Anti-claudin-18.2 antibodies and uses thereof

Abstract

Provided are antibodies or antigen-binding fragments thereof that specifically bind to CLDN-18.2 and compositions comprising the same. Also provided are 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, as well as therapeutic or diagnostic methods and uses of the antibodies or antigen-binding fragments thereof.

Description

Technical Field

[0001] This invention provides antibodies or antigen-binding fragments thereof that specifically bind to CLDN-18.2 and compositions comprising them. It also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments of this invention, expression vectors and host cells for expressing the antibodies or antigen-binding fragments of this invention, and methods and uses for the treatment or diagnosis of the antibodies or antigen-binding fragments of this invention. Background Technology

[0002] Stomach cancer is one of the most common cancers worldwide. According to statistics from the World Health Organization's Cancer Control Program, up to 7 million people die from cancer globally each year, with stomach cancer accounting for 700,000 of those deaths. Compared to conventional stomach cancer treatments, antibody-based therapies have significant potential due to their high specificity and low side effects.

[0003] Claudin, also known as CLDN, is a family of cell surface proteins that establish paracellular barriers and control intercellular molecular flow; at least 26 species have been identified to date. Claudin protein family members are important structural components of tight junctions, playing crucial roles in maintaining epithelial cell polarity, controlling paracellular diffusion, and regulating cell growth and differentiation. Claudin molecules cross the cell membrane four times, with both their N-terminus and C-terminus located in the cytoplasm. Different Claudin members are expressed in different tissues, and alterations in their function are associated with cancer development. Changes in the expression levels of Claudin 1, Claudin 18, and Claudin 10 are associated with colorectal 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 in trace amounts in normal short-lived gastric epithelial cells, but in tumor cells, Claudin 18.2 is strongly expressed in various cancer types, such as 75% of gastric cancer patients, 50% of pancreatic cancer patients, and 30% of esophageal cancer patients. It is also highly expressed in lung cancer and other cancers.

[0005] Although Ganymed's Claudiximab (IMAB362) is currently in a phase II clinical trial for advanced gastric and esophageal cancer (WO2007059997), there is still a need for novel anti-CLDN18.2 antibodies that improve upon known antibodies in terms of affinity and specificity. Summary of the Invention

[0006] This invention provides an anti-CLDN-18.2 antibody or its antigen-binding fragment thereof, which has advantages such as high affinity and high specificity against human CLDN-18.2. The anti-CLDN-18.2 antibody or its antigen-binding fragment provided by this invention can be used as a standalone therapy or in combination with other therapies and / or other anticancer agents for the treatment of diseases such as cancer.

[0007] In one aspect, the present invention provides an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,

[0008] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein

[0009] The HCDR1 sequence is selected from sequences such as SEQ ID NO:1, 10, 16, 22, 28.

[0010] The amino acid sequences shown in 34 and 37;

[0011] The HCDR2 sequence is selected from sequences such as SEQ ID NO: 62, 63, 17, 23, 29.

[0012] The amino acid sequences shown in SEQ ID NO: 62 are: X1 is C or S, X2 is T or S.

[0013] In HCDR2 shown in ID NO:63, X3 is D or G, and X4 is K or T;

[0014] and

[0015] The HCDR3 sequence is selected from sequences such as SEQ ID NO:3, 12, 18, 24, 30,

[0016] The amino acid sequences shown are 36, 39, 73, and 75; and

[0017] The light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein

[0018] The sequence of LCDR1 is selected from the amino acid sequences shown in SEQ ID NO:4, 7, 13, 19, 25 and 31;

[0019] The sequence of LCDR2 is selected from the amino acid sequences shown in SEQ ID NO: 5, 8, 14, 20, 26 and 32; and

[0020] The sequence of LCDR3 is selected from the amino acid sequences shown in SEQ ID NO:6, 9, 15, 21, 27 and 33.

[0021] In some embodiments, the heavy chain variable region of the present invention comprises:

[0022] (I) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or

[0023] (II) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or

[0024] (III) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; or

[0025] (IV) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; or

[0026] (V) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; or

[0027] (VI) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or

[0028] (VII) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively; or

[0029] (VIII) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:40, and SEQ ID NO:12, respectively; or

[0030] (IX) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:41, and SEQ ID NO:3, respectively; or

[0031] (X) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:72, and SEQ ID NO:73, respectively; or

[0032] (XI) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:72, and SEQ ID NO:3, respectively; or

[0033] (XII) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:74, and SEQ ID NO:75, respectively; or

[0034] (XIII) The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:74, and SEQ ID NO:30, respectively; and

[0035] The light chain variable region includes:

[0036] (I) The amino acid sequences are as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, for LCDR1, LCDR2 and LCDR3; or

[0037] (II) The amino acid sequences are as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, for LCDR1, LCDR2 and LCDR3; or

[0038] (III) The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, for LCDR1, LCDR2 and LCDR3; or

[0039] (IV) The amino acid sequences are as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, for LCDR1, LCDR2 and LCDR3; or

[0040] (V) The amino acid sequences are as shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively, for LCDR1, LCDR2 and LCDR3; or

[0041] (VI) The amino acid sequences are LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively.

[0042] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises:

[0043] (I) Heavy chain variable region, comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain variable region, comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; or

[0044] (II) Heavy chain variable region, comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain variable region, comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or

[0045] (III) Heavy chain variable region, comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; and light chain variable region, comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or

[0046] (IV) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; or

[0047] (V) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; or

[0048] (VI) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or

[0049] (VII) A heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; and a light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; or

[0050] (VIII) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or

[0051] (IX) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; or

[0052] (X) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or

[0053] (XI) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or

[0054] (XII) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:40, and SEQ ID NO:12, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or

[0055] (XIII) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:41, and SEQ ID NO:3, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or

[0056] (XⅣ) Heavy chain variable region, comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:72, and SEQ ID NO:73, respectively; and light chain variable region, comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or

[0057] (XV) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:72, and SEQ ID NO:3, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or

[0058] (XVI) Heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:74, and SEQ ID NO:30, respectively; and light chain variable region comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or

[0059] (XⅦ) Heavy chain variable region, comprising amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:74 and SEQ ID NO:75 respectively; and light chain variable region, comprising amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 respectively.

[0060] In some embodiments, the amino acid sequence of the heavy chain variable region of the present invention comprises a sequence selected from those shown in SEQ ID NO: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 with any of the sequences shown in SEQ ID NO:42, 45, 47, 49, 51, 53, 54, 76, 78, 82, and 83; and

[0061] The amino acid sequence of the light chain variable region comprises an amino acid sequence selected from those shown in SEQ ID NO:43, 44, 46, 48, 50 and 52, or having at least 95%, 96%, 97%, 98% or 99% identity with any of the sequences shown in SEQ ID NO:43, 44, 46, 48, 50 and 52.

