WO2020228806A1 - 针对密蛋白18a2的抗体及其应用 - Google Patents

针对密蛋白18a2的抗体及其应用 Download PDF

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WO2020228806A1
WO2020228806A1 PCT/CN2020/090427 CN2020090427W WO2020228806A1 WO 2020228806 A1 WO2020228806 A1 WO 2020228806A1 CN 2020090427 W CN2020090427 W CN 2020090427W WO 2020228806 A1 WO2020228806 A1 WO 2020228806A1
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seq
antibody
antigen
cells
cancer
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French (fr)
Inventor
杨莹莹
李高
王亚宁
安振明
赵树雍
刘玉雪
刘世聪
张美娟
姜锦锦
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Qilu Pharmaceutical Co Ltd
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Qilu Pharmaceutical Co Ltd
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Priority to CA3138414A priority Critical patent/CA3138414A1/en
Priority to AU2020273695A priority patent/AU2020273695B2/en
Priority to CN202080036234.5A priority patent/CN113825771B/zh
Priority to US17/611,551 priority patent/US12415853B2/en
Priority to BR112021023024A priority patent/BR112021023024A2/pt
Priority to JP2021568260A priority patent/JP7407841B2/ja
Priority to EP20805122.7A priority patent/EP3971208A4/en
Priority to KR1020217040715A priority patent/KR102958621B1/ko
Publication of WO2020228806A1 publication Critical patent/WO2020228806A1/zh
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Definitions

  • the present invention belongs to the field of immunology. More specifically, the present invention relates to antibodies or antigen-binding fragments thereof against claudin 18.2 (Claudin 18.2, CLDN18A2, CLDN18.2), derivatives and drugs containing the antibodies or antigen-binding fragments thereof The composition and its related application in the treatment of cancer.
  • claudin 18.2 Claudin 18.2, CLDN18A2, CLDN18.2
  • derivatives and drugs containing the antibodies or antigen-binding fragments thereof The composition and its related application in the treatment of cancer.
  • Claudin is an integral membrane protein containing the main structural proteins of tight junctions, which are the top cell-cell adhesion junctions in polarized cell types such as those found in epithelial cells or endothelial cell layers. Tight junctions are composed of multi-chain network proteins. These multi-chain network proteins form a continuous seal around the cell, providing a physical barrier for the transportation of solute and water in the space around the cell, but the physical barrier is adjustable.
  • the claudin family in humans contains at least 23 members, ranging in size from 22-34kDa. Although claudins are important for the function and stability of normal tissues, tumor cells often exhibit abnormal tight junction functions.
  • Individual claudin family members can be up-regulated in some cancer types, but down-regulated in other cancer types.
  • Claudin 18 is an intrinsic membrane protein located in the tight junctions of epithelium and endothelium, with a molecular weight of about 27.9KD. It forms tight junctions between cells together with other tight junction proteins, regulating the communication of tissue molecules and ions between cells. To maintain the stability of the environment within the organization.
  • claudin 18A1, CLDN18.1 GenBank registration number is NP_057453, NM016369, and splice variant 2 (CLDN18A2, CLDN18.2): GenBank registration number is NM_001002026, NP_001002026.
  • CLDN18A1 is selectively expressed in the epithelium of the lung, while CLDN18A2 is specifically expressed in differentiated cells of normal gastric epithelium, but not expressed in gastric epithelial stem cells with cell division activity.
  • CLDN18A2 is overexpressed in a variety of cancer types. For example, 75% of gastric cancer patients have high expression of CLDN18A2, 50% of pancreatic cancer patients have high expression of CLDN18A2, and 30% of esophageal cancer patients have high expression of CLDN18A2. There are also high expressions in other cancer types. Therefore, finding antibodies that specifically bind to CLDN18A2 but not CLDN18A1 is of great significance for cancer treatment and detection.
  • the existing CLDN18A2 antibody IMAB362 has entered the clinical research stage.
  • the clinical results show that in gastric cancer patients with high expression of CLDN18.2 (>70% of tumor cells with CLDN18.2 expression ⁇ 2+), chemotherapy + IMAB362 has no effect compared to chemotherapy alone.
  • CAR-T prepared against the CLDN18.2 target has also entered clinical research.
  • these antibodies (or antigen-binding fragments in CAR-T) that have entered the clinical stage have a weak affinity for claudin 18.2. Therefore, it is still necessary to continue to screen and prepare CLDN18.2 antibodies with higher affinity to produce stronger pharmacodynamic effects at the same dosage.
  • the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof specifically binds to CLDN 18.2 and does not significantly bind to CLDN 18.1.
  • the CLDN18.2 is a peptide with GenBank accession number NP_001002026 (mRNA: NM_001002026).
  • the CLDN18.1 is a peptide with GenBank accession number NP_057453 (mRNA: NM_016369).
  • the antibody or antigen-binding fragment thereof of the present invention does not show significant binding to CLDN 18.1.
  • the binding level of the antibody or antigen-binding fragment thereof to CLDN 18.1 does not exceed 20% of its binding level to CLDN 18.2.
  • the binding level may be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% of the binding level of the antibody or antigen-binding fragment to CLDN 18.2 , 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1%.
  • the level of binding of the antibody or antigen-binding fragment thereof of the present invention to CLDN18.2 is 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than 10 times.
  • the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof can specifically bind to CLDN18.2, comprising: a heavy chain variable region, the heavy chain variable The region contains 3 HCDRs selected from the following sequences: SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85; and/or, the light chain variable region, said light chain variable region comprising 3 selected from the LCDR shown in the following sequence: SEQ ID NO: 86 , 87, 88, 89, 90, 91, 92, 93
  • the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a variable region selected from SEQ ID NO: 37, 40, 43, 45, 49, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83; and selected from SEQ ID NO: 38, 41 , 46, 48, 50, 52, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84; and, selected from SEQ ID NO: 39, 42, 44, 47 , 51, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85; and/or, the light chain variable region comprises SEQ ID NO: 86, 87 ,88,89,90,91,92,111,112,113; and, selected from the LCDR2 shown in SEQ ID NO:93
  • the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising a selection HCDR1, HCDR2 and HCDR3 from the following group:
  • SEQ ID NO: 40 SEQ ID NO: 41 and SEQ ID NO: 42 or
  • the light chain variable region is selected from the group consisting of LCDR1, LCDR2 and LCDR3:
  • SEQ ID NO: 112 SEQ ID NO: 93 and SEQ ID NO: 107; or
  • SEQ ID NO: 112 SEQ ID NO: 93 and SEQ ID NO: 110; or
  • SEQ ID NO: 113 SEQ ID NO: 93 and SEQ ID NO: 110.
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the variable region includes any of the following 6 CDR, the 6 CDRs of each group are arranged in sequence according to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3:
  • the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region having the following The sequence has at least 80%, 85%, 90%, 95% or 100% sequence identity: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 , 27, 29, 31, 33, 35; and/or, the light chain variable region has at least 80%, 85%, 90%, 95% or 100% sequence identity with the following sequence: SEQ ID NO : 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,114,115,116,117,118,119.
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises at least 80% to 100% of the heavy chain variable region and light chain variable region of any one of the following groups Sequence identity of:
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention is a murine antibody, a chimeric antibody or a humanized antibody.
  • the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having at least the following sequence 80%, 85%, 90%, 95% or 100% sequence identity: SEQ ID NO: 120, 122, 125, 128; and/or, the light chain variable region has at least 80% sequence identity with the following sequence , 85%, 90%, 95% or 100% sequence identity: SEQ ID NO: 121, 123, 124, 126, 127.
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof comprises at least 80%, 85% of the heavy chain variable region and light chain variable region of any one of the following groups: , 90%, 95% or 100% sequence identity:
  • the anti-CLDN18.2 antibody is a monoclonal antibody.
  • the anti-CLDN18.2 antibody or antigen-binding fragment thereof further includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region includes Fc Or variant Fc, Fc is derived from murine or human.
  • the anti-CLDN18.2 antibody is a full-length antibody.
  • an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention is in the form of IgG1, IgG2, IgG3 or IgG4.
  • the antigen-binding fragments of the present invention are Fab, Fv, scFv, F(ab') 2 , linear antibodies, single domain antibodies.
  • the present invention provides a conjugate formed by coupling the aforementioned antibody or antigen-binding fragment thereof with a capture label or a detection label.
  • the detection label includes, but is not limited to, radionuclides, luminescent substances (such as fluorescein), colored substances or enzymes.
  • the present invention provides a bispecific antibody or multispecific antibody, one of the antigen binding domains in the bispecific antibody or multispecific antibody comprises the anti-CLDN18.2 antibody or Its antigen-binding fragment.
  • the present invention provides an antibody-drug conjugate containing the antibody or antigen-binding fragment thereof as described above.
  • the structure of the antibody-drug conjugate is well-known in the art, and it is formed by interconnecting antibody-linker-drug (toxin).
  • the present invention provides a chimeric antigen receptor whose extracellular recognition unit comprises the aforementioned antibody or antigen-binding fragment thereof.
  • the present invention provides nucleic acids encoding any of the aforementioned antibodies or antigen-binding fragments thereof. According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid.
  • the present invention provides a host cell comprising the expression vector or genome of the present invention integrated with the nucleic acid encoding the antibody or antigen-binding fragment thereof.
  • the host cell may be a prokaryotic cell, such as E. coli; it may also be a eukaryotic cell, such as yeast or mammalian cells, or a mammalian cell such as CHO cells or HEK293 cells.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment thereof, comprising: culturing the host cell of the present invention under suitable conditions, and purifying the expression product from the cell.
  • the present invention provides the use of the antibody or antigen-binding fragment thereof for the preparation of drugs specifically targeting tumor cells expressing CLDN18.2, such as monoclonal antibody drugs, antibody drug conjugates , Bispecific antibody or multispecific antibody; or used to prepare chimeric antigen receptor-modified immune cells; or used to prepare reagents for diagnosing tumors expressing CLDN18.2; in some embodiments, said expressing CLDN18.2
  • the tumors include: gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer and their metastases, such as Kukenberg's tumor.
  • the present invention provides a method for detecting the expression of CLDN18.2 in a sample, comprising: contacting the sample with the aforementioned anti-CLDN18.2 antibody or antigen-binding fragment thereof; detecting the anti-CLDN18.2 antibody or its The formation of a complex of the antigen-binding fragment and CLDN18.2; optionally, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is detectably labeled.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof of the present invention, or an effective amount of nucleic acid encoding the antibody or antigen-binding fragment thereof, or An effective amount of a recombinant vector containing an encoding nucleic acid, or an effective amount of a host cell containing an encoding nucleic acid, or an effective amount of the antibody drug conjugate of the present invention, or an effective amount of the chimeric antigen receptor of the present invention, Or it contains an effective amount of the bispecific antibody or multispecific antibody of the present invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more additional other therapeutic agents.
  • additional therapeutic agents include: cytotoxic agents, cytostatic agents, anti-angiogenesis agents, tumor reducing agents, chemotherapeutics, radiotherapeutics, targeted anti-cancer agents, biological response modifiers, cancer vaccines, cells Factors, hormones, anti-metastatic agents and immunotherapeutics.
  • the present invention provides a kit or kit, which includes a container, and the pharmaceutical composition of the present invention in the container.
  • the invention provides a method of inducing the death of a cell expressing CLDN 18.2, comprising contacting the cell with a pharmaceutical composition of the invention.
  • the cells are contacted with the pharmaceutical composition in vitro.
  • the cells are contacted with the pharmaceutical composition in vivo.
  • the cell is a cancer cell.
  • the cell is a solid tumor cell.
  • the cell is selected from: gastric cancer cells, esophageal cancer cells, colon cancer cells, pancreatic cancer cells, Wilms tumor cells, lung cancer cells, ovarian cancer cells, colon cancer cells, rectal cancer cells, liver cancer cells , Head and neck cancer cells, chronic myelogenous leukemia cells and gallbladder cancer cells.
  • the present invention provides a method of treating a disease associated with expression of CLDN 18.2 in a subject, the method comprising administering the pharmaceutical composition of the present invention to a subject in need thereof.
  • the disease is a tumor.
  • the tumor is preferably gastric cancer, esophageal cancer, bowel cancer, pancreatic cancer, Wilms tumor, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, chronic myelogenous leukemia, or gallbladder cancer.
  • the method further includes administering an additional therapeutic agent to the subject.
  • the antibody of the present invention can be administered in combination with another additional therapeutic agent, including but not limited to chemotherapeutic agents, cytotoxic agents, radiotherapeutics, cancer vaccines, tumor reducing agents, targeted anticancer agents, and antiangiogenic agents , Biological response modifiers, cytokines, hormones, anti-metastatic agents and immunotherapeutics.
  • additional therapeutic agent including but not limited to chemotherapeutic agents, cytotoxic agents, radiotherapeutics, cancer vaccines, tumor reducing agents, targeted anticancer agents, and antiangiogenic agents , Biological response modifiers, cytokines, hormones, anti-metastatic agents and immunotherapeutics.
  • the targeted anticancer agent includes, but is not limited to, large molecule targeted drugs, small molecule targeted drugs, and the like.
  • macromolecular targeted drugs include, but are not limited to, targeted epidermal growth factor drugs, including cetuximab, panitumumab, and nimotuzumab; HER-2 or HER-3 Signal pathway inhibitors, including trastuzumab, pertuzumab and T-DM1; anti-vascular endothelial growth factor drugs, including VEGF-TRAP, bevacizumab and ramucirumumab; and, Drugs that target other targets, including but not limited to PI3K, PARP, PI3K ⁇ , PKB/AKT and STAT3.
  • targeted epidermal growth factor drugs including cetuximab, panitumumab, and nimotuzumab
  • HER-2 or HER-3 Signal pathway inhibitors including trastuzumab, pertuzumab and T-DM1
  • anti-vascular endothelial growth factor drugs including VEGF-TRAP, bevacizumab and ramucirumumab
  • small molecule targeted drugs include but are not limited to drugs that target epidermal growth factor, including erlotinib or gefitinib, etc.; HER-2 or HER-3 signaling pathway inhibitors, including Lapa Tinib or afatinib, etc.; Tyrosine kinase inhibitors, including imatinib or sunitinib, etc.; anti-vascular endothelial growth factor drugs, including sorafenib, regatinib, and pazotinib , Recombinant human endostatin and apatinib, etc.; targeted c-Met/ROS1 drugs, including crizotinib, etc.; and, other targeted drugs, including but not limited to vorinostat and masstatin Etc.; drugs targeting mTOR, including everolimus, etc.; and drugs targeting other targets, including but not limited to targets such as PI3K ⁇ , PKB/AKT and STAT3.
  • the immunotherapeutic drugs include but are not limited to immunosuppressants and agonists, and targets include PD-1/PD-L1, PD-L2, CTLA-4, LAG-3, IDO, TIM3, TIGIT , CD47, SIRP ⁇ , 4-1BB, CSF-1/CSF1R, GITR, OX40, CD40, CD27, CD28, B7H4, B7H3, TGF ⁇ , BTLA, VISTA, ICOS, CD39, CD73, A2AR, KIR and NKG2A, etc.; and Cell therapy related to immunotherapy.
  • targets include PD-1/PD-L1, PD-L2, CTLA-4, LAG-3, IDO, TIM3, TIGIT , CD47, SIRP ⁇ , 4-1BB, CSF-1/CSF1R, GITR, OX40, CD40, CD27, CD28, B7H4, B7H3, TGF ⁇ , BTLA, VISTA, ICOS, CD39, CD73, A2AR, K
  • immune checkpoint inhibitors targeting PD-1/PD-L1 include, but are not limited to, macromolecular drugs, such as pambrolizumab, nivolumab, atezolizumab, avitizumab And Sintilimab, Cemiplimab and Durvalumab, etc.; and, small molecule drugs.
  • macromolecular drugs such as pambrolizumab, nivolumab, atezolizumab, avitizumab And Sintilimab, Cemiplimab and Durvalumab, etc.
  • small molecule drugs such as pambrolizumab, nivolumab, atezolizumab, avitizumab And Sintilimab, Cemiplimab and Durvalumab, etc.
  • immune checkpoint inhibitors that target CTLA-4 include, but are not limited to, ipilimumab, etc.; cytokines include, but are not limited to, IL-10, IL-15, IL4, and IL13, etc.; those that target BRAF Inhibitors include but are not limited to Binimetinib and the like.
  • the other therapeutic agent is selected from oncolytic viruses, such as parvovirus, adenovirus, herpes virus, poxvirus, poliovirus, reovirus, alphavirus, Malaba virus, retrovirus, and Cossackie virus, etc.; or, another therapeutic agent is selected from cancer vaccines or protease inhibitors, such as bortezomib and the like.
  • oncolytic viruses such as parvovirus, adenovirus, herpes virus, poxvirus, poliovirus, reovirus, alphavirus, Malaba virus, retrovirus, and Cossackie virus, etc.
  • another therapeutic agent is selected from cancer vaccines or protease inhibitors, such as bortezomib and the like.
  • Figures 1-5 show the use of flow cytometry to detect the binding of the supernatants of the 18 hybridoma cell lines of the present invention to HEK293 cells stably transfected with hCLDN18.2.
  • Figures 6-11 show the binding of 18 chimeric antibodies of the present invention to HEK293 cells stably expressing hCLDN18.2.
  • Figures 12-17 show the binding of 18 chimeric antibodies of the present invention to cells derived from gastric cancer tumor tissues that naturally express hCLDN18.2.
  • Figures 18-23 show the ADCC results of 18 chimeric antibodies of the present invention on CHO-K1 cells stably expressing hCLDN18.2.
  • Figures 24-29 show the CDC results of the 18 chimeric antibodies of the present invention on CHO-K1 cells stably expressing hCLDN18.2.
