An anti-Claudin18.2 monoclonal antibody and its application

Abstract

The present invention belongs to the fields of biotechnology and biopharmaceutical technology, and particularly relates to an anti-Claudin18.2 monoclonal antibody and its application. The heavy chain variable region of the monoclonal antibody of the present invention comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO.1 to SEQ ID NO.3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO.4 to SEQ ID NO.6. The anti-Claudin18.2 monoclonal antibody of the present invention has a higher affinity than the existing commercial IMB362 antibody, and the anti-Claudin18.2 monoclonal antibody can specifically bind to tumor cells and can target and bind to tumors with high expression of Claudin18.2 in mice.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology and biomedicine, and particularly relates to an anti-Claudin18.2 monoclonal antibody and its application. Background Art

[0002] Claudin18 is a membrane protein that forms tight junctions and is encoded by the human CLDN18 gene, belonging to the Claudins family members. Claudin-18 is specifically expressed in lung and gastric tissues, has a complete membrane localization on lung and gastric epithelial cells, and electron microscopy shows that it is mainly concentrated at the boundaries of these cells. These characteristics indicate that Claudin-18 may play an important role in the structure and function of tight junctions in the lung and stomach.

[0003] Claudin18 has two subtypes, Claudin18.1 and Claudin18.2. The Claudin18.2 subtype is a gastric-specific subtype. Claudin18.2 is a highly selective molecule and is only widely expressed in cancer cells, making it an ideal target. Claudin18.2 is usually buried in the gastric mucosa and monoclonal antibodies in normal tissues can hardly contact it. The occurrence of malignant tumors will cause the disruption of tight junctions, exposing the Claudin18.2 epitopes on the surface of tumor cells, becoming specific targets. Therefore, Claudin18.2 confers specificity to targeted therapy. And recent studies have found that the expression of Claudin18.2 in pancreatic cancer (50%), esophageal cancer and lung cancer also shows the potential for diagnosing and treating other tumors.

[0004] Based on the diagnostic and therapeutic advantages of Claudin18.2 in tumors, the research on anti-Claudin18.2 antibodies has gradually become popular and extensive, and a series of murine and human antibodies against Claudin18.2 have been produced. In particular, human antibodies obtained by modifying murine antibodies have good tumor binding ability and inhibitory activity. However, many murine antibodies have the problem of low affinity after humanization. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-Claudin18.2 monoclonal antibody and its application, and the anti-Claudin18.2 monoclonal antibody has higher affinity compared with existing anti-Claudin18.2 monoclonal antibodies.

[0006] The present invention provides an anti-Claudin18.2 monoclonal antibody, including a heavy chain variable region and a light chain variable region;

[0007] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively;

[0008] The light chain variable region includes LCDR1, LCDR2 and LCDR3, and the amino acid sequences of LCDR1 and LCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.5 respectively, and the amino acid sequence of LCDR2 is WAS.

[0009] Preferably, the heavy chain variable region includes a heavy chain hypervariable region and a heavy chain framework region, and the sequence of the heavy chain framework region is derived from a murine antibody or a human antibody;

[0010] The light chain variable region includes a light chain hypervariable region and a light chain framework region, and the sequence of the light chain framework region is derived from a murine antibody or a human antibody;

[0011] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9;

[0012] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13.

[0013] Preferably, the anti-Claudin18.2 monoclonal antibody includes a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9, and a light chain variable region with the amino acid sequence shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13.

[0014] Preferably, the anti-Claudin18.2 monoclonal antibody includes the following combinations of heavy chain variable region and light chain variable region:

[0015] 1) The heavy chain variable region shown in SEQ ID NO.6 and the light chain variable region shown in SEQ ID NO.10;

[0016] 2) The heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.11;

[0017] 3) The heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.12;

[0018] 4) The heavy chain variable region shown in SEQ ID NO.9 and the light chain variable region shown in SEQ ID NO.13;

[0019] Preferably, when the anti-Claudin18.2 monoclonal antibody comprises 1) the said combination, the anti-Claudin18.2 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO.14, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO.15;

[0020] Preferably, when the anti-Claudin18.2 monoclonal antibody comprises 2), 3) or 4) the said combination, the anti-Claudin18.2 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO.26, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO.27.

[0021] Preferably, the anti-Claudin18.2 monoclonal antibody comprises a single-chain antibody, a chimeric antibody, a multimeric antibody or a CDR-grafted antibody.

[0022] The present invention also provides a biological material, which comprises an expression cassette, a recombinant vector, a recombinant engineering bacterium or a recombinant cell line constructed from a nucleotide sequence or an amino acid sequence;

[0023] The amino acid sequence comprises the amino acid sequence of the anti-Claudin18.2 monoclonal antibody described in the above technical solution;

[0024] The nucleotide sequence comprises the nucleotide sequence encoding the anti-Claudin18.2 monoclonal antibody described in the above technical solution.