[0062] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises:

[0063] (I) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:42 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:43; or

[0064] (II) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:42 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:44; or

[0065] (III) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:45 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:46; or

[0066] (IV) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:47 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:48; or

[0067] (V) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:49 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:50; or

[0068] (VI) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:51 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:52; or

[0069] (VII) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:53 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:50; or

[0070] (VIII) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:54 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:52; or

[0071] (IX) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:45 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:48; or

[0072] (X) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:51 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:46; or

[0073] (XI) The heavy chain variable region with amino acid sequences as shown in SEQ ID NO:49 and the light chain variable region with amino acid sequences as shown in SEQ ID NO:46.

[0074] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0075] The amino acid sequence of the heavy chain variable region comprises a sequence selected from those shown in SEQ ID NO: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 with any of the sequences shown in SEQ ID NO:55, 57, 58, 59, 60, 76, 78, 79, 82, and 83; and

[0076] The amino acid sequence of the light chain variable region comprises sequences selected from those shown in SEQ ID NO:56, 61, 77, 80 and 81, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity with the sequences shown in SEQ ID NO:56, 61, 77, 80 and 81.

[0077] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 55, 57, 60, 76, 78, 79, 82 or 83; and the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 56, 61, 77, 80 or 81.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:55, 57, 82 or 83; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:56.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:60, 76 or 78; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:61.

[0080] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0081] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:55; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:56; or

[0082] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:57; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:56; or

[0083] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:58; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:56; or

[0084] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:59; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:56; or

[0085] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:60; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:61; or

[0086] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:76; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:77; or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:79; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:80; or

[0087] The variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO:55; and the variable region of the light chain comprises the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain, the amino acid sequence of which is shown in SEQ ID NO:64 or a variant thereof or SEQ ID NO:68 or a variant thereof, and a light chain, the amino acid sequence of which is shown in SEQ ID NO:65 or a variant thereof or SEQ ID NO:69 or a variant thereof, wherein the variant comprises 1, 2, 3, 4 or 5 amino acid variations in its variable region;

[0088] Preferably, the variant of SEQ ID NO:64 includes an amino acid change at position 50 or 63 or a combination thereof; and / or the variant of SEQ ID NO:68 includes an amino acid change at position 63 or 65 or a combination thereof;

[0089] Preferably, variants of SEQ ID NO:64 comprise S50C or S63T or a combination thereof; and / or variants of SEQ ID NO:68 comprise G63D or T65K or a combination thereof.

[0090] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises: a heavy chain and a light chain, wherein

[0091] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:64, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:64; and the light chain comprises the amino acid sequence shown in SEQ ID NO:65, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:65; or

[0092] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:68; and the light chain comprises the amino acid sequence shown in SEQ ID NO:69, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:69; or

[0093] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:84, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:84; and the light chain comprises the amino acid sequence shown in SEQ ID NO:85, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:85; or

[0094] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:86, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:86; and the light chain comprises the amino acid sequence shown in SEQ ID NO:87, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:87; or

[0095] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:88, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:88; and the light chain comprises the amino acid sequence shown in SEQ ID NO:89, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:89; or

[0096] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:90, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:90; and the light chain comprises an amino acid sequence as shown in SEQ ID NO:91, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:91; or

[0097] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:92, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:92; and the light chain comprises the amino acid sequence shown in SEQ ID NO:93, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:93; or

[0098] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:94, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:94; and the light chain comprises the amino acid sequence shown in SEQ ID NO:95, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:95; or

[0099] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:96, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:96; and the light chain comprises the amino acid sequence shown in SEQ ID NO:97, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:97; or

[0100] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:98, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:98; and the light chain comprises an amino acid sequence as shown in SEQ ID NO:99, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:99; or

[0101] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:100, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:100; and the light chain comprises an amino acid sequence as shown in SEQ ID NO:101, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:101.

[0102] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises:

[0103] (I) The heavy chain with an amino acid sequence as shown in SEQ ID NO:64 and the light chain with an amino acid sequence as shown in SEQ ID NO:65; or

[0104] (II) The heavy chain with an amino acid sequence as shown in SEQ ID NO:68 and the light chain with an amino acid sequence as shown in SEQ ID NO:69.

[0105] In some embodiments, the antibody described in this invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0106] In some embodiments, the antigen-binding fragment of the present invention is Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, sdAb, or a double antibody.

[0107] In some embodiments, the antibody of the present invention is any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4; preferably, the antibody is low or non-fucosylated.

[0108] In some embodiments, the antibody described in this invention is hypofucosylated.

[0109] In some embodiments, the antibody described in this invention is unfucosylated.

[0110] In another aspect, the present invention provides an isolated anti-CLDN-18.2 antibody or its antigen-binding fragment thereof, having one or more of the following properties:

[0111] (1) Epitopes of human CLDN-18.2 protein that bind to the same or completely or partially overlapping anti-CLDN-18.2 antibody or its antigen-binding fragment described herein;

[0112] (2) Competes with the anti-CLDN-18.2 antibody or its antigen-binding fragment described herein for binding to the epitope of human CLDN-18.2 protein;

[0113] (3) It binds to human CLDN-18.2 protein, but does not bind to human CLDN-18.1 protein;

[0114] (4) Inducing ADCC effect in cells expressing human CLDN-18.2 protein; and

[0115] (5) Inducing the CDC effect in cells expressing human CLDN-18.2 protein.

[0116] In another aspect, the present invention provides a polynucleotide encoding an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein.

[0117] In another aspect, the present invention provides an expression vector comprising polynucleotides as described herein, preferably a eukaryotic expression vector.

[0118] In another aspect, the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, or expressing an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein, preferably a eukaryotic cell, more preferably a mammalian cell.

[0119] In another aspect, the present invention provides a method for preparing an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein, the method comprising culturing a host cell as described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0120] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, or a host cell as described herein, and a pharmaceutically acceptable carrier or excipient.

[0121] In another aspect, the present invention provides the use of antibodies or antigen-binding fragments thereof as described herein, polynucleotides as described herein, expression vectors as described herein, host cells as described herein, or pharmaceutical compositions as described herein in the preparation of medicaments for treating and / or preventing CLDN-18.2-mediated diseases or conditions, preferably cancer.

[0122] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof as described herein, polynucleotides as described herein, expression vectors as described herein, host cells as described herein, or pharmaceutical compositions as described herein for the treatment and / or prevention of CLDN-18.2-mediated diseases or conditions, preferably cancer.

[0123] In another aspect, the present invention provides a method for treating and / or preventing CLDN-18.2-mediated diseases or conditions, comprising administering to a subject in need an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, preferably, the disease or condition being cancer.

[0124] In some embodiments, the cancer is selected from drugs for 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.

[0125] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0126] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, preferably further comprising a drug delivery device.