  • Figure 30 shows the binding of the humanized antibody hu299B2-S32A of the present invention to HEK293 cells stably expressing hCLDN18.2.
  • Figure 31 shows the binding of the humanized antibody hu253C4-N31Q of the present invention to HEK293 cells stably expressing hCLDN18.2.
  • Figure 32 shows the binding of the humanized antibody hu299B2-S32A of the present invention to gastric cancer tumor tissue-derived cells that naturally express hCLDN18.2.
  • Figure 33 shows the binding of the humanized antibody hu253C4-N31Q of the present invention to cells derived from gastric cancer tumor tissue that naturally express hCLDN18.2.
  • Figure 34 shows the ADCC results of the humanized antibody hu299B2-S32A of the present invention on CHO-K1 cells stably expressing hCLDN18.2.
  • Figure 35 shows the ADCC results of the humanized antibody hu253C4-N31Q of the present invention on CHO-K1 cells stably expressing hCLDN18.2.
  • Figure 36 shows the CDC results of the humanized antibody hu299B2-S32A of the present invention on CHO-K1 cells stably expressing hCLDN18.2.
  • Figure 37 shows the CDC results of the humanized antibody hu253C4-N31Q of the present invention on CHO-K1 cells stably expressing hCLDN18.2.
  • the term "about” means to include ⁇ 20% of the specified value, or in some cases ⁇ 10%, or in some cases ⁇ 5%, or In some cases ⁇ 1%, or in some cases ⁇ 0.1% change.
  • anti-Claudin18.2 antibody refers to such An antibody that can bind to the CLDN18.2 protein or fragments thereof with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting CLDN18.2.
  • Human-derived CLDN18.2 protein is expressed as hCLDN18.2.
  • anti-human Claudin18.2 antibody specifically refers to the ability to bind human CLDN18.2 protein or fragments thereof with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting human CLDN18.2.
  • antibody typically refers to a Y-type tetramer comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions.
  • Natural IgG antibodies have this structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and constant region.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • the corresponding heavy chain constant domains are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ respectively.
  • IgG and IgA can be further divided into different For example, IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two distinct types based on the amino acid sequence of their constant domains, called kappa and lambda.
  • the constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4).
  • CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length proline and cysteine-rich segment.
  • Each type of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine residues.
  • variable region or “variable domain” shows a significant change in the amino acid composition from one antibody to another, and is mainly responsible for antigen recognition and binding.
  • the variable region of each light chain/heavy chain pair forms an antibody binding site, so that a complete IgG antibody has two binding sites (ie, it is bivalent).
  • the variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, called the hypervariable region (HVR), or more generally, the Complementarity determining region (CDR), VH and VL each have 4 framework regions FR, represented by FR1, FR2, FR3, FR4, respectively.
  • the CDR and FR sequences usually appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3 -HCDR3(LCDR3)-FR4.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Fc is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also called the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • antibodies in a broad sense can include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR grafted antibodies (CDR- grafted antibody), human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies ( Including mutant proteins and their variants) and so on.
  • full-length antibody can be used interchangeably herein and refer to an antibody whose structure is substantially similar to that of a natural antibody or has an Fc region.
  • monoclonal antibody refers to a substantially homogeneous antibody produced by a single cell clone and only directed against a specific epitope.
  • Monoclonal antibodies can be prepared using a variety of technologies known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of the above technologies.
  • chimeric antibody is a construct in which a part of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, and this or these The remainder of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, and fragments of such antibodies.
  • chimeric antibodies comprise all or most of the selected murine heavy and light chain variable regions operably linked to human light and heavy chain constant regions.
  • the constant region sequence or its variants or derivatives can be operably associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide anti-CLDN18 that can be used or incorporated into the present invention. 2 full-length antibodies.
  • humanized antibody is a hybrid immunoglobulin, immunoglobulin chain or fragments thereof containing minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody is a human immunoglobulin (acceptor antibody), in which the residues from the CDR of the acceptor are derived from a non-human species with the required specificity, affinity and performance (donor antibody) ) Residues in the CDR, such as mouse, rat, rabbit or primate.
  • donor antibody a human immunoglobulin
  • the framework region residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • back mutations can be introduced into a humanized antibody, in which residues in one or more FRs of the variable region of the recipient human antibody are replaced by corresponding residues from the donor antibody of the non-human species. replace. Such back mutations can help maintain the proper three-dimensional configuration of one or more grafted CDRs and thus improve affinity and antibody stability.
  • Antibodies from various donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates.
  • the humanized antibody may contain new residues not found in the recipient antibody or in the donor antibody in order to further improve antibody performance.
  • CDR and FR of the variable region of the monoclonal antibody of the present invention is determined according to the Kabat definition.
  • Other naming and numbering systems such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, based on the monoclonal antibody sequence of the present invention, a humanized antibody containing one or more CDRs derived from any naming system is clearly maintained within the scope of the present invention.
  • sequence identity or “sequence similarity” or “sequence homology” means that when the sequences are aligned (and gaps are introduced when necessary) to obtain the maximum percent sequence identity, and any After conservative substitutions are considered as part of sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence.
  • sequence alignment can be used for sequence alignment to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the measurement alignment, including any algorithm required to obtain the maximum alignment over the entire length of the sequence being compared.
  • antibody fragment encompasses at least a part of an intact antibody.
  • fragment of the antibody molecule includes the "antigen-binding fragment” of the antibody, and the term “antigen-binding fragment” refers to an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or its epitope
  • polypeptide fragments, or the fusion protein products further derived from the fragments, such as single-chain antibodies, extracellular binding regions in chimeric antigen receptors, etc.
  • Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab') 2 fragments, Fd fragments, Fv fragments, Single domain antibodies, linear antibodies, single chain antibodies (scFv), and bispecific antibodies or multispecific antibodies formed from antibody fragments.
  • VL variable light chain fragments
  • VH variable heavy chain fragments
  • Fab fragments F(ab') 2 fragments
  • Fd fragments Fv fragments
  • Single domain antibodies linear antibodies, single chain antibodies (scFv), and bispecific antibodies or multispecific antibodies formed from antibody fragments.
  • Fab fragment includes the variable region of the heavy chain and the variable region of the light chain, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain, which is a monovalent antibody fragment.
  • F(ab') 2 fragment includes 2 Fab fragments and a hinge region, which are bivalent antibody fragments.
  • Fd fragment generally includes the variable region of the heavy chain and the constant region CH1; the term “Fv fragment” contains the variable region of the heavy chain and the variable region of the light chain of an antibody, but does not have a constant region, and has a minimum of all antigen binding sites. Antibody fragments.
  • scFv refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH or may include VH-linker-VL.
  • multispecific antibody refers to the functional connection of an antibody (such as chemical coupling, gene fusion, non-covalent binding or other methods) to one or more other binding molecules, thereby forming a non-localized antibody. Spot and/or target binding to new antibody constructs.
  • bispecific antibodies are more commonly used, which specifically refer to antibody constructs with specificity to two different antigens.
  • bispecific antibodies or multispecific antibodies include at least two antigen binding domains.
  • an antigen refers to a substance recognized and specifically bound by an antibody or antibody-binding fragment.
  • an antigen can include any immunogenic fragment or determinant of the selected target, including single epitopes, multiple epitopes, and single structures. Domain, multi-domain, or complete extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, parts and combinations thereof can all constitute antigens.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens and the like.
  • Antigen can also refer to a molecule that triggers an immune response. Any form of antigen or cells or preparations containing the antigen can be used to generate antibodies specific for the epitope.
  • the antigen may be an isolated full-length protein, a cell surface protein (for example, immunized with cells expressing at least a part of the antigen on its surface), or a soluble protein (for example, only the ECD portion of the protein is used for immunization) or a protein Construct (e.g., Fc antigen).
  • the antigen can be produced in genetically modified cells. Any of the aforementioned antigens can be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art.
  • the DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA), and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform the cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation.
  • Methods for determining the epitope bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length may be linear, cyclic or branched, it may contain modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • modified amino acid polymers such as those that have been processed by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolysis, prenylation, and elimination.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a designated protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a designated nucleic acid sequence.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions in the polypeptide sequence.
  • amino acid substitution or “substitution” herein means to replace an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • substitution S32A refers to the replacement of serine at position 32 with alanine.
  • sequence identity or homology of the humanized antibody variable region and the human receptor variable region can be determined as discussed herein, and when so measured, will preferably share at least 60% or 65% of the sequence Identity, more preferably at least 70%, 75%, 80%, 85% or 90% sequence identity, even more preferably at least 93%, 95%, 98% or 99% sequence identity.
  • residue positions that are not identical differ due to conservative amino acid substitutions.
  • a "conservative substitution” is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue in a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions will not substantially change the functional properties of the protein.
  • the art has defined families of amino acid residues with similar side chains.
  • amino acids containing basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged acute side chains e.g. , Glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains for example, alanine, valine, leucine, isoleucine
  • Acid proline, phenylalanine, methionine
  • ⁇ -branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, benzene Alanine, tryptophan, histidine
  • one or more amino acid residues in the CDR region or framework region of the antibody of the present invention can be replaced with other amino acid residues of similar side chains.
  • the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution.
  • PTM post-translational modification
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • complement-dependent cytotoxicity refers to the cytotoxicity of complement, that is, through specific antibodies binding to the corresponding antigen on the cell membrane surface to form a complex to activate the classical pathway of complement, the formed attack complex against target cells Play a cracking effect.
  • the antibodies of the present invention may also include substitutions or modifications of constant regions (such as Fc), including but not limited to amino acid residue substitutions, mutations and/or modifications, which produce compounds with the following preferred characteristics, which include but not Limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered Fc ligand binding to Fc receptors (FcR), enhanced or weakened ADCC or CDC, altered glycosylation and/or disulfide bonds, and modified binding specificity.
  • constant regions such as Fc
  • Fc constant regions
  • affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen).
  • K D refers to the dissociation constant of a specific antibody-antigen interaction.
  • Various techniques known in the art can be used to determine binding affinity, such as surface plasmon resonance, biological layer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical overspeed Centrifugation and flow cytometry, etc.
  • competitive binding or “competitive antibody” generally refers to an antibody that binds to the same epitope as the antibody of the present invention, and its binding causes the binding of the antibody of the present invention to the epitope to be inhibited or blocked, in a competition assay The degree of competitive inhibition can be obtained.
  • composition refers to a formulation that exists in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered .
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle administered with the therapeutic agent.
  • the term "effective amount" refers to the dose of the pharmaceutical preparation of the antibody or fragment of the present invention, which produces the desired effect in the treated patient after being administered to the patient in single or multiple doses.
  • the effective amount can be easily determined by the attending physician as a person skilled in the art by considering the following factors: such as ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; The specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations.
  • the progeny of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.
  • transfection refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • stable transfection refers to the introduction and integration of exogenous nucleic acid, DNA or RNA into the genome of a transfected cell.
  • stable transfectant refers to a cell that stably integrates foreign DNA into genomic DNA.
  • nucleic acid molecule encoding refers to the sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Therefore, the nucleic acid sequence encodes an amino acid sequence.
  • the methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15.
  • the antibody or its antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to a non-human CDR region.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the Journal of Immunoglobulin, (2001) ISBN: 012441351.
  • the engineered antibody or antigen-binding fragment thereof of the present invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the term "individual” or “subject” as used herein refers to any animal, such as a mammal or a marsupial. Individuals of the present invention include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any species Of poultry.
  • non-human primates such as cynomolgus monkeys or rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cows, sheep, rats, and any species Of poultry.
  • ADC antibody drug conjugate
  • ADC antibody drug conjugate
  • ADC refers to an antibody that has been covalently coupled to a therapeutically active substance or active pharmaceutical ingredient (API), so that the therapeutically active substance or active pharmaceutical ingredient (API) can be targeted to the binding target of the antibody To show its pharmacological functions.
  • the therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing the cells targeted by the ADC, preferably malignant or cancer cells.
  • Covalent attachment of therapeutically active substances, active pharmaceutical ingredients or cytotoxins can be carried out in a non-site-specific manner using standard chemical linkers coupling the payload to lysine or cysteine residues, or, preferably, conjugation It is done in a site-specific manner, which allows complete control of the conjugation site and the ratio of drug to antibody of ADC produced.
  • CAR chimeric antigen receptor
  • CAR-engineered T cells are genetically engineered T cells equipped with chimeric receptors.
  • the extracellular recognition unit contains antibody-derived recognition domains and its intracellular region is derived from From the lymphocyte stimulation part.
  • the structure of the prototype CAR is modular and designed to accommodate various functional domains, thereby enabling selection of specificity and control of T cell activation.
  • the preferred antibody-derived recognition unit is a single chain variable fragment (scFv) that combines the specificity and binding residues of the heavy and light chain variable regions of a monoclonal antibody.
  • the most common lymphocyte activation part includes a T-cell costimulatory (for example CD28) domain in series with a T-cell trigger (for example CD3 ⁇ ) part.
  • T-cell costimulatory for example CD28
  • T-cell trigger for example CD3 ⁇
  • the engineered cell redirection has a predefined specificity for any desired target antigen, in a non-HLA-restricted manner.
  • a retroviral or lentiviral vector or transposon is used to introduce the CAR construct ex vivo into T cells from peripheral lymphocytes of a given patient.
  • the therapeutic targets of the CAR method include cancer and HIV-infected cells, or autoimmune effector cells.
  • tumor refers to a disease characterized by the pathological proliferation of cells or tissues, and subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organ, or tissue or corresponding cells.
  • Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).
  • the tumor of the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, Acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin’s disease, non-Hodgkin’s disease), primary macroglobulinemia, severe Chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphan
  • the "tumor” includes but is not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • disease or “disorder” or “disorder” as used herein refers to any change or disorder that damages or interferes with the normal function of cells, tissues or organs.
  • disease includes but is not limited to: tumors, pathogen infections, autoimmune diseases, T cell dysfunction diseases, or immune tolerance defects (such as transplant rejection).
  • treatment refers to clinical intervention in the process of trying to change a person or treating a disease caused by cells, which can be used for prevention or intervention in the clinical pathological process.
  • the therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, reducing the symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progression of the disease, improving or relieving the condition, relieving or improving the prognosis, etc.
  • kits or “kit” includes an effective amount of one or more unit dosage forms of the pharmaceutical composition of the present invention.
  • the kits may contain sterile containers; such containers may be boxes, ampoules, bottles, vials, tubes, bags, blister packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding medicines.
  • the kit also includes instructions for administering the pharmaceutical composition of the present invention to an individual. The instructions usually include methods of using the pharmaceutical composition of the present invention to treat diseases.
  • mice of different strains were immunized with CHO-K1 cells (CHO-K1/hCLDN18.2) expressing hCLDN18.2 stably and a DNA vector encoding hCLDN18.2 as immunogens.
  • mice were euthanized with carbon dioxide, and the feeder cells were collected separately with a syringe, and the feeder cell suspension was spread in a prepared 96-well plate. A certain number of myeloma cells and spleen cells are mixed in proportion to perform cell fusion. Add HAT medium (1mL 100 ⁇ HT supplement+1mL aminopterin+10mL FBS+88mL DMEM) to the fused cells, mix well to make a cell suspension. Then pour the cell suspension into a petri dish and mix well, and spread the cell suspension into a 96-well feeder cell plate with a multichannel pipette.
  • HAT medium (1mL 100 ⁇ HT supplement+1mL aminopterin+10mL FBS+88mL DMEM
  • the hybridoma cell lines secreting monoclonal antibodies obtained by the above screening are cultivated, and total RNA is extracted from the cells by conventional biological methods.
  • total RNA as template, using PrimeScript TM 1st Strand cDNA Synthesis Kit (TAKARA), reverse transcription to synthesize cDNA.
  • TAKARA PrimeScript TM 1st Strand cDNA Synthesis Kit
  • cDNA as a template, the antibody constant region primers are used for amplification. After the PCR products were separated by agarose gel electrophoresis, the DNA fragments were purified and recovered, and the amino acid sequences of the 18 monoclonal antibody variable regions of the present invention were obtained by sequencing. The results are shown in Table 1:
  • VH Light chain variable region
  • VL Light chain variable region
  • VH Light chain variable region
  • VL Light chain variable region
  • SEQ ID NO: 1 SEQ ID NO: 2 48H1 SEQ ID NO: 3
  • SEQ ID NO: 4 43F5 SEQ ID NO: 5
  • SEQ ID NO: 6 40G7 SEQ ID NO: 7
  • SEQ ID NO: 8 76D11 SEQ ID NO: 9
  • SEQ ID NO: 10 32G8 SEQ ID NO: 11 SEQ ID NO: 12 37G8 SEQ ID NO: 13
  • SEQ ID NO: 14 39C8 SEQ ID NO: 15 SEQ ID NO: 16 42D2 SEQ ID NO: 17
  • SEQ ID NO: 18 126A11 SEQ ID NO: 19
  • SEQ ID NO: 20 239E9 SEQ ID NO: 21
  • SEQ ID NO: 24 252F11 SEQ ID NO: 25
  • SEQ ID NO: 26 299B2 SEQ
  • the CDR and FR of the antibody variable region are divided using Kabat numbering rules.
  • the 6 CDR sequences of each antibody are composed as shown in Table 2 below.
  • the numbers in parentheses in Table 2 indicate the sequence number, for example ( ) Represents SEQ ID NO: 37.
  • the sequenced monoclonal antibody variable region of the present invention is spliced with the human IgG1 constant region and the target gene fragment is cloned into a pcDNA3.4 expression vector to prepare a transfection grade expression plasmid.
  • Expi293F TM cells (Thermo Fisher Scientific) were cultured in a serum-free medium, and the cells were seeded in shake flasks (Corning Inc.), and cultured on a shaker at 37° C., 8% CO 2 . Adjust the cell density, mix the recombinant expression vector containing the target gene fragment and ExpiFectamine TM 293 transfection reagent in an appropriate ratio, and add it to the cell culture shake flask.