[0025] The present invention also provides the use of the anti-Claudin18.2 monoclonal antibody described in the above technical solution or the biological material described in the above technical solution in the preparation of tumor-related products;

[0026] Preferably, the tumor-related products include products with one or more functions of tumor diagnosis, prevention, treatment and prognosis.

[0027] Preferably, the tumor includes gastric cancer.

[0028] The present invention also provides a biological product, and the active ingredient in the biological product comprises the anti-Claudin18.2 monoclonal antibody described in the above technical solution or the biological material described in the above technical solution.

[0029] Beneficial effects:

[0030] The present invention provides an anti-Claudin18.2 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively; the light chain variable region comprises LCDR1 and LCDR3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.5 respectively, and the amino acid sequence of LCDR2 is WAS. The anti-Claudin18.2 monoclonal antibody containing the heavy chain variable region and the light chain variable region of the present invention has a higher affinity than the existing commercial IMB362 antibody, and the anti-Claudin18.2 monoclonal antibody can specifically bind to tumor cells and has a relatively high binding force with tumors. Brief Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0032] Figure 1 It is a specific detection result diagram of m1D6 with different dilution ratios for Claudin18.2-CHO cells in Example 2;

[0033] Figure 2 It is the specific detection result of m1D6 for NUGC4 cells in Example 2;

[0034] Figure 3 It is the detection result of the binding force of m1D6 to the cell membrane surface of NUGC4 cells in Example 2;

[0035] Figure 4 It is the in vivo imaging result after injecting m1D6 into mice in Example 2;

[0036] Figure 5 It is the affinity result of detecting humanized antibodies h1D6-1, h1D6-2, h1D6-3 by flow cytometry in Example 3;

[0037] Figure 6 It is the affinity result of detecting humanized antibodies h1D6-1, h1D6-2, h1D6-3 by ELISA method in Example 3. Detailed Embodiments

[0038] The present invention provides an anti-Claudin18.2 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region;

[0039] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO.1 to SEQ ID NO.3 respectively;

[0040] The light chain variable region includes LCDR1, LCDR2, and LCDR3. The amino acid sequences of LCDR1 and LCDR3 are shown as SEQ ID NO.4 to SEQ ID NO.5 respectively, and the amino acid sequence of LCDR2 is WAS.

[0041] In the present invention, HCDR1, HCDR2, and HCDR3 constitute the heavy chain hypervariable region in the heavy chain variable region. The amino acid sequence shown as SEQ ID NO.1 in the present invention is specifically GYTFTSYR, the amino acid sequence shown as SEQ ID NO.2 is specifically IDPSNSYT, and the amino acid sequence shown as SEQ ID NO.3 is specifically ARLGRGNTLGY.

[0042] In the present invention, LCDR1, LCDR2, and LCDR3 constitute the light chain hypervariable region in the light chain variable region. The amino acid sequence of SEQ ID NO.4 in the present invention is specifically QSLLNSGNQKNY, and the amino acid sequence of SEQ ID NO.5 is specifically QNGYSYPFT.

[0043] In the present invention, the heavy chain variable region preferably includes a heavy chain hypervariable region and a heavy chain framework region, and the sequence of the heavy chain framework region preferably originates from a murine antibody or a human antibody. On this basis, the amino acid sequence of the heavy chain variable region in the present invention is preferably shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, or SEQ ID NO.9.

[0044] The amino acid sequence shown as SEQ ID NO.6 in the present invention is specifically The amino acid sequences of HCDR1, HCDR2, and HCDR3 are successively represented by the three underlined and bold amino acid sequences from left to right; the four non-underlined contents from left to right represent the four heavy chain framework region sequences in the heavy chain variable region shown as SEQ ID NO.6, and the four heavy chain framework region sequences originate from a murine antibody.

[0045] The amino acid sequence shown as SEQ ID NO.7 in the present invention is specifically: The three underlined and bolded amino acid sequences from left to right represent the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively; the four non-underlined contents from left to right represent four heavy chain framework region sequences in the heavy chain variable region shown in SEQ ID NO.7, and the four heavy chain framework region sequences are derived from a human antibody. The nucleotide sequence of the present invention for encoding the amino acids of the heavy chain variable region shown in SEQ ID NO.7 is as shown in SEQ ID NO.20, specifically 5’-CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGAGATCGACCCCAGCAACAGCTACACCAACTACAACCAGAACTTCAGGGGCAGGAGCACCCTGACCGTGGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGCTGGGCAGGGGCAACACCCTGGGCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC-3’.

[0046] The amino acid sequence shown in SEQ ID NO.8 of the present invention is specifically: The three underlined and bolded amino acid sequences from left to right represent the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively; the four non-underlined contents from left to right represent the four heavy chain framework region sequences in the heavy chain variable region shown in SEQ ID NO.8, and the four heavy chain framework region sequences are derived from a human antibody. The nucleotide sequences for encoding the amino acids of the heavy chain variable region shown in SEQ ID NO.8 of the present invention are respectively shown in SEQ ID NO.21, specifically 5’-CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGAGATCGACCCCAGCAACAGCTACACCAACTACAACCAGAACTTCAGGGGCAGGAGCACCCTGACCGTGGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGCTGGGCAGGGGCAACACCCTGGGCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC-3’.

