WO2021008463A1 - Cldn18.2抗体及其用途 - Google Patents

Cldn18.2抗体及其用途 Download PDF

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WO2021008463A1
WO2021008463A1 PCT/CN2020/101383 CN2020101383W WO2021008463A1 WO 2021008463 A1 WO2021008463 A1 WO 2021008463A1 CN 2020101383 W CN2020101383 W CN 2020101383W WO 2021008463 A1 WO2021008463 A1 WO 2021008463A1
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antibody
seq
antigen
binding fragment
amino acid
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PCT/CN2020/101383
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English (en)
French (fr)
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靳照宇
李芸
李锋
霍耐凡
金秀梅
王飞
陈江涛
任丽
闫哲贤
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明济生物制药（北京）有限公司
北京明济生物科技有限公司
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Priority to KR1020227003966A priority Critical patent/KR20220032077A/ko
Priority to CN202211067908.5A priority patent/CN116063504A/zh
Priority to CA3147122A priority patent/CA3147122A1/en
Priority to EP20840176.0A priority patent/EP3992209A4/en
Priority to US17/626,757 priority patent/US20220235129A1/en
Priority to JP2022528357A priority patent/JP7440122B2/ja
Priority to AU2020314119A priority patent/AU2020314119A1/en
Priority to CN202211071490.5A priority patent/CN116041512A/zh
Priority to CN202080046257.4A priority patent/CN114026125B/zh
Publication of WO2021008463A1 publication Critical patent/WO2021008463A1/zh

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Definitions

  • the present disclosure relates to the field of immunotherapy. Specifically, the present disclosure relates to antibodies that bind to CLDN 18.2 and their defucosylated forms, and their applications in the treatment of diseases, particularly cancer.
  • Upper gastrointestinal tumors including gastric cancer and esophageal cancer, are a common malignant tumor with poor prognosis worldwide.
  • Gastric cancer is particularly high in China. Nearly 42% of new cases of gastric cancer worldwide are concentrated in China. Due to the lack of early diagnosis, about 80% of patients with gastric cancer are already at an advanced stage. As gastric cancer is not sensitive enough to conventional chemotherapy drugs, the five-year survival rate of advanced gastric cancer is extremely low, and it has become the third leading cause of death in China. Therefore, research in recent years is devoted to finding specific targeted therapy for gastric cancer.
  • Claudin family proteins are the main components of tight junction structures widely distributed in epithelial cells. Similar in structure to other occluded protein family proteins, CLDN18.2 is a membrane protein with a molecular weight of about 27.8kd, four transmembrane regions, and two shorter extracellular regions EC1 and EC2. Different from other occluded protein family proteins, CLDN18.2 is very specifically expressed only in the highly differentiated epithelial cells of the stomach in normal tissues (Tureci O et al., Gene, 2011). Among tumors derived from gastric epithelial cells, a high percentage of tumor cells express CLDN 18.2 on their surface.
  • High levels of CLDN18.2 expression can also be detected in the lymph nodes and other tissue metastatic tumors of gastric cancer cells (Woll S. et al., Int. J. Cancer, 2013).
  • gastric cancer there are also a high percentage of tumor cells such as cholangiocarcinoma, esophageal cancer and pancreatic cancer that express CLDN 18.2.
  • As a specific surface marker, CLDN18.2 has become a potential target for cancer treatment.
  • the development of high-efficiency antibody drugs against CLDN18.2 will provide the possibility for the treatment of a variety of cancers, with huge application potential and market value.
  • ADCC effect antibody-dependent cell-mediated cytotoxicity
  • Fab antigen recognition segment
  • Fc constant region
  • the binding power of the antibody to the antigen, the abundance of antigen expression and the binding power of the FC region of the antibody and the FC receptor on the surface of the effector cell can all affect the strength of the ADCC effect, which in turn affects the therapeutic effect of cancer (Jefferis and Lund, 2002 ).
  • the present disclosure provides new antibodies against CLDN 18.2.
  • the aforementioned antibodies can bind to CLDN 18.2 with high affinity and specificity, and mediate the killing of effector cells on target cells expressing CLDN 18.2 (for example, tumor cells).
  • target cells expressing CLDN 18.2 for example, tumor cells.
  • a defucosylated form of the above-mentioned antibody was also generated, which can better bind to Fc ⁇ RIIa and induce ADCC effects than ordinary antibodies.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to CLDN 18.2, the antibody having the following heavy chain CDRs (CDRH) and light chain CDRs (CDRL):
  • the antibody has the following CDRH and CDRL:
  • the antibody has the following VH and VL:
  • VH comprising the amino acid sequence of SEQ ID NO: 1 and VL comprising the amino acid sequence of SEQ ID NO: 6;
  • VH comprising the amino acid sequence of SEQ ID NO: 11
  • VL comprising the amino acid sequence of SEQ ID NO: 16
  • VH comprising the amino acid sequence of SEQ ID NO: 21, and VL comprising the amino acid sequence of SEQ ID NO: 26;
  • VH comprising the amino acid sequence of SEQ ID NO: 31
  • VL comprising the amino acid sequence of SEQ ID NO: 36;
  • VH comprising the amino acid sequence of SEQ ID NO: 41
  • VL comprising the amino acid sequence of SEQ ID NO: 46
  • VH comprising the amino acid sequence of SEQ ID NO: 55
  • VL comprising the amino acid sequence of SEQ ID NO: 60.
  • the antibody may have a heavy chain constant region sequence as shown in SEQ ID NO: 51 and/or a light chain constant as shown in SEQ ID NO: 53 District sequence.
  • the antibodies have an Fc region.
  • the antibody may have a glycosyl structural modification at Asn297, where numbering is based on the EU numbering system.
  • the proportion of the glycosyl structure having fucose in the glycosyl structure is 50% or less.
  • the proportion of the glycosyl structure with fucose in the glycosyl structure is not higher than 30%, such as not higher than 20%, not higher than 10%, not higher than 5%, not higher Less than 2% or not higher than 1%.
  • the proportion of the fucose-containing glycosyl structure in the antibody is 0%-1%.
  • the antibodies are produced by Fut8 knockout cells.
  • the cell may be selected from CHO cells and HEK293 cells.
  • the antibodies have increased Fc ⁇ RIIIa binding activity compared to antibodies produced in cells without Fut8 gene knockout. In some embodiments, the antibody has increased ADCC activity compared to an antibody produced in a cell that does not have a Fut8 gene knockout.
  • the antibodies may be monoclonal antibodies. In other embodiments, the antibody may be a bispecific antibody or a multispecific antibody.
  • the antibodies may be selected from IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibody may be selected from IgG1, IgG2, IgG3, and IgG4 subtypes.
  • the antigen-binding fragment may be selected from Fab fragment, Fab' fragment, F(ab') 2 fragment, Fd fragment, Fd' fragment, Fv fragment, scFv Fragments, ds-scFv fragments, dAb fragments, single chain fragments, bivalent antibodies and linear antibodies.
  • the present disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the present disclosure or an antigen-binding fragment thereof.
  • the present disclosure relates to a vector comprising the nucleic acid molecule of the present disclosure.
  • the present disclosure relates to a host cell comprising the nucleic acid molecule or vector of the present disclosure.
  • the present disclosure relates to a conjugate comprising any antibody or antigen-binding fragment thereof of the present disclosure conjugated to a therapeutic agent, diagnostic agent, or imaging agent.
  • the present disclosure relates to a composition
  • a composition comprising the antibody of the present disclosure or its antigen-binding fragment or conjugate, and one or more pharmaceutically acceptable carriers, excipients and/ Or thinner.
  • the composition further comprises one or more additional therapeutic agents.
  • the therapeutic agent may be selected from antibodies, chemotherapeutics, and small molecule drugs.
  • the chemotherapeutic drug may be selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil (5-FU).
  • the present disclosure relates to a method for treating a disease associated with the expression of CLDN18.2 in a subject, the method comprising administering to the subject the antibody or antigen-binding fragment or conjugate of the present disclosure Or composition step.
  • the disease is cancer.
  • the cancer may be selected from gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.
  • the method further includes the step of administering to the subject one or more additional therapies, such as cancer therapy.
  • the additional therapy is selected from chemotherapy, radiation therapy, immunotherapy, and surgical treatment.
  • the immunotherapy is selected from the group consisting of therapy directed against immune checkpoint molecules, CAR-T cell therapy, and CAR-NK cell therapy.
  • the chemotherapy is selected from a combination chemotherapy regimen including epirubicin, oxaliplatin, and 5-fluorouracil.
  • the present disclosure relates to the use of the antibodies or antigen-binding fragments, conjugates or compositions thereof of the present disclosure in the treatment of diseases associated with the expression of CLDN 18.2 in a subject.
  • the present disclosure relates to the use of the antibody or antigen-binding fragment, conjugate or composition thereof of the present disclosure in the preparation of a medicament for treating a disease related to the expression of CLDN 18.2 in a subject.
  • the disease is cancer.
  • the cancer may be selected from gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.
  • the present disclosure relates to a polypeptide having an amino acid sequence selected from SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.
  • the present disclosure relates to a nucleic acid molecule having a nucleoside selected from SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56 and 61 Acid sequence.
  • the present disclosure also relates to a vector containing the aforementioned nucleic acid molecule.
  • Figure 1 shows the results of detecting CLDN18.2 expression in CHO, CT26, SNU601 cells and the constructed CHO-CLDN18.2, CT26-CLDN18.2, and SNU601-CLDN18.2 cells by flow cytometry.
  • Figure 2 shows the results of detection of control M13 phage by flow cytometry and the binding of the phage library after the first, second and third rounds of screening to CHO cells and CHO-CLDN18.2 cells.
  • Figure 3 shows the results of binding of phage clones A1, A2, A3, A4, A5 ( Figure 3A) and A6 ( Figure 3B) selected by flow cytometry to CHO cells and CHO-CLDN18.2 cells.
  • Figure 4 shows the detection of different concentrations of recombinant antibodies 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5 (Figure 4A), 18.2-A5F and 18.2-A6F ( Figure 4B) by flow cytometry The result of binding to CT26-CLDN18.2 cells and EC50 value.
  • Figure 5 shows the results of detecting the binding of CLDN18.2 antibody to 293t cells expressing CLDN18.1 (18.1-flag) or CLDN18.2 (18.2-flag) by flow cytometry.
  • Figure 6 shows the results of detection of CLDN18.2 antibody binding to the surface of SNU601 cells expressing CLDN18.2 and endocytosis by cells under incubation conditions of 4°C and 37°C.
  • Figure 7 shows the results of detecting the binding of the antibody CLDN18.2 antibody and the defucosylated antibody to CT26-CLDN18.2 cells by flow cytometry.
  • Figure 8 shows the results of CLDN18.2-A1 and CLDN18.2-A1F binding to Fc ⁇ RIIIa detected by the Biacore method.
  • Figure 8A shows the fitted curve of the antibody binding FcyRIIIa
  • Figure 8B shows the Ka, Kd, KD and tc values of the antibody binding FcyRIIIa.
  • Figure 9 shows the results of detecting the binding of CLDN18.2 antibody and defucosylated antibody to Fc ⁇ RIIIa expressed on the cell surface by flow cytometry.
  • Figure 9A shows the result of antibody binding to FcyRIIIa-Jurkat cells.
  • Figure 9B shows the result of antibody binding to NK92MI cells.
  • Figure 10 shows that the CLDN18.2 antibody and the defucosylated antibody activate Fc ⁇ RIIIa in the presence of SNU601-CLDN18.2 cells ( Figure 10A) or CT26-CLDN18.2 ( Figure 10B) detected by the luciferase reporter system. Results of Fc ⁇ RIIIa receptor in Jurkat cells.
  • Figure 11 shows the results of detecting the expression level of CLDN18.2 on KATOIII cells by flow cytometry.
  • Figure 12 shows that the combination of CLDN18.2 antibody ( Figure 12A) and CLDN18.2 antibody antibody and the chemotherapy drug EOF ( Figure 12B) activates Fc ⁇ RIIIa in the presence of SNU601-CLDN18.2 cells detected by the luciferase reporter system -Results of Fc ⁇ RIIIa receptor in Jurkat cells.
  • Figure 13 shows in the presence of PBMC from healthy human donor 1 (Figure 13A), donor 2 ( Figure 13B) or donor 3 ( Figure 13C), CLDN18.2 antibody and defucosylated antibody mediation The killing effect of the guided ADCC pathway on SNU601-CLDN18.2 target cells.
  • Figure 14 shows the killing effect of CLDN18.2 antibody and defucosylated antibody-mediated CDC pathway on CHO-CLDN18.2 target cells in the presence of complement.
  • Figure 15 shows the results of different doses of CLDN18.2 antibody 18.2-A1F inhibiting tumor growth in a mouse xenograft model of SNU601-CLDN18.2 cells.
  • Figure 16 shows the flow cytometry results of CLDN18.2 highly expressed KATOIII cells (KATOIII-18.2High) used in a xenograft model.
  • Figure 17 shows the results of the combination of CLDN18.2 antibody 18.2-A1F and 18.2-A1F and the chemotherapeutic drug EOF inhibiting tumor growth in a mouse xenograft model of KATOIII-18.2High cells.
  • Antibody antibody, Ab; immunoglobulin: immunoglobulin, Ig;
  • Heavy chain heavy chain, HC
  • light chain light chain, LC
  • Heavy chain variable domain heavy chain variable domain, VH;
  • Heavy chain constant domain heavy chain constant domain, CH;
  • Light chain variable domain light chain variable domain, VL;
  • Light chain constant domain light chain constant domain, CL;
  • Complementarity determining region complementarity determining region, CDR;
  • Fab fragment antigen binding fragment, Fab
  • Fc area fragment crystallizable region, Fc;
  • Monoclonal antibody monoclonal antibody, mAb
  • Antibody-dependent cytotoxicity antibody-dependent cell-mediated cytotoxicity, ADCC;
  • Complement-dependent cytotoxicity complement dependent cytotoxicity, CDC.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to CLDN18.2, the antibody having the following heavy chain CDRs (CDRH) and light chain CDRs (CDRL):
  • binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen.
  • an antibody that specifically binds to a certain antigen refers to an antibody with a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less affinity (K D ) binds the antigen.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • antibody refers to an immunoglobulin molecule that contains at least one antigen recognition site and can specifically bind to an antigen.
  • antigen refers to a substance that can induce an immune response in the body and specifically bind to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the foregoing substances.
  • the binding of antibody and antigen is mediated by the interaction between the two, including hydrogen bond, van der Waals force, ionic bond and hydrophobic bond.
  • the area on the surface of an antigen that binds to an antibody is an "antigenic determinant" or "epitope".
  • each antigen can have multiple epitopes.
  • epitope can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes usually include at least 3, more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • An “epitope” includes a structural unit normally bound by an immunoglobulin VH/VL pair. The epitope defines the smallest binding site of an antibody and therefore represents the specific target of the antibody or antigen-binding fragment thereof.
  • antibody mentioned in the present disclosure is understood in its broadest sense, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and those containing at least two different antigen-binding domains. Multispecific antibodies (e.g., bispecific antibodies). Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. Antibodies may contain additional changes, such as unnatural amino acids, mutations in Fc effector functions, and mutations in glycosylation sites.
  • Antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules containing at least one antigen-binding domain.
  • variable region refers to a pair of light chain and heavy chain domain portions that directly participate in the binding of antibody and antigen.
  • Each VH and VL region consists of three hypervariable regions or complementarity determining regions (CDR) and four framework regions (FR) arranged from N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4.
  • CDR refers to the complementarity determining region within the variable sequence of an antibody.
  • CDR1, CDR2, and CDR3 are three CDRs in each variable region of the heavy chain and light chain.
  • CDR1, CDR2, and CDR3 CDR1, CDR2, and CDR3.
  • the exact boundaries of these CDRs are defined differently according to different systems.
  • the system described by Kabat et al. Kabat et al. (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides clear residue numbers for antibody variable regions
  • the system also provides residue boundaries that define the three CDRs. These CDRs can be called Kabat CDRs.
  • Each complementarity determining region can contain the amino acid residues of the "complementarity determining region" defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that some sub-parts in Kabat CDR adopt almost the same peptide skeleton image, although There is diversity at the level of amino acid sequence. These sub-parts are called L1, L2 and L3 or H1, H2 and H3, respectively, where "L” and “H” represent light chain and heavy chain regions, respectively. These regions can be called Chothia CDR It has a boundary that overlaps with Kabat CDR.
