CLDN18.2-TARGETING ANTIBODY, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

Provided are a CLDN18.2-targeting antibody or an antigen-binding fragment thereof, a preparation method therefor, and the use thereof. The antibody comprises a light chain variable region and/or a heavy chain variable region, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. Compared with the prior art, the antibody has significant advantages in terms of binding affinity, ADCC, CDC, inhibitory effects on growth, endocytic activity, etc.

Claims

1. An antibody targeting CLDN18.2 or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein the HCDR1 has an amino acid sequence of SEQ ID NO: 8 or variant 1 thereof, the HCDR2 has an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and variant 2 thereof, and SEQ ID NO: 18 and variant 3 thereof, the HCDR3 has an amino acid sequence of any one of SEQ ID NO: 26-29, the LCDR1 has an amino acid sequence of SEQ ID NO: 42 or variant 4 thereof, the LCDR2 has an amino acid sequence of SEQ ID NO: 47 or variant 5 thereof, the LCDR3 has an amino acid sequence of SEQ ID NO: 55 or variant 6 thereof; the variant has substitution, deletion or addition of 1, 2 or 3 amino acids to the original amino acid sequence, and antibody or antigen-binding fragment comprising the variant retains binding ability to CLDN18.2.

2. The antibody or the antigen-binding fragment thereof of claim 1, wherein, the variant 1 has a mutation occurring at least at position 6 or position 7 of the amino acid sequence of SEQ ID NO: 8; the variant 2 has a mutation occurring at least at position 5 of the amino acid sequence of SEQ ID NO: 16; the variant 3 has a mutation occurring at least at position 3 of the amino acid sequence of SEQ ID NO: 18; the variant 4 has a mutation occurring at least at position 8 or position 9 of the amino acid sequence of SEQ ID NO: 42; the variant 5 has a mutation occurring at least at position 1 or position 4 of the amino acid sequence of SEQ ID NO: 47; the variant 6 has a mutation occurring at least at one or more of positions 3-5 of the amino acid sequence of SEQ ID NO: 55; preferably, the variant 1 comprises mutation S6G or Y7F, the variant 2 comprises mutation G5R, the variant 3 comprises mutation D3E, the variant 4 comprises mutation S8R or N9Y, the variant 5 comprises mutation G1D or T4N, and the variant 6 comprises one or more of mutations Y3R/N, N4S and N5Y; more preferably, amino acid sequence of the variant 1 is SEQ ID NO: 6 or 7; amino acid sequence of the variant 2 is SEQ ID NO: 17; amino acid sequence of the variant 3 is SEQ ID NO: 19; amino acid sequence of the variant 4 is SEQ ID NO: 40 or 41; amino acid sequence of the variant 5 is SEQ ID NO: 48; amino acid sequence of the variant 6 is any one of SEQ ID NO: 56-58.

3. The antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 2, wherein, amino acid sequence of the HCDR1 is SEQ ID NO: 7, amino acid sequence of the HCDR2 is SEQ ID NO: 17, and amino acid sequence of the HCDR3 is SEQ ID NO: 27; amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 18, and amino acid sequence of the HCDR3 is SEQ ID NO: 28; amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 16, and amino acid sequence of the HCDR3 is SEQ ID NO: 29; or, amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 19, and amino acid sequence of the HCDR3 is SEQ ID NO: 28; amino acid sequence of the LCDR1 is SEQ ID NO: 41, amino acid sequence of the LCDR2 is SEQ ID NO: 48, and amino acid sequence of the LCDR3 is SEQ ID NO: 56: amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 57: amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 55: or, amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 58; preferably, the heavy chain variable region has a framework region, which is a framework region of the heavy chain variable region of a human antibody, gene encoding the framework region of the heavy chain variable region is preferably derived from germline V gene IGHV3-23; or, the light chain variable region has a framework region, which is a framework region of the light chain variable region of a human antibody, and gene encoding the framework region of the light chain variable region is preferably derived from germline V gene IGKV3-11 or IGKV3-15; more preferably, in the framework region of the heavy chain variable region, HFR1 comprises an amino acid sequence of any one of SEQ ID NO: 2-4 or a variant thereof, HFR2 comprises an amino acid sequence of any one of SEQ ID NO: 10-14 or a variant thereof, HFR3 comprises an amino acid sequence of any one of SEQ ID NO: 21-24 or a variant thereof, and HFR4 comprises an amino acid sequence of any one of SEQ ID NO: 31-33 or a variant thereof; or in the framework region of the light chain variable region, LFR1 comprises an amino acid sequence of any one of SEQ ID NO: 35-38 or a variant thereof, LFR2 comprises an amino acid sequence of SEQ ID NO: 44 or 45 or a variant thereof, LFR3 comprises an amino acid sequence of any one of SEQ ID NO: 50-53 or a variant thereof, and LFR4 comprises an amino acid sequence of SEQ ID NO: 60 or 61 or a variant thereof.

4. (canceled)

5. The antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 3, wherein, amino acid sequence of the HCDR1 is SEQ ID NO: 7, amino acid sequence of the HCDR2 is SEQ ID NO: 17, amino acid sequence of the HCDR3 is SEQ ID NO: 27, amino acid sequence of the LCDR1 is SEQ ID NO: 41, amino acid sequence of the LCDR2 is SEQ ID NO: 48, and amino acid sequence of the LCDR3 is SEQ ID NO: 56; amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 18, amino acid sequence of the HCDR3 is SEQ ID NO: 28, amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 57; amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 16, amino acid sequence of the HCDR3 is SEQ ID NO: 29, amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 55; amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 16, amino acid sequence of the HCDR3 is SEQ ID NO: 29, amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 58; or, amino acid sequence of the HCDR1 is SEQ ID NO: 8, amino acid sequence of the HCDR2 is SEQ ID NO: 19, amino acid sequence of the HCDR3 is SEQ ID NO: 28, amino acid sequence of the LCDR1 is SEQ ID NO: 42, amino acid sequence of the LCDR2 is SEQ ID NO: 47, and amino acid sequence of the LCDR3 is SEQ ID NO: 57; preferably, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 64 or a variant thereof, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 71 or a variant thereof; the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 67 or a variant thereof, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 73 or a variant thereof; the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 65 or a variant thereof, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 72 or a variant thereof; the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 68 or a variant thereof, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 74 or a variant thereof; or, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 66 or a variant thereof, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 72 or a variant thereof; wherein, the variant retains at least function of pre-mutated sequence, and the variant has at least 85%, preferably at least 90%, more preferably at least 95%, further more preferably at least 99% identity to the pre-mutated sequence.

6. (canceled)

7. The antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1, wherein, the antibody targeting CLDN18.2 further comprises an antibody heavy chain constant region and an antibody light chain constant region; preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4 or variants thereof, and the light chain constant region is selected from κ chain or λ chain of a human antibody or variants thereof, more preferably, the heavy chain constant region is hIgG1, and the light chain constant region is κ chain of a human antibody; more preferably, the antibody targeting CLDN18.2 is a full-length antibody, a Fab, a Fab′, a F(ab′).sub.2, a Fv, a scFv, a bispecific antibody, a multispecific antibody, a heavy-chain antibody or a single-domain antibody, or a monoclonal antibody or a polyclonal antibody derived from the antibody as defined above.