[0047] The amino acid sequence shown in SEQ ID NO.9 of the present invention is specifically as follows: The three underlined and bolded amino acid sequences from left to right represent the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively; the four non-underlined contents from left to right represent the four heavy chain framework region sequences in the heavy chain variable region shown in SEQ ID NO.9, and the four heavy chain framework region sequences are derived from a human antibody. The nucleotide sequence for encoding the amino acids of the heavy chain variable region shown in SEQ ID NO.9 in the present invention is as shown in SEQ ID NO.22, specifically 5’-CAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTACACCTTCACCAGCTACAGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGAGATCGACCCCAGCAACAGCTACACCAACTACAACCAGAACTTCAGGGGCAGGAGCACCCTGAGCGTGGACAAGGCCAAGAGCAGCGCCTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCTGGGCAGGGGCAACACCCTGGGCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC-3’。

[0048] In the present invention, the light chain variable region includes a light chain hypervariable region and a light chain framework region, and the light chain framework region sequence is derived from a murine antibody or a human antibody. On this basis, the amino acid sequence of the light chain variable region in the present invention is as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, or SEQ ID NO.13.

[0049] The specific amino acid sequence shown in SEQ ID NO.10 in the present invention is: The three underlined and bolded amino acid sequences from left to right represent the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively; the four non-underlined contents from left to right represent the four light chain framework region sequences in the light chain variable region shown in SEQ ID NO.10, and the four light chain framework region sequences are derived from a murine antibody.

[0050] The specific amino acid sequence shown in SEQ ID NO.11 in the present invention is: The amino acid sequences in bold with triple underlines from left to right represent the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively; the four pieces of content without underlines from left to right represent the four light chain framework region sequences in the light chain variable region shown in SEQ ID NO.11, and the four light chain framework region sequences are derived from a human antibody. The nucleotide sequence of the present invention for encoding the amino acids of the light chain variable region shown in SEQ ID NO.11 is as shown in SEQ ID NO.23, specifically 5’-GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGAGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGGGAGAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGGCTACAGCTACCCCTTCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAG-3’。

[0051] The amino acid sequence shown in SEQ ID NO.12 of the present invention is specifically: The amino acid sequences of LCDR1, LCDR2, and LCDR3 are represented by the three segments of underlined and bolded amino acid sequences from left to right; the four segments without underlining from left to right represent the four light chain framework region sequences in the light chain variable region shown in SEQ ID NO.12, and the four light chain framework region sequences are derived from a human antibody. The nucleotide sequence of the present invention for encoding the amino acids of the light chain variable region shown in SEQ ID NO.12 is as shown in SEQ ID NO.24, specifically 5’-GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGAACGGCTACAGCTACCCCTTCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAG-3’.

[0052] The amino acid sequence shown in SEQ ID NO.13 of the present invention is specifically:

[0053] The three underlined and bolded amino acid sequences from left to right represent the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively; the four non-underlined contents from left to right represent the four light chain framework region sequences in the light chain variable region shown in SEQ ID NO. 13, and the four light chain framework region sequences are derived from a human antibody. The nucleotide sequence for encoding the amino acids of the light chain variable region shown in SEQ ID NO. 13 in the present invention is as shown in SEQ ID NO. 25, specifically 5’-GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGAACGGCTACAGCTACCCCTTCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAG-3’.

[0054] In the present invention, the anti-Claudin 18.2 monoclonal antibody preferably comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9, and a light chain variable region having the amino acid sequence shown in SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, or SEQ ID NO. 13, and more preferably comprises a combination of the heavy chain variable region and the light chain variable region as described in 1) to 4) below: 1) the heavy chain variable region shown in SEQ ID NO. 6 and the light chain variable region shown in SEQ ID NO. 10; 2) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 7 and the light chain variable region having the amino acid sequence shown in SEQ ID NO. 11; 3) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 8 and the light chain variable region having the amino acid sequence shown in SEQ ID NO. 12; 4) the heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 9 and the light chain variable region having the amino acid sequence shown in SEQ ID NO. 13.

[0055] In the present invention, the anti-Claudin18.2 monoclonal antibody preferably includes a single-chain antibody, a chimeric antibody, a multimeric antibody or a CDR-grafted antibody, more preferably includes a single-chain antibody or a chimeric antibody, and even more preferably is a chimeric antibody.

