Anti-CLDN18.2 antibody and its uses

Abstract

The present invention provides murine, human, chimeric or humanized antibodies or antigen-binding fragments thereof that specifically bind to CLDN18.2, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, and expression vectors and host cells for expressing the antibodies or antigen-binding fragments thereof, as well as methods of using the antibodies or antigen-binding fragments thereof, including for treating diseases associated with CLDN18.2 positivity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims the priority and benefit of Chinese patent application No. 202111307619.3, filed on November 5, 2021. The contents disclosed in said application are incorporated herein by reference in their entirety. [Technical field]

[0002] The present application is in the field of biotechnology, and specifically relates to antibodies and antigen-binding fragments thereof that bind to tight junction protein 18.2 (CLDN18.2), and methods of using such antibodies and antigen-binding fragments. [Background technology]

[0003] Tight junction protein 18 (CLDN18 or Claudin18) belongs to the family of cellular tight junction proteins, which participate in the formation of tight junctions between cells, control the flow of intercellular molecules, and play an important role in maintaining cell polarity, permeability, and intercellular signaling. CLDN18 is a four-transmembrane protein with both N- and C-termini inside the cell and two loop regions outside the cell. Due to alternative splicing of RNA, two different isoforms exist in tissues, namely, tight junction protein 18.1 (CLDN18.1, for example, the Uniprot accession number of human CLDN18.1 is P56856-1) and tight junction protein 18.2 (CLDN18.2, for example, the Uniprot accession number of human CLDN18.2 is P56856-2).

[0004] Although the protein sequences of CLDN18.1 and CLDN18.2 are highly similar, their expression distributions are different: CLDN18.1 is selectively expressed in normal lung tissues, whereas CLDN18.2 is highly restricted in normal tissues and specifically expressed in differentiated epithelial cells of the gastric mucosa. Studies have shown that CLDN18.2 is highly expressed in 70% of primary gastric cancers and their metastases, and that CLDN18.2 is frequently ectopically activated in various malignant tumors, including pancreatic cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer, and ENT tumors (Niimi et al., (2001) Mol Cell Biol 21(21):7380-7390; Sahin, U. et al., (2008) Clin Cancer Res.14,7624-34; Tanaka et al., (2011) J Histochem Cytochem 59(10):942-952; Micke et al., (2014) Int J Cancer 135(9):2206-2214; Shimobaba et al., (2016) Biochim Biophys Acta 1863(6Pt A):1170-1178, Sing h et al., (2017) J Hema tol Oncol 10(1):105, Tokumitsu et al., (2017) Cytopathology 28(2):116-121).

[0005] Various CLDN18.2 antibodies have already been used in cancer treatment research, including the CLDN18.2 chimeric IgG1 antibody developed by Ganymed, Claudiximab (IMAB362) (Sahin et al., (2018) Eur J Cancer 100:17-26), and the CLDN18.2 humanized antibody disclosed in PCT application WO2020211792. There is still a great need for antibodies against CLDN18.2 in clinical practice. Summary of the Invention [Means for solving the problem]

[0006] In one aspect, the present application provides an isolated antibody or antigen-binding fragment thereof that binds to CLDN18.2. In some embodiments, the antibody or antigen-binding fragment thereof binds to human CLDN18.2. In some embodiments, the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a monospecific antibody, a multispecific antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a dAb, an isolated CDR, a single chain Fv molecule, a nanobody, a recombinant polypeptide, a fusion protein, an immunoconjugate, or a combination thereof.

[0007] In some embodiments, an anti-CLDN18.2 antibody, or antigen-binding fragment thereof, provided herein comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein: (1) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 11, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 11, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 11; (2) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 12, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12; (3) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 13; (4) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 14, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 14, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 14; (5) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 15, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 15, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 15; (6) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO:29, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO:29, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO:29; (7) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 30, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30; (8) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 31, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 31, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 31; (9) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 32, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 32, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 32; (10) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 33, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 33, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 33; (11) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 43, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 43, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 43; (12) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 51, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 51, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 51; or (13) The heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO:59, the heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO:59, and the heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO:59.

[0008] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, wherein: (1) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:3, 4, or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:3, 4, or 7, (2) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:3, 5, or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:3, 5, or 7, (3) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:3, 6, or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:3, 6, or 7, (4) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 19, 20, or 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 20, or 24, (5) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 19, 21, or 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 21, or 24, (6) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 19, 22, or 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 22, or 24, (7) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 19, 23, or 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 23, or 24, (8) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 37, 38, or 39, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 37, 38, or 39, (9) the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 45, 46, or 47, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 45, 46, or 47, respectively; or (10) The heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:53, 54, or 55, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:53, 54, or 55, respectively.

[0009] In some embodiments, the anti-CLDN18.2 antibody, or antigen-binding fragment thereof, provided herein comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), wherein: (1) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 16, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 16; (2) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (3) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (4) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 34, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 34, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 34; (5) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (6) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (7) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 44, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 44, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 44; (8) the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 52, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 52, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 52; or (9) The light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO:60, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO:60, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO:60.

[0010] In some embodiments, the anti-CLDN18.2 antibody, or antigen-binding fragment thereof, provided herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein: (1) the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 8, 9, or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10, (2) the light chain CDR1, light chain CDR2, and light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 25, 26, or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 26, or 27, (3) the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 25, 26, or 28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, 26, or 28, (4) the light chain CDR1, light chain CDR2, and light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 40, 41, or 42, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 40, 41, or 42, (5) the light chain CDR1, light chain CDR2, and light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 48, 49, or 50, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 48, 49, or 50, respectively; or (6) The light chain CDR1, light chain CDR2, and light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:56, 57, or 58, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:56, 57, or 58, respectively.

[0011] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, with the proviso that (1) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 11, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 11, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 11, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 16, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 16; (2) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 12, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (3) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 13, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (4) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 14, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 14, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 14, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (5) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 15, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 15, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 15, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (6) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 12, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (7) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 13, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (8) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 14, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 14, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 14, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (9) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 15, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 15, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 15, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (10) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 29, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 29, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 29, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 34, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 34, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 34; (11) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 30, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (12) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 31, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 31, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 31, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (13) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 32, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 32, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 32, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (14) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 33, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 33, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 33, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (15) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 30, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (16) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 31, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 31, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 31, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (17) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 32, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 32, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 32, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (18) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 33, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 33, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 33, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (19) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 43, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 43, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 43, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 44, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 44, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 44; (20) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 51, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 51, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 51, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 52, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 52, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 52; or (21) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 59, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 59, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 59, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 60, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 60, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 60.

[0012] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, with the proviso that (1) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:3, 4, 7, 8, 9, or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:3, 4, 7, 8, 9, or 10, (2) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 8, 9, or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 8, 9, or 10, (3) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 3, 6, 7, 8, 9, or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 3, 6, 7, 8, 9, or 10, (4) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO: 19, 20, 24, 25, 26, or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, 20, 24, 25, 26, or 27, (5) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 21, 24, 25, 26, or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 21, 24, 25, 26, or 27, (6) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 22, 24, 25, 26, or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 22, 24, 25, 26, or 27, (7) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 23, 24, 25, 26, or 27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 23, 24, 25, 26, or 27, (8) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 20, 24, 25, 26, or 28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 20, 24, 25, 26, or 28, (9) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 21, 24, 25, 26, or 28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 21, 24, 25, 26, or 28, (10) the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 22, 24, 25, 26, or 28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 22, 24, 25, 26, or 28, (11) The heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 19, 23, 24, 25, 26, or 28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 19, 23, 24, 25, 26, or 28, respectively; (12) The heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence represented by SEQ ID NO: 37, 38, 39, 40, 41, or 42, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence represented by SEQ ID NO: 37, 38, 39, 40, 41, or 42, respectively; (13) The heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence shown in SEQ ID NO: 45, 46, 47, 48, 49, or 50, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 45, 46, 47, 48, 49, or 50, respectively; or (14) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each comprise an amino acid sequence set forth in SEQ ID NO:53, 54, 55, 56, 57, or 58, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:53, 54, 55, 56, 57, or 58, respectively.

[0013] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, with the proviso that the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, 19, 37, 45 or 53; the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, 5, 6, 20, 21, 22, 23, 38, 46, or 54; and the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, 24, 39, 47 or 55; the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, 25, 40, 48 or 56; the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, 26, 41, 49 or 57; and The light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, 27, 28, 42, 50 or 58.

[0014] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, with the proviso that the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, 19 or 53; the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, 5, 6, 20, 21, 22, 23, or 54; the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, 24 or 55; the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, 25 or 56; the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, 26 or 57; and The light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, 27, 28 or 58.

[0015] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 11, 12, 13, 14, 15, 29, 30, 31, 32, 33, 43, 51, or 59, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, 12, 13, 14, 15, 29, 30, 31, 32, 33, 43, 51, or 59.

[0016] In some embodiments, an anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 16, 17, 18, 34, 35, 36, 44, 52, or 60, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, 34, 35, 36, 44, 52, or 60.

[0017] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region and a light chain variable region, wherein: (1) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 11 or 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 11 or 16, (2) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 12 or 17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12 or 17, (3) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 13 or 17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 17, (4) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 14 or 17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 14 or 17, (5) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 15 or 17, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15 or 17, (6) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 12 or 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12 or 18, (7) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 13 or 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 18, (8) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 14 or 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 14 or 18, (9) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 15 or 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15 or 18, (10) the heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 29 or 34, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 29 or 34, (11) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 30 or 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 30 or 35, (12) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 31 or 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 31 or 35, (13) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 32 or 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 32 or 35, (14) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 33 or 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 33 or 35, (15) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 30 or 36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 30 or 36, (16) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 31 or 36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 31 or 36, (17) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 32 or 36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 32 or 36, (18) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 33 or 36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 33 or 36, (19) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 43 or 44, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 43 or 44, (20) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO:51 or 52, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:51 or 52, respectively; or (21) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO:59 or 60, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:59 or 60, respectively.

[0018] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11, 12, 13, 14, 15, 29, 30, 31, 32, 33, 43, 51 or 59, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, 34, 35, 36, 44, 52 or 60.

[0019] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11, 12, 13, 14, 15, 29, 30, 31, 32, 33 or 59, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, 34, 35, 36 or 60.

[0020] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the above-mentioned heavy chain variable region and light chain variable region, respectively, and the heavy chain constant region comprises a human IgG1, IgG2, IgG4 constant region or a variant thereof, preferably a human IgG1 constant region or a variant thereof, and the light chain constant region comprises a human κ constant region, a human λ constant region or a variant thereof, preferably a human κ constant region or a variant thereof. Exemplary variants include variants of the heavy chain constant region of IgG1, IgG2 or IgG4 in which site-specific modifications and amino acid substitutions have been made to the heavy chain constant region, such as the AAA mutation, the DLE mutation (Shields et al., 2002; Lazar et al., 2006), the YTE mutation, the LS mutation (Ghetie et al., 1997; Zalevsky et al., 2010) known in the art.

