Cldn18.2 antibodies and uses thereof

Abstract

Provided are antibodies that bind CLDN18.2 and afucosylated forms thereof. Also provided are methods of making the above antibodies, conjugates and compositions comprising the antibodies, and uses thereof in treating diseases, e.g., cancer.

Description

[0001] This application is a divisional application of the application with the application number 202080046257.4, the priority date of July 12, 2019, the filing date of July 10, 2020, and the invention name of "CLDN18.2 antibody and its use".

[0002] This application claims the priority of the Chinese patent application with the application number CN 201910628018.9, the filing date of July 12, 2019, and the invention name of "CLDN18.2 antibody and its use", the disclosure of which is incorporated herein in its entirety as a part of this application. TECHNICAL FIELD

[0003] The present disclosure relates to the field of immunotherapy. In particular, the present disclosure relates to antibodies binding to CLDN18.2 and their afucosylated forms, and their use in the treatment of diseases, in particular cancer. BACKGROUND

[0004] Upper gastrointestinal tumors, including gastric cancer and esophageal cancer, are a common global malignant tumor with poor prognosis. Gastric cancer is particularly prevalent in China, with nearly 42% of new cases worldwide concentrated in China. Due to the lack of early diagnosis, about 80% of gastric cancer patients are found to be in the advanced stage. Because gastric cancer is not sensitive to conventional chemotherapy drugs, the five-year survival rate of advanced gastric cancer is extremely low and has become the third leading cause of death in China. Therefore, recent research has focused on finding specific targeted therapy for gastric cancer.

[0005] Claudin family proteins are the main components of tight junction structures widely distributed in epithelial cells. Similar to other claudin family proteins, CLDN18.2 is a membrane protein with a molecular weight of about 27.8 kd and four transmembrane regions, and has two shorter extracellular regions EC1 and EC2. Unlike other claudin family proteins, CLDN18.2 is very specifically expressed only in the highly differentiated epithelial cells of the stomach in normal tissues (Tureci O et al., Gene, 2011). In tumors derived from gastric epithelial cells, a high proportion of tumor cells express CLDN18.2 on the surface. High levels of CLDN18.2 expression can also be detected in lymph node and other tissue metastatic tumors of gastric cancer cells (Woll S. et al., Int. J. Cancer, 2013). In addition to gastric cancer, a high proportion of tumor cells in cholangiocarcinoma, esophageal cancer, and pancreatic cancer also express CLDN18.2. As a specific surface marker, CLDN18.2 becomes a potential target for cancer treatment. The development of high-efficiency antibody drugs targeting CLDN18.2 will provide possibilities for the treatment of various cancers, with great application potential and market value.

[0006] One of the main mechanisms of action of therapeutic monoclonal antibodies to kill tumor cells is the ADCC effect (antibody dependent cell mediated cytotoxicity). After the antigen recognition segment (Fab) of the therapeutic antibody binds to the specific antigen on the surface of the tumor cell, the cell killing activity of the effector cell is activated by using the antibody constant region (Fc) to bind to the effector cell expressing the Fc receptor (FcγR), thereby secreting cytotoxic mediators including granulysin and perforin, etc., ultimately leading to the lysis and destruction of the target cells (tumor cells). The strength of the binding of the antibody to the antigen, the expression abundance of the antigen, and the strength of the binding of the Fc region of the antibody to the Fc receptor on the surface of the effector cell can all affect the strength of the ADCC effect, and thus affect the therapeutic effect of cancer (Jefferis and Lund, 2002). SUMMARY

[0007] The present disclosure provides new antibodies against CLDN18.2. The above-mentioned antibodies are capable of binding to CLDN18.2 with high affinity and specificity, and mediate the killing of target cells (e.g., tumor cells) expressing CLDN18.2 by effector cells. In addition, a defucosylated form of the above-mentioned antibodies is also generated, which is capable of binding to FcγRIIa better than the ordinary antibody and inducing the ADCC effect.

[0008] Accordingly, in one aspect, the present disclosure relates to an antibody or an antigen binding fragment thereof that binds to CLDN18.2, the antibody having the following heavy chain CDRs (CDRHs) and light chain CDRs (CDRLs):

[0009] a. CDRH1, CDRH2 and CDRH3 in a heavy chain variable region (VH) as set forth in SEQ ID NO: 1; and CDRL1, CDRL2 and CDRL3 in a light chain variable region (VL) as set forth in SEQ ID NO: 6;

[0010] b. CDRH1, CDRH2 and CDRH3 in a VH as set forth in SEQ ID NO: 11; and CDRL1, CDRL2 and CDRL3 in a VL as set forth in SEQ ID NO: 16;

[0011] c. CDRH1, CDRH2 and CDRH3 in a VH as set forth in SEQ ID NO: 21; and CDRL1, CDRL2 and CDRL3 in a VL as set forth in SEQ ID NO: 26;

[0012] d. CDRH1, CDRH2, and CDRH3 in a VH as shown in SEQ ID NO: 31; and CDRL1, CDRL2, and CDRL3 in a VL as shown in SEQ ID NO: 36;

[0013] e. CDRH1, CDRH2, and CDRH3 in a VH as shown in SEQ ID NO: 41; and CDRL1, CDRL2, and CDRL3 in a VL as shown in SEQ ID NO: 46; or

[0014] f. CDRH1, CDRH2, and CDRH3 in a VH as shown in SEQ ID NO: 55; and CDRL1, CDRL2, and CDRL3 in a VL as shown in SEQ ID NO: 60.

[0015] In some embodiments of the antibody or antigen binding fragment thereof of the present disclosure, the antibody has the following CDRHs and CDRLs:

[0016] a. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NOs: 3-5; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NOs: 8-10;

[0017] b. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NOs: 13-15; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NOs: 18-20;

[0018] c. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NOs: 23-25; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NOs: 28-30;

[0019] d. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NOs: 33-35; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NOs: 38-40;

[0020] e. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NOs: 43-45; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NOs: 48-50; or

[0021] f. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NOs: 57-59; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NOs: 62-64.

[0022] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody has the following VH and VL:

[0023] a. a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 6;

[0024] b. a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0025] c. a VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 26;

[0026] d. a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VL comprising the amino acid sequence of SEQ ID NO: 36;

[0027] e. a VH comprising the amino acid sequence of SEQ ID NO: 41, and a VL comprising the amino acid sequence of SEQ ID NO: 46;

[0028] f. a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VL comprising the amino acid sequence of SEQ ID NO: 60.

[0029] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody can have a heavy chain constant region sequence as set forth in SEQ ID NO: 51 and / or a light chain constant region sequence as set forth in SEQ ID NO: 53.

[0030] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody has a Fc region. In some embodiments, the antibody can have a glycosylation structure modification at Asn297, wherein numbering is according to the EU numbering system. In some embodiments, the proportion of glycosylation structures having a fucose in the glycosylation structure is 50% or less. In some embodiments, the proportion of glycosylation structures having a fucose in the glycosylation structure is not higher than 30%, for example, not higher than 20%, not higher than 10%, not higher than 5%, not higher than 2%, or not higher than 1%.

[0031] In preferred embodiments, the proportion of glycosylation structures having a fucose in the antibody is 0-1%.

[0032] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody is produced by a cell that is Fut8 gene knockout. In some embodiments, the cell can be selected from the group consisting of CHO cells and HEK293 cells.

[0033] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody has increased FcyRIIIa binding activity as compared to an antibody produced in a cell that does not have a Fut8 gene knockout. In some embodiments, the antibody has increased ADCC activity as compared to an antibody produced in a cell that does not have a Fut8 gene knockout.

[0034] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody can be a monoclonal antibody. In other embodiments, the antibody can be a bispecific antibody or a multispecific antibody.

[0035] In some embodiments of the antibody or antigen-binding fragment thereof of the present disclosure, the antibody can be selected from the group consisting of IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibody can be selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 subtypes.

[0036] In any embodiment of the antibody or antigen-binding fragment thereof of the present disclosure, the antigen-binding fragment can be selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a scFv fragment, a ds-scFv fragment, a dAb fragment, a single chain fragment, a diabody, and a linear antibody.

[0037] In one aspect, the present disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0038] In another aspect, the present disclosure relates to a vector comprising a nucleic acid molecule of the present disclosure.

[0039] In yet another aspect, the present disclosure relates to a host cell comprising a nucleic acid molecule or a vector of the present disclosure.

[0040] In one aspect, the present disclosure relates to a conjugate comprising any antibody or antigen-binding fragment thereof of the present disclosure conjugated to a therapeutic agent, a diagnostic agent, or an imaging agent.

[0041] In another aspect, the present disclosure relates to a composition comprising an antibody or antigen-binding fragment thereof or conjugate of the present disclosure, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents. In some embodiments, the composition further comprises one or more additional therapeutic agents.

[0042] In some embodiments, the therapeutic agent can be selected from an antibody, a chemotherapeutic drug, and a small molecule drug. In some embodiments, the chemotherapeutic drug can be selected from one or more of Epirubicin, Oxaliplatin, and 5-Fluorouracil (5-FU).

[0043] In one aspect, the disclosure relates to a method of treating a disease associated with expression of CLDN18.2 in a subject, the method comprising the step of administering to the subject an antibody or antigen-binding fragment thereof, conjugate, or composition of the disclosure.

[0044] In some embodiments, the disease is cancer. For example, the cancer can be selected from gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.

[0045] In some embodiments, the method further comprises the step of administering to the subject one or more additional therapies, such as cancer therapies. In some embodiments, the additional therapy is selected from chemotherapy, radiotherapy, immunotherapy, and surgical treatment.

[0046] In some embodiments, the immunotherapy is selected from therapy against immune checkpoint molecules, CAR-T cell therapy, and CAR-NK cell therapy.

[0047] In some embodiments, the chemotherapy is selected from a combination chemotherapy regimen comprising Epirubicin, Oxaliplatin, and 5-Fluorouracil.

[0048] In one aspect, the disclosure relates to the use of an antibody or antigen-binding fragment thereof, conjugate, or composition of the disclosure in the treatment of a disease associated with expression of CLDN18.2 in a subject.

[0049] In another aspect, the disclosure relates to the use of an antibody or antigen-binding fragment thereof, conjugate, or composition of the disclosure in the manufacture of a medicament for the treatment of a disease associated with expression of CLDN18.2 in a subject.

[0050] In some embodiments of the use of the disclosure, the disease is cancer. For example, the cancer can be selected from gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.

[0051] In one aspect, the disclosure relates to a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.

[0052] In another aspect, the present disclosure relates to a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56, and 61. The present disclosure also relates to a vector comprising the aforementioned nucleic acid molecule.

