Anti-CLDN-18.2 antibody and its uses

Abstract

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CLDN-18.2, and compositions comprising the same. The present invention also provides nucleic acid molecules encoding the antibody or antigen-binding fragment thereof, expression vectors and host cells for expressing the antibody or antigen-binding fragment thereof, and therapeutic or diagnostic methods and uses of the antibody or antigen-binding fragment thereof.

Description

[Technical Field]

[0001] The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CLDN-18.2, and compositions comprising the same. The present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, expression vectors and host cells for expressing the antibodies or antigen-binding fragments thereof, and therapeutic or diagnostic methods and uses of the antibodies or antigen-binding fragments thereof. [Background technology]

[0002] Gastric cancer is one of the most common cancers worldwide. According to statistics from the World Health Organization's Cancer Control Project, 7 million cancer patients die each year worldwide, of which 700,000 are gastric cancer patients. Compared to conventional gastric cancer treatments, antibody-based therapies have high specificity and low side effects, making them highly applicable.

[0003] Claudins, also known as CLDNs, are a family of cell surface proteins that establish the paracellular barrier and regulate the flow of intercellular molecules. At least 26 members have been identified to date. Members of the claudin protein family are tightly linked structural elements that play important roles in maintaining epithelial cell polarity, controlling paracellular diffusion, and regulating cell proliferation and differentiation. Claudin molecules span the cell membrane four times, with both their N- and C-termini located in the cytoplasm. Different claudin members are expressed in different tissues, and alterations in their function are associated with cancer formation. Altered expression levels of claudin 1, claudin 18, and claudin 10 have been associated with intestinal cancer, gastric cancer, and hepatocellular carcinoma, respectively.

[0004] Claudin 18 (CLDN18) has two alternative splicing variants, CLDN-18.1 and CLDN-18.2. Claudin 18.1 (CLDN-18.1) is selectively expressed in normal lung and gastric epithelium. Claudin 18.2 (CLDN-18.2) is expressed at low levels in normal short-lived gastric epithelial cells. However, Claudin 18.2 is highly expressed in tumor cells across a variety of cancer types. For example, Claudin 18.2 is highly expressed in 75% of gastric cancer patients, 50% of pancreatic cancer patients, and 30% of esophageal cancer patients. Claudin 18.2 is also highly expressed in lung cancer and other cancers.

[0005] Claudiximab (IMAB362), developed by Ganymed, is currently undergoing phase II clinical trials for late-stage gastroesophageal cancer (WO2007059997). However, there remains a need for novel anti-CLDN18.2 antibodies that are improved in terms of affinity, specificity, etc. compared to known antibodies. Summary of the Invention

[0006] The present invention provides an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof that has advantages such as high affinity and high specificity for human CLDN-18.2. The anti-CLDN-18.2 antibody or an antigen-binding fragment thereof according to the present invention can be used in the treatment of cancer, etc., as an independent therapy or in combination with other therapies and / or other anti-cancer agents.

[0007] In one aspect, the invention provides an anti-CLDN-18.2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein: the sequence of said HCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs: 1, 10, 16, 22, 28, 34 and 37; the HCDR2 sequence is selected from the amino acid sequences shown in SEQ ID NO: 62, 63, 17, 23, 29, 35, 38, 72 and 74, wherein in the HCDR2 shown in SEQ ID NO: 62, X1 is C or S, and X2 is T or S; and in the HCDR2 shown in SEQ ID NO: 63, X3 is D or G, and X4 is K or T; the sequence of said HCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs: 3, 12, 18, 24, 30, 36, 39, 73 and 75; The light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein: the LCDR1 sequence is selected from the amino acid sequences set forth in SEQ ID NOs: 4, 7, 13, 19, 25 and 31; the LCDR2 sequence is selected from the amino acid sequences set forth in SEQ ID NOs: 5, 8, 14, 20, 26 and 32; The LCDR3 sequence is selected from the amino acid sequences shown in SEQ ID NOs: 6, 9, 15, 21, 27 and 33.

[0008] In some embodiments, the heavy chain variable region according to the invention comprises: (I) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; or (II) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; or (III) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively; or (IV) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively; or (V) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively; or (VI) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; or (VII) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, respectively; or (VIII) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO: 10, SEQ ID NO: 40 and SEQ ID NO: 12, respectively; or (IX) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:1, SEQ ID NO:41 and SEQ ID NO:3, respectively; or (X) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:1, SEQ ID NO:72 and SEQ ID NO:73, respectively; or (XI) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:1, SEQ ID NO:72 and SEQ ID NO:3, respectively; or (XII) HCDR1, HCDR2 and HCDR3, whose amino acid sequences are set forth in SEQ ID NO: 28, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; or (XIII) the amino acid sequences of which comprise HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 28, SEQ ID NO: 74, and SEQ ID NO: 30, respectively; The light chain variable region (I) LCDR1, LCDR2 and LCDR3, whose amino acid sequences are set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or (II) LCDR1, LCDR2 and LCDR3, whose amino acid sequences are set forth in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or (III) LCDR1, LCDR2 and LCDR3, whose amino acid sequences are set forth in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively; or (IV) LCDR1, LCDR2 and LCDR3, whose amino acid sequences are set forth in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively; or (V) LCDR1, LCDR2 and LCDR3, whose amino acid sequences are set forth in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively; or (VI) comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (I) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or (II) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or (III) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or (IV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or (V) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; or (VI) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or (VII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or (VIII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; or (IX) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or (X) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively; or (XI) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or (XII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 10, SEQ ID NO: 40, and SEQ ID NO: 12, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or (XIII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 1, SEQ ID NO: 41, and SEQ ID NO: 3, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; or (XIV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 1, SEQ ID NO: 72, and SEQ ID NO: 73, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; or (XV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO:1, SEQ ID NO:72, and SEQ ID NO:3, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or (XVI) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 28, SEQ ID NO: 74, and SEQ ID NO: 30, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or (XVII) A heavy chain variable region including HCDR1, HCDR2, and HCDR3, whose amino acid sequences are set forth in SEQ ID NO:28, SEQ ID NO:74, and SEQ ID NO:75, respectively, and a light chain variable region including LCDR1, LCDR2, and LCDR3, whose amino acid sequences are set forth in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region described herein comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 42, 45, 47, 49, 51, 53, 54, 76, 78, 82, and 83, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of the sequences set forth in SEQ ID NOs: 42, 45, 47, 49, 51, 53, 54, 76, 78, 82, and 83; The amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 43, 44, 46, 48, 50, and 52, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of the sequences set forth in SEQ ID NOs: 43, 44, 46, 48, 50, and 52.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (I) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 42 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 43, or (II) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 42 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 44, or (III) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 45 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 46, or (IV) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 47 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 48, or (V) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 49 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 50; or (VI) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 51 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 52, or (VII) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 53 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 50, or (VIII) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 54 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 52, or (IX) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 45 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 48, or (X) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 51 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 46, or (XI) A heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO:49 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO:46.

[0012] In some embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 55, 57, 58, 59, 60, 76, 78, 79, 82, and 83, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of the sequences set forth in SEQ ID NOs: 55, 57, 58, 59, 60, 76, 78, 79, 82, and 83; The light chain variable region amino acid sequence comprises an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 56, 61, 77, 80, and 81, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth in SEQ ID NOs: 56, 61, 77, 80, and 81.