[0127] In another aspect, the present invention provides a method for detecting the presence of CLDN-18.2 in a sample using an antibody or an antigen-binding fragment thereof as described herein. Attached Figure Description

[0128] Figure 1 Cellular affinity of chimeric anti-CLDN-18.2 antibody as determined by flow cytometry.

[0129] Figure 2 a and 2b: ADCC activity of chimeric anti-CLDN-18.2 antibody as determined by reporter gene assay.

[0130] Figure 3 CDC activity of chimeric anti-CLDN-18.2 antibody as determined by flow cytometry.

[0131] Figure 4a , 4b 4c: Cellular affinity of humanized anti-CLDN-18.2 antibody as determined by flow cytometry.

[0132] Figure 5a , 5b 5c: ADCC activity of humanized anti-CLDN-18.2 antibody as determined by reporter gene assay.

[0133] Figure 6a , 6b 6c: CDC activity of humanized anti-CLDN-18.2 antibody as determined by flow cytometry.

[0134] Figure 7 Inhibitory effect of humanized anti-CLDN-18.2 antibody on the growth of human gastric cancer MKN45hClaudin18.2Mixeno tumor transplanted into M-NSG mice.

[0135] Figure 8 Inhibitory effect of humanized anti-CLDN-18.2 antibody on the growth of human pancreatic cancer hCLDN18.2 MIAPaCa-2 tumors transplanted into CB-17SCID mice. Detailed Implementation

[0136] definition

[0137] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0138] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0139] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0140] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0141] The term “CLDN-18.2” or “Claudin18.2” is one of two splicing 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 rodent (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length” unprocessed CLDN-18.2 as well as any form of CLDN-18.2 or any fragment thereof produced by intracellular processing. The term also includes variants of naturally occurring CLDN-18.2, such as splicing variants or allelic variants. In a preferred embodiment, CLDN-18.2 refers to the full-length or fragment thereof (such as the mature fragment lacking the signal peptide) of CLDN-18.2 from humans and cynomolgus monkeys.

[0142] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0143] 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, which results in selective damage, destruction, or clearance from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation.

[0144] The terms "signal transduction pathway" or "signal transduction activity" refer to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of a signal from one part of the cell to another. Generally, this transmission involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that induce signal transduction. The penultimate process typically involves nuclear events that lead to changes in gene expression.

[0145] The terms “activity” or “biological activity”, or “biological property” or “biological characteristic”, are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize the activity of CLDN-18.2 in vivo or in vitro, IC50, etc. 50 The in vivo stability and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics are observed, measured, or evaluated using techniques known in the art, including but not limited to ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0146] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is 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 camel-derived single-domain antibodies.

[0147] The term "isolated antibody" refers to the purified state of a binding compound, and in this context, it means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the binding compound described herein.

[0148] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0149] The term "full-length antibody" refers to an immunoglobulin molecule that, in its natural state, contains four peptide chains: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains (approximately 25 kDa in full length) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins 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.

[0150] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the C-terminus. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIHP Publication 91-3242.

[0151] The term "antigen-binding fragment" of an antibody ("parental antibody") includes fragments or derivatives of the antibody, typically comprising at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When the antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of an antibody may include conserved or non-conserved amino acid substitutions (referred to as "conserved variants" or "functionally conserved variants" of the antibody) that do not significantly alter its biological activity. The term "binding compound" refers to both the antibody and its binding fragment.

[0152] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing both VH and VL domains, which are located within a single polypeptide chain. Fv polypeptides typically also include a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding.

[0153] The term "domain antibody" refers to an immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0154] The term "bivalent antibody" refers to an antibody with two antigen-binding sites. In some cases, both binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.

[0155] The term "dual antibody" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker too short to allow pairing between two domains on the same chain, the linker is forced to pair with a complementary domain of the other chain, creating two antigen-binding sites.

[0156] The term "chimeric antibody" refers to an antibody possessing a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies originate from different species. Typically, the variable domain is derived from antibodies from rodents, etc. ("parental antibodies"), while the constant domain sequence is derived from human antibodies, making the resulting chimeric antibody less likely to induce an adverse immune response in human subjects compared to parental rodent antibodies.

[0157] The term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, humanized antibodies contain at least one, and usually two, variable domains, where all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or almost all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant region (Fc) of human immunoglobulins.

[0158] The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulin proteins. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, a fully human antibody may contain mouse glycans. Similarly, a "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, a fully human antibody may contain rat glycans. Likewise, a "rat antibody" refers to an antibody that contains only the sequence of rat immunoglobulins.

[0159] "Isotype" antibodies refer to antibody classes provided by heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been generated to alter Fc function, such as to enhance or weaken effector function or binding to the Fc receptor.

[0160] The term "epitope" refers to the antigenic region to which an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein.

[0161] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be determined by the equilibrium dissociation constant (K). D The equilibrium dissociation constant represents the dissociation rate constant and the binding rate constant (k, k, and k, respectively). dis and k on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0162] The term "non-binding" protein or cell refers to proteins or cells that do not bind to each other, or do not bind to them with high affinity; that is, K, the protein or cell that binds to. D 1.0×10 -6 M or higher, more preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0163] For IgG antibodies, the term "high affinity" refers to the affinity for the antigen's K+. D 1.0×10 -6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding in the IgM subtype refers to K... D 10 -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0164] The terms “antibody-dependent cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to cell-mediated immune defense, in which immune system effector cells actively bind to cell membrane surface antigens with antibodies, such as Claudin18.2 antibody, and lyse target cells, such as cancer cells.

[0165] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies, which, upon binding to surface antigens, triggers a typical complement pathway, including the formation of a membrane attack complex and target cell lysis. The antibody of this invention, upon binding to Claudin 18.2, triggers CDC against cancer cells.

[0166] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (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)).

[0167] "Construction" refers to any recombinant polynucleotide molecule (such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules), derived from any source, capable of integrating with or autonomously replicating the genome, constituting a polynucleotide molecule in which one or more polynucleotide molecules are functionally linked (i.e., operably linked). Recombinant constructs typically contain polynucleotides of the present invention operably linked to transcription initiation regulatory sequences that guide the transcription of the polynucleotide in the host cell. Expression of the nucleic acids of the present invention can be guided using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0168] "Vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, into which an additional DNA segment can be ligated into the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. These vectors are referred to herein as "expression vectors."

[0169] As used in this article, "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene when transformed, transfected, or transduced into host cells. Expression vectors contain one or more phenotypic selection markers and origins of replication to ensure the maintenance of the vector and to provide amplification within the host when needed.