[0053] The present invention relates to the following:

[0054] 1. An antibody or antigen-binding fragment thereof that binds CLDN18.2, the antibody having the following heavy chain CDRs (CDRHs) and light chain CDRs (CDRLs):

[0055] a. CDRH1, CDRH2, and CDRH3 in a heavy chain variable region (VH) as set forth in SEQ ID NO: 1; and CDRL1, CDRL2, and CDRL3 in a light chain variable region (VL) as set forth in SEQ ID NO: 6;

[0056] b. CDRH1, CDRH2, and CDRH3 in a VH as set forth in SEQ ID NO: 11; and CDRL1, CDRL2, and CDRL3 in a VL as set forth in SEQ ID NO: 16;

[0057] c. CDRH1, CDRH2, and CDRH3 in a VH as set forth in SEQ ID NO: 21; and CDRL1, CDRL2, and CDRL3 in a VL as set forth in SEQ ID NO: 26;

[0058] d. CDRH1, CDRH2, and CDRH3 in a VH as set forth in SEQ ID NO: 31; and CDRL1, CDRL2, and CDRL3 in a VL as set forth in SEQ ID NO: 36;

[0059] e. CDRH1, CDRH2, and CDRH3 in a VH as set forth in SEQ ID NO: 41; and CDRL1, CDRL2, and CDRL3 in a VL as set forth in SEQ ID NO: 46; or

[0060] f. CDRH1, CDRH2, and CDRH3 in a VH as set forth in SEQ ID NO: 55; and CDRL1, CDRL2, and CDRL3 in a VL as set forth in SEQ ID NO: 60.

[0061] 2. The antibody or antigen-binding fragment thereof of item 1, the antibody having the following CDRHs and CDRLs:

[0062] a. CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 3-5; and CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 8-10;

[0063] b. CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 13-15; and CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 18-20;

[0064] c. CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 23-25; and CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 28-30;

[0065] d. CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 33-35; and CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 38-40;

[0066] e. CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 43-45; and CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 48-50;

[0067] f. CDRH1, CDRH2, and CDRH3 as set forth in SEQ ID NOs: 57-59; and CDRL1, CDRL2, and CDRL3 as set forth in SEQ ID NOs: 62-64.

[0068] 3. The antibody or antigen-binding fragment thereof of item 1 or 2, wherein the antibody has the following VH and VL:

[0069] a. a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 6;

[0070] b. a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0071] c. a VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 26;

[0072] d. a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VL comprising the amino acid sequence of SEQ ID NO: 36;

[0073] e. a VH comprising the amino acid sequence of SEQ ID NO: 41, and a VL comprising the amino acid sequence of SEQ ID NO: 46;

[0074] f. a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VL comprising the amino acid sequence of SEQ ID NO: 60.

[0075] 4. The antibody or antigen-binding fragment thereof of any one of clauses 1-3, wherein the antibody has an Fc region.

[0076] 5. The antibody or antigen-binding fragment thereof of clause 4, wherein the antibody has a glycosyl structure modification at Asn297, wherein numbering is according to the EU numbering system.

[0077] 6. The antibody or antigen-binding fragment thereof of clause 5, wherein the proportion of glycosyl structures having a fucose in the glycosyl structure is 50% or less.

[0078] 7. The antibody or antigen-binding fragment thereof of clause 6, wherein the proportion of glycosyl structures having a fucose in the glycosyl structure is 30% or less, e.g., 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less.

[0079] 8. The antibody or antigen-binding fragment thereof of clause 6, wherein the proportion of glycosyl structures having a fucose in the glycosyl structure is 0-1%.

[0080] 9. The antibody or antigen-binding fragment thereof of any one of clauses 6-8, wherein the antibody is produced by a cell that is Fut8 gene knockout.

[0081] 10. The antibody or antigen-binding fragment thereof of clause 9, wherein the cell is selected from the group consisting of a CHO cell and a HEK293 cell.

[0082] 11. The antibody or antigen-binding fragment thereof of clause 9 or 10, wherein the antibody has increased FcyRIIIa binding activity compared to an antibody produced in a cell that does not have a Fut8 gene knockout.

[0083] 12. The antibody or antigen-binding fragment thereof of any one of clauses 9-11, wherein the antibody has increased ADCC activity compared to an antibody produced in a cell that does not have a Fut8 gene knockout.

[0084] 13. The antibody or antigen-binding fragment thereof of any one of clauses 1-12, wherein the antibody is a monoclonal antibody.

[0085] 14. The antibody or antigen-binding fragment thereof of any one of clauses 1-12, wherein the antibody is a bispecific antibody or a multispecific antibody.

[0086] 15. The antibody or antigen-binding fragment thereof of any one of clauses 1-14, wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE, and IgD isotypes.

[0087] 16. The antibody or antigen-binding fragment thereof of any one of clauses 1-14, wherein the antibody is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 subtypes.

[0088] 17. The antibody or antigen-binding fragment thereof of any one of clauses 1-16, wherein the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fd fragment, a Fd’ fragment, a Fv fragment, a scFv fragment, a ds-scFv fragment, a dAb fragment, a single chain fragment, a diabody, and a linear antibody.

[0089] 18. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of clauses 1-17.

[0090] 19. A vector comprising the nucleic acid molecule of clause 18.

[0091] 20. A host cell comprising the nucleic acid molecule of clause 18 or the vector of clause 19.

[0092] 21. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of clauses 1-17 conjugated to a therapeutic agent, a diagnostic agent, or an imaging agent.

[0093] 22. A composition comprising the antibody or antigen-binding fragment thereof of any one of clauses 1-17 or the conjugate of clause 21, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents.

[0094] 23. The composition of clause 22, wherein the composition further comprises one or more additional therapeutic agents.

[0095] 24. The composition of clause 23, wherein the therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic drug, and a small molecule drug.

[0096] 25. The composition of clause 24, wherein the chemotherapeutic drug is selected from one or more of Epirubicin, Oxaliplatin, and 5-Fluorouracil (5-FU).

[0097] 26. A method of treating a disease associated with expression of CLDN18.2 in a subject, the method comprising the step of administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-17, the conjugate of claim 21, or the composition of any one of claims 22-25.

[0098] 27. The method of claim 26, wherein the disease is cancer.

[0099] 28. The method of claim 27, wherein the cancer is selected from the group consisting of gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.

[0100] 29. The method of any one of claims 26-28, wherein the method further comprises the step of administering to the subject one or more additional therapies.

[0101] 30. The method of claim 29, wherein the additional therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, and surgical treatment.

[0102] 31. The method of claim 30, wherein the immunotherapy is selected from the group consisting of therapy against an immune checkpoint molecule, CAR-T cell therapy, and CAR-NK cell therapy.

[0103] 32. The method of claim 30, wherein the chemotherapy is selected from the group consisting of a combination chemotherapy regimen comprising epirubicin, oxaliplatin, and 5-fluorouracil.

[0104] 33. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-17, the conjugate of claim 21, or the composition of any one of claims 22-25 in the treatment of a disease associated with expression of CLDN18.2 in a subject.

[0105] 34. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-17, the conjugate of claim 21, or the composition of any one of claims 22-25 in the manufacture of a medicament for the treatment of a disease associated with expression of CLDN18.2 in a subject.

[0106] 35. The use of claim 33 or 34, wherein the disease is cancer.

[0107] 36. The use of claim 35, wherein the cancer is selected from the group consisting of gastric cancer, cholangiocarcinoma, esophageal cancer, and pancreatic cancer.

[0108] 37. A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.

[0109] 38. A nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56, and 61.

[0110] 39. A vector comprising the nucleic acid molecule of item 38. BRIEF DESCRIPTION OF DRAWINGS

[0111] Figure 1 Results of detection of CLDN18.2 expression of CHO, CT26, SNU601 cells and constructed CHO-CLDN18.2, CT26-CLDN18.2 and SNU601-CLDN18.2 cells by flow cytometry are shown.

[0112] Figure 2 Results of detection of binding of control M13 phage and phage library after first, second and third round of screening to CHO cells and CHO-CLDN18.2 cells by flow cytometry are shown.

[0113] Figure 3 Results of detection of binding of screened phage clones A1, A2, A3, A4, A5( Figure 3 A) and A6( Figure 3 B) to CHO cells and CHO-CLDN18.2 cells by flow cytometry are shown.

[0114] Figure 4 Results of detection of binding of different concentrations of recombinant antibodies 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5( Figure 4 A), 18.2-A5F and 18.2-A6F( Figure 4 B) to CT26-CLDN18.2 cells and EC50 values by flow cytometry are shown.

[0115] Figure 5 Results of detection of binding of CLDN18.2 antibodies to 293t cells expressing CLDN18.1 (18.1-flag) or CLDN18.2 (18.2-flag) by flow cytometry are shown.

[0116] Figure 6 Results of detection of surface binding and endocytosis of CLDN18.2 antibodies by SNU601 cells expressing CLDN18.2 under incubation conditions at 4°C and 37°C are shown.

[0117] Figure 7Results of the binding of CLDN18.2 antibodies and afucosylated antibodies to CT26-CLDN18.2 cells detected by flow cytometry are shown.

[0118] Figure 8 Results of the binding of CLDN18.2-A1 and CLDN18.2-A1 F to FcyRIIIa detected by Biacore method are shown. Figure 8 A shows the fitted curves of antibody binding to FcyRIIIa, and Figure 8 B shows the Ka, Kd, KD and tc values of antibody binding to FcyRIIIa.

[0119] Figure 9 Results of the binding of CLDN18.2 antibodies and afucosylated antibodies to cell surface expressed FcyRIIIa detected by flow cytometry are shown. Figure 9 A shows the results of antibody binding to FcyRIIIa-Jurkat cells. Figure 9 B shows the results of antibody binding to NK92MI cells.

[0120] Figure 10 Results of the activation of FcyRIIIa receptor in FcyRIIIa-Jurkat cells by CLDN18.2 antibodies and afucosylated antibodies in the presence of SNU601-CLDN18.2 cells Figure 10 A) or CT26-CLDN18.2 Figure 10 B) detected by luciferase reporter system are shown.

[0121] Figure 11 Results of the detection of CLDN18.2 expression level on KATOIII cells by flow cytometry are shown.

[0122] Figure 12 shows results of the activation of FcyRIIIa receptor in FcyRIIIa-Jurkat cells by CLDN18.2 antibodies Figure 12A ) and CLDN18.2 antibody antibody in combination with chemotherapeutic drug EOF Figure 12B ) detected by luciferase reporter system in the presence of SNU601-CLDN18.2 cells are shown.