  • CDRH1, CDRH2, and CDRH3 in the VH shown in SEQ ID NO:1 have SEQ ID NO: 3 (SSWLI), SEQ ID NO: 4 (TIVPSDSYTNYNQKFKD) and SEQ ID, respectively
  • the amino acid sequence of NO: 5 FRTGNSFDY
  • CDRL1, CDRL2, and CDRL3 in VL have SEQ ID NO: 8 (KSSQSVLNSGNQKNYLT), SEQ ID NO: 9 (WAVARQS) and SEQ ID NO: 10 (QNSIAYPFT) amino acid sequence;
  • CDRH1, CDRH2, and CDRH3 in the VH have the amino acid sequences of SEQ ID NO: 13 (SFWVG), SEQ ID NO: 14 (NVSPSDSYTNYNQKFKD) and SEQ ID NO: 15 (LSSGNSFDY), respectively,
  • CDRL1, CDRL2, and CDRL3 in the VL shown in SEQ ID NO: 16 have the amino acid sequences of SEQ ID NO: 18 (KSSQSVLNSGNQKNYLT), SEQ ID NO: 19 (WSSTKQS) and SEQ ID NO: 20 (QNAFSFPFT), respectively ;
  • CDRH1, CDRH2 and CDRH3 in the VH have the amino acid sequences of SEQ ID NO: 23 (SYWLN), SEQ ID NO: 24 (SMYPSDSYTNYNQKFKD) and SEQ ID NO: 25 (FSRGNSFDY), respectively,
  • SEQ ID NO: 26 CDRL1, CDRL2, and CDRL3 in the VL respectively have the amino acid sequence of SEQ ID NO: 28 (KSSQSLLESGNQKNYLT), SEQ ID NO: 29 (WSWAKNS) and SEQ ID NO: 30 (QNAYAFPFT) ;
  • CDRH1, CDRH2 and CDRH3 in the VH have the amino acid sequences of SEQ ID NO: 33 (SFWIS), SEQ ID NO: 34 (NILPSDSYTNYNQKFKD) and SEQ ID NO: 35 (YWRGNSFDY), respectively, And as shown in SEQ ID NO: 36, CDRL1, CDRL2, and CDRL3 in the VL respectively have the amino acid sequence of SEQ ID NO: 38 (KSSQSIINSGNQKNYLT), SEQ ID NO: 39 (WGGTRHS) and SEQ ID NO: 40 (QNGYYSPFT) ;
  • CDRH1, CDRH2, and CDRH3 in the VH have the amino acid sequences of SEQ ID NO: 43 (SSWVG), SEQ ID NO: 44 (NSYPSDSYTNYNQKFKD) and SEQ ID NO: 45 (LGRGNSFDY), respectively,
  • SEQ ID NO: 46, CDRL1, CDRL2, and CDRL3 in VL have the amino acid sequences of SEQ ID NO: 48 (KSSQSLIHSGNQKNYLT), SEQ ID NO: 49 (WGLSKNS) and SEQ ID NO: 50 (QNSIYYPFT), respectively ;
  • CDRH1, CDRH2, and CDRH3 in the VH have the amino acid sequences of SEQ ID NO: 57 (SYWLG), SEQ ID NO: 58 (IIYPSDSYTNYNQKFKD) and SEQ ID NO: 59 (FWRGNSFDY), respectively,
  • SEQ ID NO: 60, CDRL1, CDRL2, and CDRL3 in VL have the amino acid sequences of SEQ ID NO: 62 (KSSQSLLESGNQKNYLT), SEQ ID NO: 63 (WAAGKES) and SEQ ID NO: 64 (QNGYSHPFT), respectively .
  • the antibody has the following CDRH and CDRL:
  • the antibody has the following VH and VL:
  • VH comprising the amino acid sequence of SEQ ID NO: 1 and VL comprising the amino acid sequence of SEQ ID NO: 6;
  • VH comprising the amino acid sequence of SEQ ID NO: 11
  • VL comprising the amino acid sequence of SEQ ID NO: 16
  • VH comprising the amino acid sequence of SEQ ID NO: 21, and VL comprising the amino acid sequence of SEQ ID NO: 26;
  • VH comprising the amino acid sequence of SEQ ID NO: 31
  • VL comprising the amino acid sequence of SEQ ID NO: 36;
  • VH comprising the amino acid sequence of SEQ ID NO: 41
  • VL comprising the amino acid sequence of SEQ ID NO: 46
  • VH comprising the amino acid sequence of SEQ ID NO: 55
  • VL comprising the amino acid sequence of SEQ ID NO: 60.
  • the antibody has the following VH and VL:
  • VH comprising an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 1
  • VL comprising an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 6;
  • VH containing an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 11
  • VL containing an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 16
  • VH containing an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 21, and a VL containing an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 26;
  • VH comprising an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 31, and a VL comprising an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 36;
  • VH containing an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 41
  • VL containing an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 46
  • a VH comprising an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 55
  • a VL comprising an amino acid sequence with one or more amino acid modifications in SEQ ID NO: 60.
  • the amino acid modification does not change the CDR sequence of the antibody, that is, the amino acid modification is performed in the framework region (FR) of the variable region.
  • the one or several amino acid modifications refer to 1-10 amino acid modifications or 1-5 amino acid modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Amino acid modifications.
  • the amino acid modification is selected from substitution, deletion, addition and/or insertion of amino acid residues. In some embodiments, the amino acid modification is an amino acid substitution, such as a conservative substitution.
  • the antibody has the following VH and VL:
  • VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and comprising a VH with SEQ ID
  • the VL of the amino acid sequence of NO: 6 having at least 80% sequence identity, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 11, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and containing VH with SEQ ID
  • the VL of the amino acid sequence of NO:16 has at least 80% sequence identity, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 21, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and comprising a VH with SEQ ID
  • the VL of the amino acid sequence of NO:26 has at least 80% sequence identity, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • a VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 31, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and comprising a VH with SEQ ID
  • the VL of the amino acid sequence of NO:36 has at least 80% sequence identity, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;
  • VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 41, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and containing VH with SEQ ID
  • the VL of the amino acid sequence of NO:46 has at least 80% sequence identity, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; or
  • VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 55, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and containing VH with SEQ ID
  • the amino acid sequence of NO:60 has a VL with an amino acid sequence of at least 80% sequence identity, for example, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • sequence identity As understood by those skilled in the art, the correlation between two amino acid sequences or between two nucleotide sequences can be described by the parameter "sequence identity".
  • sequence identity The percentage of sequence identity between two sequences can be determined, for example, by using a mathematical algorithm.
  • sequence identity between two sequences can be determined by, for example, using a mathematical algorithm.
  • Non-limiting examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smith et al.
  • Such programs include, but are not limited to, the PC/Gene program CLUSTAL, the ALIGN program (Version 2.0), and the Wisconsin genetics software package GAP, BESTFIT, BLAST, FASTA, and TFASTA.
  • the alignment using these programs can be implemented, for example, by using initial parameters.
  • the antibody may have a heavy chain constant region sequence as shown in SEQ ID NO: 51 and/or a light chain constant as shown in SEQ ID NO: 53 District sequence.
  • the antibodies have an Fc region.
  • the antibody has a glycosyl structural modification at Asn297, where numbering is based on the Eu numbering system.
  • “Asn297” according to the present disclosure means asparagine located at position 297 of the Fc region of an antibody according to the Eu numbering system. Based on the subtle sequence changes of specific antibodies, Asn297 can also be located a few amino acids upstream or downstream of position 297.
  • the term "Fc region of an antibody” or "human immunoglobulin Fc region” includes antibody constant region polypeptides other than heavy chain constant region 1 (CH1), such as human immunoglobulin IgA, IgD, IgG heavy chain
  • CH1 heavy chain constant region 1
  • the boundaries of the Fc region can vary, the Fc region of a human IgG heavy chain is generally defined as containing residues starting from A231 to its carboxy terminus.
  • the Fc region of immunoglobulin is the functional domain for antibodies to exert immune effects.
  • the Fc of an IgG antibody can interact with a variety of receptors, the most important of which is the Fc ⁇ receptor (Fc ⁇ R) family.
  • Fc ⁇ R Fc ⁇ receptor
  • One of the main mechanisms of therapeutic monoclonal antibody killing tumor cells is the ADCC effect (antibody-dependent cell-mediated cytotoxicity).
  • the Fc region of the antibody is used to bind to killer cells expressing Fc ⁇ R and activate the cell killing activity of effector cells, thereby secreting cytotoxic mediators, including granzymes Perforin, etc., eventually lead to the lysis and destruction of target cells (such as tumor cells).
  • Fc can also bind to the complement protein C1q, resulting in a CDC effect (complement dependent cytotoxicity, CDC).
  • the glycosylation modification level and form of the Fc region of an antibody can affect the binding ability of the Fc region and its receptor Fc ⁇ R, thereby affecting the strength of the antibody's ADCC effect.
  • the glycosylation modification of the Fc region of an antibody as used herein generally refers to the glycosylation modification at Asn297. During the production of the antibody, it undergoes glycosylation in the cell's ER and Golgi network. In the related research of antibody glycosylation, it is found that reducing the fucosylation level of the antibody helps to increase the binding of the Fc region to Fc ⁇ RIIIa, thereby enhancing the ADCC effect of the antibody. Therefore, changing the glycosylation metabolic pathway of antibody-expressing cells can effectively change the fucose level of the expressed antibody, thereby regulating the antibody-mediated ADCC effect (Jefferis R. 2009, NAT. REV. Drug. DISC).
  • the antibodies have reduced fucosylation levels.
  • the proportion of the glycosyl structure having fucose in the glycosyl structure at Asn297, is 60% or less, such as 50% or less For example, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less.
  • the proportion of the glycosyl structure with fucose is 0%-10%, for example, 0%-5%, 0%-2%, or 0%-1%.
  • the proportion of the glycosyl structure having fucose is 0.1% or less. In other embodiments, there is no detectable fucose in the glycosyl structure at Asn297.
  • antibodies can be expressed in cells that are defective or lack the ability to fucosylate.
  • cell lines with Fut8 gene knockout can be used to produce antibodies with reduced fucosylation levels.
  • antibodies or antigen-binding fragments with reduced or no-fucose content can be produced by, for example: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) translation After removal of fucose (for example, with fucosidase); (iii) post-translational addition of desired carbohydrates, for example after recombinant expression of non-glycosylated glycoprotein; or (iv) purification of glycoprotein to choose not to be Fucosylated antibodies or antigen-binding fragments thereof.
  • the antibodies are produced by Fut8 gene knockout cells to obtain antibodies with reduced fucosylation levels.
  • the cells are Chinese Hamster Ovary (CHO) cells, such as CHO-K1 cells, CHOS cells, or other CHO-derived cells.
  • the cell is a Fut8 knockout CHO cell.
  • the cells are human embryonic kidney (HEK) 293 cells, such as HEK293, HEK293A, HEK293T, HEK293F, or other HEK293-derived cells.
  • HEK human embryonic kidney
  • the cell is a Fut8 knockout HEK 293 cell.
  • the antibodies are produced by Fut8 gene knockout cells, and wherein the antibodies are produced in cells without Fut8 gene knockout, for example, the same sequence
  • the control antibody has increased Fc ⁇ RIIIa binding activity.
  • antibodies produced by Fut8 gene knockout cells have an increased ability to induce Fc ⁇ RIIIa activity compared to antibodies produced in cells that do not have Fut8 gene knockout, such as a control antibody of the same sequence.
  • the EC50 value of an antibody produced by Fut8 knockout cells that induces Fc ⁇ RIIIa activity is increased by at least 2-fold, for example, at least 5-fold, compared to antibodies produced in cells without Fut8 knockout Or at least 10 times, for example 10-20 times.
  • the term "EC50" refers to the concentration of antibody that causes 50% of the maximum effect.
  • the antibodies are produced by Fut8 gene knockout cells, and wherein the antibodies are produced in cells without Fut8 gene knockout, for example, the same sequence
  • the control antibody has increased ADCC activity.
  • the ADCC activity of antibodies produced by Fut8 knockout cells is increased by at least 2-fold, for example, at least 5-fold or at least 10 times, compared to antibodies produced in cells without Fut8 knockout. Times, for example 10-20 times.
  • the antibodies are monoclonal antibodies.
  • the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is the same, except for possible naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations which usually include different antibodies directed against different determinants (epitopes), each antibody in a monoclonal preparation is directed against the same single determinant on the antigen. As used herein, the term “monoclonal antibody” is not limited to antibodies produced by hybridoma technology, and the modifier "monoclonal antibody” should not be interpreted as requiring the production of antibodies by any specific method.
  • the antibodies are bispecific antibodies or multispecific antibodies.
  • the bispecific antibody or multispecific antibody has a first antigen binding region that binds to an epitope on CLDN 18.2, and a second antigen binding region that binds to another epitope, wherein the first antigen binds
  • the region has CDRH1, CDRH2, and CDRH3, CDRL1, CDRL2, and CDRL3; or VH and VL sequences of the antibody or antigen-binding fragment thereof as described in the present disclosure, and the second antigen-binding region binds to the first antigen-binding region Different epitopes.
  • the second antigen binding region binds to another antigen binding epitope on the CLDN18.2 molecule. In other embodiments, the second antigen binding region binds another antigen. In some embodiments, the another antigen is selected from tumor-associated antigens and immune checkpoint molecules.
  • tumor-associated antigen refers to an antigen that is differentially expressed by cancer cells and therefore can be used to target cancer cells.
  • Tumor-associated antigens are antigens that can potentially stimulate obvious tumor-specific immune responses. Some of these antigens are encoded by normal cells, but not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (ie not expressed) in normal cells, those that are expressed only during certain stages of differentiation, and those that are expressed over time, such as embryonic and fetal antigens.
  • cancer antigens are encoded by mutant cell genes such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations.
  • cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and/or commercialized, and can also be produced by those skilled in the art.
  • Immune checkpoint protein receptors and their ligands mediate the suppression of T cell-mediated cytotoxicity, and are usually expressed on tumors or non-reactive T cells in the tumor microenvironment. And allow the tumor to evade immune attack. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow the tumor's cytotoxic T cell attack.
  • immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103.
  • the modulation (including inhibition) of the activity of such proteins can be accomplished by immune checkpoint modulators, which can include, for example, antibodies, aptamers, small molecules, and soluble forms of checkpoint receptor proteins that target checkpoint proteins.
  • immune checkpoint modulators can include, for example, antibodies, aptamers, small molecules, and soluble forms of checkpoint receptor proteins that target checkpoint proteins.
  • Antibodies specific for PD-1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103 are known and/or commercialized, and can also be produced by those skilled in the art.
  • immunoglobulins can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, which can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.
  • the amino acid sequence of the light chain the light chain can be classified as a lambda chain or a kappa chain.
  • the antibodies of the present disclosure can be of any of the aforementioned classes or subclasses.
  • the antibodies of the present disclosure are selected from IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibodies of the present disclosure are IgG, for example selected from IgG1, IgG2, IgG3, and IgG4 subtypes.
  • the term "antigen-binding fragment” includes but is not limited to: Fab fragments, which have VL, CL, VH and CH1 domains; Fab' fragments, which have one or more cysteine at the C-terminus of CH1 domains Fab fragments of residues; Fd fragments, which have VH and CH1 domains; Fd' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of CH1 domains; Fv fragments and scFv, which VL and VH domains with a single arm of an antibody; dAb fragments, which are composed of VH domains or VL domains; isolated CDR regions; F(ab') 2 fragments, which contain two disulfide bridges connected at the hinge region A bivalent fragment of a Fab'fragment; a single-chain antibody molecule (such as a single-chain Fv; scFv); a "diabody" with two antigen-bind
  • the antigen-binding fragment is selected from Fab fragment, Fab' fragment, F(ab') 2 fragment, Fd fragment, Fd' fragment, Fv fragment, scFv fragment , Ds-scFv fragments, dAb fragments, single chain fragments, bivalent antibodies and linear antibodies.
  • the present disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the present disclosure or an antigen-binding fragment thereof.
  • the present disclosure also relates to a vector comprising the nucleic acid molecule of the present disclosure.
  • Vector refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and then the genetic material elements it carries can be expressed in the host cell.
  • the vector is recognized by those skilled in the art, including but not limited to: (1) plasmid; (2) phagemid; (3) cosmid; (4) artificial chromosome, such as yeast artificial chromosome, bacterial artificial chromosome or P1 source (5) bacteriophage such as lambda bacteriophage or M13 bacteriophage and (6) animal virus, such as retrovirus, adenovirus, adeno-associated virus, sporangia virus, poxvirus, baculovirus.
  • a vector can contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes; in addition, the vector can also contain replication initiation sites.
  • the present disclosure relates to a host cell comprising the nucleic acid molecule or vector of the present disclosure.
  • the host cell is a CHO cell, such as a CHO-K1 cell, CHOS cell, or other CHO-derived cell.
  • the host cell is a HEK293 cell, such as HEK293, HEK293A, HEK293T, HEK293F or other HEK293-derived cells.
  • the present disclosure relates to a conjugate comprising the antibody of the present disclosure or an antigen-binding portion thereof conjugated to a therapeutic agent, diagnostic agent, or imaging agent.
  • the therapeutic agent may be selected from cytotoxins and radioisotopes.
  • the diagnostic agent or imaging agent may be selected from fluorescent markers, luminescent substances, chromogenic substances, and enzymes.
  • the present disclosure relates to a composition
  • a composition comprising the antibody or antigen-binding fragment thereof, or conjugate of the present disclosure, and one or more pharmaceutically acceptable carriers, excipients, and / Or thinner.
  • phrases “pharmaceutically acceptable” means that it is suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response or other problems or complications, and reasonable Those compounds, materials, compositions and/or dosage forms that have a commensurate benefit/risk ratio.
  • pharmaceutically acceptable carriers, excipients and/or diluents refers to pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients Agents, solvents, media, encapsulating materials, manufacturing aids or solvent encapsulating materials, which are related to maintaining the stability, solubility or activity of the antibody or antigen-binding fragment thereof of the present disclosure.
  • compositions of the present disclosure can be formulated for administration to a subject in solid, liquid or gel form.
  • the composition of the present disclosure may be formulated for parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, a sterile solution or suspension or a sustained release formulation.
  • the composition further comprises one or more additional therapeutic agents.
  • the additional therapeutic agent is selected from antibodies, chemotherapeutics, and small molecule drugs.
  • the therapeutic agent targets a tumor-associated antigen, such as the tumor-associated antigen described above.
  • the therapeutic agent targets immune checkpoint molecules, for example, immune checkpoint molecules as described above.
  • chemotherapeutic agent refers to any chemical agent that has therapeutic usefulness in treating diseases characterized by abnormal cell growth.
  • Chemotherapeutic agents as used herein include chemical agents and biological agents. These agents function to inhibit the cell activity that cancer cells depend on for continued survival.
  • the categories of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and various anti-new biological drugs.
  • the additional therapeutic agent is a chemotherapeutic agent
  • the chemotherapeutic agent may be wherein the chemotherapeutic agent may be selected from epirubicin (Epirubicin), oxaliplatin (Oxaliplatin) and 5-fluorouracil ( One or more of 5-FU).
  • the present disclosure relates to a method for treating a disease associated with the expression of CLDN18.2 in a subject, the method comprising administering to the subject the antibody or antigen-binding fragment or conjugate of the present disclosure Or composition step.
  • treatment refers to therapeutic treatment in which the purpose is to reverse, reduce, ameliorate, inhibit, slow down, or stop the progression or severity of the condition associated with the disease or disorder.
  • treatment includes reducing or alleviating at least one side effect or symptom of a disease or condition. If one or more symptoms or clinical markers are reduced, the treatment is usually “effective.” Or, if the progression of the disease is reduced or stopped, the treatment is “effective”, that is, “treatment” includes not only the improvement of symptoms, but also the cessation of the progression or worsening of symptoms expected in the absence of treatment, At least slow it down.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, reduction of disease severity, stabilization (ie not worsening) of disease state, delay or slowing down of disease progression, improvement or alleviation of disease state, and remission (whether partial or All), whether detectable or undetectable.
