Anti-CLDN6 antibody and application thereof

Abstract

Provided are an anti-CLDN6 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-CLDN6 antibody or the antigen-binding fragment thereof, and a use of the anti-CLDN6 antibody or the antigen-binding fragment thereof in the treatment and / or prevention of tumors, thereby providing a brand-new means for treating tumors.

Description

Antibodies against CLDN6 and their applications

[0001] This application claims priority to Chinese patent application 2024118234958, filed on 2024 / 12 / 11. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This article belongs to the field of immunology, and more specifically, it relates to antibodies against CLDN6 or antigen-binding fragments thereof, derivatives comprising said antibodies or antigen-binding fragments thereof, pharmaceutical compositions and their related applications in the treatment of CLDN6-related diseases. Background Technology

[0003] CLDN6, a member of the claudin family of tight junction proteins, is a crucial component of the structure and function of tight junctions. It works synergistically with other structural and signaling proteins to play a vital role in maintaining the structure and function of tight junctions. Aberrant expression of claudin disrupts the epithelial permeability barrier, leading to loss of cell polarity, decreased cell adhesion, and ultimately, the development and progression of various tumors. The human CLDN6 gene is located on chromosome 16p13.3, consists of 220 amino acids, has a molecular weight of 23 kDa, and contains three exons. Consistent with other CLDNs, CLDN6 possesses four transmembrane domains, a short cytoplasmic N-terminal domain, a C-terminal cytoplasmic domain, two extracellular domains (the larger ECL1 and the smaller ECL2), and a short intracellular loop. This loop structure forms a vital structural basis for tight junctions.

[0004] CLDN6 expression is dynamically regulated by multiple factors. CLDN6 is generally expressed in fetal tissues such as the stomach, pancreas, lung, and kidney, but not in corresponding adult tissue samples. It is specifically highly expressed in various solid tumors, including the ovary, endometrium, lung, stomach, and testis. It has been reported that over 55% of ovarian cancers and over 90% of testicular cancers express CLDN6. Furthermore, CLDN6 expression increases with tumor progression, and high CLDN6 expression is associated with poor tumor prognosis. Therefore, CLDN6 could serve as a target for tumor therapy.

[0005] Therapies targeting CLDN6 for cancer treatment include monoclonal antibodies, bispecific antibodies (BsAbs), and antibody-drug conjugates (ADCs). Currently, there are seven projects in clinical trials: one monoclonal antibody, two bispecific antibodies, three ADCs, and two CAR-T therapies. ASP1650, a CLDN6 monoclonal antibody developed by Astellas, did not show clinically meaningful monoclonal antibody activity in ovarian and testicular cancers in Phase I and II clinical trials, and this pipeline has been discontinued. BNT211 comprises two parts: an autologous CAR-T cell therapy targeting CLDN6 and a CLDN6-encoded mRNA vaccine (CARVac) developed using BioNTech's proprietary mRNA-lipoplex technology. This therapy aims to induce a robust immune response against CLDN6-positive solid tumors. Phase I / II clinical results showed that, as of March 2023, in 17 evaluable patients receiving BNT211 with or without CARVac, the overall objective response rate was 41%, and the disease control rate was 65%. One testicular cancer patient who received automated BNT211 treatment achieved complete remission after surgery. AMG794 is a bispecific antibody with an extended half-life developed using a bispecific T-cell adaptor technology platform. It redirects T cells to kill tumor cells expressing CLDN6. It is currently in Phase I clinical trials recruiting patients. DS9606 is an ADC drug targeting CLDN6 / CLDN9, constructed by Daiichi Sankyo based on a benzodiazepine derivative (PBD). Preclinical data showed that this technology platform-constructed CLDN6-targeting ADC exhibited significant anti-tumor activity. It is currently in Phase I clinical trials. Preliminary data were presented at ESMO in 2024, showing preliminary efficacy in the ≥0.072 mg / kg dose group, with four patients achieving confirmed objective responses, including two GCT patients, one gastric / esophageal cancer patient, and one NSCLC patient. Of the 7 evaluable GCT patients, 2 achieved confirmed objective response after more than 6 months of treatment, and 5 patients showed a ≥90% reduction in alpha-fetoprotein and human chorionic gonadotropin (hCG) tumor marker levels. As of the data cutoff on June 14, 2024, 21 of the 53 patients were still receiving DS-9606 treatment. No dose-limiting toxicities were observed. TOR-1-23 is an ADC developed by TORL BioTherapeutics, generated by conjugating an anti-CLDN6 monoclonal antibody with the microtubule inhibitor MMAE via a cleavable linker. In vitro studies have shown that TORL-1-23 is highly selective, binding strongly to CLDN6-overexpressing cell lines but not to other tight protein-overexpressing cell lines.The Phase I clinical trial results presented at ESMO 2024 showed that in CLDN6-positive ovarian cancer patients, the objective response rate (ORR) was 21% (3 / 14) for 0.2-2.0 mg / kg, 67% (4 / 6) for 2.4 mg / kg, and 50% (6 / 12) for 3.0 mg / kg. 26 patients are still receiving treatment, with one continuing treatment for over 100 weeks. The treatment demonstrated some tolerability and preliminary antitumor activity. Although CAR-T and ADCs that have entered clinical trials have shown some efficacy, considering that CAR-T requires autologous cells and is expensive, and that ADC molecules have strong MMAE and PBD toxicity, the overall therapeutic window is relatively small. Therefore, it is still necessary to screen for suitable CLDN6 antibodies and appropriate treatment methods. Summary of the Invention

[0006] This article provides an anti-CLDN6 antibody or its antigen-binding fragment that can bind to CLDN6 and induce the killing of CLDN6-expressing cells (e.g., tumor cells) via ADCC and / or CDC.

[0007] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3), and the light chain variable region comprises light chain complementarity-determining region 1 (LCDR1), light chain complementarity-determining region 2 (LCDR2), and light chain complementarity-determining region 3 (LCDR3).

[0008] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment comprises, i) a heavy chain complementarity-determining region 1 (HCDR1) containing any of the following amino acid sequences: SEQ ID NO: 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, or 123; ii) a heavy chain complementarity-determining region 2 (HCDR2) containing any of the following amino acid sequences: SEQ ID NO: 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, or 124; iii) a heavy chain complementarity-determining region 3 (HCDR3) containing any of the following amino acid sequences: SEQ ID NO: 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, or 123; iii) a heavy chain complementarity-determining region 3 (HCDR3) containing any of the following amino acid sequences: SEQ ID NO: 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 118, or 124; SEQ ID NO: 45, 51, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, or 124. NO: 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, or 125; iv) Light chain complementarity-determining region 1 (LCDR1) contains any of the following amino acid sequences: SEQ ID NO: 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, or 126; v) Light chain complementarity-determining region 2 (LCDR2) contains any of the following amino acid sequences: SEQ ID NO: 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, or 127; and vi) Light chain complementarity-determining region 3 (LCDR3) contains any of the following amino acid sequences: SEQ ID NO: 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122 or 128.

[0009] In some embodiments, preferably, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise any of the following amino acid sequences: a) SEQ ID NO: 57, 58, 59, 60, 61, and 62; b) SEQ ID NO: 45, 46, 47, 48, 49, and 50; c) SEQ ID NO: 51, 52, 53, 54, 55, and 56; d) SEQ ID NO: 63, 64, 65, 66, 67, and 68; e) SEQ ID NO: 69, 70, 71, 72, 73, and 74; f) SEQ ID NO: 75, 76, 77, 78, 79, and 80; g) SEQ ID NO: 81, 82, 83, 84, 85, and 86; h) SEQ ID NO: 87, 88, 89, 90, 91, and 92; i) SEQ ID NO: 57, 58, 59, 60, 61, and 62; SEQ ID NO: 93, 94, 95, 96, 97 and 98; j) SEQ ID NO: 99, 100, 101, 102, 103 and 104; k) SEQ ID NO: 105, 106, 107, 108, 109 and 110; l) SEQ ID NO: 111, 112, 113, 114, 115 and 116; m) SEQ ID NO: 117, 118, 119, 120, 121 and 122; or n) SEQ ID NO: 123, 124, 125, 126, 127 and 128.