[0170] The terms “activation,” “stimulation,” and “treatment” used for cells or receptors can have the same meaning, such as activating, stimulating, or treating cells or receptors with ligands, unless the context otherwise specifies. “Ligand” includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and antibody-derived binding compounds. “Ligand” also includes small molecules, such as peptide mimics of cytokines and peptide mimics of antibodies. “Activation” can refer to cell activation regulated by internal mechanisms as well as external or environmental factors. “Response” refers to responses of cells, tissues, organs, or organisms, including changes in biochemical or physiological behaviors (e.g., concentrations, densities, adhesion or migration, gene expression rates, or differentiation states within biological compartments) that are related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0171] As used herein, the term “treatment” or “cure” for any disease or condition, in one embodiment, means improving the disease or condition (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treatment” or “cure” means alleviating or improving at least one bodily parameter, including those physical parameters that may not be identifiable by the patient. In yet another embodiment, “treatment” or “cure” means regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or in both ways. Unless explicitly described herein, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0172] "Subjects" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cyno-eating macaque" refers to the cyno-eating macaque.

[0173] "Combined" administration of one or more other therapeutic agents includes simultaneous (co-) administration and consecutive administration in any order.

[0174] "Therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the CLDN-18.2 antibody or its antigen-binding fragment thereof, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to the amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or increases the rate of treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10% in diagnostic criteria or parameters; typically at least 20%; preferably at least about 30%; more preferably at least 40%; and most preferably at least 50%.

[0175] “Cancer” and “cancerous” refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. This definition includes both 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 this type of cancer include 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, stomach 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 cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small aneuploid NHL, and bulky lymphoma). Diseases including NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0176] Anti-CLDN-18.2 antibody

[0177] In one aspect, the present invention provides an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof. The terms "anti-CLDN-18.2 antibody," "anti-CLDN-18.2," "CLDN-18.2 antibody," or "antibody binding to CLDN-18.2" refer to antibodies capable of binding with sufficient affinity to the CLDN-18.2 protein or a fragment thereof such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CLDN-18.2.

[0178] The antibodies of the present invention can be generated using any suitable method for antibody production. Any suitable form of CLDN-18.2 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any variant of CLDN-18.2 or a fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells that produce mouse monoclonal anti-human CLDN-18.2 antibodies can be generated by methods known in the art. Antibodies derived from rodents (such as mice) may cause unwanted antibody immunogenicity when used in vivo as therapeutic agents, and repeated use may lead to immune responses against the therapeutic antibodies in the human body, which at least result in loss of therapeutic efficacy and, in severe cases, potentially fatal anaphylactic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies in which a mouse variable region is fused with a human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the retention of the intact rodent variable region in chimeric antibodies may still cause harmful immunogenicity in patients. Transplanting the complementarity-determining region (CDR) loop of rodent variable domains onto human scaffolds (i.e., humanization) has been used to further reduce rodent sequences to a minimum (Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0179] In some embodiments, the chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the prepared mouse monoclonal hybridoma antibody. The DNA encoding the heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to include non-mouse (e.g., human) immunoglobulin sequences.

[0180] In some embodiments, the chimeric CLDN-18.2 antibody of the present invention can be prepared by effectively linking the variable regions of the hybridoma-derived immunoglobulin heavy and light chains with the constant regions of human IgG using methods known in the art (see, for example, 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 antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.

[0181] In some embodiments, the chimeric CLDN-18.2 antibody of the present invention can be obtained by transfecting expression cells with a chimeric light chain and a chimeric heavy chain expression plasmid in a “mixed and matched” manner. The CLDN-18.2 binding of such “mixed and matched” antibodies can be tested using the binding assays described above and other conventional binding assays (e.g., ELISA).

[0182] The precise amino acid sequence boundaries of the variable region CDR of the antibody of the present invention can be determined using any of many well-known schemes, including those based on the three-dimensional structure of the antibody and the topology of the CDR loop (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)) and those based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids). Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering of a large number of crystal structures. The CDR of the antibodies of the present invention can be determined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0183] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined with the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0184] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs. However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0185] The humanized antibody described in this invention can be used to insert a murine CDR region into a human germline framework region using methods known in the art. See U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0186] In some embodiments, amino acid changes include amino acid deletions, insertions, or substitutions. In some embodiments, the anti-CLDN-18.2 antibody of the present invention, or its antigen-binding fragment, comprises those antibodies having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still possess at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the antibodies described above (particularly in the CDR region depicted in the above sequence). In some embodiments, when the antibody of the present invention is compared with the CDR region depicted in the specific sequence, the number of amino acid mutations in the CDR region that have been mutated by amino acid deletions, insertions, or substitutions does not exceed 1, 2, 3, 4, or 5.

[0187] In some embodiments, the polynucleotide encoding the antibody of the present invention comprises a polynucleotide that has been mutated by nucleotide deletion, insertion or substitution, but still has at least about 60, 70, 80, 90, 95 or 100% identity with the coding region corresponding to the CDR depicted in the sequence described above.

[0188] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to produce an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0189] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0190] In some embodiments, antibodies can be modified to increase or decrease their degree of glycosylation and / or alter their glycosylation pattern. The addition or deletion of glycosylation sites on an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions can be performed to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site. Antibodies with altered types of glycosylation can be prepared, such as low- or non-fucosylated antibodies with reduced amounts of fucosylated residues or antibodies with increased isomeric GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies.

[0191] In some preferred embodiments, the present invention provides antibodies that are low- or non-fucosylated, thereby significantly increasing the binding affinity of the antibody to receptors expressed on effector cells, resulting in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The amount of fucose can be determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297 using MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to an aspartic acid residue located approximately at position 297 (EU number of Fc region residues) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acid positions upstream or downstream of position 297, i.e., between positions 294 and 300. See, for example, US2003 / 0157108; US2004 / 0093621. Such antibody variants can be generated in cell lines capable of producing defucosylated or hypofucosylated antibodies. Examples of such cells include Lecl3 CHO cells with protein fucosylation defects (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986):533-545; US2003 / 0157108). In some embodiments, fucosidase is used to cleave the fucose residues of the antibody. In some embodiments, a glycotype modulator is used to control the fucose residues of the antibody. The glycotype modulator may be CDFS01, commercially available from Shanghai Aopumai Biotechnology Co., Ltd. Defucosylation can be performed using conventional methods known in the art.

[0192] The level of fucosylation in an antibody can be structurally defined. As described herein, "non-fucosylated" or "unfucosylated" means that the fucose content in the antibody is less than 5%, for example, approximately 0%, less than 1%, less than 2%, less than 3%, or less than 4%. The term "low fucosylation" means that the fucose content in the antibody is approximately 5% or greater and less than 30%; for example, the fucose content in antibodies is approximately 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 10%-20%, 20%-30%, or 10%-30%. The term "low fucosylation or unfucosylated" means that the fucose content in the antibody is less than 30%.

[0193] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0194] antibody expression

[0195] In another aspect, the present invention provides a polynucleotide encoding an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein. The polynucleotide may comprise a polynucleotide encoding an amino acid sequence encoding the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide comprising an amino acid sequence encoding the light chain and / or the heavy chain of the antibody.

