隆包括：3A6、7E3、5E6、14E12和17B10共5株单克隆细胞。
表2.杂交瘤克隆的流式筛选
Figure PCTCN2022084922-appb-000002
Figure PCTCN2022084922-appb-000003
实施例4 CLDN18.2鼠源抗原结合蛋白可变区测序
采用简并引物扩增法测序获得CLDN18.2鼠源抗原结合蛋白重链可变区和轻链可变区序列，所用引物参阅Lotta von Boehmer发表的PROTOCOL(doi:10.1038/nprot.2016.102)，委托苏州金唯智公司进行引物合成。具体来说，收集实施例3中5个CLDN18.2阳性的杂交瘤单克隆细胞，采用QIAGEN RNeasy Plus Mini Kit试剂盒提取细胞总RNA，1％琼脂糖凝胶电泳检测RNA完整性，NanoDrop核酸定量分析仪测定RNA浓度。使用RNA反转录试剂盒，将1ug的RNA逆转录成cDNA，-20℃保存待用。以5ul cDNA为模板，按照PCR高保真酶(北京全式金，AP231)的操作说明配置PCR反应体系和设置PCR程序，其中退火温度采用梯度降温的方法，然后直接以1ul第一轮PCR产物为模板进行第二轮PCR，PCR反应体系和程序同上，第二轮PCR产物全部上样进行1％琼脂糖凝胶电泳，切下合适大小的特异目的条带，进行胶回收，连接平末端克隆载体(北京全式金，CB501)，转化Trans1-T1感受态细胞，涂2YT平板，37℃培养箱倒置培养12-16h，在超净工作台中用枪头挑取单克隆菌落，菌落活化后委托苏州金唯智公司进行测序，测序后获得5株杂交瘤克隆的VH和VL基因序列，如表3所示。
表3. 5株杂交瘤克隆的VH和VL序列
Figure PCTCN2022084922-appb-000004
实施例5 CLDN18.2抗原结合蛋白的制备
以实施例4中测序得到的5个重轻链可变区序列为模板分别设计引物，委托苏州金唯智生物科技有限公司进行引物合成，采用高保真酶进行PCR扩增、琼脂糖凝胶电泳和胶回收， 回收得到的质粒DNA同源重组(Vazyme,C112)到经酶切的带有人IgG1恒定区的真核表达载体(pCMV-IgG1NDL和pCMV-κ)中并委托苏州金唯智生物科技有限公司进行测序鉴定正确的阳性重组载体。测序正确后进行质粒中提，重轻链共转染Expi293细胞，5天后离心收集细胞培养上清，进行ProteinA亲和层析纯化，获得完整的人鼠IgG1，Igκ抗原结合蛋白。这5个抗原结合蛋白分别命名为c5E6、c7E3、c3A6、c14E12、c17B10。
实施例6 CLDN18.2抗原结合蛋白与CLDN18.2特异性结合活性测定
通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液检测上述嵌合抗体与靶细胞的特异性结合活性，选择三种靶细胞：表达人CLDN18.2的稳转细胞株、表达人CLDN18.1的稳转细胞株以及肿瘤细胞系，分别进行结合活性的测定。
1.实施例5中的抗原结合蛋白与高表达人CLDN18.2细胞的流式结合活性检测
细胞为实施例2构建的293T-人CLDN18.2、CHO-人CLDN18.2和SP2/0-人CLDN18.2细胞，细胞消化计数，使用流式缓冲液重悬细胞并调整至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，抗体以30ug/ml浓度起始，用流式缓冲液按两或三倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照，阳性对照抗体为实施例1中纯化获得的zolbetuximab；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593)，30ul/孔，4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。将原始数据代入GraphPad8.0软件作图并计算，结果如图1。
2.实施例5中的抗原结合蛋白与高表达人CLDN18.1细胞的流式结合活性检测
阳性对照抗体为市售抗CLDN18抗体(Anti-Claudin18 antibody)(abcam,Cat#ab203563)，其抗原结合位点位于CLDN18.2四次胯膜蛋白的胞内部分，需要进行流式胞内染色分析。具体地，细胞为实施例1构建的293T-人CLDN18.1和SP2/0-人CLDN18.1细胞，细胞消化计数后进行固定破膜处理，处理后的细胞用流式缓冲液重悬至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，抗体以30ug/ml浓度起始，用流式缓冲液按三倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照，阳性对照抗体稀释条件同上；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593和Cat#ab150079)，30ul/孔，4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。将原始数据代入GraphPad8.0软件作图并计算，结果如图2。
3.实施例5中的抗原结合蛋白与肿瘤细胞系的流式结合活性检测
选择按照实施例2所示方法构建获得的稳定高表达人CLDN18.2的肿瘤细胞MC38-人 CLDN18.2为靶细胞，细胞消化计数，使用流式缓冲液重悬细胞并调整至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，抗体以30ug/ml浓度起始，用流式缓冲液按三倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照，阳性对照抗体为实施例1中纯化获得的zolbetuximab，稀释条件同上；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593)，30ul/孔，4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。将原始数据代入GraphPad8.0软件作图并计算，结果如图3。