[0010] In some embodiments, the heavy chain variable region comprises any amino acid sequence selected from SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 129, 133, 134, 135, 136, 140, 141, 142, 143, 147, 148, 149, 150, 155, 156, 157, or 158, or any amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0011] In some embodiments, the light chain variable region comprises any amino acid sequence selected from SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 130, 131, 132, 137, 138, 139, 144, 145, 146, 151, 152, 153, or 154, or any amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0012] In some embodiments, the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences selected from any of the following or having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity: 1) SEQ ID NO:150 and SEQ ID NO:152; 2) SEQ ID NO:17 and SEQ ID NO:18; 3) SEQ ID NO:19 and SEQ ID NO:20; 4) SEQ ID NO:21 and SEQ ID NO:22; 5) SEQ ID NO:23 and SEQ ID NO:24; 6) SEQ ID NO:25 and SEQ ID NO:26; 7) SEQ ID NO:27 and SEQ ID NO:28; 8) SEQ ID NO:29 and SEQ ID NO:20; 9) SEQ ID NO:20 and SEQ ID NO:21; 19) SEQ ID NO:20 and 10 ... SEQ ID NO:29 and SEQ ID NO:30; 9) SEQ ID NO:31 and SEQ ID NO:32; 10) SEQ ID NO:33 and SEQ ID NO:34; 11) SEQ ID NO:35 and SEQ ID NO:36; 12) SEQ ID NO:37 and SEQ ID NO:38; 13) SEQ ID NO:39 and SEQ ID NO:40; 14) SEQ ID NO:41 and SEQ ID NO:42; 15) SEQ ID NO:43 and SEQ ID NO:44; 16) SEQ ID NO:129 and SEQ ID NO:130; 17) SEQ ID NO:129 and SEQ ID NO:131; 18) SEQ ID NO:129 and SEQ ID NO:132; 19) SEQ ID NO:133 and SEQ ID NO:130; 20) SEQ ID NO:133 and SEQ ID NO:131; 21) SEQ ID NO:133 and SEQ ID NO:30; 22) SEQ ID NO:132; 23) SEQ ID NO:134 and SEQ ID NO:130; 24) SEQ ID NO:134 and SEQ ID NO:131; 25) SEQ ID NO:135 and SEQ ID NO:130; 26) SEQ ID NO:135 and SEQ ID NO:131; 27) SEQ ID NO:135 and SEQ ID NO:132; 28) SEQ ID NO:136 and SEQ ID NO:137;29) SEQ ID NO:136 and SEQ ID NO:138; 30) SEQ ID NO:136 and SEQ ID NO:139; 31) SEQ ID NO:140 and SEQ ID NO:137; 32) SEQ ID NO:140 and SEQ ID NO:138; 33) SEQ ID NO:140 and SEQ ID NO:139; 34) SEQ ID NO:141 and SEQ ID NO:137; 35) SEQ ID NO:141 and SEQ ID NO:138; 36) SEQ ID NO:141 and SEQ ID NO:139; 37) SEQ ID NO:142 and SEQ ID NO:137; 38) SEQ ID NO:142 and SEQ ID NO:138; 39) SEQ ID NO:142 and SEQ ID NO:139; 40) SEQ ID NO:143 and SEQ ID NO:144; 41) SEQ ID NO:143 and SEQ ID NO:138; 42) SEQ ID NO:145; 43) SEQ ID NO:147 and SEQ ID NO:144; 44) SEQ ID NO:147 and SEQ ID NO:145; 45) SEQ ID NO:147 and SEQ ID NO:146; 46) SEQ ID NO:148 and SEQ ID NO:144; 47) SEQ ID NO:148 and SEQ ID NO:145; 48) SEQ ID NO:148 and SEQ ID NO:146; 49) SEQ ID NO:149 and SEQ ID NO:144; 50) SEQ ID NO:149 and SEQ ID NO:145; 51) SEQ ID NO:149 and SEQ ID NO:146; 52) SEQ ID NO:150 and SEQ ID NO:151; 53) SEQ ID NO:150 and SEQ ID NO:153; 54) SEQ ID NO:150 and SEQ ID NO:153; ID NO:154; 55) SEQ ID NO:155 and SEQ ID NO:151; 56) SEQ ID NO:155 and SEQ ID NO:152; 57) SEQ ID NO:155 and SEQ ID NO:153; 58) SEQ ID NO:155 and SEQ ID NO:154; 59) SEQ ID NO:156 and SEQ ID NO:151; 60) SEQ ID NO:156 and SEQ ID NO:152;61) SEQ ID NO:156 and SEQ ID NO:153; 62) SEQ ID NO:156 and SEQ ID NO:154; 63) SEQ ID NO:157 and SEQ ID NO:151; 64) SEQ ID NO:157 and SEQ ID NO:152; 65) SEQ ID NO:157 and SEQ ID NO:153; 66) SEQ ID NO:157 and SEQ ID NO:154; 67) SEQ ID NO:158 and SEQ ID NO:151; 68) SEQ ID NO:158 and SEQ ID NO:152; 69) SEQ ID NO:158 and SEQ ID NO:153; or 70) SEQ ID NO:158 and SEQ ID NO:154.

[0013] The anti-CLDN6 antibody or its antigen-binding fragment described herein further comprises a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region comprises an Fc; more preferably, the Fc is derived from a mouse or a human; even more preferably, the sequence of the Fc is natural or modified.

[0014] The anti-CLDN6 antibody or its antigen-binding fragment in this article can be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a humanized antibody, a chimeric antibody, a fully human antibody, a full-length antibody, Fab, Fv, scFv, F(ab')2, a linear antibody, or a single-domain antibody.

[0015] The anti-CLDN6 antibody or its antigen-binding fragment mentioned in this article may be in the form of IgG1, IgG2, IgG3 or IgG4.

[0016] This article also provides a conjugate formed by conjugating the anti-CLDN6 antibody or its antigen-binding fragment described herein with a capture marker or a detection marker; preferably, the detection marker includes a radionuclide, a luminescent substance, a colored substance, or an enzyme.

[0017] This article also provides an antibody-drug conjugate (ADC), which is formed by conjugating the anti-CLDN6 antibody or its antigen-binding fragment described herein with other bioactive molecules; preferably, the other bioactive molecules are small molecule drugs; preferably, the anti-CLDN6 antibody or its antigen-binding fragment is connected to the other bioactive molecules through a linker.

[0018] This document also provides a nucleic acid encoding the present anti-CLDN6 antibody or its antigen-binding fragment, a recombinant vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the recombinant vector. Preferably, the host cell is a prokaryotic cell (preferably Escherichia coli) or a eukaryotic cell (preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell or a HEK293 cell).

[0019] This article also provides a method for preparing the anti-CLDN6 antibody or its antigen-binding fragment thereof, the method comprising: culturing the host cells described above under suitable conditions, and purifying the expression product from the host cells.

[0020] This article also provides a pharmaceutical composition comprising an effective amount of the present article's anti-CLDN6 antibody or its antigen-binding fragment, or an effective amount of the present article's antibody-drug conjugate, or an effective amount of the present article's nucleic acid, or an effective amount of the present article's recombinant vector, or an effective amount of the present article's host cell.

[0021] This document also provides a pharmaceutical composition comprising the anti-CLDN6 antibody or its antigen-binding fragment as described herein, or comprising the antibody-drug conjugate as described herein, or comprising the nucleic acid, recombinant vector or host cell as described herein; preferably, it further comprises a pharmaceutically acceptable vector.

[0022] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0023] This document also provides a kit or reagent kit comprising a container and a pharmaceutical composition of this disclosure located within the container.

[0024] This document also provides the use of the anti-CLDN6 antibody or its antigen-binding fragment or pharmaceutical composition described herein in the preparation of medicaments for the treatment and / or prevention of diseases; preferably, the diseases are tumors.

[0025] This document also provides the use of the anti-CLDN6 antibody or its antigen-binding fragment described herein, or the drug conjugate described herein, or the pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing a disease; preferably, the disease is a tumor.

[0026] In one embodiment, the drug targets tumor cells that abnormally express CLDN6. In one embodiment, the tumor is selected from: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms.

[0027] This article also provides a method for treating and / or preventing diseases associated with CLDN6 expression in a subject, comprising administering to the subject an effective amount of the anti-CLDN6 antibody of this article or its antigen-binding fragment or pharmaceutical composition; preferably, the disease associated with CLDN6 expression is a tumor; more preferably, the tumor is selected from: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms.

[0028] This article also provides a method for treating and / or preventing CLDN6-related diseases in subjects of need, comprising administering to the subject an effective amount of the anti-CLDN6 antibody described herein or its antigen-binding fragment, or the drug conjugate described herein, or the drug composition described herein; preferably, the CLDN6-related disease is a tumor; more preferably, the tumor is selected from: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms.

[0029] In some embodiments, the anti-CLDN6 antibody or its antigen-binding fragment treats and / or prevents disease through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis induction, and proliferation inhibition.

[0030] In some embodiments, the tumor is a tumor that abnormally expresses CLDN6; preferably, the tumor is selected from any of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms.

[0031] This document also provides the use of the anti-CLDN6 antibody or its antigen-binding fragment described herein, or the conjugates described herein, in the preparation of detection or diagnostic reagents.

[0032] In one embodiment, the detection reagent is used to detect the expression of CLDN6; the diagnostic reagent is used to diagnose tumors; preferably, the tumors are selected from: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms.

[0033] This article also provides a method for detecting CLDN6 expression in a sample, the method comprising:

[0034] (1) Contact the sample with the anti-CLDN6 antibody or its antigen-binding fragment as described herein;

[0035] (2) Detect the formation of a complex of the anti-CLDN6 antibody or its antigen-binding fragment; optionally, the anti-CLDN6 antibody or its antigen-binding fragment is detectably labeled.