[0196] In another aspect, the present invention provides an expression vector comprising a polynucleotide as described herein, preferably a eukaryotic expression vector. In some embodiments, the polynucleotide as described herein is contained in one or more expression vectors.

[0197] In another aspect, the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, preferably a eukaryotic cell, more preferably a mammalian cell.

[0198] In another aspect, the present invention provides a method for preparing an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0199] This invention provides mammalian host cells for expressing the recombinant antibodies of this invention, including a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammal host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines exhibit high expression levels.

[0200] In one embodiment, the present invention provides a method for preparing an anti-CLDN-18.2 antibody, wherein the method includes, when an expression vector is introduced into mammalian host cells, producing the antibody by culturing the host cells for a sufficient period of time to allow the antibody to be expressed in the host cells, or more preferably by secreting the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0201] Antibodies expressed in different cell lines or in transgenic animals may have different glycosylations. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are part of the invention, regardless of their glycosylation. Similarly, in some embodiments, non-fucosylated antibodies are advantageous because they generally have stronger efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structure is a normal component of native human serum IgG.

[0202] Pharmaceutical compositions and pharmaceutical preparations

[0203] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, and a pharmaceutically acceptable carrier or excipient.

[0204] It should be understood that the anti-CLDN-18.2 antibody or its pharmaceutical composition provided by the present invention can integrate suitable carriers, excipients and other reagents in the formulation for co-administration, thereby providing improved transfer, delivery, tolerability, etc.

[0205] The term "pharmaceutical composition" refers to a formulation that allows the biologically effective form of the active ingredient contained therein to be present, and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation.

[0206] Pharmaceutical formulations containing the anti-CLDN-18.2 antibody described herein can be prepared by mixing the anti-CLDN-18.2 antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)). Preferably, the formulation is in the form of an aqueous solution or a lyophilized preparation.

[0207] The pharmaceutical compositions or formulations of the present invention may further comprise one or more other active ingredients required for the specific indication being treated, preferably those 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, wherein the active ingredients are suitably combined in an amount effective for the intended use. In some embodiments, the pharmaceutical compositions of the present invention further comprise a composition encoding a polynucleotide of an anti-CLDN-18.2 antibody.

[0208] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0209] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, preferably further comprising a drug delivery device.

[0210] Medical Use

[0211] In another aspect, the present invention provides the use of antibodies or antigen-binding fragments thereof as described herein, polynucleotides as described herein, expression vectors as described herein, host cells as described herein, or pharmaceutical compositions as described herein in the preparation of medicaments for treating and / or preventing CLDN-18.2-mediated diseases or conditions, preferably cancer.

[0212] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof as described herein, polynucleotides as described herein, expression vectors as described herein, host cells as described herein, or pharmaceutical compositions as described herein for the treatment and / or prevention of CLDN-18.2-mediated diseases or conditions, preferably cancer.

[0213] In another aspect, the present invention provides a method for treating and / or preventing CLDN-18.2-mediated diseases or conditions, comprising administering to a subject in need an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, preferably, the disease or condition being cancer.

[0214] In some embodiments, the cancer is selected from drugs for 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.

[0215] In some embodiments, the administration methods of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-articular, intra-articular (e.g., in arthritic joints), inhalation, aerosol delivery, or intratumoral administration.

[0216] In some embodiments, the present invention provides the combined administration of therapeutically effective amounts of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. In some embodiments, the therapies include surgical treatment and / or radiation therapy.

[0217] In some embodiments, the methods or uses provided by the present invention further include administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to an individual. 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 additional therapeutic agent, such as a PD-1 antibody, a PD-L1 antibody, a LAG-3 antibody, and / or a CTLA-4 antibody.

[0218] In one embodiment, the cancer treatment method of the present invention further includes administration of a CLDN-18.2 expression stabilizer or enhancer. CLDN-18.2 expression is preferably on the cell surface of cancer cells. The CLDN-18.2 expression stabilizer or enhancer may be oxaliplatin and / or 5-FU.

[0219] Methods for diagnosis and detection

[0220] In another aspect, the present invention provides a method for detecting the presence of CLDN-18.2 in a sample using an antibody or an antigen-binding fragment thereof as described herein. The term "detection" as used herein includes both quantitative and 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.

[0221] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific embodiments indicate preferred embodiments of the invention, these descriptions and embodiments are provided by way of illustration only, as 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. For all purposes, all disclosures, patents, and patent applications cited herein, including in quotation marks, are incorporated herein by reference in their entirety.

[0222] Example

[0223] The following examples are provided to demonstrate and further explain some preferred embodiments and aspects of the invention, and should not be construed as limiting its scope.

[0224] Example 1: Preparation of recombinant human CLDN-18.2 (hCLDN-18.2) protein for anti-CLDN-18.2 antibody preparation Preparation

[0225] Plasmid HG20047-U containing the human CLDN-18 gene cDNA sequence was purchased from Sinocare. A partial human CLDN-18 gene fragment was amplified by PCR using the forward primer 5'-GTACgctagccaccTgaagagcggatccgctcttctTGCCCTGAAATGCATCCGCA TTGGCAGC-3' and the reverse primer 5'-GATCgcggccgccctgcaggTTACACATAGTCGTGCTTGGAAGGATAAG-3'. After double digestion with NheI and SbfI, the amplified fragment was cloned into a eukaryotic expression plasmid system (HXP) to obtain the construct HXP-CLDN18P. The partial hCLDN-18.2 gene sequence was synthesized at Nanjing GenScript Genetics, and the gene sequence was cloned into HXP-CLDN18P by BSPQI digestion, finally obtaining the construct HXP-hCLDN-18.2.

[0226] Example 2: Preparation of mouse hybridoma cells

[0227] 2.1 Immunized Animals

[0228] The HXP-hCLDN-18.2 obtained in Example 1 was mixed with an equal volume of immune adjuvant (Freud's adjuvant), and five 8-week-old female BALB / c mice were intramuscularly immunized. For the initial immunization, each mouse received 100 μg of DNA plasmid. Booster immunizations were then performed every 2 or 3 weeks for a total of 5 times, with each mouse receiving 50 μg of DNA plasmid per mouse. For the final booster immunization, stable CHO cells overexpressing CLDN-18.2 were injected intraperitoneally into each mouse, with 1e+7 cells administered.

[0229] 2.2 Cell Fusion

[0230] Four days after the final booster immunization, inguinal lymph nodes, popliteal lymph nodes, and spleen were harvested from mice. The lymphocyte-rich suspension was then collected by grinding the cells in DMEM medium and fused with mouse myeloma cells Sp2 / 0 (ATCC) using standard electroporation methods. The fusion product was cultured for 5 days in DMEM complete medium containing 1:50 HAT (hypoxanthine, methotrexate, and thymidine) to screen for successfully fused cells (i.e., hybridoma cells). The medium was then switched to DMEM complete medium containing 1:50 HT (hypoxanthine and thymidine) until screening was complete.