通过以上操作步骤，表达纯化获得5个人鼠抗原结合蛋白，流式验证其抗原结合活性：如图1所示，5个抗原结合蛋白均呈现和人CLDN18.2浓度依赖性结合活性，且大部分强于zolbetuximab阳性抗体，3种细胞的检测结果保持一致；如图2所示，5个抗原结合蛋白均能特异性与人CLDN18.2结合而不与人CLDN18.1结合；如图3所示，5个抗原结合蛋白均与稳定高表达人CLDN18.2的小鼠结肠癌MC38细胞强结合，结合活性强度呈浓度依赖性，部分强于zolbetuximab阳性抗体。
实施例7 CLDN18.2抗原结合蛋白的种属交叉结合活性分析
通过流式细胞荧光分选技术(FACS)，使用CytoFLEX流式细胞仪(购自BECKMAN COULTER公司)，用含有0.1％BSA的PBS作为缓冲液检测上述抗原结合蛋白与小鼠(mouseCLDN18.2)、食蟹猴(macacaCLDN18.2)的种属交叉结合活性。具体而言，293T-小鼠CLDN18.2和293T-食蟹猴CLDN18.2细胞为实施例2构建获得的稳转细胞株，细胞消化计数，用流式缓冲液重悬至细胞密度1×10 6/ml，30ul/孔加入V底96孔板中；一抗用流式缓冲液按三倍比梯度稀释，起始浓度为10ug/ml，形成6个梯度，每种抗体设PBS阴性对照，阳性对照抗体为zolbetuximab，稀释条件同上；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗(abcam,Cat#ab98593)，30ul/孔，4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入30ul/孔流式缓冲液，上机检测。将原始数据代入GraphPad8.0软件作图并计算，结果如图4，5个CLDN18.2嵌合抗原结合蛋白和阳性对照抗体zolbetuximab，均和小鼠(mouseCLDN18.2)、食蟹猴(macacaCLDN18.2)存在结合，且结合活性呈现梯度依赖。说明这5个嵌合抗原结合蛋白和zolbetuximab不仅特异性结合人CLDN18.2(humanCLDN18.2)，而且具有人、小鼠、食蟹猴的种属交叉结合活性。
实施例8 CLDN18.2抗原结合蛋白的CDC活性
使用细胞毒性检测试剂盒(Promega，Cat#G1780)，检测CLDN18.2抗原结合蛋白针对SP2/0-人CLDN18.2细胞引发CDC效应的能力，具体过程如下：
(1)配置培养基(A:DMEM+2％FBS+1％双抗，B:DMEM+2％FBS+1％双抗+10％兔补体)；
(2)靶细胞SP2/0-人CLDN18.2于400g离心5分钟，然后用上述培养基A重悬细胞，使靶细胞密度为4×10 5个细胞/ml，100ul/孔加入96孔细胞培养板中；
(3)抗体用上述培养基B稀释成三种不同浓度，分别为10ug/ml、2ug/ml和0.4ug/ml，100ul/孔加入检测孔，各个浓度点设两个复孔，zolbetuximab作为阳性对照抗体，人IgG-Fc作为阴性对照抗体；
(4)补体为购自PelFreez Bio公司的兔补体Rabbit Complent 3-4 week(Cat#31061-3)，补体所用实验终浓度为5％；
(5)按试剂盒要求设置对照孔，获得的混合物于37℃培养箱共孵育4小时后，酶标仪记录490nM吸光值，利用试剂盒给出的公式计算靶细胞裂解百分比，GraphPad prism 8分析处理数据。
结果如图5a所示，以SP2/0-人CLDN18.2细胞做为杀伤靶细胞时，所有检测抗原结合蛋白均能以剂量依赖方式诱导强CDC效应，即均具有显著的CDC活性，其中c5E6、c7E3和c17B10表现出比zolbetuximab更高的CDC活性。
对c5E6抗原结合蛋白进一步检测其针对稳定过表达人CLDN18.2的CHO细胞引发CDC效应的能力。简单而言，CHO-人CLDN18.2细胞如实施例1所示通过本公司慢病毒转染系统构建获得，操作方法同上所述，以1.2×10 4个细胞每孔的密度铺板，兔补体终浓度为5％，抗原结合蛋白以五倍比梯度稀释，即10ug/ml、2ug/ml、0.4ug/ml、0.08ug/ml、0.016ug/ml、0.0032ug/ml、0.64ng/ml，各个浓度设两个复孔，zolbetuximab作为阳性对照抗体，人IgG-Fc作为同型对照抗体，酶标仪检测共孵育4h后的吸光值，计算靶细胞裂解百分比，GraphPad prism 8分析处理数据。
结果如图5b所示，c5E6抗原结合蛋白能以剂量依赖方式对CHO-人CLDN18.2细胞诱导强CDC效应，且表现出比zolbetuximab更高的CDC活性。
实施例9 CLDN18.2抗原结合蛋白的竞争结合活性分析
用生物素(EZ-Link Sulfo-NHS-LC-Biotin,Thermo,A39257)标记c5E6和zolbetuximab，各标记500ug，生物素化的抗原结合蛋白分子使用分子相互作用分析仪OctetRED384(sartorius)来测定标记效果,选择SA传感器固化抗体，抗体浓度为100nM,标记180s，标记完成的生物素化抗原结合蛋白命名为生物素化-c5E6(Biotinylated-c5E6)、生物素化-c7E3(Biotinylated-c7E3)、生物素化-c17B10(Biotinylated-c17B10)、生物素化-zolbetuximab(Biotinylated- zolbetuximab)。
通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液检测上述抗原结合蛋白与CHO-人CLDN18.2细胞(实施例2获得)的表位竞争结合活性，具体过程如下：
(1)使用缓冲液配置浓度为1×10 6cells/ml的CHO-人CLDN18.2细胞，加入V底96孔板(corning 3894)，每孔30μl；
(2)使用缓冲液分别配置受试抗原结合蛋白，使生物素化-c5E6(Biotinylated-c5E6)抗原结合蛋白终浓度为0.5μg/ml，30ul/孔加入V底96孔板，生物素化-zolbetuximab(Biotinylated-zolbetuximab)抗原结合蛋白终浓度为5μg/ml，30ul/孔加入V底96孔板；