  • ExpiFectamine TM 293 Transfection Enhancer 1 With ExpiFectamine TM 293 Transfection Enhancer 2 the supernatant was collected and purified after cell culture for 6 days, and the final purified chimeric antibody was analyzed by SDS-PAGE purity analysis and A280 concentration determination. Chimeric antibodies are named by adding the prefix ch- on the basis of the original hybridoma clone.
  • FACS was used to detect the binding of anti-CLDN18.2 chimeric antibody to HEK293 cells stably expressing hCLDN18.2 (HEK293-hCLDN18.2), and gastric cancer tumor tissue-derived cells that naturally express hCLDN18.2 (PDX-hCLDN18.2).
  • HEK293-hCLDN18.2 or PDX-hCLDN18.2 cells Collect HEK293-hCLDN18.2 or PDX-hCLDN18.2 cells, resuspend them in PBS, adjust the cell concentration, and add serially diluted antibodies.
  • the negative control is irrelevant human IgG
  • the positive control is from The chimeric antibody ch-175D10 disclosed in the patent CN103509110B.
  • Table 3 and Figures 6-17 show the affinity results of the chimeric antibody of the present invention and the reference antibody ch-175D10 with HEK293-hCLDN18.2 cells and PDX-hCLDN18.2 cells, respectively.
  • the experimental results show that the highest average fluorescence intensity of the chimeric antibody of the present invention and HEK293-hCLDN18.2 cells is between 5586-12203, and the reference antibody ch-175D10 binds to HEK293-hCLDN18.2 under the same reaction conditions The highest average fluorescence intensity is only 5451.
  • the highest average fluorescence intensity of the chimeric antibody binding to PDX-hCLDN18.2 is between 7616-19876, and the half-binding concentration (EC 50 ) is between 4.554-96.23nM.
  • the reference antibody ch-175D10 is in the same reaction conditions with The highest average fluorescence intensity of PDX-hCLDN18.2 binding was only 7160, and the half binding concentration (EC 50 ) was only 63.85 nM. It can be seen that most of the chimeric antibodies of the present invention have stronger binding ability to hCLDN18.2 antigen than ch-175D10.
  • FACS was used to detect the binding of the chimeric antibody of the present invention to HEK293 cells stably expressing murine CLDN18.2 (HEK293-mCLDN18.2) and HEK293 cells stably expressing human CLDN18.1 (HEK293-hCLDN18.1).
  • the negative control is irrelevant human IgG
  • the positive control reference antibody
  • Table 4 shows the affinity results of the chimeric antibody of the present invention and the reference antibody ch-175D10 with HEK293-mCLDN18.2 cells and HEK293-hCLDN18.1 cells, respectively.
  • the experimental results show that the chimeric antibody of the present invention is the same as the reference antibody ch-175D10, and both bind to the mCLDN18.2 antigen.
  • the highest average fluorescence intensity of the chimeric antibody combined with HEK293-mCLDN18.2 is between 6093-14768, half
  • the binding concentration (EC 50 ) is between 0.573 and 1.763 nM.
  • the highest average fluorescence intensity of the reference antibody ch-175D10 combined with HEK293-mCLDN18.2 under the same reaction conditions is 4187, and the half binding concentration (EC 50 ) is 1.014 nM, chimeric antibody bound to the reference antibody EC 50 comparable maximum binding than the reference antibody. Moreover, the chimeric antibody is the same as the reference antibody ch-175D10, and neither binds to the hCLDN18.1 antigen.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • CHO-K1/hCLDN18.2 was used as the target cell, and NK cells (NK92/FcR ⁇ 3a.158V/V) transfected with 158V/V Fc ⁇ RIIIa gene were used as effector cells.
  • the cytotoxicity detection kit (Roche) was used to detect cell lactate Hydrogenase (LDH) release amount, and use this as an indicator of cell killing effect.
  • the CHO-K1/hCLDN18.2 cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in ADCC buffer and the cell density was adjusted, and then transferred to a 96-well experimental plate. Transfer different concentration gradients of chimeric antibody, control sample working solution or ADCC buffer to 96-well plate, incubate in a cell incubator (37°C/5%CO 2 ) for about 30 minutes, transfer effector cells, ADCC buffer or Transfer the cell lysate to a 96-well experiment plate, and incubate for about 6 hours in a cell incubator (37°C/5% CO 2 ).
  • % Cell lysis 100% ⁇ (sample release-target cell/effector cell mixed release)/(maximum release—target cell release), where the maximum release is the absorbance value produced by the wells of the target cells treated by Triton X-100, target cells/
  • the mixed release of effector cells is the absorbance value produced by the mixed wells of target cells and effector cells
  • the target cell release is the absorbance value produced in the wells containing only target cells
  • the sample release is the absorbance produced by the mixed wells of the chimeric antibody and target cells and effector cells.
  • Table 5 and Figures 18-23 show the ADCC results of the chimeric antibody of the present invention and the reference antibody ch-175D10 on CHO-K1/hCLDN18.2 cells.
  • Experimental results show that the maximum ADCC effect of the chimeric antibody of the present invention on CHO-K1/hCLDN18.2 cells is between 29.06% and 48.46%, and the ADCC effect of the reference antibody ch-175D10 under the same reaction conditions is 31.81%.
  • Chimeric antibody of the present invention produce concentration (EC 50) 50% ADCC effect between 0.010-0.064 ⁇ g / mL, ch-175D10 concentration producing 50% ADCC effect (EC 50) under the same reaction conditions 0.021 ⁇ g /mL.
  • concentration (EC 50) 50% ADCC effect between 0.010-0.064 ⁇ g / mL
  • ch-175D10 concentration producing 50% ADCC effect (EC 50) under the same reaction conditions 0.021 ⁇ g /mL.
  • the above results prove that the chimeric antibody of
  • CHO-K1/hCLDN18.2 Take CHO-K1/hCLDN18.2 as the target cell and mixed healthy human serum (PNHS) as the source of complement.
  • Chemiluminescence cell viability analysis kit detects cell viability.
  • CHO-K1/hCLDN18.2 cells Collect CHO-K1/hCLDN18.2 cells, resuspend them in CDC buffer and adjust the cell density, inoculate the cells into a 384-well cell plate, 20 ⁇ L per well, prepare a 4-fold concentration sample solution, and transfer the solution (the control group is CDC Buffer) into the corresponding well of the 384-well cell plate, 10 ⁇ L per well, incubate at room temperature for about 30 minutes, and use CDC buffer to dilute and mix Pooled Normal Human Serum (PNHS) to 4 times the working concentration.
  • PNHS Pooled Normal Human Serum
  • the cell lysis rate caused by the chimeric antibody in the CDC test is calculated using the following formula:
  • the experimental control is: serum control well: only serum (ie 30 ⁇ L buffer + 10 ⁇ L diluted serum).
  • Cell+serum well Add serum (ie 20 ⁇ L cell suspension+10 ⁇ L buffer+10 ⁇ L diluted serum) to the well of CHO-K1/hCLDN18.2 cell suspension.
  • Test hole Add serum and chimeric antibody (ie 20 ⁇ L cell suspension + 10 ⁇ L antibody + 10 ⁇ L diluted serum) to the CHO-K1/hCLDN18.2 cell suspension hole.
  • Table 6 and Figures 24-29 show the CDC results of the chimeric antibody of the present invention and the reference antibody ch-175D10 on CHO-K1/hCLDN18.2 cells.
  • Experimental results show that the maximum CDC effect of the chimeric antibody of the present invention on CHO-K1/hCLDN18.2 is between 89.08% and 97.11%, while the maximum CDC effect of the reference antibody ch-175D10 under the same reaction conditions is 87.41%.
  • Chimeric antibody of the present invention produces a concentration of 50% CDC effect (EC 50) between 0.038-0.380 ⁇ g / mL, while ch-175D10 concentration producing 50% CDC effect under the same reaction conditions (EC 50) higher than 1 ⁇ g/mL. It can be seen that the CDC activity of most antibodies is stronger than that of the reference antibody.
  • the amino acid sequence of the heavy chain variable region of 299B2-N31Q, 299B2-S32A and 299B2-G33A is the same as that of 299B2, and the amino acid sequence of the heavy chain variable region of 253C4-N31Q, 253C4-S32A and 253C4-G33A is the same as that of 253C4.
  • the variable region CDR sequences and the heavy chain and light chain variable region sequences of the above 6 variants are shown in Table 7 and Table 8, respectively.
  • Example 3 the target gene fragments generated by splicing the variable regions of the above 6 variants with the constant regions of human IgG1 were subcloned into an expression vector to construct the chimeric antibody of the variant, and the chimeric antibody of the variant.
  • the naming is based on the "hybridoma clone-mutation site" by adding the prefix ch-.
  • the chimeric antibody of 299B2-N31Q is named ch-299B2-N31Q.
  • HEK293-hCLDN18.2 cells were used for affinity detection, and monoclonal cell supernatants were screened by FACS.
  • the affinity results of antibody ch-299B2 and ch-253C4 variants are shown in Table 9 .
  • Clone number EC 50 (ng/mL) The highest average fluorescence intensity (Top MFI) ch-175D10 155.9 2836 ch-299B2-N31Q -* -* ch-299B2-S32A 130.7 6504 ch-299B2-G33A 157.0 6869 ch-253C4-N31Q 175.0 6565 ch-253C4-S32A 206.7 6894 ch-253C4-G33A 186.7 6837
  • the antibody germline with the highest similarity to 299B2 was selected as the antibody template.
  • IMGT database IGHV1-46*01 was selected as the antibody template of the 299B2-S32A heavy chain, IGKV4-1*01
  • the CDR regions of the light and heavy chains of 299B2-S32A were replaced with the CDR regions of the antibody template.
  • the PDB antibody database was searched for the best modeling template based on the sequence of the mouse anti-variable region, and 2GKI with 74% homology was selected as the template.
  • the amino acid residues in the framework region of the CDR grafted sequence are backmutated according to the following criteria: 1. The classic residues in the framework region will be selected for backmutation; 2. Residues in the hydrophobic core region of the framework region Will be selected for back mutation; 3. Heavy chain/light chain interaction interface residues will be selected for back mutation; 4. Similar residues can also be used as low priority back mutations.
  • 299B2-S32A has been humanized and modified to obtain 4 humanized antibodies hu299B2-S32A-1, hu299B2-S32A-2, hu299B2-S32A-3, hu299B2-S32A-4, all of the above-mentioned humanized antibody sequences of 299B2-S32A See Table 10.
  • the target gene fragment generated by splicing the variable region of the humanized antibody hu299B2-S32A with the constant region of human IgG1 was subcloned into the pcDNA3.4 expression vector, and transfected with ExpiFectamine TM 293
  • the dye reagent was transiently transfected into Expi293F TM cells in logarithmic growth phase, and the culture supernatant was collected and subjected to affinity purification.
  • the final purified antibody was subjected to SDS-PAGE purity analysis and A280 concentration determination.
  • the antibody germline with the highest similarity to 253C4 was selected as the antibody template.
  • IMGT database IGKV4-1*01 was selected as the antibody template for the 253C4-N31Q heavy chain
  • IGHV1-2*06 As an antibody template for the light chain of 253C4-N31Q
  • the light chain and heavy chain CDR regions of 253C4-N31Q were replaced with the CDR regions of the antibody template.
  • the PDB antibody database was searched for the best modeling template based on the sequence of the mouse anti-variable region, and 2GKI with 74% homology was selected as the template.
  • the amino acid residues in the framework region of the CDR grafted sequence are backmutated according to the following criteria: 1. The classic residues in the framework region will be selected for backmutation; 2. Residues in the hydrophobic core region of the framework region Will be selected for back mutation; 3. Heavy chain/light chain interaction interface residues will be selected for back mutation; 4. Similar residues can also be used as low priority back mutations.
  • 253C4-N31Q was humanized to obtain 3 humanized antibodies, hu253C4-N31Q-1, hu253C4-N31Q-2, and hu253C4-N31Q-3. See Table 10 for the sequences of all the above-mentioned humanized 253C4-N31Q antibodies.
  • the target gene fragment generated by splicing the variable region of the humanized antibody hu253C4-N31Q with the constant region of human IgG1 was subcloned into the pcDNA3.4 expression vector and transfected with ExpiFectamine TM 293
  • the dye reagent was transiently transfected into Expi293F TM cells in logarithmic growth phase, and the culture supernatant was collected and subjected to affinity purification.
  • the final purified antibody was subjected to SDS-PAGE purity analysis and A280 concentration determination.
  • Example 4A for binding activity determination, using HEK293-hCLDN18.2 and PDX-hCLDN18.2 cells, the negative control is an irrelevant human IgG, and the positive control (reference antibody) is from the chimera disclosed in patent CN103509110B Antibody ch-175D10.
  • MFI average fluorescence intensity of each concentration
  • EC 50 half binding concentration
  • Top MFI highest average fluorescence intensity
  • Table 11 and Figures 30-33 show the affinity results of the humanized antibody of the present invention and the reference antibody ch-175D10 with HEK293-hCLDN18.2 cells and PDX-hCLDN18.2 cells, respectively.
  • the experimental results show that the highest average fluorescence intensity of the humanized antibody hu299B2-S32A of the present invention and HEK293-hCLDN18.2 cells is between 29529-39986, while the reference antibody ch-175D10 reacts with HEK293- under the same reaction conditions.
  • the highest average fluorescence intensity bound by hCLDN18.2 is only 26921.
  • the affinity of humanized antibody hu299B2-S32A and HEK293-hCLDN18.2 is better than that of reference antibody ch-175D10.
  • the highest average fluorescence intensity of the binding between hu253C4-N31Q and HEK293-hCLDN18.2 is comparable to that of the reference antibody ch-175D10, and the EC 50 is better than that of the reference antibody ch-175D10.
  • Hu299B2-S32A present invention hu253C4-N31Q highest mean fluorescence intensity in combination with PDX-hCLDN18.2 between 15585-25189, binding half concentration (EC 50) between 11.42-29.68nM, reference antibody ch-175D10 Under the same reaction conditions, the highest average fluorescence intensity combined with PDX-hCLDN18.2 was only 5703, and the half-binding concentration (EC 50 ) was 24.89 nM. It can be seen that the maximum binding of the humanized antibody of the present invention to the natural hCLDN18.2 antigen is stronger than ch-175D10, and the EC50 is equivalent to ch-175D10.
  • FACS was used to detect the binding of the humanized antibody of the present invention to HEK293-mCLDN18.2 cells and HEK293-hCLDN18.1 cells.
  • the negative control is irrelevant human IgG
  • the positive control is still ch-175D10.
  • Table 12 shows the affinity results of the humanized antibody of the present invention and the reference antibody ch-175D10 to HEK293-mCLDN18.2 cells and HEK293-hCLDN18.1 cells, respectively.
  • the experimental results show that the humanized antibody of the present invention is the same as the reference antibody ch-175D10, and both bind to the mCLDN18.2 antigen.
  • the highest average fluorescence intensity of the humanized antibody combined with HEK293-mCLDN18.2 is between 36841-102785 .
  • the half-binding concentration (EC 50 ) is between 0.026-0.342 ⁇ g/mL
  • the highest average fluorescence intensity of the reference antibody ch-175D10 binding to HEK293-mCLDN18.2 under the same reaction conditions is 86407
  • the half-binding concentration (EC 50 ) is 0.136 ⁇ g/mL
  • the binding of the humanized antibody to mCLDN18.2 is equivalent to that of the reference antibody.
  • the humanized antibody is the same as the reference antibody ch-175D10, and neither binds to the hCLDN18.1 antigen.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • CHO-K1/hCLDN18.2 was used as target cells
  • NK cells NK92/FcR ⁇ 3a.158V/V
  • cytotoxicity detection reagents were used Roche detects the release of lactate dehydrogenase (LDH) from cells and uses this as an indicator of cell killing.
  • % Cell lysis 100% ⁇ (sample release-target cell/effector cell mixed release)/(maximum release—target cell release), where the maximum release is the absorbance value produced by the wells of Triton X-100 treated target cells, target cells/ The mixed release of effector cells is the absorbance value produced by the mixed wells of target cells and effector cells, the target cell release is the absorbance produced in the wells containing only target cells, and the sample release is the absorbance produced by the mixed wells of humanized antibody and target cells and effector cells. Value, GraphPad software calculated EC50 and maximum lysis, the results are shown in Table 13.
  • Table 13 and Figures 34-35 show the ADCC results of the humanized antibody of the present invention and the reference antibody ch-175D10 on CHO-K1/hCLDN18.2 cells.
  • Experimental results show that the maximum ADCC effect of the humanized antibody of the present invention on CHO-K1/hCLDN18.2 cells is between 54.85% and 70.26%, and the ADCC effect of the reference antibody ch-175D10 under the same reaction conditions is about 50% .
  • the present invention is to produce a humanized antibody concentrations (EC 50) 50% ADCC effect between 0.022-0.038 ⁇ g / mL, ch-175D10 concentration producing 50% ADCC effect under the same reaction conditions (EC 50) is about 0.040 ⁇ g/mL.
  • the above results prove that the humanized antibody of the present invention is comparable to the reference antibody ch-175D10 in terms of ADCC activity.
  • CHO-K1/hCLDN18.2 was used as the target cell, and mixed healthy human serum (PNHS) was used as the source of complement.
  • PNHS mixed healthy human serum
  • Chemiluminescence cell viability analysis kit detects cell viability, and reads the results using PHERAstar Plus software.
  • the cell lysis rate caused by humanized antibodies in the CDC test is calculated using the following formula:
  • the experimental control is: serum control well: only serum (ie 30 ⁇ L buffer + 10 ⁇ L diluted serum).