8. (canceled)

9. The antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 7, which is a full-length antibody comprising a heavy chain and a light chain; the heavy chain comprises an amino acid sequence of any one of SEQ ID NO: 77-90, and the light chain comprises an amino acid sequence of any one of SEQ ID NO: 93-96; preferably, the heavy chain comprises an amino acid sequence of SEQ ID NO: 77, and the light chain comprises an amino acid sequence of SEQ ID NO: 93; the heavy chain comprises an amino acid sequence of SEQ ID NO: 78, and the light chain comprises an amino acid sequence of SEQ ID NO: 94; the heavy chain comprises an amino acid sequence of SEQ ID NO: 79, and the light chain comprises an amino acid sequence of SEQ ID NO: 93; the heavy chain comprises an amino acid sequence of SEQ ID NO: 85, and the light chain comprises an amino acid sequence of SEQ ID NO: 93; the heavy chain comprises an amino acid sequence of SEQ ID NO: 83, and the light chain comprises an amino acid sequence of SEQ ID NO: 93; the heavy chain comprises an amino acid sequence of SEQ ID NO: 84, and the light chain comprises an amino acid sequence of SEQ ID NO: 93; the heavy chain comprises an amino acid sequence of SEQ ID NO: 81, and the light chain comprises an amino acid sequence of SEQ ID NO: 95; the heavy chain comprises an amino acid sequence of SEQ ID NO: 82, and the light chain comprises an amino acid sequence of SEQ ID NO: 96; the heavy chain comprises an amino acid sequence of SEQ ID NO: 80, and the light chain comprises an amino acid sequence of SEQ ID NO: 94; the heavy chain comprises an amino acid sequence of SEQ ID NO: 86, and the light chain comprises an amino acid sequence of SEQ ID NO: 95; the heavy chain comprises an amino acid sequence of SEQ ID NO: 87, and the light chain comprises an amino acid sequence of SEQ ID NO: 96; the heavy chain comprises an amino acid sequence of SEQ ID NO: 88, and the light chain comprises an amino acid sequence of SEQ ID NO: 94; the heavy chain comprises an amino acid sequence of SEQ ID NO: 89, and the light chain comprises an amino acid sequence of SEQ ID NO: 95; or, the heavy chain comprises an amino acid sequence of SEQ ID NO: 90, and the light chain comprises an amino acid sequence of SEQ ID NO: 96.

10. A biomaterial encoding the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1, wherein, the biomaterial is selected from the group consisting of: (i) an isolated nucleic acid; (ii) a recombinant expression vector comprising the isolated nucleic acid of (i); preferably, the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenovirus vector or an adeno-associated virus vector; (iii) a transformant, which is a host cell comprising the recombinant expression vector of (ii); preferably, the host cell is an E. coli TG1, a BL21 cell or a CHO-K1 cell.

11-12. (canceled)

13. A medicament comprising the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1, wherein, the biological medicament is selected from the group consisting of: (i) a chimeric antigen receptor; (ii) a genetically modified cell; preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably an immune cell such as a T cell, or an NK cell; (iii) an antibody drug conjugate: wherein the antibody drug conjugate further comprises a cytotoxic agent: preferably, the cytotoxic agent is MNAF or MNAE; (iv) a pharmaceutical composition, wherein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier: preferably, the pharmaceutical composition further comprises one or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

14. (canceled)

15. A method for preparing the antibody targeting CLDN18.2 or the antigen-binding fragment thereof, which comprises: culturing the transformant of claim 10 and obtaining an antibody targeting CLDN18.2 or an antigen-binding fragment thereof from culture.

16-18. (canceled)

19. A kit comprising the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1; preferably, the kit further comprises (i) a device for administering an antibody or an antigen binding fragment thereof, or an antibody drug conjugate or a pharmaceutical composition; or (ii) instructions for use.

20. A kit combination comprising kit A and kit B, wherein, the kit A comprises the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1; the kit B comprises other anti-tumor antibodies or a pharmaceutical composition containing the other anti-tumor antibodies, or one or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

21. A method for diagnosing, treating or preventing a CLDN18.2-mediated disease or symptom, which comprises: administering to a subject in need of a therapeutically effective amount of the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1; the disease or symptom is a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma or leukemia.

22. (canceled)

23. A method of immunoassaying or measuring CLDN18.2, which comprises using the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1; preferably, the immunoassaying is for non-diagnostic or therapeutic purposes.

24. A combination therapy, which comprises: administering the antibody targeting CLDN18.2 or the antigen-binding fragment thereof of claim 1 and a second therapeutic agent, respectively, to a subject in need; the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical composition containing the other anti-tumor antibodies, or one or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0131] FIG. 1 shows the binding affinity of antibodies HBM1029 and PR002727 to HEK293 hCLDN18.2 cells (A), NUGC4_D8 cells (B), and HEK293 hCLDN18.1 cells (C), respectively.

[0132] FIG. 2 shows the binding affinity of PR003197, PR003292, PR003293 and PR003340 antibodies to NUGC4_D8 cells (A) and HEK293 hCLDN18.2 cells (B); the binding affinity of PR003197, PR003292, PR003293, PR003340 and PR002725 antibodies to HEK293 hCLDN18.1 cells (C).

[0133] FIG. 3 shows the binding affinity of PR003240, PR003291, PR003289 and HBM1029 antibodies to NUGC4_D8 (A) and HEK293 hCLDN18.1 (B) cells, respectively.

[0134] FIG. 4 shows the binding affinity of PR003890, PR003891, PR003894, PR003897 and PR003898 antibodies to NUGC4_D8 cells (A) and HEK293 hCLDN18.2 cells (B), respectively. The binding affinity of PR003890, PR003891, PR003894, PR003897, PR003898, PR002725 and HBM1029 antibodies to HEK293 hCLDN18.1 cells (C).

[0135] FIG. 5 shows that HBM1029 antibody exhibited ADCC activity to NUGC4_D8 cells, HEK293 hCLDN 18.1 cells by human PBMC.

[0136] FIG. 6 shows that PR003894, PR003240, PR003340, PR003197 and HBM1029 exhibited ADCC activity to NUGC4_D8 cells by human PBMC.

[0137] FIG. 7 shows that HBM1029, PR003894, PR003240, PR003340, PR003197, PR003891 and PR003898 exhibited ADCC activity to NUGC4_D8 cells by reporter cells.

[0138] FIG. 8 shows HBM1029 antibody triggered CDC effect in HEK293 hCLDN18.2 cells, HEK293 hCLDN18.1 cells and NUGC4_D8 cells.

[0139] FIG. 9 shows that HBM1029, PR003197 and PR003340 antibodies triggered CDC effect in HEK293 hCLDN18.2 cells.

[0140] FIG. 10 shows the growth inhibitory activity triggered by HBM1029 antibody in HEK293 hCLDN18.1 cells and HEK293 hCLDN18.2 cells.