(3)使用缓冲液配置竞争抗原结合蛋白(c5E6、c7E3、c17B10和zolbetuximab)，抗体终浓度333ug/ml起始，按4倍比梯度稀释抗体，形成10个浓度梯度，设PBS为阴性对照孔，将配置好的不同浓度的抗体按30μl/孔加入已铺好靶细胞和受试抗体的V底96孔板中混匀；
(4)4℃冰箱孵育1小时；
(5)每孔加110μl缓冲液，500g离心5分钟，弃上清后将细胞震松散；
(6)重复步骤(5)；
(7)使用缓冲液按1:500配比配置荧光二抗(BD phamingen APC Streptavidin,Cat#554067)，每孔30μl加入细胞中混匀，4℃冰箱孵育30分钟；
(8)每孔加170μl缓冲液，500g离心5分钟，弃上清后将细胞震松散；
(9)重复步骤(8)；
(10)每孔加25μl缓冲液混匀后用流式仪器检测。
流式表位竞争结合活性检测的分析结果如图6所示，本申请的分离的抗原结合蛋白c17B10/c7E3和生物素化-c5E6(Biotinylated-c5E6)都存在竞争，且c17B10/c7E3抗原结合蛋白在高浓度下(333ug/ml)完全竞争了生物素化-c5E6(Biotinylated-c5E6)和细胞抗原的结合，推测它们互为同一抗原结合表位；相比之下，zolbetuximab和生物素化-c5E6(Biotinylated-c5E6)存在微弱竞争，随zolbetuximab抗体浓度增加，Median信号值有所下降，且呈现一定的浓度依赖性，但当zolbetuximab抗体浓度提高为333ug/ml时，它的竞争活性和力度也依然远远弱于其它抗原结合蛋白，无法完全竞争生物素化-c5E6(Biotinylated-c5E6)抗原结合蛋白，推测zolbetuximab和c5E6的抗原结合表位不完全一致，呈现交叉或包含(zolbetuximab抗体的表 位包含在c5E6内)的关系。
实施例10 CLDN18.2抗原结合蛋白的体内抗肿瘤活性
选择实施例5中的抗原结合蛋白(c5E6、c7E3、c17B10)，阳性对照抗体zolbetuximab和同型对照抗体人IgG-Fc，测试其在接种小鼠结肠癌细胞的C57BL/6小鼠体内的抗肿瘤活性。利用本公司Expi293瞬时转染系统，纯化获得以上六种抗原结合蛋白，抗原结合蛋白内毒素控制在4EU/mg以下。具体实施方案如下：
115只雌性C57BL/6小鼠在右腹皮下接种MC38-人CLDN18.2细胞，接种细胞量为1.1×10 6，接种后7天，选取肿瘤大小为31.33-116.31mm 3(平均肿瘤大小为71.59mm 3)的小鼠80只，按肿瘤体积分为10组，每组8只小鼠，把小鼠随机分组日定义为Day0。分组后进行小鼠尾静脉给药，每周给药一次，连续给药四周，分30mg/kg和7.5mg/kg两组给药剂量，小鼠分组和给药方案如表4。在给药期间每周称重和测量肿瘤大小三次，并通过公式TV＝(长×宽 2)/2计算肿瘤体积，然后用肿瘤大小计算肿瘤生长抑制率(TGI)和T/C值。当肿瘤体积达到2000mm 3时停止实验，小鼠实施安乐死。
表4.CLDN18.2抗原结合蛋白体内药效实验的分组和给药方案
Figure PCTCN2022084922-appb-000005
通过皮下MC38-人CLDN18.2小鼠结肠癌模型，在雌性C57BL/6小鼠中评价尾静脉途径给药zolbetuximab、c17B10、c5E6和c7E3的抗肿瘤效果。结果显示在MC38-人CLDN18.2荷瘤 C57BL/6小鼠中，所有给药小鼠均耐受良好，体重保持稳定，无任何不良反应，小鼠体重变化和相对变化(％)分别见图7和图8；分析Day17前的肿瘤体积变化曲线和肿瘤生长抑制曲线，相比PBS对照组，c5E6和zolbetuximab高剂量给药组表现出较好的抑瘤效果，抑瘤效果呈现显著性差异(****，P<0.0001)，见图9和图10；分析Day40前的Kaplan-Meier生存曲线，相比PBS对照组，c5E6低剂量、c5E6高剂量和zolbetuximab高剂量给药组均表现出较高的小鼠生存百分比，且呈现显著性差异(*，P<0.05)，其中c5E6抗原结合蛋白的抗肿瘤活性最强，结果见图11。
实施例11 CLDN18.2单链抗体的生成和抗原结合活性鉴定
本实施例利用慢病毒感染和流式分选获得稳定表达人CLDN18.2的细胞，即SP2/0-hCLDN18.2细胞。对Balb/c小鼠进行细胞免疫，免疫原为SP2/0-hCLDN18.2细胞，利用小鼠杂交瘤技术，流式筛选并亚克隆获得CLDN18.2阳性的单克隆细胞，体外培养获得IgG类CLDN18.2鼠源单抗。采用简并引物扩增法测序获得重链可变区(V H)和轻链可变区(V L)序列，通过人工合成的连接肽(Linker)基因将抗体重链可变区(V H)和轻链可变区(V L)连接成重组基因，由该重组基因表达的抗体为CLDN18.2单链抗体，即5E6-scFv(SEQ ID NO:90)。同时慢病毒感染和流式分选获得稳定表达人CLDN18.1的细胞SP2/0-hCLDN18.1。
采用流式细胞术的方法鉴定5E6-scFv单链抗体和Standard抗体(VH包含SEQ ID NO:92所示的氨基酸序列，VL包含SEQ ID NO:93所示的氨基酸序列)的特异性结合活性：SP2/0-hCLDN18.2细胞计数，使用流式缓冲液重悬细胞并调整至1×10 6/ml，30ul/孔加入V底96孔板；30ul/孔加入一抗，一抗以80ug/ml工作浓度起始，用流式缓冲液按4倍比梯度稀释，形成7个梯度，每种抗体设PBS阴性对照；4℃孵育1小时，流式缓冲液清洗一遍，加入二抗：山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2022084922-appb-000006
650)(abcam,Cat#ab98593)，30ul/孔,4℃孵育30分钟；流式缓冲液清洗两遍，细胞震松散，加入25ul/孔流式缓冲液，等待上机。同时SP2/0-hCLDN18.1细胞计数进行铺板，一抗工作浓度从10ug/ml起始，3倍比梯度稀释，共形成5个浓度梯度，二抗为山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2022084922-appb-000007