[0056] In the present invention, when the anti-Claudin18.2 monoclonal antibody includes 1) the combination, the anti-Claudin18.2 monoclonal antibody preferably further includes a heavy-chain constant region and a light-chain constant region; the heavy-chain variable region and the heavy-chain constant region form the heavy chain of the anti-Claudin18.2 monoclonal antibody; the light-chain variable region and the light-chain constant region form the light chain of the anti-Claudin18.2 monoclonal antibody; the heavy chain and the light chain of the anti-Claudin18.2 monoclonal antibody thus formed are respectively the heavy chain and the light chain of a murine anti-Claudin18.2 monoclonal antibody.

[0057] The amino acid sequence of the heavy chain constant region of the present invention is shown in SEQ ID NO. 14, specifically AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK: The amino acid sequence of the light chain constant region is shown in SEQ ID NO. 15, specifically RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC, that is, the heavy chain is composed of the heavy chain variable region shown in SEQ ID NO. 6 and the heavy chain constant region shown in SEQ ID NO. 14, and the amino acid sequence of the heavy chain is preferably as SEQ ID NO.As shown in , specifically: MGWSCIILFLVSTATGVHSQVQLQQPGPELVMPGASVKLSCKASGYTFTSYRMHWVKQRPGQGLEWIGEIDPSNSYTNYNQNFRGRSTLTVDKSSSTAYMQLSSLTSGDSAVYYCARLGRGNTLGYWGQGTSVTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK*; the nucleotide sequence encoding the heavy chain is preferably as shown in SEQ ID NO. 17, specifically. The bold underlined part is the nucleotide sequence encoding the heavy chain constant region shown in SEQ ID NO. 14, and the remaining part is the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO. 6; the light chain variable region shown in SEQ ID NO. 10 and the light chain constant region shown in SEQ ID NO. 15 form the light chain. The amino acid sequence of the light chain is preferably as shown in SEQ ID NO. 18, specifically: MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTVGCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIRSVQAEDLAVYYCQNGYSYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*; the nucleotide sequence encoding the light chain is preferably as shown in SEQ ID NO. 19, specifically The nucleotide sequence of the coding light chain constant region is in bold and underlined, and the remaining part is the nucleotide sequence of the coding light chain variable region shown in SEQ ID NO.7.

[0058] When the anti-Claudin18.2 monoclonal antibody comprises the combination described in 2), 3) or 4), the anti-Claudin18.2 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region. The heavy chain of the anti-Claudin18.2 monoclonal antibody is composed of the heavy chain constant region and the heavy chain variable region; the light chain of the anti-Claudin18.2 monoclonal antibody is composed of the light chain constant region and the light chain variable region; the heavy chain of the anti-Claudin18.2 monoclonal antibody and the light chain of the anti-Claudin18.2 monoclonal antibody are respectively the heavy chain and the light chain of a human-derived anti-Claudin18.2 monoclonal antibody. The amino acid sequence of the heavy chain constant region (H-GAMMA-1) of the present invention is preferably as shown in SEQ ID NO.26, specifically ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and the nucleotide sequence encoding the amino acid of the heavy chain constant region shown in SEQ ID NO.26 is preferably as shown in SEQ ID NO.28,Specifically, 5’-GCCTCTACAAAGGGCCCCAGCGTGTTTCCACTGGCCCCCTCTAGCAAGTCTACCAGCGGCGGCACAGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGTCTTGGAACAGCGGCGCCCTGACCTCCGGGGTGCACACATTTCCAGCCGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGCTCCGTGGTGACCGTGCCCTCTAGCTCCCTGGGCACCCAGACATACATCTGCAACGTGAATCACAAGCCAAGCAATACAAAGGTGGACAAGAAGGTCGAGCCCAAGAGCTGTGATAAGACCCACACATGCCCCCCTTGTCCTGCTCCAGAGCTGCTGGGCGGCCCTAGCGTGTTCCTGTTTCCACCCAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCCGAGGTGACATGCGTGGTGGTGGACGTGTCTCACGAGGATCCTGAGGTGAAGTTTAACTGGTATGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTATAACTCCACCTACCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGCCCTGCCCGCCCCTATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTAGAGAGCCACAGGTGTATACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTACCCTTCTGATATCGCCGTGGAGTGGGAGAGCAATGGCCAGCCAGAGAACAATTATAAGACCACACCCCCTGTGCTGGACTCCGATGGCTCTTTCTTTCTGTACAGCAAGCTGACCGTGGATAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGTTCTGTGATGCACGAAGCCCTGCATAATCACTATACTCAGAAATCCCTGTCACTGTCACCTGGAAAG-3’.