[0123] Figure 13 Killing effect of CLDN18.2 antibodies and afucosylated antibodies mediated ADCC pathway on SNU601-CLDN18.2 target cells in the presence of PBMCs from healthy human donor 1 Figure 13 A), donor 2 Figure 13 B) or donor 3 Figure 13 C) and are shown.

[0124] Figure 14 The killing effect of CLDN18.2 antibody and afucosylated antibody mediated CDC pathway on CHO-CLDN18.2 target cells in the presence of complement is shown.

[0125] Figure 15 The results of different doses of CLDN18.2 antibody 18.2-A1F inhibiting tumor growth in a mouse xenograft model of SNU601-CLDN18.2 cells are shown.

[0126] Figure 16 The flow cytometry results of KATOIII cells (KATOIII-18.2High) with high expression of CLDN18.2 for xenograft model are shown.

[0127] Figure 17 The results of CLDN18.2 antibody 18.2-A1F and the combination of 18.2-A1F and chemotherapy drug EOF inhibiting tumor growth in a mouse xenograft model of KATOIII-18.2High cells are shown. DETAILED DESCRIPTION

[0129] Unless otherwise defined herein, scientific and technical terms and their abbreviations used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which the present application belongs. Some of the terms and abbreviations used herein are listed below.

[0130] Antibody: antibody, Ab; Immunoglobulin: immunoglobulin, Ig;

[0131] Heavy chain: heavy chain, HC; Light chain: light chain, LC;

[0132] Heavy chain variable region: heavy chain variable domain, V H ;

[0133] Heavy chain constant region: heavy chain constant domain, C H ;

[0134] Light chain variable region: light chain variable domain, V L ;

[0135] Light chain constant region: light chain constant domain, C L ;

[0136] Complementarity determining region: complementarity determining region, CDR;

[0137] Fab fragment: antigen-binding fragment, Fab;

[0138] Fc region: fragment crystallizable region, Fc;

[0139] Monoclonal antibody: mAb;

[0140] Antibody-dependent cell-mediated cytotoxicity (ADCC);

[0141] Complement-dependent cytotoxicity (CDC).

[0142] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to CLDN18.2, said antibody having the following heavy chain CDR (CDRH) and light chain CDR (CDRL):

[0143] a. CDRH1, CDRH2 and CDRH3 in the heavy chain variable region (VH) as shown in SEQ ID NO:1; and CDRL1, CDRL2 and CDRL3 in the light chain variable region (VL) as shown in SEQ ID NO:6;

[0144] b. CDRH1, CDRH2 and CDRH3 in VH as shown in SEQ ID NO:11; and CDRL1, CDRL2 and CDRL3 in VL as shown in SEQ ID NO:16;

[0145] c. CDRH1, CDRH2 and CDRH3 in VH as shown in SEQ ID NO:21; and CDRL1, CDRL2 and CDRL3 in VL as shown in SEQ ID NO:26;

[0146] d. CDRH1, CDRH2 and CDRH3 in VH as shown in SEQ ID NO:31; and CDRL1, CDRL2 and CDRL3 in VL as shown in SEQ ID NO:36;

[0147] e. CDRH1, CDRH2, and CDRH3 in VH as shown in SEQ ID NO:41; and CDRL1, CDRL2, and CDRL3 in VL as shown in SEQ ID NO:46; or

[0148] f. CDRH1, CDRH2 and CDRH3 in VH as shown in SEQ ID NO:55; and CDRL1, CDRL2 and CDRL3 in VL as shown in SEQ ID NO:60.

[0149] As used herein, the terms "binding" or "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or lower affinity (K) D This antigen binds to the antigen. The term "K" as used in this article... D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0150] As used herein, the term "antibody" refers to an immunoglobulin molecule that contains at least one antigen recognition site and can specifically bind to an antigen. The term "antigen" is a substance in the body that can induce an immune response and specifically binds to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the above. The binding of an antibody to an antigen is mediated by interactions between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the antigen surface where the antibody binds is called an "antigenic determinant" or "epitope," and generally, each antigen can have multiple epitopes.

[0151] As used herein, the term "epitope" can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes typically comprise at least three, more commonly at least five, about nine, or about eight to ten amino acids in a distinctive spatial conformation. An epitope comprises a structural unit that is typically bound by immunoglobulin VH / VL pairs. An epitope defines a minimal binding site for an antibody and thus represents a specific target of the antibody or its antigen-binding fragment.

[0152] The term "antibody" as used in this disclosure is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding domains. Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. Antibodies may contain additional modifications, such as non-natural amino acids, Fc effector function mutations, and glycosylation site mutations. Antibodies also include post-translational modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing at least one antigen-binding domain.

[0153] As used in this article, "variable regions" (heavy chain variable region VH and light chain variable region VL) refer to paired light and heavy chain domains that directly participate in antibody-antigen binding. Each VH and VL region consists of three hypervariable or complementarity-determining regions (CDRs) and four framework regions (FRs) arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0154] As used herein, the term “CDR” refers to the complementarity-determining region within the antibody variable sequence. For each variable region, there are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a definitive residue numbering system applicable to antibody variable regions but also residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Each complementarity-determining region may contain the amino acid residues of the “complementarity-determining region” defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that Kabat… Certain sub-regions within a CDR adopt nearly identical peptide skeletons, despite diversity at the amino acid sequence level. These sub-regions are referred to as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" represent the light and heavy chain regions, respectively. These regions may be called Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other CDR boundary definitions may not strictly adhere to one of the systems described above, but will still overlap with Kabat CDRs. The methods used herein can utilize CDRs defined according to any of these systems, although preferred embodiments use CDRs defined by Kabat or Chothia.

[0155] When the Kabat system is used to define CDR sequences, CDRH1, CDRH2 and CDRH3 in VH as shown in SEQ ID NO:1 have amino acid sequences of SEQ ID NO:3 (SSWLI), SEQ ID NO:4 (TIVPSDSYTNYNQKFKD) and SEQ ID NO:5 (FRTGNSFDY), respectively, and CDRL1, CDRL2 and CDRL3 in VL as shown in SEQ ID NO:6 have amino acid sequences of SEQ ID NO:8 (KSSQSVLNSGNQKNYLT), SEQ ID NO:9 (WAVARQS) and SEQ ID NO:10 (QNSIAYPFT), respectively;

[0156] CDRH1, CDRH2, and CDRH3 in VH as shown in SEQ ID NO:11 have the amino acid sequences of SEQ ID NO:13 (SFWVG), SEQ ID NO:14 (NVSPSDSYTNYNQKFKD), and SEQ ID NO:15 (LSSGNSFDY), respectively; and CDRL1, CDRL2, and CDRL3 in VL as shown in SEQ ID NO:16 have the amino acid sequences of SEQ ID NO:18 (KSSQSVLNSGNQKNYLT), SEQ ID NO:19 (WSSTKQS), and SEQ ID NO:20 (QNAFSFPFT), respectively.

[0157] CDRH1, CDRH2, and CDRH3 in VH as shown in SEQ ID NO:21 have the amino acid sequences of SEQ ID NO:23 (SYWLN), SEQ ID NO:24 (SMYPSDSYTNYNQKFKD), and SEQ ID NO:25 (FSRGNSFDY), respectively; and CDRL1, CDRL2, and CDRL3 in VL as shown in SEQ ID NO:26 have the amino acid sequences of SEQ ID NO:28 (KSSQSLLESGNQKNYLT), SEQ ID NO:29 (WSWAKNS), and SEQ ID NO:30 (QNAYAFPFT), respectively.

[0158] CDRH1, CDRH2, and CDRH3 in VH as shown in SEQ ID NO:31 have the amino acid sequences of SEQ ID NO:33 (SFWIS), SEQ ID NO:34 (NILPSDSYTNYNQKFKD), and SEQ ID NO:35 (YWRGNSFDY), respectively; and CDRL1, CDRL2, and CDRL3 in VL as shown in SEQ ID NO:36 have the amino acid sequences of SEQ ID NO:38 (KSSQSIINSGNQKNYLT), SEQ ID NO:39 (WGGTRHS), and SEQ ID NO:40 (QNGYYSPFT), respectively.

[0159] CDRH1, CDRH2, and CDRH3 in VH, as shown in SEQ ID NO:41, have the amino acid sequences SEQ ID NO:43 (SSWVG), SEQ ID NO:44 (NSYPSDSYTNYNQKFKD), and SEQ ID NO:45 (LGRGNSFDY), respectively; and CDRL1, CDRL2, and CDRL3 in VL, as shown in SEQ ID NO:46, have the amino acid sequences SEQ ID NO:48 (KSSQSLIHSGNQKNYLT), SEQ ID NO:49 (WGLSKNS), and SEQ ID NO:50 (QNSIYYPFT), respectively.

[0160] CDRH1, CDRH2, and CDRH3 in VH, as shown in SEQ ID NO:55, have the amino acid sequences SEQ ID NO:57 (SYWLG), SEQ ID NO:58 (IIYPSDSYTNYNQKFKD), and SEQ ID NO:59 (FWRGNSFDY), respectively. Similarly, CDRL1, CDRL2, and CDRL3 in VL, as shown in SEQ ID NO:60, have the amino acid sequences SEQ ID NO:62 (KSSQSLLESGNQKNYLT), SEQ ID NO:63 (WAAGKES), and SEQ ID NO:64 (QNGYSHPFT), respectively.

[0161] Accordingly, in some embodiments of the antibody or its antigen-binding fragment disclosed herein, the antibody has the following CDRH and CDRL:

[0162] a. CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO:3-5; and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO:8-10;

[0163] b. CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO:13-15; and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO:18-20;

[0164] c. CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO:23-25; and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO:28-30;

[0165] d. CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO:33-35; and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO:38-40;

[0166] e. CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NO:43-45; and CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NO:48-50; or

[0167] f. CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO:57-59; and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO:62-64.

[0168] In some embodiments of the antibody or its antigen-binding fragment disclosed herein, the antibody has the following VH and VL:

[0169] a. VH containing the amino acid sequence of SEQ ID NO:1, and VL containing the amino acid sequence of SEQ ID NO:6;

[0170] b. VH containing the amino acid sequence of SEQ ID NO:11, and VL containing the amino acid sequence of SEQ ID NO:16;

[0171] c. VH containing the amino acid sequence of SEQ ID NO:21, and VL containing the amino acid sequence of SEQ ID NO:26;

[0172] d. VH containing the amino acid sequence of SEQ ID NO:31, and VL containing the amino acid sequence of SEQ ID NO:36;

[0173] e. VH containing the amino acid sequence of SEQ ID NO:41, and VL containing the amino acid sequence of SEQ ID NO:46; or

[0174] f. VH containing the amino acid sequence of SEQ ID NO:55, and VL containing the amino acid sequence of SEQ ID NO:60.