650)(abcam,Cat#ab98593)，将原始数据代入GraphPad8.0软件作图并计算，结果如图12：5E6-scFv单链抗体能特异性结合人CLDN18.2，同Standard抗体。
实施例12 CLDN18.2单链抗体与非靶细胞的结合活性鉴定
本实施例利用流式细胞荧光分选技术(FACS)，鉴定5E6-scFv单链抗体与人体组织中多种非靶细胞的结合活性。细胞分别为人包皮成纤维细胞(HFF)、人类永生化表皮细胞(HaCaT)、 人正常肝细胞(LO2)、间充质干细胞(MSC)、人正常表皮细胞(KERA)、腺癌人类肺泡基底上皮细胞(A549)、人乳腺癌细胞(MCF-7)、人皮肤成纤维细胞(BJ)。抗体从30ug/ml的工作浓度起始，3倍比梯度稀释，共形成7个浓度梯度，二抗为山羊F(ab')2抗人IgG-Fc(
Figure PCTCN2022084922-appb-000008
650)(abcam,Cat#ab98593)，孵育后离心，用流式缓冲液重悬后上机，结果代入GraphPad8.0软件作图。结果如图13，5E6-scFv单链抗体与人体组织中多种非靶细胞不存在非特异结合，同Standard抗体，初步验证其对肿瘤靶标的特异性，即不存在“脱靶效应”现象。
实施例13 CLDN18.2抗原结合蛋白在人胃癌小鼠模型中的抗肿瘤活性
如实施例10，选择人CLDN18.2抗原结合蛋白(c5E6)，阳性对照抗体zolbetuximab和同型对照抗体人IgG-Fc，测试其在接种人胃癌细胞(NUGC-4)的雌性裸鼠体内的抗肿瘤活性。具体实施方案如下：
28只雌性裸鼠皮下接种NUGC-4细胞，接种细胞量为3x10 6，接种后7天，选取平均肿瘤大小为80mm 3的小鼠20只，按肿瘤体积平均分为4组，每组5只小鼠，把小鼠随机分组日定义为Day0。分组后进行小鼠腹腔给药，每周给药两次，连续给药三周，小鼠分组和给药方案如表5。在给药期间每周称重和测量肿瘤大小三次，并通过公式TV＝(长x宽 2)/2计算肿瘤体积。当肿瘤体积达到3000mm 3时停止实验，小鼠实施安乐死。
表5.人胃癌小鼠模型体内药效实验的分组和给药方案
Figure PCTCN2022084922-appb-000009
利用皮下NUGC-4人胃癌肿瘤模型，在雌性裸鼠中评价腹腔给药zolbetuximab和c5E6抗体的抗肿瘤效果，实验观察至给药后的25天，结果显示在NUGC-4荷瘤雌性裸鼠中，所有给药小鼠均耐受良好，体重保持稳定，无任何不良反应，小鼠体重变化见图14A；在观察期间，小鼠肿瘤体积变化曲线见图14B，G3：zolbetuximab，10mg/kg组和G4：c5E6，10mg/kg组小鼠相对G2同型对照组均显示出一定的抑瘤活性，均具有统计学上的显著性差异(p＜0.05)；G3和G4之间的抑瘤活性无显著差异，具有相似的抑瘤水平。
实施例14 CLDN18.2特异性CAR在T细胞中稳定表达
如图15A所示，本实施例中的CLDN18.2特异性CAR结构是由人CD8信号肽(SEQ ID NO:88)、抗人CLDN18.2单链抗体(SEQ ID NO:90)、人CD8绞链区(SEQ ID NO:83)、人CD8跨膜区(SEQ ID NO:84)、人4-1BB胞内共刺激域(SEQ ID NO:85)、人CD3ζ胞内激活域(SEQ ID NO:86)以及增加的Ori新元件(SEQ ID NO:91)共同组成。首先，本实施例探究了CLDN18.2特异性CAR在人T细胞中的表达情况和体外常规培养条件下的CART细胞扩增倍数。具体方法如下：
1)37℃水浴复苏液氮冻存的人PBMC细胞，用11mlPBS+1mlPBMC的体系离心重悬润洗三次(500g，5min；400g，5min；300g，5min)，润洗后的人PBMC细胞通过和CD3 MicroBeads,human(美天尼，130-050-101)混合，利用磁力架进行CD3阳选，即分离并保留CD3 +T细胞；用含有(4％FBS+X-VIVO(Lonza公司)+20ng/ml因子1+10ng/ml因子2)培养基重悬CD3 +T细胞至细胞密度为1×10 6cells/ml，按1:3的比例(细胞:磁珠)加入CD3/CD28磁珠(赛默飞40203D，磁珠用培养基清洗两次，用磁力架吸住，静置1min)对T细胞进行激活；T细胞加入磁珠中，混匀，12孔板中每孔补加培养基至700ul，细胞数为7×10 5/孔，密度1×10 6cells/ml，分选当天记为Day0。
2)激活的T细胞静置在37℃CO 2培养箱中培养20小时后，按病毒感染复数(MOI)为4的比例加入对应的病毒上清，加polybrene至终浓度为10ug/ml，吹打混匀后用水平离心机1200rpm离心1小时，将孔板放回37℃CO 2培养箱中培养24小时，感染当天记为Day1。
3)24h后，把12孔板中每孔细胞反复吹打均匀，转移至1.5mlEP管中，400g离心5分钟，去上清，并用新鲜的X-VIVO完全培养基1ml重悬细胞至细胞密度为7×10 5cells/ml，置于37℃CO 2培养箱中培养，培养基变黄需补加培养基；每2天对细胞进行计数，并补加新鲜的X-VIVO完全培养基将细胞密度调回7×10 5cells/ml；记录统计计数结果，用GraphPad8.0软件作图并计算常规培养下的CART细胞扩增倍数。
4)培养9-14天的CAR-T细胞检测细胞阳性率：用于感染细胞的病毒中携带Myc-tag，所以病毒感染细胞后通过流式细胞仪检测Myc阳性率从而得到CAR表达阳性率，所用直标检测抗体为Myc-Tag(9B11)Mouse mAb(Alexa
Figure PCTCN2022084922-appb-000010
488Conjugate)(Cell Signaling,2279S)。