[0036] This article also provides a method for detecting CLDN6 expression in a sample, the method comprising:

[0037] (1) The sample is contacted with the anti-CLDN6 antibody or its antigen-binding fragment described herein, or the conjugate described herein, wherein the anti-CLDN6 antibody or its antigen-binding fragment, or the conjugate, forms a complex with CLDN6 in the sample;

[0038] (2) Detect the formation of a complex of the anti-CLDN6 antibody or its antigen-binding fragment, or conjugate, with CLDN6; optionally, the anti-CLDN6 antibody or its antigen-binding fragment is detectably labeled;

[0039] Preferably, the detection is not for diagnostic or therapeutic purposes.

[0040] This article also provides a method for inducing cell death in cells expressing CLDN6, the method comprising contacting the cells with an anti-CLDN6 antibody or an antigen-binding fragment thereof described herein or a pharmaceutical composition described herein, wherein the CLDN6-expressing cells are tumor cells.

[0041] This article also provides one or more immune effector functions of anti-CLDN6 antibodies or their antigen-binding fragments against CLDN6 cells with native conformation, wherein the immune effector function is preferably selected from complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis induction, and proliferation inhibition, with preferred effector functions being ADCC and / or CDC.

[0042] This document also provides an anti-CLDN6 antibody or its antigen-binding fragment thereof, or a drug conjugate or pharmaceutical composition thereof, for the treatment and / or prevention of diseases associated with CLDN6 expression.

[0043] In some embodiments, the disease is a tumor; preferably, the anti-CLDN6 antibody or its antigen-binding fragment treats and / or prevents the disease through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis induction, and proliferation inhibition; preferably, the tumor is a tumor cell with abnormal CLDN6 expression.

[0044] In some implementations, the tumor is selected from any of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms. Attached Figure Description

[0045] The accompanying drawings further illustrate the novel features disclosed herein. Referring to these drawings will provide a better understanding of the features and advantages disclosed herein; however, it should be understood that these drawings are for illustrative purposes only, illustrating specific embodiments of the principles disclosed herein, and are not intended to limit the scope of this document.

[0046] Figure 1 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN6.

[0047] Figure 2 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN9.

[0048] Figure 3 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN4.

[0049] Figure 4 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN3.

[0050] Figure 5 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN6.

[0051] Figure 6 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN9.

[0052] Figure 7 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN4.

[0053] Figure 8 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing human CLDN3.

[0054] Figure 9 shows the binding of an exemplary chimeric antibody to HEK293 overexpressing human CLDN6.

[0055] Figure 10 shows the binding of an exemplary chimeric antibody to PA-1 tumor cells.

[0056] Figure 11 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing rat.

[0057] Figure 12 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing mouse CLDN6.

[0058] Figure 13 shows the binding of an exemplary chimeric antibody to HEK293 cells overexpressing cyno CLDN6.

[0059] Figure 14 shows the internalization of an exemplary chimeric antibody in HEK293 cells overexpressing human CLDN6.

[0060] Figure 15 shows the internalization of an exemplary chimeric antibody in tumor cells PA-1.

[0061] Figure 16 shows the ADCC results of an exemplary chimeric antibody in PA-1 tumor cells.

[0062] Figure 17 shows the CDC results of an exemplary chimeric antibody in PA-1 tumor cells.

[0063] Figures 18-25 show the binding of exemplary humanized antibodies to PA-1 tumor cells.

[0064] Figure 26 shows the binding of an exemplary humanized antibody overexpressing human CLDN3 to HEK293 cells.

[0065] Figure 27 shows the binding of an exemplary humanized antibody overexpressing human CLDN4 to HEK293 cells.

[0066] Figure 28 shows the binding of an exemplary humanized antibody overexpressing human CLDN6 to HEK293 cells.

[0067] Figure 29 shows the binding of an exemplary humanized antibody overexpressing human CLDN9 to HEK293 cells.

[0068] Figure 30 shows the binding of an exemplary humanized antibody to HEK293 cells overexpressing rat CLDN6.

[0069] Figure 31 shows the binding of an exemplary humanized antibody to HEK293 cells overexpressing cyno CLDN6.

[0070] Figure 32 shows the binding of an exemplary humanized antibody to HEK293 cells overexpressing mouse CLDN6.

[0071] Figure 33 shows the internalization of an exemplary humanized antibody in tumor cells PA-1 as detected by the DT3C method.

[0072] Figure 34 shows the internalization of an exemplary humanized antibody in tumor cells PA-1 as detected by the DT3C method.

[0073] Figure 35 shows the internalization of an exemplary humanized antibody in PA-1 tumor cells detected by the DT3C method.

[0074] Figure 36 shows the internalization of an exemplary humanized antibody in PA-1 tumor cells detected by the DT3C method.

[0075] Figure 37 shows the internalization of an exemplary humanized antibody in PA-1 tumor cells detected by the DT3C method.

[0076] Figure 38 shows the detection of the internalization of an exemplary humanized antibody in tumor cells PA-1 using the pHrodo method.

[0077] Figure 39 shows the internalization of an exemplary humanized antibody in OVCAR3 cells as detected by the pHrodo method.

[0078] Figure 40 shows the ADCC results of an exemplary humanized antibody in PA-1 cells.

[0079] Figure 41 shows the CDC results of an exemplary humanized antibody in PA-1 cells. Detailed Implementation

[0080] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each publication, patent or patent application has been specifically and individually indicated to be incorporated herein by reference.

[0081] Before describing this document in detail below, it should be understood that this document is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing specific implementations only and is not intended to limit the scope of this document. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this document pertains.

[0082] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0083] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0084] the term

[0085] The term "CLDN (Claudin)" refers to the cleaudins family, a crucial component of tight junctions (TJs). The human cleaudins family comprises at least 23 members ranging in size from 22 to 34 kDa. Claudins share similar structures; each cleaudin molecule has four transmembrane domains, two extracellular loops (EC1 and EC2), and two intracellular tails. Typically, EC1 is approximately 50-60 amino acids in size, while EC2 is smaller, around 25 amino acids. CLDN6 is located on chromosome 16p3.3 and has a molecular weight of approximately 20-40 kDa. As an important molecule in cytoskeleton formation and regulation of cell signaling pathways, CLDN6 can influence cell growth processes.

[0086] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, extracellular domains (ECDs), etc. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions and combinations thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing such antigen, can be used to generate antibodies specific to the antigenic determinant. The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response.

[0087] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes typically exist in a unique spatial conformation and comprise at least 3-15 amino acids. Methods for determining the epitope bound to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0088] The terms “antibody” or “antigen-binding fragment” used in this article may include complete antibodies (e.g., full-length monoclonal antibodies) or any antigen-binding fragment (i.e., antigen-binding part) or their single chains, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0089] In one embodiment, an antibody typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have this structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and a constant region (CH).

[0090] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be assigned to one of two distinctly different types based on the amino acid sequence of its constant domain, termed κ and λ.

[0091] In the case of IgG, IgA, and IgD antibodies, this constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0092] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site of the antibody, giving the complete IgG antibody two binding sites. The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Both VH and VL have four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence within the heavy chain variable domain (or light chain variable domain): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0093] The term "Fc" is used to define the C-terminal region of the immunoglobulin heavy chain, which includes at least a portion of the constant region, including the native sequence Fc region and the variant Fc region.

[0094] In this article, "antibody" can be used in the broadest sense, including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0095] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, or combinations of the above techniques.

[0096] It should be noted that the division of the CDR and FR in the variable region of the antibody in this article is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, based on the monoclonal antibody sequence in this article, humanized antibodies containing one or more CDRs derived from any nomenclature system are explicitly kept within the scope of this article.

[0097] The term "chimeric antibody" is a construct in which a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, while the remainder of this or these chains is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, or in fragments of such antibodies. In a narrower sense, a chimeric antibody comprises all or most of selected murine heavy and light chain variable regions operatively linked to human light and heavy chain constant regions. Constant region sequences, or variants or derivatives thereof, can be operatively associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide a full-length anti-CLDN6 antibody that can be used on its own or incorporated into this document.

[0098] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mouse, rat, rabbit, or primate. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the receptor human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Additionally, humanized antibodies may contain novel residues not found in the receptor antibody or the donor antibody to further improve antibody performance.

[0099] The terms "bispecific binding molecule" and "multispecific binding molecule" refer to binding molecules (e.g., antibodies or molecules containing antibody fragments) that are specific to two or more different antigens (or epitopes), with bispecific antibodies being preferred.

[0100] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its immunogenicity-determining moiety, or a fusion protein product further derived from such fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0101] When using the variable regions described herein to prepare antibodies, antigen-binding molecules, bispecific binding molecules, or multispecific binding molecules, the constant regions are not particularly limited. Constant regions known to those skilled in the art or obtained independently can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fcγ receptors or FcRn) can also be introduced into the constant regions.