[0059] The amino acid sequence of the light chain constant region (L-KAPPA) of the present invention is as shown in SEQ ID NO.27, specifically TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; the nucleotide sequence encoding the amino acid sequence of the light chain constant region shown in SEQ ID NO.27 is preferably as shown in SEQ ID NO.29, specifically 5’-ACAGTGGCCGCCCCATCCGTGTTCATCTTTCCACCCTCTGACGAGCAGCTGAAGTCTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAGGCCAAGGTCCAGTGGAAGGTGGATAACGCCCTGCAGTCCGGCAATTCTCAGGAGAGCGTGACCGAGCAGGACTCCAAGGATTCTACATATAGCCTGTCTAGCACCCTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTATGCCTGTGAAGTCACTCATCAGGGGCTGTCATCACCTGTCACTAAGTCATTCAATAGAGGCGAATGC-3’。

[0060] The present invention also provides a biological material, which includes an expression cassette, a recombinant vector, a recombinant engineering bacterium or a recombinant cell line constructed from a nucleotide sequence or an amino acid sequence; the amino acid sequence includes the amino acid sequence of the anti-Claudin18.2 monoclonal antibody described in the above technical solution; the nucleotide sequence includes the nucleotide sequence encoding the anti-Claudin18.2 monoclonal antibody described in the above technical solution.

[0061] The initial vector in the recombinant vector of the present invention preferably includes a plasmid vector or a lentivirus, and more preferably a plasmid vector. The present invention has no special limitation on the construction method of the biological material, and conventional genetic engineering means in the art can be used.

[0062] The anti-Claudin18.2 monoclonal antibody containing the heavy chain variable region and the light chain variable region of the present invention has a higher affinity than the existing commercial IMB362 antibody, and the anti-Claudin18.2 monoclonal antibody can specifically bind to tumor cells, and both the tumor activity and the binding force are relatively high.

[0063] Based on the above advantages, the present invention also provides the use of the anti-Claudin18.2 monoclonal antibody described in the above technical solution or the biological material described in the above technical solution in the preparation of tumor-related products. The tumor-related products of the present invention preferably include products with one or more functions of tumor diagnosis, prevention, treatment, and prognosis, more preferably products with one or more of tumor diagnosis, prevention, and treatment, and even more preferably products with tumor diagnosis and / or treatment. The tumors of the present invention preferably include gastric cancer, pancreatic cancer, or colon cancer, and more preferably gastric cancer.

[0064] The present invention also provides a biological product, and the active ingredient in the biological product includes the anti-Claudin18.2 monoclonal antibody described in the above technical solution or the biological material described in the above technical solution.

[0065] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] Example 1

[0067] The preparation and screening of the anti-Claudin18.2 monoclonal antibody are as follows:

[0068] 1. Mouse immunization and acquisition of murine monoclonal antibody sequences:

[0069] The Claudin18.2 gene knockout C57 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized subcutaneously at multiple points with 60 μg of the pCDNA3.4 plasmid containing the full-length hClaudin18.2 gene per mouse per time, once a week. After 4 weeks of immunization, the mice with a blood antibody titer reaching more than 1k were sacrificed, and spleen cells were fused with SP2.0 cells. The fusion cells were subjected to titer detection, and positive fusion cells were selected and monoclonal cells were picked by the limiting dilution method.

[0070] Flow cytometry was used to detect the specificity of the antibody in the supernatant of monoclonal cells. The genes of the finally screened murine monoclonal antibody were extracted and antibody light and heavy chain expression plasmids were constructed for fermentation and purification of the antibody. The specific steps were as follows: The constructed antibody light and heavy chain expression plasmids were paired, and PEI reagent was used as the transfection reagent. At the same time, a pair of light and heavy chain expression plasmids were transiently transfected into 293T cells for antibody protein fermentation. The antibody light and heavy chains were naturally paired during the fermentation process, and the culture medium was collected. The culture medium was filtered through a 0.22 μm sterile filter membrane, and the culture supernatant was obtained and purified by affinity chromatography.

[0071] The purification method is as follows: (1) Equilibrate a 1 mL Protein A affinity column with 20 mM PB, pH 7.0 at a flow rate of 1.0 mL / min; (2) Load 50 mL of the culture solution at a flow rate of 1 mL / min and equilibrate with the equilibration solution; (3) Elute with 0.1 M glycine-HCl, pH 2.7 at a flow rate of 1 mL / min and collect the absorption peak at 280 nm; (4) Equilibrate a Superdex 200 Increase 10 / 300 GL column with 20 - 50 mM PB, 150 mM NaCl, pH 6.7 at a flow rate of 1 mL / min; (5) Load the sample and collect 2 mL of the absorption peak at 280 nm.

[0072] Perform HPLC analysis on the purified sample, detect the received peak, detect the concentration of the purified eluted peak, and calculate the protein amount.

[0073] The amino acid sequence of the heavy chain of the obtained anti-Claudin 18.2 mouse monoclonal antibody m1D6 is shown in SEQ ID NO.16, wherein the amino acids of the heavy chain variable region and the heavy chain constant region are shown in SEQ ID NO.6 and SEQ ID NO.14 respectively; the amino acid sequence of the light chain is shown in SEQ ID NO.18, wherein the amino acids of the light chain variable region and the heavy chain constant region are shown in SEQ ID NO.10 and SEQ ID NO.15 respectively.