按照MOI＝4分别感染人CLDN18.2序列病毒，包括5E6序列和Standard序列病毒。结果如图15B，CART细胞体外培养第12天时，流式检测CAR阳性率，5E6-CART的阳性率为72.6％，Positive control CAR-T的阳性率为65.4％。本实施例共采用了6个不同的PBMC donar 进行CAR阳性率验证，图15C即显示了5E6-CART在6个不同的PBMC donar中的阳性率均保持在60％左右，同Positive control CAR-T。图15D显示了体外常规培养12天的5E6-CART细胞累积扩增倍数约240倍，与Standard-CART的扩增倍数之间无显著性差异。
实施例15 CLDN18.2特异性CAR在体外可以特异性杀伤靶细胞
本实施例首先通过慢病毒感染并流式分选获得稳定高表达人CLDN18.2的CHO-hCLDN18.2细胞，和稳定高表达人CLDN18.1的CHO-hCLDN18.1细胞。本实施例进一步使用细胞毒性检测试剂盒(Promega，Cat#G1780)，利用LDH法评估CAR-T细胞在体外的特异性杀伤能力，步骤如下：
将上述实施例14中常规培养了9天的CAR-T和Mock T细胞，分别离心后用空白的X-VIVO培养基重悬，使细胞密度为1×10 5/ml；靶细胞为CHO、CHO-hCLDN18.2和CHO-hCLDN18.1三种，将三种靶细胞分别进行消化后计数，然后用空白的X-VIVO培养基进行重悬，使细胞密度为5×10 5/ml；然后按照每孔100ul靶细胞悬液+100ul CAR-T/Mock T细胞悬液的体积体系混合加入无菌v底96孔板中。按试剂盒要求设置对照孔，获得的混合物于37℃培养箱共孵育24小时后，酶标仪记录490nM吸光值，利用试剂盒给出的公式计算靶细胞裂解百分比，Graphpad prism 8分析处理数据。结果如图16，5E6-CART在体外特异性诱导CLDN18.2阳性靶细胞的裂解，对阴性及CLDN18.1阳性细胞无裂解作用，同Standard-CART，提示5E6-CART细胞在体外具有人CLDN18.2高特异性靶细胞杀伤活性。
实施例16 CLDN18.2特异性CAR的因子分泌情况
本实施例分别采用Human IFN-γELISA试剂盒(R&D，DY285B)和Human IL-2 ELISA试剂盒(R&D，DY202)分析CAR-T在杀伤靶细胞的过程中对IFN-γ和IL-2的分泌量。具体如下，按照1×10 4个细胞每孔的细胞量将高表达CLDN18.2的靶细胞(CHO-hCLDN18.2)、高表达CLDN18.1的对照细胞(CHO-hCLDN18.1)和阴性细胞CHO分别接种于无菌的96孔板，分别向靶细胞中按效应细胞(Effector)：靶细胞(Target)＝5:1的比例加入CAR-T细胞(5E6-CART和Positive control CAR-T)和未加修饰的T细胞(Mock T cell)等效应细胞。孵育24h后，按照试剂盒说明书进行操作，取上清用酶联免疫法(ELISA)检测IL-2和IFN-γ含量。结果如图17A和17B所示，5E6-CART与CLDN18.2阳性细胞共孵育时分泌较高的因子水平，与阴性及CLDN18.1阳性细胞共孵育下无显著因子分泌，同Standard-CART。因此5E6-CART细胞对高表达CLDN18.2的肿瘤细胞具有特异细胞因子诱导的作用。
实施例17 CLDN18.2特异性CAR-T细胞在小鼠结肠癌CDX模型中的抑瘤作用
本实施例首先将人CLDN18.2基因通过慢病毒的方式导入MC38细胞中，再通过流式分选仪分选出高表达人CLDN18.2的小鼠结肠癌细胞，即MC38-hCLDN18.2细胞。利用雌性B-NDG重度免疫小鼠，通过皮下注射途径构建小鼠结肠癌肿瘤模型，验证CLDN18.2特异性CAR-T细胞在小鼠体内的抑瘤作用。按照1.5×10 6/只的剂量接种MC38-hCLDN18.2细胞，小鼠接瘤后8天，选取肿瘤大小41.68-120.6mm 3(平均肿瘤大小80.01mm 3)的小鼠随机分为3组，每组8只，尾静脉回输已培养10天的CAR-T(或Mock T)细胞，其中5E6-CART和Standard-CART组的CAR-T回输剂量为3×10 6细胞/只小鼠，空白对照Mock T细胞组的回输剂量为1×107细胞/只小鼠。每周测量小鼠瘤体大小和体重3次，观察4周，如图18A。小鼠尾静脉回输CAR-T(或Mock T)细胞后，5E6-CART组小鼠在D13体重减重10％以上，D15时单只小鼠体重相对变化为-34.51％，D13出现第一只小鼠的死亡；Standard-CART组小鼠在D16体重减重10％以上，D16时单只小鼠体重相对变化为-36.93％，D18出现第一只小鼠的死亡，由于小鼠体重下降严重，所以PG-D22即结束实验，结束实验时暂未观察到明显的抑瘤作用，见图18B和18C。对小鼠安乐死后进行解剖，发现5E6-CART组和Standard-CART组的小鼠均出现胃脏受损出血的异常现象，如图18D。综上所述，5E6-CART组小鼠在此肿瘤模型中表现为不耐受问题及明显副作用，具体表现为小鼠体重下降、背毛竖立、蜷缩不动且胃部出血等；Mock T组小鼠表现耐受，小鼠体重等正常。

Claims (87)

  1. 分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:67所示的氨基酸序列。
  2. 根据权利要求1中所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:4、SEQ ID NO:12和SEQ ID NO:18中任一项所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:68或SEQ ID NO:11所示的氨基酸序列。
  4. 根据权利要求3所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:3或SEQ ID NO:17所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:2、SEQ ID NO:10和SEQ ID NO:16中任一项所示的氨基酸序列。
  6. 根据权利要求5所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:72所示的氨基酸序列。