[0102] There are no particular limitations on the methods used to obtain the binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules disclosed herein, and they can be obtained by any method, such as Cold Spring Harbor's Guide to Laboratory Antibody Techniques, Chapters 5-8 and 15. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0103] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0104] The term "biological activity" refers to the ability of an antibody to bind to an antigen and cause a measurable biological response, which can be measured in vitro or in vivo.

[0105] The terms "sequence identity," "sequence similarity," or "sequence homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0106] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated to a therapeutic active substance or active pharmaceutical ingredient (API), such that the therapeutic active substance or active pharmaceutical ingredient (API) can target the antibody's binding target to exert its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent linking of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC. The ADCs described herein can be used to deliver cytotoxic agents or other payloads to target sites (e.g., tumorigenic cells and / or cells expressing CLDN). As used herein, the terms "drug" and "warhead" are used interchangeably and will refer to a biologically active or detectable molecule or compound, including anticancer agents as described below. A "payload" may comprise a drug or warhead in combination with an optional linker compound. The warhead on the conjugate can contain peptides, polypeptides, proteins, prodrugs that are metabolized in vivo into active agents, polymers, nucleic acid molecules, small molecules, binders, mimics, synthetic drugs, inorganic molecules, organic molecules, and radioactive isotopes.

[0107] The term "pharmaceutical composition" refers to a formulation which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation.

[0108] The term “pharmaceutical carrier” or “pharmaceuticalally acceptable carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, auxiliary agent, or medium that is administered with a therapeutic agent.

[0109] The binding molecules or antigen-binding fragments described in this article can be used in combination with other drugs. The active ingredients can be mixed together to form a single drug delivery unit, or they can be used independently as drug delivery units.

[0110] The term "effective dose" refers to the dosage of a pharmaceutical preparation containing the active ingredient described herein, which, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by considering a number of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered preparation; the chosen dosing regimen; and the use of any concomitant therapies.

[0111] The terms "pharmaceutical kit" or "reagent kit" include an effective amount of one or more unit dosage forms of the pharmaceutical composition described herein. In some embodiments, the kit may contain a sterile container; such a container may be a box, ampoule, bottle, vial, tube, bag, blister pack, or other suitable container form known in the art. Furthermore, the kit includes instructions for administering the pharmaceutical composition described herein to an individual.

[0112] The terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals in this article include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

[0113] The term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in the clinicopathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and mitigating or improving prognosis.

[0114] The terms "disease," "symptom," or "disorder" refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. "Disease" includes, but is not limited to, tumors.

[0115] The term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is usually uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells.

[0116] The term "immune effector function" includes any function mediated by components of the immune system that inhibits tumor growth and / or tumorigenesis, including the suppression of tumor spread and metastasis. Immune effector functions result in the killing of tumor cells. In this article, immune effector functions refer to antibody-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), induction of apoptosis in cells carrying tumor-associated antigens (e.g., through antibody binding to surface antigens), and / or inhibition of the proliferation of cells carrying tumor-associated antigens.

[0117] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to the binding of antibodies to antigenic epitopes of infected cells or tumor cells, whose Fc fragments bind to Fc receptors (FcRs) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0118] The term "complement-dependent cytotoxicity (CDC)" refers to cytotoxic effects involving complement, which involve antibodies binding to corresponding antigens on the cell membrane surface, forming complexes that activate the classical complement pathway and exert a lytic effect on target cells.

[0119] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of generation. Progeny may not be identical to parental cells in terms of nucleic acid content, but may contain mutations. A host cell is any type of cell system that can be used to produce the bispecific antigen-binding molecule described herein. Host cells include cultured cells, such as mammalian cultured cells including CHO cells, HEK293 cells, HepG2 cells, Hep3B cells, and Huh-7 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0120] Example

[0121] The following specific examples further illustrate this article. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this article. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or as recommended by the manufacturer.

[0122] Example 1: CLDN6 and its related sequence information

[0123] This embodiment introduces CLDN6 and other related sequence information. The full-length amino acid information of these sequences is used for the construction of various stable cell lines required for hybridoma, chimeric, and humanized antibody affinity screening, specificity, species cross-fertilization, and related functional activity verification. This sequence information is also used for the preparation of animal immunogens (proteins, cells, and DNA). Detailed sequence information is shown in Table 1.

[0124] Table 1 Sequence Information

[0125] Example 2: Information related to positive control antibodies

[0126] Table 2. Amino acid sequences of the variable region of positive antibodies.

[0127] Example 3: Animal Immunization

[0128] To generate antibodies against CLDN6, standard biological protocols were followed in the experimental procedures. In this experiment, protein (hCLDN6-VLP), cells (HEK293 cells stably transfected with hCLDN6), and DNA (a DNA vector encoding hCLDN6) were used as immunogens to immunize different strains of mice.

[0129] Plasma samples were obtained by collecting blood from the inner canthal vein during the later stages of immunization. The titer of the immune serum was determined by ELISA and FACS to assess the animal's immune response. After immunization, mice with an immune response were euthanized to prepare hybridoma cells.

[0130] Example 4: Hybridoma cells producing anti-CLDN6 monoclonal antibody

[0131] To generate hybridoma cells with anti-CLDN6 monoclonal antibodies, immune-responsive mice were euthanized with carbon dioxide, and feeder cells were collected using a syringe. The feeder cell suspension was then seeded into prepared 96-well plates. A certain number of myeloma cells and spleen cells were mixed in a specific ratio for cell fusion. HAT medium (1 mL 100×HT supplement + 1 mL aminopterin + 10 mL FBS + 88 mL DMEM) was added to the fused cells and mixed thoroughly to prepare a cell suspension. The cell suspension was then poured into culture dishes and thoroughly mixed, and seeded into 96-well feeder cell plates using a multichannel pipette. The fused feeder cell plates were placed in an incubator and cultured at 37°C with 5.5% CO2 for 7–10 days. Positive clones against CLDN6 were then screened using ELISA and FACS. The selected positive clones were subcloned using limiting dilution to obtain stable, monoclonal hybridoma cells. In this experiment, cell supernatants from subclones were screened using FACS technology in CHO-K1 cells overexpressing hCLDN6 and HKE293 cells overexpressing hCLDN9. The results are shown in Tables 3-1 and 3-2. After two rounds of fusion, 16 hybridoma cell lines producing antibodies specifically binding to CHO-K1-hCLDN6 were finally obtained. Among them, some antibodies were screened to bind to both CHO-K1-hCLDN6 and HKE293-hCLDN9, possibly due to the high homology of the amino acid sequences of CLDN6 and CLDN9.

[0132] Table 3-1 Binding of hybridoma antibodies to overexpressing cell lines

[0133] Table 3-2 Binding of hybridoma antibodies to overexpressing cell lines

[0134] Hybridoma cell lines secreting monoclonal antibodies, obtained through the above screening process, were cultured, and total RNA was extracted from the cells using standard biological methods. Using the total RNA as a template, PrimeScript was employed... TM The 1st Strand cDNA Synthesis Kit (TAKARA) was used for reverse transcription to synthesize cDNA. Using cDNA as a template, amplification was performed using primers for the constant region of the antibody. After separation by agarose gel electrophoresis, the DNA fragments were purified and sequenced. Hybridoma cells obtained through screening yielded 14 specific sequences; the amino acid sequences of their variable regions are shown in Table 4.

[0135] Table 4. Amino acid sequences of the variable regions of 14 monoclonal antibodies

[0136] Based on the above amino acid sequence, the CDR and FR of the antibody variable region are divided using the Kabat numbering rule. The composition of the 6 CDR sequences of each antibody is shown in Table 5 below. The numbers in parentheses in Table 5 represent the sequence number. For example, (45) represents SEQ ID NO:45.

[0137] Table 5. CDR sequences of 14 antibody variable regions

[0138] Example 5: Construction and expression of anti-CLDN6 chimeric antibody plasmid

[0139] Sequencing fragments of the hybridoma monoclonal antibody light and heavy chain variable regions were cloned into expression vector pTT5 containing the antibody light and heavy chain constant regions, respectively. Expression vectors with correct sequences were selected for large-scale amplification to prepare expression plasmids suitable for transfection. The antibody was expressed using Expi293F cells after resuscitation. TM (Thermo Fisher Scientific) Cells were seeded at a specific density in shake flasks (Corning Inc.) and cultured on a shaker at 37°C with 8% CO2. Once the cells showed good growth, the cell density was adjusted, and transfection was performed after 24 hours of shake-flask culture. The successfully constructed light chain plasmid and heavy chain plasmid were mixed in Opti-MEM at a specific ratio. TMCells were cultured in IReduced Serum Medium. PEI MAX transfection reagent was added, mixed, and incubated at room temperature for 10 minutes. The plasmid-PEI mixture was slowly added dropwise to a shake flask. After 16-18 hours of transfection, 10× Feed Solution and VPA solution were added. After 6 days of cell culture, the supernatant was collected and purified. The purified chimeric antibody was subjected to SDS-PAGE purity analysis and A280 concentration determination. The chimeric antibody was named by adding the prefix CH- to the original hybridoma clone.