  • Cell+serum well Add serum (ie 20 ⁇ L cell suspension+10 ⁇ L buffer+10 ⁇ L diluted serum) to the well of CHO-K1/hCLDN18.2 cell suspension.
  • Test hole Add serum and chimeric antibody (ie 20 ⁇ L cell suspension + 10 ⁇ L antibody + 10 ⁇ L diluted serum) to the CHO-K1/hCLDN18.2 cell suspension hole.
  • Table 14 and Figures 36-37 show the CDC results of the humanized antibody of the present invention and the reference antibody ch-175D10 on CHO-K1/hCLDN18.2 cells.
  • the experimental results show that the humanized antibody of the present invention has a maximum CDC effect on CHO-K1/hCLDN18.2 between 96.11% and 99.87%, while the reference antibody ch-175D10 produces the maximum CDC effect under the same reaction conditions. It is 87.76%.
  • Chimeric antibody of the present invention produces a concentration of 50% CDC effect (EC 50) between 0.092-0.175 ⁇ g / mL, while ch-175D10 concentration producing 50% CDC effect under the same reaction conditions (EC 50) higher than 1 ⁇ g/mL. It can be seen that the CDC activity of the humanized antibody is stronger than that of the reference antibody.

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Abstract

提供抗CLDN18.2的抗体或其抗原结合片段、包含所述抗体或其抗原结合片段的衍生物以及药物组合物，以及所述抗体或其抗原结合片段在治疗癌症和检测诊断方面的相关应用。

Description

针对密蛋白18A2的抗体及其应用 技术领域
本发明属于免疫学领域，更具体地，本发明涉及针对密蛋白18.2(Claudin 18.2，CLDN18A2，CLDN18.2)的抗体或其抗原结合片段、包含所述抗体或其抗原结合片段的衍生物、药物组合物及其在治疗癌症方面的相关应用。
背景技术
密蛋白(Claudin)为包含紧密连接的主要结构蛋白的整合膜蛋白，紧密连接为诸如上皮细胞或内皮细胞层中所见的极化细胞类型中的最顶端细胞-细胞黏着连接。紧密连接是由多链网状蛋白质组成，这些多链网状蛋白质在细胞周围形成连续密封，为溶质及水在细胞旁空间中的输送提供物理屏障，但该物理屏障可调节。人类中的密蛋白家族包含至少23名成员，大小在22-34kDa范围内。虽然密蛋白对正常组织的功能及稳定很重要，但肿瘤细胞经常展现异常的紧密连接功能。这可能与由于肿瘤细胞的反分化所致的密蛋白的失调表达和/或定位或迅速生长的癌组织有效吸收血管形成异常的肿瘤块内的营养物的要求有关(Morin，2005，PMID：16266975)。个别密蛋白家族成员可在某些癌症类型中上调，但在其他癌症类型中下调。
密蛋白18(Claudin 18，CLDN18)是位于上皮和内皮的紧密连接中的内在膜蛋白，分子量约为27.9KD，与其他紧密连接蛋白共同形成细胞间紧密连接，调节组织分子和离子在细胞间隙通透，维持组织内环境的稳定。密蛋白18已知存在2个亚型，剪接变体1(CLDN18A1、CLDN18.1)：GenBank登记号为NP_057453、NM016369，以及剪接变体2(CLDN18A2、CLDN18.2)：GenBank登记号为NM_001002026、NP_001002026。在正常细胞中，CLDN18A1在肺的上皮中选择性表达，而CLDN18A2则特异性表达于正常胃上皮已分化细胞，在具有细胞分裂活性的胃上皮干细胞中无表达。但在肿瘤细胞中，CLDN18A2在多种癌症类型中均呈现过度表达，如75％的胃癌患者高表达CLDN18A2，50％的胰腺癌患者高表达CLDN18A2，30％的食管癌患者高表达CLDN18A2，在肺癌等癌症类型中也有高表达。因此，找到特异性结合CLDN18A2而不结合CLDN18A1的抗体，对癌症的治疗和检测具有重要意义。
现有CLDN18A2抗体IMAB362已经进入临床研究阶段，临床结果显示CLDN18.2高表达的胃癌患者中(大于等于70％的肿瘤细胞内CLDN18.2表达≥2+)，化疗+IMAB362相比单纯化疗，无进展生存期由6.1个月延长至9.1个月，HR＝0.46；总生存期从9.3个月延长到16.6个月，HR＝0.44。除抗体IMAB362以外，针对CLDN18.2靶点制备的CAR-T也 已进入临床研究。然而，这些已进入临床阶段的抗体(或CAR-T中的抗原结合片段)与密蛋白18.2的亲和力较弱。因此，仍然需要继续筛选和制备亲和力较高的CLDN18.2抗体，才能在相同给药剂量下产生更强的药效作用。
发明概述
本发明提供了一种抗CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性地结合CLDN18.2，并且不显著结合CLDN18.1。
在一些实施方案中，所述CLDN18.2是具有GenBank登记号NP_001002026的肽(mRNA:NM_001002026)。所述CLDN18.1是具有GenBank登记号NP_057453的肽(mRNA:NM_016369)。
在一些实施方案中，本发明的抗体或其抗原结合片段不显示与CLDN18.1的显著结合。在一些实例中，所述抗体或其抗原结合片段与CLDN18.1的结合水平不超过其与CLDN18.2的结合水平的20％。例如，结合水平可以是所述抗体或抗原结合片段与CLDN18.2的结合水平的20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或小于1％。在一些实施方案中，本发明的抗体或其抗原结合片段与CLDN18.2的结合水平是其与CLDN18.1的结合水平的1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或多于10倍。
本发明提供了一种抗CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合CLDN18.2，其包含：重链可变区，所述的重链可变区包含3个选自如以下序列所示的HCDR：SEQ ID NO:37，38，39，40，41，42，43，44，45，46，47，48，49，50，51，52，53，54，55，56，57，58，59，60，61，62，63，64，65，66，67，68，69，70，71，72，73，74，75，76，77，78，79，80，81，82，83，84，85；和/或，轻链可变区，所述的轻链可变区包含3个选自如以下序列所示的LCDR：SEQ ID NO:86，87，88，89，90，91，92，93，94，95，96，97，98，99，100，101，102，103，104，105，106，107，108，109，110，111，112，113。
在一些实施方案中，本发明提供了一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和/或轻链可变区，所述重链可变区包含选自SEQ ID NO:37，40，43，45，49，53，56，59，62，65，68，71，74，77，80，83所示的HCDR1；以及，选自SEQ ID NO:38，41，46，48，50，52，54，57，60，63，66，69，72，75，78，81，84所示的HCDR2；以及，选自SEQ ID NO:39，42，44，47，51，55，58，61，64，67，70，73，76，79，82，85所示的HCDR3；和/或，所述轻链可变区包含选自SEQ ID NO:86，87，88，89，90，91，92，111，112，113所示的LCDR1；以及，选自SEQ ID NO:93，94，95，96所示的 LCDR2；以及，选自SEQ ID NO:97，98，99，100，101，102，103，104，105，106，107，108，109，110所示的LCDR3。
在一个优选的实施方案中，本发明提供了一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和/或轻链可变区，所述重链可变区包含选自下组的HCDR1、HCDR2和HCDR3：
SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39或
SEQ ID NO:40、SEQ ID NO:41和SEQ ID NO:42或
SEQ ID NO:43、SEQ ID NO:41和SEQ ID NO:44或
SEQ ID NO:45、SEQ ID NO:46和SEQ ID NO:47或
SEQ ID NO:37、SEQ ID NO:48和SEQ ID NO:39或
SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51或
SEQ ID NO:49、SEQ ID NO:52和SEQ ID NO:51或
SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55或
SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58或
SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61或
SEQ ID NO:62、SEQ ID NO:63和SEQ ID NO:64或
SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67或
SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70或
SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73或
SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76或
SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79或
SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82或
SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85；
和/或，所述轻链可变区选自下组的LCDR1、LCDR2和LCDR3：
SEQ ID NO:86、SEQ ID NO:93和SEQ ID NO:97；或
SEQ ID NO:87、SEQ ID NO:94和SEQ ID NO:98；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:99；或
SEQ ID NO:87、SEQ ID NO:95和SEQ ID NO:100；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:97；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:101；或
SEQ ID NO:89、SEQ ID NO:93和SEQ ID NO:102；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:100；或
SEQ ID NO:90、SEQ ID NO:93和SEQ ID NO:103；或
SEQ ID NO:91、SEQ ID NO:96和SEQ ID NO:104；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:98；或
SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:105；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:106；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:107；或
SEQ ID NO:87、SEQ ID NO:93和SEQ ID NO:108；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:109；或
SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:110；或
SEQ ID NO:111、SEQ ID NO:93和SEQ ID NO:107；或
SEQ ID NO:112、SEQ ID NO:93和SEQ ID NO:107；或
SEQ ID NO:113、SEQ ID NO:93和SEQ ID NO:107；或
SEQ ID NO:111、SEQ ID NO:93和SEQ ID NO:110；或
SEQ ID NO:112、SEQ ID NO:93和SEQ ID NO:110；或
SEQ ID NO:113、SEQ ID NO:93和SEQ ID NO:110。
根据本发明的一个方面，所述一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和轻链可变区，所述可变区包含如下任一一组的6个CDR，每组的6个CDR按照HCDR1，HCDR2，HCDR3，LCDR1，LCDR2，LCDR3依次排列：
(1)SEQ ID NO:37，38，39，86，93，97；
(2)SEQ ID NO:40，41，42，87，94，98；
(3)SEQ ID NO:43，41，44，88，93，99；
(4)SEQ ID NO:45，46，47，87，95，100；
(5)SEQ ID NO:37，48，39，88，93，97；
(6)SEQ ID NO:49，50，51，88，93，101；
(7)SEQ ID NO:49，52，51，89，93，102；
(8)SEQ ID NO:53，54，55，88，93，100；
(9)SEQ ID NO:56，57，58，90，93，103；
(10)SEQ ID NO:59，60，61，91，96，104；
(11)SEQ ID NO:62，63，64，88，93，98；
(12)SEQ ID NO:65，66，67，92，93，105；
(13)SEQ ID NO:68，69，70，88，93，106；
(14)SEQ ID NO:71，72，73，88，93，107；
(15)SEQ ID NO:74，75，76，88，93，106；
(16)SEQ ID NO:77，78，79，87，93，108；
(17)SEQ ID NO:80，81，82，88，93，109；
(18)SEQ ID NO:83，84，85，88，93，110；
(19)SEQ ID NO:71，72，73，111，93，107；
(20)SEQ ID NO:71，72，73，112，93，107；
(21)SEQ ID NO:71，72，73，113，93，107；
(22)SEQ ID NO:83，84，85，111，93，110；
(23)SEQ ID NO:83，84，85，112，93，110；
(24)SEQ ID NO:83，84，85，113，93，110。
在一些实施方案中，本发明提供了一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和/或轻链可变区，所述重链可变区具有与下述序列至少80％、85％、90％、95％或100％的序列同一性：SEQ ID NO:1，3，5，7，9，11，13，15，17，19，21，23，25，27，29，31，33，35；和/或，所述轻链可变区具有与下述序列至少80％、85％、90％、95％或100％的序列同一性：SEQ ID NO:2，4，6，8，10，12，14，16，18，20，22，24，26，28，30，32，34，36，114，115，116，117，118，119。
根据本发明的一个方面，所述一种抗CLDN18.2的抗体或其抗原结合片段，其包含与如下任一一组的重链可变区和轻链可变区具有至少80％至100％的序列同一性：
(1)SEQ ID NO:1和2；
(2)SEQ ID NO:3和4；
(3)SEQ ID NO:5和6；
(4)SEQ ID NO:7和8；
(5)SEQ ID NO:9和10；
(6)SEQ ID NO:11和12；
(7)SEQ ID NO:13和14；
(8)SEQ ID NO:15和16；
(9)SEQ ID NO:17和18；
(10)SEQ ID NO:19和20；
(11)SEQ ID NO:21和22；
(12)SEQ ID NO:23和24；
(13)SEQ ID NO:25和26；
(14)SEQ ID NO:27和28；
(15)SEQ ID NO:29和30；
(16)SEQ ID NO:31和32；
(17)SEQ ID NO:33和34；
(18)SEQ ID NO:35和36；
(19)SEQ ID NO:27和114；
(20)SEQ ID NO:27和115；
(21)SEQ ID NO:27和116；
(22)SEQ ID NO:35和117；
(23)SEQ ID NO:35和118；
(24)SEQ ID NO:35和119。
在一些优选的实施方案中，本发明所述的抗CLDN18.2的抗体或其抗原结合片段为鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，本发明提供了一种抗CLDN18.2的抗体或其抗原结合片段，包括重链可变区和轻链可变区，所述重链可变区具有与下述序列至少80％、85％、90％、95％或100％的序列同一性：SEQ ID NO:120，122，125，128；和/或，所述轻链可变区具有与 下述序列至少80％、85％、90％、95％或100％的序列同一性：SEQ ID NO:121，123，124，126，127。
根据本发明的一个方面，所述一种抗CLDN18.2的抗体或其抗原结合片段，其包含与如下任一一组的重链可变区和轻链可变区具有至少80％、85％、90％、95％或100％的序列同一性：
(1)SEQ ID NO:120和121；
(2)SEQ ID NO:120和123；
(3)SEQ ID NO:120和124；
(4)SEQ ID NO:122和121；
(5)SEQ ID NO:125和126；
(6)SEQ ID NO:125和127；
(7)SEQ ID NO:128和126。
在一些优选的实施方案中，所述抗CLDN18.2的抗体为单克隆抗体。
在一些优选的实施方案中，所述一种抗CLDN18.2的抗体或其抗原结合片段，还进一步包括重链恒定区和/或轻链恒定区，优选的，所述重链恒定区包括Fc或变体Fc，Fc来源于鼠或人。
在一些优选的实施方案中，所述抗CLDN18.2的抗体为全长抗体。
在一些优选的实施方案中，本发明的一种抗CLDN18.2的抗体或其抗原结合片段，为IgG1、IgG2、IgG3或IgG4形式。
在一些优选的实施方案中，本发明所述的抗原结合片段为Fab、Fv、scFv、F(ab’) 2、线性抗体、单结构域抗体。
在一些实施方案中，本发明提供一种偶联物，将前述抗体或其抗原结合片段与捕获标记物或检测标记物偶联形成。所述的检测标记物包括但不限于放射性核素、发光物质(例如荧光素)、有色物质或酶。