  7. 根据权利要求6所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:34、SEQ ID NO:43和SEQ ID NO:49中任一项所示的氨基酸序列。
  8. 根据权利要求5-7中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:73所示的氨基酸序列。
  9. 根据权利要求8所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:35或SEQ ID NO:44所示的氨基酸序列。
  10. 根据权利要求3-9中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:80或SEQ ID NO:45所示的氨基酸序列。
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:36或SEQ ID NO:42所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:74所示的氨基酸序列。
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:46或SEQ ID NO:37所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包含VH，所述VH包含 SEQ ID NO:78所示的氨基酸序列。
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:15和SEQ ID NO:19中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含LCDR3，且所述LCDR3包含SEQ ID NO:69所示的氨基酸序列。
  17. 根据权利要求16所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:8、SEQ ID NO:14、SEQ ID NO:23和SEQ ID NO:25中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含LCDR2，且所述LCDR2包含SEQ ID NO:70所示的氨基酸序列。
  19. 根据权利要求18所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:7或SEQ ID NO:22所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包含LCDR1，且所述LCDR1包含SEQ ID NO:71所示的氨基酸序列。
  21. 根据权利要求20所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:6或SEQ ID NO:21所示的氨基酸序列。
  22. 根据权利要求20-21中任一项所述的分离的抗原结合蛋白，其包含L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:75所示的氨基酸序列。
  23. 根据权利要求22所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:38、SEQ ID NO:47、SEQ ID NO:50和SEQ ID NO:53中任一项所示的氨基酸序列。
  24. 根据权利要求20-23中任一项所述的分离的抗原结合蛋白，其包含L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:39所示的氨基酸序列。
  25. 根据权利要求18-24中任一项所述的分离的抗原结合蛋白，其包含L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:76所示的氨基酸序列。
  26. 根据权利要求25所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:40、SEQ ID NO:48和SEQ ID NO:51中任一项所示的氨基酸序列。
  27. 根据权利要求16-26中任一项所述的分离的抗原结合蛋白，其包含L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:77所示的氨基酸序列。
  28. 根据权利要求27所述的分离的抗原结合蛋白，其中所述L-FR4包含SEQ ID NO:41、SEQ ID NO:52和SEQ ID NO:54中任一项所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的分离的抗原结合蛋白，其包含VL，且所述VL包含SEQ ID NO:79所示的氨基酸序列。
  30. 根据权利要求29所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:20和SEQ ID NO:24中任一项所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其包含选自下述的任一组VH和VL：
    1)所述VH包含SEQ ID NO:1所示的氨基酸序列，且所述VL包含SEQ ID NO:5所示的氨基酸序列；
    2)所述VH包含SEQ ID NO:15所示的氨基酸序列，且所述VL包含SEQ ID NO:5所示的氨基酸序列；
    3)所述VH包含SEQ ID NO:9所示的氨基酸序列，且所述VL包含SEQ ID NO:13所示的氨基酸序列；
    4)所述VH包含SEQ ID NO:19所示的氨基酸序列，且所述VL包含SEQ ID NO:20所示的氨基酸序列；以及
    5)所述VH包含SEQ ID NO:19所示的氨基酸序列，且所述VL包含SEQ ID NO:24所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区。
