Antibody capable of binding to CLDN18.2 or antigen binding fragment thereof and application of antibody or antigen binding fragment

Abstract

The invention relates to a novel humanized anti-human CLDN18.2 antibody and an antigen binding fragment of the novel humanized anti-human CLDN18.2 antibody. The invention also relates to a preparation method of the antibody. The invention further relates to a method for treating and / or preventing tumors by targeted binding of the antibody or the antigen binding fragment thereof to CLDN18.2.

Description

An antibody or antigen-binding fragment thereof that can bind to CLDN18.2 and its applications

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antibody or antigen-binding fragment thereof that can bind to CLDN18.2. The present invention also relates to the applications of the antibody.

[0002] Claudins are important members of the tight junction protein family and play an important role in cell-cell and cell-matrix junctions. Claudin18 is encoded by the gene CLDN18, is a four-transmembrane protein with two extracellular regions, and is widely expressed in tissues such as the stomach, pancreas, and lung. Claudin18 is considered to be a diagnostic marker and a therapeutic target (Krause, G., Winkler, L., Mueller, S. L., Haseloff, R. F., Piontek, J., & Blasig, I. E. (2008). Structure and function of claudins. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1778(3), 631-645.).

[0003] The Claudin18 family contains two variants, Claudin18.1 (CLDN18.1) and Claudin18.2 (CLDN18.2). Among them, CLDN18.1 is specifically expressed on alveolar epithelial cells (Yasui, W., Sentani, K., Sakamoto, N., Anami, K., Naito, Y., & Oue, N. (2011). Molecular pathology of gastric cancer: Research and practice. Pathology-Research and Practice, 207(10), 608-612.), and it differs from CLDN18.2 by only 21 amino acids (Sahin et al., 2008). CLDN18.2 is widely present in gastric tumor tissues; it has recently been found to be expressed in pancreatic cancer, esophageal cancer, and lung cancer as well (Jovov et al., 2007; Karanjawala et al., 2008). In normal cells, due to the tight cell-cell junctions, antibodies cannot access the Claudin18.2 expressed on the cells; while in tumor cells, due to cell lesions, the cell-cell junctions become loose, thus exposing CLDN18.2, and antibodies can bind to these exposed CLDN18.2 molecules. Therefore, CLDN18.2 has become an ideal drug target molecule (Klamp, T., Schumacher, J., Huber, G., Kuhne, C., Meissner, U., Selmi, A., Sahin, U. (2011). Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases. Cancer Res, 71(2), 516-527.).

[0004] Zolbetuximab (IMAB362) is the first chimeric antibody drug developed against the CLDN18.2 target. It is not humanized and can specifically bind to CLDN18.2 on the surface of tumor cells, thereby triggering antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibiting cell proliferation (Woll, S., Schlitter, A.M., Dhaene, K., Roller, M., Esposito, I., Sahin, U., & Tureci, O. (2014). Claudin 18.2 is a target for IMAB362 antibody in pancreatic neoplasms. Int J Cancer, 134(3), 731 - 739). Preclinical studies have successfully demonstrated the powerful ability of Zolbetuximab to eliminate cancer cells and control the disease; subsequently, its clinical efficacy and safety have been evaluated through multiple phase I / II trials, but there are still unmet clinical needs in the treatment of gastric cancer targeting CLDN18.2 (Lordick, F., Al-Batran, S.E., Ganguli, A., Morlock, R., Sahin, U., & Tureci, O. (2021). Patient-reported outcomes from the phase II FAST trial of zolbetuximab plus EOX compared to EOX alone as first-line treatment of patients with metastatic CLDN18.2+ gastroesophageal adenocarcinoma. Gastric Cancer, 24(3), 721 - 730). Therefore, the development of monoclonal antibodies that specifically bind to CLDN18.2 but not to CLDN18.1, and have highly humanized and low immunogenicity antibodies is of great significance in the diagnosis and development of tumors such as gastric cancer.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is to obtain an antibody that can specifically bind to CLDN18.2, does not bind to CLDN18.1, and has high affinity, low immunogenicity, and good clinical prospects. For this purpose, the inventors have prepared an antibody against human CLDN18.2 or its antigen-binding fragment. Compared with the prior art, the antibody or its antigen-binding fragment obtained in the present invention has higher affinity and lower immunogenicity.

[0007] The antibodies of the present invention can have various uses, including detecting Claudin 18.2 protein, diagnosing, treating or preventing Claudin18.2-related cancers, etc.

[0008] Accordingly, in one aspect, the present invention relates to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, which comprises: a light chain complementarity-determining region LC-CDR1 as shown in the amino acid sequence SEQ ID NO: 5, a light chain complementarity-determining region LC-CDR2 as shown in the amino acid sequence SEQ ID NO: 7, and a light chain complementarity-determining region LC-CDR3 as shown in the amino acid sequence SEQ ID NO: 9; and a heavy chain complementarity-determining region HC-CDR1 as shown in the amino acid sequence SEQ ID NO: 11, a heavy chain complementarity-determining region HC-CDR2 as shown in the amino acid sequence SEQ ID NO: 13, and a heavy chain complementarity-determining region HC-CDR3 as shown in the amino acid sequence SEQ ID NO: 15.

[0009] In some embodiments, the amino acid sequences of its heavy chain and light chain variable regions are respectively as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0010] In some embodiments, the present invention also provides a humanized antibody or an antigen-binding fragment thereof of the above-mentioned antibody or its antigen-binding fragment.

[0011] In some embodiments, the humanized antibody is characterized in that the amino acid sequence of its light chain variable region is as shown in SEQ ID NO: 17; and the amino acid sequence of its heavy chain variable region is as shown in SEQ ID NO: 19.

[0012] In one aspect, the present invention thus provides a nucleic acid encoding the antibody or its antigen-binding portion described above.

[0013] In one aspect, the present invention also provides an expression vector and / or a host cell comprising the nucleic acid.

[0014] In one aspect, the present invention provides a pharmaceutical composition comprising the antibody or its antigen-binding portion described above, the nucleic acid, the expression vector or the host cell.