在一些实施方案中，本发明提供一种双特异性抗体或多特异性抗体，所述双特异性抗体或多特异性抗体中的一个抗原结合结构域包含本发明的抗CLDN18.2的抗体或其抗原结合片段。
在一些实施方案中，本发明提供一种抗体药物缀合物，含有如前所述的抗体或其抗原结合片段。所述的抗体药物缀合物的结构是本领域公知的，其由抗体-接头-药物(毒素)相互 连接形成。
在一些实施方案中，本发明提供一种嵌合抗原受体，其胞外识别单元包含如前所述的抗体或其抗原结合片段。
在一些实施方案中，本发明提供了编码前述任一抗体或其抗原结合片段的核酸。根据本发明的另一个方面，本发明提供了包含所述核酸的重组载体。
在一些实施方案中，本发明提供了一种宿主细胞，其包含本发明所述的表达载体或基因组中整合有编码所述抗体或其抗原结合片段的核酸。在一些优选的方案中，宿主细胞可以是原核细胞，例如大肠杆菌；也可以是真核细胞，例如酵母或哺乳动物细胞，哺乳动物细胞例如CHO细胞或HEK293细胞。
在一些实施方案中，本发明提供了制备所述抗体或其抗原结合片段的方法，包括：在适合的条件下培养本发明的宿主细胞，并从所述细胞中纯化获得表达产物。
在一些实施方案中，本发明提供了所述抗体或其抗原结合片段的用途，其用于制备特异性靶向表达CLDN18.2的肿瘤细胞的药物，例如单克隆抗体药物、抗体药物缀合物，双特异性抗体或多特异性抗体；或用于制备嵌合抗原受体修饰的免疫细胞；或用于制备诊断表达CLDN18.2肿瘤的试剂；在一些实施方案中，所述表达CLDN18.2的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌，卵巢癌，结肠癌，肝癌，头颈癌和胆囊癌及其转移，所述胃癌转移例如库肯勃氏瘤。
在一些实施方案中，本发明提供了一种检测样品中CLDN18.2表达的方法，包括：将样品与前述抗CLDN18.2的抗体或其抗原结合片段接触；检测抗CLDN18.2的抗体或其抗原结合片段和CLDN18.2的复合物的形成；任选地，抗CLDN18.2的抗体或其抗原结合片段是被可检测地标记的。
在一些实施方案中，本发明提供了一种药物组合物，其包含有效量的本发明的抗体或其抗原结合片段、或包含有效量的编码所述抗体或其抗原结合片段的核酸、或包含有效量的含有编码核酸的重组载体、或包含有效量的含有编码核酸的宿主细胞、或包含有效量的本发明的抗体药物缀合物、或包含有效量的本发明的嵌合抗原受体、或包含有效量的本发明的双特异性抗体或多特异性抗体。在一些实施方案中，所述药物组合物还包含药学上可接受的载体。
在一些优选的实施方案中，所述药物组合物还包含一种或多种额外的其他治疗剂。所述额外的治疗剂包括：细胞毒性剂、细胞生长抑制剂、抗血管生成剂、减瘤剂、化学治疗 剂、放射性治疗剂、靶向性抗癌剂、生物反应修饰剂、癌症疫苗、细胞因子、激素、抗转移剂和免疫治疗剂。
在一些实施方案中，本发明提供了一种药盒或试剂盒，其包括容器，以及位于容器中的本发明的药物组合物。
在一些实施方案中，本发明提供诱导表达CLDN18.2的细胞死亡的方法，包括使所述细胞与本发明的药物组合物接触。在一些实施方案中，使所述细胞与药物组合物在体外接触。在一些实施方案中，使所述细胞与药物组合物在体内接触。在一些实施方案中，所述细胞是癌细胞。在一些实施方案中，所述细胞是实体瘤细胞。在一些实施方案中，所述细胞选自：胃癌细胞、食道癌细胞、肠癌细胞、胰腺癌细胞、肾母细胞瘤细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、直肠癌细胞、肝癌细胞、头颈癌细胞、慢性骨髓性白血病细胞和胆囊癌细胞。
在一些实施方案中，本发明提供治疗受试者中与表达CLDN18.2相关的疾病的方法，所述方法包括向有此需要的受试者施用本发明的药物组合物。在一些实施方案中，所述疾病是肿瘤。在一些实施方案中，所述肿瘤优选胃癌、食道癌、肠癌、胰腺癌、肾母瘤、肺癌、卵巢癌、结肠癌、直肠癌、肝癌、头颈癌、慢性骨髓性白血病或胆囊癌。在一些实施方案中，所述方法还包括向所述受试者给予额外的治疗剂。
本发明的抗体可以与另一额外的治疗剂联合给药，包括但不限于化学治疗剂、细胞毒性剂、放射性治疗剂、癌症疫苗、减瘤剂、靶向性抗癌剂、抗血管生成剂、生物反应修饰剂、细胞因子、激素、抗转移剂和免疫治疗剂。
在一些优选的实施方案中，可以与本发明的抗体或其抗原结合片段联合应用的化学治疗剂包括但不限于有丝分裂抑制剂，包括长春新碱、长春花碱、长春地辛和诺维宾；拓扑异构酶I抑制剂，例如喜树碱化合物，包括伊立替康、托泊替康和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂，例如顺铂、卡铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、丝裂霉素、肉瘤霉素C、放线菌素D、罗红霉素、阿霉素、雷帕霉素及其衍生物和道诺霉素；以及其它化疗药物，包括但不限于紫杉醇、多西他赛、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些优选的实施方案中，所述额外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。
在一些实施方案中，所述靶向性抗癌剂包括但不限于大分子靶向药物和小分子靶向药 物等。
在一些优选的实施方案中，大分子靶向药物包括但不限于靶向表皮生长因子药物，包括西妥昔单抗、帕尼单抗和尼妥珠单抗等；HER-2或HER-3信号通路抑制剂，包括曲妥珠单抗、帕妥珠单抗和T-DM1等；抗血管内皮生长因子药物，包括VEGF-TRAP、贝伐珠单抗和雷莫芦单抗等；以及，靶向其他靶点的药物，包括但不限于PI3K、PARP、PI3Kα、PKB/AKT和STAT3等靶点。
在一些实施方案中，小分子靶向药物包括但不限于靶向表皮生长因子的药物，包括厄洛替尼或吉非替尼等；HER-2或HER-3信号通路抑制剂，包括拉帕替尼或阿法替尼等；酪氨酸激酶抑制剂，包括伊马替尼或舒尼替尼等；抗血管内皮生长因子药物，包括索拉非尼、瑞戈替尼、帕唑替尼、重组人血管内皮抑制素和阿帕替尼等；靶向c-Met/ROS1药物，包括克唑替尼等；以及，其他靶向药物，包括但不限于伏立诺他和马马司他等；靶向mTOR药物，包括依维莫司等；以及其他靶点的药物，包括但不限于PI3Kα、PKB/AKT和STAT3等靶点。
在一些实施方案中，所述免疫治疗药物包括但不限于免疫抑制剂和激动剂，靶点包括PD-1/PD-L1、PD-L2、CTLA-4、LAG-3、IDO、TIM3、TIGIT、CD47、SIRPα、4-1BB、CSF-1/CSF1R、GITR、OX40、CD40、CD27、CD28、B7H4、B7H3、TGFβ、BTLA、VISTA、ICOS、CD39、CD73、A2AR、KIR和NKG2A等；以及与免疫治疗相关的细胞疗法。
在一些实施方案中，靶向PD-1/PD-L1的免疫检查点抑制剂包括但不限于大分子药物，例如帕姆单抗、纳武单抗、阿特珠单抗、阿维单抗和信迪利单抗、Cemiplimab和Durvalumab等；以及，小分子药物。
在一些实施方案中，靶向CTLA-4的免疫检查点抑制剂包括但不限于伊匹单抗等；细胞因子包括但不限于IL-10、IL-15、IL4和IL13等；靶向BRAF的抑制剂包括但不限于Binimetinib等。
在一些实施方案中，另一治疗剂选自溶瘤病毒，例如细小病毒、腺病毒、疱疹病毒、痘病毒、脊髓灰质炎病毒、呼肠孤病毒、甲病毒、马拉巴病毒、逆转录病毒和科萨基病毒等；或，另一治疗剂选自癌症疫苗或蛋白酶抑制剂，例如硼替佐米等。
附图说明
附图更进一步说明了本说明书所公开的新特性。参照这些附图将能更好地理解本说明书中所公开的特性和优点，但应当理解，这些附图仅用于说明本文所公开原理的具体的实施方案，而非意欲对所附权利要求的范围加以限制。
图1-5显示了采用流式细胞术检测本发明的18个杂交瘤细胞株的上清与稳转hCLDN18.2的HEK293细胞的结合情况。
图6-11显示了本发明的18个嵌合抗体与稳转表达hCLDN18.2的HEK293细胞的结合情况。
图12-17显示了本发明的18个嵌合抗体与天然表达hCLDN18.2的胃癌肿瘤组织来源细胞的结合情况。
图18-23显示了本发明的18个嵌合抗体对稳转表达hCLDN18.2的CHO-K1细胞的ADCC结果。
图24-29显示了本发明的18个嵌合抗体对稳转表达hCLDN18.2的CHO-K1细胞的CDC结果。
图30显示了本发明的人源化抗体hu299B2-S32A与稳转表达hCLDN18.2的HEK293细胞的结合情况。
图31显示了本发明的人源化抗体hu253C4-N31Q与稳转表达hCLDN18.2的HEK293细胞的结合情况。
图32显示了本发明的人源化抗体hu299B2-S32A与天然表达hCLDN18.2的胃癌肿瘤组织来源细胞的结合情况。
图33显示了本发明的人源化抗体hu253C4-N31Q与天然表达hCLDN18.2的胃癌肿瘤组织来源细胞的结合情况。
图34显示了本发明的人源化抗体hu299B2-S32A对稳转表达hCLDN18.2的CHO-K1细胞的ADCC结果。
图35显示了本发明的人源化抗体hu253C4-N31Q对稳转表达hCLDN18.2的CHO-K1细胞的ADCC结果。
图36显示了本发明的人源化抗体hu299B2-S32A对稳转表达hCLDN18.2的CHO-K1细胞的CDC结果。
图37显示了本发明的人源化抗体hu253C4-N31Q对稳转表达hCLDN18.2的CHO-K1细胞的CDC结果。
具体实施方式
术语
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文，所述引用的 程度就如同已特定地和个别地指示将各个别公布、专利或专利申请以引用的方式并入一般。
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
本文所公开的某些实施方案包含了数值范围，并且本发明的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本发明的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。例如，从1至6的范围的描述应当被认为具体公开了从1至3、1至4、1至5、2至4、2至6、3至6等的子范围，以及在这些范围内的具体的数值点，例如1、2、3、4、5、6。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时，术语“约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
本文所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
本文所用的术语“抗Claudin18.2的抗体”、“抗Claudin18.2抗体”、“抗CLDN18.2的抗体”、“抗CLDN18.2抗体”、“针对CLDN18.2的抗体”是指这样的抗体，所述抗体能够以足够的亲合力结合CLDN18.2蛋白或其片段以致所述抗体可以用作靶向CLDN18.2的诊断剂和/或治疗剂。人来源的CLDN18.2蛋白表示为hCLDN18.2，因此，“抗人Claudin18.2的抗体”、“抗人Claudin18.2抗体”、“抗hCLDN18.2的抗体”、“抗hCLDN18.2抗体”、“针对hCLDN18.2的抗体”是特指能够以足够的亲合力结合人CLDN18.2蛋白或其片段以致所述抗体可以用作靶向人CLDN18.2的诊断剂和/或治疗剂。
本文所用的术语“抗体”，典型是指包含通过共价二硫键和非共价相互作用保持在一起的两条重(H)多肽链和两条轻(L)多肽链的Y型四聚蛋白。天然IgG抗体即具有这样的结构。每条轻链由一个可变结构域(VL)和一个恒定结构域(CL)组成。每条重链包含一个可变结构域(VH)和恒定区。
本领域已知五个主要类别的抗体：IgA，IgD，IgE，IgG和IgM，对应的重链恒定结构域分别被称为α，δ，ε，γ和μ，IgG和IgA可以进一步分为不同的亚类，例如IgG可分为 IgG1，IgG2，IgG3，IgG4，IgA可分为IgA1和IgA2。来自任何脊椎动物物种的抗体的轻链基于其恒定结构域的氨基酸序列可以被分配到两种明显相异的类型之一，称为κ和λ。
在IgG、IgA和IgD抗体的情形中，该恒定区包含称为CH1、CH2和CH3的三个结构域(IgM和IgE具有第四结构域CH4)。在IgG、IgA和IgD类别中，CH1和CH2结构域被柔性铰链区分离，该铰链区是可变长度的富含脯氨酸和半胱氨酸的区段。每类抗体进一步包含由配对半胱氨酸残基形成的链间和链内二硫键。
术语“可变区”或“可变结构域”显示出从一种抗体到另一种抗体的氨基酸组成的显著变化，并且主要负责抗原识别和结合。每个轻链/重链对的可变区形成抗体结合位点，使得完整的IgG抗体具有两个结合位点(即它是二价的)。重链的可变区(VH)和轻链的可变区(VL)结构域各包含具有极端变异性的三个区域，被称为高变区(HVR)，或更通常地，被称为互补决定区(CDR)，VH和VL各有4个骨架区FR，分别用FR1，FR2，FR3，FR4表示。因此，CDR和FR序列通常出现在重链可变结构域(VH)(或轻链可变结构域(VL))的以下序列中：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
术语“Fc”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5thEd.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
如在此使用的，广义上的“抗体”的类型可包括如多克隆抗体(polyclonal antibodies)、单克隆抗体、嵌合抗体、人源化抗体及灵长类化抗体、CDR移植抗体(CDR-grafted antibody)、人类抗体(包括重组产生的人类抗体)、重组产生的抗体、胞内抗体、多特异性抗体、双特异性抗体、单价抗体、多价抗体、抗个体基因型抗体、合成抗体(包括突变蛋白及其变体)等等。
术语“全长抗体”、“完整的抗体”和“完整抗体”在本文中可以被交换地替代使用，指结构与天然抗体结构基本相似或具有包含Fc区的抗体。
术语“单克隆抗体”(或称“单抗”)指由单一细胞克隆产生的基本均质、仅针对某一特定抗原表位的抗体。单克隆抗体可以使用本领域中已知的多种技术制备，包括杂交瘤技术、重组技术、噬菌体展示技术、转基因动物、合成技术或上述技术的组合等。
术语“嵌合抗体”是一种构建体，其中重链和/或轻链的一部分与来自特定物种的或属于 特定抗体类别或亚类的抗体中的相应序列相同或同源，而这个或这些链的剩余部分与来自另一物种的或属于另一个抗体类别或亚类的抗体、以及这类抗体的片段中的相应序列相同或同源。狭义的，嵌合抗体包含与人类轻链和重链恒定区可操作地连接的全部或大多数的所选鼠类重链和轻链可变区。恒定区序列或其变体或衍生物可以使用标准分子生物学技术可操作地与所披露的重链和轻链可变区缔合，以提供本身可以使用或可以掺入本发明的抗CLDN18.2的全长抗体。
术语“人源化抗体”是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。在大多数情况下，人源化抗体是人免疫球蛋白(受体抗体)，其中来自受体的CDR的残基被来自具有所需的特异性、亲和力和性能的非人物种(供体抗体)的CDR的残基替代，例如小鼠、大鼠、兔子或灵长类动物。在一些情况下，人免疫球蛋白的框架区残基被相应的非人残基代替。在某些情况下，“回复突变”可以引入到人源化抗体中，其中受体人类抗体的可变区的一个或多个FR中的残基被来自非人类物种供体抗体的相应残基替换。这样的回复突变可以有助于保持一种或多种嫁接CDR的适当三维构型并因此改进亲和性和抗体稳定性。可以使用来自各种供体物种的抗体，这些供体物种包括但不限于小鼠、大鼠、兔或非人类灵长类动物。另外，人源化抗体可以包含在受体抗体中或在供体抗体中未发现的新残基，以为进一步改善抗体性能。
需说明的是，本发明的单克隆抗体可变区的CDR和FR的划分是根据Kabat定义确定的。而其他命名和编号系统，例如Chothia、IMGT或AHo等，也是本领域技术人员已知的。因此，以本发明的单抗序列为基础，包含任何命名系统衍生的一种或多种CDR的人源化抗体均明确地保持在本发明的范围内。
术语“序列同一性”或“序列相似性”或“序列同源性”，是指在将所述序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守置换视为序列同一性的部分之后，候选序列中的氨基酸残基与参比多肽序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
术语“抗体片段”包含完整抗体的至少一部分。如在此所使用，抗体分子的“片段”包括抗体的“抗原结合片段”，并且术语“抗原结合片段”是指免疫球蛋白或抗体中与所选抗原或其抗原表位特异性结合或反应的多肽片段，或由此片段进一步衍生的融合蛋白产物，例如单链抗体，嵌合抗原受体中的胞外结合区等。示例性的抗体片段或其抗原结合片段包括但 不限于：可变轻链片段(VL)、可变重链片段(VH)、Fab片段、F(ab’) 2片段、Fd片段、Fv片段、单结构域抗体、线性抗体、单链抗体(scFv)及由抗体片段形成的双特异性抗体或多特异性抗体等。
术语“Fab片段”包括重链可变区和轻链可变区，并且还包括轻链的恒定区和重链的第一恒定区CH1，其为单价的抗体片段。术语“F(ab’) 2片段”包含2个Fab片段以及铰链区，其为二价的抗体片段。
术语“Fd片段”一般包含重链可变区和恒定区CH1；术语“Fv片段”含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
术语“多特异性抗体”是指，将抗体通过功能连接(例如化学偶联、基因融合、非共价结合或其他方法)至一个或多个其他结合分子上，从而形成与两个以上不同位点和/或靶点结合的新的抗体构建体。其中，使用较多的是“双特异性抗体”，其特指针对两种不同抗原具有特异性的抗体构建体。通常，双特异性抗体或多特异性抗体至少包括2个抗原结合结构域。
术语“抗原”是指被抗体或抗体结合片段识别并特异性结合的物质，广义上，抗原可以包括所选靶标的任何免疫原性片段或决定簇，包括单表位、多表位、单结构域、多结构域、或完整的胞外结构域(ECD)或蛋白质。肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合均可构成抗原。非限制性示例性抗原包括肿瘤抗原或病原体抗原等。“抗原”也可以指引发免疫反应的分子。任何形式的抗原或含有该抗原的细胞或制剂都可以用于生成对抗原决定簇具有特异性的抗体。抗原可以是分离的全长蛋白质、细胞表面蛋白(例如，用在其表面上表达至少一部分抗原的细胞进行免疫的)、或可溶性蛋白质(例如，仅用该蛋白质的ECD部分进行免疫的)或蛋白质构建体(例如，Fc抗原)。该抗原可以在基因修饰的细胞中产生。前述任何抗原可以单独或与本领域已知的一种或多种免疫原性增强佐剂组合使用。编码该抗原的DNA可以是基因组的或非基因组的(例如，cDNA)，并且可以编码足以引起免疫原性应答的至少一部分ECD。可以使用任何载体来转化其中表达抗原的细胞，所述载体包括但不限于腺病毒载体、慢病毒载体、质粒以及非病毒载体如阳离子脂质。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨 基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。由给定的抗体确定其结合的表位的方法是本领域熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用以指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“氨基酸修饰”(或“修饰的氨基酸”)包括在多肽序列中的氨基酸取代、插入和/或缺失。本文中的“氨基酸取代”或“取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代S32A指第32位的丝氨酸被丙氨酸替换。