[0140] Example 6: Binding assay of anti-CLDN6 chimeric antibody

[0141] A. Cell-binding specificity of anti-CLDN6 chimeric antibody

[0142] The binding of the chimeric antibody described in this study to HEK293 cells overexpressing human CLDN6, CLDN9, CLDN4, and CLDN3 was detected using FACS.

[0143] HEK293-hCLDN6, HEK293-hCLDN9, HEK293-hCLDN4, and HEK293-hCLDN3 cells were collected, resuspended in 2% FBS phosphate buffer, and cell concentrations were adjusted. Serially diluted chimeric antibodies were added, with unrelated human IgG as the negative control and PC-2 as the positive control. Cells were incubated at 4°C for 1 h, washed twice with 2% FBS phosphate buffer, and 100 μL of fluorescently labeled anti-human IgG secondary antibody was added to each well. Cells were incubated at 4°C for 0.5–1 h, washed twice with 2% FBS phosphate buffer, and the prepared samples were analyzed by flow cytometry. The mean fluorescence intensity (MFI) for each concentration was calculated using Flowjo software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Tables 6-1 and 6-2.

[0144] Table 6-1 Binding of anti-CLDN6 chimeric antibody to hCLDN6

[0145] Table 6-2 Binding of anti-CLDN6 chimeric antibody to hCLDN6

[0146] Table 6 and Figures 1-8 show the binding affinity of 14 chimeric antibodies to HEK293-hCLDN6, HEK293-hCLDN9, HEK293-hCLDN4, and HEK293-hCLDN3 overexpressing cells, respectively. The results show that the chimeric antibody CH-1 bound to both CLDN6 and CLDN9 with strong binding affinity. CH-2 and CH-6 bound to both CLDN6 and CLDN9, but their binding affinity was lower than that of CH-1. CH-4 bound to CLDN6 more strongly than CLDN9, showing some selectivity, but this antibody also bound to CLDN4 to some extent. CH-5 showed good specificity, binding only to CLDN6. CH-8 and CH-9 bound to CLDN6 weakly, while CH-7 and CH-14 bound to CLDN6 more strongly than CLDN9, showing some selectivity, but CH-14 bound to CLDN4 more strongly than CH-7. CH-10, CH-11, CH-12, and CH-13 are all dual-bindings of CLDN6 and CLDN9. CH-10 and CH-13 are weakly bound to CLDN3, while CH-11 and CH-12 are strongly bound to CLDN4. Finally, CH-1, CH-4, CH-5, CH-7, and CH-14 were selected for further verification.

[0147] B. Binding of anti-CLDN6 chimeric antibody to cells with high hCLDN6 expression

[0148] The binding of anti-CLDN6 chimeric antibody to HEK293 cells stably expressing hCLDN6 (HEK293-hCLDN6) and PA-1 cells derived from ovarian cancer tissue that naturally express hCLDN6 was detected by FACS.

[0149] HEK293-hCLDN6 and PA-1 cells were collected, resuspended in PBS, and the cell concentration was adjusted. Serially diluted antibodies were added, with unrelated human IgG as the negative control and PC-2 as the positive control. The cells were incubated at 4°C for 1 h, washed twice by centrifugation with 2% FBS phosphate buffer, and 100 μL of fluorescently labeled anti-human IgG secondary antibody was added to each well. The cells were incubated at 4°C for 0.5–1 h, washed twice by centrifugation with 2% FBS phosphate buffer, and then the prepared samples were analyzed by flow cytometry. The mean fluorescence intensity (MFI) for each concentration was calculated using Flowjo software, and the half-maximal binding concentration (EC50) was calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 7.

[0150] Table 7. Binding of anti-CLDN6 chimeric antibody to hCLDN6

[0151] Table 7 and Figures 9-10 show the affinity assay results of the chimeric antibody and positive control antibody PC-2 in HEK293-hCLDN6 and PA-1 cells. The results show that the chimeric antibody exhibits significant binding ability in both the overexpressing cell line HEK293-hCLDN6 and the tumor cell line PA-1. In HEK293-hCLDN6 cells, the affinity of the chimeric antibody CH-1 is weaker than that of the positive antibody PC-2, but in PA-1 cells, the affinity of CH-1 is essentially the same as that of PC-2. CH-14 shows the weakest affinity in both cell types, while the affinity of other candidate antibodies falls between that of CH-1 and CH-14.

[0152] C. Species cross-reactivity of anti-CLDN6 chimeric antibodies

[0153] The binding of the chimeric antibody presented in this study to HEK293-ratCLDN6 cells overexpressing rat CLDN6, HEK293-mCLDN6 cells overexpressing mouse CLDN6, and HEK293-cynoCLDN6 cells overexpressing monkey CLDN6 was detected using FACS.

[0154] HEK293-ratCLDN6, HEK293-mCLDN6, and HEK293-cynoCLDN6 cells were collected, resuspended in 2% FBS phosphate buffer, and cell concentrations were adjusted. Serially diluted chimeric antibodies were added, with unrelated human IgG as the negative control and PC-2 as the positive control. After incubation at 4°C for 1 h, the cells were washed twice by centrifugation with 2% FBS phosphate buffer. 100 μL of fluorescently labeled anti-human IgG secondary antibody was added to each well, and the cells were incubated at 4°C for 0.5–1 h. After washing twice by centrifugation with 2% FBS phosphate buffer, the prepared samples were analyzed by flow cytometry. The mean fluorescence intensity (MFI) for each concentration was calculated using Flowjo software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The binding status was determined by the highest average fluorescence intensity (Top MFI). The results are shown in Table 8.

[0155] Table 8. Species Cross-Reactivity of Candidate Chimeric Antibodies

[0156] Table 8 and Figures 9 and 11-13 show the affinity results of the chimeric antibody and positive control PC-2 with CLDN6-overexpressing cells of different species. The experimental results show that all candidate chimeric antibodies and positive controls in this study bound to CLDN6-overexpressing cells in humans, rats, mice, and cynomolgus monkeys, and the affinity trends of the chimeric antibodies on different cell lines were basically consistent.

[0157] Example 7: Internalization assay of anti-CLDN6 chimeric antibody

[0158] A. Internalization assay of anti-CLDN6 chimeric antibody (DT3C)

[0159] PA-1 and HEK293-hCLDN6 cells in good growth condition were collected by centrifugation and seeded into 96-well white transparent plates at a certain cell density for overnight culture. The candidate antibody and positive control were mixed with DT3C at a certain ratio and incubated at 37°C for 30 minutes to form mAb-DT3C conjugates. The mAb-DT3C conjugates were then serially diluted. The diluted mAb-DT3C conjugates were added to 96-well white transparent plates and incubated at 37°C in a 5% CO2 incubator for 3 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of [unspecified ingredient] was added to each well. Reagent was incubated at room temperature with shaking for 10 min, and the luminescence signal was detected using a microplate reader. The internalizing killing ability of each candidate antibody was calculated using the following formula:

[0160] The median cytotoxicity (EC50) was calculated using GraphPad software. 50 The internalization ability was determined by the maximum damage (Top value). The results are shown in Table 9.

[0161] Table 9 Results of internalization detection of anti-CLDN6 chimeric antibody

[0162] Table 9 and Figures 14-15 show the internalization results of the chimeric antibody and the positive control PC-2 in two types of CLDN6 cells with high expression. The results show that in HEK293-hCLDN6 cells, the candidate antibodies CH-1, CH-7, and CH-14 exhibited better internalization ability than the positive antibody PC-2, while CH-5 also showed good internalization ability. CH-4's internalization ability was weaker than PC-2. In PA-1 cells, CH-1 showed stronger internalization ability than PC-2, while the other chimeric antibodies showed weaker internalization ability than PC-2.

[0163] Example 8: In vitro functional assay of anti-CLDN6 chimeric antibody

[0164] A. Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0165] PA-1 was used as the target cell, and normal human NK cells were used as effector cells. The Promega chemiluminescent cell viability assay kit detects cell viability and uses it as an indicator of cell killing activity.

[0166] PA-1 cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in ADCC buffer and the cell density was adjusted before being transferred to 96-well plates. Different concentration gradients of chimeric antibodies and positive control samples were added to the 96-well plates, and the plates were incubated in a cell culture incubator (37℃ / 5% CO2) for approximately 30 minutes. Effector cells were then added to the 96-well plates at a specific ratio, and the plates were incubated in a cell culture incubator (37℃ / 5% CO2) for approximately 24 hours. After incubation, the 96-well plates were removed and allowed to equilibrate at room temperature for 30 minutes. A certain amount of... Reagent was incubated at room temperature with shaking for 10 min, and the luminescence signal was detected using a microplate reader. The ADCC killing ability of each candidate antibody was calculated using the following formula:

[0167] The median cytotoxicity (EC50) was calculated using GraphPad software. 50 The ADCC (Advanced Driver Classification) capability was determined by the maximum damage (Top value). The results are shown in Table 10.