[0013] In some embodiments, an antibody or antigen-binding fragment thereof described in the present invention 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 any one of SEQ ID NOs: 55, 57, 60, 76, 78, 79, 82, or 83, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 56, 61, 77, 80, or 81.

[0014] In some embodiments, an antibody or antigen-binding fragment thereof described in the present invention 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:55, 57, 82, or 83, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:56.

[0015] In some embodiments, an antibody or antigen-binding fragment thereof described in the present invention 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:60, 76, or 78, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61.

[0016] In some embodiments, an antibody or antigen-binding fragment thereof according to the invention 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:55 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:56; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:56; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:58 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:56; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:56; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:60 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:76 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:77; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:79 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:80; or The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:81. In some embodiments, the antibody or antigen-binding fragment thereof described in the invention comprises a heavy chain having an amino acid sequence set forth in SEQ ID NO:64 or a variant thereof or SEQ ID NO:68 or a variant thereof, and a light chain having an amino acid sequence set forth in SEQ ID NO:65 or a variant thereof or SEQ ID NO:69 or a variant thereof, wherein the variants comprise 1, 2, 3, 4, or 5 amino acid changes in the variable region; Preferably, the variant of SEQ ID NO:64 comprises an amino acid change at position 50 or 63 or a combination thereof, and / or the variant of SEQ ID NO:68 comprises an amino acid change at position 63 or 65 or a combination thereof; Preferably, the variant of SEQ ID NO:64 comprises S50C or S63T or a combination thereof, and / or the variant of SEQ ID NO:68 comprises G63D or T65K or a combination thereof.

[0017] In some embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain and a light chain, wherein: the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:64 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:64, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:65 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:65; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:68, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:68, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:69; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:84 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:84, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:85 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:85; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:86, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:86, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:87, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:87; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:88, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:88, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:89, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:89; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:90 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:90, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:91 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:91; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:92, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:92, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:93, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:93; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:94 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:94, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:95 or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:95; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:96, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:96, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:97, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:97; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:98, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:98, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:99, or an amino acid sequence with at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:99; or The heavy chain comprises the amino acid sequence set forth in SEQ ID NO:100 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:100, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:101 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:101.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (I) a heavy chain having an amino acid sequence set forth in SEQ ID NO: 64 and a light chain having an amino acid sequence set forth in SEQ ID NO: 65, or (II) A heavy chain having the amino acid sequence set forth in SEQ ID NO:68 and a light chain having the amino acid sequence set forth in SEQ ID NO:69.

[0019] In some embodiments, an antibody according to the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0020] In some embodiments, the antigen-binding fragment according to the invention is a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv or sdAb or a diabody.

[0021] In some embodiments, the antibody according to the invention is of any IgG subtype, for example, IgG1, IgG2, IgG3 or IgG4, and preferably said antibody is hypofucosylated or afucosylated.

[0022] In some embodiments, the antibodies according to the invention are hypofucosylated.

[0023] In some embodiments, the antibodies according to the invention are afucosylated.

[0024] In another aspect, the present invention provides an isolated anti-CLDN-18.2 antibody or antigen-binding fragment thereof, (1) binds to the same epitope on the human CLDN-18.2 protein as, or a complete or partial overlap with, an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein; and (2) competes with an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein for binding to an epitope on human CLDN-18.2 protein; and (3) binds to human CLDN-18.2 protein but does not bind to human CLDN-18.1 protein; (4) Inducing ADCC effect in cells expressing human CLDN-18.2 protein; and (5) Inducing CDC effect in cells expressing human CLDN-18.2 protein.

[0025] In yet another aspect, the invention provides polynucleotides encoding the anti-CLDN-18.2 antibodies or antigen-binding fragments thereof described herein.

[0026] In yet another aspect, the present invention provides an expression vector comprising a polynucleotide described herein, preferably said vector is a eukaryotic expression vector.

[0027] In yet another aspect, the present invention provides a host cell comprising a polynucleotide described herein or an expression vector described herein, or expressing an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described in the present invention, preferably wherein the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0028] In yet another aspect, the invention provides a method for producing an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein, the method comprising culturing a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0029] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, or a host cell described herein, and a pharmaceutically acceptable vector or excipient.

[0030] In yet another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein in the production of a medicament for treating and / or preventing a disease or condition mediated by CLDN-18.2, preferably, the disease or condition is cancer.

[0031] In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein for treating and / or preventing a disease or condition mediated by CLDN-18.2, preferably wherein the disease or condition is cancer.

[0032] In yet another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by CLDN-18.2, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, wherein preferably the disease or condition is cancer.

[0033] In some embodiments, the cancer is selected from gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, intestinal cancer and bladder cancer.

[0034] In yet another aspect, the invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0035] In yet another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, preferably further comprising an administration device.

[0036] In yet another aspect, the invention provides methods for detecting the presence of CLDN-18.2 in a sample using the antibodies or antigen-binding fragments thereof described herein. [Brief explanation of the drawings]

[0037] [Figure 1] 1 shows the cellular affinity of chimeric anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 2] 2a and 2b show the ADCC activity of chimeric anti-CLDN-18.2 antibodies measured by a reporter gene. [Figure 3] CDC activity of chimeric anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 4a] 1 shows the cellular affinity of humanized anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 4b] 1 shows the cellular affinity of humanized anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 4c] 1 shows the cellular affinity of humanized anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 5a] 1 shows the ADCC activity of humanized anti-CLDN-18.2 antibodies measured by a reporter gene. [Figure 5b] 1 shows the ADCC activity of humanized anti-CLDN-18.2 antibodies measured by a reporter gene. [Figure 5c] 1 shows the ADCC activity of humanized anti-CLDN-18.2 antibodies measured by a reporter gene. [Figure 6a] 1 shows the CDC activity of humanized anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 6b] 1 shows the CDC activity of humanized anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 6c] 1 shows the CDC activity of humanized anti-CLDN-18.2 antibodies measured by flow cytometry. [Figure 7] 1 shows the inhibitory effect of humanized anti-CLDN-18.2 antibodies on the growth of human gastric cancer MKN45 hClaudin18.2 Mixeno tumors transplanted into M-NSG mice. [Figure 8]1 shows the inhibitory effect of humanized anti-CLDN-18.2 antibody on the growth of human pancreatic cancer hCLDN18.2 MIA PaCa-2 tumors transplanted into CB-17 SCID mice. DETAILED DESCRIPTION OF THE INVENTION

[0038] definition The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0039] In order to make the present invention easier to understand, some technical terms are specifically defined as follows. All scientific and technical terms used herein have the meanings commonly understood by those skilled in the art of the present invention, unless specifically defined elsewhere in this specification. For definitions and terms in the art, those skilled in the art can specifically refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used in this specification (including the claims), singular forms include the corresponding plural forms unless the context clearly dictates otherwise.

[0040] The term "about," when used in conjunction with a number or value, is meant to cover a range of numbers or values from a lower limit of 5% less than the specified number or value to an upper limit of 5% greater than the specified number or value.