[0015] The present invention also provides a kit comprising the antibody or its antigen-binding portion described above, the nucleic acid, the expression vector or the host cell.

[0016] The present invention further provides the use of the antibody or its antigen-binding portion described above, the nucleic acid, the expression vector or the host cell in the preparation of a drug or reagent for diagnosing, treating or preventing tumors.

[0017] Further, the tumor is a tumor associated with CLDN18.2.

[0018] Further, the tumor is gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, or breast cancer.

[0019] The antibody or antigen-binding fragment thereof of the present invention has high affinity, low immunogenicity, can specifically bind to CLDN18.2, and has endocytosis activity, CDC activity, ADCC activity, and ADCP phagocytosis.

[0020] Definition

[0021] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures used herein are all widely used terms and conventional procedures in the relevant fields. At the same time, to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0022] As used herein and unless otherwise stated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. In cases where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0023] Provided herein are antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to CLDN18.2. In a specific aspect, provided herein are antibodies that specifically bind to CLDN18.2 (e.g., human CLDN18.2). The term "CLDN18.2" refers to any CLDN18.2 receptor known to those skilled in the art. For example, the CLDN18.2 can be from a mammal, such as the CLDN18.2 can be from a human or a cynomolgus monkey.

[0024] As used herein, a full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. An "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody, but which comprises at least the portion of the variable region of the antibody that binds the antigen (e.g., one or more CDRs and / or one or more antibody-binding sites), and thus retains the binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. The heavy chain constant region is composed of three domains, namely CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by relatively conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. Each heavy and light chain of an antibody molecule has 3 CDRs (i.e., the heavy chain has HC-CDR1, HC-CDR2, HC-CDR3; the light chain has LC-CDR1, LC-CDR2, LC-CDR3). CDRs are also called hypervariable regions and are present in the variable regions of each heavy and light chain of an antibody, and have very highly variable sites in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, CDR3 from the amino terminus of the amino-terminal sequence of the heavy chain, which have the same meaning as HC-CDR1, HC-CDR2, HC-CDR3, and the CDRs of the light chain are represented by CDR1, CDR2, CDR3 from the amino terminus of the amino-terminal sequence of the light chain, which have the same meaning as LC-CDR1, LC-CDR2, LC-CDR3. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0025] Antibodies include antibody fragments, examples of antibody fragments include but are not limited to Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (Welschof & Krauss, 2003). Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), acquires the ability to bind immunospecifically (i.e., exhibits at least or at least about 10 7 -10 8An antibody against the Ka antigen of M-1. A "functional fragment" or "analogue of an anti-CLDN18.2 antibody" is a fragment or analogue that can prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an "antibody fragment", and in the context of an antibody, can refer to a fragment that can prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, such as an Fv, Fab, F(ab')2, etc. An "Fv" fragment consists of a dimer (VH-VL dimer) formed by non-covalent binding of the variable domain of a heavy chain and the variable domain of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target-binding site on the surface of the VH-VL dimer, as in the case of a full antibody. The six CDRs together confer the target-binding specificity of the full antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind a target.

[0026] In this document, the three-letter or single-letter abbreviations for amino acids have been used in their conventional meanings, as shown in the following table:

[0027] As used herein, a "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the others. This property is contrary to that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods. For example, monoclonal antibodies can be prepared by immortalizing B cells, such as by fusing them with myeloma cells to produce a hybridoma cell line or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence encoding the antibody.

[0028] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0029] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0030] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, the mutations in the CDR are generally no more than one or two. Therefore, the humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 amino acid mutations in the CDR.

[0031] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0032] As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present in a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present in an amount of about 1×10 7 M -1 or 1x10 8 M -1 or greater affinity constant K a (or 1x10 -7 M or 1×10 -8 M or lower dissociation constant (K d )) binds to the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, for example, immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available.

[0033] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0034] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying the polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0035] As used herein, a "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0036] As used herein, a "vector" is a replicable nucleic acid that, when the vector is transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Regarding vectors, those include vectors into which nucleic acids encoding a polypeptide or a fragment thereof can typically be introduced by restriction enzyme digestion and ligation. Regarding vectors, those also include vectors containing nucleic acids encoding a polypeptide. Vectors are used to introduce nucleic acids encoding a polypeptide into host cells for amplifying the nucleic acid or for expressing / displaying the polypeptide encoded by the nucleic acid. Vectors generally remain free, but can be designed to integrate a gene or a portion thereof into the chromosomes of the genome. Vectors of artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0037] As used herein, a vector also includes a "viral vector" or "vector of a virus". A vector of a virus is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0038] As used herein, "expression vector" includes a vector capable of expressing DNA, wherein the DNA is operably linked to regulatory sequences capable of affecting the expression of such DNA fragments, such as a promoter region. Such additional fragments may include promoter and terminator sequences and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally are derived from plasmid or viral DNA or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells and expression vectors that remain episomal or integrate into the host cell genome.

[0039] As used herein, "pharmaceutical composition" refers to a pharmaceutically acceptable composition that includes, for example, one or more of the therapeutic agents described herein, such as two, three, four, five, six, seven, eight, or more, formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. The carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0040] As used herein, "treating" an individual having a disease or medical condition means that the symptoms of the individual are partially or completely alleviated or remain unchanged after treatment. Thus, treatment includes prevention, therapy, and / or cure. Prevention refers to preventing a potential disease and / or preventing the worsening of symptoms or the progression of a disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein and any composition provided herein.

[0041] "Diagnosing" refers to assessing the likelihood that an individual has a disease or disorder or is at risk of developing a disease or disorder. In particular, as will be understood by those skilled in the art, such an assessment, while preferably being correct for 100% of the subjects to be diagnosed, generally is not. In one embodiment, the term requires that a statistically significant portion of the subjects can be identified as having the disease or being predisposed thereto.

[0042] As used herein, "efficacy" refers to the effect resulting from the treatment of an individual, which modifies, typically ameliorates or improves, the symptoms of a disease or medical condition, or cures the disease or medical condition.