[0175] In other embodiments of the antibody or its antigen-binding fragment disclosed herein, the antibody has the following VH and VL:

[0176] a. VH containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:1, and VL containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:6;

[0177] b. VH containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:11, and VL containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:16;

[0178] c. VH containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:21, and VL containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:26;

[0179] d. VH containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:31, and VL containing an amino acid sequence modified with one or more amino acids in SEQ ID NO:36;

[0180] e. VH containing an amino acid sequence modified with one or more amino acids as described in SEQ ID NO:41, and VL containing an amino acid sequence modified with one or more amino acids as described in SEQ ID NO:46; or

[0181] f. VH containing an amino acid sequence modified with one or more amino acids as described in SEQ ID NO:55, and VL containing an amino acid sequence modified with one or more amino acids as described in SEQ ID NO:60.

[0182] In some embodiments, the amino acid modification does not alter the CDR sequence of the antibody, i.e., the amino acid modification is performed in the frame region (FR) of the variable region.

[0183] In some embodiments, the one or more amino acid modifications refer to 1-10 amino acid modifications or 1-5 amino acid modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0184] In some embodiments, the amino acid modification is selected from the substitution, deletion, addition, and / or insertion of amino acid residues. In some embodiments, the amino acid modification is an amino acid substitution, such as a conserved substitution.

[0185] In some embodiments of the antibody or its antigen-binding fragment disclosed herein, the antibody has the following VH and VL:

[0186] a. A VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a VL comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:6, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0187] b. A VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:11, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a VL comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:16, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0188] c. A VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:21, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a VL comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:26, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0189] d. A VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:31, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a VL comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:36, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0190] e. A VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:41, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a VL comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:46, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; or

[0191] f. A VH comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:55, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a VL comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:60, such as at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0192] As those skilled in the art will understand, the correlation between two amino acid sequences or two nucleotide sequences can be described by the parameter "sequence identity". The percentage of sequence identity between two sequences can be determined, for example, using mathematical algorithms. Non-restrictive examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the homology comparison algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the method for searching homology of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and a modified form of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, described in the algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Sequence comparisons (i.e., alignments) for determining sequence identity can be performed using programs based on such mathematical algorithms. These programs can be appropriately executed by a computer. Examples of such programs include, but are not limited to, the CLUSTAL and ALIGN programs (Version 2.0) of the PC / Gene program, and the GAP, BESTFIT, BLAST, FASTA, and TFASTA programs of the Wisconsin genetics software package. Alignments using these programs can be performed, for example, by using initial parameters.

[0193] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody may have a heavy chain constant region sequence as shown in SEQ ID NO:51 and / or a light chain constant region sequence as shown in SEQ ID NO:53.

[0194] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody has an Fc region. In some embodiments, the antibody has a glycosyl modification at Asn297, wherein it is numbered according to the Eu numbering system. According to this disclosure, "Asn297" refers to asparagine located at position 297 of the antibody's Fc region according to the Eu numbering system. Depending on subtle sequence variations specific to the antibody, Asn297 may also be located a few amino acids upstream or downstream of position 297.

[0195] As used herein, the term "antibody Fc region" or "human immunoglobulin Fc region" encompasses the constant region polypeptide of an antibody other than the heavy chain constant region 1 (CH1), such as the two constant region domains CH2 and CH3 at the carboxyl terminus of the heavy chain constant region of human immunoglobulin IgA, IgD, and IgG, and the three constant region domains CH2, CH3, and CH4 at the carboxyl terminus of the heavy chain constant region of human immunoglobulin IgE and IgM, and also includes the flexible hinge region at the amino terminus of these domains. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as containing residues from A231 to its carboxyl terminus.

[0196] The Fc region of immunoglobulins is the functional domain in which antibodies exert their immune effects. The Fc region of IgG antibodies can interact with various receptors, the most important of which is the Fcγ receptor (FcγR) family. A major mechanism by which therapeutic monoclonal antibodies kill tumor cells is ADCC (antibody-dependent cell-mediated cytotoxicity). After the antigen recognition region of the therapeutic antibody binds to specific antigens on the surface of tumor cells, the antibody's Fc region binds to FcγR-expressing killer cells, activating the cytotoxic activity of effector cells. This leads to the secretion of cytotoxic mediators, including granzymes and perforin, ultimately resulting in the lysis and destruction of target cells (e.g., tumor cells). In addition, Fc can also bind to complement protein C1q, producing the CDC (complement-dependent cytotoxicity) effect.

[0197] The level and form of glycosylation modification in the antibody Fc region can affect the binding ability of the Fc region to its receptor FcγR, thereby affecting the strength of the antibody's ADCC effect. The glycosylation modification of the antibody Fc region used in this article typically refers to the glycosylation modification at Asn297. During antibody production, glycosylation occurs in the cellular ER and Golgi network. Studies on antibody glycosylation have found that reducing the level of fucosylation in antibodies helps to increase the binding of the Fc region to FcγRIIIa, thus enhancing the antibody's ADCC effect. Therefore, altering the glycosylation metabolic pathway in antibody-expressing cells can effectively change the fucosylation level of expressed antibodies, thereby regulating the antibody-mediated ADCC effect (Jefferis R. 2009, NAT.REV.Drug.DISC).

[0198] Accordingly, in some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody has a reduced level of fucosylation. For example, in some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the proportion of the glycosyl structure containing fucose in the glycosyl structure at Asn297 is 60% or less, for example 50% or less, for example 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less. In some embodiments, the proportion of the glycosyl structure containing fucose is 0%-10%, for example 0%-5%, 0%-2%, or 0%-1%. In some embodiments, the proportion of the glycosyl structure containing fucose is 0.1% or less. In other embodiments, there is no detectable fucose in the glycosyl structure at Asn297.

[0199] Such defucosylated antibodies can be generated using techniques known to those skilled in the art. For example, the antibody can be expressed in cells that are deficient in or lack fucosylation capacity. In some embodiments, for example, cell lines with the Fut8 gene knockout can be used to generate antibodies with reduced fucosylation levels. Alternatively, antibodies or antigen-binding fragments with reduced or no fucosylation content can be generated by, for example: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) post-translational removal of fucosylation (e.g., with fucosidase); (iii) post-translational addition of desired carbohydrates, for example, after recombinant expression of a non-glycosylated glycoprotein; or (iv) purification of the glycoprotein to select for antibodies or antigen-binding fragments thereof that are not fucosylated.

[0200] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody is produced from Fut8 gene knockout cells to obtain an antibody with reduced fucosylation levels. In some embodiments, the cells are Chinese hamster ovary (CHO) cells, such as CHO-K1 cells, CHOS cells, or other CHO-derived cells. In some embodiments, the cells are Fut8 gene knockout CHO cells.

[0201] In other embodiments, the cells are human embryonic kidney (HEK)293 cells, such as HEK293, HEK293A, HEK293T, HEK293F, or other HEK293-derived cells. In some embodiments, the cells are Fut8 gene knockout HEK 293 cells.

[0202] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody is produced from Fut8 gene knockout cells, and wherein the antibody has increased FcγRIIIa binding activity compared to an antibody produced in cells without Fut8 gene knockout, such as a sequence-identical control antibody. In some embodiments, the antibody produced from Fut8 gene knockout cells has increased ability to induce FcγRIIIa activity compared to an antibody produced in cells without Fut8 gene knockout, such as a sequence-identical control antibody. For example, in some embodiments, the EC50 value of FcγRIIIa activity induced by the antibody produced from Fut8 gene knockout cells is at least 2-fold, for example, at least 5-fold or at least 10-fold, for example, 10-20-fold, compared to an antibody produced in cells without Fut8 gene knockout. As used herein, the term “EC50” refers to the antibody concentration that produces a 50% maximum effect.

[0203] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody is produced from Fut8 gene knockout cells, and wherein the antibody has increased ADCC activity compared to an antibody produced in cells without Fut8 gene knockout, such as a control antibody with the same sequence. For example, in some embodiments, the ADCC activity of the antibody produced from Fut8 gene knockout cells is increased by at least 2-fold, such as at least 5-fold or at least 10-fold, such as 10-20-fold, compared to an antibody produced in cells without Fut8 gene knockout.

[0204] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody is a monoclonal antibody.

[0205] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the antibodies constituting the population are identical, except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigen. Furthermore, unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each antibody in a monoclonal formulation targets the same single determinant on the antigen. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology, and the modifier "monoclonal antibody" should not be construed as requiring the antibody to be produced by any particular method.

[0206] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antibody is a bispecific antibody or a multispecific antibody.

[0207] For example, the bispecific or multispecific antibody has a first antigen-binding region that binds to an epitope on CLDN18.2 and a second antigen-binding region that binds to another antigen epitope, wherein the first antigen-binding region has CDRH1, CDRH2, and CDRH3 and CDRL1, CDRL2, and CDRL3 of the antibody or its antigen-binding fragment as described in this disclosure; or VH and VL sequences, and the second antigen-binding region binds to a different antigen epitope than the first antigen-binding region.

[0208] In some embodiments, the second antigen-binding region binds to another antigen-binding epitope on the CLDN18.2 molecule. In other embodiments, the second antigen-binding region binds to another antigen. In some embodiments, the other antigen is selected from tumor-associated antigens and immune checkpoint molecules.

[0209] Numerous tumor-associated antigens have been identified in this art as being associated with specific cancers. As used herein, the term "tumor-associated antigen" refers to an antigen that is differentially expressed by cancer cells and can therefore be used to target cancer cells. Tumor-associated antigens are antigens that can potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells but are not necessarily expressed by normal cells. These antigens can be characterized as those that are typically silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogenes), repressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Other cancer antigens may be encoded by viral genes, such as genes carried on RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be produced by those skilled in the art.

[0210] Immune checkpoint protein receptors and their ligands (collectively referred to herein as immune checkpoint molecules) mediate the suppression of T cell-mediated cytotoxicity and are typically expressed on tumors or on unresponsive T cells in the tumor microenvironment, allowing tumors to evade immune attack. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow cytotoxic T cell attack from the tumor. Examples of immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103. Regulation (including inhibition) of the activity of such proteins can be accomplished by immune checkpoint modulators, which may include, for example, antibodies, aptamers, small molecules, and soluble forms of checkpoint receptor proteins targeting checkpoint proteins. Antibodies specific to PD-1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103 are known and / or commercially available, and may also be produced by those skilled in the art.