[0041] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0042] The term "CLDN-18.2" or "Claudin 18.2" refers to one of the two splice variants of Claudin 18. The term refers to any naturally occurring CLDN-18.2 from any vertebrate (including mammals such as primates (e.g., humans)) and rodents (e.g., mice and rats) unless otherwise specified. The term covers "full-length," unprocessed CLDN-18.2 and any form of CLDN-18.2 produced by intracellular processing, or any fragment thereof. The term further includes naturally occurring variants of CLDN-18.2, such as splice variants or allelic variants. In a preferred embodiment, CLDN-18.2 refers to full-length CLDN-18.2 from humans and cynomolgus monkeys, or a fragment thereof (e.g., a mature fragment lacking its signal peptide).

[0043] The term "percent (%) amino acid sequence identity" or abbreviated "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to those in a reference amino acid sequence when the amino acid sequences are aligned (introducing gaps, if necessary) to achieve the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Sequence alignment can be performed using various methods known in the art to measure percent amino acid sequence identity, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0044] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver to selectively damage, destroy, or remove from the body invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in autoimmune or pathological inflammatory conditions.

[0045] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship, usually triggered by a protein-protein interaction, such as the binding of a growth factor to a receptor, that results in signal transduction from one part of a cell to another part of the cell. Generally, transduction involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that triggers signal transduction. The penultimate process usually involves a nuclear event, thereby resulting in a change in gene expression.

[0046] The terms "activity" or "biological activity," or "biological property" or "biological characteristic," are used interchangeably herein and refer to epitope / antigen affinity and specificity, the ability to neutralize or antagonize CLDN-18.2 activity in vivo or in vitro, IC 50 These include, but are not limited to, the antibody's in vivo stability and its immunogenicity. Other identifiable biological properties or characteristics of antibodies that are well known in the art include, for example, cross-reactivity (i.e., cross-reactivity, usually with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high expression levels of the protein in mammalian cells. The above-mentioned properties or characteristics can be observed, measured, or assessed using techniques well known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasma resonance analysis, open-ended neutralization assays in vitro or in vivo, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections of different origins (including human, primate, or any other origin).

[0047] The term "antibody" refers to any form of antibody having the requisite biological activity, and as such is used in the broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies of camel origin.

[0048] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biological molecules, e.g., nucleic acids, proteins, lipids, sugars, or other materials, such as cell debris and growth medium. The term "isolated" does not refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless they are present in amounts that would clearly interfere with the experimental or therapeutic use of the binding compounds described herein.

[0049] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.

[0050] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally occurring, comprises four peptide chains: two heavy (H) chains (total length approximately 50-70 kDa) and two light (L) chains (total length approximately 25 kDa) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions may be further subdivided into highly variable complementarity-determining regions (CDRs) and separate, more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0051] The term "Fc region" is intended to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Numbering of amino acid residues in the Fc region or constant region, unless otherwise specified herein, is based on the EU numbering system, also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0052] The term "antigen-binding fragment" of an antibody ("parent antibody") includes antibody fragments or derivatives, typically comprising at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody, which retain at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as sc-Fv, nanobodies composed of antibody fragments, and multispecific antibodies. When the antigen-binding activity of an antibody is expressed on a molar basis, a binding fragment or derivative typically retains at least 10% of the antigen-binding activity of the antibody. Preferably, a binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that an antigen-binding fragment of an antibody may contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of an antibody) that do not significantly alter its biological activity. The term "binding compound" refers to both antibodies and binding fragments thereof.

[0053] The terms "single-chain Fv" or "scFv" antibody refer to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the required structure for antigen binding.

[0054] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only a heavy or light chain variable region. In some cases, two or more VH regions and a peptide linker are covalently linked to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0055] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody may be bispecific.

[0056] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, said fragment comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains can pair with the complementary domains of another chain, producing two antigen-binding sites.

[0057] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, where the first and second antibodies are from different species. Typically, the variable domains are derived from an antibody (the "parent antibody"), such as a rodent, while the constant domain sequences are derived from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0058] The term "humanized antibody" refers to antibody forms containing sequences derived from human and non-human (e.g., murine, rat) antibodies. Generally, humanized antibodies contain at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, while all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally also contain at least a portion of a human immunoglobulin constant region (Fc).

[0059] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. In a mouse, a fully human antibody may contain mouse glycosylation if produced in a mouse cell or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Or, if produced in a rat, a rat cell, or a hybridoma derived from a rat cell, a fully human antibody may contain rat glycosylation. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0060] An "isotype" of an antibody refers to the antibody class (e.g., IgM, IgE, IgG, such as IgG1, IgG2, or IgG4) conferred by the heavy chain constant region genes. Isotypes also include modified forms of one of these classes, where modifications have been produced to alter Fc function, e.g., to enhance or weaken effector function or binding to an Fc receptor.

[0061] The term "epitope" refers to the region of an antigen bound by an antibody. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0062] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y is typically determined by the dissociation and association rate constants (k dis and kon The equilibrium dissociation constant (K D ) Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0063] The term "non-binding" protein or cell refers to a protein or cell that does not bind or does not bind with high affinity, i.e., the K that binds to the protein or cell. D is 1.0 x 10 -6 M or more, and more preferably 1.0 × 10 -5 M or more, and more preferably 1.0 × 10 -4 M or more, 1.0 × 10 -3 M or more, and more preferably 1.0 × 10 -2 M or above.

[0064] The term "high affinity" refers to the K D is 1.0×10 -6 M or less, preferably 5.0 x 10 -8 M or less, and more preferably 1.0 × 10 -8 M or less, 5.0 × 10 -9 M or less, and more preferably 1.0 × 10 -9 For other antibody subtypes, "high affinity" binding can vary. For example, "high affinity" binding for IgM subtypes is defined as a K D is 10 -6 M or less, preferably 10 -7 M or less, and more preferably 10 -8 It means that it is less than M.

[0065] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to cell-mediated immune defense in which immune system effector cells actively lyse target cells, e.g., cancer cells, whose cell membrane surface antigens are bound by antibodies, e.g., Claudin18.2 antibodies.

[0066] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies that, upon binding to surface antigens, trigger the classic complement pathway, including membrane attack complex formation and target cell lysis. The antibodies of the present invention, upon binding to Claudin 18.2, trigger CDC against cancer cells.

[0067] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see U.S. Pat. No. 8,278,036 to Kariko et al., which disclosed mRNA molecules in which uridine is replaced with pseudouridine, methods for synthesizing said mRNA molecules, and methods for in vivo delivery of therapeutic proteins). Unless otherwise specified, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, and the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0068] "Construct" refers to any recombinant polynucleotide molecule (e.g., a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from any source, which may constitute the following polynucleotide molecules by genomic integration or autonomous replication, of which it is already linked in a functionally operating manner (i.e., operably linked) to one or more polynucleotide molecules. Recombinant constructs typically comprise a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell. Expression of a nucleic acid of the invention can be induced using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0069] A "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA region segments can be ligated. Another type of vector is a viral vector, in which additional DNA region segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and free mammalian vectors). After introduction into a host cell, other vectors (e.g., non-free mammalian vectors) are integrated into the genome of the host cell and are therefore replicated along with the host genome. Additionally, some vectors can direct the expression of genes to which they are operably linked. Herein, such vectors are referred to as "expression vectors."

[0070] As used herein, the term "expression vector" refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if necessary, to provide for propagation within the host.