[0043] The term "EC 50 ", also known as the half maximal effective concentration, refers to the antibody concentration that elicits 50% of the maximal effect.

[0044] Figure 1 shows the binding activity of hybridoma supernatant on 293T-hCLDN18.2 cells.

[0045] Figures 2A-2G show the binding activity of the H7E12-2 humanized antibody and human CLDN18.2. Among them:

[0046] Figure 2A shows the binding activity of the humanized antibody H7E12-2 on 293T-hCLDN18.2 cells;

[0047] Figure 2B shows the comparison of the expression intensity of human CLDN18.2 in different cell lines;

[0048] Figure 2C shows the binding activity on CT-26-hCLDN18.2 cells;

[0049] Figure 2D shows the binding activity on MC-38-hCLDN18.2-C4 cells;

[0050] Figure 2E shows the binding activity on 293T-hCLDN18.2 cells;

[0051] Figure 2F shows the binding activity on MC-38-hCLDN18.2-A11 cells;

[0052] Figure 2G shows the binding activity on KATOIII-hCLDN18.2 cells.

[0053] Figure 3 shows the activity of the H7E12-2 antibody cross-binding human CLDN18.1.

[0054] Figure 4 shows the activity of the H7E12-2 antibody cross-binding mouse CLDN18.2.

[0055] Figure 5 shows the endocytosis activity of the H7E12-2 antibody.

[0056] Figure 6 shows the CDC activity of the H7E12-2 antibody.

[0057] Figures 7A-7C show the activity of the H7E12-2 antibody on the ADCC reporter cell line (Jurkat-NFAT-Luc2-CD16a-V158). Among them:

[0058] Figure 7A shows MC38-hCLDN18.2 as the target cell;

[0059] Figure 7B shows 293T-hCLDN18.2-A11 as the target cell;

[0060] Figure 7C shows KATOIII-hCLDN18.2 as the target cell.

[0061] Figure 8 shows the ADCC activity mediated by the H7E12-2 antibody in PBMC-derived NK cells.

[0062] Figure 9 shows the ADCP activity mediated by macrophages of the H7E12-2 antibody. Specific implementation

[0063] The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0064] The experimental methods in the following examples are all conventional methods unless otherwise specified. The reagents, raw materials, etc. used in the following examples are all commercially available products unless otherwise specified. Zolbetuximab (IMAB362): Ganymed Pharmaceuticals, see NW_004504382.1.

[0065] Example 1 Preparation of hybridoma cells

[0066] Immunize mice with the CHO-K1 Human CLDN18.2 Cell Line (kyinno KC-1180) that stably transfected and expressed the CLDN18.2 gene (from Genbank accession number NM_001002026.3, whose sequence is as SEQ ID NO: 1) as the immunogen. Five mice were immunized intraperitoneally, and the adjuvant used was Quick antibody 5W water-soluble adjuvant. The titer was measured 2 weeks after the booster immunization. Two mice with high titers were selected for immune boosting, and cell fusion described below was carried out 3 days later.

[0067] Take the two mice ready for fusion, take serum, dissect the spleen after dissection, isolate spleen cells, fuse the spleen cells with cultured myeloma cells, plate in 96-well plates, and at the same time add selective medium for screening. Change the medium after 7 days, and perform ELISA detection after 10 days. Select those with an OD value more than 10 times that of the negative control and then perform flow cytometry detection.

[0068] Select double-positive cells, perform subcloning plating by the method of limited dilution of cells, and select monoclonal cells. Take the culture supernatant of the selected monoclonal cells, perform ELISA detection and flow cytometry detection, and select double-positive cells for expansion culture.

[0069] Example 2 ELISA detection of the binding of the culture supernatant of hybridoma cells to CLDN18.2

[0070] Use the Bxpc-3 Human CLDN18.2 Cell Line containing the CLDN18.2 gene (from Genbank accession number NM_001002026.3), purchased from Kangyuan Bochuang, cell number KC-1272, and add 1x10 4 cells / well, 100 μL / well into a 96-well cell culture plate, and incubate overnight at 37°C in 5% CO 2 Discard the liquid in the wells and wash 3 times with the washing solution. Add 200 μL of blocking solution to each well and incubate at 4°C overnight for 2 hours.

[0071] Add 50 - 100 μL of the culture supernatant of the hybridoma cells to be tested to each well. At the same time, set up a positive control (add the serum of the fused mice), a negative control (add the serum of normal mice), and a blank control (add the culture medium). Incubate at 37°C for 1 - 2 hours and wash. Then add the enzyme-labeled secondary antibody to each well, which is horseradish peroxidase-labeled goat anti-mouse IgG (SIGMA, catalog number A9044-2ml) diluted 1:10000, 50 - 100 μL per well, incubate at 37°C for 0.5 - 1 hour, and wash. Add 50 - 100 μL of freshly prepared substrate chromogenic solution TMB to each well and incubate at 37°C for 10 - 30 minutes.

[0072] Add 2 mol / L H 2 SO 4 to terminate the reaction and read the OD value on an enzyme-linked immunosorbent assay reader.

[0073] Result determination: Consider positive when P / N > 2:1 (P represents the positive value, N represents the value of normal mouse serum). If the negative control wells are colorless or nearly colorless and the positive control wells show clear color development, the results can be directly observed with the naked eye.