[0021] In some specific embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:61 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to the amino acid sequence set forth in SEQ ID NO:61, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:62 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to the amino acid sequence set forth in SEQ ID NO:62.

[0022] As used herein, the amino acid substitution may be a conservative amino acid substitution, i.e., a substitution with another amino acid of the same type (having similar chemical properties or function).For example, based on side chain properties, amino acids can be classified as follows: (1) non-polar amino acids: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M); (2) uncharged polar amino acids: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q); (3) acidic amino acids: Asp(D), Glu(E); (4) basic amino acids: Lys(K), Arg(R), His(H). Alternatively, amino acids can be classified based on common side chain properties as follows: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; and (6) aromatic amino acids: Trp, Tyr, Phe.

[0023] As used herein, various variants of the above anti-CLDN18.2 antibodies or antigen-binding fragments thereof retain the ability to specifically bind to the antigen CLDN18.2.

[0024] In some embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present application comprises two heavy chains (H) and two light chains (L), or consists of two heavy chains and two light chains, the heavy and light chains being connected to each other by disulfide bonds, and each heavy chain comprises the above-mentioned heavy chain variable region (VH) and heavy chain constant region, and the heavy chain variable region (VH) comprises framework regions (FR) and the above-mentioned heavy chain complementarity determining region (HCDR), and each light chain comprises the above-mentioned light chain variable region (VL) and light chain constant region, and the light chain variable region (VL) comprises FR and the above-mentioned light chain complementarity determining region (LCDR), and the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region and the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region.

[0025] In some embodiments, the antibodies of the present application may be full-length antibodies, e.g., full-length antibodies of the IgG1 isotype. In other embodiments, the antibodies of the present application may be single chain antibodies (scFv), nanobodies, or antibody fragments (e.g., Fab, F(ab')2 fragments, Fd fragments, Fv fragments, dAbs, or isolated CDRs).

[0026] The antibody or antigen-binding fragment thereof provided by the present application binds to CLDN18.2, thereby inducing antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity against cells expressing CLDN18.2. In some embodiments, the antibody or antigen-binding fragment thereof provided by the present application binds to human CLDN18.2, thereby inducing ADCC and / or CDC against cells expressing human CLDN18.2.

[0027] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein do not substantially bind to CLDN18.1. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein do not substantially bind to human CLDN18.1. "Does not substantially bind" to a protein or cell refers to not binding to a protein or cell or non-specific binding to a protein or cell.

[0028] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein do not induce ADCC and / or CDC against cells expressing CLDN18.1. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein do not induce ADCC and / or CDC against cells expressing human CLDN18.1.

[0029] In one aspect, the present application provides an isolated antibody or antigen-binding fragment thereof that binds to CLDN18.2, wherein the antibody or antigen-binding fragment thereof is produced by a hybridoma, wherein the hybridoma is selected from the group consisting of hybridomas designated herein as 28G3, 40F6, 22F12, 34G6, and 10D8. Accordingly, the present application further includes an antibody or antigen-binding fragment thereof produced by any of hybridomas 28G3, 40F6, 22F12, 34G6, 10D8, and hybridomas that produce the antibodies disclosed herein.

[0030] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that binds to the same epitope on human CLDN18.2 as any of the exemplary anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that binds to human CLDN18.2 competitively with any of the exemplary anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application.

[0031] In another aspect, the present application also provides a multispecific antibody comprising one or more of the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application and at least one antibody or antigen-binding fragment thereof having a different specificity from the antibodies or antigen-binding fragments of the present application, said multispecific antibody being capable of binding to at least two different binding sites or targets. As used herein, "multispecific antibody" covers antibodies having two (i.e., bispecific antibodies), three (i.e., trispecific antibodies), four (i.e., tetraspecific antibodies) or more specificities. These multispecific antibodies may be produced by methods well known in the art.

[0032] In another aspect, the present application also provides recombinant polypeptides or fusion proteins comprising one or more of the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application, wherein the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application are linked to at least one other functional fragment, which includes, but is not limited to, another peptide, protein, cytokine, or receptor ligand. These recombinant polypeptides and fusion proteins may be produced by methods well known in the art.

[0033] In another aspect, the present application also provides immunoconjugates comprising one or more of the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application, wherein the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application are linked to at least one other therapeutic agent (e.g., a cytotoxic drug, a radioisotope, an immunoadhesion molecule, or an imaging agent, etc.), e.g., an antibody-drug conjugate (ADC). These immunoconjugates may be produced by methods well known in the art.

[0034] In some embodiments, the connection is a covalent connection. In some embodiments, the connection is a non-covalent connection. In some embodiments, the connection is a direct connection. In some embodiments, the connection is connected by a linker. Suitable linkers include, but are not limited to, amino acid and polypeptide linkers.

[0035] In another embodiment, the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present application may be part of a chimeric antigen receptor (CAR). The present application also provides an immune cell, e.g., a T cell, comprising the chimeric antigen receptor (i.e., a CAR-T cell).

[0036] The present application also provides a gene vector, which comprises a gene encoding the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present application, and the gene can be introduced into a mammalian cell (preferably a human cell) and expressed therein. These gene vectors include, but are not limited to, naked plasmid vectors, yeast vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, poxvirus vectors, rhabdovirus vectors, or baculovirus vectors.

[0037] In one aspect, the present application also provides an isolated nucleic acid molecule encoding an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present application. The present application also provides an expression vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the expression vector.

[0038] In one aspect, the present application also provides a method for producing an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, comprising the steps of (i) expressing the anti-CLDN18.2 antibody or an antigen-binding fragment thereof in a host cell, and (ii) isolating the anti-CLDN18.2 antibody or an antigen-binding fragment thereof from the host cell or a cell culture thereof.

[0039] In one aspect, the present application provides a pharmaceutical composition comprising an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present application and one or more pharma- ceutically acceptable carriers or excipients. In another embodiment, the present application also provides a pharmaceutical composition comprising a recombinant polypeptide, fusion protein, multispecific antibody, immunoconjugate, chimeric antigen receptor, nucleic acid molecule, or gene vector of the present application and a pharma- ceutically acceptable carrier or excipient.

[0040] In another aspect, the present application provides a method for preventing, alleviating, or treating a disease associated with CLDN18.2 positivity in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CLDN18.2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof of the present application. In another aspect, the present application provides a use of an anti-CLDN18.2 antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof of the present application in the manufacture of a medicament for preventing, alleviating, or treating a disease associated with CLDN18.2 positivity. Alternatively, the present application provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof for preventing, alleviating, or treating a disease associated with CLDN18.2 positivity. In some embodiments, the disease associated with CLDN18.2 positivity is cancer. In some embodiments, the subject is a human.

[0041] Thus, in some embodiments, the present application provides a method of preventing, alleviating, or treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present application, or a pharmaceutical composition thereof. In some embodiments, the subject is a human.

[0042] The term "cancer" as used herein refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" may include tumors. Cancers that can be treated by the methods of the present application include, but are not limited to, carcinomas, sarcomas, lymphomas, blastomas, and leukemias. The term "cancer" includes, but is not limited to, primary cancers that begin at a particular site in the body, metastatic cancers that have spread from their origin to other parts of the body, recurrent cancers after the original primary cancer has been alleviated, and secondary cancers (new primary cancers that arise in patients with a history of a different type of cancer).

[0043] In some embodiments, the cancer includes hematological tumors and solid tumors. Hematological tumors include, for example, leukemia, lymphoma, and myeloma. In some embodiments, the cancer is a CLDN18.2 positive cancer. In some embodiments, the cancer includes, but is not limited to, gastric cancer, esophageal cancer, gastrointestinal cancer (including colon cancer and rectal cancer), pancreatic cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), bronchial cancer, mesothelioma, renal cancer, ovarian cancer (including Krukenberg's tumor), breast cancer, bladder cancer, uterine cancer (including endometrial cancer), prostate cancer, testicular cancer, anal cancer, vaginal cancer, bile duct cancer, gallbladder cancer, head and neck cancer, spinal tumors, otorhinolaryngological tumors, thyroid cancer, mesothelioma, bone cancer, skin cancer, melanoma, adenocarcinoma, sarcoma, neuroblastoma, glioblastoma, brain cancer, central nervous system cancer, neuroendocrine tumors, leukemia, lymphoma, and myeloma. The cancer may be pre-invasive, metastatic, recurrent, and / or refractory.

[0044] The anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application may be administered alone or in combination with one or more other therapeutic agents, which may be administered before, after, or simultaneously with the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application.

[0045] In some embodiments, the other therapeutic agent is an antibody or antigen-binding fragment thereof having a different specificity than the antibody or antigen-binding fragment of the present application, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, a CTLA-4 antibody, or an antigen-binding fragment thereof. In some embodiments, the other therapeutic agent is a cytokine, such as interleukin 2. In some embodiments, the other therapeutic agent is an agent that stimulates γδ T cells, which may be Vγ9Vδ2 T cells, and the agent that stimulates γδ T cells includes, but is not limited to, a bisphosphonic acid, or a salt thereof, or an ester thereof, such as, but not limited to, zoledronic acid, dichloromethylene bis(phosphonic acid), ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid, and salts thereof. In some embodiments, the other therapeutic agent is a chemotherapeutic agent, including, but not limited to, alkylating agents, podophyllums, camptothecins, taxanes, antimetabolites, and antibiotics, such as platinum-based drugs (cisplatin, carboplatin, oxaliplatin), anthracyclines (e.g., adriamycin or epirubicin), taxanes (e.g., paclitaxel), fluoropyrimidine derivatives (e.g., 5-fluorouracil or gemcitabine), and prodrugs thereof. In some embodiments, the other therapeutic agent is an agent that stabilizes or increases the expression of CLDN18.2, such as epirubicin, oxaliplatin, 5-fluorouracil, folin, irinotecan, docetaxel, cisplatin, gemcitabine, and prodrugs thereof.

[0046] "Treatment" refers to a method of alleviating the progression or severity of a symptom, condition, course, or disease. Cancer in a subject can be treated by administering to the subject an anti-CLDN18.2 antibody or antigen-binding fragment thereof, a nucleic acid molecule, a pharmaceutical composition, a recombinant polypeptide, a fusion protein, a multispecific antibody, an immunoconjugate, a chimeric antigen receptor, or a gene vector of the present application. In some embodiments, the subject is a human.