  • the terms “subject”, “patient” and “individual” are used interchangeably herein, and refer to animals, such as humans.
  • the term subject also includes “non-human mammals” such as, for example, rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.
  • the subject is a human subject.
  • the disease is cancer.
  • cancer include, but are not limited to: basal cell carcinoma, cholangiocarcinoma; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; cholangiocarcinoma; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer Endometrial cancer; Esophageal cancer; Eye cancer; Head and neck cancer; Gastric cancer; Glioblastoma; Liver cancer; Kidney cancer; Laryngeal cancer; Leukemia; Liver cancer; Lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung gland Cancer and lung squamous cell carcinoma); lymphoma, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retina Blastoma; Rhabdomyo
  • the method further includes the step of administering one or more additional therapies.
  • the therapy is selected from chemotherapy, radiation therapy, immunotherapy, and surgical therapy.
  • the immunotherapy is selected from the group consisting of therapy directed against immune checkpoint molecules, CAR-T cell therapy, and CAR-NK cell therapy.
  • the immune checkpoint molecule may be selected from PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT and CD103.
  • the chemotherapy is selected from a combination chemotherapy regimen including epirubicin, oxaliplatin, and 5-fluorouracil.
  • the present disclosure relates to the use of the antibodies or antigen-binding fragments, conjugates or compositions thereof of the present disclosure in the treatment of diseases associated with the expression of CLDN 18.2 in a subject.
  • the present disclosure relates to the use of the antibody or antigen-binding fragment, conjugate or composition thereof of the present disclosure in the preparation of a medicament for treating a disease related to the expression of CLDN 18.2 in a subject.
  • the disease is cancer, such as the type of cancer described above.
  • the cancer is selected from gastric cancer, cholangiocarcinoma, esophageal cancer and pancreatic cancer.
  • the subject is a human.
  • the present disclosure relates to a polypeptide having an amino acid sequence selected from SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.
  • the present disclosure relates to a nucleic acid molecule having a nucleoside selected from SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56 and 61 Acid sequence.
  • the present disclosure also relates to a vector containing the aforementioned nucleic acid molecule.
  • antibody drugs are currently mainly achieved by humanizing antibodies derived from other species or directly screening fully human antibodies through transgenic animals and in vitro screening technology, which further reduces the immunogenicity of antibodies in humans and reduces the corresponding side effects , And at the same time improve the druggability, is an important trend in the development of antibody drugs.
  • Phage display technology uses molecular biology to insert exogenous gene fragments into phage specific proteins such as the gene of gIII, and express the protein or polypeptide encoded by the exogenous gene through phage, maintaining the relative spatial structure and biology of the recombinant fusion protein Active and displayed on the surface of phage.
  • the constructed diverse phage library was incubated with the target protein, and non-target protein-binding phage strains were removed by biological panning.
  • the phage pool is large enough, high-affinity and high-specificity phage clones can be obtained through multiple rounds of collection, amplification and enrichment.
  • the protein sequences encoded by these phage clones can be identified by gene sequencing for further research.
  • the phage antibody library is a diverse phage library formed by phage display of antibody genes.
  • the quality of the phage antibody library is mainly determined by the capacity and diversity of the library.
  • the phage antibody library needs to be as large as possible under the premise of ensuring diversity.
  • the number of antibody fragments displayed on the phage surface represents the size of the pool, and the diversity of displayed fragments represents the diversity of the antibody library.
  • the larger the storage capacity of the phage antibody library the higher the affinity of the antibodies obtained by screening.
  • RNA was reverse transcribed to synthesize single-stranded cDNA, and then the VH, V ⁇ and V ⁇ genes were amplified using variable region primers for different subgroups of antibodies. After mixing the amplified products according to a certain ratio, the heavy chain and light chain genes were linked into single-chain antibodies (scFv) by PCR and cloned into phage plasmids by double enzyme digestion. The phage plasmid carrying scFv gene was used to electroporate SS320 E. coli competent cells. After SS320 proliferated to the logarithmic phase, add helper phage infection.
  • scFv single-chain antibodies
  • a CHO cell line stably expressing CLDN18.2 (CHO-CLDN18.2) was prepared for cell-based phage display screening.
  • the specific experimental process is as follows: clone the cDNA sequence of human CLDN18.2 into the pCDH lentiviral vector, and then co-transfect it with the lentiviral packaging vector into 293t cells. After culturing for 48 or 72 hours, collect the cell supernatant rich in lentiviral particles and directly infect CHO cells, CT26 cells or SNU601 cells. The cells infected by the lentivirus were screened and cultured with puromycin. After 2-3 weeks, the CLDN18.2 specific antibody (IMAB362, Ganymed) was used for flow cytometry to detect the expression of CLDN18.2 in the cells. 1 shown.
  • IMAB362 IMAB362, Ganymed
  • CHO cells named CHO-CLDN18.2 cell line
  • CLDN18.2 CHO-CLDN18.2 cell line
  • CT26 cells CT26-CLDN18.2
  • SNU601 cells SNU601-CLDN18.2
  • the experimental process mainly refers to the following documents: Targeting membrane proteins for antibody discovery using phage display Jones ML et al. Scientific Reports 2016.
  • the specific process is briefly described as follows: incubate the phage library with CHO-K1 cells, centrifuge and take the supernatant. The supernatant was then incubated with CHO-CLDN18.2 cells to allow phage specific for CLDN18.2 to bind to the cells. After centrifugation, the cell pellet was collected, and the cells were washed and collected. The phage library bound to the cells was then eluted with 75 mM sodium citrate buffer. After neutralizing the phage library, the M13K07 helper phage was used to amplify the 100-fold screened phage library, and then the second and third rounds of screening were performed in a similar manner to the first round of screening.
  • the enrichment of phage is monitored by the initial phage dosage of each round of screening and the phage titer collected after screening, and the phage library and CHO or CHO-CLDN18.2 stable expression cell lines are used for flow cytometric detection.
  • the results shown in Figure 2 show that from the second round of enrichment screening, the phage display library can specifically bind to CHO-CLDN 18.2, and its binding strength increases with the number of screenings.
  • an enriched phage library was obtained. After using the library to infect bacteria, spread it on an agarose plate for culture. Monoclonal colonies are picked and placed in a 96-well deep-well plate with 2YT medium containing ampicillin and kanamycin, shaking culture at 37°C to obtain a supernatant containing monoclonal phage. The monoclonal phage supernatant was incubated with CHO-CLDN18.2 cells or CT26-CLDN18.2 cells at 4°C for 1 hour, and then washed with PBS (flow cytometry buffer) containing 1mM EDTA and 0.5% BSA.
  • PBS flow cytometry buffer
  • PE-labeled anti-M13 antibody purchased from Yiqiao Shenzhou was added and incubated at 4°C for 30 minutes. Subsequently, flow cytometry was used to detect the binding of phage and cells, and the results are shown in Figure 3.
  • the cDNA sequences of the heavy and light chain variable regions of the monoclonal phage were cloned into the pcDNA3.4 vector (Invitrogen) which already contained the antibody constant region, and a total of 6 monoclonal antibody heavy and light chain expression plasmids were obtained.
  • the plasmid was transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged and the supernatant was collected. The supernatant was purified by protein A to obtain purified antibodies.
  • the above 6 monoclonal antibodies were named 18.2-A1 (A1), 18.2-A2 (A2), 18.2-A3 (A3), 18.2-A4 (A4), 18.2-A5 (A5) and 18.2-A6 (A6), respectively .
  • the amino acid sequences and coding sequences of the variable regions of the heavy and light chains of these antibodies are shown in Table 1 and Table 2 below, and the CDR sequences of the antibodies determined by the Kabat CDR system are shown in Table 3.
  • the heavy and light chain constant region sequences of the recombinant antibodies are shown in Table 4.
  • the binding activity of recombinant monoclonal antibodies to CLDN 18.2 was tested. Specifically, a well-growing CT26-CLDN18.2 stably expressing cell line was taken. After the cells were washed with PBS, they were mixed with the antibody diluted in multiples and incubated at 4°C for 1 hour. The cells were washed once with flow cytometry buffer, PE-labeled anti-human IgG antibody was added, and incubated at 4°C for 30 minutes. The supernatant was removed by centrifugation, washed with flow cytometry buffer, and then the fluorescence intensity on the cell surface was measured with a flow cytometer. The average fluorescence intensity was used to calculate the relative binding activity of each antibody.
  • Human Claudin18 protein has two homologous isomers, namely CLDN18.1 and CLDN18.2, these two homologous isomers share other exons except for exon 1, and CLDN18.1 and CLDN18.2 Exon 1 sequence is highly similar, with only 8 different amino acids in the first extracellular region.
  • CLDN18.1 and CLDN18.2 exon 1 are specifically expressed in different tissues driven by different promoters. Among them, CLDN18.1 is specifically expressed in lung epithelial cells, and CLDN18.2 is specifically expressed in gastric epithelial cells. In order to identify the specificity of the CLDN18.2 antibody selected, the binding of the antibody to CLDN18.1 and CLDN18.2 was tested.
  • the supernatant was removed by centrifugation, washed twice with flow cytometry buffer, and the cells were fixed in a fixative containing 1% paraformaldehyde and 0.5% triton-X100 and the cell membrane was permeated. Subsequently, the APC-labeled anti-flag antibody was incubated with the cells for 30 minutes, and the detection was performed with a flow cytometer.
  • the results are shown in Figure 5.
  • the flag-positive cell population can be detected in 293t cells expressing CLDN18.1-flag or CLDN18.2-flag exogenously, indicating that the transfected CLDN18.1 or CLDN18.2 protein is good in the cells Expression, the positive rate of expression is about 20%.
  • CLDN18.2 antibody 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5, and 18.2-A6 can bind to the positive 293t cells transfected with CLDN18.2-flag well, but all 6 antibodies Does not bind to CLDN18.1 positive cells.
  • the above results indicate that these 6 CLDN18.2 antibodies can specifically bind to CLDN18.2 but not to CLDN18.1.
  • the CLDN18.2 antibody 18.2-A1, 18.2-A3, 18.2-A4, 18.2-A6 or IMAB362 antibody (as described in Example 2) at a concentration of 10 ⁇ g/ml, a blank control (PBS) and the same Isotype control (ISO) and cells were incubated at 4°C or 37°C for 4 hours. Subsequently, washing was performed at 4°C and staining with PE-labeled anti-human IgG antibody. After 4% formalin fixation, the antibody binding on the cell surface was detected by flow cytometry.
  • IMAB362 antibody and 18.2-A1, 18.2-A3, 18.2-A4 and 18.2-A6 antibodies can all bind well to SNU601 cells expressing CLDN18.2.
  • IMAB362 antibody was endocytosed by the cells, and the antibody staining on the cell surface was significantly reduced; while 18.2-A1, 18.2-A3, 18.2-A4 and 18.2-A6 antibodies did not undergo obvious endocytosis, at 37°C It can still bind well to cells under conditions.
  • IMAB362 antibody can cause endocytosis, which may down-regulate the expression level of CLDN18.2 antigen on the cell surface.
  • endocytosis can also cause changes in antibody metabolism and accelerate the elimination of antibodies in the body.
  • the CLDN18.2 antibody of the present disclosure does not cause significant endocytosis, and therefore does not down-regulate the level of the target antigen CLDN18.2 on the cell surface, nor does it affect the metabolism of the antibody in the body.
  • the defucosylated form of CLDN18.2 antibody was further prepared. Specifically, the heavy and light chain coding sequences of the antibody are cloned into a mammalian GS expression vector that can be stably expressed, and the heavy and light chain coding sequences are both driven by the CMV promoter.
  • the expression vector was transfected into CHO-K1 cells domesticated by serum-free and suspension culture and domesticated CHO-K1 cell lines with Fut8 gene knockout. Use MSX to screen and select stable expression cell lines. After obtaining the stable expression cell line, culture it in a shake flask for 12-14 days, and supplement the feed medium as needed.
  • the culture supernatant was collected, filtered and passed through a protein A chromatography column to capture the expressed antibody.
  • the antibody was eluted and dialyzed with PBS to obtain a purified antibody.
  • the 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5 and 18.2-A6 antibodies expressed in the Fut8 gene knockout CHO-K1 cell line were named 18.2-A1F and 18.2-A2F, respectively , 18.2-A1F, 18.2-A4F, 18.2-A5F and 18.2-A6F.
  • glycosylation modification of the defucosylated antibody expressed in the Fut8 knockout CHO-K1 cell line and the common antibody produced in the CHO-K1 cell was analyzed.
  • the glycosyl groups of the antibody were obtained by purifying 100 ⁇ g of the antibody after digestion by trypsin and glycosyl peptidase, and then subjected to fluorescence tandem mass spectrometry analysis. The identification of various sugar groups depends on their mass-to-charge ratio m/z, and the percentage of sugar groups is calculated from the area percentage detected by fluorescence.
  • N-acetylneuraminic acid N-acetylgalactosamine Fucose Mannose Galactose N-glycolylneuraminic acid
  • glycosyl number 5 numbers respectively express the number of different sugars: six carbon sugars (galactose, mannose, or glucose), N-acetylhexosamine (GlcNA or GalNAc), fucose (Fucose, FUC for short) ), N-acetylneuraminic acid (Neu5Ac), and N-glycolylneuraminic acid (Neu5Gc).
  • the third number indicates the number of fucose. As shown in Table 4 below:
  • Glycan number Fucose proportion% 33000 no 0.2 33100 Have 0.3 34000 no 4.8 34100 Have 45.0 44000 no 3.3 44100 Have 37.7 45,000 no 0.3 45100 Have 0.2 54100 Have 7.0 54110 Have 0.3 54101 Have 0.2 or 0 54120 Have 0.3
  • Fc ⁇ RIIIa (Sino Biological Inc, 10389-H08C1) was diluted with HBS-EP buffer to 0.1 ⁇ g/ml as the ligand, and 18.2-A1F and 18.2-A1 antibody samples were diluted to 360 ⁇ g/ml, 120 ⁇ g, respectively /ml, 40 ⁇ g/ml, 13.3 ⁇ g/ml and 4.4 ⁇ g/ml as analytes.
  • indirect capture method to immobilize the ligand Fc ⁇ RIIIa, first use 50 ⁇ g/ml of Anti-His IgG to covalently bind to the surface of the CM5 chip through amino coupling, and then bind the ligand and analyte.
  • Biacore Wizard mode a multi-cycle method is used, with Fc ⁇ RIIIa as the ligand and 18.2-A1F and 18.2-A1 antibody samples as the analyte, and affinity analysis experiments are performed.
  • the test of each sample includes 3 Startups, 1 zero concentration control, 5 gradient concentration samples and 1 repeated sample (reference product).
  • the chip is regenerated with 10mM glycine-HCl, pH 1.5 regeneration solution.
  • Each analyte concentration cycle is set to capture time 60s, ligand solution flow rate 10 ⁇ l/min; ligand and analyte binding time 180s, analyte solution flow rate 30 ⁇ l/min; dissociation time 180s.
  • the Fc ⁇ RIIIa receptor can activate the NF-AT transcription factor pathway in effector cells after binding to the FC region of the antibody. Therefore, the detection of NF-AT-mediated reporter gene intensity can reflect the activation intensity of Fc ⁇ RIIIa receptor.
  • a promoter containing an NF-AT binding site was inserted into the luciferase reporter gene expression vector and stably transfected into the Jurkat cell line.
  • Fc ⁇ RIIIa (V158) with high FC binding was also stably transfected into Jurkat cells to construct a functional cell line (Fc ⁇ RIIIa-Jurkat) that can sense the activation intensity of Fc ⁇ RIIIa receptor.
  • SNU601 gastric cancer cells (SNU601-CLDN18.2) stably expressing CLDN18.2, dilute to 4x10 5 /ml, and mix with Fc ⁇ RIIIa-Jurkat cells at a ratio of 1:6.
  • Example 14 Combining chemotherapy drug EOF to enhance CLDN18.2 antibody-induced Fc ⁇ RIIIa receptor activation
  • Combination chemotherapy including EOF is the main existing treatment for gastric cancer.
  • EOF sensitive cell lines will undergo different degrees of cell division cycle blockade, proliferation inhibition and apoptosis after EOF treatment.
  • KATOIII cells are a human gastric epithelial cancer cell line that expresses a very low level of CLDN 18.2, and only about 5.2% of the cells are significantly positive in flow cytometry (Figure 11). Due to the low level of CLDN18.2 expression, in the Fc ⁇ RIIIa receptor activation experiment induced by antibodies co-incubated with KATOIII cells and Fc ⁇ RIIIa-Jurkat cells (experimental methods are as described in Example 13), 18.2-A1F and 18.2-A6F antibodies Does not cause Fc ⁇ RIIIa receptor activation (Figure 12A).
  • EOF epirubicin: 300 nM; oxaliplatin: 130 nM; 5-fluorouracil: 561.3 nM
  • the cell volume was found to increase by microscopic observation and flow cytometry. Larger, rounded, and decreased cells in the division phase. Indicates that the cell stays in the division phase. But the cells are still alive and there is no obvious apoptosis (results not shown).
  • the EOF-treated KATOIII cells were incubated with Fc ⁇ RIIIa-Jurkat cells and CLDN18.2 antibodies of different concentrations. The results indicate that the 18.2-A1F and 18.2-A6F antibodies can induce significant Fc ⁇ RIIIa receptor activation compared to the isotype control (ISO) ( Figure 12B).
  • PBMC Peripheral blood leukocytes
  • donor 1 and donor 2 Peripheral blood leukocytes
  • Ficoll-Paque Plus GE Healthcare
  • SNU601 gastric cancer cells SNU601-CLDN18.2
  • CLDN18.2 gastric cancer cells
  • dilute PBMC 5x10 6 /ml
  • mix the two in equal volumes and the ratio of effector cells to target cells is 50 :1.