[0141] FIG. 11 shows the endocytic activity of HBM1029 antibody in NUGC4_D8 cells. The results showed that: (A) NUGC4_D8 cells were mixed and incubated with 200 nM antibody for different time periods, and (B) NUGC4_D8 cells were mixed and incubated with different concentrations of antibody for 1 hour.

[0142] FIG. 12 shows the survival rate of target cells when HBM1029 antibody was co-cultured with anti-human IgG antibody-conjugated MMAF.

[0143] FIG. 13 shows the competitive binding affinity of HBM1029 antibody to IMAB362-FITC analogue in HEK293 hCLDN18.2 cells. Different concentrations of HBM1029 antibody were mixed and incubated with 20 nM IMAB362-FITC analogue and HEK293 hCLDN18.2 cells.

[0144] FIG. 14 shows the pharmacokinetic profile of the IMAB362 analogue and HBM1029.

[0145] FIG. 15 shows a study on in vivo pharmacokinetics of the IMAB362 analogue and HBM1029.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0146] The present invention will be further illustrated by examples described below, which, however, are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are selected according to conventional methods and conditions, or according to the commercial instruction.

Example 1: Antigen Preparation, Mouse Immunization and Hybridoma Preparation

[0147] a. Preparation of an Expression Vector for Immunized Mice

[0148] The preparation method of the human CLDN18.2 expression vector for immunizing the fully humanized transgenic mice is as follows: a cDNA sequence encoding human CLDN18.2 (Uniprot ID P56856-iso2) is synthesized, and the encoding sequence of the above gene is cloned into a pCAGGS plasmid (YOUBIO, VT1076) by enzymatic cleavage.

b. Preparation of Stably Transfected Cell Line

[0149] The construction of HEK293 (ATCC, Cat #: CRL-1573) cell line stably expressing human CLDN18.1 or CLDN18.2 is as follows: HEK293 cells were transfected with a plasmid encoding human CLDN18.1 (GenScript, OHu29174D) or CLDN18.2 (GenScript, OHu03374D) to generate stable cell line overexpressing human CLDN18.1 or CLDN18.2. The expression of CLDN18.1 and CLDN18.2 was detected by fluorescence-activated cell sorting (FACS). Specifically, 20,000 transfected cells were plated in each well of a 96-well plate and followed by addition of a commercially available rabbit anti-human CLDN18 antibody (LifeSpan Bio, Cat #: LS-C168812-400). After incubation for 1 h at 4° C., the plate was washed 2 times with PBS, and then AF-680-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Cat #: A21091) was added. After incubation at 4° C. for 1 hour, the plate was washed 3 times with PBS, then the cell fluorescence was monitored using a FACS machine (IntelliCytiQue Plus BR).

Example 2: Production and Screening of CLDN18.2 Monoclonal Antibody

[0150] Fully humanized transgenic mice (HarbourH2L2 mice, commercially available mice, purchased from Harbour Biomed) were immunized with the human CLDN18.2 expression vector and the HEK293 cells (HEK293 hCLDN18.2 cells) expressing human CLDN18.2 prepared above. The human CLDN18.2 vector and gold powder were prepared into the bullets of a gene gun, and the mice were immunized with the gene gun at multiple points on the abdomen. Expression vector DNA was used to immunize with a dosage of 50 μg each time, with an interval of 2 weeks each time, after immunization for three times, HEK293 hCLDN18.2 cells were used to immunize, with 4×10.sup.6 cells each time for each mouse, with an interval of 2 weeks each time, then blood samples were collected and titer was measured after cell immunization for 2 times. The binding affinity of mouse serum was detected by FACS using CHO K1 cells expressing human CLDN18.2 (CHOK1 hCLDN18.2, purchased from kyinno (KC-1180)) or CHO K1 cells expressing CLDN18.1 (CHO K1 hCLDN18.1, purchased from kyinno (KC-1181)). According to the results of serum titer test of immunized mice, mice were selected for hybridoma fusion; three days before fusion, HEK293 hCLDN18.2 cells was used for booster immunization at a dose of 4×10.sup.6 cells for the mice. Spleen cells and lymph node cells of the mouse and mouse myeloma cells SP2/0 were mixed at a ratio of 2:1 (cell number ratio), and the mixed cells were fused with an electrofusion instrument (BTX ECM2001); the fused cells were placed on a 96-well cell culture plate, and incubated in a CO.sub.2 incubator at 37° C. for 10 days before the primary screening of hybridomas. Primary screening was performed by Mirrorball using CHO KI cells expressing human CLDN18.2, as follows: the cells were resuspended with a medium (F12K 10% FBS); the cell density was adjusted to 5×10.sup.4 cells/ml, and 40 μl cell suspension was added into each well of a 384-well plate and incubated in a CO.sub.2 incubator at 37° C. overnight. After staining the wells with the nuclear dye DRAQ5, the supernatant of the well plates was discarded, and 50 μl of the supernatant of the hybridoma culture plate was taken out and added into a 384-well microplate; after incubation at 4° C. for two hours, AF488-conjugated fluorescent secondary antibody (invitrogen, Cat #: A11066) was added and incubated at 4° C. overnight, then the 384-well microplate was detected by Mirrorball. Positive hybridomas were selected and transferred from the 96-well plate to a 24-well plate for expansion culture. 5 days later, supernatants from 24-well plates were rescreened. The rescreening was performed by FACS using CHOK1hCLDN18.1 cells and CHOK1hCLDN18.2 cells. Cells were centrifuged at 300 g for 5 minutes and then resuspended in FACS buffer (PBS containing 2% FBS). The density of cells was adjusted to 10.sup.6 cells/ml, and 50 μl of cell suspension was added into each well of a 96-well plate. After incubation at 4° C. for 2 hours, the plate was washed 2 times with FACS buffer. Subsequently, FACS buffer containing APC-conjugated goat anti-rat IgG secondary antibody (Biolegend, Cat #: 405407) was added. After incubation at 4° C. for 1 hour, the plate was washed 2 times with FACS buffer. Cells were resuspended with fixative for monitoring the fluorescence of the cells using a FACS machine (ACEA NovoCyte). The hybridomas with good specificity were subcloned by limiting dilution analysis, and primary screening of subclones was conducted after incubating the hybridomas in a CO.sub.2 incubator at 37° C. for 7 days. The primary screening of subclones was detected by Mirrorball using CHO K1 cells expressing human CLDN18.2. According to the test results and observation under a microscope, clones that were both monoclonal and positively bound to CHOK1/CLDN18.2, were selected for expansion in a 24-well plate, and the supernatants in the wells were rescreened after incubation in a CO.sub.2 incubator at 37° C. for 3 days. Rescreening was performed by FACS (the same rescreening procedure as described above) using CHOK1hCLDN18.1 and CHOK1hCLDN18.2 cell lines. Monoclonal clones with specific binding were identified using subtype identification kit (invitrogen, Cat #: 88-50640-88). Cells with antibody subtype IgG2b were selected for sequencing (sequencing company: GENEWIZ Biotechnology Co., Ltd.).