[0047] The contents of the disclosure and other features and advantages will become more apparent from the following specific description and examples, which should not be construed as limiting. The contents of all publications, Genbank records, patents and disclosed patent applications referenced in this application are expressly incorporated herein by reference. [Brief description of the drawings]

[0048] [Figure 1] Figure 1 shows the binding activity of chimeric antibodies xi40F6, xi34G6, xi10D8, xi22F12, and xi28G3 against U2OS-CLDN18.2 cells (A to C), NUGC-4 cells (D to F), and KATOIII cells (G to I). [Diagram 2] Figure 2 shows the ADCC activity of chimeric antibodies xi22F12, xi28G3, xi40F6, and xi34G6, where Jurkat-CD16a v158-NFAT-luciferase is the effector cell and KATOIII (A), NUGC-4 (B), and U2OS-CLDN18.2 (C) are the target cells. [Diagram 3] FIG. 3 shows the ADCC activity of chimeric antibodies xi22F12, xi28G3, xi40F6, and xi34G6, in which PBMCs are effector cells and KATOIII (A) and NUGC-4 (B) are target cells. [Figure 4] Figure 4 shows the CDC activity of chimeric antibodies xi22F12, xi28G3, xi40F6, xi34G6, and xi10D8, where KATOIII (A) and U2OS-CLDN18.2 (B) are target cells. [Diagram 5] FIG. 5 shows the endocytosis activity of chimeric antibodies xi28G3 and xi40F6, with NUGC-4 (A) and NIH-3T3-CLDN18.2 (B) as target cells. [Figure 6]Figure 6 shows the non-specific ADCC activity of chimeric antibodies xi22F12, xi28G3, xi40F6, xi34G6, and xi10D8, where Jurkat-CD16a v158-NFAT-luciferase is the effector cell and NIH-3T3-CLDN18.1 is the target cell. [Figure 7] Figure 7 shows the binding activity of chimeric antibody xi28G3 and humanized antibodies hz28G3-1.1, hz28G3-1.2, hz28G3-1.3, hz28G3-1.4, hz28G3-2.1, hz28G3-2.2, hz28G3-2.3, and hz28G3-2.4 to NUGC-4 cells (A) and KATOIII cells (B). [Figure 8] Figure 8 shows the binding activity of chimeric antibody xi40F6 and humanized antibodies hz40F6-1.1, hz40F6-1.2, hz40F6-1.3, hz40F6-1.4, hz40F6-2.1, hz40F6-2.2, hz40F6-2.3, and hz40F6-2.4 to NUGC-4 cells (A) and KATOIII cells (B). [Figure 9] Figure 9 shows the ADCC activity of chimeric antibody xi28G3 and humanized antibodies hz28G3-1.1, hz28G3-1.2, hz28G3-1.3, hz28G3-1.4, hz28G3-2.1, hz28G3-2.2, hz28G3-2.3, and hz28G3-2.4, where Jurkat-CD16a v158-NFAT-luciferase is the effector cell and U2OS-CLDN18.2 (A) and KATOIII (B) are the target cells. [Figure 10] Figure 10 shows the ADCC activity of chimeric antibody xi40F6 and humanized antibodies hz40F6-1.1, hz40F6-1.2, hz40F6-1.3, hz40F6-1.4, hz40F6-2.1, hz40F6-2.2, hz40F6-2.3, and hz40F6-2.4, where Jurkat-CD16a v158-NFAT-luciferase is the effector cell and NIH-3T3-CLDN18.2 (A) and NUGC-4 (B) are the target cells. [Figure 11]FIG. 11 shows the ADCC activity of chimeric antibody xi28G3 and humanized antibodies hz28G3-1.1, hz28G3-1.3, and hz40F6-1.4, where PBMC are effector cells and NIH-3T3-CLDN18.2 (A), KATOIII (B), and NUGC-4 (C) are target cells. [Figure 12] Figure 12 shows the CDC activity of chimeric antibody xi28G3 and humanized antibodies hz28G3-1.1, hz28G3-1.3, hz40F6-1.1, hz40F6-1.2, hz40F6-1.3, hz40F6-1.4, hz40F6-2.1, hz40F6-2.2, hz40F6-2.3, and hz40F6-2.4, where NIH-3T3-CLDN18.2 is the target cell. [Figure 13] FIG. 13 shows the endocytosis activity of chimeric antibody xi28G3 and humanized antibodies hz28G3-1.1 and hz28G3-1.3, where U2OS-CLDN18.2 (A) and NUGC-4 (B) are target cells. [Figure 14] FIG. 14 shows the non-specific ADCC activity of chimeric antibody xi28G3 and humanized antibodies hz28G3-1.1 and hz28G3-1.3, where PBMCs are effector cells and NIH-3T3-CLDN18.1 are target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] It should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] The term "antibody" refers to a binding protein having at least one antigen (e.g., CLDN18.2) binding domain. The antibody or antigen-binding fragment thereof of the present application may be a whole antibody or any fragment thereof, including a monoclonal antibody or fragment thereof and an antibody variant or fragment thereof. Examples of antibodies or antigen-binding fragments thereof include monoclonal antibodies, monospecific antibodies, multispecific antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAbs, isolated CDR regions, single chain Fvs (scFvs), nanobodies, and any of the antibody fragments known in the art. The anti-CLDN18.2 antibodies or antigen-binding fragments thereof disclosed herein may be of IgG1, IgG2, IgG3, or IgG4 isotype. The term "isotype" refers to the class of antibody encoded by the heavy chain constant region genes. In some embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof disclosed herein are of IgG1 isotype. The anti-CLDN18.2 antibodies or antigen-binding fragments thereof disclosed herein may be derived from any species, including, but not limited to, mouse, rat, rabbit, non-human primate (e.g., chimpanzee, cynomolgus monkey, spider monkey, macaque), llama, and human. The anti-CDCLDN18.2 antibodies or antigen-binding fragments thereof disclosed herein may be murine, chimeric, humanized, or human. Unless otherwise specified, the term "antibody" as used herein includes full-length antibodies and any antigen-binding portion (or "antigen-binding fragment") thereof or single chains.

[0051] A typical "full-length antibody" is a glycoprotein comprising two heavy (H) chains and two light (L) chains, where the heavy and light chains are connected by disulfide bonds, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region, the heavy chain constant region being composed of three domains, namely CH1, CH2, and CH3, each light chain is composed of a light chain variable region (VL) and a light chain constant region, the light chain constant region being composed of one domain, CL, the VH and VL regions may be further divided into hypervariable regions (i.e., complementarity determining regions (CDRs)) and sequence-conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of an antibody comprises a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells), the first component of the classical pathway of complement (C1q).

[0052] An "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen (e.g., a CLDN18.2 protein). It is recognized that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples covered by the term "antigen-binding portion / fragment" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of an antibody single arm; (v) a dAb fragment consisting of the VH domain (see Ward et al., Nature. 341:544-546 (1989)); (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, which is an antibody comprising one heavy chain variable domain and two constant domains but no light chains. In addition, the two domains VL and VH of the Fv fragment are encoded by different genes, but VH and VL may be linked by a linker by recombinant methods to form a single protein chain, and among them, a monovalent molecule formed by matching VL and VH is called a single chain Fv (scFv), see Bird et al., Science. 242: 423-426 (1988), Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883 (1988), and these single chain antibodies are also covered by the term "antigen-binding portion / fragment". In addition, recombinant polypeptides, fusion proteins and immunoconjugates containing the antigen-binding portion / fragment are also covered by the term "antigen-binding portion / fragment". These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments may be subjected to functional screening in the same manner as full-length antibodies.

[0053] A "murine antibody" or "rodent antibody" refers to an antibody in which both the framework and CDR regions of the variable region are derived from mouse germline immunoglobulin sequences, and, if the antibody contains a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. A murine antibody of the present application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or point mutation in vitro or by somatic mutation in vivo), however, the term "murine antibody" or "rodent antibody" does not include antibodies in which mouse framework sequences have germline CDR sequences from other mammals inserted into them.

[0054] A "chimeric antibody" is an antibody that is formed by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response induced by murine antibodies. To produce a chimeric antibody, first prepare a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the hybridoma cell, and optionally clone the constant region gene of a human antibody, and then connect the mouse variable region gene and the human constant region gene to obtain a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody is expressed in a eukaryotic or prokaryotic system. In this application, the chimeric antibody is also referred to as "xi".

[0055] A "humanized antibody" is an antibody that contains complementarity determining regions (CDRs) derived from a non-human antibody, framework regions and constant regions derived from a human antibody. For example, a humanized antibody that binds CLDN18.2 provided herein may contain CDRs derived from one or more murine antibodies, human framework regions, and a human constant region. Thus, in some embodiments, a humanized antibody provided herein binds to the same epitope on CLDN18.2 as the murine antibody from which the CDRs of said antibody are derived. This specification provides exemplary humanized antibodies. Other CLDN18.2-binding humanized antibodies or variants thereof that contain heavy chain CDRs and light chain CDRs provided herein may be produced using any of the human framework sequences and are also included in this application. In some embodiments, framework sequences suitable for use in this application include those framework sequences that are structurally similar to the framework sequences provided herein. Other modifications in the framework regions may be made to alter the properties of the antibodies provided herein. Such other framework modifications may include chemical modifications, point mutations to reduce immunogenicity or eliminate T-cell epitopes, or backmutations to residues in the original germline sequence. In some embodiments, such other modifications include those modifications that correspond to the exemplary mutations herein, such as backmutations to the germline sequence. For example, in some embodiments, one or more amino acids in the human framework regions of the VH and / or VL of the humanized antibodies provided herein are backmutated to the corresponding amino acid in the parent murine antibody. In this application, humanized antibodies are also referred to as "hz."

[0056] As used herein, "derived from," when used in reference to other molecules or polypeptides that bind to a reference antibody or protein, means a molecule or polypeptide that is capable of specifically binding to the same epitope as the reference antibody or other binding protein.

[0057] "Isolated" refers to a compound of interest (eg, an antibody, antigen-binding fragment, or nucleic acid molecule) that has been separated from its natural environment.

[0058] "Identity" refers to the similarity between two or more nucleic acid sequences or between two or more polypeptide sequences. The sequence identity of the present application may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sequence comparison and identity percentage determination between two sequences may be performed with the default settings of the BLASTN / BLASTP algorithm on the website of the National Center for Biotechnology Information.

[0059] An antibody that "competitively binds" refers to an antibody that partially or completely blocks another antibody from binding to a target. Whether two antibodies competitively bind to a target, i.e., whether and to what extent one antibody blocks another antibody from binding to a target, may be determined using competitive assays known in the art, such as direct or indirect radioimmunoassays (RIA) using solid-phase methods, direct or indirect enzyme immunoassays (EIA) using solid-phase methods, competitive assays using sandwich methods, etc. In some embodiments, an antibody competitively binds to a target with another antibody and blocks the binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0060] Two or more antibodies "bind to the same epitope" means that the antibodies bind to the same section of amino acid residues as measured by a given method. Techniques for determining whether an antibody binds to the "same epitope on CLDN18.2" as an antibody described herein include, for example, epitope mapping methods, such as epitope mapping by yeast surface display, X-ray analysis of crystals of antigen-antibody complexes, and hydrogen deuterium exchange mass spectrometry (HDX-MS).

[0061] The term “EC 50 " is also called the median effective concentration and refers to the antibody concentration that can reach 50% of its maximum effect after a certain exposure time. 50 EC, also known as the half-maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% compared to the absence of the antibody. 50 and IC 50 Both can be measured by either ELISA or FACS analysis or other methods known in the art.

[0062] A "patient" or "subject" includes either a human or a non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, preferably mammals, e.g., non-human primates, sheep, dogs, cats, cows, horses.