[0071] "Activation," "stimulation," and "treatment" for a cell or receptor may have the same meaning, e.g., activation, stimulation, or treatment of a cell or receptor by a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes binding compounds derived from natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and antibodies. "Ligand" further includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cellular activation regulated by internal mechanisms and external or environmental factors. "Responses," e.g., responses of cells, tissues, organs, or organisms, include alterations in biochemical or physiological behavior (e.g., concentration, density, adhesion, or migration within a biological compartment, gene expression rate, or differentiation state), where the alterations are related to activation, stimulation, or treatment, or are related to internal mechanisms, e.g., genetic programming.

[0072] As used herein, the term "treatment" or "medical treatment" of any disease or condition refers, in one embodiment, to ameliorating the disease or condition (i.e., slowing, arresting, or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "medical treatment" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be discernible to the patient. In another embodiment, "treatment" or "medical treatment" refers to modulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for assessing the treatment and / or prevention of a disease are generally commonly known in the art unless expressly set forth herein.

[0073] A "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the terms "cyno" or "cynomolgus monkey" refer to cynomolgus monkeys.

[0074] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0075] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the CLDN-18.2 antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, effectively prevents or ameliorates the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose further refers to the amount of the antibody or antigen-binding fragment thereof sufficient to lead to an improvement in symptoms, e.g., an amount that treats, cures, prevents, or ameliorates the associated medical condition, or increases the rate of treatment, cure, prevention, or amelioration of such a condition. When an individual is administered a single active ingredient, the therapeutically effective dose refers to that ingredient alone. In the case of combined administration, the therapeutically effective dose refers to the combined amount of active ingredients that leads to a therapeutic effect, regardless of combined, sequential, or simultaneous administration. An effective amount of a therapeutic agent can improve a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0076] "Cancer" and "cancerous" refer to or describe a physiological disorder in a mammal, typically characterized by uncontrolled cell growth. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers are squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer. , liver cancer, prostate cancer, vulvar cancer, thyroid cancer, cancer of the liver, and various types of head and neck cancer, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky storage diseases These include: NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's hypergammaglobulinemia; chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD); and abnormal blood vessel growth associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0077] Anti-CLDN-18.2 antibody In one aspect, the present invention provides an anti-CLDN-18.2 antibody or antigen-binding fragment thereof. The terms "anti-CLDN-18.2 antibody," "anti-CLDN-18.2," "CLDN-18.2 antibody," or "antibody that binds to CLDN-18.2" refer to an antibody that can bind to the CLDN-18.2 protein or a fragment thereof with sufficient affinity such that it can be used as a diagnostic and / or therapeutic agent targeting CLDN-18.2.

[0078] The antibodies of the present invention can be produced using any suitable method for producing antibodies. Any suitable form of CLDN-18.2 can be used as an immunogen (antigen) to produce antibodies. By way of example and not limitation, any CLDN-18.2 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing mouse monoclonal anti-human CLDN-18.2 antibodies can be produced by methods well known in the art. Antibodies derived from rodents (e.g., mice) can cause undesirable antibody immunogenicity when used as therapeutic drugs in vivo, and repeated use can cause an immune response against the therapeutic antibody in the human body. Such an immune response can result in at least a loss of therapeutic efficacy and, in severe cases, potentially fatal allergic reactions. One method for reducing the immunogenicity of rodent antibodies involves producing chimeric antibodies, in which mouse variable regions are fused with human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, preservation of the complete rodent variable region in a chimeric antibody may still cause adverse immunogenicity in patients. Grafting of complementarity-determining region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has already been used to further minimize rodent sequences (Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0079] In some embodiments, chimeric or humanized antibodies of the invention can be made based on the sequences of mouse monoclonal hybridoma antibodies produced. DNA encoding heavy and light immunoglobulin chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to include non-mouse (e.g., human) immunoglobulin sequences.

[0080] In some embodiments, chimeric CLDN-18.2 antibodies described herein can be produced by effectively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant regions included in the chimeric antibodies of the present invention can be selected from any human IgG subtype, e.g., IgG1, IgG2, IgG3, or IgG4, preferably IgG4.

[0081] In some embodiments, chimeric CLDN-18.2 antibodies of the present invention can be obtained by transfecting expression cells with expression plasmids for the chimeric light chain and chimeric heavy chain in a "mixed and matched" manner, and the CLDN-18.2 binding of such "mixed and matched" antibodies can be tested in the above binding assays and other common binding assays (e.g., ELISA).

[0082] The precise amino acid sequence boundaries of the variable region CDRs of the antibody of the present invention can be determined using any of a number of well-known schemes, including Chothia, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat, which is based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and affinity propagation clustering, which utilizes multiple crystal structures. The CDRs of the antibodies of the present invention may be defined by those skilled in the art based on any scheme known in the art (e.g., different assignment systems or combinations).

[0083] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different allocation systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different allocation systems will be different. Therefore, when an antibody is defined by the specific CDR sequences defined in the present invention, the scope of the antibody further covers the following antibodies: whose variable region sequences contain the specific CDR sequences described above, but whose described CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of a different scheme (e.g., a different allocation system or combination).

[0084] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, while CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. A "minimum binding unit" for antigen binding can be provided by determining the minimal overlap region using at least two of the Kabat, Chothia, AbM, Contact, and North methods. The minimum binding unit may be a subpart of a CDR. As will be apparent to those skilled in the art, residues in other portions of the CDR sequence may be determined depending on the antibody structure and protein folding. Therefore, the present invention also contemplates variants of any CDR presented herein. For example, in one CDR variant, the amino acid residues of the minimum binding unit may remain unchanged, while other CDR residues defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0085] Humanized antibodies according to the invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, see U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0086] In some embodiments, the amino acid changes comprise amino acid deletions, insertions, or substitutions. In some embodiments, the anti-CLDN-18.2 antibodies or antigen-binding fragments thereof of the present invention include antibodies that have been mutated by amino acid deletions, insertions, or substitutions, but still have an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the antibodies described above (particularly in the CDR regions set forth in the sequences above). In some embodiments, the antibodies of the present invention have no more than 1, 2, 3, 4, or 5 amino acids mutated by amino acid deletions, insertions, or substitutions in the CDR regions compared to the CDR regions set forth in the specific sequences.

[0087] In some embodiments, polynucleotides encoding antibodies of the invention include polynucleotides that have been mutated by nucleotide deletion, insertion or substitution, but still have at least about 60, 70, 80, 90, 95, or 100% identity to the coding regions corresponding to the CDRs set forth in the sequences above.

[0088] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0089] In some embodiments, it may be desirable to produce cysteine engineered antibodies, e.g., "ThioMAbs," in which one or more residues of the antibody are substituted with cysteine residues.

[0090] In some embodiments, antibodies can be modified to increase or decrease their degree of glycosylation and / or alter their glycosylation pattern. Addition or deletion of glycosylation sites in an antibody can be readily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites. For example, glycosylation at one or more glycosylation sites can be eliminated by making one or more amino acid substitutions to eliminate those sites. Antibodies with altered types of glycosylation can be generated, for example, hypo- or non-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to improve the ADCC ability of antibodies.