  • LDH lactate dehydrogenase
  • Control group target cell + effector cell + negative control antibody
  • Killing rate (%) (experimental group-minimum release group)/(maximum release group-minimum release group)% ⁇ 100%
  • Negative control group target cell + negative control antibody + complement
  • Killing rate (%) (experimental group-minimum release group) / (maximum release group-minimum release group) ⁇ 100%
  • Example 17 In vivo tumor killing activity of CLDN18.2 antibody
  • SNU601-CLDN18.2 cells and human PBMC cells isolated from Ficoll were subcutaneously inoculated into NPG immunodeficient mice to establish a mouse xenograft model of SNU601 cells.
  • Example 18 In vivo tumor killing activity of CLDN18.2 antibody and chemotherapeutic drugs
  • KATOIII expressed very low levels of CLDN 18.2 when cultured in IMDM medium in vitro, and only about 5.2% of the cells showed significant positive in flow cytometry ( Figure 11). After flow cytometry sorting, KATOIII cells (KATOIII-18.2High) with high expression of CLDN18.2 were selected to inoculate NPG immunodeficient mice for xenotransplantation model experiments ( Figure 16).
  • KATOIII-18.2High cells and human PBMC cells isolated from Ficoll were subcutaneously inoculated into NPG immunodeficient mice to establish a xenotransplantation model of KATOIII-18.2High cells.
  • ISO isotype control antibody
  • EOF epirubicin: 1 mg/kg
  • oxaliplatin 3 mg/kg
  • 5-Fluorouracil 30mg/kg
  • 18.2-A1F antibody at a dose of 10mg/kg
  • the drug was administered every 3 days after tumor inoculation, and the tumor volume in mice was measured.

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Abstract

提供了结合CLDN18.2的抗体及其去岩藻糖化的形式。还提供了制备上述抗体的方法，包含抗体的缀合物和组合物，及其在治疗疾病例如癌症中的用途。

Description

CLDN18.2抗体及其用途
本申请要求于2019年07月12日递交的申请号为CN 201910628018.9，发明名称为“CLDN18.2抗体及其用途”的中国专利申请的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本公开涉及免疫治疗领域。具体而言，本公开涉及结合CLDN18.2的抗体及其去岩藻糖化的形式，及其在治疗疾病特别是癌症中的应用。
技术背景
包括胃癌及食道癌在内的上消化道肿瘤是一种全球常见，预后较差的恶性肿瘤。胃癌在中国尤其高发，全球有近42％的胃癌新发病例集中在中国。由于缺乏早期诊断，约80％胃癌患者发现时已是晚期。由于胃癌对常规化疗药物不够敏感，晚期胃癌的五年生存率极低，并已成为中国第三大致死的恶性肿瘤。因此，近年的研究致力于寻找胃癌特异性的靶向治疗。
封闭蛋白(Claudin)家族蛋白是广泛分布于上皮细胞中的紧密连接结构的主要组成部分。和其它封闭蛋白家族蛋白的结构类似，CLDN18.2是分子量约27.8kd，具有四个跨膜区的膜蛋白，且具有两个较短的胞外区EC1和EC2。与其它封闭蛋白家族蛋白不同的是，在正常组织中CLDN18.2非常特异地仅表达于胃部高度分化的上皮细胞中(Tureci O et al.,Gene,2011)。在来源于胃上皮细胞的肿瘤中，有很高比例的肿瘤细胞表面表达CLDN18.2。在胃癌细胞的淋巴结和其它组织转移肿瘤中，也可以检测到高水平的CLDN18.2表达(Woll S.et al.,Int.J.Cancer,2013)。除胃癌外，也有很高比例的胆管癌、食道癌和胰腺癌等肿瘤细胞中表达CLDN18.2。作为特异性表面标志物，CLDN18.2成为癌症治疗的潜在靶点。研发针对CLDN18.2的高效抗体药物将为多种癌症的治疗提供可能，具有巨大的应用潜力和市场价值。
治疗性单克隆抗体杀伤肿瘤细胞的主要作用机制之一是ADCC效应(抗体依赖性细胞介导的细胞毒性，antibody dependent cell mediated cytotoxicity)。通过治疗性抗体的抗原识别段(Fab)与肿瘤细胞表面的特异性抗原结合后，利用抗体恒定区(Fc)与表达FC受体(FcγR)的效应细胞结合来激活 效应细胞的细胞杀伤活性，从而分泌细胞毒性介质包括颗粒酶和穿孔素等，最终导致靶细胞(肿瘤细胞)的溶解破坏。抗体对抗原的结合力强弱，抗原表达丰度以及抗体FC区和效应细胞表面的FC受体的结合力强弱均可影响ADCC效应的强度，进而影响癌症的治疗效果(Jefferis and Lund,2002)。
发明概述
本公开提供了新的抗CLDN18.2的抗体。上述抗体能够以高亲和力和特异性结合CLDN18.2，并介导效应细胞对表达CLDN18.2的靶细胞(例如肿瘤细胞)的杀伤。此外，还生成了上述抗体的去岩藻糖化形式，其能够比普通抗体更好地结合FcγRIIa并诱导ADCC效应。
相应地，在一方面，本公开涉及一种结合CLDN18.2的抗体或其抗原结合片段，所述抗体具有以下重链CDR(CDRH)和轻链CDR(CDRL)：
a.如SEQ ID NO:1中所示的重链可变区(VH)中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:6中所示的轻链可变区(VL)中的CDRL1、CDRL2和CDRL3；
b.如SEQ ID NO:11中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:16中所示的VL中的CDRL1、CDRL2和CDRL3；
c.如SEQ ID NO:21中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:26中所示的VL中的CDRL1、CDRL2和CDRL3；
d.如SEQ ID NO:31中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:36中所示的VL中的CDRL1、CDRL2和CDRL3；
e.如SEQ ID NO:41中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:46中所示的VL中的CDRL1、CDRL2和CDRL3；或
f.如SEQ ID NO:55中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:60中所示的VL中的CDRL1、CDRL2和CDRL3。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有以下CDRH和CDRL：
a.如SEQ ID NO:3-5中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:8-10中所示的CDRL1、CDRL2和CDRL3；
b.如SEQ ID NO:13-15中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:18-20中所示的CDRL1、CDRL2和CDRL3；
c.如SEQ ID NO:23-25中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:28-30中所示的CDRL1、CDRL2和CDRL3；
d.如SEQ ID NO:33-35中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:38-40中所示的CDRL1、CDRL2和CDRL3；
e.如SEQ ID NO:43-45中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:48-50中所示的CDRL1、CDRL2和CDRL3；或
f.如SEQ ID NO:57-59中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:62-64中所示的CDRL1、CDRL2和CDRL3。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有以下VH和VL：
a.包含SEQ ID NO:1的氨基酸序列的VH，和包含SEQ ID NO:6的氨基酸序列的VL；
b.包含SEQ ID NO:11的氨基酸序列的VH，和包含SEQ ID NO:16的氨基酸序列的VL；
c.包含SEQ ID NO:21的氨基酸序列的VH，和包含SEQ ID NO:26的氨基酸序列的VL；
d.包含SEQ ID NO:31的氨基酸序列的VH，和包含SEQ ID NO:36的氨基酸序列的VL；
e.包含SEQ ID NO:41的氨基酸序列的VH，和包含SEQ ID NO:46的氨基酸序列的VL；
f.包含SEQ ID NO:55的氨基酸序列的VH，和包含SEQ ID NO:60的氨基酸序列的VL。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体可以具有如SEQ ID NO:51所示的重链恒定区序列和/或如SEQ ID NO:53所示的轻链恒定区序列。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有Fc区。在一些实施方案中，所述抗体可以在Asn297处具有糖基结构修饰，其中根据EU编号系统编号。在一些实施方案中，所述糖基结构中具有岩藻糖的糖基结构的比例为50％或更少。在一些实施方案中，所述糖基结构中具有岩藻糖的糖基结构的比例为不高于30％，例如不高于20％，不高于10％，不高于5％，不高于2％或不高于1％。
在优选的实施方案中，所述抗体中具有岩藻糖的糖基结构的比例为0％-1％。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体由Fut8基因敲除的细胞产生。在一些实施方案中，所述细胞可以选自CHO细胞和HEK293细胞。
在本公开的抗体或其抗原结合片段的一些实施方案中，相比于在不具有Fut8基因敲除的细胞中产生的抗体，所述抗体具有增加的FcγRIIIa结合活性。在一些实施方案中，相比于在不具有Fut8基因敲除的细胞中产生的抗体，所述抗体具有增加的ADCC活性。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体可以是单克隆抗体。在另一些实施方案中，所述抗体可以是双特异性抗体或多特异性抗体。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体可以选自IgG、IgA、IgM、IgE和IgD同种型。在一些实施方案中，所述抗体可以选自IgG1、IgG2、IgG3和IgG4亚型。
在本公开的抗体或其抗原结合片段的任何实施方案中，所述抗原结合片段可以选自Fab片段、Fab’片段、F(ab’) 2片段、Fd片段、Fd’片段、Fv片段、scFv片段、ds-scFv片段、dAb片段、单链片段、二价抗体和线性抗体。
在一个方面，本公开涉及一种核酸分子，所述核酸分子包含编码本公开的抗体或其抗原结合片段的核苷酸序列。
在另一个方面，本公开涉及一种载体，所述载体包含本公开的核酸分子。
在再一个方面，本公开涉及一种宿主细胞，所述宿主细胞包含本公开的核酸分子或载体。
在一个方面，本公开涉及一种缀合物，所述缀合物包含与治疗剂、诊断剂或显像剂缀合的本公开的任何抗体或其抗原结合片段。
在另一个方面，本公开涉及一种组合物，所述组合物包含本公开的抗体或其抗原结合片段或缀合物，以及一种或多种药学上可接受的载体、赋形剂和/或稀释剂。在一些实施方案中，所述组合物还包含一种或多种另外的治疗剂。
在一些实施方案中，所述治疗剂可以选自抗体、化疗药物和小分子药物。在一些实施方案中，所述化疗药物可以选自表柔比星(Epirubicin)、奥沙利铂 (Oxaliplatin)和5-氟尿嘧啶(5-FU)的一种或多种。
在一个方面，本公开涉及一种治疗受试者中与CLDN18.2的表达相关的疾病的方法，所述方法包括对所述受试者施用本公开的抗体或其抗原结合片段、缀合物或组合物的步骤。
在一些实施方案中，所述疾病是癌症。例如，所述癌症可以选自胃癌、胆管癌、食道癌和胰腺癌。
在一些实施方案中，所述方法还包括对所述受试者施用一种或多种另外的疗法例如癌症疗法的步骤。在一些实施方案中，所述另外的疗法选自化学疗法、放射疗法、免疫疗法和手术治疗。
在一些实施方案中，所述免疫疗法选自针对免疫检查点分子的疗法、CAR-T细胞治疗和CAR-NK细胞治疗。
在一些实施方案中，所述化学疗法选自包括表柔比星、奥沙利铂和5-氟尿嘧啶的联合化疗方案。
在一个方面，本公开涉及本公开的抗体或其抗原结合片段、缀合物或组合物在治疗受试者中与CLDN18.2的表达相关的疾病中的用途。
在另一个方面，本公开涉及本公开的抗体或其抗原结合片段、缀合物或组合物在制备用于治疗受试者中与CLDN18.2的表达相关的疾病的药物中的用途。
在本公开的用途的一些实施方案中，所述疾病是癌症。例如，所述癌症可以选自胃癌、胆管癌、食道癌和胰腺癌。
在一个方面，本公开涉及一种多肽，所述多肽具有选自SEQ ID NO:1、6、11、16、21、26、31、36、41、46、55和60的氨基酸序列。
在另一个方面，本公开涉及一种核酸分子，所述核酸分子具有选自SEQ ID NO:2、7、12、17、22、27、32、37、42、47、56和61的核苷酸序列。本公开还涉及包含上述核酸分子的载体。
附图说明
图1显示了通过流式细胞法检测CHO、CT26、SNU601细胞以及构建的CHO-CLDN18.2、CT26-CLDN18.2和SNU601-CLDN18.2细胞的CLDN18.2表达的结果。
图2显示了通过流式细胞法检测对照M13噬菌体以及经过第一轮、第二 轮和第三轮筛选的噬菌体文库与CHO细胞和CHO-CLDN18.2细胞结合的结果。
图3显示了通过流式细胞法检测筛选的噬菌体克隆A1、A2、A3、A4、A5(图3A)和A6(图3B)与CHO细胞和CHO-CLDN18.2细胞结合的结果。
图4显示了通过流式细胞法检测不同浓度的重组抗体18.2-A1、18.2-A2、18.2-A3、18.2-A4、18.2-A5(图4A)、18.2-A5F和18.2-A6F(图4B)与CT26-CLDN18.2细胞结合的结果以及EC50值。
图5显示了通过流式细胞法检测CLDN18.2抗体与表达CLDN18.1(18.1-flag)或CLDN18.2(18.2-flag)的293t细胞结合的结果。
图6显示了在4℃和37℃孵育条件下检测CLDN18.2抗体与表达CLDN18.2的SNU601细胞表面结合和被细胞内吞的结果。
图7显示了通过流式细胞法检测抗体CLDN18.2抗体以及去岩藻糖化的抗体与CT26-CLDN18.2细胞结合的结果。
图8显示了通过Biacore方法检测的CLDN18.2-A1和CLDN18.2-A1F与FcγRIIIa结合的结果。图8A显示了抗体结合FcγRIIIa的拟合曲线，且图8B显示了抗体结合FcγRIIIa的Ka、Kd、KD和tc值。
图9显示了通过流式细胞法检测CLDN18.2抗体以及去岩藻糖化的抗体与细胞表面表达的FcγRIIIa的结合结果。图9A显示了抗体与FcγRIIIa-Jurkat细胞结合的结果。图9B显示了抗体与NK92MI细胞结合的结果。
图10显示了通过荧光素酶报告系统检测在SNU601-CLDN18.2细胞(图10A)或CT26-CLDN18.2(图10B)的存在下，CLDN18.2抗体以及去岩藻糖化的抗体活化FcγRIIIa-Jurkat细胞中的FcγRIIIa受体的结果。
图11显示了通过流式细胞检测KATOIII细胞上的CLDN18.2表达水平的结果。
图12显示了显示了通过荧光素酶报告系统检测在SNU601-CLDN18.2细胞的存在下，CLDN18.2抗体(图12A)和CLDN18.2抗体抗体与化疗药物EOF的联合(图12B)活化FcγRIIIa-Jurkat细胞中的FcγRIIIa受体的结果。
图13显示了在来自健康人供体1(图13A)、供体2(图13B)或供体3(图13C)和的PBMC的存在下，CLDN18.2抗体以及去岩藻糖化的抗体介导的ADCC途径对SNU601-CLDN18.2靶细胞的杀伤效果。
图14显示了在补体的存在下，CLDN18.2抗体以及去岩藻糖化的抗体介 导的CDC途径对CHO-CLDN18.2靶细胞的杀伤效果。
图15显示了在SNU601-CLDN18.2细胞的小鼠异种移植模型中，不同剂量的CLDN18.2抗体18.2-A1F抑制肿瘤生长的结果。
图16显示了用于异种移植模型的CLDN18.2高表达的KATOIII细胞(KATOIII-18.2High)的流式细胞结果。
图17显示了在KATOIII-18.2High细胞的小鼠异种移植模型中，CLDN18.2抗体18.2-A1F以及18.2-A1F和化疗药物EOF的联合抑制肿瘤生长的结果。
发明详述
除非本文另有定义，与本发明结合使用的科学和技术术语及其缩略语应具有本发明所属领域的普通技术人员通常理解的含义。以下列举了本文中使用的部分术语和缩略语。
抗体：antibody，Ab；免疫球蛋白：immunoglobulin，Ig；
重链：heavy chain，HC；轻链：light chain，LC；
重链可变区：heavy chain variable domain，VH；
重链恒定区：heavy chain constant domain，CH；
轻链可变区：light chain variable domain，VL；
轻链恒定区：light chain constant domain，CL；
互补决定区：complementarity determining region，CDR；
Fab片段：antigen binding fragment，Fab；
Fc区：fragment crystallizable region，Fc；
单克隆抗体：monoclonal antibody，mAb；
抗体依赖性细胞毒作用：antibody-dependent cell-mediated cytotoxicity，ADCC；
补体依赖性细胞毒性作用：complement dependent cytotoxicity，CDC。
在一方面，本公开涉及一种结合CLDN18.2的抗体或其抗原结合片段，所述抗体具有以下重链CDR(CDRH)和轻链CDR(CDRL)：