[0074] Example 3 FACS detection of the binding of the culture supernatant of hybridoma cells to human CLDN18.2

[0075] The human CLDN18.2 gene (from Genbank accession number NM_001002026.3) was constructed into the PLVX virus packaging vector (clontech, virus package mix, catalog number 631275). 293T cells were transfected to package the virus, and the virus was used to infect 293T cells. The cells were screened with puromycin to obtain a drug-resistant cell line, namely 293T cells stably expressing the human CLDN18.2 gene (subsequently denoted as 293T-hCLDN18.2, cell number KC-0986, purchased from Kangyuan Botech (Beijing) Co., Ltd., hereinafter referred to as Kangyuan Botech). The 293T-hCLDN18.2 cells were prepared into a cell suspension with a cell concentration of 10^7 cells / ml in PBS containing 2% FBS. 50 μL of the cell suspension was added to each flow tube (sample tube), and then 50 μL of the culture supernatant of the hybridoma cells to be tested was added, and incubated at 4°C for 60 minutes. 1 ml of flow buffer was added to each flow tube, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the washing was repeated three times. At the same time, control tube 1 (without adding the culture supernatant and the secondary antibody below, only adding the cell suspension) and control tube 2 (without adding the culture supernatant, only adding the cell suspension and the secondary antibody below) were set up.

[0076] Then, 100 μL of flow buffer was added to each flow tube for resuspension, and 5 μL of the PE-labeled anti-mouse Fc tag secondary antibody (Biolegend, catalog number 409304) was added according to the experimental requirements, and incubated at 4°C in the dark for 30 minutes. Then, 1 ml of flow buffer was added, centrifuged at 1200 rpm for 5 minutes at room temperature, the supernatant was discarded, and the washing was repeated three times. 250 μL of flow buffer was added to each flow tube, resuspended and mixed evenly, and then detected by flow cytometry. The detection results of the binding of the target antibody to human CLDN18.2 are shown in Figure 1.

[0077] The corresponding murine antibody 7E12 was obtained from the above hybridoma cells. The variable region sequences of its heavy chain and light chain are SEQ ID NO: 3 and SEQ ID NO: 4 respectively. According to the Kabat definition method, the heavy chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 11 (RYGVH), SEQ ID NO: 13 (VIWAGGSTNYNSALMS), and SEQ ID NO: 15 (EGLRHTMDY) respectively, and the light chain CDR1, CDR2, and CDR3 sequences are SEQ ID NO: 5 (KS SQSLFNSGNQKNYLA), SEQ ID NO: 7 (GASTRES), and SEQ ID NO: 9 (QNDHSFPFT) respectively.

[0078] Example 4 Humanization of the 7E12 murine antibody

[0079] For the murine anti-7E12, the following human germline sequences were selected as templates for the heavy and light chains respectively: IGHV4-39 and IGKV1-39. Homology modeling was performed on the murine anti-7E12 to simulate the structure of the Fab region. After homology modeling calculations, the predicted Fab structure of the 7E12 antibody was finally obtained.

[0080] By aligning and analyzing the predicted Fab structure, heavy chain and the IGHV4-39 sequence, the amino acids 2V, 37V, 48L, 67L, 71K, 73N, 76R, 78V in the CDR region of the heavy chain were retained as the amino acids of the original mouse, and the other mouse amino acids were replaced with the corresponding human amino acids of the IGHV4-39 template.

[0081] By aligning and analyzing the predicted Fab structure, light chain and the IGKV1-39 sequence, except for the amino acids in the CDR region of the VL which were retained as the amino acids of the original mouse, the other mouse amino acids were replaced with the corresponding human amino acids of the IGKV1-39 template. The humanized antibody H7E12-2 obtained by recombining the humanized heavy and light chains has heavy and light chain sequences of SEQ ID NO: 19 and SEQ ID NO: 17 respectively.

[0082] Using the sequence shown in SEQ ID NO: 23 as the heavy chain constant region and the sequence shown in SEQ ID NO: 21 as the light chain constant region, the murine antibody and its humanized variable region sequences were combined pairwise to prepare chimeric antibodies and humanized antibodies. Among them, the coding genes of the corresponding antibody sequences were synthesized, ligated into eukaryotic expression vectors, transformed into DH5alpha competent cells, and cultured overnight in a 37°C constant temperature incubator. Single clone strains were picked for sequencing and identification. Strains with correct sequences were selected, plasmids were extracted, and transfected into mammalian expression cells HEK-293F, and placed in a 37°C, 5% CO 2 incubator for 7 days of expression culture.

[0083] The expression supernatant was collected, centrifuged, filtered, and a protein G affinity chromatography column was selected for purification. The purity of the purified antibody was detected by SDS-PAGE electrophoresis, the antibody concentration was detected using a BCA protein detection kit, aliquoted, and stored in a -80°C refrigerator for later use. The sequences of the obtained chimeric antibodies and humanized antibodies (hereinafter referred to as H7E12-2) are shown in Table 1.

[0084] Table 1 Sequences of chimeric antibodies and humanized antibodies

[0085] Example 5 FACS detection of the binding of H7E12-2 antibody to human CLDN18.2

[0086] To determine the binding activity of the humanized H7E12-2 antibody to human CLDN18.2 protein, the binding activity was first detected using the engineered cell line 293T-hCLDN18.2 cells (cell number - KC-0986, purchased from Kangyuan Bochuang) overexpressing human CLDN18.2 membrane protein. Collect 293T-hCLDN18.2 cells in the logarithmic growth phase, wash them once with flow buffer, and centrifuge at 1000 rpm for 4 minutes. Resuspend the cells with flow buffer, adjust the cell density to 4×10^6 / mL, and plate them in a 96-well plate in a 50 μL system. Set the wells without adding the antibody to be tested and the secondary antibody as the blank control group and the wells with only the secondary antibody added as the secondary antibody control group.