[0151] It is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., sequences of proteins of immunological interest, fifth edition, national institutes of health, Bethesda, Md. (1991)) and Chothia definition rule based on the location of a structural loops region (see J Mol Biol 273:927-48, 1997). In the present application, amino acid residues in variable domain sequences may also be determined using a Combined definition rule that incorporates both Kabat definition and Chothia definition. The Combined definition rule refers to the combination of the ranges of Kabat definition and Chothia definition, based on which a larger range is taken, see Table 1-1 in the content of the present invention. The germline gene analysis and PTM site analysis obtained after sequencing in this embodiment are shown in Table 2 below. The design information of mutation sites of antigen-binding protein is shown in Table 3 below. The sequence number of antigen-binding protein is shown in Table 4 below.

TABLE-US-00004 TABLE 2 germline gene analysis and PTM site analysis of antibodies subtypes of VH VL Recom- recom- germ- germ- binant binant Clone line line VH VL anti- anti- number V gene V gene PTM PTM bodies bodies 11C12- VH3-23 VK3-15 None None PR002725 Human 13C2 IgG1 13E6F4 VH3-23 VK3-11 None None PR002726 Human IgG1 31H3E2 VH3-30 VK3-15 DG None PR002727 Human (HCDR2), IgG1 DG (HCDR3) 205A7F1D3 VH3-23 VK3-15 None None PR003289 Human IgG1 214C4G11 VH3-23 VK3-15 None None PR003291 Human IgG1

TABLE-US-00005 TABLE 3 design of antigen-binding protein mutation sites Primary Variable region subtypes of recombinant antibodies Variants mutations antibodies Fc mutations PR002726 PRO03197 None Human IgG1 S239D, I332E PR002726 PR003292 None Human IgG1 M252Y, S254T, T256E PR002726 PR003293 None Human IgG1 S239D, 1332E, M252Y, S254T, T256E PR002726 PR003340 None Human IgG1 S239D, I332E, K274Q, Y300F, L309V, Y296F, A339T, V397M PR003289 PR003890 None Human IgG1 S239D, I332E PR003289 PR003897 None Human IgG1 S239D, I332E, K274Q, Y300F, L309V, Y296F, A339T, V397M PR003291 PR003891 None Human IgG1 S239D, I332E PR003291 PR003898 None Human IgG1 S239D, I332E, K274Q, Y300F, L309V, Y296F, A339T, V397M PR002727 PR003240 VH:D54E Human IgG1 S239D, I332E PR002727 PRO03894 VH:D54E Human IgG1 S239D, I332E, K274Q, Y300F, L309V, Y296F, A339T, V397M

TABLE-US-00006 TABLE 4 antigen-binding protein sequence number table Antibody Light Heavy number chain chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR000400 91 75 69 62 39 46 54 5 15 25 PR002725 92 76 70 63 40 47 55 6 16 26 PR002726 93 77 71 64 41 48 56 7 17 27 PR002727 94 78 72 65 42 47 57 8 18 28 PR003197 93 79 71 64 41 48 56 7 17 27 PR003340 93 85 71 64 41 48 56 7 17 27 PR003292 93 83 71 64 41 48 56 7 17 27 PR003293 93 84 71 64 41 48 56 7 17 27 PR003289 95 81 73 67 42 47 55 8 16 29 PR003291 96 82 74 68 42 47 58 8 16 29 PR003240 94 80 72 66 42 47 57 8 19 28 PR003890 95 86 73 67 42 47 55 8 16 29 PR003891 96 87 74 68 42 47 58 8 16 29 PR003894 94 88 72 66 42 47 57 8 19 28 PR003897 95 89 73 67 42 47 55 8 16 29 PR003898 96 90 74 68 42 47 58 8 16 29 Note: PR002726C mentioned in the above table is HBM1029, and in the present invention, both numbers refer to one same antibody; PR000400 is an analogue of IMAB362, and both numbers refer to one same antibody in the present invention.

Example 3: Expression, Purification and Characterization of Full-Length CLDN18.2 Monoclonal Antibody

[0152] After obtaining the nucleic acid encoding light and heavy chain variable domain sequences of the antibody molecules, the light and heavy chain variable domain sequences and corresponding human antibody light and heavy chain constant domain sequences can be fused and expressed by the conventional recombinant DNA technology to obtain the recombinant antibody molecules. In this example, the gene encoding antibody heavy chain variable domain sequence (VH) is synthesized and cloned into a mammalian cell expression plasmid vector encoding a human IgG1 antibody heavy chain constant domain sequence to encode a full-length heavy chain of an IgG1 antibody. The gene encoding antibody light chain variable domain sequence (VL) is synthesized and cloned into a mammalian cell expression plasmid vector encoding a human antibody Igκ light chain constant domain sequence to encode a full-length light chain of an antibody. In this example, since the sequence of the variable domain of the monoclonal antibody molecule obtained from immunized Harbour H2L2 mice is a human antibody sequence, this example yields a fully human anti-CLDN18.2 recombinant IgG1 antibody.

[0153] Plasmid (Genscript US) encoding heavy chain of antibody and plasmid (Genscript US) encoding light chain of antibody are simultaneously transfected into a mammalian host cell (e.g. human embryonic kidney cell HEK293), and purified recombinant antibody with correct paired assembly of heavy chain and light chain can be obtained by conventional recombinant protein expression and purification technology. Specifically, HEK293 cells were expanded and cultured in FreeStyle™ F17 Expression Medium (Thermo, Cat #: A1383504). Prior to start of transient transfection, cell concentration was adjusted to 6-8×10.sup.5 cells/ml, and the cells were incubated in a shaker at 37° C. in 8% CO.sub.2 for 24 hours, the cell concentration reached 1.2×10.sup.6 cells/ml. 30 ml of cultured cells were prepared. The plasmid encoding the heavy chain of the antibody and the plasmid encoding the light chain of the antibody were mixed in a ratio of 2:3 (mass ratio), and a total of 30 μg of plasmid was dissolved in 1.5 ml Opti-MEM serum reduction medium (Thermo, Cat #: 31985088) and sterilized by filtration through a 0.22 μm filter membrane. Then 120 μl of 1 mg/ml PEI (Polysciences, Cat #: 23966-2) was dissolved in 1.5 ml Opti-MEM and allowed to stand for 5 minutes. The PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes; the plasmid PEI mixture solution was slowly added dropwise into the flask while shaking, then the solution was incubated at 37° C. in 8% CO.sub.2 for 5 days, followed by observing cell viability. The culture was collected and centrifuged at 3300 g for 10 minutes, and then supernatant was taken; the supernatant was then centrifuged at high speed to remove impurities. Gravity column (Bio-Rad, Cat #: 7311550) containing MabSelect™ (GE Healthcare Life Science, Cat #: 71-5020-91 AE) was equilibrated and rinsed with PBS (pH 7.4) of 2-5 times of the column volume. The column was loaded with the supernatant sample, followed by washing with PBS of 5-10 times of the column volume. Then the target protein was eluted with 0.1 M glycine at pH 3.5, which was later adjusted to neutral with Tris-HCl at pH 8.0, and finally concentrated and changed into PBS buffer using an ultrafiltration tube (Millipore, Cat #: UFC901024) to obtain a purified antibody solution. Finally, the concentration was measured by NanoDrop (Thermo Scientific™ NanoDrop™ One), separately packed and stored for later use.