人源化抗体可变区与人类受体可变区的序列同一性或同源性可以如在此所论述的进行测定，并且当这样测量时，将优选地共享至少60％或65％的序列同一性，更优选至少70％、75％、80％、85％或90％的序列同一性，甚至更优选至少93％、95％、98％或99％的序列同一性。优选地，不相同的残基位置因保守性氨基酸置换而不同。“保守取代”是一个氨基酸残基被具有类似化学特性(例如，电荷或疏水性)的侧链(R基团)的另一个氨基酸残基替换的氨基酸取代。一般而言，保守氨基酸取代不会实质上改变蛋白质的功能特性。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的急性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因而，可以用其他相似侧链的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基。在两个或更多个氨基酸序列因保守取代而彼此不同的情形中，序列同一性百分比或相似性程度可以向上调整以校正该取代的保守性质。
在单克隆抗体生产过程中，不同的理化因素易产生各种翻译后修饰(post-translational modification，PTM)变异体，如糖基化、氧化、糖化、脱酰胺、异构化及端基环化等，这些PTM可能引起抗体理化性质变化，改变与抗体Fc受体的相互反应，影响与目标抗原的结合活性；一些PTM的发生甚至可能降低抗体稳定性、引起免疫原性等(JARASCH等，JOURNAL OF PHARMACEUTICAL SCIENCES，2015)。可以通过对PTM位点的氨基酸修饰，例如保守取代，来消除其所带来的负面影响。基于改造PTM的目的对抗体CDR进行氨基酸取代，也均明确地保持在本发明的范围内。
术语“抗体依赖性细胞介导的细胞毒性”(ADCC)指抗体结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的Fc受体(FcR)结合，介导杀伤细胞直接杀伤靶细胞。
术语“补体依赖的细胞毒性”(CDC)指补体参与的细胞毒作用，即通过特异性抗体与细胞膜表面相应抗原结合，形成复合物而激活补体经典途径，所形成的攻膜复合物对靶细胞发挥裂解效应。
本发明的抗体还可以包括恒定区(例如Fc)的取代或修饰，包括但不限于氨基酸残基取代、突变和/或修饰，它们产生具有以下优选的特征的化合物，这些优选的特征包括但不限于：改变的药物代谢动力学、增加的血清半衰期、增加的结合亲和力、降低的免疫原性、增加的产量、与Fc受体(FcR)的改变的Fc配体结合、增强或减弱的ADCC或CDC、改变的糖基化和/或二硫键以及修饰的结合特异性。
术语“亲和力”或“结合亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。术语“K D”是指特定的抗体-抗原相互作用的解离常数。可以使用本领域已知的各种技术来确定结合亲和力，例如表面等离子体共振、生物层干涉法、双极化干涉法、静态光散射、动态光散射、等温滴定量热法、ELISA、分析超速离心和流式细胞术等。
术语“竞争结合”或“竞争性抗体”通常是指，与本发明的抗体结合相同表位的抗体，其结合导致本发明的抗体与抗原表位的结合被抑制或阻断，在竞争测定中可以得到竞争性抑制的程度。
术语“药物组合物”指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
术语“药用载体”或“药学上可接受的载体”指与治疗剂一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂或媒介物。
术语“有效量”指本发明的抗体或片段的药物制剂的剂量，其以单一或多次剂量施用患者后，在治疗的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如人种差异；体重、年龄和健康状况；涉及的具体疾病；疾病的严重程度；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸含量上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括与在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
本文所用的术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳转”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。发明所述的抗体或其抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，(2001)ISBN：012441351上获得。
本发明工程化的抗体或其抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可 以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。
本文所用的术语“个体”或“受试者”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如食蟹猴或恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
术语“抗体药物缀合物”(ADC)是指已共价偶联治疗活性物质或活性药物成分(API)的抗体，从而治疗活性物质或活性药物成分(API)可以靶向至抗体的结合靶标以表现出其药理学功能。治疗活性物质或活性药物成分可以是能够杀死ADC靶向的细胞的细胞毒素，优选恶性或癌细胞。治疗活性物质、活性药物成分或细胞毒素的共价连接可以以非位点特异性方式利用偶联有效载荷至赖氨酸或半胱氨酸残基的标准化学接头进行，或者优选地，缀合以位点特异性方式进行，其允许完全控制缀合位点以及产生的ADC的药物比抗体比例。
术语“嵌合抗原受体”(CAR)是工程化的受体，其将任意特异性移植至免疫效应细胞上。通常，这些受体用来将单克隆抗体的特异性移植至T细胞上；通过逆转录病毒或慢病毒载体或者通过转座子促进它们编码序列的转移。CAR-工程化的T细胞(也缩写为CAR-T细胞)是装有嵌合受体的遗传工程化的T细胞，其胞外识别单元包含抗体衍生的识别结构域，并且其胞内区源自淋巴细胞刺激部分。原型CAR的结构是模块化的，设计为容纳各种功能结构域，从而使得能够选择特异性并控制T细胞的激活。优选的抗体衍生的识别单元是组合单克隆抗体的重链和轻链可变区的特异性和结合残基的单链可变片段(scFv)。最常见的淋巴细胞激活部分包括与T-细胞触发(例如CD3ζ)部分串联的T-细胞共刺激(例如CD28)结构域。通过用这类嵌合受体装备效应子淋巴细胞(如T细胞和自然杀伤细胞)，工程化的细胞重新定向具有预先定义的对任何期望靶抗原的特异性，以非HLA限制性方式。利用逆转录病毒或慢病毒载体或者转座子将CAR构建体离体引入来自给定患者的外周淋巴细胞的T细胞。将所得的CAR工程化的T细胞输注回患者之后，它们运输，到达它们的靶位点，并且在与它们的靶细胞或组织相互作用时，它们经历激活并发挥它们预先定义的效应子功能。CAR方法的治疗靶标包括癌症和HIV感染的细胞，或者自身免疫效应细胞。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的，不诱导或抑制正常细胞增殖。肿瘤可影响多种细胞、组织或器官，包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经 组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、尿道、输尿管、尿道、子宫、阴道器官，或组织或相应的细胞。肿瘤包括癌症，如肉瘤，癌，或浆细胞瘤(浆细胞的恶性肿瘤)。本发明所述的肿瘤，可包括，但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)，淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症，重链病，实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。较佳地，所述的“肿瘤”包括但不限于：胰腺癌、肝癌、肺癌、胃癌、食管癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、多发性骨髓瘤、卵巢癌、宫颈癌和胶质瘤。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如，所述的“疾病”包括但不限于：肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的的疾病过程中的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“药盒”或“试剂盒”包括有效量的一种或多种单位剂型的本发明的药物组合物。在一些实施方案中，药盒可含有无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。此外，药盒还包括将本发明的药物组合物给予个体的说明书。说明书中通常包含使用本发明的药物组合物来治疗疾病的方法。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1动物免疫
为产生抗CLDN18.2的抗体，采用标准生物学方案进行试验操作。使用稳转表达hCLDN18.2的CHO-K1细胞(CHO-K1/hCLDN18.2)和编码hCLDN18.2的DNA载体作为免疫原免疫不同品系的小鼠共15只。
免疫后期通过内眦静脉采血获得血浆样品，通过ELISA和FACS对免疫血清进行效价检测，确定动物的免疫应答情况，4轮免疫后，选择6只小鼠进行安乐死，制备杂交瘤细胞。
实施例2抗CLDN18.2单克隆抗体的杂交瘤细胞产生
为产生抗CLDN18.2单克隆抗体的杂交瘤细胞，用二氧化碳将6只小鼠安乐死后，用注射器分别采集饲养细胞，将饲养细胞悬液铺入准备好的96孔板中。取一定数量的骨髓瘤细胞和脾细胞按比例混合，进行细胞融合。将HAT培养基(1mL 100×HT supplement+1mL氨基喋呤+10mL FBS+88mL DMEM)加入到融合后的细胞中，混匀制成细胞悬液。然后将细胞悬液倒入培养皿中充分混匀，用多道移液器将细胞悬液铺入96孔饲养细胞板中。将融合后的饲养细胞板放入培养箱，5.5％CO 2，37℃恒温培养7-10天。然后通过ELISA和FACS筛选抗CLDN18.2的阳性克隆。筛选的阳性克隆利用有限稀释法将细胞进行亚克隆，进而获得稳定单一的杂交瘤细胞。使用稳转表达hCLDN18.2的HEK293细胞(HEK293-hCLDN18.2)，通过FACS对亚克隆的细胞上清进行筛选。如说明书附图1-5所示，最终获得18个产生特异性结合hCLDN18.2抗体的杂交瘤细胞株。
培养上述筛选得到的分泌单克隆抗体的杂交瘤细胞株，采用常规的生物学方法从细胞中提取总RNA。以总RNA为模板，使用PrimeScript TM 1st Strand cDNA Synthesis Kit(TAKARA)，逆转录合成cDNA。以cDNA为模板，使用抗体的恒定区引物扩增。PCR产物经琼脂糖凝胶电泳分离后，纯化回收DNA片段，经测序获得本发明的18个单克隆抗体可变区的氨基酸序列，结果如表1所示：
表1 18个单克隆抗体可变区的氨基酸序列
克隆号 重链可变区(VH) 轻链可变区(VL)
59F9 SEQ ID NO:1 SEQ ID NO:2
48H1 SEQ ID NO:3 SEQ ID NO:4
43F5 SEQ ID NO:5 SEQ ID NO:6
40G7 SEQ ID NO:7 SEQ ID NO:8
76D11 SEQ ID NO:9 SEQ ID NO:10
32G8 SEQ ID NO:11 SEQ ID NO:12
37G8 SEQ ID NO:13 SEQ ID NO:14
39C8 SEQ ID NO:15 SEQ ID NO:16
42D2 SEQ ID NO:17 SEQ ID NO:18
126A11 SEQ ID NO:19 SEQ ID NO:20
239E9 SEQ ID NO:21 SEQ ID NO:22
229C9 SEQ ID NO:23 SEQ ID NO:24
252F11 SEQ ID NO:25 SEQ ID NO:26
299B2 SEQ ID NO:27 SEQ ID NO:28
241F7 SEQ ID NO:29 SEQ ID NO:30
251H11 SEQ ID NO:31 SEQ ID NO:32
287F12 SEQ ID NO:33 SEQ ID NO:34
253C4 SEQ ID NO:35 SEQ ID NO:36
在上述氨基酸序列的基础上，利用Kabat编号规则划分抗体可变区的CDR和FR，每个抗体的6个CDR序列组成如下表2所示，表2中括号内数字表示序列号，例如(37)即代表SEQ ID NO:37。
Figure PCTCN2020090427-appb-000001
Figure PCTCN2020090427-appb-000002
实施例3抗CLDN18.2嵌合抗体的构建及其在真核细胞中的瞬时转染表达
将测序完成的本发明的单抗可变区与人IgG1恒定区拼接后生成的目的基因片段克隆到pcDNA3.4表达载体中，制备转染级别的表达质粒。在无血清培养基中培养Expi293F TM细胞(Thermo Fisher Scientific)，将细胞接种在摇瓶(Corning Inc.)中，并在37℃，8％CO 2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和ExpiFectamine TM 293转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，转染16-18小时后，添加ExpiFectamine TM 293 Transfection Enhancer 1和ExpiFectamine TM 293 Transfection Enhancer 2，细胞培养6天后收集上清纯化，对最终纯化的嵌合抗体进行SDS-PAGE纯度分析和A280浓度测定。嵌合抗体以在原杂交瘤克隆的基础上增加前缀ch-的方式来进行命名。
实施例4抗CLDN18.2嵌合抗体的结合试验
A.抗CLDN18.2嵌合抗体与表达hCLDN18.2细胞的结合
使用FACS检测抗CLDN18.2嵌合抗体与稳转表达hCLDN18.2的HEK293细胞(HEK293-hCLDN18.2)、天然表达hCLDN18.2的胃癌肿瘤组织来源细胞(PDX-hCLDN18.2)的结合情况。
收集HEK293-hCLDN18.2或PDX-hCLDN18.2细胞，用PBS重悬，调整细胞浓度，加入梯度稀释后的抗体，其中，阴性对照为不相关的人IgG，阳性对照(参比抗体)为来自专利CN103509110B中公开的嵌合抗体ch-175D10。4℃摇床孵育50min-1h后，用磷酸盐缓冲液离心洗涤2次，加入荧光标记的抗人IgG二抗，每孔100μL，4℃摇床孵育40min-1h后，用磷酸盐缓冲液离心洗涤2次，然后将制备好的样品在流式细胞仪上进行检测，通过软件计算每个浓度的平均荧光强度(以下简称MFI)，然后通过GraphPad软件计算半数结合浓度(以下简称EC 50)和最高平均荧光强度(Top MFI)，结果如表3所示。
表3抗CLDN18.2嵌合抗体与hCLDN18.2的结合
Figure PCTCN2020090427-appb-000003
Figure PCTCN2020090427-appb-000004
表3与附图6-17展示的是本发明的嵌合抗体和参比抗体ch-175D10分别与HEK293-hCLDN18.2细胞和PDX-hCLDN18.2细胞的亲和力结果。实验结果显示：本发明的嵌合抗体与HEK293-hCLDN18.2细胞的结合最高平均荧光强度在5586-12203之间，而参比抗体ch-175D10在同样的反应条件下与HEK293-hCLDN18.2结合的最高平均荧光强度仅为5451。嵌合抗体与PDX-hCLDN18.2结合的最高平均荧光强度在7616-19876之间，半数结合浓度(EC 50)在4.554-96.23nM之间，参比抗体ch-175D10在同样的反应条件下与PDX-hCLDN18.2结合的最高平均荧光强度仅为7160，半数结合浓度(EC 50)仅为63.85nM。由此可见，本发明的大多数嵌合抗体与hCLDN18.2抗原的结合能力均强于ch-175D10。
B.抗CLDN18.2嵌合抗体的结合选择性
使用FACS检测本发明的嵌合抗体与稳转表达鼠CLDN18.2的HEK293细胞(HEK293-mCLDN18.2)和稳转表达人CLDN18.1的HEK293细胞(HEK293-hCLDN18.1)的结合情况。
分别收集HEK293-mCLDN18.2和HEK293-hCLDN18.1细胞，用PBS重悬并调整细胞浓度，加入梯度稀释后的嵌合抗体，其中，阴性对照为不相关的人IgG，阳性对照(参比 抗体)仍然是ch-175D10。4℃摇床孵育50分钟后，用磷酸盐缓冲液离心洗涤2次，加入荧光标记的抗人IgG二抗，每孔100μL，4℃摇床孵育40分钟后，用磷酸盐缓冲液离心洗涤2次，将制备好的样品在流式细胞仪上进行检测，然后通过GraphPad软件计算半数结合浓度(EC 50)和最高平均荧光强度(Top MFI)，结果如表4所示。
表4抗CLDN18.2嵌合抗体分别与mCLDN18.2和hCLDN18.1的结合
Figure PCTCN2020090427-appb-000005
表4展示了本发明的嵌合抗体和参比抗体ch-175D10分别与HEK293-mCLDN18.2细胞和HEK293-hCLDN18.1细胞的亲和力结果。实验结果显示：本发明的嵌合抗体与参比抗体ch-175D10相同，均与mCLDN18.2抗原结合，嵌合抗体与HEK293-mCLDN18.2结合的最高平均荧光强度在6093-14768之间，半数结合浓度(EC 50)在0.573-1.763nM之间，参 比抗体ch-175D10在同样的反应条件下与HEK293-mCLDN18.2结合的最高平均荧光强度为4187，半数结合浓度(EC 50)为1.014nM，嵌合抗体的结合EC 50与参比抗体相当，最大结合高于参比抗体。并且，嵌合抗体与参比抗体ch-175D10相同，均不与hCLDN18.1抗原结合。
实施例5抗CLDN18.2嵌合抗体的体外功能试验
A.抗体依赖性细胞介导的细胞毒性(ADCC)
以CHO-K1/hCLDN18.2为靶细胞，以转染158V/V型FcγRⅢa基因的NK细胞(NK92/FcRγ3a.158V/V)为效应细胞，使用细胞毒性检测试剂盒(Roche)检测细胞乳酸脱氢酶(LDH)释放量，并以此作为细胞杀伤作用的指标。
离心收集CHO-K1/hCLDN18.2细胞，弃上清后用ADCC缓冲液重悬并调整细胞密度，转移到96孔实验板中。转移不同浓度梯度的嵌合抗体、对照样品工作液或ADCC缓冲液至96孔板中，于细胞培养箱(37℃/5％CO 2)内孵育约30分钟，转移效应细胞、ADCC缓冲液或细胞裂解液至96孔实验板中，于细胞培养箱(37℃/5％CO 2)内继续孵育约6小时。孵育结束后，取出96孔实验板，低转速离心后，小心吸取上清，转移至新的96孔实验板中，加入LDH检测工作液后，于室温孵育约10-30分钟，在酶标仪上检测OD值，检测波长为492nm，参比波长为650nm。
ADCC效应引起的细胞裂解百分比采用以下公式进行计算：
％细胞裂解＝100％×(样品释放-靶细胞/效应细胞混合释放)/(最大释放–靶细胞释放)，其中最大释放为Triton X-100处理靶细胞的孔产生的吸光度值，靶细胞/效应细胞混合释放为靶细胞和效应细胞混合孔产生的吸光度值，靶细胞释放为仅含有靶细胞的孔中产生的吸光度值，样品释放为嵌合抗体与靶细胞、效应细胞混合孔产生的吸光度值，GraphPad软件计算EC 50和最大裂解，结果如表5所示。
表5抗CLDN18.2嵌合抗体的ADCC活性
克隆号 最大裂解(％) EC 50(μg/mL)
ch-175D10 31.81 0.021
ch-59F9 29.37 0.023
ch-48H1 44.73 0.011
ch-43F5 48.46 0.064
ch-40G7 32.72 0.018
ch-76D11 45.60 0.021
ch-32G8 46.83 0.019
ch-37G8 31.71 0.022
ch-39C8 34.45 0.022
ch-42D2 39.12 0.020
ch-126A11 36.65 0.010
ch-239E9 36.68 0.021
ch-229C9 29.06 0.023
ch-252F11 31.09 0.022
ch-299B2 33.52 0.047
ch-241F7 36.57 0.027
ch-251H11 38.79 0.024
ch-287F12 35.46 0.017