[0087] In the experimental group, the antibody H7E12-2 to be tested and the positive control antibody Zolbetuximab (WO2014 / 146778A1) were started at a concentration of 20 μg / mL and diluted 10 gradient points by 3.16-fold. 50 μL of the system was added to the plated cells, and the total system was 100 μL. Incubate at 4°C in the dark for 1 hour; wash the cells 2 times with flow buffer, 200 μL each time, and centrifuge at 1500 rpm for 4 minutes. Prepare a staining solution of PE-labeled secondary antibody (PE anti-human IgG Fc Antibody, Biolegend) with a dilution ratio of 1:100 using flow buffer. Add 100 μL to the experimental group and the secondary antibody control group, and add an equal volume of flow buffer to the blank control wells; after mixing, incubate at 4°C in the dark for 1 hour; wash the cells 2 times with flow buffer, 200 μL each time, and centrifuge at 1500 rpm for 4 minutes. Resuspend in a 100 μL system, and use a Guava easyCyte6HT (Millipore) flow cytometer to detect the mean fluorescence intensity of the positive signal. Analyze the data using GraphPad Prism 7.0 software, and use a four-parameter fit of the nonlinear S curve Y = Bottom+(Top - Bottom) / (1 + 10^((LogEC 50 -X)*HillSlope)) regression to fit the data and obtain the dose-response curve, where Bottom represents the lower limit of the fluorescence signal, representing the lower asymptote of the S curve, and Top represents the upper limit of the fluorescence signal, representing the upper asymptote of the S curve. HillSlope is the absorbance increase rate parameter, equivalent to the slope of the curve, and the EC 50 value was calculated from this, and the experimental results are shown in Figure 2A and Table 2.

[0088] Table 2. FACS detection results of the binding of different CLDN18.2 antibodies to human CLDN18.2

[0089] The experimental results showed that H7E12-2 humanized monoclonal antibody bound to the EC of human CLDN18.2 antigen overexpressed on 293T-hCLDN18.2 cells. 50 The drug Zolbetuximab (EC 50 The affinity was 1.71 μg / mL, and it increased by 3.16 times.

[0090] Furthermore, the inventors compared the differences in the expression of CLDN18.2 on the cell membrane surface of 5 different genetically engineered cell lines, including the human CLDN18.2 overexpressing engineered cell line CT26-hCLDN18.2 (purchased from Nanjing Bowang), MC38-hCLDN18.2-C4 (purchased from Nanjing Bowang), MC38-hCLDN18.2-A11 (purchased from Nanjing Bowang), KATO III-hCLDN18.2 (cell number KC-1453, purchased from Kangyuan Bochuang), and 293T-hCLDN18.2 cells (cell number KC-0986, purchased from Kangyuan Bochuang). 8x 10^4 of the above different cells were incubated with 1 μg / mL of H7E12-2 antibody, and flow staining and data processing were performed according to the above binding activity experimental steps, and the average fluorescence intensity of H7E12-2 expressed by different cells was statistically shown in Figure 2B and Table 3. The results showed that the expression levels of CLDN18.2 on the membrane surface of these five cell lines were ranked as follows: CT26-hCLDN18.2 <MC38-hCLDN18.2-C4<293T-hCLDN18.2< KATOIII-hCLDN18.2<MC38-hCLDN18.2-A11。

[0091] Table 3. Comparison of CLDN18.2 expression levels in different engineered cell lines

[0092] Next, these cell lines carrying different membrane surface CLDN18.2 molecular densities were used to perform comprehensive binding activity tests on H7E12-2 and the positive control antibody Zolbetuximab. The experimental steps were as described above, except that the concentration of the H7E12-2 antibody started from 100 μg / mL and was diluted 3.16 times to 8 concentrations. The negative controls were hIgG1 and blank secondary antibody controls. The PE signal was detected by NovoCyteQuanteon flow cytometer (Agilent), and the data were analyzed using GraphPad Prism 7.0 software. Nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, from which the EC was calculated. 50 The experimental results are shown in Figures 2C-2G and Table 4. Among them, EC 50 The unit is μg / mL.

[0093] Table 4. Summary of the binding activity of H7E12-2 in cell lines with different antigen intensities of human CLDN18.2

[0094] In the cell CT26-hCLDN18.2 with the lowest fluorescence intensity of human CLDN18.2, an obvious affinity advantage of the H7E12-2 antibody can be seen. Although no effective EC 50 was obtained, it can be seen that the binding activity of H7E12-2 shows a concentration-gradient-dependent increase, while the positive control antibody Zolbetuximab has no binding activity at low antigen density. In the cell MC38-hCLDN18.2-C4 with low antigen density, the EC 50 of the binding activities of H7E12-2 and Zolbetuximab are 0.2 μg / mL and 2.7 μg / mL respectively, and the affinity is increased by 13.5 times.

[0095] In two experiments on the cell 293T-hCLDN18.2 with medium antigen density, due to the different laser intensities of the flow cytometer, there are differences in the absolute values of MFI in Figures 2A and 2E. However, the EC 50 of H7E12-2 binding to the cell surface antigen of 293T-hCLDN18.2 is very close in the two experiments, being 0.54 μg / mL and 0.8 μg / mL respectively, while the EC 50 of the positive control antibody Zolbetuximab are 1.7 μg / mL and 1.8 μg / mL respectively. Both experiments show that the binding activity of H7E12-2 in 293T-hCLDN18.2 cells is enhanced by 2.25 to 3.16 times.

[0096] In the cell KATOIII-hCLDN18.2 with high antigen density, the EC 50 of the binding activities of H7E12-2 and Zolbetuximab are 0.7 μg / mL and 1.5 μg / mL respectively, and the affinity difference is 2.1 times; in another cell MC38-hCLDN18.2-A11 with high antigen density, the EC 50 of the binding activities of H7E12-2 and Zolbetuximab are 0.7 μg / mL and 8.7 μg / mL respectively, and the affinity is increased by 12.7 times.

[0097] In summary, the H7E12-2 antibody in the present invention has a significant affinity improvement compared with the positive control antibody Zolbetuximab in cell lines with different antigen expression intensities. Especially in the case of low antigen abundance, it will provide an important theoretical basis for its subsequent applications.

[0098] Example 6 FACS Detection of the Binding of H7E12-2 Antibody to Human CLDN18.1

[0099] CLDN18.1 and CLDN18.2 both belong to the Claudins family, with a sequence homology of approximately 92% (240 / 261), and there are only 21 amino acid differences in the first transmembrane region at the N-terminus (Sahin et al., 2008). To further study the specificity of the H7E12-2 antibody, the binding activity of the H7E12-2 antibody to human CLDN18.1 antigen was tested. The experimental method referred to Example 5, with the difference that the cells used were the engineered cell line 293T-hCLDN18.1 cells that highly express human CLDN18.1 membrane protein (purchased from Kangyuan Bochuang, cell number - KC-0990). GraphPad Prism 7.0 software was used to analyze the data, and nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, and the EC 50 value was calculated. The experimental results are shown in Figure 3. As shown in the figure, H7E12-2 and the positive control antibody Zolbetuximab did not show cross-binding activity with human CLDN18.1 antigen at concentrations up to 100 μg / mL, indicating that H7E12-2 has high specificity.