[0154] Appropriate amounts of the above purified samples were separately loaded onto analytical SEC column TSKgel G3000SWxl (HPLC machine model: Agilent 1260 Infinity II) to detect the purity of the samples, ensuring that the purity of the homogeneous samples was more than 95%. The condition of the method was: mobile phase of 1×PBS, pH 7.4 (Biotech, Cat #: E607016), room temperature, flow rate of 1.0 ml/min, sample concentration of 1 mg/ml, injection volume of 20 μl and the detection wavelength of 280 nm. After collection, the chromatograms were integrated by ChemStation software and relevant data were calculated. Appropriate amounts of the above purified samples were separately loaded onto analytical HIC column TSKGE 1BUTTY1-NPR 4.6*35 (HPLC machine model: Agilent 1260 Infinity II) to detect the purity and hydrophobicity thereof. The method consists of a linear gradient over 16 minutes from 100% mobile phase A (20 mM PB, 1.8 M (NH.sub.4).sub.2SO.sub.4, pH 6.0) to 100% mobile phase B (20 mM PB, pH 6.0). The flow rate was set to 0.7 ml/min, the sample concentration was 1 mg/ml, the injection volume was 20 μl, and the detection wavelength was 280 nm. After collection, the chromatograms were integrated by ChemStation software and relevant data were calculated. Differential Scanning Fluorimetry (DSF) is a common high-throughput method to determine the thermal stability of proteins. It monitors the change in the fluorescence intensity of the dye bound to the unfolded protein molecule using a real-time fluorescent quantitative PCR machine to reflect the denaturation process of protein, thereby reflecting the thermal stability of the protein molecule. In this example, the DSF method was used to determine the thermal denaturation temperature (Tm) of protein molecules. 10 g of protein was added to a 96-well PCR plate (Thermo, Cat #: AB-0700/W), followed by 2 μl of 100× diluted dye SYPRO™ (Invitrogen, 2008138), and then buffer was added to make a final volume of 40 μl per well. The PCR plate was sealed and placed on a real-time fluorescent PCR machine (Bio-Rad CFX96 PCR System). The PCR plate was first incubated at 25° C. for 5 min, followed by a gradual increase in temperature from 25° C. to 95° C. with a gradient of 0.2° C./0.2 min and a decrease in temperature to 25° C. at the end of the test. FRET scanning mode and Bio-Rad CFX Maestro software were used to analyze the data and calculate the TM of the samples. The results are shown in table 5 below.

TABLE-US-00007 TABLE 5 HIC-HPLC HIC-HPLC SEC-HPLC purity retention time Tm1 Tm2 purity (%) (%) (min) (° C.) (° C.) HBM1029 98 100 15.653 62.8 68.2

Example 4: Binding Affinity of CLDN18.2 Monoclonal Antibody

[0155] Antibody binding affinity was measured by FACS using HEK293 cells expressing human CLDN18.2 or CLDN18.1 and NUGC4_D8 cells endogenously expressing human CLDN18.2 (the NUGC cell line was purchased from JCRB, Cat #: JCRB0834), and selected by limiting dilution analysis to obtain NUGC4_D8 subclone cells), specifically: the cells were centrifuged at 300 g for 5 min and then resuspended in FACS buffer (PBS with 2% FBS). The cell density was adjusted to 10.sup.6 cells/ml, and 50 μl of cell suspension was added to each well of a 96-well plate. Antibodies were diluted to different concentrations with FACS buffer and 50 μl of antibody dilution was added to each well of the 96-well plate. After incubation at 4° C. for 2 hours, the plate was washed 2 times with FACS buffer. Subsequently, FACS buffer (final concentration of 1.5 μg/ml, Jackson, Cat #: 109-605-098) containing APC-conjugated goat anti-human IgG secondary antibody was added. After incubation at 4° C. for 1 hour, the plate was washed 2 times with FACS buffer. The cells were resuspended with fixative for monitoring the fluorescence of the cells using a FACS machine (ACEA NovoCyte). IMAB362 analogue (IMAB362 analogue was home-made, with amino acid sequence of heavy chain shown in SEQ ID NO: 75 and amino acid sequence of light chain shown in SEQ ID NO: 91 (synthesized by Genscript Biotechnology Co., Ltd.), which is identical to the variable region of IMAB362, with only individual amino acids different in the constant region, and both have similar activity) was used as positive control, and antibody of human Iso IgG1 (CrownBio, Cat #: C00001-4) was used as negative control. FIG. 1 shows the binding affinity of HBM1029 antibody to NUGC4_D8 cells endogenously expressing CLDN18.2, and HEK293 cells overexpressing human CLDN18.2 (HEK293 hCLDN 18.2) or HEK293 cells overexpressing human CLDN18.1 (HEK293 hCLDN18.1). PR002727 antibody was able to bind to NUGC4_D8 cells in a dose-dependent manner. HBM1029 antibody was able to bind to HEK293 hCLDN18.2 and NUGC4_D8 cells in a dose-dependent manner. The binding affinity of HBM1029 antibody to HEK293 hCLDN18.2 cells was comparable to that of the IMAB362 analogue; HBM1029 antibody showed higher binding affinity to NUGC4_D8 cells endogenously expressing CLDN18.2 compared to the IMAB362 analogue. The EC.sub.50 values of HBM1029 are shown in Table 7, and HBM1029 showed lower EC.sub.50 values for binding affinity to NUGC4_D8 cells endogenously expressing CLDN18.2 compared to the IMAB362 analogue. HBM1029 had a low binding affinity to HEK293 hCLDN18.1 cells. At the same time, it can be inferred that HBM1029 binds to ECL1 (Extracellular loop 1) of human CLDN18.2 protein instead of ECL2. FIG. 2, FIG. 3 and FIG. 4 show the binding affinity of CLDN18.2 antibody to NUGC4_D8 cells endogenously expressing CLDN18.2, HEK293 cells overexpressing human CLDN18.2 (HEK293 hCLDN 18.2) and HEK293 cells overexpressing human CLDN18.1 (HEK293 hCLDN18.1). Results show that: compared with the IMAB362 analogue, HBM1029, PR003197, PR003340, PR003292, PR003293, PR003240, PR003291, PR003289, PR003890, PR003891, PR003894, PR003897 and PR003898 antibodies presented higher binding affinity to NUGC4_D8 cells endogenously expressing CLDN18.2 and lower binding affinity to HEK293 hCLDN18.1 cells, while PR002725 showed high affinity to HEK293 hCLDN18.1.