[0231] The DMEM complete culture medium is prepared as follows: 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U / mL penicillin and streptomycin + 1:100 OPI (oxaloacetic acid, pyruvate, and insulin). The incubator conditions are 8% CO2 and 37°C.

[0232] Example 3: Screening of mouse hybridoma cells and performance testing of the obtained anti-CLDN-18.2 mouse antibody

[0233] Among 3840 different polyclonal hybridoma cell lines, hybridoma cell lines that could bind to human CLDN-18.2 protein but not human CLDN18.1 protein were screened based on cellular-level binding reactions. After obtaining monoclonal hybridoma cell lines through subcloning, functional screening was performed based on in vitro ADCC and CDC activities, ultimately yielding 7 monoclonal hybridoma cell lines. The corresponding expressed antibodies 1H17, 2B19, 4G3, 9J24, 9O24, 10L8, and 10N10 specifically bound to recombinant human CLDN-18.2 protein but not recombinant human CLDN-18.1 protein, and exhibited strong ADCC and CDC activities.

[0234] Example 4: Determination of the variable region sequence of anti-CLDN-18.2 murine antibody (represented by Kabat or IMGT).

[0235] The DNA coding sequence corresponding to the variable region of the anti-CLDN-18.2 murine antibody was determined using a degenerate primer-based PCR method. Candidate hybridoma cells were cultured, centrifuged at 1000 rpm to collect cells, and total RNA was extracted with Trizol. First-strand cDNA was synthesized using this as a template, and then the corresponding variable region DNA coding sequence was 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 frame region and constant region of the antibody's variable region (Larrick, JW et al., 1990, Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). In a 50 μl reaction system, add 1 μl cDNA, 5 μl 10× PCR buffer, 1 μl each of upstream and downstream primers (25 pmol), 1 μl dNTP, 1 μl 25 mmol PL MgCl2, and 39 μl H2O. Pre-denature at 95 °C for 10 min, add 1 μl Taq enzyme, and cycle for PCR amplification. The reaction conditions are: denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, extension at 72 °C for 15 s, for a total of 32 cycles, followed by incubation at 72 °C for 10 min. The PCR product is recovered and purified. Sequencing of the amplified product yields the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-CLDN-18.2 mouse antibody.

[0236] Common sequences were searched in phylogenetic and rearranged Ig variable region sequence databases using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ). Based on Kabat (Wu, TT, and Kabat, EA 1970 J. Exp. Med., 132: 211-250) and the IMGT system (Lefranc M.-P. et al., 1999 Nucleic Acids Research, 27, 209-212), the amino acid sequences of complementarity-determining regions (CDRs) were determined by sequence annotation and by Internet-based sequence analysis (http: / / www.Imgt.org / IMGT_vquest / share / textes / index.html and http: / / www.ncbi.nlm.nih.gov / igblast / ).

[0237] The amino acid sequences of the light chain and heavy chain variable regions and CDRs of the anti-CLDN-18.2 murine antibody are shown in the table below:

[0238] Table 1. CDR and variable region amino acid sequences of anti-CLDN-18.2 murine antibody (KABAT protocol)

[0239]

[0240]

[0241] Example 5: Construction of anti-CLDN-18.2 chimeric antibody

[0242] Considering the expression level, activity, and type of antibodies expressed in hybridoma cells, the following mouse antibodies against CLDN-18.2 were selected for further analysis: 1H17, 1H17-2, 2B19, 4G3, 9J24, and 9O24.

[0243] The coding sequences for the Fc region of the heavy chain and the κ region of the light chain were cloned from human blood cells (from the Beijing Institute of Blood) and introduced into the pCDNA3.1 plasmid. The coding sequences for the variable regions of the heavy and light chains of the aforementioned anti-CLDN-18.2 murine antibodies were synthesized by Genscript. The coding sequences for the variable regions of the heavy and light chains of various anti-CLDN-18.2 murine antibodies were digested with Bspq I and introduced into the pCDNA3.1 plasmid containing the constant region coding sequences in various combinations shown in Table 2. Sequencing confirmed the correct cloning. Various chimeric heavy and light chain expression plasmids were mixed and paired for transfection into expression cells to obtain five anti-CLDN-18.2 chimeric antibodies, whose numbers and corresponding variable region amino acid sequences are shown in Table 2. Subsequent experimental materials were obtained from cells transfected with these plasmids.

[0244] Table 2. Numbering of anti-CLDN-18.2 chimeric antibodies and the origin of their heavy and light chain variable regions.

[0245] Chimeric antibody numbering VH VL Chi-JS012-2 1H17 VH 1H17-2 VL Chi-JS012-9 2B19 VH 2B19 VL Chi-JS012-10 2B19 VH 4G3 VL Chi-JS012-21 9J24 VH 2B19 VL Chi-JS012-27 9O24 VH 2B19 VL

[0246] Example 6: Detection of chimeric antibodies

[0247] 6.1 Antibody binding affinity:

[0248] The gastric cancer cell line NUGC4-CLDN-18.2, overexpressing human CLDN-18.2, was incubated with serially diluted concentrations of the aforementioned 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. After washing, the cells were incubated with fluorescently labeled secondary antibodies. Finally, fluorescence intensity was detected using a BD Canto II flow cytometer. A stronger fluorescence signal indicates higher antibody affinity for the target. Antibody dose-dependent binding curves were fitted using GraphPad. Figure 1 And calculate EC 50(Table 3) The positive control and negative control were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0249] like Figure 1 As shown in 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 surface of the gastric cancer cell line NUGC4-CLDN-18.2.

[0250] Table 3. Cellular-level affinity (EC50) of anti-CLDN-18.2 chimeric antibodies 50 )

[0251] chimeric antibodies <![CDATA[EC 50 (ng / mL)]]> Chi-JS012-2 4023 Chi-JS012-9 3218 Chi-JS012-10 3195 Chi-JS012-21 2373 Chi-JS012-27 2920

[0252] 6.2 Antibody-dependent cell-mediated cytotoxicity (ADCC):

[0253] The aforementioned anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21, and Chi-JS012-27, serially diluted to a series of concentrations, were co-incubated at 37°C for 6 h with target cells CHO-CLDN-18.2 (50,000 cells) overexpressing human CLDN-18.2 and effector cells Jurkat ADCC (100,000 cells) expressing NFAT-Luc and FcγRIIIaR. Then, the substrate one-glo was added, and the luciferase signal was detected using a microplate reader. Higher fluorescence readings indicate a stronger ADCC effect. Antibody dose-dependent ADCC effect curves were fitted using GraphPad. Figure 2 a) and 2b), the positive control and negative control were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0254] like Figure 2 As shown in a and 2b, Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21, and Chi-JS012-27 mediate the ECG effect of ADCC. 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, compared with the positive control IMAB362 ( Figure 2 a: 35.98; Figure 2 b: 34.41 ng / mL) is equivalent.