[0091] In some preferred embodiments, the present invention provides antibodies as follows, which are hypofucosylated or afucosylated, thereby significantly increasing the binding affinity of the antibody to a receptor expressed on an effector cell, thereby resulting in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The amount of fucose can be determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans linked to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to an aspartic acid residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acid positions upstream or downstream of position 297, i.e., positions 294 to 300. See, e.g., US2003 / 0157108; US2004 / 0093621. Such antibody variants can be produced in cell lines capable of producing defucosylated or hypofucosylated antibodies. Examples of such cells include Lecl3 CHO cells, which are deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986):533-545, US2003 / 0157108). In some embodiments, fucosidases are used to cleave off fucose residues on the antibody. In some embodiments, glycoregulators are used to control the fucose residues on the antibody. The glycoregulator can be CDFS01, available from Shanghai Aupumai Biotechnology Co., Ltd. Defucosylation can be performed by common methods well known in the art.

[0092] The level of antibody fucosylation can be defined structurally. As described herein, "nonfucosylated" or "afucosylated" refers to an antibody having a fucose content of less than 5%, for example, about 0%, less than 1%, less than 2%, less than 3%, or less than 4%. The term "low fucosylation" refers to an antibody having a fucose content of at least about 5% but less than 30%, for example, an antibody having a fucose content of about 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 10% to 20%, 20% to 30%, or 10% to 30%. The term "low fucosylated or afucosylated" refers to an antibody having a fucose content of less than 30%.

[0093] In some embodiments, the antibodies provided herein may be further modified to contain other nonproteinaceous moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol polymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0094] Antibody expression In yet another aspect, the invention provides a polynucleotide encoding an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein, which may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide encoding the amino acid sequence of the light chain and / or the heavy chain of the antibody.

[0095] In yet another aspect, the invention provides expression vectors comprising the polynucleotides described herein, preferably wherein the vectors are eukaryotic expression vectors. In some embodiments, the polynucleotides described herein are comprised in one or more expression vectors.

[0096] In yet another aspect, the present invention provides a host cell comprising a polynucleotide described herein or an expression vector described herein, preferably wherein the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0097] In yet another aspect, the present invention provides a method for producing an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0098] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including several immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NS0, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.

[0099] In one embodiment, the invention provides a method for producing an anti-CLDN-18.2 antibody, the method comprising introducing an expression vector into mammalian host cells and culturing the host cells for a period of time sufficient to allow expression of the antibody within the host cells, or more preferably, to allow secretion of the antibody into the medium in which the host cells are grown, to produce the antibody. The antibody can be recovered from the medium using standard protein purification methods.

[0100] Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules herein or comprising the amino acid sequences herein are components of the present invention, regardless of the glycosylation of the antibody. Similarly, in some embodiments, non-fucosylated antibodies are advantageous because they typically have stronger potency in vitro and in vivo than their fucosylated counterparts and are less likely to be immunogenic because their glycostructure is a normal component of native human serum IgG.

[0101] Drug Compositions and Drug Formulations In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-CLDN-18.2 antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, and a pharmaceutically acceptable vector or excipient.

[0102] It should be understood that the anti-CLDN-18.2 antibody or pharmaceutical composition thereof according to the present invention can be incorporated into a formulation and administered in combination with appropriate vectors, excipients and other reagents to provide improved metastasis, delivery, tolerance, etc.

[0103] The term "drug composition" refers to a formulation that allows the active ingredient contained therein to be present in a biologically effective form and that does not contain other ingredients that have unacceptable toxicity to the subject to whom said formulation is administered.

[0104] Pharmaceutical formulations containing the anti-CLDN-18.2 antibodies described herein can be prepared by mixing the anti-CLDN-18.2 antibodies of the present invention having the desired purity with one or more optional pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0105] The pharmaceutical compositions or formulations of the present invention may further comprise one or more other active ingredients as needed for the particular indication being treated, preferably with those active ingredients having complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known antitumor or anticancer agents, and the active ingredients are suitably present in combination in amounts effective for the intended purpose. In some embodiments, the pharmaceutical compositions of the present invention further comprise a composition of a polynucleotide encoding an anti-CLDN-18.2 antibody.

[0106] In yet another aspect, the invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0107] In yet another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, preferably further comprising an administration device.

[0108] Medical uses In yet another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein in the production of a medicament for treating and / or preventing a disease or condition mediated by CLDN-18.2, preferably, the disease or condition is cancer.

[0109] In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein for treating and / or preventing a disease or condition mediated by CLDN-18.2, preferably wherein the disease or condition is cancer.

[0110] In yet another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by CLDN-18.2, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein; preferably, the disease or condition is cancer.

[0111] In some embodiments, the cancer is selected from gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, intestinal cancer and bladder cancer.

[0112] In some embodiments, modes of administration of the present invention include, but are not limited to, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intra-articular administration (e.g., in an arthritic joint), administration by inhalation, aerosol delivery, or intratumoral administration.

[0113] In some embodiments, the present invention provides one or more therapies (e.g., therapeutic regimens and / or other therapeutic agents) co-administered to a subject in therapeutically effective amounts, hi some embodiments, the therapies include surgical therapy and / or radiation therapy.

[0114] In some embodiments, the methods or uses according to the present invention further comprise administering to the individual one or more therapies (e.g., therapeutic regimens and / or other therapeutic agents). The antibodies of the present invention can be used alone or in combination with other therapeutic agents in a therapy. For example, they can be co-administered with at least one other therapeutic agent, such as a PD-1 antibody, a PD-L1 antibody, a LAG-3 antibody, and / or a CTLA-4 antibody.

[0115] In one embodiment, the method for treating cancer disease of the present invention further comprises administering an agent for stabilizing or increasing CLDN-18.2 expression. CLDN-18.2 is preferably expressed on the cell surface of cancer cells. The agent for stabilizing or increasing CLDN-18.2 expression may be oxaliplatin and / or 5-FU.

[0116] Methods for diagnosis and detection In yet another aspect, the present invention provides methods for detecting the presence of CLDN-18.2 in a sample using an antibody or antigen-binding fragment thereof described herein. The term "detection," as used herein, includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue.

[0117] The present invention includes all combinations of the specific embodiments enumerated. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description provided below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including references thereto, are hereby incorporated by reference in their entirety for all purposes.

[0118] Example The following examples are presented to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope.

[0119] Example 1: Production of recombinant human CLDN-18.2 (hCLDN-18.2) protein for producing anti-CLDN-18.2 antibodies Plasmid HG20047-U containing the human CLDN-18 cDNA sequence was purchased from Yiqiao Shenzhou Co., Ltd., and a fragment of the human CLDN-18 gene was amplified by PCR using the forward primer 5'-GTACgctagccaccTgaagagcggatccgctcttctTGCCCTGAAATGCATCCGCATTGGCAGC-3' and the reverse primer 5'-GATCgcggccgccctgcaggTTACACATAGTCGTGCTTGGAAGGATAAG-3'. The amplified fragment was double-digested with NheI and SbfI and then cloned into the eukaryotic expression plasmid system (HXP) to obtain the construct HXP-CLDN18P. A part of the gene sequence of hCLDN-18.2 was sent to Nanjing Jinsirui Co., Ltd. for gene synthesis, and the gene sequence was cloned into HXP-CLDN18P by BSPQI digestion, finally obtaining the construct HXP-hCLDN-18.2.