  33. 根据权利要求32所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG重链恒定区。
  34. 根据权利要求32-33中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区源自人IgG1重链恒定区。
  35. 根据权利要求1-34中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区。
  36. 根据权利要求35所述的分离的抗原结合蛋白，其中所述抗体轻链恒定区源自人Igκ恒定区。
  37. 根据权利要求1-36中任一项所述的分离的抗原结合蛋白，其包含抗体或其抗原结合片段。
  38. 根据权利要求37所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
  39. 根据权利要求37-38中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组中的 一种或多种：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  40. 根据权利要求1-39中任一项所述的分离的抗原结合蛋白，其中，在FACS中验证，其基本上不与参比抗体竞争结合CLDN18.2，其中所述参比抗体包含重链可变区(VH)和轻链可变区(VL)，所述参比抗体的VH包含SEQ ID NO:55所示的氨基酸序列，所述参比抗体的VL包含SEQ ID NO:56所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的分离的抗原结合蛋白，其能够特异性结合CLDN18.2，且基本上不结合CLDN18.1。
  42. 根据权利要求40-41中任一项所述的分离的抗原结合蛋白，其中所述CLDN18.2包含小鼠CLDN18.2、食蟹猴CLDN18.2和/或人CLDN18.2。
  43. 根据权利要求1-42中任一项所述的分离的抗原结合蛋白，其具有CDC活性。
  44. 根据权利要求1-43中任一项所述的分离的抗原结合蛋白，其能够抑制肿瘤生长和/或肿瘤细胞的增殖。
  45. 嵌合抗原受体，其包含靶向部分，所述靶向部分包含权利要求1-44中任一项所述的抗原结合蛋白。
  46. 根据权利要求45所述的嵌合抗原受体，其包含共刺激域，所述共刺激域包含源自选自下组中的一种或多种蛋白的共刺激域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  47. 根据权利要求45-46中任一项所述的嵌合抗原受体，其中所述共刺激域为源自4-1BB的胞内共刺激信号区域。
  48. 根据权利要求46-47中任一项所述的嵌合抗原受体，其中所述共刺激域包含SEQ ID NO:85所示的氨基酸序列。
  49. 根据权利要求45-48中任一项所述的嵌合抗原受体，其包含胞内信号传导域，所述胞内信号传导域包含源自选自下组中的一种或多种蛋白的胞内信号传导域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14 Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
  50. 根据权利要求49所述的嵌合抗原受体，其中所述胞内信号传导域为源自CD3ζ的信号传导域。
  51. 根据权利要求49-50中任一项所述的嵌合抗原受体，其中所述胞内信号传导域包含SEQ ID NO:86所示的氨基酸序列。
  52. 根据权利要求45-51中任一项所述的嵌合抗原受体，其包含跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  53. 根据权利要求52所述的嵌合抗原受体，其中所述跨膜区为源自CD8的跨膜区。
  54. 根据权利要求52-53中任一项所述的嵌合抗原受体，其中所述跨膜区包含SEQ ID NO:84所示的氨基酸序列。
  55. 根据权利要求45-54中任一项所述的嵌合抗原受体，其在靶向部分和跨膜区之间包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  56. 根据权利要求55所述的嵌合抗原受体，其中所述铰链区为源自CD8的铰链区。
  57. 根据权利要求55-56中任一项所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:83所示的氨基酸序列。
  58. 根据权利要求45-57中任一项所述的嵌合抗原受体，其还包含信号肽。
  59. 根据权利要求58所述的嵌合抗原受体，其中所述信号肽源自CD8蛋白的信号肽。
  60. 根据权利要求58-59中任一项所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:88所示的氨基酸序列。
  61. 根据权利要求45-60中任一项所述的嵌合抗原受体，其还包含低密度脂蛋白受体相关蛋白或其片段。
  62. 根据权利要求61所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  63. 根据权利要求61-62中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:91所示的氨基酸序列。
  64. 多肽分子，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白或权利要求45-63中任一项所述的嵌合抗原受体。
  65. 根据权利要求64所述的多肽分子，其包含融合蛋白。
  66. 免疫缀合物，其包含权利要求1-46中任一项所述的分离的抗原结合蛋白。
  67. 分离的一种或多种核酸分子，其编码权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45-63中任一项所述的嵌合抗原受体或权利要求64-65中任一项所述的多肽分子。