a.如SEQ ID NO:1中所示的重链可变区(VH)中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:6中所示的轻链可变区(VL)中的CDRL1、CDRL2和CDRL3；
b.如SEQ ID NO:11中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:16中所示的VL中的CDRL1、CDRL2和CDRL3；
c.如SEQ ID NO:21中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:26中所示的VL中的CDRL1、CDRL2和CDRL3；
d.如SEQ ID NO:31中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:36中所示的VL中的CDRL1、CDRL2和CDRL3；
e.如SEQ ID NO:41中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:46中所示的VL中的CDRL1、CDRL2和CDRL3；或
f.如SEQ ID NO:55中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:60中所示的VL中的CDRL1、CDRL2和CDRL3。
如本文所用，术语“结合”或“特异性结合”是指两种分子间的非随机的结合反应，如抗体及其所针对的抗原之间的反应。在某些实施方案中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M、或10 -10M或更小的亲和力(K D)结合该抗原。本文所用“K D”是指，特定抗体-抗原相互作用的解离平衡常数，用于描述抗体与抗原间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
如本文所用，术语“抗体”指包含至少一个抗原识别位点并能特异性结合抗原的免疫球蛋白分子。术语“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质，如蛋白质、多肽、肽、碳水化合物、多聚核苷酸、脂质、半抗原或上述物质的组合。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原可以具有多个表位。
如本文所用，术语“表位”可以由连续氨基酸或通过蛋白质的三级折叠并置的非连续氨基酸形成。表位通常在独特的空间构象中包括至少3个，更通常至少5个、约9个、或约8-10个氨基酸。“表位”包括通常由免疫球蛋白VH/VL对结合的结构单元。表位定义抗体的最小结合位点，因此代表抗体或其抗原结合片段特异性的靶物。
本公开所提及的术语“抗体”以其最广泛的含义理解，并包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同的抗 原结合结构域的多特异性抗体(例如，双特异性抗体)。抗体还包括鼠源抗体、人源化抗体、嵌合抗体、人抗体以及其它来源的抗体。抗体可以含有另外的改变，如非天然氨基酸，Fc效应功能突变和糖基化位点突变。抗体还包括翻译后修饰的抗体、包含抗体的抗原决定簇的融合蛋白，以及包含对抗原识别位点的任何其它修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。换句话说，抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合结构域的分子。
如本文所用，“可变区”(重链可变区VH和轻链可变区VL)表示成对的轻链和重链结构域部分，其直接参与抗体和抗原的结合。每个VH和VL区由以下顺序从N末端到C末端排列的三个高变区或互补决定区(CDR)和四个框架区(FR)组成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
如本文所用，术语“CDR”是指抗体可变序列内的互补决定区。对于每个可变区，在重链和轻链的每个可变区中有三个CDR，其称为CDR1、CDR2和CDR3。这些CDR的确切边界根据不同的系统而不同定义。Kabat等人(Kabat et al,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987)和(1991))描述的系统不仅提供了适用于抗体可变区的明确的残基编号系统，而且还提供了限定三个CDR的残基边界。这些CDR可以称为Kabat CDR。每个互补决定区可以包含由Kabat定义的“互补决定区”的氨基酸残基。Chothia等人(Chothia&Lesk,J.Mol.Biol,196:901-917(1987)和Chothia et al.,Nature 342:877-883(1989))发现，Kabat CDR内的某些子部分采用几乎相同的肽骨架构象，尽管在氨基酸序列水平上具有多样性。这些子部分分别称为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别表示轻链和重链区。这些区域可以称为Chothia CDR，其具有与Kabat CDR重叠的边界。还有其它CDR边界定义可以不严格遵循上述系统之一，但是仍将与Kabat CDR重叠。本文使用的方法可以利用根据任何这些系统定义的CDR，尽管优选实施方案使用Kabat或Chothia定义的CDR。
当使用Kabat系统来定义CDR序列时，如SEQ ID NO:1中所示的VH中的CDRH1、CDRH2和CDRH3分别具有SEQ ID NO:3(SSWLI)、SEQ ID NO:4(TIVPSDSYTNYNQKFKD)和SEQ ID NO:5(FRTGNSFDY)的氨基酸序列，且如SEQ ID NO:6中所示的VL中的CDRL1、CDRL2和CDRL3分别具有SEQ ID NO:8(KSSQSVLNSGNQKNYLT)、SEQ ID NO:9(WAVARQS)和SEQ ID  NO:10(QNSIAYPFT)的氨基酸序列；
如SEQ ID NO:11中所示的VH中的CDRH1、CDRH2和CDRH3分别具有SEQ ID NO:13(SFWVG)、SEQ ID NO:14(NVSPSDSYTNYNQKFKD)和SEQ ID NO:15(LSSGNSFDY)的氨基酸序列，且如SEQ ID NO:16中所示的VL中的CDRL1、CDRL2和CDRL3分别具有SEQ ID NO:18(KSSQSVLNSGNQKNYLT)、SEQ ID NO:19(WSSTKQS)和SEQ ID NO:20(QNAFSFPFT)的氨基酸序列；
如SEQ ID NO:21中所示的VH中的CDRH1、CDRH2和CDRH3分别具有SEQ ID NO:23(SYWLN)、SEQ ID NO:24(SMYPSDSYTNYNQKFKD)和SEQ ID NO:25(FSRGNSFDY)的氨基酸序列，且如SEQ ID NO:26中所示的VL中的CDRL1、CDRL2和CDRL3分别具有SEQ ID NO:28(KSSQSLLESGNQKNYLT)、SEQ ID NO:29(WSWAKNS)和SEQ ID NO:30(QNAYAFPFT)的氨基酸序列；
如SEQ ID NO:31中所示的VH中的CDRH1、CDRH2和CDRH3分别具有SEQ ID NO:33(SFWIS)、SEQ ID NO:34(NILPSDSYTNYNQKFKD)和SEQ ID NO:35(YWRGNSFDY)的氨基酸序列，且如SEQ ID NO:36中所示的VL中的CDRL1、CDRL2和CDRL3分别具有SEQ ID NO:38(KSSQSIINSGNQKNYLT)、SEQ ID NO:39(WGGTRHS)和SEQ ID NO:40(QNGYYSPFT)的氨基酸序列；
如SEQ ID NO:41中所示的VH中的CDRH1、CDRH2和CDRH3分别具有SEQ ID NO:43(SSWVG)、SEQ ID NO:44(NSYPSDSYTNYNQKFKD)和SEQ ID NO:45(LGRGNSFDY)的氨基酸序列，且如SEQ ID NO:46中所示的VL中的CDRL1、CDRL2和CDRL3分别具有SEQ ID NO:48(KSSQSLIHSGNQKNYLT)、SEQ ID NO:49(WGLSKNS)和SEQ ID NO:50(QNSIYYPFT)的氨基酸序列；
如SEQ ID NO:55中所示的VH中的CDRH1、CDRH2和CDRH3分别具有SEQ ID NO:57(SYWLG)、SEQ ID NO:58(IIYPSDSYTNYNQKFKD)和SEQ ID NO:59(FWRGNSFDY)的氨基酸序列，且如SEQ ID NO:60中所示的VL中的CDRL1、CDRL2和CDRL3分别具有SEQ ID NO:62(KSSQSLLESGNQKNYLT)、SEQ ID NO:63(WAAGKES)和SEQ ID NO:64(QNGYSHPFT)的氨基酸序列。
相应地，在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有以下CDRH和CDRL：
a.如SEQ ID NO:3-5中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:8-10中所示的CDRL1、CDRL2和CDRL3；
b.如SEQ ID NO:13-15中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:18-20中所示的CDRL1、CDRL2和CDRL3；
c.如SEQ ID NO:23-25中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:28-30中所示的CDRL1、CDRL2和CDRL3；
d.如SEQ ID NO:33-35中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:38-40中所示的CDRL1、CDRL2和CDRL3；
e.如SEQ ID NO:43-45中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:48-50中所示的CDRL1、CDRL2和CDRL3；或
f.如SEQ ID NO:57-59中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:62-64中所示的CDRL1、CDRL2和CDRL3。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有以下VH和VL：
a.包含SEQ ID NO:1的氨基酸序列的VH，和包含SEQ ID NO:6的氨基酸序列的VL；
b.包含SEQ ID NO:11的氨基酸序列的VH，和包含SEQ ID NO:16的氨基酸序列的VL；
c.包含SEQ ID NO:21的氨基酸序列的VH，和包含SEQ ID NO:26的氨基酸序列的VL；
d.包含SEQ ID NO:31的氨基酸序列的VH，和包含SEQ ID NO:36的氨基酸序列的VL；
e.包含SEQ ID NO:41的氨基酸序列的VH，和包含SEQ ID NO:46的氨基酸序列的VL；或
f.包含SEQ ID NO:55的氨基酸序列的VH，和包含SEQ ID NO:60的氨基酸序列的VL。
在本公开的抗体或其抗原结合片段的另一些实施方案中，所述抗体具有以下VH和VL：
a.包含在SEQ ID NO:1中进行了一个或几个氨基酸修饰的氨基酸序列 的VH，和包含在SEQ ID NO:6中进行了一个或几个氨基酸修饰的氨基酸序列的VL；
b.包含在SEQ ID NO:11中进行了一个或几个氨基酸修饰的氨基酸序列的VH，和包含在SEQ ID NO:16中进行了一个或几个氨基酸修饰的氨基酸序列的VL；
c.包含在SEQ ID NO:21中进行了一个或几个氨基酸修饰的氨基酸序列的VH，和包含在SEQ ID NO:26中进行了一个或几个氨基酸修饰的氨基酸序列的VL；
d.包含在SEQ ID NO:31中进行了一个或几个氨基酸修饰的氨基酸序列的VH，和包含在SEQ ID NO:36中进行了一个或几个氨基酸修饰的氨基酸序列的VL；
e.包含在SEQ ID NO:41中进行了一个或几个氨基酸修饰的氨基酸序列的VH，和包含在SEQ ID NO:46中进行了一个或几个氨基酸修饰的氨基酸序列的VL；或
f.包含在SEQ ID NO:55中进行了一个或几个氨基酸修饰的氨基酸序列的VH，和包含在SEQ ID NO:60中进行了一个或几个氨基酸修饰的氨基酸序列的VL。
在一些实施方案中，所述氨基酸修饰不改变抗体的CDR序列，即在可变区的框架区(FR)中进行所述氨基酸修饰。
在一些实施方案中，所述一个或几个氨基酸修饰是指1-10个氨基酸修饰或1-5个氨基酸修饰，例如1、2、3、4、5、6、7、8、9或10个氨基酸修饰。
在一些实施方案中，所述氨基酸修饰选自氨基酸残基的取代、缺失、添加和/或插入。在一些实施方案中，所述氨基酸修饰是氨基酸取代，例如保守取代。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有以下VH和VL：
a.包含与SEQ ID NO:1的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH，和包含与SEQ ID NO:6的氨基酸序列的具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VL；
b.包含与SEQ ID NO:11的氨基酸序列具有至少80％序列同一性，例如 至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH，和包含与SEQ ID NO:16的氨基酸序列的具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VL；
c.包含与SEQ ID NO:21的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH，和包含与SEQ ID NO:26的氨基酸序列的具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VL；
d.包含与SEQ ID NO:31的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH，和包含与SEQ ID NO:36的氨基酸序列的具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VL；
e.包含与SEQ ID NO:41的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH，和包含与SEQ ID NO:46的氨基酸序列的具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VL；或
f.包含与SEQ ID NO:55的氨基酸序列具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VH，和包含与SEQ ID NO:60的氨基酸序列的具有至少80％序列同一性，例如至少90％、至少95％、至少98％或至少99％序列同一性的氨基酸序列的VL。
如本领域技术人员所理解的，两个氨基酸序列之间或两个核苷酸序列之间的相关性可以通过参数“序列同一性”描述。出可以通过例如使用数学算法测定两种序列间的序列同一性的百分比。可以通过例如使用数学算法测定两种序列间的序列同一性的百分比。此类数学算法的非限制性实例包括Myers和Miller(1988)CABIOS 4:11-17的算法、Smith等(1981)Adv.Appl.Math.2:482的局部同源性算法、Needleman和Wunsch(1970)J.Mol.Biol.48:443-453的同源性比对算法、Pearson和Lipman(1988)Proc.Natl.Acad.Sci.85:2444-2448的用于搜索同源性的方法、和Karlin和Altschul(1990)Proc.Natl.Acad.Sci.USA 87:2264的算法的修改形式，记载于Karlin和Altschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-5877的算法。通过使用基于此类数学算法的程序，可以实施用于测定序列同一性的序列比较(即比对)。程序可以由计算机适当执行。此类程序的实例包括但不限于PC/Gene程序的CLUSTAL、 ALIGN程序(Version 2.0)、和Wisconsin遗传学软件包的GAP、BESTFIT、BLAST、FASTA、和TFASTA。可以例如通过使用初始参数实施使用这些程序的比对。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体可以具有如SEQ ID NO:51所示的重链恒定区序列和/或如SEQ ID NO:53所示的轻链恒定区序列。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有Fc区。在一些实施方案中，所述抗体在Asn297处具有糖基结构修饰，其中根据Eu编号系统编号。根据本公开的“Asn297”意指根据Eu编号系统位于抗体Fc区的297位的天冬酰胺。基于具体抗体的细微序列变化，Asn297也可以位于297位上游或下游几个氨基酸。
如本文所用，术语“抗体的Fc区”或“人免疫球蛋白Fc区”包含抗体除重链恒定区1(CH1)之外的恒定区多肽，例如人免疫球蛋白IgA、IgD、IgG重链恒定区羧基端的两个恒定区结构域CH2及CH3，以及人免疫球蛋白IgE和IgM重链恒定区羧基端的三个恒定区结构域CH2、CH3及CH4，并且还包括这些结构域氨基端的柔性铰链区。虽然Fc区的边界可以变化，但人IgG重链Fc区通常定义为包含从A231开始到其羧基末端的残基。
免疫球蛋白Fc区是抗体发挥免疫效应的功能域。IgG抗体的Fc能够与多种受体相互作用，其中最重要的是Fcγ受体(FcγR)家族。治疗性单克隆抗体杀伤肿瘤细胞的一个主要作用机制是ADCC效应(抗体依赖性细胞介导的细胞毒性，antibody dependent cell mediated cytotoxicity)。通过治疗性抗体的抗原识别区与肿瘤细胞表面的特异性抗原结合后，利用抗体Fc区与表达FcγR的杀伤细胞结合并激活效应细胞的细胞杀伤活性，从而分泌含有细胞毒性介质，包括颗粒酶，穿孔素等，最终导致靶细胞(例如肿瘤细胞)的溶解破坏。除此之外，Fc还能与补体蛋白C1q结合，产生CDC效应(补体依赖性细胞毒性complement dependent cytotoxicity，CDC)。
抗体Fc区的糖基化修饰水平和形式可以影响Fc区与其受体FcγR的结合能力，从而影响抗体的ADCC效应的强弱。如本文所用的抗体Fc区的糖基化修饰通常是指在Asn297处的糖基化修饰。在抗体的产生过程中其经历细胞的ER和高尔基体网中的糖基化。在抗体糖基化的相关研究中发现，降低抗体的岩藻糖化水平有助于提高Fc区与FcγRIIIa的结合，进而提高抗体的ADCC 效应。因此，改变抗体表达细胞的糖基化代谢途径，可以有效改变表达抗体的岩藻糖水平，从而调节抗体介导的ADCC效应(Jefferis R.2009,NAT.REV.Drug.DISC)。
相应地，在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体具有降低的岩藻糖化水平。例如，在本公开的抗体或其抗原结合片段的一些实施方案中，在Asn297处的糖基结构中，具有岩藻糖的糖基结构的比例为60％或更少，例如50％或更少，例如40％或更少，30％或更少，20％或更少，10％或更少，5％或更少，2％或更少，或1％或更少。在一些实施方案中，具有岩藻糖的糖基结构的比例为0％-10％，例如0％-5％，0％-2％或0％-1％。在一些实施方案中，具有岩藻糖的糖基结构的比例为0.1％或更少。在另一些实施方案中，在Asn297处的糖基结构中没有可检测的岩藻糖。