[0120] Example 2: Preparation of mouse hybridoma cells 2.1, Immunized animals The HXP-hCLDN-18.2 obtained in Example 1 was mixed with an equal volume of immunoadjuvant (Freund's adjuvant) and immunized intramuscularly in five 8-week-old female BALB / c mice. For the initial immunization, 100 μg of DNA plasmid per mouse was used. Subsequently, booster immunizations were performed five times, every two or three weeks, using 50 μg of DNA plasmid per mouse. For the final booster immunization, stabilized CHO cells overexpressing CLDN-18.2 were used as a second booster, and 1e+7 cells were injected intraperitoneally into each mouse.

[0121] 2.2, Cell fusion Four days after the final booster immunization, the inguinal lymph nodes, popliteal lymph nodes, and spleens of the mice were removed and crushed in DMEM medium. A rich suspension of lymphocytes was obtained and fused with mouse myeloma cells Sp2 / 0 (ATCC) by conventional electroporation. The fusion products were cultured in DMEM complete medium containing 1:50 HAT (hypoxanthine, methotrexate, and thymidine) for 5 days, and successfully fused cells (i.e., hybridoma cells) were screened. The medium was then replaced with DMEM complete medium containing 1:50 HT (hypoxanthine and thymidine) until screening was complete.

[0122] The DMEM complete medium was formulated as 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U / mL streptomycin + 1:100 OPI (oxaloacetate, pyruvate, and insulin), and the culture chamber conditions were 8% CO2, 37°C.

[0123] Example 3: Screening of mouse hybridoma cells and performance detection of the obtained anti-CLDN-18.2 mouse antibody We screened 3,840 different polyclonal hybridoma cell lines for those expressing antibodies that bind to human CLDN-18.2 protein but not to human CLDN18.1 protein based on cell-level binding activity. After subcloning to obtain monoclonal hybridoma cell lines, we performed functional screening based on in vitro ADCC and CDC. Finally, we obtained seven monoclonal hybridoma cell lines. The corresponding antibodies, 1H17, 2B19, 4G3, 9J24, 9O24, 10L8, and 10N10, specifically bind to human CLDN-18.2 recombinant protein but not to human CLDN-18.1 recombinant protein, and exhibited high ADCC and CDC activity.

[0124] Example 4. Assay of variable region sequences of anti-CLDN-18.2 murine antibodies (expressed according to Kabat or IMGT) A method based on degenerate primer PCR was used to determine the DNA coding sequence corresponding to the variable region of the anti-CLDN-18.2 murine antibody. Candidate hybridoma cells were cultured and harvested by centrifugation at 1000 rpm. Total RNA was extracted with Trizol. First-strand cDNA was synthesized using this as a template, and the corresponding variable region DNA coding sequence was then amplified by PCR using the first-strand cDNA as a subsequent template. The primer sequences used in the amplification reaction were complementary to the first framework region and constant region of the antibody variable region (Larrick, JW et al., 1990, Scand. J. Immunol., 32, 121-128; Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). To a 50 μl reaction mixture, 1 μl of cDNA, 5 μl of 10x PCR buffer, 1 μl each of upstream and downstream primers (25 pmol), 1 μl of dNTPs, 1 μl of 25 mmol / L MgCl2, and 39 μl of HO were added. The mixture was pre-denatured at 95°C for 10 min, followed by the addition of 1 μl of Taq enzyme and temperature cycling for PCR amplification. The reaction conditions were denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and elongation at 72°C for 15 s, for a total of 32 cycles, followed by incubation at 72°C for 10 min. The PCR products were recovered and purified. The amplified products were sequenced to obtain the amino acid sequences of the heavy and light chain variable regions of the anti-CLDN-18.2 mouse antibody.

[0125] The NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ) was used to search for shared sequences in the germline and rearranged Ig variable region sequence database. The complementarity-determining region (CDR) amino acid sequences were determined by sequence analysis and internet-based sequence analysis (http: / / www.Imgt.org / IMGT_vquest / share / textes / index.html and http: / / www.ncbi.nlm.nih.gov / igblast / ) based on the Kabat (Wu, TT, and Kabat, EA 1970 J. Exp. Med., 132:211-250) and IMGT systems (Lefranc M.-P. et al., 1999 Nucleic Acids Research, 27, 209-212).

[0126] The amino acid sequences of the light and heavy chain variable regions and CDRs of the anti-CLDN-18.2 murine antibody are shown in the table below:

[0127] [Table 1]

[0128] Example 5. Construction of anti-CLDN-18.2 chimeric antibody Considering the expression level, activity, and type of antibody expressed by the hybridoma cells, the anti-CLDN-18.2 mouse antibodies 1H17, 1H17-2, 2B19, 4G3, 9J24, and 9O24 were selected for further analysis.

[0129] The coding sequences for the heavy chain constant region Fc and light chain constant region kappa were cloned from human blood cells (from the Beijing Institute of Blood Research) and introduced into the pCDNA3.1 plasmid. The coding sequences for the heavy and light chain variable regions of the aforementioned anti-CLDN-18.2 murine antibody were synthesized by Genscript. The coding sequences for the heavy and light chain variable regions of various anti-CLDN-18.2 murine antibodies were digested with BspqI and then introduced into the pCDNA3.1 plasmid containing the constant region coding sequences in various combinations shown in Table 2. The correct clones were confirmed by sequencing. Various chimeric heavy and light chain expression plasmids were mixed and combined with transfected expression cells to obtain five anti-CLDN-18.2 chimeric antibodies. The numbers and corresponding variable region amino acid sequences are specifically shown in Table 2. All subsequent experimental materials were obtained by extracting from cells transfected with this series of plasmids.

[0130] [Table 2]

[0131] Example 6: Detection of chimeric antibodies 6.1 Antibody binding affinity: Gastric cancer cell line NUGC4-CLDN-18.2 cells overexpressing human CLDN-18.2 were incubated with serially diluted anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21, and Chi-JS012-27 at 4°C for 30 min, washed, and then incubated with fluorescently labeled secondary antibodies. Finally, fluorescence intensity was detected using a BD Canto II flow cytometer. A stronger fluorescent signal indicates a higher affinity between the antibody and its target. The antibody dose-dependent binding graph (Figure 1) was fitted using GraphPad to calculate the EC 50 (Table 3) was calculated, and the positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0132] As shown in Figure 1 and Table 3, Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21, and Chi-JS012-27 can all bind to human CLDN-18.2, which is highly expressed on the cell surface of the gastric cancer cell line NUGC4-CLDN-18.2.

[0133] [Table 3]

[0134] 6.2 Antibody-dependent cell-mediated cytotoxicity (ADCC): Serially diluted anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21, and Chi-JS012-27 were incubated with target cells CHO-CLDN-18.2 overexpressing human CLDN-18.2 (50,000 cells) and effector Jurkat ADCC cells expressing NFAT-Luc and FcγRIIIaR (100,000 cells) at 37°C for 6 hours. Then, the substrate One-glo was added, and luciferase signals were detected using a microplate reader. Higher fluorescence readings indicate stronger ADCC effects. Antibody dose-dependent ADCC effect graphs (Figures 2a and 2b) were fitted using GraphPad. IMAB362 and anti-KLH hu-IgG1 antibody were used as positive and negative controls, respectively.