[0255] 6.3 Complement-dependent cytotoxicity (CDC):

[0256] 100,000 CHO-CLDN-18.2 target cells overexpressing human CLDN-18.2 were mixed with serially diluted concentrations of the aforementioned anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, and Chi-JS012-27, respectively. Ten-fold diluted complement serum (Quidel, cat#A113) was added, and the mixture was incubated at 37°C for 1 h. Finally, the cells were resuspended in PBS, and PI dye was added. After incubation at 4°C for 5 min, fluorescence intensity was detected using a BD Canto II flow cytometer. The antibody dose-dependent complement killing curve was fitted using GraphPad. Figure 3 The positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0257] like Figure 3 As shown, Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, and Chi-JS012-27 mediate the ECG CDC effect. 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).

[0258] Example 7: Humanization of the antibody variable region

[0259] To reduce the immunogenicity of antibodies, the variable region of the antibody was humanized and optimized. The optimization results are as follows:

[0260] HCDR2:CISSGSGTIYYADTVKG(SEQ ID NO:2) optimized to S ISSGSGTIYYAD S VKG (SEQ ID NO: 41) or YISSGSGTIYYADSVKG (SEQ ID NO: 72);

[0261] HCDR2: WINTYTGESTYADDFKG (SEQ ID NO:11) is optimized to WINTYTGESTYAD G F T G(SEQ IDNO:40),

[0262] HCDR2:TISGGDSYTYYPDSVRG (SEQ ID NO:29) is optimized to TISGGDSYTYYPDSV K G(SEQ IDNO:74);

[0263] HCDR3:AYYGNGFSF (SEQ ID NO:3) optimized to AYYGN A FSF (SEQ ID NO:73);

[0264] HCDR3:SYNGNSLPY (SEQ ID NO:30) optimized to SYN A NSLPY (SEQ ID NO:75);

[0265] This resulted in a series of humanized anti-CLDN-18.2 antibodies.

[0266] 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 were used for subsequent experiments. The CDR / variable region sequences of the above antibodies are shown in Tables 4-1 and 4-2, and the amino acid sequences and nucleotide coding sequences of the heavy / light chains are shown in Table 5.

[0267] The heavy and light chain variable region coding sequences of the humanized anti-CLDN-18.2 antibody 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 introduced into pCDNA3.1 plasmids already containing constant region coding sequences. Sequencing confirmed the correct clones. Various humanized heavy and light chain expression plasmids were mixed and paired for transfection into expression cells (CHOK1 18, Suzhou Junmeng). The expressed antibodies were recovered by centrifugation and purified using standard methods to obtain the humanized anti-CLDN-18.2 antibodies listed in Tables 4-1 and 4-2.

[0268] Among them, the humanized antibody JS012-Chi2-hu2-v3-2 underwent process optimization by adding a glycoform regulator (CDFS01, Shanghai Aopumai Biotechnology Co., Ltd.) to cell culture, resulting in the defucosylated antibody JS012-Chi2-hu2-v3-2a. Antibody JS012-Chi2-hu2-v3-2a is a humanized anti-CLDN-18.2 antibody with fucosylation below 30%.

[0269] Table 4-1. Variable region and CDR sequence of humanized anti-CLDN-18.2 antibody (KABAT protocol)

[0270]

[0271]

[0272] Table 4-2. Variable region and CDR sequence of humanized anti-CLDN-18.2 antibody (KABAT protocol)

[0273]

[0274] Table 5. Amino acid and nucleotide coding sequences of the heavy and light chains of the humanized anti-CLDN-18.2 antibody

[0275]

[0276]

[0277] Example 8: Detection of humanized anti-CLDN-18.2 antibody

[0278] 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 CLDN-18.2 expressed on the cell surface was detected by FACS. NUGC4-CLDN-18.2 cells were incubated with serially diluted concentrations of the aforementioned humanized anti-CLDN-18.2 antibodies, then washed and incubated with fluorescently labeled secondary antibodies. Fluorescence signals were detected by FACS; stronger fluorescence signals indicated higher antibody affinity, i.e., higher target binding activity. Antibody binding curves were fitted using GraphPad. Figure 4a , 4b (and 4c), the positive control and negative control were IMAB362 and anti-KLHhu-IgG1 antibody, respectively.

[0279] like Figure 4a , 4b As shown in 4c, JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2, and JS012-Chi27-hu6-v3-2 can all bind to human CLDN-18.2 highly expressed on the surface of the gastric cancer cell line NUGC4. Among them, the EC50 of JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, and JS012-Chi2-hu2-v3-2 is... 50 Comparable to the positive control.

[0280] Example 9: ADCC of humanized anti-CLDN-18.2 antibody

[0281] Humanized anti-CLDN-18.2 antibody is a human IgG1 subtype that mediates ADCC (antibody-dependent cell-mediated death and cancer cell death). The antibody's Fc terminus binds to the FcγIIIaR receptor on the surface of NK cells, activating NK cells to kill target cells. The in vivo ADCC effect was mimicked using a reporter gene system via the NFAT pathway luciferase system expressing FcγIIIaR. Humanized anti-CLDN-18.2 antibody was co-incubated with target cells (CHO-CLDN-18.2 cells) and Jurkat ADCC effector cells. Signals were detected using a microplate reader after the addition of the substrate one-glo. Data were analyzed using GraphPad to compare dose-dependent antibody ADCC effects. Figure 5a , 5b (and 5c). The positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0282] like Figure 5a , 5b As shown in 5c, JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2 and JS012-Chi27-hu6-v3-2 mediate the ECG of ADCC effect. 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 significantly better than the positive control IMAB362.

[0283] Example 10: Humanized anti-CLDN-18.2 antibody in CDC

[0284] Humanized anti-CLDN-18.2 antibody can also mediate the CDC effect, killing target cells by forming a membrane attack complex. Complement serum (1:10 dilution) was co-incubated with humanized anti-CLDN-18.2 antibody and target cells (CHO-CLDN-18.2, 100,000 cells) at 37°C for 1 hour. Cell survival and killing were assessed 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 by the relative killing rate. Figure 6a , 6b (and 6c). The positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.

[0285] like Figure 6a , 6bAs shown in 6c, JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2 and JS012-Chi27-hu6-v3-2 mediate the ECG of the CDC effect. 50 The values ​​were 134.2 ng / mL, 86.04 ng / mL, 168.9 ng / mL, 166.5 ng / mL and 714.9 ng / mL, respectively. Among them, JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2 and JS012-Chi2-hu2-v3-2 were significantly better than the positive control IMAB362.

[0286] Example 11: Humanized anti-CLDN-18.2 antibody against human gastric cancer transplanted in M-NSG mice Inhibitory effect of MKN45hClaudin18.2Mixeno on tumor growth

[0287] 1. Test Objective

[0288] The antitumor effect of the present invention JS012-Chi2-hu2 v3-2a in the human gastric cancer MKN45 hClaudin18.2Mixeno subcutaneous transplantation model was evaluated.