[0168] Table 10 ADCC activity of anti-CLDN6 chimeric antibody

[0169] Table 10 and Figure 16 show the ADCC results of the chimeric antibody and the control antibody PC-2 in this study on PA-1 cells. The experimental results show that both the chimeric antibody and the positive control antibody have significant ADCC function in PA-1 cells.

[0170] B. Complement-dependent cytotoxicity (CDC)

[0171] Using PA-1 as the target cell and mixed healthy human serum (PNHS) as the complement source, through... The Promega chemiluminescent cell viability assay kit was used to detect cell viability, which is then used as an indicator of cell-killing activity.

[0172] PA-1 cells were collected, resuspended in CDC buffer, and cell density was adjusted. Cells were seeded into 96-well cell culture plates and cultured overnight. Different concentration gradients of chimeric antibodies and control samples were transferred to the 96-well plates and incubated in a cell culture incubator (37℃ / 5% CO2) for approximately 30 minutes. Human serum was diluted to a specific ratio, and the 96-well plates were incubated in a cell culture incubator (37℃ / 5% CO2) for approximately 24 hours. After incubation, the 96-well plates were removed and allowed to equilibrate at room temperature for approximately 30 minutes. A certain amount of... Reagent was incubated at room temperature with shaking for 10 min, and the luminescence signal was detected using a microplate reader. The CDC killing ability of each candidate antibody was calculated using the following formula:

[0173] The median cytotoxicity (EC50) was calculated using GraphPad software. 50 The CDC lethality was determined by the maximum lethality (Top value). The results are shown in Table 11.

[0174] Table 11 CDC activity of anti-CLDN6 chimeric antibody

[0175] Table 11 and Figure 17 show the CDC killing results of the chimeric antibody and the positive control PC-2 on PA-1. The experimental results show that all the chimeric antibodies in this paper have significant CDC activity, among which CH-1 has stronger CDC activity than PC-2.

[0176] Example 9: Antibody Humanization Design

[0177] A. Humanization design of antibodies

[0178] Based on the above experiments, CH1, CH5, and CH7 were selected for humanization design. Through sequence similarity comparison, antibody lines with the highest similarity to CH1, CH5, and CH7 were selected as antibody templates. After grafting the CDR region of the anti-CLDN6 antibody onto the selected human antibody variable region framework, the mutated amino acids were restored according to the structure to obtain humanized antibodies. In this embodiment, IGHV1_69D_01.human and IGKV1D_12_01.human from the IMGT database were selected as antibody templates for the CH1 heavy and light chains; IGHV4_4_08.human and IGKV1D_33_01.human from the IMGT database were selected as antibody templates for the CH7 heavy and light chains; IGHV4_30_4_01.human and IGKV1_NL1_01.human from the IMGT database were selected as antibody templates for the CH5 H1-H4 and CH5 L1-L3 heavy and light chains; IGHV4-28*02, IGHV6-1*01, IGHV2-70*01 and IGHV3-11*01 were selected as antibody templates for the CH5 H6-H9 heavy chains, respectively; and IGKV4-1*01, IGKV3-11*01 and IGKV1-27*01 were selected as antibody templates for the CH5 heavy and light chains, respectively. Antibody templates for the L4-L6 light chains. The humanized antibody numbers and their corresponding relationships are shown in Table 12.

[0179] Table 12 Variable region sequences of humanized antibodies

[0180] Example 10: Binding activity assay of humanized antibody

[0181] A. Binding of CH-1 humanized antibody to PA-1 cells

[0182] The binding activity of the CH-1 humanized antibody was determined according to Example 6A, using CLDN6-overexpressing PA-1 cells. Unrelated human IgG was used as the negative control, and PC-2 as the positive control. Flow cytometry was employed, and the mean fluorescence intensity (MFI) for each concentration was calculated using software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Tables 13-1 and 13-2.

[0183] Table 13-1 Binding of CH-1 humanized antibody to PA-1

[0184] Table 13-2 Binding of CH-1 humanized antibody to PA-1

[0185] Table 13 and Figures 18-19 show the affinity results of the humanized CH-1 antibody and its chimeric antibody in PA-1 cells with high expression in CLDN6 cells. The experimental results show that the Top value of the humanized antibody is slightly lower than that of the chimeric antibody CH-1, while the humanized antibody EC... 50 The affinity is also slightly lower than that of chimeric antibodies. In summary, the affinity of humanized antibodies and chimeric antibodies is basically the same.

[0186] B. Binding of CH-5 humanized antibody to PA-1 cells

[0187] The binding activity of the humanized CH-5 antibody was determined according to Example 6A using CLDN6-overexpressing PA-1 cells. Unrelated human IgG was used as the negative control, and PC-2 was used as the positive control. Flow cytometry was employed, and the mean fluorescence intensity (MFI) for each concentration was calculated using software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 14.

[0188] Table 14 Binding of CH-5 humanized antibody to PA-1

[0189] Table 14 and Figures 20-21 show the affinity results of the humanized CH-5 antibody and its chimeric antibody in PA-1 cells with high CLDN6 expression. The results show that the Top value of the humanized antibody was significantly lower than that of the chimeric antibody CH-5, indicating that the affinity of the humanized antibody was significantly lower than that of the chimeric antibody, necessitating re-humanization of the chimeric antibody. Meanwhile, studies have shown that antibody affinity and internalization ability are not correlated; further investigation will verify the differences in internalization ability between some humanized antibodies and chimeric antibodies.

[0190] Binding of C.CH-7 humanized antibody to PA-1 cells

[0191] The binding activity of the CH-7 humanized antibody was determined according to Example 6A using CLDN6-overexpressing PA-1 cells. Unrelated human IgG was used as the negative control, and PC-2 was used as the positive control. Flow cytometry was employed, and the mean fluorescence intensity (MFI) for each concentration was calculated using software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 15.

[0192] Table 15 Binding of anti-CLDN6 humanized antibody to PA-1

[0193] Table 15 and Figures 22-23 show the affinity results of the humanized antibodies and chimeric antibodies in PA-1 cells with high expression in CLDN6. The results show that the humanized antibodies CH7-H3L2, CH7-H3L3, CH7-H4L2, and CH7-H4L3 have significantly higher affinity than the chimeric antibody CH-7, while the affinity of other humanized antibodies is lower than that of their chimeric counterparts. Further investigation will verify the changes in the internalization ability of some humanized antibodies and chimeric antibodies.

[0194] D. Binding of humanized CH-5 antibody to PA-1 expressing cells

[0195] The binding activity of the humanized CH-5 antibody was determined according to Example 6A using CLDN6-overexpressing PA-1 cells. Unrelated human IgG was used as the negative control, and PC-2 was used as the positive control. Flow cytometry was employed, and the mean fluorescence intensity (MFI) for each concentration was calculated using software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Tables 16-1 and 16-2.

[0196] Table 16-1 Binding of CH-5 humanized antibody to PA-1

[0197] Table 16-2 Binding of CH-5 humanized antibody to PA-1

[0198] Table 16 and Figures 24-25 show the affinity results of the humanized CH-5 antibodies and their chimeric antibodies in PA-1 cells with high CLDN6 expression. The results show that the humanized antibodies CH5-5-H6L5, CH5-5-H7L5, CH5-5-H8L5, CH5-5-H9L5, and CH5-5-H10L5 have essentially the same affinity as the chimeric CH-5 antibody. The affinity of other humanized antibodies is weaker than that of their chimeric counterparts. Further investigation will be conducted to verify the changes in the internalization ability of some humanized antibodies and chimeric antibodies.

[0199] E. Binding selectivity of anti-CLDN6 humanized antibody

[0200] Binding activity was determined as described in Example 6A using HEK293 cells overexpressing human CLDN3, CLDN4, CLDN6, and CLDN9. Unrelated human IgG was used as a negative control, and PC-2 and TORL-23 were used as positive controls. Flow cytometry was employed, and the mean fluorescence intensity (MFI) for each concentration was calculated using software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 17.

[0201] Table 17 Binding Specificity of Anti-CLDN6 Humanized Antibody

[0202] Table 17 and Figures 26-29 show the binding of the humanized antibodies and control antibodies hPC-2 and TORL-23 to human CLDN3, CLDN4, CLDN6 and CLDN9, respectively.

[0203] Experimental results showed that CH5-5-H6L5 exhibited good specificity, not binding to hCLDN3, hCLDN4, and hCLDN9, but specifically binding to hCLDN6. Its binding specificity was similar to that of TORL23. CH5-H2L2 did not bind to hCLDN3, but showed weak binding to hCLDN4. CH1-H4L2 did not bind to either hCLDN3 or hCLDN4, but specifically bound to hCLDN6 and hCLDN9. Its binding specificity was similar to that of hPC-2.