[0135] As shown in Figures 2a and 2b, the EC of ADCC effect mediated by Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21, and Chi-JS012-27. 50 The values were 56.08 ng / mL, 43.6 ng / mL, 49.51 ng / mL, 63.09 ng / mL, and 43.21 ng / mL, respectively, which were comparable to the positive control IMAB362 (Figure 2a: 35.98, Figure 2b: 34.41 ng / mL).

[0136] 6.3 Complement-dependent cytotoxicity (CDC): CHO-CLDN-18.2 cells (100,000 cells) overexpressing human CLDN-18.2 were mixed with serially diluted anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, and Chi-JS012-27, respectively, and then 10-fold diluted complement serum (Quidel, cat# A113) was added and incubated at 37°C for 1 h. Finally, the cells were resuspended in PBS, and PI dye was added. After 5 min of incubation at 4°C, fluorescence intensity was detected using a BD Canto II flow cytometer. The antibody dose-dependent complement killing graph (Figure 3) was fitted using GraphPad. IMAB362 and anti-KLH hu-IgG1 antibody were used as positive and negative controls, respectively.

[0137] As shown in Figure 3 , the EC of the CDC effect mediated by Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, and Chi-JS012-27 50 The values were 461.2 ng / mL, 316 ng / mL, 358.3 ng / mL, and 264.5 ng / mL, respectively, which were comparable to the positive control IMAB362 (353.6 ng / mL).

[0138] Example 7: Humanization of antibody variable regions To reduce the immunogenicity of the antibody, humanized modification and optimization of the antibody variable region was performed. The results are as follows: JPEG0007726978000004.jpg98161This resulted in a series of humanized anti-CLDN-18.2 antibodies.

[0139] Subsequent experiments were performed using the humanized anti-CLDN-18.2 antibodies JS012-Chi9-hu7, JS012-Chi9-hu7-v2, JS012-Chi27-hu3-v2, JS012-Chi27-hu6-v3-2, and JS012-Chi2-hu2-v3-2. The CDR / variable region sequences of these antibodies are shown in Tables 4-1 and 4-2, and the heavy / light chain amino acid sequences and nucleotide coding sequences are shown in Table 5.

[0140] The heavy and light chain variable region coding sequences of the humanized anti-CLDN-18.2 antibodies were synthesized by Genscript. The heavy and light chain variable region coding sequences of various synthesized humanized anti-CLDN-18.2 antibodies were digested with Bspq I and then inserted into the pCDNA3.1 plasmid containing the constant region coding sequence. The correct clones were confirmed by sequencing. The various humanized heavy and light chain expression plasmids were mixed and coupled with transfected expression cells (CHOK1 18, Suzhou Junmeng). The expressed antibodies were collected by centrifugation and then purified using standard methods to obtain the humanized anti-CLDN-18.2 antibodies listed in Tables 4-1 and 4-2.

[0141] Here, we performed process optimization on the humanized antibody JS012-Chi2-hu2-v3-2 by adding a carbohydrate regulator (CDFS01, Shanghai Aopuma Biotechnology Co., Ltd.) during cell culture to obtain defucosylated antibody JS012-Chi2-hu2-v3-2a, a defucosylated anti-CLDN-18.2 antibody with less than 30% fucosylation.

[0142] [Table 4-1]

[0143] [Table 4-2]

[0144] [Table 5]

[0145] Example 8: Detection of humanized anti-CLDN-18.2 antibodies The binding activity of five humanized anti-CLDN-18.2 antibodies, JS012-Chi9-hu7, JS012-Chi9-hu7-v2, JS012-Chi27-hu3-v2, JS012-Chi27-hu6-v3-2, and JS012-Chi2-hu2-v3-2, to cell surface-expressed CLDN-18.2 was detected by FACS. NUGC4-CLDN-18.2 cells were incubated with serially diluted humanized anti-CLDN-18.2 antibodies at different concentrations, washed, and then incubated with a fluorescently labeled secondary antibody. Fluorescent signals were detected by FACS; a stronger fluorescent signal indicated higher antibody affinity, i.e., higher target binding activity. Antibody binding graphs (Figures 4a, 4b and 4c) were fitted by GraphPad, with positive and negative controls being IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0146] As shown in Figures 4a, 4b, and 4c, JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2, and JS012-Chi27-hu6-v3-2 could all bind to human CLDN-18.2, which was highly expressed on the cell surface of the gastric cancer cell line NUGC4. The EC 50 was comparable to the positive control.

[0147] Example 9. ADCC of humanized anti-CLDN-18.2 antibodies The humanized anti-CLDN-18.2 antibody, a human IgG1 subtype, mediates ADCC. The antibody Fc tail binds to the FcγIIIaR receptor on the surface of NK cells, activating NK cells and killing target cells. Using a reporter gene system, we mimicked in vivo ADCC using an NFAT-transduced luciferase system expressing FcγIIIaR. Humanized anti-CLDN-18.2 antibody was incubated with target CHO-CLDN-18.2 cells and Jurkat ADCC effector cells. After addition of the substrate one-glomerate, the signal was detected using a microplate reader. Data were analyzed using GraphPad to compare the dose-dependent antibody ADCC effects (Figures 5a, 5b, and 5c). The positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0148] As shown in Figures 5a, 5b, and 5c, the EC of ADCC effect mediated by JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2, and JS012-Chi27-hu6-v3-2. 50 The values were 22.5 ng / mL, 23.63 ng / mL, 35.75 ng / mL, 14.18 ng / mL and 74.24 ng / mL, respectively, all of which were much higher than the positive control IMAB362.

[0149] Example 10. CDC of humanized anti-CLDN-18.2 antibodies Humanized anti-CLDN-18.2 antibody can also mediate CDC effect and kill target cells by forming a membrane attack complex. Complement serum (1:10 dilution) was incubated with humanized anti-CLDN-18.2 antibody and target cells CHO-CLDN-18.2 (100,000 cells) in an incubator at 37°C for 1 h. The viability and killing status of target cells was detected by propidium iodide (PI) staining, and the relative killing rate was calculated. The dose-dependent CDC effect of humanized anti-CLDN-18.2 antibody was reflected in the relative killing rate (Figures 6a, 6b, and 6c). The positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0150] As shown in Figures 6a, 6b and 6c, the EC of CDC effect mediated by JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2 and JS012-Chi27-hu6-v3-2. 50 The values were 134.2ng / mL, 86.04ng / mL, 168.9ng / mL, 166.5ng / mL and 714.9ng / mL, respectively, where JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2 and JS012-Chi2-hu2-v3-2 were significantly superior to the positive control IMAB362.

[0151] Example 11: Inhibitory effect of humanized anti-CLDN-18.2 antibody on the growth of human gastric cancer MKN45 hClaudin18.2 Mixeno tumors transplanted into M-NSG mice 1. Purpose of the test The antitumor effect of JS012-Chi2-hu2 v3-2a of the present invention will be evaluated in a human gastric cancer MKN45 hClaudin18.2 Mixeno subcutaneous transplant model.