[0074] 2. Humanization of the antibody and preparation of humanized antibodies:

[0075] Perform humanization design on the framework regions of the variable regions of the mouse antibody sequence to obtain the heavy chain variable regions SEQ ID NO.7 - 9 and the light chain variable regions SEQ ID NO.11 - 13. Ligate the designed nucleotide sequences of the humanized antibody light chain variable region and heavy chain variable region with the human IgG1 constant region sequence, the heavy chain constant region SEQ ID NO.26 and the light chain constant region SEQ ID NO.27 of IgG1. Synthesize the complete antibody sequence by total gene synthesis method, ligate it into the expression vector pcDNA3.4 respectively, and transfect it into HEK293F cells for expression to obtain the applicant's humanized antibodies h1D6-1, h1D6-2, h1D6-3 respectively; among them, the humanized antibodies h1D6-1, h1D6-2, h1D6-3 correspond in sequence to humanized antibody 1 containing a variable region composed of the heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.11, a humanized antibody containing a variable region composed of the heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.12, and a humanized antibody containing a variable region composed of the heavy chain variable region shown in SEQ ID NO.9 and the light chain variable region shown in SEQ ID NO.13.

[0076] Example 2

[0077] 1. Activity identification of anti-Claudin18.2 mouse monoclonal antibody m1D6 in Example 1

[0078] 1.1 Detection of the affinity of m1D6 monoclonal antibody to cells by flow cytometry:

[0079] The Claudin18.2-CHO high-expression cell line and Claudin18.1-CHO high-expression cell line were constructed, expressed and cultured in this laboratory. The construction and culture methods of the Claudin18.2-CHO high-expression cell line and Claudin18.1-CHO high-expression cell line are as follows:

[0080] The base sequences expressing mClaudin18.1 and mClaudin18.2 were cloned into the PHBLV lentiviral vector backbone and placed under the promoter of EF1α (EF-1α) to form PHBLV-EF1α-mClaudin18.1 and PHBLV-EF1α-mClaudin18.2. The plasmids of PHBLV-EF1α-mClaudin 18.1 and PHBLV-EF1α-mClaudin18.2 were transiently expressed in CHO cells using Lipofectamine3000; after 72 h, the cell positive rate was detected. Finally, CHO cells transiently expressing mClaudin18.1 or mClaudin18.2 were obtained and named CHO-mClaudin18.1 and CHO-mClaudin18.2 for antibody species specificity detection. All the above cell media: were cultured using DMEM medium. 10% (v / v) fetal bovine serum was added to all media.

[0081] During the experiment, cells of the Claudin18.2-CHO high-expression cell line in the logarithmic growth phase were collected separately, and 1E+06 cells were prepared in each test tube. After centrifugation at 300 g for 5 min, the cells were collected and washed and centrifuged 3 times with an equal volume of staining solution (precooled PBS containing 1% BSA). Each type of cell was aliquoted into flow cytometry tubes at a volume of 100 μL per tube. Anti-Claudin18.2 mouse monoclonal antibody m1D6 diluted at different ratios was added, and incubated at 4 °C for 30 min. After washing 3 times with the staining solution, anti-mouse IgGFc-FITC-labeled secondary antibody was incubated at 4 °C for 30 min. After washing 3 times with the staining solution, it was used for flow cytometry to detect the fluorescence intensity. The flow cytometry results showed that m1D6 at different dilution ratios had a good specific reaction to Claudin18.2-CHO cells, and the results were as Figure 1 shown.

[0082] Collect human gastric cancer NUGC4 cells in the logarithmic growth phase and prepare 2×10⁶ cells. After centrifugation at 300 g for 5 min, collect the cells and wash and centrifuge them 3 times with an equal volume of staining solution (precooled PBS containing 1% BSA). The cells are aliquoted into two flow cytometry tubes at 100 μL per tube. Add isotype control (recombinant mIgG, purchased from Sino Biological) and m1D6 antibody, incubate at 4 °C for 30 min, wash 3 times with the staining solution, and then incubate with anti-mouse IgG Fc-FITC labeled secondary antibody at 4 °C for 30 min. After washing 3 times with the staining solution, it is used for flow cytometry to detect the fluorescence intensity. The flow cytometry results show that m1D6 has a good specific reaction to NUGC4 cells, and the results are as Figure 2 shown, in Figure 2 Isotopy represents mouse isotype control antibody, anti-CLDN18.2 represents m1D6, and unstaining represents unstained sample (without adding detection antibody).

[0083] 1.2 Detection of the affinity of anti-Claudin18.2 monoclonal antibody m1D6 by immunofluorescence method:

[0084] Digest NUGC4 cells in the logarithmic growth phase in the cell culture flask with trypsin, inoculate 1×10⁴ cells in a glass-bottomed confocal dish for culture. After it adheres to the wall, add 4% paraformaldehyde for fixation. After rinsing 3 times with PBS, add isotype control and m1D6 antibody respectively, incubate at room temperature for 30 min, wash 3 times with PBS, then incubate with anti-mouse IgG Fc-FITC labeled secondary antibody at room temperature in the dark for 30 min, wash 1 time with PBS, add Hoest33342 for nuclear counterstaining, wash 3 times with PBS, and observe with a fluorescence microscope. The results are as Figure 3 shown, m1D6 has a strong binding ability to the cell membrane surface of NUGC4.