[0063] "Effective dose" or "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. A therapeutically "effective amount" or "effective dose" of a drug or therapeutic agent refers to an amount sufficient to prevent or ameliorate the associated symptoms of a disease or condition, preferably an amount that causes a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of symptom-free periods, or prevents damage or functional impairment caused by the disease, when used alone or in combination with another therapeutic agent. The therapeutically effective amount is related to the disease being treated, although the actual effective amount can be easily determined by one of ordinary skill in the art.

[0064] As used herein, "about" means within an acceptable range of error as determined by a person skilled in the art for a particular value, which is determined in part from the limits of the measurement method, i.e., the measurement system, of the value. For example, according to common knowledge in the art, "about" represents up to 1 or more than 1 standard deviation. Alternatively, "about" represents a range of up to ±5%, for example, varying up to ±2% of the indicated numerical range, up to ±1%, or up to ±0.5%. When a particular value is indicated in the present specification or claims, unless otherwise specified, the meaning of "about" is understood to be within the acceptable error range for the particular value. In the present specification, unless otherwise specified, the value indicated as a parameter or condition of a step is considered to be "about".

[0065] Unless otherwise specified, the terms "a" or "one" mean "at least one" or "at least one type," the term "at least one" is equivalent to "one or more," and "and / or" is used in the sense of "and" or "or."

[0066] Unless otherwise stated, any percentage range, ratio range, or integer range set forth herein should be understood to include any of the integers within the recited range.

[0067] In this specification, unless the context dictates otherwise, the terms "comprise", "include", and "containing" are understood to mean the inclusion of the step or element or series of steps or elements, but not the exclusion of any other step or element or series of steps or elements. "Consisting of" means including and limited to what is defined by the phrase "consisting of". Thus, the phrase "consisting of" means that the recited elements are necessary or essential, and that no other elements are present. "Consisting essentially of" means that the recited elements are included, and are limited to other elements that do not interfere with or contribute to the activity or action of the recited elements as detailed herein. Thus, the phrase "consisting essentially of" means that the recited elements are necessary or essential, but that other elements are optional, and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0068] As used herein, unless otherwise specified, the terms "nucleic acid," "nucleic acid sequence," and "gene" may be used interchangeably to refer to a product obtained by linking two or more nucleotides together via phosphodiester bonds.

[0069] Unless the context clearly dictates otherwise, singular terms cover plural referents and vice versa.

[0070] Several aspects of the present application will now be described in more detail.

[0071] The amino acid sequence IDs (SEQ ID NOs) of the heavy chain variable regions, light chain variable regions and CDRs of exemplary antibodies or antigen-binding fragments thereof of the present application are provided in Table 1 below. Some antibodies have the same CDRs and some antibodies have the same VH or VL. The heavy chain constant region of the antibody may be a human IgG1, IgG2, IgG4 constant region or a variant thereof, preferably a human IgG1 constant region or a variant thereof, for example comprising the amino acid sequence set forth in SEQ ID NO: 61 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions and additions compared to the amino acid sequence set forth in SEQ ID NO: 61. The light chain constant region of the antibody may be a human κ constant region, a human λ constant region or a variant thereof, preferably a human κ constant region or a variant thereof, for example comprising the amino acid sequence set forth in SEQ ID NO: 62 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions and additions compared to the amino acid sequence set forth in SEQ ID NO: 62. These antibodies may comprise a mouse IgG1 heavy chain constant region and / or a mouse κ constant region. [Table 1]

[0072] The term "complementarity determining region," "CDR," or "hypervariable region" refers to one of the six hypervariable regions within the variable region of an antibody that are primarily responsible for antigen binding. From the amino acid sequence of an antibody variable region, one of skill in the art can determine the amino acid sequence boundaries of the CDRs using a variety of well-known schemes, including the "Kabat" numbering rule (see Kabat et al., (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule (see Al-Lazikani et al., (1997) JMB273:927-948), the ImMunoGenTics (IMGT) numbering rule (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), the AbM numbering rule (Oxford The numbering rules include the following: the numbering rules defined by AbM antibody modeling software from IMGT, Inc. (as defined by AbM antibody modeling software from IMGT, Inc.), the Contact numbering rule (based on the analysis of available complex crystal structures), and combinations of two or more of the numbering rules of Kabat, Chothia, IMGT, AbM, Contact, etc. For example, in the case of the classical format, according to the Kabat rules, the CDR amino acid residue numbers in the heavy chain variable region (VH) are 31 to 35 (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3), and the CDR amino acid residue numbers in the light chain variable region (VL) are 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3). For example, according to the Chothia rules, the CDR amino acid residue numbers in VH are 26 to 32 (HCDR1), 52 to 56 (HCDR2), and 95 to 102 (HCDR3), and the CDR amino acid residue numbers in VL are 26 to 32 (LCDR1), 50 to 52 (LCDR2), and 91 to 96 (LCDR3).Combining both the Kabat and Chothia rules, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. When the IMGT rules are followed, the CDR amino acid residue numbers in VH are approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). When the IMGT rules are followed, the CDRs of the antibody may be determined using the program IMGT / DomainGapAlign.

[0073] The boundaries of the amino acid sequences of the CDRs vary depending on the numbering system. As will be understood by one of skill in the art, unless otherwise specified, the term "CDR" or "complementarity determining region" of an antibody or a region thereof (e.g., a variable region) covers the complementarity determining region as defined by any known method. Although the CDR sequences shown in Tables 1 and 11 are defined according to one of the numbering rules above, corresponding amino acid sequences according to other CDR numbering rules are also within the scope of the present application.

[0074] The VH and / or VL sequences (or CDR sequences) of other antibodies that bind human CLDN18.2 can be "mixed and matched" with the VH and / or VL sequences (or CDR sequences) of the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present application. Preferably, when VH and VL chains (or their CDRs) are mixed and matched, the VH sequence in a particular VH / VL pairing may be replaced with a structurally similar VH sequence. Similarly, preferably, the VL sequence in a particular VH / VL pairing is replaced with a structurally similar VL sequence.

[0075] Thus, in one embodiment, the antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable region comprising an amino acid sequence set forth in Table 1; (b) a light chain variable region comprising an amino acid sequence set forth in Table 1, or the VL of another anti-CLDN18.2 antibody, provided that the antibody or antigen-binding fragment thereof specifically binds to human CLDN18.2.

[0076] In another embodiment, the antibody or antigen-binding fragment thereof of the present application comprises: (a) HCDR1, HCDR2 and HCDR3 as set forth in Table 1; (b) comprises LCDR1, LCDR2 and LCDR3 set forth in Table 1, or the CDRs of the light chain variable region of another anti-CLDN18.2 antibody, provided that the antibody or antigen-binding fragment thereof specifically binds to human CLDN18.2.

[0077] In another embodiment, the antibody or antigen-binding fragment thereof of the present application comprises an HCDR2 as set forth in Table 1 and a CDR of another anti-CLDN18.2 antibody, e.g., an HCDR1 and / or HCDR3 of another anti-CLDN18.2 antibody, and / or an LCDR1, LCDR2 and / or LCDR3 of another anti-CLDN18.2 antibody.

[0078] Furthermore, as is well known in the art, the CDR3 domain is independent of the CDR1 and / or CDR2 domains and can independently determine the binding specificity of an antibody to the same antigen, and it is predicted that multiple types of antibodies having the same binding specificity can be produced from the CDR3 sequence.

[0079] In another embodiment, the antibody or antigen-binding fragment thereof of the present application comprises an HCDR2 listed in Table 1, an HCDR3 listed in Table 1 and / or an LCDR3 listed in Table 1, and the CDRs of other anti-CLDN18.2 antibodies, provided that the antibody or antigen-binding fragment thereof specifically binds to human CLDN18.2. In another embodiment, the antibody or antigen-binding fragment thereof of the present application further comprises an LCDR2 listed in Table 1, and the CDRs of other anti-CLDN18.2 antibodies, provided that the antibody or antigen-binding fragment thereof specifically binds to human CLDN18.2. In another embodiment, the antibody or antigen-binding fragment thereof of the present application further comprises an HCDR1 listed in Table 1 or an LCDR1 listed in Table 1, and the CDRs of other anti-CLDN18.2 antibodies, provided that the antibody or antigen-binding fragment thereof specifically binds to human CLDN18.2. Preferably, these antibodies (a) competitively bind to human CLDN18.2, (b) retain the same functional characteristics, (c) bind to the same epitope, and / or (d) have similar binding affinity as the anti-CLDN18.2 antibodies of the present application.

[0080] In another embodiment, the heavy and / or light chain variable regions of the antibodies or antigen-binding fragments thereof of the present application may contain one or more conservative modifications, although as will be understood in the art, some conservative modifications do not abolish the antigen-binding properties of the antibody.

[0081] Thus, in one embodiment, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region each comprising CDR1, CDR2, and CDR3, respectively, with the proviso that: (a) the HCDR1 sequence comprises a sequence set out in Table 1, and / or a conservative modification thereof; and / or (b) the HCDR2 sequence comprises a sequence set forth in Table 1, and / or a conservative modification thereof; and / or (c) the HCDR3 sequence comprises a sequence set forth in Table 1, and / or a conservative modification thereof; and / or (d) the LCDR1 and / or LCDR2 and / or LCDR3 sequences comprise a sequence set forth in Table 1 and / or a conservative modification thereof; and (e) The antibody or antigen-binding fragment thereof specifically binds to human CLDN18.2.

[0082] As used herein, the term "conservative modifications" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as point mutations, PCR-mediated mutagenesis. A conservative amino acid substitution refers to one in which an amino acid residue is replaced with an amino acid residue having similar structural or chemical properties (e.g., similar side chains). Families of amino acid residues having similar side chains are known in the art. These amino acid residue families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the present anti-CLDN18.2 antibodies or antigen-binding fragments thereof can be substituted with other amino acid residues from the same side chain family, and the resulting antibodies can be tested for functionality in the functional assays described herein.

[0083] The antibodies or antigen-binding fragments thereof of the present application may include modifications to the variable regions (including the CDR regions and / or framework regions) or the antibodies or antigen-binding fragments thereof of the present application may further include Fc modifications, e.g., those that improve the effector functions of the antibody.

[0084] Thus, the isolated anti-CLDN18.2 monoclonal antibody or antigen-binding fragment thereof provided by one embodiment of the present application comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the above sequence of the present application, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the above sequence of the present application, and also comprises a framework different from the framework sequence in the above sequence of the present application. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. Such framework sequences are preferably structurally similar to the framework sequences used in the anti-CLDN18.2 antibodies of the present application. For example, in some cases, mutations to residues in the framework regions are beneficial, which can maintain or enhance the antigen-binding ability of the antibody (see, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370).

[0085] Another type of variable region modification is to improve one or more properties (e.g., affinity, physicochemical properties) of the antibody of interest by mutating amino acid residues in the CDR1, CDR2 and / or CDR3 regions. Mutations can be introduced by point mutation or PCR-mediated mutation, and the effect of the mutation on antibody binding or other functional properties can be evaluated by in vitro or in vivo assays known in the art. Amino acid substitutions, additions or deletions can be used, and substitutions are preferred. Also, generally, no more than 1, 2, 3, 4 or 5 residues in each CDR region are changed.