[0100] Example 7 FACS Detection of the Binding of H7E12-2 Antibody to Mouse CLDN18.2

[0101] Next, the cross-recognition activity of H7E12-2 in recognizing mouse CLDN18.2 antigen was verified. Cross-recognition activity will greatly facilitate the evaluation of animal pharmacodynamic experiments. The cross-recognition experimental method referred to Example 5, with the difference that the cells used were 293T-mCLDN18.2 cells in the logarithmic growth phase (purchased from Kangyuan Bochuang, cell number - KC-1014), which overexpress mouse CLDN18.2 antigen. GraphPad Prism 7.0 software was used to analyze the data, and nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, and the EC 50 value was calculated. The experimental results are shown in Figure 4 and Table 5.

[0102] Table 5. FACS Detection Results of the Binding of H7E12-2 Antibody to 293T-mCLDN18.2 Cells

[0103] The experimental results of cross-recognition showed that both H7E12-2 and the positive control antibody Zolbetuximab have the ability to cross-recognize mouse CLDN18.2, and the EC 50They were 0.7 μg / mL and 2.8 μg / mL respectively. The affinity of H7E12-2 for murine CLDN18.2 was 4-fold higher than that of the positive control antibody Zolbetuximab.

[0104] Example 8 Endocytosis assay of H7E12-2 antibody

[0105] Receptor-mediated endocytosis is an important pathway for many membrane protein-mediated antibodies and antibody-drug conjugates to enter cells. Therefore, the endocytic activity of H7E12-2 was evaluated. Logarithmically growing 293T-hCLDN18.2 cells (cell number KC-0986, purchased from Kangyuan Bochuang) were collected, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS, resuspended with flow buffer for cell counting, adjusted to a density of 5×106 cells / mL and added to flow tubes. The test antibody was added at a final working concentration of 10 μg / mL, and incubated at 4°C in the dark for 1 hour. The cells were washed twice with pre-chilled flow buffer, resuspended with 100 μL of wash buffer, and 2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc Antibody) was added, then incubated at 4°C in the dark for 40 minutes. The cells were washed twice with pre-chilled flow buffer, and resuspended with 100 μL of wash buffer in 200 μL of complete medium (DMEM + 10% FBS), and incubated in a 37°C incubator for 2 h, 1 h, 0.5 h, 0 h at 0 h, 1 h, 1.5 h, 2 h respectively. Dissociation: The cells were washed once with flow buffer, resuspended with 200 μL of surface antibody dissociation buffer, and incubated at room temperature for 7 min. Neutralization: Neutralization buffer was added and centrifuged, and finally the cells were resuspended with 100 μL of flow buffer. The signal of PE was detected using a NovoCyte Quanteon flow cytometer (Agilent). The experimental results are shown in Figure 5 and Table 6.

[0106] Table 6. Summary of endocytic activity of H7E12-2 antibody

[0107] The experimental results showed that compared with the positive control antibody Zolbetuximab, H7E12-2 showed significant enhanced endocytic activity, with an endocytosis rate of up to 63% after 2 hours, while Zolbetuximab was only 32.5%.

[0108] Example 9 CDC activity detection of H7E12-2 humanized antibody

[0109] Complement-dependent cytotoxicity (CDC) is the mechanism by which many antibodies cause tumor killing. When the Fab end of the antibody binds to the antigen, the Fc end of the antibody binds to complement C1q in the serum, activating the complement system to form a membrane attack complex that exerts a lytic effect on the target cells. Therefore, the CDC activities of H7E12-2 and the positive control antibody Zolbetuximab were further compared. The 293T-hCLDN18.2 cells (cell number KC-0986, purchased from Kangyuan Bochuang) were centrifuged and counted, and seeded at 1x10^5 cells per well into a U-bottom 96-well plate. After adding the antibody to be tested, inactivated human AB serum at 56°C for 30 minutes and non-inactivated human AB serum were added respectively, and incubated at 37°C for 4 hours. The cells were washed twice with flow buffer, and 100 μL of PBS solution containing 1 μL of the nucleic acid dye 7-AAD (559925, BD) that binds to the DNA of apoptotic cells was added to each well for staining, and incubated at room temperature in the dark for 10 minutes. The activity of the target cells was detected using a NovoCyte Quanteon flow cytometer (Agilent). The data were analyzed using GraphPad Prism 7.0 software, and the dose-effect curve was obtained by fitting the data using non-linear S-curve regression, and the EC 50 value was calculated from this. The experimental results are shown in Figure 6 and Table 7.

[0110] Table 7. Summary of the CDC activity of the H7E12-2 antibody

[0111] The results showed that the H7E12-2 antibody had strong CDC activity, with an EC 50 of 2.1 μg / mL, while the positive control antibody Zolbetuximab had no CDC activity at all.