可以使用本领域技术人员已知的技术来产生此类去岩藻糖化的抗体。例如，抗体可以在缺陷的或缺乏岩藻糖基化能力的细胞中表达。在一些实施方案中，例如，具有Fut8基因敲除的细胞系可用于产生具有降低的岩藻糖化水平的抗体。或者，可以产生具有降低的岩藻糖含量或无岩藻糖含量的抗体或抗原结合片段，其通过例如：(i)在阻止或减少岩藻糖基化的条件下培养细胞；(ii)翻译后除去岩藻糖(例如，用岩藻糖苷酶)；(iii)期望碳水化合物的翻译后添加，例如重组表达非糖基化糖蛋白后；或(iv)糖蛋白的纯化，以选择不被岩藻糖基化的抗体或其抗原结合片段。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体由Fut8基因敲除的细胞产生，以获得具有降低的岩藻糖化水平的抗体。在一些实施方案中，所述细胞是中国仓鼠卵巢(CHO)细胞，例如CHO-K1细胞、CHOS细胞或其它CHO来源的细胞。在一些实施方案中，所述细胞是Fut8基因敲除的CHO细胞。
在另一些实施方案中，所述细胞是人胚胎肾(HEK)293细胞，例如HEK293、HEK293A、HEK293T、HEK293F或其它HEK293来源的细胞。在一些实施方案中，所述细胞是Fut8基因敲除的HEK 293细胞。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体由Fut8基因敲除的细胞产生，且其中相比于在不具有Fut8基因敲除的细胞中产生的抗体例如序列相同的对照抗体，所述抗体具有增加的FcγRIIIa结合活性。在一些实施方案中，相比于在不具有Fut8基因敲除的细胞中产生的抗体例如序 列相同的对照抗体，由Fut8基因敲除的细胞产生的抗体具有增加的诱导FcγRIIIa活性的能力。例如，在一些实施方案中，相比于在不具有Fut8基因敲除的细胞中产生的抗体，由Fut8基因敲除的细胞产生的抗体诱导FcγRIIIa活性的EC50值提高至少2倍，例如至少5倍或至少10倍，例如10-20倍。如本文所用，术语“EC50”是指引起50％最大效应所对应的抗体浓度。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体由Fut8基因敲除的细胞产生，且其中相比于在不具有Fut8基因敲除的细胞中产生的抗体例如序列相同的对照抗体，所述抗体具有增加的ADCC活性。例如，在一些实施方案中，相比于在不具有Fut8基因敲除的细胞中产生的抗体，由Fut8基因敲除的细胞产生的抗体的ADCC活性提高至少2倍，例如至少5倍或至少10倍，例如10-20倍。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体是单克隆抗体。
如本文所用，术语“单克隆抗体”是指从基本均质的抗体群体获得的抗体，即构成群体的各抗体是相同的，除了可以少量存在的可能的天然存在的突变外。单克隆抗体是高度特异性的，针对单一抗原。此外，与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆制剂中的每种抗体针对抗原上相同的单一决定簇。如本文使用，术语“单克隆抗体”不限于通过杂交瘤技术产生的抗体，并且修饰语“单克隆抗体”不应被解释为需要通过任何特定方法生产抗体。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗体是双特异性抗体或多特异性抗体。
例如，所述双特异性抗体或多特异性抗体具有结合CLDN18.2上的表位的第一抗原结合区，以及结合另一抗原表位的第二抗原结合区，其中所述第一抗原结合区具有如本公开所述的抗体或其抗原结合片段的CDRH1、CDRH2和CDRH3以及CDRL1、CDRL2和CDRL3；或VH和VL序列，且所述第二抗原结合区与所述第一抗原结合区结合不同的抗原表位。
在一些实施方案中，所述第二抗原结合区结合CLDN18.2分子上的另一抗原结合表位。在另一些实施方案中，所述第二抗原结合区结合另一抗原。在一些实施方案中，所述另一抗原选自肿瘤相关抗原和免疫检查点分子。
本领域已经鉴定出与特定癌症相关的许多肿瘤相关抗原。如本文所 用，术语“肿瘤相关抗原”是指由癌细胞差异表达，因此可以利用以靶向癌细胞的抗原。肿瘤相关抗原是可以潜在地刺激明显的肿瘤特异性免疫应答的抗原。这些抗原中的一些由正常细胞编码，但不一定由正常细胞表达。这些抗原可以表征为在正常细胞中通常是沉默(即不表达)的那些抗原、仅在某些分化阶段表达的那些抗原和那些在时间上表达的那些抗原，如胚胎和胎儿抗原。其它癌症抗原由突变细胞基因如癌基因(例如激活的ras癌基因)、抑制基因(例如突变型p53)和由内部缺失或染色体易位产生的融合蛋白编码。其它癌症抗原可以由病毒基因编码，如RNA和DNA肿瘤病毒上携带的基因。许多其它的肿瘤相关抗原及针对其的抗体是已知的和/或商品化的，并且也可以由本领域技术人员产生。
免疫检查点蛋白受体及其配体(本文统称为免疫检验点分子)介导T细胞介导的细胞毒性的抑制，并且通常由肿瘤或在肿瘤微环境中的无反应性T细胞上表达，并允许肿瘤逃避免疫攻击。免疫抑制检查点蛋白受体及其配体的活性的抑制剂可以克服免疫抑制性肿瘤环境，以允许肿瘤的细胞毒性T细胞攻击。免疫检查点蛋白的实例包括但不限于PD-1、PD-L1、PD-L2、CTLA4、OX40、LAG3、TIM3、TIGIT和CD103。此类蛋白质的活性的调节(包括抑制)可以通过免疫检查点调节剂完成，其可以包括例如靶向检查点蛋白的抗体、适体、小分子和检测点受体蛋白质的可溶性形式。对PD-1、PD-L2、CTLA4、OX40、LAG3、TIM3、TIGIT和CD103特异性的抗体是已知的和/或商品化的，并且也可以由本领域技术人员产生。
根据抗体重链恒定区的氨基酸序列，可以将免疫球蛋白分为5类(同种型)：IgA、IgD、IgE、IgG和IgM，其还可以进一步分成不同的亚型，如IgG1、IgG2、IgG3、IgG4、IgA1、IgA2等。根据轻链氨基酸序列，可将轻链分类为λ链或κ链。本公开的抗体可以是任何上述种类或亚类。
在一些实施方案中，本公开的抗体选自IgG、IgA、IgM、IgE和IgD同种型。在一些实施方案中，本公开的抗体是IgG，例如选自IgG1、IgG2、IgG3和IgG4亚型。
如本文所用，术语“抗原结合片段”包括但不限于：Fab片段，其具有VL、CL、VH和CH1域；Fab'片段，其是在CH1域的C端具有一个或多个半胱氨酸残基的Fab片段；Fd片段，其具有VH和CH1域；Fd'片段，其具有VH和CH1域和在CH1域的C端的一个或多个半胱氨酸残基；Fv片段和scFv，其 具有抗体的单一臂的VL和VH域；dAb片段，其由VH域或VL域组成；分离的CDR区；F(ab') 2片段，其是包含由铰链区处的二硫桥连接的两个Fab'片段的二价片段；单链抗体分子(例如单链Fv；scFv)；具有两个抗原结合位点的"二价抗体(diabody)"，其包含同一多肽链中与轻链可变域(VL)连接的重链可变域(VH)；"线性抗体"，其包含一对串联Fd区段(VH-CH1-VH-CH1)，该区段与互补的轻链多肽一起形成一对抗原结合区；和任何前述物质的修饰的形式，其保留了抗原结合活性。
在本公开的抗体或其抗原结合片段的一些实施方案中，所述抗原结合片段选自Fab片段、Fab’片段、F(ab’) 2片段、Fd片段、Fd’片段、Fv片段、scFv片段、ds-scFv片段、dAb片段、单链片段、二价抗体和线性抗体。
在另一个方面，本公开涉及一种核酸分子，所述核酸分子包含编码本公开的抗体或其抗原结合片段的核苷酸序列。本公开还涉及一种载体，所述载体包含本公开的核酸分子。
本文所用“载体”是指可以将多聚核苷酸插入其中的一种核酸运载工具。而当载体能使插入的多核苷酸编码的蛋白获得表达时，该载体称为表达载体。载体可以通过转化、转导或者转染等方法导入宿主细胞，继而使其携带的遗传物质元件在宿主细胞内获得表达。载体是本领域技术人员公认的、包括但不限于：(1)质粒；(2)噬菌粒；(3)柯斯质粒；(4)人工染色体，如酵母人工染色体、细菌人工染色体或P1来源的人工染色体；(5)噬菌体如λ噬菌体或M13噬菌体及(6)动物病毒，如逆转录酶病毒、腺病毒、腺相关病毒、孢疹病毒、痘病毒、杆状病毒。一种载体可以含有多种控制表达的元件，包括但不局限于，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；此外，载体还可以含有复制起始位点。
在一个方面，本公开涉及一种宿主细胞，所述宿主细胞包含本公开的核酸分子或载体。在一些实施方案中，所述宿主细胞是CHO细胞，例如CHO-K1细胞、CHOS细胞或其它CHO来源的细胞。在另一些实施方案中，所述宿主细胞是HEK 293细胞，例如HEK293、HEK293A、HEK293T、HEK293F或其它HEK293来源的细胞。
在一个方面，本公开涉及一种缀合物，所述缀合物包含与治疗剂、诊断剂或显像剂缀合的本公开的抗体或其抗原结合部分。在一些实施方案中，所述治疗剂可以选自细胞毒素和放射性同位素。在一些实施方案中，所述诊断 剂或显像剂可以选自荧光标记物、发光物质、显色物质和酶。
在另一个方面，本公开涉及一种组合物，所述组合物包含本公开的抗体或其抗原结合片段，或缀合物，以及一种或多种药学上可接受的载体、赋形剂和/或稀释剂。
短语“药学上可接受的”是指在合理的医学判断的范围内适合用于与人和动物的组织接触而没有过度毒性、刺激性、变应性应答或其它问题或并发症，与合理的益处/风险比相称的那些化合物、材料、组合物和/或剂型。如本文中所用，短语“药学上可接受的载体、赋形剂和/或稀释剂”是指药学上可接受的材料、组合物或媒介物，如液体或固体填充剂、稀释剂、赋形剂、溶剂、介质、包封材料、制造助剂或溶剂包封材料，其涉及维持本公开的抗体或其抗原结合片段的稳定性、溶解度或活性。
本公开的组合物可以经配制用于以固体、液体或凝胶形式向受试者施用。例如，本公开的组合物可以经配制用于肠胃外施用，例如通过皮下、肌肉内、静脉内或硬膜外注射，作为例如无菌溶液或悬浮液或持续释放配制剂。
在一些实施方案中，所述组合物还包含一种或多种另外的治疗剂。在一些实施方案中，所述另外的治疗剂选自抗体、化疗药物和小分子药物。在一些实施方案中，所述治疗剂靶向肿瘤相关抗原，例如如上文中所述的肿瘤相关抗原。在另一些实施方案中，所述治疗剂靶向免疫检查点分子，例如如上文中所述免疫检查点分子。
如本文所用，术语“化疗药物”是指具有在治疗以异常细胞生长为特征的疾病中的治疗有用性的任何化学试剂。如本文所用的化学治疗剂包括化学剂和生物剂。这些试剂发挥功能以抑制癌细胞实现持续存活所依赖的细胞活性。化疗剂的类别包括烷化/生物碱剂、抗代谢物、激素或激素类似物，以及各种抗新生物药物。
在一些实施方案中，所述另外的治疗剂是化疗药物，且所述化疗药物可以其中所述化疗药物可以选自表柔比星(Epirubicin)、奥沙利铂(Oxaliplatin)和5-氟尿嘧啶(5-FU)的一种或多种。
在一个方面，本公开涉及一种治疗受试者中与CLDN18.2的表达相关的疾病的方法，所述方法包括对所述受试者施用本公开的抗体或其抗原结合片段、缀合物或组合物的步骤。
如本文所用，术语“治疗”是指治疗性处理，其中目的是反转、减轻、改善、抑制、减缓或停止与疾病或病症相关的状况的进展或严重性。术语“治疗”包括减少或减轻疾病或病症的至少一种副作用或症状。如果减少一种或多种症状或临床标志物，则治疗通常是“有效的”。或者，如果疾病的进展减少或停止，则治疗是“有效的”，也就是说，“治疗”不仅包括症状的改善，而且还包括在缺乏治疗的情况下预期的症状的进展或恶化的停止，至少减慢。有益或期望的临床结果包括但不限于减轻一种或多种症状、减少疾病程度，稳定(即不恶化)疾病状态、延迟或减缓疾病进展、改善或缓解疾病状态、和缓解(不管是部分还是全部)，无论是可检测的还是检测不到的。
如本文所用，术语“受试者”、“患者”和“个体”在本文中可互换使用，并且是指动物，例如人类。术语受试者还包括“非人哺乳动物”，例如如大鼠、小鼠、兔、绵羊、猫、狗、牛、猪和非人灵长类动物。在优选的实施方案中，所述受试者是人类受试者。
在上述方法的一些实施方案中，所述疾病是癌症。癌症的具体实例包括但不限于：基底细胞癌、胆管癌；膀胱癌；骨癌；乳腺癌；腹膜癌；宫颈癌；胆管癌；绒毛膜癌；结肠和直肠癌；结缔组织癌；消化系统癌症；子宫内膜癌；食道癌；眼癌；头颈癌；胃癌；胶质母细胞瘤；肝癌；肾癌；喉癌；白血病；肝癌；肺癌(例如，小细胞肺癌、非小细胞肺癌、肺腺癌和肺鳞状细胞癌)；淋巴瘤，包括霍奇金淋巴瘤和非霍奇金淋巴瘤；黑色素瘤；骨髓瘤；神经母细胞瘤；口腔癌；卵巢癌；胰腺癌；前列腺癌；视网膜母细胞瘤；横纹肌肉瘤；直肠癌；呼吸系统癌；唾液腺癌；肉瘤；皮肤癌；鳞状细胞癌；睾丸癌；甲状腺癌；子宫或子宫内膜癌；泌尿系统癌症；B细胞淋巴瘤；慢性淋巴细胞性白血病(CLL)；急性成淋巴细胞性白血病(ALL)；毛细胞白血病；慢性成髓细胞性白血病等。在优选的实施方案中，所述癌症选自胃癌、胆管癌、食道癌和胰腺癌。
在上述方法的一些实施方案中，所述方法还包括施用一种或多种另外的疗法的步骤。例如，在一些实施方案中，所述疗法选自化学疗法、放射疗法、免疫疗法和手术疗法。
在一些实施方案中，所述免疫疗法选自针对免疫检查点分子的疗法、CAR-T细胞疗法和CAR-NK细胞疗法。例如，所述免疫检查点分子可以选自PD-1、PD-L1、PD-L2、CTLA4、OX40、LAG3、TIM3、TIGIT和CD103。
在一些实施方案中，所述化学疗法选自包括表柔比星、奥沙利铂和5-氟尿嘧啶的联合化疗方案。
在一个方面，本公开涉及本公开的抗体或其抗原结合片段、缀合物或组合物在治疗受试者中与CLDN18.2的表达相关的疾病中的用途。在另一个方面，本公开涉及本公开的抗体或其抗原结合片段、缀合物或组合物在制备用于治疗受试者中与CLDN18.2的表达相关的疾病的药物中的用途。
在上述用途的一些实施方案中，所述疾病是癌症，例如如上文中所述的癌症类型。在优选的实施方案中，所述癌症选自胃癌、胆管癌、食道癌和胰腺癌。
在上述用途的一些实施方案中，所述受试者是人。
在一个方面，本公开涉及一种多肽，所述多肽具有选自SEQ ID NO:1、6、11、16、21、26、31、36、41、46、55和60的氨基酸序列。
在另一个方面，本公开涉及一种核酸分子，所述核酸分子具有选自SEQ ID NO:2、7、12、17、22、27、32、37、42、47、56和61的核苷酸序列。本公开还涉及包含上述核酸分子的载体。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1.天然噬菌体库的构建
抗体药物开发目前主要通过人源化源自其它物种的抗体或通过转基因动物及体外筛选技术直接筛选全人源抗体来实现,其进一步降低了抗体在人中的免疫原性,减轻了相应的副作用，同时提高了成药性,是抗体药物发展的一个重要趋势。
体外筛选技术中噬菌体展示抗体库技术是获取全人源抗体的主要手段之一。噬菌体展示技术(phage display technology)利用分子生物学手段将外 源基因片段插入噬菌体特定蛋白如gIII的基因,通过噬菌体表达外源基因编码的蛋白或多肽,保持重组融合蛋白的相对空间结构和生物学活性并呈现于噬菌体表面。将构建的多样噬菌体库与靶蛋白共同孵育,经生物淘洗去除非靶蛋白结合型噬菌体株。在噬菌体库容足够大的情况下，通过多轮收集、扩增及富集获得高亲和力、高特异性的噬菌体克隆株，采用基因测序鉴定这些噬菌体克隆编码的蛋白序列可用于进一步的研究。
噬菌体抗体库是抗体基因经噬菌体展示后形成的多样噬菌体库。噬菌体抗体库的质量主要由库容量及多样性决定。为获得高亲和力抗体,噬菌体抗体库在保证多样性的前提下还需要尽可能增大库容。噬菌体表面展示的抗体片段数量代表库容大小，而展示的片段的多样性则代表抗体库多样性。理论上来说，噬菌体抗体库的库容越大，筛选获得的抗体亲和力越高。
在本实施例中，为了避免由于个体差异导致抗体库的偏向性,同时尽可能保证库的多样性,共获取、收集和提取了120份来自成年健康个体外周血及脾脏，以及新生儿脐带血的单个核细胞的总RNA。将所述RNA逆转录合成单链cDNA,再使用针对抗体不同亚群的可变区引物分别扩增VH、Vκ和Vλ基因。将扩增产物按照一定比例混合后,分别将重链与轻链基因以PCR法链接成为单链抗体(scFv)，并用双酶切法克隆到噬菌体质粒中。使用携带scFv基因的噬菌体质粒电击穿孔转化SS320大肠杆菌感受态细胞。待SS320增殖到对数期后，加入辅助噬菌体感染。
最终获得库容为1.2×10 12、阳性率为88％的天然抗体噬菌体抗体库。通过随机挑选克隆进行测序,发现该抗体库基因家族与天然分布接近,CDR3区的氨基酸个数为3-20分布。测序中，未发现抗体重复序列，且88％的基因序列具有正确的读码框。上述结果表明，该噬菌体抗体库多样性良好，有效库容率很高。
实施例2.CLDN18.2稳定表达细胞株的制备
在本实施例中，制备了稳定表达CLDN18.2的CHO细胞株(CHO-CLDN18.2)以用于基于细胞的噬菌体展示筛选。其具体实验过程如下：将人CLDN18.2的cDNA序列克隆到pCDH慢病毒载体中，然后和慢病毒包装载体共同转染到293t细胞中。培养48或72小时后，收集富含慢病毒颗粒的细胞上清，直接感染CHO细胞、CT26细胞或SNU601细胞。将慢病毒感染后的 细胞用嘌呤霉素进行筛选培养，2-3周后使用CLDN18.2特异性抗体(IMAB362,Ganymed)进行流式细胞法来检测细胞的CLDN18.2表达，其结果如图1所示。
上述结果表明，经慢病毒转染和嘌呤霉素筛选，获得了稳定表达CLDN18.2的CHO细胞(命名为CHO-CLDN18.2细胞系)、CT26细胞(CT26-CLDN18.2)和SNU601细胞(SNU601-CLDN18.2)。
实施例3.从抗体噬菌体展示文库中筛选抗CLDN18.2抗体
利用CHO-CLDN18.2稳定表达细胞株，进行了三轮噬菌体展示文库的淘选。实验流程主要参考以下文献：Targeting membrane proteins for antibody discovery using phage display Jones ML et al.Scientific Reports 2016。
具体流程简述如下：将噬菌体文库和CHO-K1细胞孵育，离心后取上清液。再将所述上清液与CHO-CLDN18.2细胞孵育，以使对CLDN18.2具有特异性的噬菌体与细胞结合。离心后收集细胞沉淀，并清洗和收集细胞。然后使用75mM的柠檬酸钠缓冲液洗脱与细胞结合的噬菌体文库。中和噬菌体文库后，使用M13K07辅助噬菌体扩增100倍筛选后的噬菌体文库，然后以类似第一轮筛选的方法进行第二轮和第三轮筛选。噬菌体的富集情况通过每一轮筛选的起始噬菌体用量和筛选后收集到的噬菌体滴度监测，并用噬菌体文库和CHO或CHO-CLDN18.2稳定表达细胞株进行流式细胞检测。如图2所示的结果表明，从第二轮富集筛选开始，噬菌体展示文库可以特异性结合CHO-CLDN18.2，其结合强度随筛选次数增多而增强。
经过三轮筛选后得到富集的噬菌体文库。使用该文库侵染细菌后，涂于琼脂糖平板培养。挑取单克隆菌落，放入96孔深孔板中用含有氨苄青霉素和卡那霉素的2YT培养基于37℃震荡培养，得到含有单克隆噬菌体的上清。将单克隆噬菌体上清与CHO-CLDN18.2细胞或CT26-CLDN18.2细胞在4℃孵育1小时，随后用含有1mM EDTA和0.5％BSA的PBS(流式细胞缓冲液)清洗。加入PE标记的抗M13抗体(购自义翘神州)，并于4℃孵育30分钟。随后，使用流式细胞法检测噬菌体和细胞的结合，其结果如图3所示。
上述结果表明，筛选得到的单克隆噬菌体A1-A6均可以很好结合CHO-CLDN18.2细胞而不结合CHO对照细胞。
实施例4.重组抗体的制备
将单克隆噬菌体的重链和轻链可变区的cDNA序列分别克隆到已含有抗体恒定区的pcDNA3.4载体(Invitrogen)中，共获得6个单克隆抗体的重链和轻链表达质粒。使用PEI法将质粒转染到EXPI-293细胞(Invitrogen)中，瞬时转染7-10天，离心并收取上清液。将上清液经过protein A纯化，获得纯化的抗体。上述6个单克隆抗体分别命名为18.2-A1(A1)、18.2-A2(A2)、18.2-A3(A3)、18.2-A4(A4)、18.2-A5(A5)和18.2-A6(A6)。这些抗体的重链和轻链可变区的氨基酸序列以及编码序列如以下表1和表2所示，通过Kabat CDR系统确定的抗体的CDR序列如表3所示。重组抗体的重链和轻链恒定区序列如表4中所示。
表1.CLDN18.2抗体的VH和VL序列
Figure PCTCN2020101383-appb-000001
Figure PCTCN2020101383-appb-000002
表2.CLDN18.2抗体的VH和VL的编码序列
Figure PCTCN2020101383-appb-000003