TABLE-US-00008 TABLE 6 amino acid sequences of IMAB362 analogue (synthesized by Genscript Biotechnoloy Co., Ltd.) amino acid sequence of heavy chain SEQ ID NO: 75 amino acid sequence of light chain SEQ ID NO: 91

TABLE-US-00009 TABLE 7 EC.sub.50 values for binding affinity of HBM1029 antibody IMAB362 Cells HBM1029 analogue HEK293 Max Binding (MFI) 976,754 1,022,190 hCLDN18.2 EC.sub.50 (nM) 3.8 4.0 Max Binding (MFI) 490,220 313,021 NUGC4_D8 EC.sub.50 (nM) 4.4 20.7

Example 5: ADCC Activity of CLDN18.2 Antibody

[0156] The activity of CLDN18.2 antibody mediating ADCC effect against NUGC4_D8 cells endogenously expressing human CLDN18.2 and HEK293 hCLDN 18.1 was assayed using CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, Cat #: G1780). Human PBMC (Miaotong) were centrifuged at 300 g for 5 minutes and incubated overnight in medium (RPMI1640+10% FBS). Target cells and human PBMC were centrifuged at 300 g for 5 minutes and then resuspended in medium (RPMI1640+2% FBS). The density of target cells was adjusted to 2×10.sup.5 cells/ml, and the cell density of PBMC was adjusted to at least 6×10.sup.6 cells/ml, then 50 μl of each type of cell was added into wells of a 96-well plate (the effective target ratio was at least 30:1). The antibodies to be tested were diluted with medium (RPMI1640+2% FBS) into different concentrations and added to each well. Samples were incubated at 37° C. for at least 4 hours, then 10 times Triton-X 100 lysate (RPMI1640+2% FBS+10% Triton-X 100) was added to the target cell maximum LDH release control wells and the volume correction control wells, mixed, and incubated at 37° C. for 0.5 hour. The 96-well plate was centrifuged at 300 g for 5 minutes, 50 μl of supernatant was removed, and then LDH chromogenic substrate was added at a concentration of 50 l/well. After the mixture was placed in the dark at room temperature for 20 minutes, the plate was read on MD StakMax (OD.sub.490). IMAB362 analogue was used as positive control, and antibody of human Iso IgG1 (CrownBio, Cat #: C00001-4) was used as negative control. Results: firstly, the correction readings were calculated, the readings of experimental wells, target cell spontaneous LDH release control wells and effector cell spontaneous LDH release control wells were subtracted from the readings of medium background control wells, secondly, the readings of the target cell maximum LDH release control wells were subtracted from the readings of the volume corrected control wells. ADCC activity (%)=(experimental well corrected reading−effector cell spontaneous release LDH well corrected reading−target cell spontaneous LDH release well corrected reading)/(target cell maximum LDH release well corrected reading−target cell spontaneous release LDH well corrected reading)×100. FIG. 5 shows the ADCC activity of HBM1029 antibody against NUGC4_D8 cells endogenously expressing CLDN18.2 and HEK293 hCLDN18.1. HBM1029 antibody specifically mediated a stronger ADCC effect in a dose-dependent manner against NUGC4_D8 than IMAB362 analogue, while no cytotoxic effect was observed on HEK-293 cells overexpressing CLDN18.1. The EC.sub.50 value of HBM1029 is shown in Table 8 and the EC.sub.50 value of HBM1029-mediated ADCC against NUGC4_D8 was lower than the IMAB362 analogue.

TABLE-US-00010 TABLE 8 ADCC activity of HBM1029 IMAB362 cell HBM1029 analogue Max Lysis (%) 20.9 26.9 NUGC4_D8 EC.sub.50 (nM) 0.05 0.91

[0157] FIG. 6 illustrates the ADCC activities of HBM1029, PR003197, PR003340, PR003240 and PR003894 against NUGC4_D8 cells endogenously expressing CLDN18.2; HBM1029, PR003197, PR003340, PR003240 and PR003894 were able to mediate stronger ADCC effect in a dose-dependent manner on NUGC4_D8 than IMAB362 analogue.

[0158] Jurkat FcγRIIIa-V158/NFAT-Luc cells were used to detect the ADCC activity mediated by CLDN18.2 antibody against NUGC4_D8 and HEK293 hCLDN18.1. NUGC4_D8 and HEK293 hCLDN18.1 were centrifuged at 300 g for 5 minutes and then resuspended in RPMI1640+4% FBS serum culture medium. The density of cells was adjusted to 6×10.sup.5 cells/ml, and 50 μl of cell suspension was added to each well of a 96-well plate, and then incubated at 37° C. overnight. Jurkat FcγRIIIa-V158/NFAT-Luc cells were centrifuged at 400 g for 4 minutes, and then resuspended in RPMI1640+4% FBS serum culture medium. The density of cells was adjusted to 3×10.sup.6 cells/ml, and 50 μl of cell suspension was added to each well of a 96-well plate. Antibodies were diluted to different concentrations with RPMI1640+4% FBS medium, and 50 μl of antibody dilution was added in each well of a 96-well plate. Cells were incubated with antibody at 37° C. for 5 hours. The 96-well plate was allowed to stand at room temperature for 30 minutes, and 60 l/well of One-Glo chromogenic substrate (Promega) at room temperature was added. The samples were then incubated in the dark at room temperature for 10 minutes. The 96-well plate was read with PE Enspire. IMAB362 analogue was used as positive control, and antibody of human Iso IgG1 (CrownBio, Cat #: C0001-4) was used as negative control. FIG. 7 shows the ADCC activity of CLDN18.2 antibody against NUGC4_D8 cells endogenously expressing CLDN18.2; HBM1029, PR003197, PR003340, PR003240, PR003894, PR003891 and PR003898 were able to mediate stronger ADCC effect in a dose-dependent manner against NUGC4_D8 than the IMAB362 analogue.

Example 6: CDC Activity of CLDN18.2 Antibody

[0159] The ability of CLDN18.2 antibodies to mediate CDC effect against HEK293 hCLDN18.1, HEK293 hCLDN18.2 and NUGC4_D8 cells was detected using the CellTiter-Glo luminescent cell viability assay kit (Promega, Cat #: G7573). Target cells HEK293 hCLDN18.1 and HEK293 hCLDN18.2 were centrifuged at 300 g for 5 minutes and then resuspended in DMEM serum-free culture medium. Target cell NUGC4_D8 was centrifuged at 300 g for 5 minutes and then resuspended in RPMI1640 serum-free culture medium. The density of the target cells was adjusted to 4×10.sup.5 cells/ml, and 25 μl of cell suspension was added to each well of a 96-well plate. Antibodies were diluted to different concentrations with serum-free culture medium, and 25 μl of antibody diluent was added to each well of a 96-well plate. 50 μl of normal human serum (GemCell, Cat #: 100-512) was added at a final concentration of 50%, and the obtained mixture was incubated at 37° C. for 24 hours. The 96-well plate was allowed to stand at room temperature for 30 minutes, and 100 μl/well of room temperature CellTiter-Glo chromogenic substrate was added. Afterwards, the samples were incubated in the dark at room temperature for 10 minutes. The 96-well plate was read with PE Enspire. CDC activity (%)=[1−(luminescent sample)/(luminescent mock control)]×100. IMAB362 analogue was used as positive control and antibody of human Iso IgG1 (CrownBio, Cat #: C0001-4) was used as negative control. FIG. 8 shows the CDC activity of HBM1029 antibody against NUGC4_D8 cells endogenously expressing CLDN18.2, HEK293 cells overexpressing human CLDN18.1, and HEK293 cells overexpressing human CLDN18.2. HBM1029 antibody mediated a stronger CDC effect in a dose-dependent manner on HEK293 hCLDN18.2 than the IMAB362 analogue, while no CDC activity was observed against NUGC4_D8 cells and HEK293 cell overexpressing human CLDN18.1. The EC.sub.50 value of HBM1029 is shown in Table 9 and the EC.sub.50 value of HBM1029-mediated CDC against HEK293 hCLDN18.2 was lower than the IMAB362 analogue.