ch-253C4 35.72 0.036
表5与附图18-23展示了本发明的嵌合抗体和参比抗体ch-175D10对CHO-K1/hCLDN18.2细胞的ADCC结果。实验结果显示：本发明的嵌合抗体对CHO-K1/hCLDN18.2细胞的最大ADCC效应在29.06％-48.46％之间，参比抗体ch-175D10在同样的反应条件下ADCC效应为31.81％。本发明的嵌合抗体产生50％ADCC效应的浓度(EC 50)在0.010-0.064μg/mL之间，ch-175D10在同样的反应条件下产生50％ADCC效应的浓度(EC 50)为0.021μg/mL。上述结果证明了本发明的嵌合抗体在ADCC活性方面与参比抗体ch-175D10相当。
B.补体依赖的细胞毒性(CDC)
以CHO-K1/hCLDN18.2为靶细胞，以混合健康人血清(PNHS)作为补体来源，通过
Figure PCTCN2020090427-appb-000006
化学发光细胞活力分析试剂盒(Promega)检测细胞活力。
收集CHO-K1/hCLDN18.2细胞，使用CDC缓冲液重悬并调整细胞密度，将细胞接种到384孔细胞板中，每孔20μL，配制4倍浓度的样品溶液，转移溶液(对照组为CDC缓冲液)至384孔细胞板对应孔中，每孔10μL，室温孵育约30分钟，使用CDC缓冲液稀释混合健康人血清(Pooled Normal Human Serum，PNHS)至4倍工作浓度。转移稀释后的PNHS至完成孵育的384孔板的对应孔中，每孔10μL，放入细胞培养箱(37℃/5％CO 2)继续孵育约4小时后，取出384孔板，用
Figure PCTCN2020090427-appb-000007
化学发光细胞活力分析试剂盒(Promega)检测，使用PHERAstar Plus软件读取结果。
CDC试验中嵌合抗体引起的细胞裂解率采用以下公式进行计算：
％细胞裂解＝100％×(1-(试验孔-血清对照孔)/(细胞+血清孔-血清对照孔))
实验对照为：血清对照孔：仅有血清(即30μL缓冲液+10μL稀释后的血清)。细胞+血清孔：向CHO-K1/hCLDN18.2细胞悬液的孔中加入血清(即20μL细胞悬液+10μL缓冲液+10μL稀释后的血清)。试验孔：向CHO-K1/hCLDN18.2细胞悬液的孔中加入血清和嵌合抗体(即20μL细胞悬液+10μL抗体+10μL稀释后的血清)。
GraphPad软件计算EC 50和最大裂解，结果如表6所示。
表6抗CLDN18.2嵌合抗体的CDC活性
克隆号 最大裂解(％) EC 50(μg/mL)
ch-175D10 87.41 1.193
ch-59F9 94.71 0.091
ch-48H1 94.27 0.170
ch-43F5 89.79 0.206
ch-40G7 89.08 0.380
ch-76D11 95.36 0.108
ch-32G8 93.85 0.108
ch-37G8 92.86 0.160
ch-39C8 94.75 0.177
ch-42D2 94.47 0.130
ch-126A11 95.02 0.065
ch-239E9 94.80 0.139
ch-229C9 96.11 0.165
ch-252F11 95.44 0.179
ch-299B2 95.53 0.124
ch-241F7 95.05 0.099
ch-251H11 97.11 0.359
ch-287F12 96.32 0.141
ch-253C4 95.23 0.038
表6与附图24-29展示了本发明的嵌合抗体和参比抗体ch-175D10对CHO-K1/hCLDN18.2细胞的CDC结果。实验结果显示：本发明的嵌合抗体对CHO-K1/hCLDN18.2 的最大CDC效应在89.08％-97.11％之间，而参比抗体ch-175D10在同样的反应条件下产生的最大CDC效应为87.41％。本发明的嵌合抗体产生50％CDC效应的浓度(EC 50)在0.038-0.380μg/mL之间，而ch-175D10在同样的反应条件下产生50％CDC效应的浓度(EC 50)高于1μg/mL。由此可见，大多数抗体的CDC活性强于参比抗体。
实施例6制备抗CLDN18.2嵌合抗体的变体
经过对本发明的单克隆抗体进行翻译后修饰(PTM)分析，发现299B2和253C4的可变区中都存在1个脱酰胺位点，对299B2轻链可变区上第31或者第32或者第33位氨基酸进行单个位点的定点突变，制备三个299B2的突变体：分别是299B2-N31Q、299B2-S32A和299B2-G33A。
以及，对253C4轻链可变区上第31或者第32或者第33位氨基酸进行单个位点的定点突变，制备三个253C4的突变体：分别是253C4-N31Q、253C4-S32A和253C4-G33A。
299B2-N31Q、299B2-S32A和299B2-G33A的重链可变区氨基酸序列与299B2相同，253C4-N31Q、253C4-S32A和253C4-G33A的重链可变区氨基酸序列与253C4相同。上述6个变体的可变区CDR序列以及重链和轻链可变区序列分别如表7和表8所示。
Figure PCTCN2020090427-appb-000008
表8抗体299B2和253C4及其变体的可变区序列
克隆号 重链可变区 轻链可变区
299B2 SEQ ID NO:27 SEQ ID NO:28
299B2-N31Q SEQ ID NO:27 SEQ ID NO:114
299B2-S32A SEQ ID NO:27 SEQ ID NO:115
299B2-G33A SEQ ID NO:27 SEQ ID NO:116
253C4 SEQ ID NO:35 SEQ ID NO:36
253C4-N31Q SEQ ID NO:35 SEQ ID NO:117
253C4-S32A SEQ ID NO:35 SEQ ID NO:118
253C4-G33A SEQ ID NO:35 SEQ ID NO:119
参照实施例3所述，将上述6个变体可变区与人IgG1恒定区拼接后生成的目的基因片段亚克隆到表达载体中构建所述变体的嵌合抗体，变体的嵌合抗体以在“杂交瘤克隆-突变位点”的基础上增加前缀ch-的方式来进行命名，例如299B2-N31Q的嵌合抗体即命名为ch-299B2-N31Q。经哺乳动物细胞系瞬转表达后，使用HEK293-hCLDN18.2细胞进行亲和力检测，通过FACS对单克隆的细胞上清进行筛选，抗体ch-299B2和ch-253C4的变体的亲和力结果参见表9。
表9抗体ch-299B2和ch-253C4的变体的亲和力
克隆号 EC 50(ng/mL) 最高平均荧光强度(Top MFI)
ch-175D10 155.9 2836
ch-299B2-N31Q -* -*
ch-299B2-S32A 130.7 6504
ch-299B2-G33A 157.0 6869
ch-253C4-N31Q 175.0 6565
ch-253C4-S32A 206.7 6894
ch-253C4-G33A 186.7 6837
*最高浓度仍未达到最大结合，曲线无法拟合。
实施例7人源化抗体的制备
A.抗体299B2-S32A的人源化设计和表达
通过序列相似性比对，选取与299B2相似度最高的抗体种系作为抗体模板，在本实施例中，选取IMGT数据库IGHV1-46*01作为299B2-S32A重链的抗体模板，IGKV4-1*01 作为299B2-S32A轻链的抗体模板，将299B2-S32A的轻链和重链CDR区替换掉抗体模板的CDR区。
对鼠抗可变区序列进行同源建模。基于鼠抗可变区的序列在PDB抗体数据库中搜索最佳的建模模板，同源性为74％的2GKI被选为模板。基于2GKI的空间结构，根据以下标准，对CDR嫁接后的序列的骨架区氨基酸残基进行回复突变：1.骨架区经典残基将选择进行回复突变；2.骨架区中疏水核心区域的残基将选择进行回复突变；3.重链/轻链相互作用界面残基将选择进行回复突变；4.类似的残基也可以作为低优先级的回复突变。
299B2-S32A经过人源化改造，获得4个人源化抗体hu299B2-S32A-1、hu299B2-S32A-2、hu299B2-S32A-3、hu299B2-S32A-4，上述所有299B2-S32A的人源化抗体序列参见表10。
通过本领域技术人员已知的标准方法，将人源化的抗体hu299B2-S32A可变区与人IgG1恒定区拼接后生成的目的基因片段亚克隆到pcDNA3.4表达载体中，采用ExpiFectamine TM 293转染试剂瞬时转染对数生长期的Expi293F TM细胞，收集培养上清并进行亲和纯化，对最终纯化的抗体进行SDS-PAGE纯度分析和A280浓度测定。
B.抗体253C4-N31Q的人源化设计和表达
通过序列相似性比对，选取与253C4相似度最高的抗体种系作为抗体模板，在本实施例中，选取IMGT数据库IGKV4-1*01作为253C4-N31Q重链的抗体模板，IGHV1-2*06作为253C4-N31Q轻链的抗体模板，将253C4-N31Q的轻链和重链CDR区替换掉抗体模板的CDR区。
对鼠抗可变区序列进行同源建模。基于鼠抗可变区的序列在PDB抗体数据库中搜索最佳的建模模板，同源性为74％的2GKI被选为模板。基于2GKI的空间结构，根据以下标准，对CDR嫁接后的序列的骨架区氨基酸残基进行回复突变：1.骨架区经典残基将选择进行回复突变；2.骨架区中疏水核心区域的残基将选择进行回复突变；3.重链/轻链相互作用界面残基将选择进行回复突变；4.类似的残基也可以作为低优先级的回复突变。
253C4-N31Q经过人源化改造，获得3个人源化抗体，hu253C4-N31Q-1、hu253C4-N31Q-2、hu253C4-N31Q-3，上述所有253C4-N31Q人源化抗体的序列参见表10。
通过本领域技术人员已知的标准方法，将人源化的抗体hu253C4-N31Q可变区与人IgG1恒定区拼接后生成的目的基因片段亚克隆到pcDNA3.4表达载体中，采用ExpiFectamine TM 293转染试剂瞬时转染对数生长期的Expi293F TM细胞，收集培养上清并进行亲和纯化，对最终纯化的抗体进行SDS-PAGE纯度分析和A280浓度测定。
表10人源化抗体hu299B2-S32A和hu253C4-N31Q的可变区序列
克隆号 重链可变区 轻链可变区
hu299B2-S32A-1 SEQ ID NO:120 SEQ ID NO:121
hu299B2-S32A-2 SEQ ID NO:120 SEQ ID NO:123
hu299B2-S32A-3 SEQ ID NO:120 SEQ ID NO:124
hu299B2-S32A-4 SEQ ID NO:122 SEQ ID NO:121
hu253C4-N31Q-1 SEQ ID NO:125 SEQ ID NO:126
hu253C4-N31Q-2 SEQ ID NO:125 SEQ ID NO:127
hu253C4-N31Q-3 SEQ ID NO:128 SEQ ID NO:126
实施例8人源化抗体hu299B2和hu253C4的结合活性
A.人源化抗体与表达hCLDN18.2细胞的结合
参照实施例4A所述进行结合活性测定，使用HEK293-hCLDN18.2和PDX-hCLDN18.2细胞，阴性对照为不相关的人IgG，阳性对照(参比抗体)为来自专利CN103509110B中公开的嵌合抗体ch-175D10。采用流式细胞术检测，通过软件计算每个浓度的平均荧光强度(以下简称MFI)，然后通过GraphPad软件计算半数结合浓度(EC 50)和最高平均荧光强度(Top MFI)，结果如表11所示。
表11抗CLDN18.2人源化抗体与hCLDN18.2的结合
Figure PCTCN2020090427-appb-000009
表11与附图30-33展示的是本发明的人源化抗体和参比抗体ch-175D10分别与HEK293-hCLDN18.2细胞和PDX-hCLDN18.2细胞的亲和力结果。实验结果显示：本发明 的hu299B2-S32A人源化抗体与HEK293-hCLDN18.2细胞的结合最高平均荧光强度在29529-39986之间，而参比抗体ch-175D10在同样的反应条件下与HEK293-hCLDN18.2结合的最高平均荧光强度仅为26921，人源化抗体hu299B2-S32A与HEK293-hCLDN18.2的亲和力优于参比抗体ch-175D10。hu253C4-N31Q与HEK293-hCLDN18.2的结合最高平均荧光强度与参比抗体ch-175D10相当，EC 50优于参比抗体ch-175D10。
本发明的hu299B2-S32A、hu253C4-N31Q与PDX-hCLDN18.2结合的最高平均荧光强度在15585-25189之间，半数结合浓度(EC 50)在11.42-29.68nM之间，参比抗体ch-175D10在同样的反应条件下与PDX-hCLDN18.2结合的最高平均荧光强度仅为5703，半数结合浓度(EC 50)为24.89nM。由此可见，本发明的人源化抗体与天然hCLDN18.2抗原的最大结合强于ch-175D10，EC50与ch-175D10相当。
B.人源化抗体的结合选择性
参照实施例4B所述方法，使用FACS检测本发明的人源化抗体与HEK293-mCLDN18.2细胞和HEK293-hCLDN18.1细胞的结合情况。
分别收集HEK293-mCLDN18.2和HEK293-hCLDN18.1细胞，用PBS重悬并调整细胞浓度，加入梯度稀释后的人源化抗体，其中，阴性对照为不相关的人IgG，阳性对照(参比抗体)仍然是ch-175D10。4℃摇床孵育50分钟后，用磷酸盐缓冲液离心洗涤2次，加入荧光标记的抗人IgG二抗，每孔100μL，4℃摇床孵育40分钟后，用磷酸盐缓冲液离心洗涤2次，将制备好的样品在流式细胞仪上进行检测，然后通过GraphPad软件计算半数结合浓度(EC 50)和最高平均荧光强度(Top MFI)，结果如表12所示。
表12抗CLDN18.2人源化抗体分别与mCLDN18.2和hCLDN18.1的结合
Figure PCTCN2020090427-appb-000010
Figure PCTCN2020090427-appb-000011
表12展示了本发明的人源化抗体和参比抗体ch-175D10分别与HEK293-mCLDN18.2细胞和HEK293-hCLDN18.1细胞的亲和力结果。实验结果显示：本发明的人源化抗体与参比抗体ch-175D10相同，均与mCLDN18.2抗原结合，人源化抗体与HEK293-mCLDN18.2结合的最高平均荧光强度在36841-102785之间，半数结合浓度(EC 50)在0.026-0.342μg/mL之间，参比抗体ch-175D10在同样的反应条件下与HEK293-mCLDN18.2结合的最高平均荧光强度为86407，半数结合浓度(EC 50)为0.136μg/mL，人源化抗体与mCLDN18.2的结合与参比抗体相当。并且，人源化抗体与参比抗体ch-175D10相同，均不与hCLDN18.1抗原结合。
实施例9人源化抗体的体外功能试验
A.抗体依赖性细胞介导的细胞毒性(ADCC)
参照实施例5A所述方法，以CHO-K1/hCLDN18.2为靶细胞，以转染158V/V型FcγRⅢa基因的NK细胞(NK92/FcRγ3a.158V/V)为效应细胞，使用细胞毒性检测试剂盒(Roche)检测细胞乳酸脱氢酶(LDH)释放量，并以此作为细胞杀伤作用的指标。
ADCC效应引起的细胞裂解百分比采用以下公式进行计算：
％细胞裂解＝100％×(样品释放-靶细胞/效应细胞混合释放)/(最大释放–靶细胞释放)，其中最大释放为Triton X-100处理靶细胞的孔产生的吸光度值，靶细胞/效应细胞混合释放为靶细胞和效应细胞混合孔产生的吸光度值，靶细胞释放为仅含有靶细胞的孔中产生的吸光度值，样品释放为人源化抗体与靶细胞、效应细胞混合孔产生的吸光度值，GraphPad软件计算EC50和最大裂解，结果如表13所示。
表13抗CLDN18.2人源化抗体的ADCC活性
克隆号 最大裂解(％) EC 50(μg/mL)
ch-175D10 51.42/49.15 0.047/0.039
hu299B2-S32A-1 57.89 0.022
hu299B2-S32A-2 68.08 0.028
hu299B2-S32A-3 69.09 0.022
hu299B2-S32A-4 63.23 0.024
hu253C4-N31Q-1 54.85 0.024
hu253C4-N31Q-2 58.80 0.037
hu253C4-N31Q-3 70.26 0.038
表13与附图34-35展示了本发明的人源化抗体和参比抗体ch-175D10对CHO-K1/hCLDN18.2细胞的ADCC结果。实验结果显示本发明的人源化抗体对CHO-K1/hCLDN18.2细胞的最大ADCC效应在54.85％-70.26％之间，参比抗体ch-175D10在同样的反应条件下ADCC效应为50％左右。本发明的人源化抗体产生50％ADCC效应的浓度(EC 50)在0.022-0.038μg/mL之间，ch-175D10在同样的反应条件下产生50％ADCC效应的浓度(EC 50)约为0.040μg/mL。上述结果证明了本发明的人源化抗体在ADCC活性方面与参比抗体ch-175D10相当。
B.补体依赖的细胞毒性(CDC)
参照实施例5B所述方法，以CHO-K1/hCLDN18.2为靶细胞，以混合健康人血清(PNHS)作为补体来源，通过
Figure PCTCN2020090427-appb-000012
化学发光细胞活力分析试剂盒(Promega)检测细胞活力，使用PHERAstar Plus软件读取结果。
CDC试验中人源化抗体引起的细胞裂解率采用以下公式进行计算：
％细胞裂解＝100％×(1-(试验孔-血清对照孔)/(细胞+血清孔-血清对照孔))
实验对照为：血清对照孔：仅有血清(即30μL缓冲液+10μL稀释后的血清)。细胞+血清孔：向CHO-K1/hCLDN18.2细胞悬液的孔中加入血清(即20μL细胞悬液+10μL缓冲液+10μL稀释后的血清)。试验孔：向CHO-K1/hCLDN18.2细胞悬液的孔中加入血清和嵌合抗体(即20μL细胞悬液+10μL抗体+10μL稀释后的血清)。
GraphPad软件计算EC 50和最大裂解，结果如表14所示。
表14抗CLDN18.2人源化抗体的CDC活性
克隆号 最大裂解(％) EC 50(μg/mL)
ch-175D10 87.76 1.549
hu299B2-S32A-1 99.15 0.125
hu299B2-S32A-2 99.74 0.129
hu299B2-S32A-3 99.83 0.092
hu299B2-S32A-4 99.87 0.123
hu253C4-N31Q-1 96.29 0.175
hu253C4-N31Q-2 97.51 0.149
hu253C4-N31Q-3 96.11 0.128
表14与附图36-37展示了本发明的人源化抗体和参比抗体ch-175D10对CHO-K1/hCLDN18.2细胞的CDC结果。实验结果显示：本发明的人源化抗体对CHO-K1/hCLDN18.2的最大CDC效应在96.11％-99.87％之间，而参比抗体ch-175D10在同样的反应条件下产生的最大CDC效应为87.76％。本发明的嵌合抗体产生50％CDC效应的浓度(EC 50)在0.092-0.175μg/mL之间，而ch-175D10在同样的反应条件下产生50％CDC效应的浓度(EC 50)高于1μg/mL。由此可见，所有人源化抗体的CDC活性强于参比抗体。
上文所述的本发明的实施方案仅为示例性的，任何本领域技术人员都可以认识到或者可以确定无数的特定化合物、材料和操作的等价物，而不需要进行超出常规的试验。所有这些等价物都是在本发明范围之内的，并且被权利要求所包含。

Claims (49)

  1. 一种抗CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够特异性结合CLDN18.2，其包含：
    (1)重链可变区，所述重链可变区包含3个选自如以下序列所示的HCDR：
    SEQ ID NO:37，38，39，40，41，42，43，44，45，46，47，48，49，50，51，52，53，54，55，56，57，58，59，60，61，62，63，64，65，66，67，68，69，70，71，72，73，74，75，76，77，78，79，80，81，82，83，84，85；和/或，
    (2)轻链可变区，所述轻链可变区包含3个选自如以下序列所示的LCDR：