[0086] In one embodiment, the antibody or antigen-binding fragment thereof provided by the present application comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising: (a) an HCDR1 comprising an HCDR1 sequence of the present application or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) an HCDR2 comprising an HCDR2 sequence of the present application or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; and (c) an HCDR3 sequence of the present application or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions. (d) an LCDR1 comprising an LCDR1 sequence of the present application or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) an LCDR2 comprising an LCDR2 sequence of the present application or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; and (f) an LCDR3 sequence of the present application or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions.

[0087] The antibodies or antigen-binding fragments thereof of the present application include modifications of the VH and / or VL framework regions that improve the properties of the antibody. Generally, such framework region modifications can reduce the immunogenicity of the antibody. For example, one or more framework residues are "backmutated" to the corresponding germline sequence. These residues can be recognized by comparing the antibody framework sequence with the germline sequence from which the antibody is derived.

[0088] Another type of framework modification involves mutating one or more residues in the framework region or one or more CDR regions to reduce the immunogenicity that the antibody may cause by removing T cell epitopes, a method also known as "deimmunization" and described in detail in U.S. Patent Disclosure No. 20030153043.

[0089] The antibodies or antigen-binding fragments thereof of the present application also include Fc modifications, which may be amino acid insertions, deletions or substitutions, and are generally used to alter one or more functional properties of the antibody, such as the serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity.

[0090] The antibodies or antigen-binding fragments thereof of the present application may also be chemically modified (e.g., by attaching one or more chemical functional groups) or modified to alter their glycosylation to alter one or more functional properties of the antibody. In other embodiments, the Fc region is modified by pegylation (e.g., reacting the antibody or fragment thereof with polyethylene glycol (PEG)). In other embodiments, the glycosylation of the antibody is altered.

[0091] The present application also provides an isolated nucleic acid molecule encoding the heavy chain variable region and / or the light chain variable region or CDR of the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present application. The nucleic acid molecule of the present application may be DNA or RNA and may or may not contain intron sequences. The nucleic acid molecule of the present application may be single-stranded or double-stranded. In a preferred embodiment, the nucleic acid molecule is a cDNA molecule.

[0092] The nucleic acids of the present application may be obtained by standard molecular biology techniques. In the case of antibodies expressed by a hybridoma, cDNAs encoding the light and heavy chains of the antibody produced in the hybridoma may be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding such antibodies may be recovered from the gene library.

[0093] Once the VH- and VL-encoding DNA fragments are obtained, they can be further manipulated by standard recombinant DNA techniques, for example, converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VH- or VL-encoding DNA fragment is operably linked to a DNA fragment encoding another protein (e.g., an antibody constant region or a flexible linker). As used in this context, the term "operably linked" refers to joining two DNA fragments together such that the amino acid sequences encoded by the two DNA fragments are both in reading frame.

[0094] The isolated DNA encoding VH can be converted into a full-length heavy chain gene by operably linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3). The sequence of the human heavy chain constant region gene is known in the art, and a DNA fragment containing the human heavy chain constant region gene can be obtained by standard PCR amplification. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, and is preferably the IgG1 constant region.

[0095] The isolated DNA encoding the VL can be converted into a full-length light chain gene by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequence of the human light chain constant region gene is known in the art, and a DNA fragment containing the human light chain constant region gene can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0096] The monoclonal antibodies (mAbs) of the present application may be produced using somatic cell fusion (hybridoma) technology as described by Kohler and Milstein (1975) Nature 256:495, which is well known in the art. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display technology. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Patent Nos. 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762, and 6,180,370, which are expressly incorporated herein by reference in their entirety.

[0097] The antibodies of the present application may also be produced in transfectomas of host cells, for example, using a method combining recombinant DNA technology and gene transfer (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors, such that the genes are operably linked to transcriptional and translational regulatory sequences. In this case, the term "operably linked" refers to the linking of the antibody gene into a vector, thereby allowing the transcriptional and translational regulatory sequences in the vector to perform their defined function of regulating the transcription and translation of the antibody gene. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene.

[0098] The antibody light chain gene and the antibody heavy chain gene may be inserted into the same expression vector or into different expression vectors. In a preferred embodiment, the variable regions are inserted into an expression vector encoding the heavy and light chain constant regions of the desired isotype to construct a full-length antibody gene, such that the VH is operably linked to the CH in the vector and the VL is operably linked to the CL in the vector. Alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is connected in reading frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0099] To express the light and heavy chains, expression vectors encoding the heavy and light chains are introduced into a host cell by standard techniques. The term "introduction" includes various techniques for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, such as, for example, electroporation, calcium phosphate co-precipitation, DEAE-dextran introduction, etc. Although it is theoretically possible to express the antibodies of the present application in prokaryotic or eukaryotic host cells, the antibodies are preferably expressed in eukaryotic cells, and most preferably in mammalian host cells, since eukaryotic cells, especially mammalian cells, are better able to assemble and secrete properly folded and immunoreactive antibodies than prokaryotic cells. Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, used with the DHFR selection marker, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, e.g., RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. After introducing a recombinant expression vector encoding an antibody gene into a mammalian host cell, the antibody is produced by culturing the host cell so that the antibody is expressed by the host cell or, preferably, secreted over a period of time into the medium in which the host cell is grown. The antibody can be recovered from the host cell or cell culture using standard protein purification methods.

[0100] In another aspect, the present application provides a pharmaceutical composition comprising an anti-CLDN18.2 antibody or antigen-binding fragment thereof, a nucleic acid molecule, a recombinant polypeptide, a fusion protein, a multispecific antibody, an immunoconjugate, a chimeric antigen receptor, or a gene vector of the present application, and a pharma- ceutically acceptable carrier or excipient. The term "pharma- ceutically acceptable carrier or excipient" refers to a carrier or excipient that can be used to make a pharmaceutical composition, which is generally safe, non-toxic, biologically inactive, and harmless, and includes carriers or excipients for veterinary and human medicines.

[0101] The pharmaceutical compositions of the present application are suitable for intravenous, intramuscular, subcutaneous, parenteral, intrathecal or epidermal administration (e.g., injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from the effects of acids and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" refers to a mode of administration other than enteral and topical administration, generally utilizing injection, and including, but not limited to, intravenous, intramuscular, intraarterial, intramedullary, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the pharmaceutical compositions of the present application may be administered by an oral route, for example, a topical, epidermal, or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0102] The pharmaceutical compositions of the present application may be in the form of sterile aqueous solutions or dispersions. They may also be prepared in microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0103] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single high dose may be administered, multiple partial doses may be administered over time, or the dose may be proportionally reduced or increased depending on the exigencies of treatment. Alternatively, the antibody may be administered as a sustained release formulation, in which case the prescribed dosing frequency is reduced.

[0104] For administration of the antibody, the dosage may be in the range of about 0.0001 to 100 mg / kg of the host body weight.

[0105] The above invention is described in detail in the examples and embodiments for clarity of understanding, but it is obvious to those skilled in the art that, according to the teachings of the present application, other slight changes and modifications may be made to the present application without departing from the spirit and scope of the claims. The present application is further described in the following examples, without being limited thereto. In addition, those skilled in the art should be aware of various non-critical parameters that can be changed or modified to obtain substantially similar results.

[0106] Unless otherwise specified, the practice of the present application employs conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art. For example, each of the reagents, materials and instruments used in the following examples may be commercially available in the art unless otherwise specified. EXAMPLES

[0107] Example 1: Production of anti-CLDN18.2 hybridoma monoclonal antibodies Construction of stable cell lines: cDNA encoding human CLDN18.2 (SEQ ID NO: 1) was obtained by gene synthesis and then subcloned into the expression vector pcDNA3.1. The expression vector was transfected into U2OS cells and NIH-3T3 cells, respectively, according to the instructions of Lipofectamine 2000 transfection reagent (Thermo, Cat. No. 11668019), to obtain stable cell lines U2OS-CLDN18.2 and NIH-3T3-CLDN18.2.

[0108] cDNA encoding human CLDN18.1 (SEQ ID NO: 2) was obtained by gene synthesis and then subcloned into the expression vector pcDNA3.1. The expression vector was transfected into U2OS cells and NIH-3T3 cells, respectively, according to the instructions of Lipofectamine 2000 transfection reagent (Thermo, Cat. No. 11668019), to obtain stable cell lines U2OS-CLDN18.1 and NIH-3T3-CLDN18.1.

[0109] Immunization of mice: BALB / c mice were immunized with the immunogen NIH-3T3-CLDN18.2 cells, and each mouse was administered 5 × 10 6 ~10×10 6 NIH-3T3-CLDN18.2 cells were injected intraperitoneally, once every 2 weeks, for a total of five immunizations. Three days before fusion, each mouse was injected with 1 × 10 7 Each mouse was immunized by intraperitoneal injection of NIH-3T3-CLDN18.2 cells. After immunization, serum from each mouse was collected to detect the titer of anti-CLDN18.2 specific antibodies. Mice with high antibody titers were selected for subsequent spleen cell fusion.

[0110] Cell fusion: Using the electrofusion method, spleen cells (1 × 10) from immunized mice were cultured using an electrofusion apparatus (BTX, ECM2001). 8 cells) and SP2 / 0 myeloma cells (5 × 10 7 After fusion, the cells were resuspended in HAT complete medium, dispensed into a 96-well plate at 0.2 mL / well, and cultured in an incubator containing 37°C and 5% CO2 for 7 to 10 days. The hybridoma cell culture supernatant was obtained and detected.

[0111] Hybridoma screening: The binding activity of the antibody against CLDN18.2 was detected by ELISA. U2OS-CLDN18.2 cells were dispensed into a 96-well plate at 10,000 cells / well and cultured overnight in an incubator containing 37°C and 5% CO2. The cell culture supernatant was discarded, and the 96-well plate was washed three times with PBS buffer and fixed with 2% paraformaldehyde for 30 minutes at room temperature. The 96-well plate was washed three times with PBS buffer, and 100 μL of hybridoma cell culture supernatant was added to each well and incubated at room temperature for 1 hour. The 96-well plate was washed three times with PBS buffer, and 100 μL of HRP-Goat Anti-Mouse IgG Fcγ (Jackson immunoresearch, Cat. No. 115-035-071) was added to each well and incubated at room temperature for 1 hour. The 96-well plate was washed three times with PBS buffer, 100 μL of TMB (Thermo, Cat. No. 00-4201-56) was added to each well and incubated for 10 min, then 50 μL of stop solution (2N H2SO4) was added to each well to stop the reaction. The OD value was read at a wavelength of 450 nm using a microplate reader (Thermo, Varioskan Flash).

[0112] Hybridoma cells that were detected as positive by ELISA were selected, and the binding activity of the antibodies in the culture supernatant of the hybridoma cells against the cell lines KATOIII and NUGC-4 that naturally express CLDN18.2 was detected by the FACS method (see Example 3 for the method).