[0211] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulins can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM. These can be further subdivided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the light chain, the light chain can be classified as a λ chain or a κ chain. The antibodies disclosed herein can be any of the above-mentioned classes or subtypes.

[0212] In some embodiments, the antibodies of this disclosure are selected from IgG, IgA, IgM, IgE, and IgD isotypes. In some embodiments, the antibodies of this disclosure are IgG, for example selected from IgG1, IgG2, IgG3, and IgG4 subtypes.

[0213] As used herein, the term "antigen-binding fragment" includes, but is not limited to: Fab fragments having VL, CL, VH, and CH1 domains; Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; Fd fragments having VH and CH1 domains; Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; Fv fragments and scFv fragments having VL and VH domains of a single arm of an antibody; dAb fragments consisting of either a VH or VL domain; isolated CDR regions; F(ab') fragments; and F(ab') fragments. The following are considered as separate, 2-component fragments: a bivalent fragment comprising two Fab' fragments connected by disulfide bridges at the hinge region; a single-chain antibody molecule (e.g., single-chain Fv; scFv); a "diabody" having two antigen-binding sites comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain; a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions; and any of the aforementioned substances in a modified form that retains antigen-binding activity.

[0214] In some embodiments of the antibody or antigen-binding fragment thereof disclosed herein, the antigen-binding fragment is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fd' fragments, Fv fragments, scFv fragments, ds-scFv fragments, dAb fragments, single-chain fragments, bivalent antibodies, and linear antibodies.

[0215] In another aspect, this disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of this disclosure. This disclosure also relates to a vector comprising the nucleic acid molecule of this disclosure.

[0216] The term "vector" as used in this article refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through methods such as transformation, transduction, or transfection, thereby enabling the expression of the genetic material elements they carry within the host cells. Vectors are those recognized by those skilled in the art, including but not limited to: (1) plasmids; (2) phage particles; (3) Cos plasmids; (4) artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes, or P1-derived artificial chromosomes; (5) bacteriophages, such as λ phage or M13 phage; and (6) animal viruses, such as retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, and baculoviruses. A vector may contain multiple elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes; in addition, vectors may also contain replication initiation sites.

[0217] In one aspect, this disclosure relates to a host cell comprising the nucleic acid molecules or vectors of this disclosure. In some embodiments, the host cell is a CHO cell, such as a CHO-K1 cell, a CHOS cell, or other CHO-derived cells. In other embodiments, the host cell is a HEK 293 cell, such as HEK293, HEK293A, HEK293T, HEK293F, or other HEK293-derived cells.

[0218] In one aspect, this disclosure relates to a conjugate comprising an antibody or antigen-binding portion thereof conjugated to a therapeutic agent, diagnostic agent, or imaging agent. In some embodiments, the therapeutic agent may be selected from cytotoxic agents and radioisotopes. In some embodiments, the diagnostic agent or imaging agent may be selected from fluorescent markers, luminescent substances, chromogenic substances, and enzymes.

[0219] In another aspect, this disclosure relates to a composition comprising an antibody or antigen-binding fragment thereof, or a conjugate thereof, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents.

[0220] The phrase "pharmaceutically acceptable" means compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, to the extent of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. As used herein, the phrase "pharmaceutically acceptable carriers, excipients, and / or diluents" means pharmaceutically acceptable materials, compositions, or media, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids, or solvent encapsulating materials, which relate to maintaining the stability, solubility, or activity of the antibodies or antigen-binding fragments of this disclosure.

[0221] The compositions disclosed herein can be formulated for administration to a subject in solid, liquid, or gel form. For example, the compositions disclosed herein can be formulated for parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, a sterile solution or suspension or a sustained-release formulation.

[0222] In some embodiments, the composition further comprises one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents are selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the therapeutic agent targets tumor-associated antigens, such as those described above. In other embodiments, the therapeutic agent targets immune checkpoint molecules, such as those described above.

[0223] As used herein, the term "chemotherapeutic agent" refers to any chemical agent that is therapeutically useful in treating diseases characterized by abnormal cell growth. Chemotherapy agents, as used herein, include both chemical and biological agents. These agents function to inhibit the cellular activity upon which cancer cells depend for sustained survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and various anti-neoplastic agents.

[0224] In some embodiments, the additional therapeutic agent is a chemotherapy drug, and the chemotherapy drug may be selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil (5-FU).

[0225] In one aspect, this disclosure relates to a method of treating a disease in a subject associated with the expression of CLDN18.2, the method comprising the step of administering to the subject an antibody of this disclosure or an antigen-binding fragment, conjugate, or composition thereof.

[0226] As used herein, the term “treatment” refers to a therapeutic approach in which the aim is to reverse, alleviate, improve, suppress, slow, or stop the progression or severity of a condition associated with a disease or symptom. The term “treatment” includes reducing or alleviating at least one side effect or symptom of a disease or symptom. Treatment is generally “effective” if it reduces one or more symptoms or clinical markers. Alternatively, treatment is “effective” if the progression of the disease is reduced or stopped; that is, “treatment” includes not only improvement of symptoms but also the cessation, or at least slowing, of the expected progression or worsening of symptoms in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, improvement or remission of the disease state, and remission (whether partial or complete), whether detectable or undetectable.

[0227] As used herein, the terms “subject,” “patient,” and “individual” are used interchangeably and refer to animals, such as humans. The term “subject” also includes “non-human mammals,” such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. In a preferred embodiment, the subject is a human subject.

[0228] In some implementations of the above methods, the disease is cancer. Specific examples of cancer include, but are not limited to: basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; bile duct cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; glioblastoma; liver cancer; kidney cancer; laryngeal cancer; leukemia; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma. Lymphoma; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; B-cell lymphoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia, etc. In a preferred embodiment, the cancer is selected from gastric cancer, bile duct cancer, esophageal cancer, and pancreatic cancer.

[0229] In some embodiments of the above method, the method further includes the step of administering one or more additional therapies. For example, in some embodiments, the therapy is selected from chemotherapy, radiotherapy, immunotherapy, and surgical therapy.

[0230] In some embodiments, the immunotherapy is selected from therapies targeting immune checkpoint molecules, CAR-T cell therapy, and CAR-NK cell therapy. For example, the immune checkpoint molecules may be selected from PD-1, PD-L1, PD-L2, CTLA4, OX40, LAG3, TIM3, TIGIT, and CD103.

[0231] In some implementations, the chemotherapy is selected from combination chemotherapy regimens including epirubicin, oxaliplatin, and 5-fluorouracil.

[0232] In one aspect, this disclosure relates to the use of the antibodies of this disclosure or their antigen-binding fragments, conjugates, or compositions in the treatment of a disease associated with CLDN18.2 expression in a subject. In another aspect, this disclosure relates to the use of the antibodies of this disclosure or their antigen-binding fragments, conjugates, or compositions in the preparation of a medicament for treating a disease associated with CLDN18.2 expression in a subject.

[0233] In some embodiments of the above-described uses, the disease is cancer, such as the cancer types described above. In a preferred embodiment, the cancer is selected from gastric cancer, bile duct cancer, esophageal cancer, and pancreatic cancer.

[0234] In some embodiments of the above-described uses, the subject is a human being.

[0235] In one aspect, this disclosure relates to a polypeptide having an amino acid sequence selected from SEQ ID NO:1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 55, and 60.

[0236] In another aspect, this disclosure relates to a nucleic acid molecule having a nucleotide sequence selected from SEQ ID NO:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 56, and 61. This disclosure also relates to a vector comprising the aforementioned nucleic acid molecule. Detailed Implementation

[0237] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0238] Example 1. Construction of a natural phage library

[0239] Antibody drug development is currently mainly achieved through humanizing antibodies derived from other species or directly screening fully human antibodies through transgenic animals and in vitro screening technology. This further reduces the immunogenicity of antibodies in humans, alleviates corresponding side effects, and improves drugability, which is an important trend in antibody drug development.

[0240] Phage display antibody library technology is one of the main methods for obtaining fully human antibodies in in vitro screening techniques. Phage display technology utilizes molecular biology techniques to insert exogenous gene fragments into the gene of a specific phage protein, such as gIII. The phage then expresses the protein or polypeptide encoded by the exogenous gene, maintaining the relative spatial structure and biological activity of the recombinant fusion protein, which is then displayed on the phage surface. The constructed diverse phage libraries are co-incubated with target proteins, and non-target protein-binding phage strains are removed through biological washing. With a sufficiently large phage library, high-affinity and high-specificity phage clones can be obtained through multiple rounds of collection, amplification, and enrichment. Gene sequencing is then used to identify the protein sequences encoded by these phage clones for further research.

[0241] A phage antibody library is a diverse phage library formed by displaying antibody genes on phages. The quality of a phage antibody library is mainly determined by its size and diversity. To obtain high-affinity antibodies, the phage antibody library needs to be as large as possible while maintaining diversity. The number of antibody fragments displayed on the phage surface represents the library size, while the diversity of the displayed fragments represents the diversity of the antibody library. Theoretically, the larger the size of the phage antibody library, the higher the affinity of the antibodies obtained through screening.

[0242] In this embodiment, to avoid bias in the antibody library due to individual differences and to ensure library diversity as much as possible, total RNA was obtained, collected, and extracted from 120 mononuclear cells from peripheral blood and spleen of healthy adults, and umbilical cord blood of newborns. The RNA was reverse transcribed into single-stranded cDNA, and the VH, Vκ, and Vλ genes were amplified using variable region primers targeting different antibody subsets. The amplification products were mixed in a certain proportion, and the heavy and light chain genes were ligated into single-stranded antibodies (scFv) using PCR, and then cloned into phage plasmids using double enzyme digestion. SS320 E. coli competent cells were transformed by electroporation using the phage plasmid carrying the scFv gene. After SS320 cells proliferated to the logarithmic growth phase, helper phages were added for infection.

[0243] The final storage capacity is 1.2 × 10⁻⁶. 12 A phage antibody library with a positive rate of 88% was obtained. Sequencing of randomly selected clones revealed that the gene family distribution of this antibody library was similar to that of the natural library, with the CDR3 region containing 3-20 amino acids. No antibody repetitive sequences were found during sequencing, and 88% of the gene sequences had correct reading frames. These results indicate that this phage antibody library has good diversity and a high effective library capacity.