Figure PCTCN2020101383-appb-000004
Figure PCTCN2020101383-appb-000005
表3.抗体的Kabat CDR序列
Figure PCTCN2020101383-appb-000006
表4.重组抗体的重链和轻链恒定区序列
Figure PCTCN2020101383-appb-000007
Figure PCTCN2020101383-appb-000008
实施例5.重组单克隆抗体的结合活性检测
检测了重组单克隆抗体对CLDN18.2的结合活性。具体而言，取生长良好的CT26-CLDN18.2稳定表达细胞株。将细胞用PBS清洗后，与倍比稀释的抗体混合，并在4℃孵育1小时。将细胞用流式细胞缓冲液清洗一次，加入PE标记抗人IgG抗体，并在4℃孵育30分钟。离心去除上清，使用流式细胞缓冲液清洗后，用流式细胞仪检测细胞表面的荧光强度。使用平均荧光强度计算每种抗体的相对结合活性。
18.2-A1(A1)、18.2-A2(A2)、18.2-A3(A3)、18.2-A4(A4)18.2-A5(A5)和18.2-A6(A6)抗体均不与对照CT26细胞株结合(结果未显示)，但可以很好地结合表达CLDN18.2的CT26细胞株，如图4所示。随着抗体的稀释，CT26-CLDN18.2的平均荧光强度逐渐减弱。图4中还显示了上述CLDN18.2抗体的相对结合活性(EC50)。
实施例6.重组抗体的结合特异性检测
人Claudin18蛋白有两个同源异构体，即CLDN18.1和CLDN18.2，这两个同源异构体共用除外显子1以外的其它外显子，并且CLDN18.1和CLDN18.2的外显子1序列高度相似，其中在第一个胞外区中仅有8个不同的氨基酸。CLDN18.1和CLDN18.2外显子1在各自不同的启动子的驱动下，特异性地在不同组织中表达。其中，CLDN18.1在肺上皮细胞中特异性表达，而CLDN18.2在胃上皮细胞中特异地表达。为了鉴定筛选出的CLDN18.2抗体的特异性，检测了抗体与CLDN18.1和CLDN18.2的结合。
具体而言，取生长良好的293t细胞，瞬时表达flag标签标记的CLDN18.1(CLDN18.1-Flag)或CLDN18.2(CLDN18.2-Flag)。转染后48小时，用胰酶消化细胞。经流式细胞缓冲液清洗后，将细胞与10μg/ml的同种型对照抗体或上述5株CLDN18.2抗体混合于200μl流式细胞缓冲液中，并在4℃孵育1小时。随后，将细胞用流式细胞缓冲液清洗一次，加入PE标记抗人IgG抗体，并在4℃孵育30分钟。离心去除上清，使用流式细胞缓冲液清洗两次，并在含有1％多聚甲醛和0.5％triton-X100的固定液中固定细胞并通透细胞膜。随后，使用APC标记的抗flag抗体与细胞孵育30分钟，并用流式细胞仪进行检测。
结果如图5所示，外源表达CLDN18.1-flag或CLDN18.2-flag的293t细胞中均可以检测到flag阳性细胞群，显示转染的CLDN18.1或CLDN18.2蛋白在细胞中良好表达，表达阳性率在20％左右。CLDN18.2抗体18.2-A1、18.2-A2、18.2-A3、18.2-A4、18.2-A5和18.2-A6均能够很好地结合CLDN18.2-flag转染的阳性293t细胞，但6株抗体均不与CLDN18.1阳性细胞结合。上述结果表明，这6株CLDN18.2抗体均能够特异性结合CLDN18.2而不结合CLDN18.1。
实施例7.CLDN18.2抗体结合细胞后被内吞的检测
已经报道了有些抗体与细胞表面的抗原结合后，可以引起抗原和抗体复合物的内吞，从而降低细胞表面抗原表达水平，并加速抗体在体内的代谢(Schrama D et al.2006,Nat Rev Drug Discov)。在本实施例中，在稳定表达CLDN18.2的SNU601细胞中检测了抗体18.2-A1、18.2-A3、18.2-A4和18.2-A6被细胞内吞的能力。具体来说，将浓度为10μg/ml的CLDN18.2抗体18.2-A1、18.2-A3、18.2-A4、18.2-A6或IMAB362抗体(如实施例2中所述)、空白对照(PBS)和同种型对照(ISO)与细胞分别在4℃或37℃孵育4小时。随后，在4℃进行洗涤，并用PE标记的抗人IgG抗体进行染色。经过4％福尔马林固定后，用 流式细胞仪检测细胞表面的抗体结合。
如图6中的结果所示，在4℃孵育条件下，IMAB362抗体和18.2-A1、18.2-A3、18.2-A4和18.2-A6抗体均可以很好地结合表达CLDN18.2的SNU601细胞。但在37℃孵育条件下，IMAB362抗体被细胞内吞，细胞表面的抗体染色明显下降；而18.2-A1、18.2-A3、18.2-A4和18.2-A6抗体没有发生明显内吞现象，在37℃条件下仍然可以很好地与细胞结合。
上述结果表明，IMAB362抗体可以引起细胞内吞作用，从而可能下调CLDN18.2抗原在细胞表面的表达水平。在体内，内吞作用还可以引起抗体代谢的改变，加速抗体在体内的清除速度。相反，本公开的CLDN18.2抗体不引起明显的细胞内吞作用，因而不会下调细胞表面的靶抗原CLDN18.2的水平，也不会影响抗体在体内的代谢。
实施例8.去岩藻糖化抗体的制备和纯化
进一步制备了CLDN18.2抗体的去岩藻糖化形式。具体而言，将抗体的重链和轻链编码序列克隆到能够稳定表达的哺乳动物GS表达载体中，重链和轻链编码序列均以CMV启动子驱动。将表达载体转染到经过无血清和悬浮培养驯化的CHO-K1细胞和Fut8基因敲除的驯化CHO-K1细胞株中。利用MSX筛选并挑选稳定表达细胞株。得到稳定表达细胞株后，用摇瓶培养12-14天，中间根据需要补充补料培养基。
随后，收集培养上清，过滤后通过protein A层析柱捕获表达的抗体。将抗体洗脱后经PBS透析，得到纯化的抗体。其中，在Fut8基因敲除的CHO-K1细胞株中表达的18.2-A1、18.2-A2、18.2-A3、18.2-A4、18.2-A5和18.2-A6抗体分别命名为18.2-A1F、18.2-A2F、18.2-A1F、18.2-A4F、18.2-A5F和18.2-A6F。
实施例9.去岩藻糖化抗体的糖基化修饰表征
随后，分析了在Fut8基因敲除的CHO-K1细胞株中表达的去岩藻糖化的抗体以及在CHO-K1细胞中产生的普通抗体的糖基化修饰。通过将100μg抗体在胰蛋白酶和糖基肽酶分解后纯化获得抗体糖基，然后进行荧光串联质谱分析。各种糖基的鉴定取决于其质荷比m/z，通过荧光检测的面积百分比计算糖基百分比。
糖基命名：
Figure PCTCN2020101383-appb-000009
Figure PCTCN2020101383-appb-000010
N-乙酰神经氨酸
Figure PCTCN2020101383-appb-000011
N-乙酰半乳糖胺
Figure PCTCN2020101383-appb-000012
岩藻糖
Figure PCTCN2020101383-appb-000013
甘露糖
Figure PCTCN2020101383-appb-000014
半乳糖
Figure PCTCN2020101383-appb-000015
N-羟乙酰神经氨酸
糖基编号定义：5个数字分别表达不同糖的个数：六碳糖(半乳糖，甘露糖，或者葡萄糖)，N-乙酰己糖胺(GlcNA或者GalNAc)，岩藻糖(Fucose，简称FUC)，N-乙酰神经氨酸(Neu5Ac)，和N-羟乙酰神经氨酸(Neu5Gc)。其中第三个数字表示岩藻糖的个数。如以下表4所示：
表4.糖基编号及其对应的糖链
Figure PCTCN2020101383-appb-000016
在CHO-K1细胞中产生的Claudin18.2-A1抗体的糖链分析结果如以下表5 中所示。
表5.Claudin18.2抗体的糖链分析结果
糖基编号 岩藻糖 比例％
33000 无 0.2
33100 有 0.3
34000 无 4.8
34100 有 45.0
44000 无 3.3
44100 有 37.7
45000 无 0.3
45100 有 0.2
54100 有 7.0
54110 有 0.3
54101 有 0.2或0
54120 有 0.3
在Fut8基因敲除的CHO-K1细胞中产生的三批去岩藻糖化的Claudin18.2抗体(18.2-A1及18.2-A4)的糖链分析结果如表6中所示。
表6.去岩藻糖化的18.2-A1及18.2-A4抗体的糖链分析结果
Figure PCTCN2020101383-appb-000017
根据以上结果，显示在CHO-K1细胞中产生的普通Claudin18.2抗体的糖基结构中，91.0％包含岩藻。而在Fut8基因敲除的CHO-K1细胞中产生的三批去岩藻糖化的Claudin18.2抗体的糖基中基本不含有可检测的岩藻糖。
实施例10.去岩藻糖化抗体的结合活性检测
通过流式细胞法检测去岩藻糖化抗体18.2-A1F、18.2-A3F和18.2-A4F相比于18.2-A1、18.2-A3和18.2-A4与CLDN18.2的结合活性，其检测方法同实施例5中所述，实验结果如图7所示。
该结果表明，去岩藻糖化的CLDN18.2抗体和普通抗体对CLDN18.2的生物学结合活性没有显著差异。
实施例11.CLDN18.2抗体及其去岩藻糖化形式与FcγRIIIa的结合活性检测
随后，使用Biacore方法检测CLDN18.2抗体及其去岩藻糖化形式与FcγRIIIa的结合活性。具体而言，将FcγRIIIa(Sino Biological Inc,10389-H08C1)用HBS-EP缓冲液稀释至0.1μg/ml作为配体，并分别将18.2-A1F和18.2-A1抗体样品稀释至360μg/ml、120μg/ml、40μg/ml、13.3μg/ml和4.4μg/ml，作为分析物。采用间接捕获的方法固定配体FcγRIIIa，首先用50μg/ml的Anti-His IgG通过氨基偶联共价结合于CM5芯片表面，之后再结合配体和分析物。在Biacore Wizard模式下使用多循环方式，以FcγRIIIa为配体和18.2-A1F和18.2-A1抗体样品为分析物，进行亲和力分析实验。
每个样品的测试包括3个Start up、1个零浓度对照、5个梯度浓度样品和1个重复样品(参考品)。每个循环结束之后用10mM甘氨酸-HCl，pH 1.5再生液使芯片再生。分析物的每个浓度循环设置捕获时间60s，配体溶液流速10μl/min；配体与分析物结合时间180s，分析物溶液流速30μl/min；解离时间180s。将偶联有Anti-His IgG的CM5芯片置于插槽内进行检测分析。将原始数据导入BIACORETM X100分析软件，扣除零浓度对照，并且扣除参比通道以消除容积效应，用亲和力分析方法以稳态模式拟合图形，整理数据。
从图8的结果可以看出，18.2-A1抗体及其去岩藻糖化形式18.2-A1F均能够有效结合FcγRIIIa。并且，18.2-A1F抗体与FcγRIIIa结合的KD值明显低于18.2-A1抗体。该结果表明，去岩藻糖化的18.2-A1F抗体与FcγRIIIa的结合活 性显著增强。
实施例12.CLDN18.2抗体及其去岩藻糖化形式与细胞表面的FcγRIIIa的结合活性检测
接下来我们进一步检测了CLDN18.2抗体及其去岩藻糖化的形式与细胞表面表达的FcγRIIIa的结合活性。具体而言，将人FcγRIIIa(V158，高FC结合亚型)基因克隆到带有嘌呤霉素筛选标记的哺乳动物细胞表达载体并转染到Jurkat细胞株中。用嘌呤霉素筛选稳定表达FcγRIIIa的细胞株(FcγRIIIa-Jurkat)。此外，NK92MI是人NK细胞株，其天然表达FcγRIIIa受体(F158，低FC结合亚型)。通过流式细胞法检测CLDN18.2抗体及其去岩藻糖化的形式与FcγRIIIa-jurkat细胞和NK92MI细胞表面表达的FcγRIIIa的结合能力。具体的流式细胞检测方法参照实施例6中所述。
流式细胞检测结果如图9所示。CLDN18.2抗体18.2-A1和18.2-A4及其去岩藻糖化形式18.2-A1F和18.2-A4F均能够结合FcγRIIIa-jurkat细胞，而不结合未转染的Jurkat细胞(结果未显示)。并且，去岩藻糖化的18.2-A1F和18.2-A4F抗体能够更好地结合FcγRIIIa-Jurkat细胞和NK92MI细胞，其EC50明显小于CLDN18.2抗体18.2-A1和18.2-A4抗体。上述结果表明，去岩藻糖化的CLDN18.2抗体具有显著改善的结合Fc受体FcγRIIIa的能力。
实施例13.CLDN18.2抗体及其去岩藻糖化形式活化FcγRIIIa受体
FcγRIIIa受体结合抗体FC区后，可以激活效应细胞内的NF-AT转录因子通路。因此，检测NF-AT介导的报告基因强度可以反映FcγRIIIa受体的激活强度。在本实施例中，在荧光素酶报告基因表达载体中插入含有NF-AT结合位点的启动子，并稳定转染到Jurkat细胞株中。同时，将高FC结合的FcγRIIIa(V158)也稳定转染到Jurkat细胞中，构建了可以感受FcγRIIIa受体激活强度的功能细胞株(FcγRIIIa-Jurkat)。
取稳定表达CLDN18.2的SNU601胃癌细胞(SNU601-CLDN18.2)，稀释至4x10 5/ml，并与FcγRIIIa-Jurkat细胞以1:6的比例混合。取100μl混合的细胞加入96孔板，然后分别加入倍比稀释的抗体，于37℃静止培养6小时。随后，用One-Glo试剂盒(promega)检测荧光素酶活性。
结果如图10所示。与在CHO-K1细胞中产生的18.2-A1和18.2-A4抗体相 比，在Fut8基因敲除的CHO细胞株中表达的去岩藻糖化的抗体18.2-A1F和18.2-A4F诱导的FcγRIIIa受体活性显著增加，其EC50值提高约10-20倍。
实施例14.联合化疗药物EOF增强CLDN18.2抗体诱导的FcγRIIIa受体激活
包括EOF(表柔比星，Epirubicin；奥沙利铂，Oxaliplatin；5-氟尿嘧啶，5-FU)在内的联合化疗方案是胃癌现有的主要治疗手段。对EOF敏感细胞株在EOF处理后，会发生不同程度的细胞分裂周期阻断，增殖抑制和凋亡。
KATOIII细胞是一种人胃上皮癌细胞株，其表达很低水平的CLDN18.2，在流式细胞检测中仅有约5.2％细胞显示明显阳性(图11)。由于低水平的CLDN18.2表达，在与KATOIII细胞和FcγRIIIa-Jurkat细胞共孵育的抗体诱导的FcγRIIIa受体活化实验中(实验方法如实施例13中所述)，18.2-A1F和18.2-A6F抗体不引起FcγRIIIa受体活化(图12A)。
进一步，使用亚致死剂量的EOF(表柔比星：300nM；奥沙利铂：130nM；5-氟尿嘧啶：561.3nM)处理KATOIII细胞48小时后，经显微镜观察和流式细胞检测，发现细胞体积增大，变圆，分裂期细胞减少。表明细胞停留在分裂期。但细胞仍然存活，未出现明显凋亡现象(结果未显示)。将EOF处理过的KATOIII细胞与FcγRIIIa-Jurkat细胞和不同浓度的CLDN18.2抗体共孵育。结果表明，相比于同种型对照(ISO),18.2-A1F和18.2-A6F抗体可以诱导显著的FcγRIIIa受体激活(图12B)。
以上结果表明，目前常用的化疗药物EOF对胃癌细胞有生长抑制作用。EOF处理后，可以增强CLDN18.2抗体诱导的FcγRIIIa受体激活。不希望受到理论束缚，该效果可能是由于化疗药物的处理通过上调细胞上的抗原表达和其它机制促进CLDN18.2抗体的ADCC效应。
实施例15.抗体对表达CLDN18.2的肿瘤细胞的杀伤
用Ficoll-Paque Plus(GE Healthcare)分离健康人(供体1和供体2)外周血白细胞(PBMC)，在37℃培养过夜。取稳定表达CLDN18.2的SNU601胃癌细胞(SNU601-CLDN18.2)并稀释至1x10 5/ml，并PBMC稀释至5x10 6/ml，将两者等体积混合，效应细胞和靶细胞的比率为50:1。取100μl混合的细胞加入96孔板，然后分别加入倍比稀释的抗体，于37℃静止培养24小时。随后，使用乳酸脱氢酶(LDH)法(promega)检测细胞活率，其通过酶标仪测定OD490下的吸光 度。杀伤率计算方法如下：
最小释放组：靶细胞单独培养
最大释放组：靶细胞+裂解液
实验组：靶细胞+效应细胞+CLDN18.2抗体
对照组：靶细胞+效应细胞+阴性对照抗体
杀伤率(％)＝(实验组-最小释放组)/(最大释放组-最小释放组)％×100％
结果如图13所示。阴性对照抗体不显示明显的细胞杀伤(结果未显示)，CLDN18.2抗体18.2-A1及去岩藻糖化的抗体18.2-A1F和18.2-A4F均能够有效地引起表达CLDN18.2的靶细胞死亡，其最大杀伤率可以达到80-90％。此外，比较去岩藻糖化的抗体和普通抗体，可以得到去岩藻糖化的抗体对靶细胞的最大杀伤率和IC50值均明显高于普通抗体。
实施例16.CLDN18.2抗体的CDC活性
取稳定表达CLDN18.2的HELA细胞，用含1％灭活补体的血清的培养基重悬计数，并将细胞浓度调整至2x10 5/ml，细胞活力大于90％。在96孔板中每孔加入50μl细胞，然后分别加入倍比稀释的抗体和50μl稀释后的人血清。将细胞于37℃孵育2小时。随后，用CCK8法测量细胞裂解率。细胞裂解率的计算方法如下：
最小释放组：靶细胞
最大释放组：靶细胞+裂解液
实验组：靶细胞+CLDN18.2抗体+补体
阴性对照组：靶细胞+阴性对照抗体+补体
杀伤率(％)＝(实验组-最小释放组)/(最大释放组-最小释放组)×100％
结果如图14所示。CLDN18.2抗体18.2-A1和18.2-A4及其去岩藻糖化形式18.2-A1F和18.2-A4F均具有明显的CDC活性，对靶细胞的最大杀伤率均大于95％。此外，去岩藻糖化形式的抗体和普通抗体相比，其CDC活性没有显著差异。
实施例17.CLDN18.2抗体的体内肿瘤杀伤活性
通过对NPG免疫缺陷小鼠皮下接种SNU601-CLDN18.2细胞和Ficoll分离的人PBMC细胞(SNU601-CLDN18.2细胞：PBMC为1:0.8)，建立SNU601细胞 的小鼠异种移植模型。随后，通过腹腔注射向接种了SNU601肿瘤细胞的NPG免疫缺陷小鼠注射10mg/kg或5mg/kg剂量的18.2-A1F抗体18.2-A1F，或空白对照(PBS)或IgG同种型对照(ISO)，每组n＝15只小鼠。在肿瘤接种后每3天给药一次，并测量小鼠体内肿瘤的体积。
如图15中的结果显示，与PBS组和IgG同种型对照抗体组相比，5mg/kg和10mg/kg剂量的18.2-A1F抗体处理组均显示出良好的肿瘤抑制效果，且5mg/kg和10mg/kg组之间没有明显的效果差别。
实施例18.CLDN18.2抗体和化疗药物联合的体内肿瘤杀伤活性
如实施例14中的结果所示，在体外EOF联合化疗可以提高CLDN18.2抗体诱导的FcγRIIIa受体激活。KATOIII在体外IMDM培养基中培养时表达很低水平的CLDN18.2，仅有约5.2％细胞在流式细胞检测中显示明显阳性(图11)。经流式细胞分选后，选择CLDN18.2高表达的KATOIII细胞(KATOIII-18.2High)接种NPG免疫缺陷小鼠，进行异种移植模型实验(图16)。
在NPG免疫缺陷小鼠皮下接种KATOIII-18.2High细胞和Ficoll分离的人PBMC细胞(KATOIII-18.2High细胞：PBMC为1:0.8)，建立KATOIII-18.2High细胞的异种移植模型。随后，通过腹腔注射向接种了KATOIII-18.2High肿瘤细胞的NPG免疫缺陷小鼠注射同种型对照抗体(ISO)、EOF(表柔比星:1mg/kg；奥沙利铂:3mg/kg；5-氟尿嘧啶:30mg/kg)、10mg/kg剂量的18.2-A1F抗体，或EOF和18.2-A1F，每组n＝6只小鼠。在肿瘤接种后每3天给药一次，并测量小鼠体内肿瘤的体积。
如图17中的结果显示，与EOF组和对照抗体组相比，18.2-A1F可以有效抑制肿瘤生长，18.2-A1F与EOF联用进一步显著降低肿瘤生长，表明其与化疗联用在癌症的临床治疗中的潜力。

Claims (39)

  1. 一种结合CLDN18.2的抗体或其抗原结合片段，所述抗体具有以下重链CDR(CDRH)和轻链CDR(CDRL)：
    a.如SEQ ID NO:1中所示的重链可变区(VH)中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:6中所示的轻链可变区(VL)中的CDRL1、CDRL2和CDRL3；