[0112] Example 10 Detection of the ADCC reporter cell line activity of the H7E12-2 antibody

[0113] Antibody-dependent cell cytotoxicity (ADCC) is the main mechanism by which many antibody drugs kill tumor cells. FcγRⅢA (CD16a) can mediate the ADCC effect of NK cells on tumor cells. Here, Jurkat-NFAT-Luc2-CD16a-V15 (constructed by Kangyuan Botech) was used as an alternative effector cell and co-incubated with three target cells overexpressing human CLDN18.2, namely MC38-hCLDN18.2-A11 (purchased from Nanjing Bowang), 293T-hCLDN18.2 (cell number KC-0986, purchased from Kangyuan Bo), and KATO III-hCLDN18.2 (cell number KC-1453, purchased from Kangyuan Botech) at an effector-to-target ratio of 1:1. The extracellular region of this effector cell overexpresses high-affinity FcγRⅢA CD16a-V158 and was co-incubated with the target cells and different concentrations of the test antibody (starting from 20 μg / mL, 5-fold dilution, 9 concentration points). The Fc region of the antibody binds to the extracellular high-affinity CD16a-V158 to activate the NFAT-luc2 luciferase reporter system. By The Plus Multifunctional Microplate Reader (BMG LABTECH) was used to detect the content of luciferase to detect the ADCC activity of the antibody. GraphPad Prism 7.0 software was used to analyze the data, and non-linear S-curve regression was used to fit the data to obtain the dose-effect curve, and the EC 50 value was calculated from this. The experimental results are shown in Figures 7A-7C and Table 8.

[0114] Table 8. Summary of the ADCC reporter cell line activity of H7E12-2 antibody

[0115] The fluorescence signal of the Jurkat-NFAT-Luc2-CD16a-V15 reporter cell line showed that the ADCC activity of the H7E12-2 antibody and the positive control antibody Zolbetuximab was significantly better than that of the positive control antibody Zolbetuximab in the high-density cell line MC38-hCLDN18.2-A11. The EC 50 values were 9.6 μg / mL and 272.7 μg / mL respectively (Figure 7A). They had similar killing activities on the 293T-hCLDN18.2 cells at the medium-density antigen level (Figure 7B), and the EC 50 values were 125.1 μg / mL and 29.7 μg / mL respectively. In another high-density cell line, KATOIII-hCLDN18.2, the ADCC activity of H7E12-2 was significantly better than that of the positive control antibody Zolbetuximab, and the EC 50They were 5.6 μg / mL and 302 μg / mL respectively (Figure 7C). In the two high-density cell lines of MC38-hCLDN18.2-A11 and KATOIII-hCLDN18.2, the ADCC activity of H7E12-2 was increased by 28.4 times and 53.9 times respectively compared with the positive control antibody Zolbetuximab, indicating that H7E12-2 has extremely strong ADCC activity and provides a basis for its potential clinical application.

[0116] Example 11 Detection of ADCC Activity of H7E12-2 Antibody

[0117] In vivo, ADCC is that the Fab end of the antibody binds to the antigen epitope of tumor cells, and its Fc end binds to FCγR on the surface of natural killer cells (NK cells). The activation of NK cells releases cytotoxic substances such as perforin and granzyme, mediating the killing of target cells by NK cells and causing apoptosis of target cells. In this experiment, we used primary NK cells derived from peripheral blood mononuclear cells PBMC as effector cells and 293T-hCLDN18.2 (cell number KC-0986, purchased from Kangyuan Bochuang) as target cells to establish a co-culture system to simulate the ADCC effect in vitro. First, label the target cells 293T-hCLDN18.2 with 1.6 μM CFSE (Carboxyfluorescein Diacetate Succinimidyl Ester, 565082, BD) for 10 minutes at room temperature in the dark, wash twice with 5-fold volume of pre-cooled serum-free medium, resuspend the labeled cells in ADCC culture medium and count. Add 5x 10^4 cells per well to a 96-well U-bottom plate, then add the test antibody for gradient dilution, with the starting concentration of 50 μg / mL, gradient dilute 3.16 times with complete medium, a total of 10 concentration points. After centrifuging at 500 rpm for 30 seconds, add 1.5x 10^5 PBMC cells per well, and the effector-to-target ratio is 30:1. Incubate the target cell-effector cell-antibody complex at 37 °C for 4 h. After incubation, wash the cells twice with PBS + 2% FBS, add 100 μL of PBS solution containing 1 μL of 7-AAD (559925, BD) to each well for staining, incubate at room temperature in the dark for 10 minutes, and detect the activity of target cells with a NovoCyte Quanteon flow cytometer (Agilent). Use GraphPad Prism 7.0 software to analyze the data, use non-linear S-curve regression to fit the data to obtain a dose-effect curve, and calculate the EC 50 value. The experimental results are shown in Figure 8 and Table 9.

[0118] Table 9. Summary of ADCC Activity Mediated by H7E12-2 Antibody in Primary NK Cells

[0119] The experimental results showed that in the ADCC killing experiment mediated by primary NK cells, similar to the results of the reporter cell line, H7E12-2 still showed strong ADCC activity, and the EC 50 was 12 ng / mL, while the EC 50 of the positive control antibody Zolbetuximab was 74 ng / mL. The ADCC activity mediated by primary NK cells of H7E12-2 was increased by 6.17-fold, indicating its superior tumor killing activity compared to Zolbetuximab.