    SEQ ID NO:86，87，88，89，90，91，92，93，94，95，96，97，98，99，100，101，102，103，104，105，106，107，108，109，110，111，112，113。
  2. 一种抗CLDN18.2的抗体或其抗原结合片段，其包含：
    (1)重链可变区，所述重链可变区包含选自SEQ ID NO:37，40，43，45，49，53，56，59，62，65，68，71，74，77，80，83所示的HCDR1；以及，选自SEQ ID NO:38，41，46，48，50，52，54，57，60，63，66，69，72，75，78，81，84所示的HCDR2；以及，选自SEQ ID NO:39，42，44，47，51，55，58，61，64，67，70，73，76，79，82，85所示的HCDR3；和/或，
    (2)轻链可变区，所述轻链可变区包含选自SEQ ID NO:86，87，88，89，90，91，92，111，112，113所示的LCDR1；以及，选自SEQ ID NO:93，94，95，96所示的LCDR2；以及，选自SEQ ID NO:97，98，99，100，101，102，103，104，105，106，107，108，109，110所示的LCDR3。
  3. 一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和/或轻链可变区，所述重链可变区包含选自下组的HCDR1、HCDR2和HCDR3：
    SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39或
    SEQ ID NO:40、SEQ ID NO:41和SEQ ID NO:42或
    SEQ ID NO:43、SEQ ID NO:41和SEQ ID NO:44或
    SEQ ID NO:45、SEQ ID NO:46和SEQ ID NO:47或
    SEQ ID NO:37、SEQ ID NO:48和SEQ ID NO:39或
    SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51或
    SEQ ID NO:49、SEQ ID NO:52和SEQ ID NO:51或
    SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55或
    SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58或
    SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61或
    SEQ ID NO:62、SEQ ID NO:63和SEQ ID NO:64或
    SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67或
    SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70或
    SEQ ID NO:71、SEQ ID NO:72和SEQ ID NO:73或
    SEQ ID NO:74、SEQ ID NO:75和SEQ ID NO:76或
    SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79或
    SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82或
    SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85；
    和/或，所述轻链可变区选自下组的LCDR1、LCDR2和LCDR3：
    SEQ ID NO:86、SEQ ID NO:93和SEQ ID NO:97；或
    SEQ ID NO:87、SEQ ID NO:94和SEQ ID NO:98；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:99；或
    SEQ ID NO:87、SEQ ID NO:95和SEQ ID NO:100；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:97；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:101；或
    SEQ ID NO:89、SEQ ID NO:93和SEQ ID NO:102；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:100；或
    SEQ ID NO:90、SEQ ID NO:93和SEQ ID NO:103；或
    SEQ ID NO:91、SEQ ID NO:96和SEQ ID NO:104；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:98；或
    SEQ ID NO:92、SEQ ID NO:93和SEQ ID NO:105；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:106；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:107；或
    SEQ ID NO:87、SEQ ID NO:93和SEQ ID NO:108；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:109；或
    SEQ ID NO:88、SEQ ID NO:93和SEQ ID NO:110；或
    SEQ ID NO:111、SEQ ID NO:93和SEQ ID NO:107；或
    SEQ ID NO:112、SEQ ID NO:93和SEQ ID NO:107；或
    SEQ ID NO:113、SEQ ID NO:93和SEQ ID NO:107；或
    SEQ ID NO:111、SEQ ID NO:93和SEQ ID NO:110；或
    SEQ ID NO:112、SEQ ID NO:93和SEQ ID NO:110；或
    SEQ ID NO:113、SEQ ID NO:93和SEQ ID NO:110。
  4. 一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和轻链可变区，所述可变区包含如下任一一组的6个CDR，每组的6个CDR按照HCDR1，HCDR2，HCDR3，LCDR1，LCDR2，LCDR3依次排列：
    (1)SEQ ID NO:37，38，39，86，93，97；
    (2)SEQ ID NO:40，41，42，87，94，98；
    (3)SEQ ID NO:43，41，44，88，93，99；
    (4)SEQ ID NO:45，46，47，87，95，100；
    (5)SEQ ID NO:37，48，39，88，93，97；
    (6)SEQ ID NO:49，50，51，88，93，101；
    (7)SEQ ID NO:49，52，51，89，93，102；
    (8)SEQ ID NO:53，54，55，88，93，100；
    (9)SEQ ID NO:56，57，58，90，93，103；
    (10)SEQ ID NO:59，60，61，91，96，104；
    (11)SEQ ID NO:62，63，64，88，93，98；
    (12)SEQ ID NO:65，66，67，92，93，105；
    (13)SEQ ID NO:68，69，70，88，93，106；
    (14)SEQ ID NO:71，72，73，88，93，107；
    (15)SEQ ID NO:74，75，76，88，93，106；
    (16)SEQ ID NO:77，78，79，87，93，108；
    (17)SEQ ID NO:80，81，82，88，93，109；
    (18)SEQ ID NO:83，84，85，88，93，110；
    (19)SEQ ID NO:71，72，73，111，93，107；
    (20)SEQ ID NO:71，72，73，112，93，107；
    (21)SEQ ID NO:71，72，73，113，93，107；
    (22)SEQ ID NO:83，84，85，111，93，110；
    (23)SEQ ID NO:83，84，85，112，93，110；
    (24)SEQ ID NO:83，84，85，113，93，110。
  5. 一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和/或轻链可变区，
    (1)所述重链可变区具有与下述序列至少80％至100％的序列同一性：SEQ ID NO:1，3，5，7，9，11，13，15，17，19，21，23，25，27，29，31，33，35；和/或，
    (2)所述轻链可变区具有与下述序列至少80％至100％的序列同一性：SEQ ID NO:2，4，6，8，10，12，14，16，18，20，22，24，26，28，30，32，34，36，114，115，116，117，118，119。
  6. 一种抗CLDN18.2的抗体或其抗原结合片段，其包含与如下任一一组的重链可变区和轻链可变区具有至少80％至100％的序列同一性：
    (1)SEQ ID NO:1和2；
    (2)SEQ ID NO:3和4；
    (3)SEQ ID NO:5和6；
    (4)SEQ ID NO:7和8；
    (5)SEQ ID NO:9和10；
    (6)SEQ ID NO:11和12；
    (7)SEQ ID NO:13和14；
    (8)SEQ ID NO:15和16；
    (9)SEQ ID NO:17和18；
    (10)SEQ ID NO:19和20；
    (11)SEQ ID NO:21和22；
    (12)SEQ ID NO:23和24；
    (13)SEQ ID NO:25和26；
    (14)SEQ ID NO:27和28；
    (15)SEQ ID NO:29和30；
    (16)SEQ ID NO:31和32；
    (17)SEQ ID NO:33和34；
    (18)SEQ ID NO:35和36；
    (19)SEQ ID NO:27和114；
    (20)SEQ ID NO:27和115；
    (21)SEQ ID NO:27和116；
    (22)SEQ ID NO:35和117；
    (23)SEQ ID NO:35和118；
    (24)SEQ ID NO:35和119。
  7. 如前述任一抗CLDN18.2的抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体或人源化抗体。
  8. 一种抗CLDN18.2的抗体或其抗原结合片段，包含重链可变区和/或轻链可变区，所述重链可变区具有与下述序列至少80％至100％的序列同一性：SEQ ID NO:120，122，125，128；和/或，所述轻链可变区具有与下述序列至少80％至100％的序列同一性：SEQ ID NO:121，123，124，126，127。
  9. 一种抗CLDN18.2的抗体或其抗原结合片段，其包含与如下任一一组的重链可变区和轻链可变区具有至少80％至100％的序列同一性：
    (1)SEQ ID NO:120和121；
    (2)SEQ ID NO:120和123；
    (3)SEQ ID NO:120和124；
    (4)SEQ ID NO:122和121；
    (5)SEQ ID NO:125和126；
    (6)SEQ ID NO:125和127；
    (7)SEQ ID NO:128和126。
  10. 如前述任一抗CLDN18.2的抗体或其抗原结合片段，其为单克隆抗体。
  11. 如前述任一抗CLDN18.2的抗体或其抗原结合片段，其还包括重链恒定区和/或轻 链恒定区，优选的，所述重链恒定区包括Fc或变体Fc，Fc来源于鼠或人。
  12. 如前述任一抗CLDN18.2的抗体，其为全长抗体。
  13. 如前述任一抗CLDN18.2的抗体或其抗原结合片段，其为IgG1、IgG2、IgG3或IgG4形式。
  14. 如前述任一抗CLDN18.2的抗体或其抗原结合片段，所述抗原结合片段为Fab、Fv、scFv、F(ab’) 2、线性抗体、单结构域抗体。
  15. 一种偶联物，将前述任一抗CLDN18.2的抗体或其抗原结合片段与捕获标记物或检测标记物偶联形成，所述的检测标记物包括放射性核素、发光物质、有色物质或酶。
  16. 一种双特异性抗体，其中的一个抗原结合结构域包含前述任一抗CLDN18.2的抗体或其抗原结合片段。
  17. 一种多特异性抗体，其中的一个抗原结合结构域包含前述任一抗CLDN18.2的抗体或其抗原结合片段。
  18. 一种抗体药物缀合物，含有前述任一抗CLDN18.2的抗体或其抗原结合片段，所述的抗体药物缀合物由抗体-接头-毒素相互连接形成。
  19. 一种嵌合抗原受体，其胞外识别单元包含前述任一抗CLDN18.2的抗体或其抗原结合片段。
  20. 编码前述任一抗CLDN18.2的抗体或其抗原结合片段的核酸。
  21. 包含权利要求20所述核酸的重组载体。
  22. 一种宿主细胞，其包含权利要求21的重组载体或基因组中整合有权利要求20的核酸。
  23. 如权利要求22所述的宿主细胞，其为原核细胞，例如大肠杆菌；或其为真核细胞，例如酵母或哺乳动物细胞，所述哺乳动物细胞例如CHO细胞或HEK293细胞。
  24. 制备前述任一抗CLDN18.2的抗体或其抗原结合片段的方法，包括：在适合的条件下培养权利要求22或23的宿主细胞，并从所述细胞中纯化获得表达产物。
  25. 前述任一抗CLDN18.2的抗体或其抗原结合片段在制备特异性靶向表达CLDN18.2的肿瘤细胞的药物中的用途。
  26. 前述任一抗CLDN18.2的抗体或其抗原结合片段在制备表达CLDN18.2的肿瘤的诊断试剂中的用途，所述表达CLDN18.2的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌，卵巢癌，结肠癌，肝癌，头颈癌和胆囊癌及其转移，所述胃癌转移例如库肯勃氏瘤。
  27. 一种检测样品中CLDN18.2表达的方法，包括：
    (1)将样品与前述任一抗CLDN18.2的抗体或其抗原结合片段接触；
    (2)检测抗CLDN18.2的抗体或其抗原结合片段和CLDN18.2的复合物的形成；任选地，抗CLDN18.2的抗体或其抗原结合片段是被可检测地标记的。
  28. 一种药物组合物，其包含有效量的前述任一抗CLDN18.2的抗体或其抗原结合片段、或包含有效量的权利要求16的双特异性抗体、或包含有效量的权利要求17的多特异性抗体、或包含有效量的权利要求18的抗体药物缀合物、或包含有效量的权利要求19的嵌合抗原受体、或包含有效量的权利要求20的核酸、或包含有效量的权利要求21的重组载体、或包含有效量的权利要求22或23的宿主细胞。
  29. 如权利要求28所述的药物组合物，还包含药学上可接受的载体。
  30. 如权利要求28或29所述的药物组合物，还包含一种或多种额外的其他治疗剂。
  31. 如权利要求29所述的药物组合物，所述一种或多种额外的其他治疗剂包括：化学治疗剂、细胞毒性剂、放射性治疗剂、癌症疫苗、减瘤剂、靶向性抗癌剂、抗血管生成剂、生物反应修饰剂、细胞因子、激素、抗转移剂和免疫治疗剂。
  32. 一种药盒或试剂盒，其包括容器，以及位于容器中的权利要求28-31任一的药物组合物。
  33. 一种诱导表达CLDN18.2的细胞死亡的方法，包括使所述细胞与权利要求28-31任一的药物组合物接触，所述表达CLDN18.2的细胞是癌细胞。
  34. 如权利要求33所述的方法，所述细胞是实体瘤细胞，选自：胃癌细胞、食道癌细胞、肠癌细胞、胰腺癌细胞、肾母细胞瘤细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、直肠癌细胞、肝癌细胞、头颈癌细胞、慢性骨髓性白血病细胞和胆囊癌细胞。
  35. 一种治疗受试者中与表达CLDN18.2相关的疾病的方法，所述方法包括向有此需要的受试者施用权利要求28或29的药物组合物。
  36. 如权利要求35所述的方法，所述疾病是肿瘤，优选胃癌、食道癌、肠癌、胰腺癌、肾母瘤、肺癌、卵巢癌、结肠癌、直肠癌、肝癌、头颈癌、慢性骨髓性白血病或胆囊癌。
  37. 如权利要求35或36所述的方法，还包括向所述受试者给予额外的治疗剂。
  38. 如权利要求35所述的方法，所述额外的治疗剂包括：化学治疗剂、细胞毒性剂、放射性治疗剂、癌症疫苗、减瘤剂、靶向性抗癌剂、抗血管生成剂、生物反应修饰剂、细胞因子、激素、抗转移剂和免疫治疗剂。
  39. 如权利要求38所述的方法，所述化学治疗剂包括：长春新碱、长春花碱、长春地辛和诺维宾、伊立替康、托泊替康、依托泊苷、替尼泊苷、米多昔佐兹、顺铂、卡铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬、达卡巴嗪、阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤、丙卡巴肼、多柔比星、博来霉素、更生霉素、柔红霉素、丝裂霉素、肉瘤霉素C、放线菌素D、罗红霉素、阿霉素、雷帕霉素、道诺霉素、紫杉醇、多西他赛、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。
  40. 如权利要求37所述的方法，所述额外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。
  41. 如权利要求38所述的方法，所述细胞因子包括IL-10、IL-15、IL4和IL13。
  42. 如权利要求38所述的方法，所述靶向性抗癌剂包括：大分子靶向药物和小分子靶向药物。
  43. 如权利要求42所述的方法，所述大分子靶向药物包括：西妥昔单抗、帕尼单抗、尼妥珠单抗、曲妥珠单抗、帕妥珠单抗、T-DM1、贝伐珠单抗、VEGF-TRAP和雷莫芦单抗。
  44. 如权利要求42所述的方法，小分子靶向药物包括：厄洛替尼、吉非替尼、拉帕替尼、阿法替尼、伊马替尼、舒尼替尼、索拉非尼、瑞戈替尼、帕唑替尼、阿帕替尼、克唑替尼、伏立诺他、马马司他和依维莫司；以及，靶向其他靶点的小分子药物，包括PI3Kα、PKB/AKT和STAT3靶点。
  45. 如权利要求38所述的方法，所述免疫治疗剂为针对下述免疫相关靶点的大分子或小分子药物：PD-1/PD-L1、PD-L2、CTLA-4、LAG-3、IDO、TIM3、TIGIT、CD47、SIRPα、4-1BB、CSF-1/CSF1R、GITR、OX40、CD40、CD27、CD28、B7H4、B7H3、TGFβ、BTLA、VISTA、ICOS、CD39、CD73、A2AR、KIR和NKG2A；以及，与免疫治疗相关的细胞疗法。
  46. 如权利要求45所述的方法，靶向PD-1/PD-L1的免疫检查点抑制剂包括帕姆单抗、纳武单抗、阿特珠单抗、阿维单抗、信迪利单抗、Cemiplimab和Durvalumab。
  47. 如权利要求45所述的方法，靶向CTLA-4的免疫检查点抑制剂包括伊匹单抗。
  48. 如权利要求37所述的方法，所述额外的治疗剂选自溶瘤病毒，所述溶瘤病毒包括细小病毒、腺病毒、疱疹病毒、痘病毒、脊髓灰质炎病毒、呼肠孤病毒、甲病毒、马拉巴病毒、逆转录病毒和科萨基病毒。
  49. 如权利要求37所述的方法，所述额外的治疗剂选自癌症疫苗或蛋白酶抑制剂，所述蛋白酶抑制剂为硼替佐米。
PCT/CN2020/090427 2019-05-16 2020-05-15 针对密蛋白18a2的抗体及其应用 Ceased WO2020228806A1 (zh)

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