[0085] 1.3 Detection of the in vivo tumor binding activity of anti-Claudin18.2 monoclonal antibody m1D6 by small animal in vivo imaging method

[0086] Collect 5×10⁶ cells each of Claudin18.2-CHO highly expressing cells and Claudin18.1-CHO highly expressing cells, resuspend them in 200 μL of serum-free medium, inoculate Claudin18.1-CHO cells under the right axilla of nude mice, and inoculate Claudin18.2-CHO cells under the left axilla of the same nude mouse. When the tumor grows to about 200 - 500 mm 3 in size, inject m1D6 labeled with Dylight680 dye into the tail vein of the mouse. Take pictures of the antibody uptake with a small animal in vivo imager at 30 min, 1 day, 2 days, 5 days, and 7 days after antibody injection. The results are as Figure 4As shown, one day after antibody injection, the fluorescently labeled antibody could be enriched on the tumor side inoculated with Claudin18.2-CHO, while no enrichment of the fluorescent antibody was observed on the Claudin18.1-CHO tumor side on the opposite side. This further indicates that m1D6 specifically binds only to the Claudin18.2-CHO tumor and does not bind to the Claudin18.1-CHO tumor, demonstrating the specificity of the antibody.

[0087] Example 3

[0088] The affinity determination of the chimeric antibody CH1D6 and the humanized antibodies h1D6-1, h1D6-2, and h1D6-3 was carried out as follows:

[0089] Among them, the chimeric antibody CH1D6 was obtained by splicing the variable region of the anti-Claudin18.2 mouse monoclonal antibody m1D6 and the human IgG1 constant region sequence, and was recombinantly expressed. The expression system and method for recombinant expression were the same as in Example 2.

[0090] 3.1 Determination of the affinity of the chimeric antibody and the humanized antibody for the Claudin18.2 antigen by Biacore

[0091] Synthesize the reference antibody IMAB362 plasmid (IMAB362 was synthesized according to the amino acid sequence disclosed in the patent application WO2014 / 146672A1), and transfect it into HEK293 cells to express and purify the IMAB362 antibody. The affinity constant of the antibody and the antigen was determined using BIACORE T200. First, the Human Claudin-18.2 Full Length Protein-VLP (purchased from ACROBiosystems) protein was conjugated to the CM-5 sensor chip, and then antibodies with different concentration gradients of IMAB362, chimeric antibody CH1D6, humanized antibody 1 (h1D6-1), humanized antibody 2 (h1D6-2), and humanized antibody 3 (h1D6-3) (the plasmids of the four antibodies, chimeric antibody CH1D6, humanized antibody 1 (h1D6-1), humanized antibody 2 (h1D6-2), and humanized antibody 3 (h1D6-3), were commissioned to NEST Biotechnology (Hangzhou) Co., Ltd. for synthesis) were injected. The antibody-antigen binding process was directly monitored on the chip surface, and the kinetic parameters of different antibodies were quickly semi-quantified through the binding curve. After the sample injection was completed, when the antibody was replaced by the buffer, the dissociation state of the complex could be observed, and the binding amount could reflect the antibody concentration and binding affinity, obtaining the reaction kinetic constant and affinity data provided by the analysis software. The original data was substituted into the GraphPad 9.0 software for plotting and calculation, and the KD values are shown in Table 1.

[0092] Table 1 KD values of each antibody

[0093]

[0094] It can be concluded from Table 1 that the KD values of the chimeric antibody and the humanized antibody are both significantly higher than those of the reference antibody IMAB362.

[0095] 3.2 Detection of the affinity of chimeric antibody and humanized antibody with Claudin18.2-CHO cells by flow cytometry

[0096] Collect Claudin18.2-CHO highly expressing cells in the logarithmic growth phase, and prepare 1E+06 cells in each test tube. After centrifugation at 300g for 5 min, collect the cells and wash and centrifuge them 3 times with the staining solution (pre-cooled PBS containing 1% BSA) in an equal volume. Then, aliquot the cells into flow cytometry tubes at a volume of 100 μL per tube. Add the antibody to be detected diluted at a 10-fold ratio, incubate at 4 °C for 30 min, wash 3 times with the staining solution, then incubate with anti-human IgG-FITC labeled secondary antibody at 4 °C for 30 min, and wash 3 times with the staining solution before detecting the fluorescence intensity by flow cytometry. The results are as Figure 5 shown; take the average fluorescence value as the original data, and use GraphPad 9.0 software to calculate the EC 50 of each antibody, as shown in Table 2.