    b.如SEQ ID NO:11中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:16中所示的VL中的CDRL1、CDRL2和CDRL3；
    c.如SEQ ID NO:21中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:26中所示的VL中的CDRL1、CDRL2和CDRL3；
    d.如SEQ ID NO:31中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:36中所示的VL中的CDRL1、CDRL2和CDRL3；
    e.如SEQ ID NO:41中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:46中所示的VL中的CDRL1、CDRL2和CDRL3；或
    f.如SEQ ID NO:55中所示的VH中的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:60中所示的VL中的CDRL1、CDRL2和CDRL3。
  2. 如权利要求1所述的抗体或其抗原结合片段，所述抗体具有以下CDRH和CDRL：
    a.如SEQ ID NO:3-5中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:8-10中所示的CDRL1、CDRL2和CDRL3；
    b.如SEQ ID NO:13-15中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:18-20中所示的CDRL1、CDRL2和CDRL3；
    c.如SEQ ID NO:23-25中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:28-30中所示的CDRL1、CDRL2和CDRL3；
    d.如SEQ ID NO:33-35中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:38-40中所示的CDRL1、CDRL2和CDRL3；
    e.如SEQ ID NO:43-45中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:48-50中所示的CDRL1、CDRL2和CDRL3；
    f.如SEQ ID NO:57-59中所示的CDRH1、CDRH2和CDRH3；和如SEQ ID NO:62-64中所示的CDRL1、CDRL2和CDRL3。
  3. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述抗体具有以下VH和VL：
    a.包含SEQ ID NO:1的氨基酸序列的VH，和包含SEQ ID NO:6的氨基酸序列的VL；
    b.包含SEQ ID NO:11的氨基酸序列的VH，和包含SEQ ID NO:16的氨基酸序列的VL；
    c.包含SEQ ID NO:21的氨基酸序列的VH，和包含SEQ ID NO:26的氨基酸序列的VL；
    d.包含SEQ ID NO:31的氨基酸序列的VH，和包含SEQ ID NO:36的氨基酸序列的VL；
    e.包含SEQ ID NO:41的氨基酸序列的VH，和包含SEQ ID NO:46的氨基酸序列的VL；
    f.包含SEQ ID NO:55的氨基酸序列的VH，和包含SEQ ID NO:60的氨基酸序列的VL。
  4. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其中所述抗体具有Fc区。
  5. 如权利要求4所述的抗体或其抗原结合片段，其中所述抗体在Asn297处具有糖基结构修饰，其中根据EU编号系统编号。
  6. 如权利要求5所述的抗体或其抗原结合片段，其中所述糖基结构中具有岩藻糖的糖基结构的比例为50％或更少。
  7. 如权利要求6所述的抗体或其抗原结合片段，其中所述糖基结构中具有岩藻糖的糖基结构的比例为30％或更少，例如20％或更少，10％或更少，5％或更少，2％或更少或1％或更少。
  8. 如权利要求6所述的抗体或其抗原能结合片段，其中所述抗体中具有岩藻糖的糖基结构的比例为0％-1％。
  9. 如权利要求6-8中任一项所述的抗体或其抗原结合片段，其中所述抗体由Fut8基因敲除的细胞产生。
  10. 如权利要求9所述的抗体或其抗原结合片段，其中所述细胞选自CHO细胞和HEK293细胞。
  11. 如权利要求9或10所述的抗体或其抗原结合片段，其中相比于在不具有Fut8基因敲除的细胞中产生的抗体，所述抗体具有增加的FcγRIIIa结合活 性。
  12. 如权利要求9-11中任一项所述的抗体或其抗原结合片段，其中相比于在不具有Fut8基因敲除的细胞中产生的抗体，所述抗体具有增加的ADCC活性。
  13. 如权利要求1-12中任一项所述的抗体或其抗原结合片段，其中所述抗体是单克隆抗体。
  14. 如权利要求1-12中任一项所述的抗体或其抗原结合片段，其中所述抗体是双特异性抗体或多特异性抗体。
  15. 如权利要求1-14中任一项所述的抗体或其抗原结合片段，其中所述抗体选自IgG、IgA、IgM、IgE和IgD同种型。
  16. 如权利要求1-14中任一项所述的抗体或其抗原结合片段，其中所述抗体选自IgG1、IgG2、IgG3和IgG4亚型。
  17. 如权利要求1-16中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段选自Fab片段、Fab’片段、F(ab’) 2片段、Fd片段、Fd’片段、Fv片段、scFv片段、ds-scFv片段、dAb片段、单链片段、二价抗体和线性抗体。
  18. 一种核酸分子，所述核酸分子包含编码如权利要求1-17中任一项的抗体或其抗原结合片段的核苷酸序列。
  19. 一种载体，所述载体包含如权利要求18所述的核酸分子。
  20. 一种宿主细胞，所述宿主细胞包含如权利要求18所述的核酸分子或如权利要求19所述的载体。
  21. 一种缀合物，所述缀合物包含与治疗剂、诊断剂或显像剂缀合的如权利要求1-17中任一项所述的抗体或其抗原结合片段。
  22. 一种组合物，所述组合物包含如权利要求1-17中任一项所述的抗体或其抗原结合片段或如权利要求21所述的缀合物，和一种或多种药学上可接受的载体、赋形剂和/或稀释剂。
  23. 如权利要求22所述的组合物，其中所述组合物还包含一种或多种另外的治疗剂。
  24. 权利要求23所述的组合物，其中所述治疗剂选自抗体、化疗药物和小分子药物。
  25. 权利要求24所述的组合物，其中所述化疗药物选自表柔比星(Epirubicin)、奥沙利铂(Oxaliplatin)和5-氟尿嘧啶(5-FU)的一种或多种。
  26. 一种治疗受试者中与CLDN18.2的表达相关的疾病的方法，所述方法包括对所述受试者施用如权利要求1-17中任一项所述的抗体或其抗原结合片段，如权利要求21所述的缀合物，或如权利要求22-25中任一项所述的组合物的步骤。
  27. 如权利要求26所述的方法，其中所述疾病是癌症。
  28. 如权利要求27所述的方法，其中所述癌症选自胃癌、胆管癌、食道癌和胰腺癌。
  29. 如权利要求26-28中任一项所述的方法，其中所述方法还包括对所述受试者施用一种或多种另外的疗法的步骤。
  30. 如权利要求29所述的方法，其中所述另外的疗法选自化学疗法、放射疗法、免疫疗法和手术治疗。
  31. 如权利要求30所述的方法，其中所述免疫疗法选自针对免疫检查点分子的疗法、CAR-T细胞治疗和CAR-NK细胞治疗。
  32. 如权利要求30所述的方法，其中所述化学疗法选自包括表柔比星、奥沙利铂和5-氟尿嘧啶的联合化疗方案。
  33. 如权利要求1-17中任一项所述的抗体或其抗原结合片段，如权利要求21所述的缀合物，或如权利要求22-25中任一项所述的组合物在治疗受试者中与CLDN18.2的表达相关的疾病中的用途。
  34. 如权利要求1-17中任一项所述的抗体或其抗原结合片段，如权利要求21所述的缀合物，或如权利要求22-25中任一项所述的组合物在制备用于治疗受试者中与CLDN18.2的表达相关的疾病的药物中的用途。
  35. 如权利要求33或34所述的用途，其中所述疾病是癌症。
  36. 如权利要求35所述的用途，其中所述癌症选自胃癌、胆管癌、食道癌和胰腺癌。
  37. 一种多肽，所述多肽具有选自SEQ ID NO:1、6、11、16、21、26、31、36、41、46、55和60的氨基酸序列。
  38. 一种核酸分子，所述核酸分子具有选自SEQ ID NO:2、7、12、17、22、27、32、37、42、47、56和61的核苷酸序列。
  39. 一种载体，所述载体包含如权利要求38所述的核酸分子。
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114539410A (zh) * 2022-03-15 2022-05-27 苏州量化细胞生物科技有限公司 Cldn18.2结合抗体、探针及在cldn18.2表达细胞的单细胞测序中的应用
WO2022140388A1 (en) 2020-12-21 2022-06-30 Allogene Therapeutics, Inc. Protease-activating cd45-gate car
WO2022165233A1 (en) 2021-01-29 2022-08-04 Allogene Therapeutics, Inc. KNOCKDOWN OR KNOCKOUT OF ONE OR MORE OF TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP AND RFXANK TO MITIGATE T CELL RECOGNITION OF ALLOGENEIC CELL PRODUCTS
US11407828B2 (en) 2019-02-01 2022-08-09 Novarock Biotherapeutics, Ltd. Anti-CLauDiN 18 antibodies and methods of use thereof
WO2022253156A1 (en) * 2021-05-31 2022-12-08 Shijiazhuang Yiling Pharmaceutical Co., Ltd. Monoclonal antibodies against cldn18.2 and fc-engineered versions thereof
WO2022266219A1 (en) * 2021-06-15 2022-12-22 Xencor, Inc. Heterodimeric antibodies that bind claudin18.2 and cd3
WO2023109953A1 (zh) * 2021-12-17 2023-06-22 信达生物制药(苏州)有限公司 靶向Claudin18.2的抗体-药物偶联物
US11739144B2 (en) 2021-03-09 2023-08-29 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CLDN6
WO2024026445A1 (en) 2022-07-29 2024-02-01 Allogene Therapeutics Inc. Engineered cells with reduced gene expression to mitigate immune cell recognition

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114790243B (zh) * 2022-06-07 2023-04-28 威海见生生物技术有限公司 一种dc瘤苗及其制备的药物组合物
WO2024067759A1 (zh) * 2022-09-29 2024-04-04 北京诺诚健华医药科技有限公司 一种能够结合cldn18.2的抗体或其抗原结合片段及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016180468A1 (en) * 2015-05-11 2016-11-17 Biontech Cell & Gene Therapies Gmbh Claudin-18.2-specific immunoreceptors and t cell epitopes
EP3099706B1 (en) * 2014-01-29 2018-10-31 BioNTech AG Peptide mimotopes of claudin 18.2 and uses thereof
CN109172820A (zh) * 2012-05-23 2019-01-11 加尼梅德药物公司 用于治疗癌症的涉及针对密蛋白18.2之抗体的联合治疗

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RS57259B1 (sr) * 2012-12-24 2018-08-31 Abbvie Inc Prolaktin receptor vezujući proteini i njihova upotreba
CN109762067B (zh) 2019-01-17 2020-02-28 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109172820A (zh) * 2012-05-23 2019-01-11 加尼梅德药物公司 用于治疗癌症的涉及针对密蛋白18.2之抗体的联合治疗
EP3099706B1 (en) * 2014-01-29 2018-10-31 BioNTech AG Peptide mimotopes of claudin 18.2 and uses thereof
WO2016180468A1 (en) * 2015-05-11 2016-11-17 Biontech Cell & Gene Therapies Gmbh Claudin-18.2-specific immunoreceptors and t cell epitopes

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
CHEN, SIYUAN ET AL.: "Advances in the application of claudins to tumor therapy", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 35, no. 6, 25 June 2019 (2019-06-25), pages 931 - 941, XP055835295 *
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 877 - 883
CHOTHIALESK, J. MOL. BIOL, vol. 196, 1987, pages 901 - 917
JEFFERIS R., NAT. REV. DRUG. DISC, 2009
JONES ML ET AL., SCIENTIFIC REPORTS, 2016
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877
MYERSMILLER, CABIOS, vol. 4, 1988, pages 11 - 17
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
PEARSONLIPMAN, PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 2444 - 2448
SCHRAMA D ET AL., NAT REV DRUG DISCOV, 2006
SMITH ET AL., ADV. APPL. MATH., vol. 2, 1981, pages 482
WOLL S. ET AL., INT. J. CANCER, 2013
XU, LIANG'E ET AL.: "Advances of CLDN18.2 protein in the therapy of malignant tumors", CHINESE JOURNAL OF CLINICAL ONCOLOGY, vol. 46, no. 6, 30 March 2019 (2019-03-30), pages 311 - 315, XP009528623 *
ZHU, G.Y. ET AL.: "Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer", SCIENTIFIC REPORTS, vol. 9, 10 June 2019 (2019-06-10), XP055630964, DOI: 20200909151430A *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11407828B2 (en) 2019-02-01 2022-08-09 Novarock Biotherapeutics, Ltd. Anti-CLauDiN 18 antibodies and methods of use thereof
WO2022140388A1 (en) 2020-12-21 2022-06-30 Allogene Therapeutics, Inc. Protease-activating cd45-gate car
WO2022165233A1 (en) 2021-01-29 2022-08-04 Allogene Therapeutics, Inc. KNOCKDOWN OR KNOCKOUT OF ONE OR MORE OF TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP AND RFXANK TO MITIGATE T CELL RECOGNITION OF ALLOGENEIC CELL PRODUCTS
US11739144B2 (en) 2021-03-09 2023-08-29 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CLDN6
WO2022253156A1 (en) * 2021-05-31 2022-12-08 Shijiazhuang Yiling Pharmaceutical Co., Ltd. Monoclonal antibodies against cldn18.2 and fc-engineered versions thereof
WO2022266219A1 (en) * 2021-06-15 2022-12-22 Xencor, Inc. Heterodimeric antibodies that bind claudin18.2 and cd3
WO2023109953A1 (zh) * 2021-12-17 2023-06-22 信达生物制药(苏州)有限公司 靶向Claudin18.2的抗体-药物偶联物
CN114539410A (zh) * 2022-03-15 2022-05-27 苏州量化细胞生物科技有限公司 Cldn18.2结合抗体、探针及在cldn18.2表达细胞的单细胞测序中的应用
CN114539410B (zh) * 2022-03-15 2023-09-05 苏州量化细胞生物科技有限公司 Cldn18.2结合抗体、探针及在cldn18.2表达细胞的单细胞测序中的应用
WO2024026445A1 (en) 2022-07-29 2024-02-01 Allogene Therapeutics Inc. Engineered cells with reduced gene expression to mitigate immune cell recognition

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