[0113] Example 2: Production of anti-CLDN18.2 chimeric antibodies Obtaining cDNA: Hybridoma cells (28G3, 40F6, 22F12, 34G6, 10D8) that were positively detected by ELISA and FACS were selected, and total RNA was isolated from the cells as a template using a total RNA extraction kit (Takara, Cat. No. 9767), and first-strand cDNA was synthesized using superscript III reverse transcriptase according to the kit's instructions (Thermo, Cat. No. 18080051). The first-strand cDNA was then used as a template to amplify the antibody heavy and light chain variable region sequences by PCR reaction using mouse IgG primers and κ primers. The PCR mixture was separated by electrophoresis in a 1% agarose / Tris(trimethylsilyl)borate gel containing 0.5 μg / mL ethidium bromide. DNA fragments of a given size were excised from the gel and purified. The purified PCR products were cloned into pMD-19T vector (Takara, Catalog No. 6013), introduced into DH5α E. coli competent cells (Takara, Catalog No. 9057), and cultured on LB solid culture plates. Single colonies were picked from the LB solid culture plates and subjected to DNA sequencing. The heavy and light chain variable region sequences of the antibodies (murine antibodies 28G3, 40F6, 22F12, 34G6, and 10D8) were obtained.

[0114] Construction and expression of chimeric antibodies: Knocking out the fucose expression-related gene FUT8 can enhance the interaction between IgG1 and FcgRIIIa, and thus enhance the ADCC activity of the antibody (Shields et al., 2002; Yamane-Ohnuki et al., 2004). In this example, a defucosylated antibody was produced using CHO-S cells in which FUT8 was knocked out (designated CHO-S Fut8- / - cells).

[0115] A chimeric light chain was constructed by connecting a mouse VL region gene fragment to a human κ chain constant region, and a chimeric heavy chain was constructed by connecting a mouse VH region gene fragment to a human IgG1 constant region. A chimeric heavy chain expression plasmid and a chimeric light chain expression plasmid corresponding to each antibody were simultaneously introduced into CHO-S Fut8- / - cells to express the protein. After the introduction, the cells were cultured under conditions including 37°C and 8% CO2. After 7 to 10 days of culture, the cells were centrifuged to obtain a cell culture supernatant, and the chimeric antibody in the cell culture supernatant was purified using a protein A column (GE healthcare). Here, the human κ light chain constant region has the amino acid sequence shown in SEQ ID NO: 62, and the human IgG1 constant region has the amino acid sequence shown in SEQ ID NO: 61.

[0116] Example 3: Measurement of binding activity of anti-CLDN18.2 antibody Based on FACS method, the binding activity of anti-CLDN18.2 antibodies to CLDN18.2 overexpressing cell line U2OS-CLDN18.2, CLDN18.2 naturally expressing cell line KATOIII (Chinese Academy of Sciences Cell Bank) and NUGC-4 (Chinese Academy of Sciences Cell Bank) was analyzed. IMAB362 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 63 and 64 of the present application) was used as a positive control, and IgG1 isotype control antibody (also referred to as IgG1 in the present examples, produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 67 and 68 of the present application) was used as a negative control.

[0117] Adjust the cell concentration with PBS and add 3 × 10 cells (U2OS-CLDN18.2, KATOIII, or NUGC-4 cells). 5Each well was dispensed into a 96-well culture plate, and then different concentrations of the test antibody were added to each well of the 96-well culture plate and incubated at 4° C. for 60 minutes. The final concentrations of xi40F6 and xi34G6 antibodies were 300nM, 100nM, 33.33nM, 11.11nM, 3.7nM, 1.23nM, 0.41nM, and 0.14nM for KATOIII and NUGC-4 cells, respectively, the final concentrations of xi10D8, xi22F12, and xi28G3 antibodies were 300nM, 60nM, 12nM, 2.4nM, 0.48nM, 0.096nM, 0.0192nM, and 0.00384nM for U2OS-CLDN18.2 cells, respectively, and the final concentrations of the test antibodies were 150nM, 50nM, 16.67nM, 5.56nM, 1.85nM, 0.62nM, 0.21nM, and 0.07nM for U2OS-CLDN18.2 cells. The supernatant was discarded, the cells were washed three times with pre-chilled PBS buffer, and 100 μL of 1:200 diluted PE-Goat Anti-Human IgG Fcγ (Jackson immunoresearch, Cat. No. 109-116-170) was added to each well and incubated at 4°C for 30 min. The cells were washed three times with pre-chilled PBS buffer, resuspended in 100 μL PBS buffer, and then the fluorescent signal was detected using a flow cytometer (Sartorius, iQUE3). The binding ability of anti-CLDN18.2 antibody to CLDN18.2 was measured from the mean fluorescence intensity (MFI) of the staining.

[0118] The binding activity of the anti-CLDN18.2 chimeric antibody to U2OS-CLDN18.2, KATOIII, and NUGC-4 cells is shown in Figure 1. The EC 50 The data are shown in Tables 2.1, 2.2 and 2.3. The binding activities of xi28G3, xi40F6, xi22F12 and xi34G6 in three different types of cells were all superior to IMAB362, while xi10D8 had the same binding activity as IMAB362. [Table 2] [Table 3] [Table 4]

[0119] Example 4: Non-specific binding of anti-CLDN18.2 antibodies The binding activity of anti-CLDN18.2 antibodies to the cell line U2OS-CLDN18.1, which overexpresses CLDN18.1 (SEQ ID NO: 2), was analyzed based on the FACS method. The cell concentration was adjusted with PBS, and U2OS-CLDN18.1 cells were cultured at 3 × 10 5 100μL / well of the antibody was added to each well of the 96-well culture plate, and then the test antibody was added to each well of the 96-well culture plate to a final antibody concentration of 200nM and incubated at 4℃ for 60min. The supernatant was discarded, the cells were washed three times with pre-chilled PBS buffer, and 100μL of 1:200 diluted PE-Goat Anti-Human IgG Fcγ (Jackson immunoresearch, Cat. No. 109-116-170) was added to each well and incubated at 4℃ for 30min. The cells were washed three times with pre-chilled PBS buffer, and the cells were resuspended in 100μL of PBS buffer, and then the fluorescent signal was detected using a flow cytometer (Sartorius, iQUE3). Nonspecific binding of anti-CLDN18.2 antibody to U2OS-CLDN18.1 cells was measured from the mean fluorescence intensity (MFI) of staining, and an IgG1 isotype control antibody (produced in-house, whose heavy and light chain amino acid sequences are shown in SEQ ID NOs: 67 and 68 of the present application) was used as a control to determine whether or not there was nonspecific binding, based on a value twice that of the IgG1 isotype control antibody.

[0120] The results are shown in Table 3. The MFI value of the anti-CLDN18.2 chimeric antibody was close to that of the control IgG1 isotype antibody, indicating that the anti-CLDN18.2 chimeric antibody did not bind nonspecifically to U2OS-CLDN18.1 cells. [Table 5]

[0121] Example 5: ADCC activity of anti-CLDN18.2 antibodies Based on the reporter gene assay, the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CLDN18.2 antibodies was measured using KATOIII, NUGC-4, and U2OS-CLDN18.2 as target cells and Jurkat cells stably transfected with CD16a v158 and NFAT-luciferase (Jurkat-CD16a v158-NFAT-luciferase) (BPS Bioscience, Cat. No. 60541) as effector cells. IMAB362 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 63 and 64 of the present application) was used as a positive control, and an IgG1 isotype control antibody (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 67 and 68 of the present application) was used as a negative control.

[0122] 2.4 × 10 KATOIII, NUGC-4, and U2OS-CLDN18.2 cells were cultured in medium. 5The cells were diluted to 100 cells / mL, and each type of cell suspension was dispensed into a 96-well plate at 100 μL / well and cultured overnight in an incubator containing 37°C and 5% CO2. The next day, the culture supernatant was discarded, and the 96-well plate was washed 1 to 3 times with PBS buffer. Next, different concentrations of the test antibody were added to the 96-well plate at 60 μL / well, and the final concentrations of the anti-CLDN18.2 antibody of the present application were set to 20 nM, 2 nM, 0.2 nM, 0.02 nM, 0.002 nM, 0.0002 nM, 0.00002 nM, and 0.000002 nM for KATOIII cells, and the final concentrations of IMAB362 were set to 2000 nM, 200 nM, 20 nM, 2 nM, 0.2 nM, 0.02 nM, 0.002 nM, and 0.0002 nM for NUGC-4 cells. The final concentrations of the anti-CLDN18.2 antibody of the present application and IMAB362 were 1333.33 nM, 133.33 nM, 13.33 nM, 1.33 nM, 0.133 nM, 0.0133 nM, 0.00133 nM, and 0.000133 nM, and the final concentrations of the anti-CLDN18.2 antibody of the present application and IMAB362 for U2OS-CLDN18.2 cells were 1.334 nM, 0.267 nM, 0.053 nM, 0.011 nM, 0.002 nM, 0.0004 nM, and 0.00009 nM. Next, Jurkat-CD16a v158-NFAT-luciferase cells were taken out, and 40 μL of Jurkat-CD16a v158-NFAT-luciferase cell suspension was added to each well of a 96-well plate at a ratio of 5:1 between the number of effector cells and the number of target cells. The effector cells and the target cells were thoroughly mixed and contacted by low-speed centrifugation, and incubated for 5-6 hours in an incubator containing 37°C and 5% CO2. Then, according to the instructions of the luciferase detection kit (Novezon, Cat. No. DD1203-03), 100 μL of detection reagent was added to each well, and the wells were left to stand for 15-30 minutes, and the fluorescent signal was detected using a microplate reader (Thermo, Varioskan Flash).

[0123] In the reporter gene assay, the ADCC activity of the anti-CLDN18.2 chimeric antibody was shown in Figure 2. 50The data is shown in Table 4. In three different cell types, the chimeric antibodies xi28G3, xi40F6, xi34G6, and xi22F12 could significantly induce the activation of NFAT signaling in Jurkat-CD16a v158-NFAT-luciferase cells, and were superior to IMAB362. [Table 6]

[0124] Based on the killing effect of PBMC, the ADCC activity of anti-CLDN18.2 antibodies was detected using KATOIII and NUGC-4 as target cells and PBMC as effector cells, respectively. IMAB362 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 63 and 64 of the present application) and 15F9 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 65 and 66 of the present application) were used as positive controls, and IgG1 isotype control antibody (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 67 and 68 of the present application) was used as negative control.