[0120] Example 12 Detection of the ADCP activity of the H7E12-2 antibody

[0121] Antibody-dependent cell-mediated phagocytosis (ADCP) is another important mechanism by which antibody-based drugs kill tumor cells. The Fab of the antibody binds to the antigen, causing a conformational change and promoting the binding of the Fc end of the antibody to FcγRIIA (CD32a) on the surface of macrophages, activating the downstream signal ITAM signal of FcγRIIA and promoting the phagocytosis of tumor antigen-positive cells by macrophages. In this experiment, macrophages differentiated from mouse bone marrow were co-cultured with 293T-hCLDN18.2 (cell number KC-0986, purchased from Kangyuan Botech) cells to evaluate the ADCP activity of the H7212-2 antibody. The femurs and tibias of mice were aseptically dissected, the surface tissues were shaved off, the two ends were cut with scissors, the bone marrow was blown out with a syringe and collected by centrifugation, resuspended with medium and counted, and BMDM was differentiated with mouse M-CSF. The induced BMDM was digested with 1 mL of Accutase, centrifuged at 1200 rpm for 5 minutes, and resuspended with 1640 + 5% FBS and counted for standby. On the day of the phagocytosis experiment, the target cells were labeled with CFSE at a final concentration of 1.6 μM / mL, stained in the dark at room temperature, resuspended with 1640 + 5% FBS and counted, and seeded at 5x10^4 cells / well onto a low-attachment 96-well U-bottom plate, 80 μL per well. After adding 40 μL / well of the test drug diluted in gradient and performing a low-speed transient centrifugation, the induced BMDM was seeded onto the low-attachment 96-well U-bottom plate according to the corresponding effector-to-target ratio of cell numbers, 80 μL per well. After mixing with a multichannel pipette, it was incubated at 37 °C for 4 hours. Washed twice with flow buffer, 100 μL of PBS containing 1 μL of APC-F4 / 80 was added, mixed well and incubated at 4 °C for 30 min. Washed twice with flow buffer, 100 μL of PBS containing 1 μL of 7-AAD was added, mixed well and incubated at room temperature for 10 minutes. The phagocytosis ratio of ADCP was detected using a NovoCyte Quanteon flow cytometer (Agilent). After the results were obtained, the phagocytosis index (%) was calculated using the formula: % (F4 / 80+CFSE+) / % (F4 / 80+) × 100%. GraphPad Prism 7.0 software was used to analyze the data, and a non-linear S-curve regression was used to fit the data to obtain a dose-effect curve, and the EC 50 value was calculated therefrom. The experimental results are shown in Figure 9. Although an effective EC 50 was not obtained, it was still possible to see an increase in the phagocytic activity of H7E12-2 showing concentration-gradient dependence, with significantly enhanced phagocytic activity at each concentration point compared to the positive control antibody Zolbetuximab.

[0122] Example 13 Preparation of H7E12-2 ADCC-enhanced antibody

[0123] Expression of H7E12-2 in HEK293 / FUT8- cells with FUT8 gene knockout. The cells were purchased from Kangyuan Botech Co., Ltd. (KC-2300). The cell line was passaged in Expi293TM expression medium for production. The passage density was 0.2 - 0.3*E6 / ml, and the passage cycle was 2 - 3 days. One day before transfection, the cells were diluted to 2*E6 / ml. On the day of transfection, the cell density should be about 6E6 / ml and the viability should be greater than 95%. On the day of transfection, transfection was carried out according to the instructions of the transfection kit. The plasmids used were 432-M147-LC and 432-M147-HC, and the molar concentration ratio of the light chain to the heavy chain was 1:1. 18 - 22 hours after transfection, Enhancer1 and Enhancer2 were added at a final concentration of 0.6% v / v. After the fifth day of transfection, the cell viability was monitored. When the cell viability was less than 70%, the cells were harvested and the protein was purified and named ICP.

[0124] Example 14 Glycosylation Analysis of Protein ICP

[0125] Take 500 μg of protein and add 4 μL of PNGaseF glycosidase. Incubate at 37 °C for 16 h. Add 300 μL of pre-cooled absolute ethanol to the sample to make up to 400 μL. Mix well and place in a -20 °C refrigerator for 1 h. After taking out, centrifuge at 12000 rpm for 10 min, and carefully aspirate the supernatant with a pipette. Put the supernatant into a vacuum centrifugal concentrator and concentrate and dry at room temperature (10 h - 15 h) until there is no liquid. Add a mixture of DMSO and acetic acid (350:150): a mixture of 2-AB: sodium cyanoborohydride = 100 μL: 5 mg: 6 mg in a ratio of 20 μL. React in a light-proof environment at 65 °C for 4 h. Add 200 μL of a mixture of acetonitrile and water (8:2), shake, centrifuge for 2 min, and take the supernatant for detection.

[0126] Chromatographic conditions: column temperature 65 °C; injection pool temperature 8 °C; injection volume 10 μL. Mobile phase A: 50 mmol·L-1 ammonium formate solution, mobile phase B: acetonitrile. The gradient is that phase B changes from 70% to 63% within 25 min and from 63% to 10% within 5 min, and the flow rate is 0.4 mL·min-1. The detection wavelength for 2-AB labeling is: excitation wavelength 330 nm, emission wavelength 330 nm. Example 15 Activity Detection of Protein ICP

[0127] The ADCC activity detection method of ICP is as described in Example 11. 293T-hCLDN18.2 cells (purchased from Kangyuan Botech, cell number KC-0986) were used as target cells and co-cultured with primary NK cells from PBMC to detect the ADCC activity mediated by the Fc end of the fusion protein.

Claims

An anti-CLDN18.2 antibody or an antigen-binding fragment thereof, comprising: a light-chain complementarity-determining region LC-CDR1 as shown in amino acid sequence SEQ ID NO: 5, a light-chain complementarity-determining region LC-CDR2 as shown in amino acid sequence SEQ ID NO: 7, and a light-chain complementarity-determining region LC-CDR3 of the light chain as shown in amino acid sequence SEQ ID NO: 9; a heavy-chain complementarity-determining region HC-CDR1 as shown in amino acid sequence SEQ ID NO: 11, a heavy-chain complementarity-determining region HC-CDR2 as shown in amino acid sequence SEQ ID NO: 13, and a heavy-chain complementarity-determining region HC-CDR3 of the heavy chain as shown in amino acid sequence SEQ ID NO: 15; The anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO: 3; and the amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:

4. The anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any one of claims 1-2, which is a humanized antibody or antigen-binding fragment, wherein the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO: 17; and the amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:

19. A nucleic acid encoding the antibody or antigen-binding portion thereof according to any one of claims 1-3. An expression vector comprising the nucleic acid according to claim 4. A host cell comprising the nucleic acid according to claim 4 or the expression vector according to claim 5. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1-3, the nucleic acid according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6. A kit comprising the antibody or antigen-binding portion thereof according to any one of claims 1-3, the nucleic acid according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6. Use of the antibody or antigen-binding portion thereof according to any one of claims 1-3, the nucleic acid according to claim 4, the expression vector according to claim 5, or the recombinant cell according to claim 6 in the preparation of a drug or reagent for diagnosing, treating, or preventing tumors. The use according to claim 9, wherein, the tumor is a tumor associated with CLDN18.2; preferably, the tumor is gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer, esophageal cancer, bronchial cancer, or breast cancer.

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