TABLE-US-00011 TABLE 9 CDC activity of HBM1029 IIVIAB362 cell HBM1029 analogue HEK293 Max Lysis (%) 100.0 100.9 hCLDN18.2 EC.sub.50 (nM) 0.42 1.07

[0160] FIG. 9 shows the CDC activities of PR003197 and PR003340 against HEK293 cells overexpressing human CLDN18.2. PR003197 and PR003340 antibodies mediated a stronger CDC effect in a dose-dependent manner on HEK293 hCLDN18.2 than the IMAB362 analogue.

Example 7: Growth Inhibitory Activity of CLDN18.2 Antibody

[0161] The ability of the CLDN18.2 antibody to induce growth inhibition against HEK293 hCLDN18.1 and HEK293 hCLDN18.2 was detected using the CellTiter-Glo luminescent cell viability assay kit (Promega, Cat #: G7573). Cells HEK293 hCLDN18.1 and HEK293 hCLDN18.2 were centrifuged at 300 g for 5 minutes and then resuspended in DMEM+0.5% FBS serum culture medium. The density of cells was adjusted to 1.2×10.sup.5 cells/ml, and 50 μl of cell suspension was added to each well of a 96-well plate, and then incubated at 37° C. overnight. Antibodies were diluted to different concentrations with medium, and 50 μl of antibody diluent was added to each well of a 96-well plate. HEK293 hCLDN18.1 and HEK293 hCLDN18.2 cells were incubated with antibodies at 37° C. for 3 days. The 96-well plate was allowed to stand at room temperature for 30 minutes, and 100 μl/well of CellTiter-Glo chromogenic substrate at room temperature was added. Afterwards, the samples were incubated in the dark at room temperature for 10 minutes. The 96-well plate was read with PE Enspire. Growth inhibitory activity (%)=[1−(luminescent sample)/(luminescent mock control)]×100. IMAB362 analogue was used as positive control and antibody of human Iso IgG1 (CrownBio, Cat #: C0001-4) was used as negative control. FIG. 10 shows the growth inhibitory activity induced by HBM1029 antibody against HEK293 hCLDN18.1 and HEK293 hCLDN18.2. HBM1029 antibody induced stronger growth inhibition in a dose-dependent manner against HEK293 hCLDN 18.2 than the IMAB362 analogue. The EC.sub.50 value of HBM1029 is shown in Table 10.

TABLE-US-00012 TABLE 10 growth inhibitory activity of HBM1029 IMAB362 cell HBM1029 analogue HEK293 Max growth 25.0 N/A hCLDN18.2 inhibition (%) EC.sub.50 (nM) 1.45 N/A

Example 8: Endocytosis (Internalization) Activity of CLDN18.2 Antibody (FACS-Based Assay)

[0162] The endocytosis activity of antibodies was detected by FACS using NUGC4_D8 cells. The cells were digested with trypsin and washed once with FACS buffer (PBS containing 2% FBS). The cells were centrifuged at 300 g for 5 minutes and then resuspended in FACS buffer. The density of cells was adjusted to 4×10.sup.6 cells/ml and pre-cooled on ice for 30 minutes. Antibodies were diluted to different concentrations in FACS buffer and pre-cooled on ice for 30 minutes. 700 μl of cell suspension and 700 μl of antibody diluent were added to the wells of the pre-cooled deep well plate. After incubation at 4° C. for 2 hours, the plate was washed 3 times with pre-cooled FACS buffer. The cells were resuspended with 250 μl pre-cooled FACS buffer, 100 μl of cell suspension and 1.1 ml of 37° C.-pre-heated FACS buffer were added to the wells of a 37° C.-pre-heated deep well plate; 100 μl of cell suspension and 1.1 ml of 4° C.-pre-cooled FACS buffer were added to the wells of a 4° C.-pre-cooled deep well plate. 50 μL of cell suspension (10.sup.5 cells/well) was taken from the obtained mixture at 0, 30, 60, 120 and 240 minutes, respectively, and placed in a pre-cooled deep well plate (containing 1.2 ml FACS buffer). The cells were centrifuged at 300 g for 5 minutes, after which pre-cooled FACS buffer containing AF647-conjugated goat anti-human IgG secondary antibody (final concentration 1.5 μg/ml, Jackson, Cat #: 109-605-088) was added. After incubation at 4° C. for 1 hour, the plate was washed 2 times with pre-cooled FACS buffer. The cells were resuspended with fixative, followed by fluorescence detection using a FACS machine (BD Canto II). Endocytosis activity (%)=(1−MFI.sub.37° C./MFI.sub.4° C.)×100. IMAB362 analogue was used as positive control and antibody of human Iso IgG1 (CrownBio, Cat #: C0001-4) was used as negative control. FIG. 11 shows the endocytosis activity of HBM1029 antibody in NUGC4_D8 cells incubated for various times. HBM1029 antibody induced approximately 50% endocytosis activity after incubation for 30 minutes. FIG. 11 also shows that HBM1029 antibody was able to induce higher endocytosis activity in a dose-dependent manner in NUGC4_D8 than the IMAB362 analogue. The EC.sub.50 value of HBM1029 is shown in Table 11 and the EC.sub.50 value of endocytosis induced by HBM1029 in NUGC4_D8 was lower than the IMAB362 analogue.

TABLE-US-00013 TABLE 11 endocytosis activity of HBM1029 IMAB362 cell HBM1029 analogue NUGC4_D8 Max 45.4 64.1 endocytosis (%) EC.sub.50 (nM) 0.05 0.40

Example 9: Endocytosis Activity of CLDN18.2 Antibody (Cytotoxicity-Based Method)