  68. 根据权利要求67所述的核酸分子，其包含SEQ ID NO:26、SEQ ID NO:27、SEQ IDNO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32和SEQ IDNO:33中任一项所示的核苷酸序列。
  69. 根据权利要求67-68中任一项所述的核酸分子，其包含选自下述的任一组核苷酸序列：
    1)SEQ ID NO:26所示的核苷酸序列，以及SEQ ID NO:27所示的核苷酸序列；
    2)SEQ ID NO:30所示的核苷酸序列，以及SEQ ID NO:27所示的核苷酸序列；
    3)SEQ ID NO:28所示的核苷酸序列，以及SEQ ID NO:29所示的核苷酸序列；
    4)SEQ ID NO:31所示的核苷酸序列，以及SEQ ID NO:32所示的核苷酸序列；以及
    5)SEQ ID NO:31所示的核苷酸序列，以及SEQ ID NO:33所示的核苷酸序列。
  70. 载体，其包含权利要求67-69中任一项所述核酸分子。
  71. 细胞，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45-63中任一项所述的嵌合抗原受体、权利要求64-65中任一项所述的多肽分子、权利要求67-69中任一项所述的核酸分子或权利要求70所述的载体。
  72. 根据权利要求71所述的细胞，其为免疫效应细胞。
  73. 根据权利要求71-72中任一项所述的细胞，其包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
  74. 根据权利要求71-73中任一项所述的细胞，其为T细胞。
  75. 根据权利要求71-74中任一项所述的细胞，其还包含和/或表达低密度脂蛋白受体相关蛋白或其片段。
  76. 根据权利要求75所述的细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  77. 根据权利要求75-76中任一项所述的细胞，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白5和/或6或其片段。
  78. 根据权利要求75-77中任一项所述的细胞，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:91所示的氨基酸序列。
  79. 药物组合物，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45-63中任一项所述的嵌合抗原受体、权利要求64-65中任一项所述的多肽分子、权利要求66所述的免疫缀合物、权利要求67-69中任一项所述的核酸分子、权利要求70所述的载体和/或权利要求71-78中任一项所述的细胞，以及任选地药学上可接受的载剂。
  80. 制备权利要求1-44中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得所述抗原结合蛋白表达的条件下，培养权利要求71所述的细胞。
  81. 权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45-63中任一项所述的嵌合抗原受体、权利要求64-65中任一项所述的多肽分子、权利要求66所述的免疫缀合物、权利要求67-69中任一项所述的核酸分子、权利要求70所述的载体、权利要求71-78中任一项所述的细胞和/或权利要求79所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病和/或病症。
  82. 根据权利要求81所述的用途，其中所述疾病和/或病症包括癌症。
  83. 根据权利要求81-82中任一项所述的用途，其中所述癌症包括实体瘤和/或血液瘤。
  84. 根据权利要求82-83中任一项所述的用途，其中所述癌症包含胃癌和/或结肠癌。
  85. 检测样品中CLDN18.2的方法，所述方法包括施用权利要求1-44中任一项所述的分离的抗原结合蛋白权利要求45-63中任一项所述的嵌合抗原受体、权利要求64-65中任一项所述的多肽分子、权利要求66所述的免疫缀合物、权利要求67-69中任一项所述的核酸分子、权利要求70所述的载体、权利要求71-78中任一项所述的细胞和/或权利要求79所述的药物组合物。
  86. 检测样品中CLDN18.2的试剂或试剂盒，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白权利要求45-63中任一项所述的嵌合抗原受体、权利要求64-65中任一项所述的多肽分子、权利要求66所述的免疫缀合物、权利要求67-69中任一项所述的核酸分子、权利要求70所述的载体、权利要求71-78中任一项所述的细胞和/或权利要求79所述的药物组合物。
  87. 权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45-63中任一项所述的嵌合抗原受体、权利要求64-65中任一项所述的多肽分子、权利要求66所述的免疫缀合物、权利要求67-69中任一项所述的核酸分子、权利要求70所述的载体、权利要求71-78中任一项所述的细胞和/或权利要求79所述的药物组合物在制备试剂盒中的用途，所述试剂盒用于检测样品中CLDN18.2的存在和/或含量。
PCT/CN2022/084922 2021-04-02 2022-04-01 Cldn18.2抗原结合蛋白及其用途 Ceased WO2022206975A1 (zh)

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CN117777306A (zh) * 2023-07-04 2024-03-29 深圳豪石生物科技有限公司 一种靶向cldn18.2的增强型嵌合抗原受体及其用途

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