[0204] F. Species cross-reactivity of anti-CLDN6 humanized antibodies

[0205] Binding activity was determined as described in Example 6A using HEK293 cells overexpressing CLDN6 from different species. Unrelated human IgG was used as a negative control, and PC-2 and TORL-23 were used as positive controls. Flow cytometry was employed, and the mean fluorescence intensity (MFI) for each concentration was calculated using software. The half-maximal binding concentration (EC50) was then calculated using GraphPad software. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 18.

[0206] Table 18. Species Cross-Reactivity of Anti-CLDN6 Humanized Antibodies

[0207] Table 18 and Figures 28 and 30-32 show the affinity results of the humanized antibody and control antibodies PC-2 and TORL-23 with CLDN6-overexpressing cells from different species. The results show that TORL-23 does not bind to rat and mouse CLDN6. CH1-H4L2, CH5-5-H6L5, and CH5-H2L2 bound to human, rat, mouse, and cynomolgus monkey CLDN6-overexpressing cells. This indicates that CH1-H4L2, CH5-5-H6L5, and CH5-H2L2 have better species cross-species compatibility, and also demonstrates that CH5-5-H6L5 and CH5-H2L2 have different binding epitopes compared to existing antibodies.

[0208] Example 11: Humanized Antibody Internalization Assay

[0209] A. CH-1 humanized antibody internalization assay (DT3C)

[0210] The internalization ability of the humanized antibody was detected using the method described in Example 7A, and the half-maximal cytotoxicity (EC50) was calculated using GraphPad software. 50 The internalization ability was determined by the maximum damage (Top value). The results are shown in Table 19.

[0211] Table 19-1 Internalization activity of CH-1 humanized antibodies

[0212] Table 19-2 Internalization activity of CH-1 humanized antibodies

[0213] Table 19 and Figures 33-34 show the internalization results of the CH-1 chimeric antibody, humanized antibody, and control antibody PC-2 in PA-1 cells. The results indicate that the internalization ability of the humanized antibody is superior to that of its chimeric antibody and positive control antibody.

[0214] B. CH-5 Humanized Antibody Internalization Assay (DT3C)

[0215] The internalization ability of the humanized antibody was detected using the method described in Example 7A, and the half-maximal cytotoxicity (EC50) was calculated using GraphPad software. 50 The internalization ability was determined by the maximum damage (Top value). The results are shown in Table 20.

[0216] Table 20 Internalization activity of CH-5 humanized antibodies

[0217] Table 20 and Figure 35 show the internalization results of the CH-5 chimeric antibody, humanized antibody, and control antibody PC-2 on PA-1 cells. The results show that the humanized antibody CH-5-5-H6L5 has a better internalization ability than the chimeric antibody CH-5, while other humanized antibodies are weaker than their chimeric counterparts.

[0218] C.CH-7 Humanized Antibody Internalization Assay (DT3C)

[0219] The internalization ability of the humanized antibody was detected according to the methods described in Examples 7 and A, and the half-maximal cytotoxic concentration (EC50) was calculated using GraphPad software. 50 The internalization ability was determined by the maximum damage (Top value). The results are shown in Table 21.

[0220] Table 21 Internalization activity of CH-7 humanized antibodies

[0221] Table 21 and Figure 36 show the internalization results of the CH-7 chimeric antibody, humanized antibody, and control antibody PC-2 in PA-1 cells.

[0222] D. Internalization assay of anti-CLDN6 humanized antibody (DT3C method)

[0223] The internalization ability of the humanized antibody was detected using the method described in Example 7A, and the half-maximal cytotoxicity (EC50) was calculated using GraphPad software. 50 The internalization ability was determined by the maximum damage (Top value). The results are shown in Table 22.

[0224] Table 22 Internalization activities of different humanized antibodies

[0225] Table 22 and Figure 37 show the internalization results of the humanized antibody and positive controls hPC-2 and TORL-23 on PA-1 cells. The results show that the humanized antibody CH1-H4L2 has better internalization ability than hPC-2 and TORL-23, CH5-5-H6L5 is between hPC-2 and TORL-23, while CH5-H2L2 is weaker than the positive control antibody.

[0226] E. Internalization assay of anti-CLDN6 humanized antibody (pHrodo method)

[0227] PA-1 cells in good growth condition were collected by centrifugation and seeded into 96-well plates at a specific cell density for overnight culture. The candidate antibodies and positive controls were mixed with pH-sensitive pHrodo fluorescent dye at a specific ratio and incubated at 37°C for 30 minutes to form conjugates. These conjugates were then serially diluted and added to 96-well plates, and incubated at 37°C with 5% CO2 for 1 day. After culture, the 96-well plates were removed, and cells were digested and collected for flow cytometry analysis. The internalizing cytotoxic activity of each candidate antibody was calculated.

[0228] Figures 38-39 show the internalization results of the humanized antibody and control antibodies hPC-2 and TORL-23 in PA-1 and OVCAR3 cells. The results show that the internalization ability of the humanized antibody CH1-H4L2 is comparable to hPC-2 and superior to TORL-23; CH5-5-H6L5 is between hPC-2 and TORL-23; while CH5-H2L2 is weaker than the positive control antibody.

[0229] Example 12: In vitro functional assay of anti-CLDN6 humanized antibody

[0230] A. Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0231] The ADCC activity of the humanized antibody was detected according to the method described in Example 8A, and the half-maximal cell killing concentration (EC50) was calculated using GraphPad software. 50 The ADCC (Advanced Driver Classification) capability was determined by the maximum damage (Top value). The results are shown in Table 23.

[0232] Table 23 ADCC activity of anti-CLDN6 humanized antibody

[0233] Table 19 and Figure 40 show the ADCC results of the humanized antibody and positive antibodies hPC-2 and TORL-23 on PA-1 cells. The results show that both the humanized antibody and the positive control antibody exhibit significant ADCC function. The ADCC activities of CH1-H4L2 and CH5-5-H6L5 are intermediate between the positive controls hPC-2 and TORL-23, while CH5-H2L2 has a weaker maximum cell-killing effect than the positive control.

[0234] B. Complement-dependent cytotoxicity (CDC)

[0235] The CDC activity of the humanized antibody was detected according to the method described in Example 8B, and the half-maximal cytotoxic concentration (EC50) was calculated using GraphPad software. 50 The CDC lethality was determined by the maximum lethality (Top value). The results are shown in Table 24.

[0236] Table 24 CDC activity of anti-CLDN6 humanized antibody

[0237] Table 24 and Figure 41 show the CDC killing results of the humanized antibody and positive controls hPC-2 and TORL-23 on PA-1. The experimental results show that both the humanized antibody and the positive antibody have significant CDC activity. Among them, the CDC activity of CH1-H4L2 is stronger than that of the positive antibodies hPC-2 and TORL-23, and the CDC activity of CH5-5-H6L5 is between that of the positive antibodies hPC-2 and TORL-23. However, the maximum CDC killing effect of CH5-H2L2 is weaker than that of the positive control antibodies hPC-2 and TORL-23.

[0238] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for extraordinary experimentation. All such equivalents are within the scope of this document and are encompassed by the claims.