[0244] Example 2. Preparation of a stable CLDN18.2 expression cell line

[0245] In this embodiment, a stable CHO cell line expressing CLDN18.2 (CHO-CLDN18.2) was prepared for cell-based phage display screening. The specific experimental procedure is as follows: The cDNA sequence of human CLDN18.2 was cloned into a pCDH lentiviral vector, and then co-transfected with the lentiviral packaging vector into 293t cells. After culturing for 48 or 72 hours, the cell supernatant rich in lentiviral particles was collected and directly used to infect CHO cells, CT26 cells, or SNU601 cells. The lentivirally infected cells were cultured with puromycin for selection. After 2-3 weeks, flow cytometry was used to detect CLDN18.2 expression in the cells using a CLDN18.2-specific antibody (IMAB362, Ganymed). The results are as follows: Figure 1 As shown.

[0246] The above results indicate that, after lentiviral transfection and puromycin selection, stable expression of CLDN18.2 was obtained in CHO cells (named CHO-CLDN18.2 cell line), CT26 cells (CT26-CLDN18.2), and SNU601 cells (SNU601-CLDN18.2).

[0247] Example 3. Screening for anti-CLDN18.2 antibodies from an antibody phage display library.

[0248] Using a stable CHO-CLDN18.2 cell line, three rounds of phage display library screening were performed. The experimental procedure mainly referred to the following literature: Targeting membrane proteins for antibody discovery using phage display Jones ML et al. Scientific Reports 2016.

[0249] The specific procedure is briefly described as follows: The phage library and CHO-K1 cells were incubated, and the supernatant was collected after centrifugation. This supernatant was then incubated with CHO-CLDN18.2 cells to allow phages specific to CLDN18.2 to bind to the cells. After centrifugation, the cell pellet was collected, washed, and the cells were collected. The phage library bound to the cells was then eluted with 75 mM sodium citrate buffer. After neutralizing the phage library, the selected phage library was amplified 100-fold using M13K07 helper phage, followed by second and third rounds of selection using a method similar to the first round. Phage enrichment was monitored by the initial phage dosage and the phage titer collected after each round of selection, and flow cytometry analysis was performed using the phage library and stable CHO or CHO-CLDN18.2 expression cell lines. Figure 2 The results show that, starting from the second round of enrichment screening, the phage display library can specifically bind to CHO-CLDN18.2, and the binding strength increases with the number of screening rounds.

[0250] After three rounds of screening, an enriched phage library was obtained. Bacteria were infected using this library and plated onto agarose plates. Single colonies were picked and placed in 96-well deep-well plates and cultured with shaking at 37°C using 2YT medium containing ampicillin and kanamycin to obtain a supernatant containing single-clone phages. The single-clone phage supernatant was incubated with CHO-CLDN18.2 cells or CT26-CLDN18.2 cells at 4°C for 1 hour, followed by washing with PBS (flow cytometry buffer) containing 1 mM EDTA and 0.5% BSA. PE-labeled anti-M13 antibody (purchased from Sinocare) was added, and the cells were incubated at 4°C for 30 minutes. Subsequently, flow cytometry was used to detect the binding of phages to cells, and the results are shown below. Figure 3 As shown.

[0251] The above results indicate that the selected monoclonal phages A1-A6 can bind well to CHO-CLDN18.2 cells but not to CHO control cells.

[0252] Example 4. Preparation of recombinant antibodies

[0253] The cDNA sequences of the heavy and light chain variable regions of monoclonal bacteriophages were cloned into pcDNA3.4 vectors (Invitrogen) containing antibody constant regions, resulting in heavy and light chain expression plasmids for six monoclonal antibodies. The plasmids were transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7–10 days, centrifuged, and the supernatant was collected. The supernatant was purified using protein A to obtain the purified antibodies. The six monoclonal antibodies were named 18.2-A1 (A1), 18.2-A2 (A2), 18.2-A3 (A3), 18.2-A4 (A4), 18.2-A5 (A5), and 18.2-A6 (A6), respectively. The amino acid sequences and coding sequences of the heavy and light chain variable regions of these antibodies are shown in Tables 1 and 2 below, and the CDR sequences of the antibodies determined by the Kabat CDR system are shown in Table 3. The heavy and light chain constant region sequences of the recombinant antibody are shown in Table 4.

[0254] Table 1. VH and VL sequences of CLDN18.2 antibody

[0255]

[0256]

[0257] Table 2. Coding sequences of VH and VL of CLDN18.2 antibody

[0258]

[0259]

[0260]

[0261] Table 3. Kabat CDR sequences of antibodies

[0262]

[0263] Table 4. Heavy and light chain constant region sequences of recombinant antibodies

[0264]

[0265]

[0266] Example 5. Detection of binding activity of recombinant monoclonal antibodies

[0267] The binding activity of recombinant monoclonal antibodies to CLDN18.2 was assessed. Specifically, well-grown, stably expressing CT26-CLDN18.2 cell lines were used. After washing the cells with PBS, they were mixed with serially diluted antibodies and incubated at 4°C for 1 hour. The cells were then washed once with flow cytometry buffer, and PE-labeled anti-human IgG antibody was added, followed by incubation at 4°C for 30 minutes. After centrifugation to remove the supernatant, the cells were washed with flow cytometry buffer, and the fluorescence intensity on the cell surface was detected by flow cytometry. The relative binding activity of each antibody was calculated using the average fluorescence intensity.

[0268] Antibodies 18.2-A1 (A1), 18.2-A2 (A2), 18.2-A3 (A3), 18.2-A4 (A4), 18.2-A5 (A5), and 18.2-A6 (A6) did not bind to the control CT26 cell line (results not shown), but they bound well to CT26 cell lines expressing CLDN18.2. Figure 4 As shown, the average fluorescence intensity of CT26-CLDN18.2 gradually decreased with antibody dilution. Figure 4 The relative binding activity (EC50) of the aforementioned CLDN18.2 antibody was also shown.

[0269] Example 6. Detection of binding specificity of recombinant antibodies

[0270] Human Claudin18 protein has two isoforms, CLDN18.1 and CLDN18.2. These two isoforms share all exons except exon 1, and the exon 1 sequences of CLDN18.1 and CLDN18.2 are highly similar, with only 8 different amino acids in the first extracellular region. Exon 1 of CLDN18.1 and CLDN18.2 are specifically expressed in different tissues driven by their respective promoters. Specifically, CLDN18.1 is specifically expressed in lung epithelial cells, while CLDN18.2 is specifically expressed in gastric epithelial cells. To identify the specificity of screened CLDN18.2 antibodies, the binding of the antibodies to CLDN18.1 and CLDN18.2 was detected.

[0271] Specifically, well-grown 293t cells were transiently expressed with either CLDN18.1 (CLDN18.1-Flag) or CLDN18.2 (CLDN18.2-Flag). Forty-eight hours post-transfection, cells were digested with trypsin. After washing with flow cytometry buffer, cells were mixed with 10 μg / ml of the allotype control antibody or one of the five CLDN18.2 antibodies mentioned above in 200 μl of flow cytometry buffer and incubated at 4°C for 1 hour. Subsequently, cells were washed once with flow cytometry buffer, and PE-labeled anti-human IgG antibody was added, followed by incubation at 4°C for 30 minutes. The supernatant was removed by centrifugation, and cells were washed twice with flow cytometry buffer. Cells were then fixed and permeabilized in a fixative solution containing 1% paraformaldehyde and 0.5% Triton-X100. Finally, cells were incubated with APC-labeled anti-flag antibody for 30 minutes and analyzed by flow cytometry.

[0272] The results are as follows Figure 5 As shown, flag-positive cell populations were detected in 293t cells expressing exogenous CLDN18.1-flag or CLDN18.2-flag, indicating that the transfected CLDN18.1 or CLDN18.2 proteins were well expressed in the cells, with a positive expression rate of approximately 20%. CLDN18.2 antibodies 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5, and 18.2-A6 all bound well to CLDN18.2-flag-transfected positive 293t cells, but none of the six antibodies bound to CLDN18.1-positive cells. These results indicate that these six CLDN18.2 antibodies specifically bind to CLDN18.2 but not CLDN18.1.

[0273] Example 7. Detection of CLDN18.2 antibody binding to cells and subsequent endocytosis

[0274] It has been reported that some antibodies, upon binding to antigens on the cell surface, can induce endocytosis of antigen-antibody complexes, thereby reducing the expression level of cell surface antigens and accelerating antibody metabolism in vivo (Schrama D et al. 2006, Nat Rev Drug Discov). In this example, the ability of antibodies 18.2-A1, 18.2-A3, 18.2-A4, and 18.2-A6 to be endocytosed in SNU601 cells stably expressing CLDN18.2 was tested. Specifically, CLDN18.2 antibodies 18.2-A1, 18.2-A3, 18.2-A4, 18.2-A6 or IMAB362 antibody (as described in Example 2), a blank control (PBS), and an isotype control (ISO) at a concentration of 10 μg / ml were incubated with cells at 4°C or 37°C for 4 hours. Subsequently, the cells were washed at 4°C and stained with PE-labeled anti-human IgG antibody. After fixation with 4% formalin, antibody binding on the cell surface was detected by flow cytometry.

[0275] like Figure 6 The results showed that, under incubation conditions at 4°C, the IMAB362 antibody and the 18.2-A1, 18.2-A3, 18.2-A4, and 18.2-A6 antibodies could bind well to SNU601 cells expressing CLDN18.2. However, under incubation conditions at 37°C, the IMAB362 antibody underwent endocytosis, and antibody staining on the cell surface decreased significantly; while the 18.2-A1, 18.2-A3, 18.2-A4, and 18.2-A6 antibodies did not exhibit significant endocytosis and could still bind well to cells at 37°C.

[0276] The above results indicate that the IMAB362 antibody can induce endocytosis, potentially downregulating the expression level of the CLDN18.2 antigen on the cell surface. In vivo, endocytosis can also alter antibody metabolism, accelerating antibody clearance. Conversely, the CLDN18.2 antibody disclosed herein does not induce significant endocytosis, and therefore does not downregulate the level of the target antigen CLDN18.2 on the cell surface, nor does it affect antibody metabolism in vivo.

[0277] Example 8. Preparation and purification of defucosylated antibodies

[0278] The defucosylated form of the CLDN18.2 antibody was further prepared. Specifically, the heavy and light chain coding sequences of the antibody were cloned into a mammalian GS expression vector capable of stable expression, with both heavy and light chain coding sequences driven by the CMV promoter. The expression vector was transfected into serum-free and suspension culture-acclimated CHO-K1 cells and Fut8 gene knockout-acclimated CHO-K1 cell lines. Stable expression cell lines were selected using MSX screening. After obtaining stable expression cell lines, they were cultured in shake flasks for 12-14 days, with feed culture medium added as needed.