[0097] Table 2 EC 50 values

[0098]

[0099] Combined with Table 2 and Figure 5 it can be concluded that each antibody to be detected has a good dose-response relationship with Claudin18.2-CHO cells.

[0100] 3.3 Detection of the affinity of chimeric antibody and humanized antibody with Claudin18.2-CHO cells by ELISA

[0101] Dilute Human Claudin-18.2 Full Length Protein-VLP with coating buffer (pH 9.6) to a concentration of 5 μg / mL, 100 μL per well, and incubate overnight at 4°C. The next day, discard the solution in the wells and wash 4 times with 300 μL of washing buffer per well (hereinafter referred to as washing). Add 300 μL of blocking solution per well, incubate at 37°C for 1.5 h, and wash. Add antibodies at each concentration gradient to each well. Dilute the chimeric antibody, humanized antibody 1, humanized antibody 2, and humanized antibody 3 to final concentrations of 300 ng / mL, 250 ng / mL, 200 ng / mL, 150 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.1 ng / mL, and 0 ng / mL respectively with PBS, incubate at 37°C for 1 h, and wash. Add goat anti-human IgG / horseradish peroxidase labeled (Zhongshan Golden Bridge) diluted 1:10,000 with dilution buffer, incubate at 37°C for 1 h, and wash. Add 100 μL of TMB Solution (TransGen Biotech) to each well, protect from light at room temperature for 30 min, add 100 μL of 0.5 M sulfuric acid to terminate, and read the OD value with an enzyme-linked immunosorbent assay (ELISA) reader. 450 Data processing: Substitute the original data into GraphPad 9.0 software for plotting and calculation. Results Figure 6 , EC 50 values are shown in Table 3.

[0102] Table 3 EC 50 values

[0103]

[0104] From Table 3 and Figure 6 it can be concluded that the affinities of both the chimeric antibody and the humanized antibody for the cells are in the nanomolar range.

[0105] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An anti-Claudin18.2 monoclonal antibody, characterized in that, It includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.1 to SEQ ID NO.3; The light chain variable region includes LCDR1, LCDR2 and LCDR3, the amino acid sequences of LCDR1 and LCDR3 are respectively shown as SEQ ID NO.4 to SEQ ID NO.5, and the amino acid sequence of LCDR2 is WAS.

2. The anti-Claudin18.2 monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region includes a heavy chain hypervariable region and a heavy chain framework region, and the heavy chain framework region sequence is derived from a murine antibody or a human antibody; The light chain variable region includes a light chain hypervariable region and a light chain framework region, and the light chain framework region sequence is derived from a murine antibody or a human antibody.

3. The anti-Claudin18.2 monoclonal antibody according to claim 2, wherein The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9; The amino acid sequence of the light chain variable region is shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.

13.

4. The anti-Claudin18.2 monoclonal antibody according to claim 3, characterized in that, The anti-Claudin18.2 monoclonal antibody includes the following combinations of heavy chain variable region and light chain variable region: 1) The heavy chain variable region shown as SEQ ID NO.6 and the light chain variable region shown as SEQ ID NO.10; 2) The heavy chain variable region shown as SEQ ID NO.7 and the light chain variable region shown as SEQ ID NO.11; 3) The heavy chain variable region shown as SEQ ID NO.8 and the light chain variable region shown as SEQ ID NO.12; 4) The heavy chain variable region shown as SEQ ID NO.9 and the light chain variable region shown as SEQ ID NO.

13.

5. The anti-Claudin18.2 monoclonal antibody according to claim 4, characterized in that, When the anti-Claudin18.2 monoclonal antibody includes the combination of 1), the anti-Claudin18.2 monoclonal antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.14, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO.

15.

6. The anti-Claudin18.2 monoclonal antibody according to claim 4, characterized in that, When the anti-Claudin18.2 monoclonal antibody includes the combinations of 2), 3) or 4), the anti-Claudin18.2 monoclonal antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.26, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO.

27.

7. The anti-Claudin 18.2 monoclonal antibody according to claim 1, characterized in that, The anti-Claudin18.2 monoclonal antibody includes a single-chain antibody, a chimeric antibody or a multimeric antibody.

8. The anti-Claudin18.2 monoclonal antibody according to claim 7, characterized in that, The chimeric antibody includes a CDR-grafted antibody.

9. A nucleotide molecule, characterized in that, The nucleotide molecule is used to encode the anti-Claudin18.2 monoclonal antibody according to any one of claims 1 to 8.

10. A biological material, characterized in that, The biological material is an expression cassette, a recombinant vector or a recombinant cell line containing the nucleotide molecule according to claim 9.

11. Use of the anti-Claudin 18.2 monoclonal antibody according to any one of claims 1 to 8 or the biological material according to claim 10 in the preparation of a product for gastric cancer diagnosis and / or treatment.

12. A biological product, characterized in that, The active ingredient in the biological product comprises the anti-Claudin 18.2 monoclonal antibody according to any one of claims 1 to 8 or the biological material according to claim 10.

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