[0152] 2. Testing process 5×10 6 MKN45 hClaudin18.2 tumor cells (Shanghai Nuoc Biotechnology) were added to 5 × 10 6 The mixture was mixed with PBMC cells (Shanghai Aoneng Bio) and subcutaneously inoculated into the right dorsal region of M-NSG mice (Shanghai Nanfang Model Bioscience and Technology Co., Ltd.) at 0.2 ml per mouse (RPMI1640 medium (Gibco) containing cells and 50% Matrigel). Fifty mice were selected based on their body weight and randomly divided into five groups, with 10 mice in each group. Anti-KLH hIgG1 negative control group, 10 mg / kg, JS012-Chi2-hu2 v3-2a treatment group, 1 mg / kg; JS012-Chi2-hu2 v3-2a treatment group, 3mg / kg; JS012-Chi2-hu2 v3-2a treatment group, 10 mg / kg; IMAB362 positive control group, 10 mg / kg.

[0153] On the day of grouping, tumor cells were inoculated and administered intraperitoneally 4 hours after the initial administration. All groups received nine consecutive doses twice weekly, with the experiment terminated three days after the final dose. Tumor volume and body weight were measured twice weekly, and mouse weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and the tumor inhibition rate (TGI%) (TGI% = [1-T / C] × 100%) was calculated. (T: mean tumor volume at the end of the experiment for the treatment group or positive control group; C: mean tumor volume at the end of the experiment for the negative control group).

[0154] As shown in Figure 7, at the end of the experiment, the mean tumor volume in the anti-KLH hIgG1 negative control group was 1276 ± 228 mm 3 JS012-Chi2-hu2a v3-2 had a mean tumor volume of 235 ± 25 mm at doses of 1, 3, and 10 mg / kg, respectively. 3 , 243±33mm 3 and 343±63mm 3 The TGI was 81.6%, 81.0%, and 73.1%, respectively, demonstrating significant tumor suppression. IMAB362 at a dose of 10 mg / kg reduced the mean tumor volume to 361 ± 73 mm 3 The TGI was 71.7%. Under the same dose condition (10 mg / kg), the tumor-suppressing activity of JS012-Chi2-hu2 v3-2a was slightly superior to that of IMAB362.

[0155] Example 12: Inhibitory effect of humanized anti-CLDN-18.2 antibody on the growth of human pancreatic cancer hCLDN18.2 MIA PaCa-2 tumors transplanted into CB-17 SCID mice 1. Purpose of the test The antitumor effect of JS012-Chi2-hu2 v3-2a of the present invention will be evaluated in a human pancreatic cancer hCLDN18.2 MIA PaCa-2 subcutaneously xenograft model.

[0156] 2. Testing process 5×10 6 hCLDN18.2 MIA PaCa-2 cells (Accurusbio-C3002) were subcutaneously inoculated into the right dorsal region of CB-17 SCID mice (Shanghai Gibco Laboratory Animal Breeding Co., Ltd.) at 0.2 ml / mouse (DMEM medium containing cells and 50% Matrigel). The average tumor volume was approximately 89 mm. 3 When the tumor volume reached 40 animals, they were randomly divided into 5 groups, 8 animals per group, and Anti-KLH hIgG1 negative control group, 3 mg / kg, IMAB362 positive control group, 3 mg / kg; JS012-Chi2-hu2 v3-2a treatment group, 0.3 mg / kg; JS012-Chi2-hu2 v3-2a treatment group, 1 mg / kg; JS012-Chi2-hu2 v3-2a treatment group, 3 mg / kg.

[0157] Mice were administered intraperitoneally on the day of group assignment. All groups received six consecutive doses, twice weekly, with the experiment terminated four days after the final dose. Tumor volume and body weight were measured three times weekly, and mouse weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and the tumor inhibition rate (TGI%) (TGI% = [1-(Ti-T0) / (Vi-V0)] × 100%) was calculated. (Ti: mean tumor volume on day i of administration for the treatment group or positive control group; TO: mean tumor volume on day 0 of administration for the treatment group or positive control group; Vi: mean tumor volume on day i of administration for the negative control group; V0: mean tumor volume on day 0 of administration for the negative control group).

[0158] As shown in Figure 8, at the end of the experiment, the mean tumor volume in the anti-KLH hIgG1 group was 2235 ± 145 mm3. IMAB362 at a dose of 3 mg / kg had a mean tumor volume of 465 ± 74 mm3, with a TGI of 82.5%. JS012-Chi2-hu2 v3-2a at doses of 0.3, 1, and 3 mg / kg had mean tumor volumes of 1455 ± 142 mm3, 673 ± 153 mm3, and 74 ± 19 mm3, respectively, with TGIs of 36.3%, 72.8%, and 100.6%, respectively, demonstrating significant tumor inhibitory activity. At the same dose (3 mg / kg), the tumor inhibitory activity of JS012-Chi2-hu2 v3-2a was significantly superior to that of IMAB362. Sequence Listing JPEG0007726978000008.jpg239156 JPEG0007726978000009.jpg244143JPEG0007726978000010.jpg255161 JPEG0007726978000011.jpg244149JPEG0007726978000012.jpg245147 JPEG0007726978000013.jpg252155 JPEG0007726978000014.jpg252156JPEG0007726978000015.jpg244149JPEG0007726978000016.jpg243153 JPEG0007726978000017.jpg218160

Claims

1. An anti-CLDN-18.2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, (I) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 42 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 43, or (III) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 45 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 46, or (IV) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 47 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 48; or (V) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 49 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 50; or (VI) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 51 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 52, or (VII) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 53 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 50, or (VIII) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 54 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 52; or (IX) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 45 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 48; or (X) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 51 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 46, or (XI) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 49 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 46; or (XII) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55 or 57, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, or (XIII) a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 60, and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 61; An anti-CLDN-18.2 antibody or an antigen-binding fragment thereof.

2. a heavy chain and a light chain, wherein (2) the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 64 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 64, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 65 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 65; or (3) the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 68 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 68, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 69 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69; or (10) The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 96 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 97 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

97.

3. The antibody or antigen-binding fragment thereof described in claim 1 or 2, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, and the antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb, or a diabody.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is of the IgG subtype.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody is IgG1, IgG2, IgG3 or IgG4.

6. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein the antibody is hypofucosylated or afucosylated.

7. A polynucleotide encoding the anti-CLDN-18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An expression vector comprising the polynucleotide of claim 7.

9. The expression vector of claim 8, which is a eukaryotic expression vector.

10. A host cell comprising the polynucleotide of claim 7, or the expression vector of claim 8 or 9, or expressing the anti-CLDN-18.2 antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

11. The host cell of claim 10 , wherein the host cell is a eukaryotic cell.

12. The host cell of claim 10 or 11, wherein the host cell is a mammalian cell.

13. A method for producing an anti-CLDN-18.2 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising culturing a host cell according to any one of claims 10 to 12 under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

14. A pharmaceutical composition comprising an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, a polynucleotide according to claim 7, an expression vector according to claim 8 or 9, or a host cell according to any one of claims 10 to 12.

15. 15. The pharmaceutical composition of claim 14, comprising a pharmaceutically acceptable vector or excipient.

16. The pharmaceutical composition according to claim 14 or 15, for treating and / or preventing a disease or condition mediated by CLDN-18.

2.

17. The pharmaceutical composition of claim 16, wherein the disease or condition is cancer.

18. The pharmaceutical composition of claim 17, wherein the cancer is selected from gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, intestinal cancer and bladder cancer.

19. A drug combination comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, the polynucleotide described in claim 7, the expression vector described in claim 8 or 9, the host cell described in any one of claims 10 to 12, or the drug composition described in claim 14 or 15, and one or more additional therapeutic agents.

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