[0279] Subsequently, the culture supernatant was collected, filtered, and the expressed antibody was captured by a protein A chromatography column. The antibody was eluted and dialyzed against PBS to obtain purified antibody. The antibodies expressed in the Fut8 gene knockout CHO-K1 cell line as 18.2-A1, 18.2-A2, 18.2-A3, 18.2-A4, 18.2-A5, and 18.2-A6 were named 18.2-A1F, 18.2-A2F, 18.2-A1F, 18.2-A4F, 18.2-A5F, and 18.2-A6F, respectively.

[0280] Example 9. Characterization of glycosylation modification of defucosylated antibody

[0281] Subsequently, the glycosylation modifications of defucosylated antibodies expressed in the Fut8 gene knockout CHO-K1 cell line and ordinary antibodies produced in CHO-K1 cells were analyzed. Antibody glycosylation was obtained by purifying 100 μg of antibody after digestion with trypsin and glycosylpeptidase, followed by fluorescence tandem mass spectrometry analysis. The identification of various glycosylation sites depended on their mass-to-charge ratio (m / z), and the percentage of glycosylation was calculated from the percentage of area detected by fluorescence.

[0282] Sugar nomenclature:

[0283]

[0284] N-acetylneuraminic acid N-acetylgalactosamine Fucose Mannose Galactose N-hydroxyacetylneuraminic acid

[0285] Glycosyl designation: Five numbers represent the number of different sugars: hexoses (galactose, mannose, or glucose), N-acetylglucosamine (GlcNA or GalNAc), fucose (FUC), N-acetylneuraminic acid (Neu5Ac), and N-hydroxyacetylneuraminic acid (Neu5Gc). The third number indicates the number of fucose molecules. See Table 4 below:

[0286] Table 4. Glycosyl numbers and their corresponding glycan chains

[0287]

[0288]

[0289] The glycan analysis results of the Claudin18.2-A1 antibody produced in CHO-K1 cells are shown in Table 5 below.

[0290] Table 5. Glycan analysis results of Claudin18.2 antibody

[0291] 33000 No 0.2 33100 Yes 0.3 34000 No 4.8 34100 Yes 45.0 44000 No 3.3 44100 Yes 37.7 45000 No 0.3 45100 Yes 0.2 54100 Yes 7.0 54110 Yes 0.3 54101 Yes 0.2 or 0 54120 Yes 0.3

[0292] The glycan analysis results of three batches of defucosylated Claudin18.2 antibodies (18.2-A1 and 18.2-A4) generated in Fut8 gene knockout CHO-K1 cells are shown in Table 6.

[0293] Table 6. Glycan analysis results of defucosylated 18.2-A1 and 18.2-A4 antibodies

[0294]

[0295]

[0296] Based on the above results, 91.0% of the glycosyl structures of ordinary Claudin18.2 antibodies produced in CHO-K1 cells contained fucose. However, the glycosyl structures of the three batches of defucosylated Claudin18.2 antibodies produced in Fut8 gene knockout CHO-K1 cells contained virtually no detectable fucose.

[0297] Example 10. Detection of binding activity of defucosylated antibodies

[0298] The binding activity of defucosylated antibodies 18.2-A1F, 18.2-A3F, and 18.2-A4F to CLDN18.2 compared to 18.2-A1, 18.2-A3, and 18.2-A4 was detected by flow cytometry, using the same method as described in Example 5. The experimental results are as follows: Figure 7 As shown.

[0299] These results indicate that there was no significant difference in the biological binding activity of defucosylated CLDN18.2 antibody and ordinary antibody to CLDN18.2.

[0300] Example 11. Detection of the binding activity of CLDN18.2 antibody and its defucosylated form with FcγRIIIa.

[0301] Subsequently, the binding activity of CLDN18.2 antibody and its defucosylated form to FcγRIIIa was detected using the Biacore method. Specifically, FcγRIIIa (Sino Biological Inc, 10389-H08C1) was diluted to 0.1 μg / ml with HBS-EP buffer as the ligand, and 18.2-A1F and 18.2-A1 antibody samples were diluted to 360 μg / ml, 120 μg / ml, 40 μg / ml, 13.3 μg / ml, and 4.4 μg / ml, respectively, as analytes. The ligand FcγRIIIa was immobilized using an indirect capture method. First, 50 μg / ml of Anti-His IgG was covalently bound to the CM5 chip surface via amino-coupling, followed by the binding of the ligand and analytes. In the Biacore Wizard mode, using a multi-cycle approach, affinity analysis was performed with FcγRIIIa as the ligand and 18.2-A1F and 18.2-A1 antibody samples as analytes.

[0302] Each sample test included 3 start-ups, 1 zero-concentration control, 5 gradient concentration samples, and 1 replicate sample (reference). After each cycle, the chip was regenerated using 10 mM glycine-HCl, pH 1.5 regeneration buffer. For each analyte concentration cycle, the capture time was set to 60 s, ligand solution flow rate to 10 μl / min; ligand-analyte binding time to 180 s, analyte solution flow rate to 30 μl / min; and dissociation time to 180 s. The CM5 chip coupled with Anti-His IgG was placed in the slot for detection and analysis. Raw data were imported into BIACORE™ X100 analysis software, the zero-concentration control was subtracted, and the reference channel was subtracted to eliminate volume effects. Affinity analysis was used to fit graphs in steady-state mode, and the data were processed.

[0303] from Figure 8 The results show that both the 18.2-A1 antibody and its defucosylated form, 18.2-A1F, can effectively bind to FcγRIIIa. Furthermore, the KD value of the 18.2-A1F antibody binding to FcγRIIIa is significantly lower than that of the 18.2-A1 antibody. This result indicates that the defucosylated 18.2-A1F antibody exhibits significantly enhanced binding activity to FcγRIIIa.

[0304] Example 12. Detection of the binding activity of CLDN18.2 antibody and its defucosylated form to FcγRIIIa on the cell surface.

[0305] Next, we further investigated the binding activity of the CLDN18.2 antibody and its defucosylated form to FcγRIIIa expressed on the cell surface. Specifically, the human FcγRIIIa (V158, high FC-binding isoform) gene was cloned into a mammalian cell expression vector carrying a puromycin selection marker and transfected into the Jurkat cell line. Cell lines stably expressing FcγRIIIa (FcγRIIIa-Jurkat) were selected using puromycin. Additionally, NK92MI is a human NK cell line that naturally expresses the FcγRIIIa receptor (F158, low FC-binding isoform). The binding ability of the CLDN18.2 antibody and its defucosylated form to FcγRIIIa expressed on the surface of FcγRIIIa-jurkat and NK92MI cells was detected by flow cytometry. The specific flow cytometry detection method is described in Example 6.

[0306] Flow cytometry results as follows Figure 9 As shown. CLDN18.2 antibodies 18.2-A1 and 18.2-A4, as well as their defucosylated forms 18.2-A1F and 18.2-A4F, all bound FcγRIIIa-jurkat cells but not untransfected Jurkat cells (results not shown). Furthermore, the defucosylated 18.2-A1F and 18.2-A4F antibodies bound FcγRIIIa-Jurkat cells and NK92MI cells better, with significantly lower EC50 values ​​than CLDN18.2 antibodies 18.2-A1 and 18.2-A4. These results indicate that the defucosylated CLDN18.2 antibodies have a significantly improved ability to bind to the Fc receptor FcγRIIIa.

[0307] Example 13. CLDN18.2 antibody and its defucosylated form activate FcγRIIIa receptor

[0308] Upon binding to the FC region of the antibody, the FcγRIIIa receptor can activate the NF-AT transcription factor pathway in effector cells. Therefore, detecting the intensity of NF-AT-mediated reporter genes can reflect the activation intensity of the FcγRIIIa receptor. In this embodiment, a promoter containing an NF-AT binding site was inserted into the luciferase reporter gene expression vector, and the vector was stably transfected into the Jurkat cell line. Simultaneously, highly FC-binding FcγRIIIa (V158) was also stably transfected into Jurkat cells, constructing a functional cell line (FcγRIIIa-Jurkat) capable of sensing the activation intensity of the FcγRIIIa receptor.

[0309] SNU601 gastric cancer cells stably expressing CLDN18.2 (SNU601-CLDN18.2) were taken and diluted to 4x10⁻⁶. 5 / ml, and mixed with FcγRIIIa-Jurkat cells at a ratio of 1:6. 100 μl of the mixed cells were added to a 96-well plate, followed by serially diluted antibody, and incubated at 37°C for 6 hours. Subsequently, luciferase activity was detected using the One-Glo kit (promega).

[0310] The results are as follows Figure 10 As shown, compared with the 18.2-A1 and 18.2-A4 antibodies produced in CHO-K1 cells, the FcγRIIIa receptor activity induced by the defucosylated antibodies 18.2-A1F and 18.2-A4F expressed in Fut8 gene knockout CHO cell lines was significantly increased, with EC50 values ​​increasing by approximately 10-20 times.

[0311] Example 14. Combined chemotherapy drug EOF enhances CLDN18.2 antibody-induced FcγRIIIa receptor activation.

[0312] Combination chemotherapy regimens, including EOF (epirarubicin, oxaliplatin, and 5-fluorouracil, 5-FU), are the main existing treatments for gastric cancer. EOF-sensitive cell lines undergo varying degrees of cell cycle arrest, proliferation inhibition, and apoptosis after EOF treatment.

[0313] KATOIII cells are a human gastric epithelial cancer cell line that expresses very low levels of CLDN18.2, with only about 5.2% of cells showing significant positivity in flow cytometry. Figure 11 Due to low levels of CLDN18.2 expression, the 18.2-A1F and 18.2-A6F antibodies did not induce FcγRIIIa receptor activation in antibody-induced FcγRIIIa receptor activation assays co-incubated with KATOIII cells and FcγRIIIa-Jurkat cells (as described in Example 13). Figure 12A ).

[0314] Furthermore, after treating KATOIII cells with sublethal doses of EOF (epirarubicin: 300 nM; oxaliplatin: 130 nM; 5-fluorouracil: 561.3 nM) for 48 hours, microscopic observation and flow cytometry revealed increased cell volume, rounding, and a decrease in mitotic cells, indicating that the cells were arrested in the mitotic phase. However, the cells remained viable, and no significant apoptosis was observed (results not shown). EOF-treated KATOIII cells were co-incubated with FcγRIIIa-Jurkat cells and different concentrations of CLDN18.2 antibody. The results showed that, compared to the isotype control (ISO), 18.2-A1F and 18.2-A6F antibodies could induce significant FcγRIIIa receptor activation. Figure 12B ).