[0289] 2. Testing Process

[0290] 5×10 6 Mixed 5 × 10 MKN45 hClaudin18.2 tumor cells (Shanghai Nuobai Biotechnology) 6 One PBMC cell (Shanghai Aoneng Biotechnology), 0.2 ml / mouse (RPMI 1640 (Gibco) medium containing cells and 50% matrix gel), was subcutaneously inoculated into the right posterior back of M-NSG mice (Shanghai Southern Model Biotechnology Co., Ltd.). Fifty mice were randomly selected based on body weight and divided into 5 groups of 10 mice each.

[0291] Anti-KLH hIgG1 negative control group, 10 mg / kg;

[0292] JS012-Chi2-hu2 v3-2a treatment group, 1 mg / kg;

[0293] JS012-Chi2-hu2 v3-2a treatment group, 3 mg / kg;

[0294] JS012-Chi2-hu2 v3-2a treatment group, 10 mg / kg;

[0295] IMAB362 positive control group, 10 mg / kg.

[0296] Four hours after tumor cell inoculation, mice were administered the drug via intraperitoneal injection on the day of grouping. The drug was administered twice weekly for nine consecutive weeks, ending the experiment three days after the last administration. Tumor volume and body weight were measured twice weekly, and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated (TGI% = [1-T / C] × 100%). (T: mean tumor volume at the experimental endpoint in the treatment group or positive control group; C: mean tumor volume at the experimental endpoint in the negative control group).

[0297] like Figure 7 As shown, at the experimental endpoint, the mean tumor volume in the anti-KLH hIgG1 negative control group was 1276±228 mm. 3 The mean tumor volume of JS012-Chi2-hu2a v3-2 at doses of 1, 3, and 10 mg / kg was 235 ± 25 mm. 3 243±33mm 3 and 343±63mm 3 The TGI values ​​were 81.6%, 81.0%, and 73.1%, demonstrating significant tumor-suppressive effects. The mean tumor volume at a dose of 10 mg / kg was 361 ± 73 mm. 3 The TGI was 71.7%. Under the same dosage conditions (10 mg / kg), JS012-Chi2-hu2 v3-2a showed slightly better antitumor activity than IMAB362.

[0298] Example 12: Humanized anti-CLDN-18.2 antibody against human pancreatic cancer hCLDN18.2 transplanted into CB-17SCID mice. MIA PaCa-2's inhibitory effect on tumor growth

[0299] 1. Test Objective

[0300] The antitumor effect of the present invention JS012-Chi2-hu2 v3-2a in a human pancreatic cancer hCLDN18.2 MIA PaCa-2 subcutaneous transplantation model was evaluated.

[0301] 2. Testing Process

[0302] 5×10 6 One hCLDN18.2 MIA PaCa-2 cell (Accurusbio-C3002) was inoculated subcutaneously into the right posterior back of CB-17SCID mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) at a dose of 0.2 ml / mouse (dMEM (Gibco) containing cells and 50% matrix gel). The tumor volume was approximately 89 mm². 3 At that time, 40 animals were randomly selected and divided into 5 groups of 8 animals each, based on tumor volume.

[0303] Anti-KLH hIgG1 negative control group, 3 mg / kg;

[0304] IMAB362 positive control group, 3 mg / kg;

[0305] JS012-Chi2-hu2 v3-2a treatment group, 0.3 mg / kg;

[0306] JS012-Chi2-hu2 v3-2a treatment group, 1 mg / kg;

[0307] JS012-Chi2-hu2 v3-2a treatment group, 3mg / kg.

[0308] Mice were administered the drug on the same day of grouping, via intraperitoneal injection in all groups. The drug was administered twice weekly for six consecutive weeks, ending the experiment four days after the last administration. Tumor volume and body weight were measured three times weekly, and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated (TGI% = [1 - (Ti - T0) / (Vi - V0)] × 100%). (Ti: mean tumor volume on day i of administration in the treatment group or positive control group; T0: mean tumor volume on day 0 of administration in the treatment group or positive control group; Vi: mean tumor volume on day i of administration in the negative control group; V0: mean tumor volume on day 0 of administration in the negative control group).

[0309] like Figure 8 As shown, at the experimental endpoint, the mean tumor volume in the anti-KLH hIgG1 group was 2235±145 mm3. The mean tumor volume of IMAB362 at a dose of 3 mg / kg was 465±74 mm3, with a TGI of 82.5%. The mean tumor volumes of JS012-Chi2-hu2v3-2a at doses of 0.3, 1, and 3 mg / kg were 1455±142 mm3, 673±153 mm3, and 74±19 mm3, respectively, with TGIs of 36.3%, 72.8%, and 100.6%, demonstrating significant tumor-suppressing effects. Under the same dose conditions (3 mg / kg), the tumor-suppressing effect of JS012-Chi2-hu2v3-2a was significantly superior to that of IMAB362.

Claims

1. An anti-CLDN-18.2 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:41, and SEQ ID NO:3, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:60; and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

61.

3. The antibody or antigen-binding fragment thereof as described in claim 1, comprising a heavy chain and a light chain, wherein, The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 64; and the light chain comprises the amino acid sequence shown in SEQ ID NO:

65.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; and the antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, scFv, or F(ab')2.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, wherein the antibody is any IgG subtype.

6. The antibody or antigen-binding fragment thereof as claimed in claim 5, wherein the antibody is IgG1, IgG2, IgG3 or IgG4.

7. The antibody or antigen-binding fragment thereof as claimed in claim 5, wherein the antibody is low- or non-fucosylated.

8. A polynucleotide encoding an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

9. An expression vector comprising the polynucleotide as described in claim 8.

10. The expression vector as described in claim 9, wherein the vector is a eukaryotic expression vector.

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

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

13. The host cell of claim 12, wherein the host cell is a mammalian cell.

14. A method for preparing an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-3, the method comprising culturing a host cell as claimed in any one of claims 11-13 under conditions suitable for expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

15. A pharmaceutical composition comprising an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-7, a polynucleotide as claimed in claim 8, an expression vector as claimed in claim 9 or 10, a host cell as claimed in any one of claims 11-13, and a pharmaceutically acceptable carrier or excipient.

16. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-7, the polynucleotide as described in claim 8, the expression vector as described in claim 9 or 10, the host cell as described in any one of claims 11-13, or the pharmaceutical composition as described in claim 15 in the preparation of a medicament for the treatment and / or prevention of gastric cancer or pancreatic cancer.

17. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-7, a polynucleotide as claimed in claim 8, an expression vector as claimed in claim 9 or 10, a host cell as claimed in any one of claims 11-13 or a pharmaceutical composition as claimed in claim 15, and one or more additional therapeutic agents.

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