[0163] The CellTiter-Glo luminescence cell viability assay kit (Promega, Cat #: G7573) was used to detect the ability of HBM1029 antibody when co-cultured with MMAF-conjugated anti-human IgG antibody (Moradec, Cat #: AH-102-AF) to induce cytotoxicity against HEK293 hCLDN18.1, HEK293 hCLDN18.2 and NUGC4_D8 cells. HEK293 hCLDN18.1 cells and HEK293 hCLDN18.2 cells were centrifuged at 300 g for 5 minutes, and then resuspended in DMEM+10% FBS serum culture medium, and the density of cells was adjusted to 4×10.sup.4 cells/ml. NUGC4_D8 cells were centrifuged at 300 g for 5 minutes, and then resuspended in RPMI1640+10% FBS serum culture medium, and the density of cells was adjusted to 2×10.sup.4 cells/ml, and 50 μl cell suspension was added to each well of a 96-well plate and incubated at 37° C. overnight. HBM1029 antibody was diluted to different concentrations with culture medium, and 25 μl of antibody diluent was added to each well in a 96-well plate. MMAF-conjugated anti-human IgG antibody was diluted with culture medium, and 25 μl of antibody diluent was added to each well in a 96-well plate at a final concentration of 6.6 nM. Cells were incubated with antibody at 37° C. for 3 days. The 96-well plate was allowed to stand at room temperature for 30 minutes, and 100 l/well of CellTiter-Glo chromogenic substrate at room temperature was added. Afterwards, the samples were then incubated in the dark at room temperature for 10 minutes. The 96-well plate was read with PE Enspire. Cell viability (%)=[(luminescent sample)/(luminescent mock control)]×100. IMAB362 analogue was used as positive control and antibody of human Iso IgG1 (CrownBio, Cat #: C0001-4) was used as negative control. FIG. 12 shows the survival rate of target cells when HBM1029 antibody was co-cultured with MMAF-conjugated anti-human IgG antibody. HBM1029 antibody co-cultured with MMAF-conjugated anti-human IgG antibody induced a stronger cytotoxic effect in a dose-dependent manner on NUGC4_D8 cells and HEK293 hCLDN18.2 cells than the IMAB362 analogue, but no cytotoxic effect on HEK293 hCLDN18.1 cells.

Example 10: Competitive Binding Activity of HBM1029 Antibody

[0164] The competitive binding affinity of antibody was detected by FACS using HEK293 cells expressing human CLDN18.2. IMAB362 analogue was conjugated as IMAB362-FITC analogue using the FITC Fluorescence Conjugation Kit (Abcam, Cat #: ab188285). Cells were centrifuged at 300 g for 5 minutes and then resuspended in FACS buffer (PBS containing 2% FBS). The density of cells was adjusted to 10.sup.6 cells/ml, and 50 μl of cell suspension was added to each well of a 96-well plate. The FITC-conjugated antibody was diluted with FACS buffer, and 50 μl of the conjugated antibody diluent was added to each well of a 96-well plate. Antibody for competitive binding was diluted to different concentrations with FACS buffer, and 50 μl of antibody dilution was added to each well of a 96-well plate. After incubation at 4° C. for 2 hours, the plate was washed 2 times with FACS buffer. The cells were resuspended with fixative, after which the fluorescence of the cells was monitored using a FACS machine (ACEA NovoCyte). Antibody of human Iso IgG1 (CrownBio, Cat #: C0001-4) was used as negative control. FIG. 13 illustrates the competitive binding affinity of HBM1029 antibody to HEK293 hCLDN 18.2 cells than the IMAB362-FITC. HBM1029 antibody was able to bind competitively to HEK293 hCLDN 18.2 cells in a dose-dependent manner, presumably with similar binding epitopes to the IMAB362 analogue. HBM1029 competed with the IMAB362 analogue and inhibit its binding to CLDN18.2-expressing cells; whereas the IMAB362 analogue is known to bind only to CLDN18.2 and not to CLDN18.1. Therefore, it is hypothesized that HBM1029 binds to ECL1 (extracellular loop 1) of human CLDN18.2 protein instead of ECL2.

Example 11: Pharmacokinetic Study of CLDN18.2 Antibody

[0165] The pharmacokinetics of CLDN18.2 antibody was determined as follows: 6 female BALB/c nude mice weighing 18-22 g were administered the antibody drug by intravenous injection at a dose of 5 mg/kg; whole blood was collected from one group of 3 mice before administration and 15 minutes, 24 hours (1 day), 4 days, and 10 days after administration, respectively, and from another group of 3 mice only before administration and 5 hours, 2 days, 7 days, and 14 days after administration. Whole blood was allowed to stand for 30 minutes to coagulate, then centrifuged at 2,000 rpm for 5 minutes at 4° C. to collect isolated serum samples which were then frozen at −80° C. until analysis. The ELISA method was used to quantify drug concentrations in mouse serum. In the ELISA method, the antibody containing human Fc in mouse serum was captured by a goat anti-human Fc polyclonal antibody coated in a 96-well plate, and then HRP-labeled goat anti-human Fc secondary antibody was added for detection. Blood concentration data were analyzed using Phoenix WinNonlin software version 8.2, and the non-atrioventricular model (NCA) was chosen to evaluate the pharmacokinetic parameters.

[0166] FIG. 14 and Table 12 show the pharmacokinetic parameters of IMAB362 analogue and HBM1029. The results showed that the half-lives of IMAB362 analogue and HBM1029 in mice were 248 and 282 hours, respectively.

TABLE-US-00014 TABLE 12 pharmacokinetic parameters of IMAB362 analogue and HBM1029 IMAB362 analogue HBM1029 T.sub.1/2 (hr) 248 282 V.sub.d (ml/kg) 112 94 AUC.sub.all (μg/ml hr) 9,542 ± 0385 12,062 ± 0418 C.sub.1 (ml/hr/kg) 0.32 0.24 C.sub.0 (μg/m1) 90 104

Example 11: In Vivo Pharmacodynamic Study of CLDN18.2 Antibody

[0167] In vivo pharmacodynamic study of CLDN18.2 antibody was performed as follows: on the day of cell inoculation, each NCG mouse was subcutaneously inoculated with 5×10.sup.6 NUGC4_D8 tumor cells, which were firstly resuspended in a mixture solution (0.1 ml) of PBS and Matrigel (1:1), and then mixed with PBMC (resuspended in 0.05 ml of PBS) and inoculated subcutaneously. When the average tumor volume of mice in each group was 90 mm.sup.3, the mice were grouped, and 18 mice were divided into three groups; administration began after the grouping; the drug administration cycle was twice a week with a total of six times of administration; the drug administration method was tail vein injection. After start of administration, the body weight of mouse was weighed and tumor volume was measured twice a week; the tumor volume was calculated as follows: tumor volume (mm.sup.3)=0.5×tumor long diameter×tumor short diameter.sup.2. The experimental observation was ended on day 21 after administration, and all mice were then euthanized. Data were analyzed by t-test. FIG. 15 shows the results of the in vivo pharmacodynamic study of IMAB362 analogue and HBM1029. The mean tumor volume of control mice was 1526 mm.sup.3 on day 21 after administration. The mean tumor volume of the test drug IMAB362 analogue (50 mg/kg) treated mice was 728 mm.sup.3 on day 21 after administration, with a significant difference compared to the vehicle control group (p-value of 0.0052) and a tumor growth inhibition (TGI) rate of 52.29%. The mean tumor volume of the test drug HBM1029 (50 mg/kg) treated mice was 618 mm.sup.3 on day 21 after administration, with a significant tumor inhibition effect compared to the vehicle control group (p-value 0.0009) and a tumor growth inhibition (TGI) rate of 59.47%. Throughout the administration period in treatment, the animals showed good drug tolerance and there were no significant body weight loss and animal death.

[0168] Although the specific embodiments of the present invention have been described above, it should be understood by those skilled in the art that these are merely illustrative examples and that a variety of changes or modifications can be made to these embodiments without departing from the principles and spirits of the present invention. Therefore, the scope of protection of the present invention is limited by the appended claims.