[0315] The above results indicate that the commonly used chemotherapy drug EOF has an inhibitory effect on the growth of gastric cancer cells. EOF treatment can enhance CLDN18.2 antibody-induced FcγRIIIa receptor activation. Without being bound by theoretical limitations, this effect may be due to the chemotherapy drug treatment promoting the ADCC effect of the CLDN18.2 antibody through upregulating antigen expression on cells and other mechanisms.

[0316] Example 15. Killing effect of antibody on tumor cells expressing CLDN18.2

[0317] Peripheral blood leukocytes (PBMCs) from healthy individuals (donor 1 and donor 2) were isolated using Ficoll-Paque Plus (GE Healthcare) and cultured overnight at 37°C. SNU601 gastric cancer cells stably expressing CLDN18.2 (SNU601-CLDN18.2) were collected and diluted to 1x10⁻⁶. 5 / ml, and diluted to 5x10 by PBMC. 6 The effector cells and target cells were mixed in equal volumes at a ratio of 50:1. 100 μl of the mixed cells were added to a 96-well plate, followed by serially diluted antibody, and incubated at 37°C for 24 hours. Cell viability was then assessed using the lactate dehydrogenase (LDH) assay (promega), measured by absorbance at OD490 using a microplate reader. The killing rate was calculated as follows:

[0318] Minimal release group: target cells cultured alone

[0319] Maximum release group: target cells + lysis buffer

[0320] Experimental group: target cells + effector cells + CLDN18.2 antibody

[0321] Control group: target cells + effector cells + negative control antibody

[0322] Lethality (%) = (Experimental group - Minimum release group) / (Maximum release group - Minimum release group)% × 100%

[0323] The results are as follows Figure 13 As shown. The negative control antibody did not show significant cell killing (results not shown). CLDN18.2 antibody 18.2-A1 and defucosylated antibodies 18.2-A1F and 18.2-A4F effectively induced cell death in target cells expressing CLDN18.2, with a maximum killing rate of 80-90%. Furthermore, comparing the defucosylated antibody with the ordinary antibody, the maximum killing rate and IC50 value of the defucosylated antibody were significantly higher than those of the ordinary antibody.

[0324] Example 16. CDC activity of CLDN18.2 antibody

[0325] HELA cells stably expressing CLDN18.2 were resuspended in culture medium containing 1% complement-inactivated serum for cell counting, and the cell concentration was adjusted to 2 x 10⁻⁶ cells / year. 5 / ml, cell viability greater than 90%. 50 μl of cells were added to each well of a 96-well plate, followed by serially diluted antibody and 50 μl of diluted human serum. Cells were incubated at 37°C for 2 hours. Cell lysis rate was then measured using the CCK8 assay. The calculation method for cell lysis rate is as follows:

[0326] Minimal release group: target cells

[0327] Maximum release group: target cells + lysis buffer

[0328] Experimental group: target cells + CLDN18.2 antibody + complement

[0329] Negative control group: target cells + negative control antibody + complement

[0330] Lethality (%) = (Experimental group - Minimum release group) / (Maximum release group - Minimum release group) × 100%

[0331] The results are as follows Figure 14 As shown, CLDN18.2 antibodies 18.2-A1 and 18.2-A4, as well as their defucosylated forms 18.2-A1F and 18.2-A4F, all exhibit significant CDC activity, with a maximum killing rate of over 95% against target cells. Furthermore, the defucosylated forms of the antibodies showed no significant difference in CDC activity compared to the standard antibodies.

[0332] Example 17. In vivo tumor-killing activity of CLDN18.2 antibody

[0333] A mouse xenograft model of SNU601 cells was established by subcutaneously inoculating NPG immunodeficient mice with SNU601-CLDN18.2 cells and Ficoll-isolated human PBMC cells (SNU601-CLDN18.2 cells:PBMCs at a ratio of 1:0.8). Subsequently, NPG immunodeficient mice inoculated with SNU601 tumor cells were intraperitoneally injected with 10 mg / kg or 5 mg / kg of 18.2-A1F antibody, or a blank control (PBS) or an IgG isotype control (ISO), with n=15 mice in each group. The drugs were administered every 3 days after tumor inoculation, and the tumor volume in the mice was measured.

[0334] like Figure 15 The results showed that, compared with the PBS group and the IgG isotype control antibody group, the 18.2-A1F antibody treatment groups at doses of 5 mg / kg and 10 mg / kg both showed good tumor suppression effects, and there was no significant difference in effect between the 5 mg / kg and 10 mg / kg groups.

[0335] Example 18. In vivo tumor-killing activity of CLDN18.2 antibody combined with chemotherapeutic drugs

[0336] As shown in Example 14, in vitro EOF combined with chemotherapy can enhance CLDN18.2 antibody-induced FcγRIIIa receptor activation. KATOIII expressed very low levels of CLDN18.2 when cultured in in vitro IMDM medium, with only about 5.2% of cells showing significant positivity in flow cytometry. Figure 11 After flow cytometry sorting, KATOIII cells with high expression of CLDN18.2 (KATOIII-18.2High) were selected and inoculated into NPG immunodeficient mice for xenotransplantation model experiments. Figure 16 ).

[0337] A xenograft model of KATOIII-18.2High cells was established by subcutaneously inoculating NPG immunodeficient mice with KATOIII-18.2High cells and Ficoll-isolated human PBMC cells (KATOIII-18.2High cells:PBMCs at a ratio of 1:0.8). Subsequently, NPG immunodeficient mice inoculated with KATOIII-18.2High tumor cells were intraperitoneally injected with an allotype control antibody (ISO), EOF (epirarubicin: 1 mg / kg; oxaliplatin: 3 mg / kg; 5-fluorouracil: 30 mg / kg), or 18.2-A1F antibody at a dose of 10 mg / kg, or EOF and 18.2-A1F, with n = 6 mice in each group. Administering the drugs every 3 days after tumor inoculation and measuring the tumor volume in the mice.

[0338] like Figure 17 The results showed that, compared with the EOF group and the control antibody group, 18.2-A1F could effectively inhibit tumor growth. The combination of 18.2-A1F and EOF further significantly reduced tumor growth, indicating its potential in the clinical treatment of cancer when used in combination with chemotherapy.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CLDN18.2, said antibody having CDRH1, CDRH2 and CDRH3 in VH as shown in SEQ ID NO: 11; and CDRL1, CDRL2 and CDRL3 in VL as shown in SEQ ID NO: 16, wherein said CDR is defined according to Kabat or Chothia numbering.

2. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody has CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO: 13-15; and CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO: 18-20.

3. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody has a VH comprising the amino acid sequence of SEQ ID NO: 11 and a VL comprising the amino acid sequence of SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, wherein the heavy chain of the antibody comprises a heavy chain variable region as shown in SEQ ID NO: 11 and a heavy chain constant region as shown in SEQ ID NO: 51, and the light chain of the antibody comprises a light chain variable region as shown in SEQ ID NO: 16 and a light chain constant region as shown in SEQ ID NO:

53.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, wherein the antibody has an Fc region.

6. The antibody or antigen-binding fragment thereof as claimed in claim 5, wherein the antibody has a glycosylation modification at Asn297, wherein the numbering is in accordance with the EU numbering system.

7. The antibody or antigen-binding fragment thereof as claimed in claim 6, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 50% or less.

8. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 30% or less.

9. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 20% or less.

10. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 10% or less.

11. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 5% or less.

12. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 2% or less.

13. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the glycosyl structure having fucose in the glycosyl structure is 1% or less.

14. The antibody or antigen-binding fragment thereof as claimed in claim 7, wherein the proportion of the fucose glycosyl structure in the antibody is 0%-1%.

15. The antibody or antigen-binding fragment thereof as described in any one of claims 7-14, wherein the antibody is composed of... Fut8 Gene knockout cells are produced.

16. The antibody or antigen-binding fragment thereof as claimed in claim 15, wherein the cell is selected from CHO cells and HEK293 cells.

17. The antibody or antigen-binding fragment thereof as described in claim 15 or 16, wherein compared to the antibody without having Fut8 Antibodies produced in gene-knockout cells, said antibodies having increased FcγRIIIa binding activity.

18. The antibody or antigen-binding fragment thereof as described in any one of claims 15-17, wherein compared to the antibody without having Fut8 Antibodies produced in gene-knockout cells, said antibodies having increased ADCC activity.

19. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-18, wherein the antibody is a monoclonal antibody.

20. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-18, wherein the antibody is a bispecific antibody or a multispecific antibody.

21. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-20, wherein the antibody is selected from IgG, IgA, IgM, IgE and IgD isotypes.

22. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-20, wherein the antibody is selected from IgG1, IgG2, IgG3 and IgG4 subtypes.

23. The antibody or antigen-binding fragment thereof as described in any one of claims 1-22, wherein the antigen-binding fragment is selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, ds-scFv fragments, and bivalent antibodies.

24. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-23.

25. A vector comprising the nucleic acid molecule as described in claim 24.

26. A host cell comprising a nucleic acid molecule as described in claim 24 or a vector as described in claim 23.

27. A conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a diagnostic agent or imaging agent as described in any one of claims 1-23.

28. A composition comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-23 or a conjugate as claimed in claim 27, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents.

29. The composition of claim 28, wherein the composition further comprises one or more additional therapeutic agents.

30. The composition of claim 29, wherein the therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

31. The composition of claim 30, wherein the chemotherapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil (5-FU).

32. The use of the antibody or antigen-binding fragment thereof as claimed in any one of claims 1-23, the conjugate as claimed in claim 27, or the composition as claimed in any one of claims 28-31 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is selected from gastric cancer, bile duct cancer, esophageal cancer, pancreatic cancer, and cervical cancer.

33. The use as described in claim 32, wherein the drug is administered in combination with one or more other therapies.

34. The use as described in claim 33, wherein the additional therapy is selected from chemotherapy, radiotherapy, immunotherapy and surgical treatment.

35. The use as described in claim 34, wherein the immunotherapy is selected from therapies targeting immune checkpoint molecules, CAR-T cell therapy, and CAR-NK cell therapy.

36. The use as described in claim 34, wherein the chemotherapy is selected from combination chemotherapy regimens comprising epirubicin, oxaliplatin and 5-fluorouracil.

37. The use as described in any one of claims 32-36, wherein the cancer is gastric cancer.

38. A polypeptide having an amino acid sequence selected from SEQ ID NO: 11 and 16.

39. A nucleic acid molecule having a nucleotide sequence selected from SEQ ID NO: 12 and 17.

40. A vector comprising the nucleic acid molecule as described in claim 39.

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