[0125] 2 × 10 KATOIII and NUGC-4 cells were cultured in medium. 5 The cells were diluted to 100 / mL and 50 μL of each cell suspension was dispensed into a 96-well plate. Different concentrations of the test antibody were then added to each well of the 96-well plate, resulting in final concentrations of 66.7 nM, 8.34 nM, 1.04 nM, 0.13 nM, 0.016 nM, 0.002 nM, 0.00025 nM, and 0.00003 nM for KATOIII cells, and 600 nM, 100 nM, 16.67 nM, 2.78 nM, 0.46 nM, 0.077 nM, 0.013 nM, and 0.002 nM for NUGC-4 cells. The PBMC cells were resuscitated, and 50 μL of the PBMC cell suspension was added to each well of the 96-well plate at a ratio of 20:1 effector cell number:target cell number (cell density was 4 × 10 6The cells were centrifuged at low speed to thoroughly mix and contact the effector cells with the target cells, and incubated for 18 hours in an incubator containing 37°C and 5% CO2. The 96-well plate was then placed in a plate centrifuge and centrifuged at 250g / min for 5 minutes, and 50μL of the supernatant was taken from each well and placed in a 96-well detection plate. The LDH working solution was prepared according to the instructions in the CytoTox96 Non-Radio kit (Promega, catalog number PR-G1780), and 50μL of the LDH working solution was added to each well of the 96-well detection plate, mixed uniformly, and incubated in the dark at room temperature for 25-35 minutes. 50μL of stop solution was added to each well, mixed uniformly, and the OD value was read at a wavelength of 490nm using a microplate reader (Thermo, Varioskan Flash).

number

[0126] As shown in Figure 3 and Table 5, in the PBMC killing assay, the ADCC activity of anti-CLDN18.2 chimeric antibodies xi22F12, xi28G3, xi40F6, and xi34G6 was superior to that of IMAB362 and 15F9. [Table 7]

[0127] Example 6: CDC activity of anti-CLDN18.2 antibodies The complement-dependent cytotoxicity (CDC) activity of the antibodies was detected using KATOIII and U2OS-CLDN18.2 as target cells according to the CellTiter-Glo Luminescent Cell Viability Assay method. IMAB362 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 63 and 64 of the present application) was used as a positive control, and an IgG1 isotype control antibody (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 67 and 68 of the present application) was used as a negative control.

[0128] KATOIII and U2OS-CLDN18.2 were cultured at 8 × 10 5 The various cell suspensions were diluted to cells / mL and dispensed into a 96-well plate at 50 μL / well. Different concentrations of the test antibodies were then added to the 96-well plate at 50 μL / well to achieve final concentrations of 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM for KATOIII cells, and 333.33 nM, 66.67 nM, 13.33 nM, 2.67 nM, 0.53 nM, 0.11 nM, 0.02 nM, and 0.004 nM for U2OS-CLDN18.2 cells. Complement (Quidel, Cat. No. A113) was then added to the 96-well plate at 50 μL / well to achieve a final complement concentration of 10%. After low-speed centrifugation, the 96-well plate was incubated at 37°C with shaking for 30 minutes to allow the complement and target cells to be thoroughly mixed and contacted. The plate was then incubated for 2 hours in a 37°C, 5% CO2 incubator. After incubation was completed, 110 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat. No. G7572) was added to each well and incubated for 5-15 minutes at room temperature in the dark. Finally, the fluorescence was measured using a microplate reader (Thermo, Varioskan Flash).

number

[0129] The results are shown in Figure 4 and Table 6. The anti-CLDN18.2 chimeric antibodies xi22F12, xi28G3, xi40F6, xi34G6, and xi10D8 were all able to induce strong CDC effects, and the activities of xi22F12, xi28G3, xi40F6 and xi34G6 were stronger than that of IMAB362. [Table 8]

[0130] Example 7: Endocytic activity of anti-CLDN18.2 antibodies According to the instructions for Antibody Internalization Human Reagent (Sartorius, catalog number 90564), the endocytosis activity of the anti-CLDN18.2 antibody was detected using NUGC-4 and NIH-3T3-CLDN18.2 as target cells, respectively.

[0131] NUGC-4 and NIH-3T3-CLDN18.2 cells were each cultured at 2 × 10 6 The cells were diluted to 100 / mL and various cell suspensions were dispensed into a 96-well plate at 20 μL / well. The test antibody and internalization reagent were mixed uniformly and incubated at 37°C in the dark for 15 minutes, and then the mixture of the test antibody and internalization reagent was added to the 96-well plate at 20 μL / well and incubated for 2 hours in a 37°C, 5% CO2 incubator to give final concentrations of the test antibody of 20 nM, 6.67 nM, 2.22 nM, 0.74 nM, 0.25 nM, 0.08 nM, 0.027 nM, and 0.009 nM. After the incubation was completed, the fluorescent signal (MFI) was detected using a flow cytometer (Sartorius, iQUE3). The endocytosis activity of the antibody was evaluated from the magnitude of the fluorescent value, and the results are shown in Figure 5.

[0132] Example 8: Non-specific ADCC activity of anti-CLDN18.2 antibodies Based on the reporter gene assay, the non-specific ADCC activity of anti-CLDN18.2 antibody against NIH-3T3CLAN18.1 cells was analyzed using NIH-3T3-CLDN18.1 cells as target cells and Jurkat-CD16a v158-NFAT-luciferase as effector cells, and the final concentrations of the tested antibodies were 333.33nM, 66.67nM, 13.33nM, 2.67nM, 0.53nM, 0.107nM, 0.021nM, and 0.004nM. The specific method was described in Example 5.

[0133] The results are shown in Figure 6. The anti-CLDN18.2 chimeric antibodies xi22F12, xi28G3, xi40F6, and xi34G6 all had no ADCC effect against NIH-3T3-CLDN18.1 cells, while xi10D8 and IMAB362 had weak ADCC activity against NIH-3T3-CLDN18.1 cells.

[0134] Example 9: Humanization of anti-CLDN18.2 monoclonal antibodies Murine antibodies 28G3 (heavy chain variable region sequence SEQ ID NO: 11, light chain variable region sequence SEQ ID NO: 16) and 40F6 (heavy chain variable region sequence SEQ ID NO: 29, light chain variable region sequence SEQ ID NO: 34) were selected for humanization design.

[0135] Humanization was performed on the murine antibodies 28G3 and 40F6 using a combination of CDR-grafting and computer-aided design, and human germline antibody sequences with the highest sequence homology were screened for by comparison and analysis of the heavy and light chain variable region sequences of the murine antibodies with protein databases. The complementarity determining regions (CDRs) of the murine antibodies 28G3 and 40F6 were grafted onto the frameworks of the screened human germline antibody sequences, and amino acid residues in the CDRs and / or frameworks were mutated to obtain more candidate variable region sequences.

[0136] A humanized light chain was constructed by linking the humanized VL region gene fragment to a human κ chain constant region, and a humanized heavy chain was constructed by linking the humanized VH region gene fragment to a human IgG1 constant region. Introduction and expression were performed according to the method of Example 2, and the humanized antibody in the cell culture supernatant was then purified using a Protein A column. Here, the human κ light chain constant region has the amino acid sequence shown in SEQ ID NO:62, and the human IgG1 constant region has the amino acid sequence shown in SEQ ID NO:61.

[0137] Example 10: Characterization of anti-CLDN18.2 humanized antibodies Binding activity: Based on FACS method, the binding activity of anti-CLDN18.2 humanized antibodies against the cell lines KATOIII (Chinese Academy of Sciences Cell Bank) and NUGC-4 (Chinese Academy of Sciences Cell Bank), which naturally express CLDN18.2, was analyzed. The final concentrations of hz28G3-1.1, hz28G3-1.2, hz28G3-1.3, hz28G3-1.4, hz28G3-2.1, hz28G3-2.2, hz28G3-2.3, hz28G3-2.4 and xi28G3 were 150nM, 50nM, 16.67nM, 5.56nM, 1.85nM, 0.62nM, 0.21nM, 0.07nM, and hz40F61. The final concentrations of 1, hz40F6-1.2, hz40F6-1.3, hz40F6-1.4, hz40F6-2.1, hz40F6-2.2, hz40F6-2.3, hz40F6-2.4 and xi40F6 were 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM and 0.14 nM, respectively, and the specific methods were as described in Example 3.

[0138] The binding activity of the anti-CLDN18.2 humanized antibody is shown in Figures 7 and 8. The EC 50 The data are shown in Tables 7.1 and 7.2. The binding activity of hz40F6-1.1 and hz40F6-2.1 was weaker than that of the chimeric antibody xi40F6, but the binding activity of the remaining humanized antibodies was equivalent to or better than that of the chimeric antibody, and the binding activity of all humanized antibodies was better than that of IMAB362. [Table 9] [Table 10]

[0139] ADCC activity: Based on the reporter gene assay, the ADCC activity of anti-CLDN18.2 humanized antibodies was analyzed using KATOIII, NUGC-4, NIH-3T3-CLDN18.2, and U2OS-CLDN18.2 as target cells and Jurkat-CD16a v158-NFAT-luciferase as effector cells, respectively. The final concentrations of the test antibodies were 13.33nM, 2.22nM, 0.37nM, 0.062nM, 0.01nM, 0.0017nM, 0.00029nM, and 0.000048nM for NIH-3T3-CLDN18.2 and U2OS-CLDN18.2 cells, and 1333.33nM, 133.33nM, 1.33nM, 0.133nM, 0.0133nM, and 0.00133nM for NUGC-4 cells. For KATOIII cells, the final concentrations of the anti-CLDN18.2 antibody of the present application were 20 nM, 2 nM, 0.2 nM, 0.02 nM, 0.002 nM, 0.0002 nM, 0.00002 nM, and 0.000002 nM, and the final concentrations of IMAB362 were 2000 nM, 200 nM, 20 nM, 2 nM, 0.2 nM, 0.02 nM, 0.002 nM, and 0.0002 nM. Specific methods were as described in Example 5.

[0140] In the reporter gene assay, the ADCC activity of the anti-CLDN18.2 humanized antibody was shown in Figures 9 and 10. 50 The data are shown in Tables 8.1 and 8.2. In two different cell types, both anti-CLDN18.2 humanized antibodies were able to significantly induce NFAT signal activation in Jurkat-CD16a v158-NFAT-luciferase cells, and the ADCC activity of the humanized antibody was comparable to that of its chimeric antibody and superior to that of IMAB362. [Table 11] [Table 12]

[0141] PBMC killing test was used to analyze the ADCC activity of anti-CLDN18.2 humanized antibodies, using KATOIII, NUGC-4, and NIH-3T3-CLDN18.2 as target cells and PBMC as effector cells, respectively. The final concentrations of the tested antibodies were 66.7nM, 8.34nM, 1.04nM, 0.13nM, 0.016nM, 0.002nM, 0.00025nM, and 0.000032nM, and the specific method was described in Example 5.

[0142] The results are shown in Figure 11 and Table 9. In the PBMC killing test, the ADCC activity of the anti-CLDN18.2 humanized antibody hz28G3-1.3 was comparable to that of xi28G3 and significantly superior to that of 15F9. [Table 13]

[0143] CDC activity: Based on the CellTiter-Glo Luminescent Cell Viability Assay method, CDC activity of the antibodies was detected using NIH-3T3-CLDN18.2 as target cells. IMAB362 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 63 and 64 of the present application) and 15F9 (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 65 and 66 of the present application) were used as positive controls, and IgG1 isotype control antibody (produced in-house, heavy and light chain amino acid sequences are shown in SEQ ID NOs: 67 and 68 of the present application) was used as negative control. NIH-3T3-CLDN18.2 cells were cultured at 8 × 10 5The cells were diluted to 100 cells / mL, and the various cell suspensions were dispensed into a 96-well plate at 50 μL / well. Different concentrations of the test antibodies were then added to the 96-well plate at 50 μL / well. The final concentrations of hz28G3-1.1, hz28G3-1.3 and xi28G3 were 100 nM, 25 nM, 6.25 nM, 1.56 nM, 0.39 nM, 0.098 nM, 0.024 nM, and 0.006 nM, and the final concentrations of hz40F6-1.1, hz40F6-1.2, hz40F6-1.3, hz40F6-1.4, The final concentrations of hz40F6-2.1, hz40F6-2.2, hz40F6-2.3, hz40F6-2.4 and xi40F6 were 333.33 nM, 66.67 nM, 13.33 nM, 2.67 nM, 0.53 nM, 0.11 nM, 0.02 nM and 0.004 nM, and the specific method was described in Example 6.

[0144] The results are shown in Figure 12 and Tables 10.1 and 10.2. All anti-CLDN18.2 humanized antibodies were able to induce strong CDC effects, and except for individual antibodies, the CDC activities of other humanized antibodies were equivalent to those of their chimeric antibodies. [Table 14] [Table 15]

[0145] Endocytic Activity: According to the instructions of Antibody Internalization Human Reagent (Sartorius, Catalog No. 90564), the endocytosis activity of anti-CLDN18.2 humanized antibody was detected using NUGC-4 and U2OS-CLDN18.2 as target cells. The final concentrations of the test antibody in NUGC-4 cells were 111.11 nM, 37.04 nM, 12.35 nM, 4.12 nM, 1.37 nM, 0.46 nM, and 0.15 nM, and the final concentrations of the test antibody in U2OS-CLDN18.2 cells were 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM, 0.046 nM, and 0.015 nM, respectively. The specific method was as described in Example 7. The strength of the endocytosis activity of the antibody was evaluated based on the magnitude of the fluorescence value, and the results are shown in FIG. 13.

[0146] Non-specific ADCC activity: Based on PBMC killing test, NIH-3T3-CLDN18.1 was used as target cell, and PBMC was used as effector cell to analyze the non-specific ADCC activity of anti-CLDN18.2 humanized antibody against NIH-3T3-CLDN18.1 cell. The final concentration of the test antibody was 66.7nM, 8.34nM, 1.04nM, 0.13nM, 0.016nM, 0.002nM, 0.00025nM, and the specific method was referred to Example 5.

[0147] The results are shown in FIG. 14, and show that the anti-CLDN18.2 humanized antibodies hz28G3-1.1 and hz28G3-1.3 had no ADCC effect on NIH-3T3-CLDN18.1 cells.

[0148] The sequence information of this application is summarized in Table 11. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6] [Table 16-7] [Table 16-8]

[0149] All patents, patent applications, and other previously established publications are expressly incorporated herein by reference for purposes of description and disclosure, and are provided as such because they were made public prior to the filing date of this application. Any statement as to the disclosure date of such documents or representation of their contents is based on the information known to the applicants and does not constitute an admission that the disclosure date of such documents or the contents thereof are correct, nor does the reference to such publications in this specification constitute common general knowledge in the art in any applicable country.

[0150] Although the present application has been described in detail above using general descriptions and specific embodiments, some amendments or improvements may be made thereto in light of the present application, which are obvious to those skilled in the art. Therefore, if such amendments or improvements are made without departing from the spirit of the present application, they all fall within the scope of protection claimed in the present application.

Claims

1. An isolated anti-CLDN18.2 antibody or antigen-binding fragment thereof, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3, with the proviso that: (1) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 14, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 14, and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 14; the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (2) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 11, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 11, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 11, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 16, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 16; (3) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 12, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (4) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 13, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (5) the heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 15, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 15, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 15, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (6) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 12, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (7) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 13, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (8) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 14, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 14, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 14, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (9) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 15, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 15, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 15, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 18, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 18, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 18; (10) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 29, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 29, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 29, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 34, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 34, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 34; (11) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 30, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (12) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 31, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 31, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 31, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (13) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 32, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 32, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 32, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (14) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 33, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 33, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 33, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 35, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 35, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 35; (15) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 30, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (16) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 31, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 31, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 31, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (17) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 32, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 32, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 32, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (18) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 33, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 33, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 33, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 36, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 36, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 36; (19) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 43, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 43, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 43, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 44, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 44, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 44; (20) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 51, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 51, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 51, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 52, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 52, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 52; or (21) The heavy chain CDR1 comprises the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 59, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 59, the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 59, the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 60, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 60, and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO: 60; or the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, with the proviso that: (1) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences represented by SEQ ID NOs: 3, 5, 7, 8, 9, and 10, respectively; (2) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences represented by SEQ ID NOs: 3, 4, 7, 8, 9, and 10, respectively; (3) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences represented by SEQ ID NOs: 3, 6, 7, 8, 9, and 10, respectively; (4) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 20, 24, 25, 26, and 27, respectively; (5) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 21, 24, 25, 26, and 27, respectively; (6) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 22, 24, 25, 26, and 27, respectively; (7) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 23, 24, 25, 26, and 27, respectively; (8) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 20, 24, 25, 26, and 28, respectively; (9) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 21, 24, 25, 26, and 28, respectively; (10) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 22, 24, 25, 26, and 28, respectively; (11) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 19, 23, 24, 25, 26, and 28, respectively; (12) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences represented by SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (13) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences represented by SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively; or (14) An antibody or antigen-binding fragment thereof, wherein heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 53, 54, 55, 56, 57, and 58, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

3. the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (1) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 11 or 16, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 11 or 16, (2) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 12 or 17, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 12 or 17, (3) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 13 or 17, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 17, (4) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 14 or 17, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 14 or 17, (5) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 15 or 17, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 15 or 17, (6) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 12 or 18, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 12 or 18, (7) The heavy chain variable region and the light chain variable region each contain an amino acid sequence set forth in SEQ ID NO: 13 or 18, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 18, (8) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 14 or 18, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 14 or 18, (9) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 15 or 18, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 15 or 18, (10) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 29 or 34, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 29 or 34, (11) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 30 or 35, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 30 or 35, (12) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 31 or 35, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 31 or 35, (13) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 32 or 35, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 32 or 35, (14) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 33 or 35, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 33 or 35, (15) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 30 or 36, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 30 or 36, (16) The heavy chain variable region and the light chain variable region each comprise an amino acid sequence set forth in SEQ ID NO: 31 or 36, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 31 or 36, (17) The heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth in SEQ ID NOs: 32 and 36, respectively, or amino acid sequences having at least 80% identity to the amino acid sequences set forth in SEQ ID NOs: 32 and 36, (18) The heavy chain variable region and the light chain variable region each comprise the amino acid sequence set forth in SEQ ID NO: 33 or 36, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 33 or 36, (19) The heavy chain variable region and the light chain variable region each comprise the amino acid sequence set forth in SEQ ID NO: 43 or 44, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 43 or 44, (20) The heavy chain variable region and the light chain variable region each comprise the amino acid sequence set forth in SEQ ID NO: 51 or 52, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 51 or 52, respectively; or (21) The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth in SEQ ID NOs: 59 and 60, respectively, or amino acid sequences having at least 80% identity to the amino acid sequences set forth in SEQ ID NOs: 59 and 60.

4. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is an IgG1, IgG2, or IgG4 isotype.

5. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 61 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to the amino acid sequence set forth in SEQ ID NO: 61, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 62 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to the amino acid sequence set forth in SEQ ID NO:

62.

6. The antibody or antigen-binding fragment thereof (a) binds to human CLDN18.2, (b) induces antibody-dependent cellular cytotoxicity (ADCC) activity against cells expressing CLDN18.2, and / or (c) induces complement-dependent cytotoxicity (CDC) activity against cells expressing CLDN18.

2. The antibody or antigen-binding fragment thereof of claim 1.

7. The antibody or antigen-binding fragment thereof may be a monoclonal antibody, a monospecific antibody, a nanobody, a Fab fragment, a F(ab') ２ 2. The antibody or antigen-binding fragment thereof of claim 1, which is selected from a fragment, an Fd fragment, an Fv fragment, a dAb fragment, an isolated CDR region, a single chain Fv molecule, or a combination thereof.

8. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid molecule described in claim 8.

10. A host cell comprising the nucleic acid molecule described in claim 8 or an expression vector containing the nucleic acid molecule.

11. A multispecific antibody comprising an antibody or an antigen-binding fragment thereof described in any one of claims 1 to 7.

12. A recombinant polypeptide or fusion protein comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7 and a functional fragment connected thereto.

13. An immunoconjugate comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7 and a therapeutic agent connected thereto.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 7 and one or more pharmaceutically acceptable carriers or excipients.

15. A pharmaceutical for preventing, alleviating, or treating a disease associated with CLDN18.2 positivity in a subject in need thereof, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof.

16. 16. A pharmaceutical for preventing, alleviating or treating a disease associated with CLDN18.2 positivity in a subject in need thereof according to claim 15, wherein the disease associated with CLDN18.2 positivity includes cancer.

17. 17. The cancer includes gastric cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, liver cancer, lung cancer, bronchial cancer, mesothelioma, kidney cancer, ovarian cancer, breast cancer, bladder cancer, uterine cancer, prostate cancer, testicular cancer, anal cancer, vaginal cancer, bile duct cancer, gallbladder cancer, head and neck cancer, spinal tumors, otorhinolaryngological tumors, thyroid cancer, mesothelioma, bone cancer, skin cancer, melanoma, adenocarcinoma, sarcoma, neuroblastoma, glioblastoma, brain cancer, central nervous system cancer, neuroendocrine tumors, leukemia, lymphoma, myeloma, 18. A pharmaceutical for preventing, alleviating, or treating a disease associated with CLDN18.2 positivity in a subject in need thereof according to claim 16.

18. A pharmaceutical agent for preventing, alleviating or treating a disease associated with CLDN18.2 positivity in a subject in need thereof as described in claim 16, wherein the cancer is pre-invasive, metastatic, recurrent, and / or refractory.

19. A pharmaceutical agent for preventing, alleviating or treating a disease associated with CLDN18.2 positivity in a subject in need thereof as described in claim 16, when administered in combination with one or more other therapeutic agents.

20. A pharmaceutical for preventing, alleviating, or treating a disease associated with CLDN18.2 positivity in a subject in need thereof as described in claim 19, wherein the other therapeutic agent includes one selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, a CTLA-4 antibody, a cytokine, a drug that stimulates γδ T cells, and a chemotherapeutic agent.

21. A method for producing an anti-CLDN18.2 antibody or its antigen-binding fragment described in any one of claims 1 to 7, comprising the steps of: (i) expressing the anti-CLDN18.2 antibody or its antigen-binding fragment in a host cell; and (ii) isolating the anti-CLDN18.2 antibody or its antigen-binding fragment from the host cell or a cell culture thereof.

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