Claims

An anti-CLDN6 antibody or its antigen-binding fragment thereof, wherein the anti-CLDN6 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3), and the light chain variable region comprises light chain complementarity-determining region 1 (LCDR1), light chain complementarity-determining region 2 (LCDR2), and light chain complementarity-determining region 3 (LCDR3); wherein, i)HCDR1 contains any of the following amino acid sequences: SEQ ID NO: 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117 or 123; ii) HCDR2 contains any of the following amino acid sequences: SEQ ID NO: 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124; iii) HCDR3 contains any of the following amino acid sequences: SEQ ID NO: 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119 or 125; iv) LCDR1 contains any of the following amino acid sequences: SEQ ID NO: 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126; v) LCDR2 contains an amino acid sequence selected from any of the following: SEQ ID NO: 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115, 121, or 127; and vi) LCDR3 contains any of the following amino acid sequences: SEQ ID NO: 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122 or 128; Preferably, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise any of the following amino acid sequences: a) SEQ ID NO: 57, 58, 59, 60, 61 and 62; b) SEQ ID NO: 45, 46, 47, 48, 49 and 50; c) SEQ ID NO: 51, 52, 53, 54, 55 and 56; d) SEQ ID NO: 63, 64, 65, 66, 67 and 68; e) SEQ ID NO: 69, 70, 71, 72, 73 and 74; f) SEQ ID NO: 75, 76, 77, 78, 79 and 80; g) SEQ ID NO: 81, 82, 83, 84, 85 and 86; h) SEQ ID NO: 87, 88, 89, 90, 91 and 92; i) SEQ ID NO: 93, 94, 95, 96, 97 and 98; j) SEQ ID SEQ ID NO: 99, 100, 101, 102, 103 and 104; k) SEQ ID NO: 105, 106, 107, 108, 109 and 110; l) SEQ ID NO: 111, 112, 113, 114, 115 and 116; m) SEQ ID NO: 117, 118, 119, 120, 121 and 122; or n) SEQ ID NO: 123, 124, 125, 126, 127 and 128. The anti-CLDN6 antibody or its antigen-binding fragment as described in claim 1, wherein, The heavy chain variable region comprises any amino acid sequence selected from SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 129, 133, 134, 135, 136, 140, 141, 142, 143, 147, 148, 149, 150, 155, 156, 157, or 158, or any amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; And / or, the light chain variable region comprises any amino acid sequence selected from SEQ ID NO:18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 130, 131, 132, 137, 138, 139, 144, 145, 146, 151, 152, 153, or 154, or any amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; Preferably, the heavy chain variable region and the light chain variable region each comprise an amino acid sequence selected from any of the following or having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity: 1) SEQ ID NO:150 and SEQ ID NO:152; 2) SEQ ID NO:17 and SEQ ID NO:18; 3) SEQ ID NO:19 and SEQ ID NO:20; 4) SEQ ID NO:21 and SEQ ID NO:22; 5) SEQ ID NO:23 and SEQ ID NO:24; 6) SEQ ID NO:25 and SEQ ID NO:26; 7) SEQ ID NO:27 and SEQ ID NO:28; 8) SEQ ID NO:29 and SEQ ID NO:30; 9) SEQ ID NO:31 and SEQ ID NO:32; 10) SEQ ID NO:33 and SEQ ID NO:34; 11) SEQ ID NO:35 and SEQ ID NO:36; 12) SEQ ID NO:37 and SEQ ID NO:38; 13) SEQ ID NO:39 and SEQ ID NO:40; 14) SEQ ID NO:41 and SEQ ID NO:42; 15) SEQ ID NO:43 and SEQ ID NO:152; NO:44; 16) SEQ ID NO:129 and SEQ ID NO:130; 17) SEQ ID NO:129 and SEQ ID NO:131; 18) SEQ ID NO:129 and SEQ ID NO:132; 19) SEQ ID NO:133 and SEQ ID NO:130; 20) SEQ ID NO:133 and SEQ ID NO:131; 21) SEQ ID NO:133 and SEQ ID NO:132; 22) SEQ ID NO:134 and SEQ ID NO:130; 23) SEQ ID NO:134 and SEQ ID NO:131; 24) SEQ ID NO:134 and SEQ ID NO:132; 25) SEQ ID NO:135 and SEQ ID NO:130; 26) SEQ ID NO:135 and SEQ ID NO:131; 27) SEQ ID NO:135 and SEQ ID NO:132; 28) SEQ ID NO:136 and SEQ ID NO:130; 29) SEQ ID NO:137; 30) SEQ ID NO:136 and SEQ ID NO:138; 31) SEQ ID NO:140 and SEQ ID NO:137; 32) SEQ ID NO:140 and SEQ ID NO:138; 33) SEQ ID NO:140 and SEQ ID NO:139;34) SEQ ID NO:141 and SEQ ID NO:137; 35) SEQ ID NO:141 and SEQ ID NO:138; 36) SEQ ID NO:141 and SEQ ID NO:139; 37) SEQ ID NO:142 and SEQ ID NO:137; 38) SEQ ID NO:142 and SEQ ID NO:138; 39) SEQ ID NO:142 and SEQ ID NO:139; 40) SEQ ID NO:143 and SEQ ID NO:144; 41) SEQ ID NO:143 and SEQ ID NO:145; 42) SEQ ID NO:143 and SEQ ID NO:146; 43) SEQ ID NO:147 and SEQ ID NO:144; 44) SEQ ID NO:147 and SEQ ID NO:145; 45) SEQ ID NO:147 and SEQ ID NO:146; 46) SEQ ID NO:148 and SEQ ID NO:139; 47) SEQ ID NO:144; 48) SEQ ID NO:148 and SEQ ID NO:145; 49) SEQ ID NO:149 and SEQ ID NO:144; 50) SEQ ID NO:149 and SEQ ID NO:145; 51) SEQ ID NO:149 and SEQ ID NO:146; 52) SEQ ID NO:150 and SEQ ID NO:151; 53) SEQ ID NO:150 and SEQ ID NO:153; 54) SEQ ID NO:150 and SEQ ID NO:154; 55) SEQ ID NO:155 and SEQ ID NO:151; 56) SEQ ID NO:155 and SEQ ID NO:152; 57) SEQ ID NO:155 and SEQ ID NO:153; 58) SEQ ID NO:155 and SEQ ID NO:154; 59) SEQ ID NO:156 and SEQ ID NO:154; ID NO: 151; 60) SEQ ID NO: 156 and SEQ ID NO: 152; 61) SEQ ID NO: 156 and SEQ ID NO: 153; 62) SEQ ID NO: 156 and SEQ ID NO: 154; 63) SEQ ID NO: 157 and SEQ ID NO: 151; 64) SEQ ID NO: 157 and SEQ ID NO: 152; 65) SEQ ID NO:157 and SEQ ID NO:153;66) SEQ ID NO:157 and SEQ ID NO:154; 67) SEQ ID NO:158 and SEQ ID NO:151; 68) SEQ ID NO:158 and SEQ ID NO:152; 69) SEQ ID NO:158 and SEQ ID NO:153; or 70) SEQ ID NO:158 and SEQ ID NO:

154. The anti-CLDN6 antibody or its antigen-binding fragment as described in claim 1 or 2 further comprises one or more of the following characteristics: i) Contains a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains Fc; more preferably, the Fc is derived from mice or humans; more preferably, the sequence of the Fc is natural or modified; ii) It is a monoclonal antibody, bispecific binding molecule, multispecific binding molecule, humanized antibody, chimeric antibody, fully human antibody, full-length antibody, Fab, Fv, scFv, F(ab')2 or linear antibody; iii) It is in the form of IgG1, IgG2, IgG3 or IgG4. A coupling, wherein, The conjugate is formed by conjugating the anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3 with a capture marker or a detection marker; preferably, the detection marker includes a radionuclide, a luminescent substance, a colored substance, or an enzyme. An antibody-drug conjugate (ADC), wherein, The antibody-drug conjugate is formed by conjugating the anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3 with other bioactive molecules; preferably, the other bioactive molecules are small molecule drugs; preferably, the anti-CLDN6 antibody or its antigen-binding fragment is connected to the other bioactive molecules through a linker. A nucleic acid encoding an anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3, or a recombinant vector containing said nucleic acid, or a host cell containing said nucleic acid or recombinant vector; preferably, said host cell is a prokaryotic cell (preferably Escherichia coli), or a eukaryotic cell (preferably a mammalian cell or yeast; more preferably, said mammalian cell is HEK293 cell). A method for preparing an anti-CLDN6 antibody or its antigen-binding fragment, the method comprising: The host cells are cultured under suitable conditions, and the expression product is purified from the host cells. The host cell is as described in claim 6. A pharmaceutical composition comprising an anti-CLDN6 antibody or an antigen-binding fragment thereof as described in any one of claims 1-3, or an antibody-drug conjugate as described in claim 5, or a nucleic acid, recombinant vector, or host cell as described in claim 6; preferably, it further comprises a pharmaceutically acceptable carrier; preferably, it further comprises one or more additional therapeutic agents. A medicine box or kit comprising a container and a pharmaceutical composition as described in claim 8 located within the container. Use of an anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3, or a drug conjugate as described in claim 5, or a pharmaceutical composition as described in claim 8, in the preparation of a medicament for treating and / or preventing diseases; Preferably, the disease is a tumor; More preferably, the tumor is a tumor cell that abnormally expresses CLDN6; More preferably, the tumor is selected from any one of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms. A method for treating and / or preventing CLDN6-related diseases in a subject in need, comprising administering to the subject an effective amount of an anti-CLDN6 antibody or an antigen-binding fragment thereof as described in any one of claims 1-3, or a drug conjugate as described in claim 5, or a pharmaceutical composition as described in claim 8; Preferably, the disease is a tumor; Preferably, the anti-CLDN6 antibody or its antigen-binding fragment treats and / or prevents disease through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis induction, and proliferation inhibition. More preferably, the tumor is a tumor that abnormally expresses CLDN6; More preferably, the tumor is selected from any one of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms. Use of an anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3, or a conjugate as described in claim 4, in the preparation of a detection reagent or diagnostic reagent; Preferably, the detection reagent is used to detect the expression of CLDN6; Preferably, the diagnostic reagent is used to diagnose tumor cells with abnormal CLDN6 expression; More preferably, the tumor is selected from any of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, uterine cancer, etc., and their metastatic forms. A method for detecting CLDN6 expression in a sample, the method comprising: (1) The sample is contacted with the anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3, or the conjugate as described in claim 4, wherein the anti-CLDN6 antibody or its antigen-binding fragment, or the conjugate, forms a complex with CLDN6 in the sample; (2) Detect the formation of a complex of the anti-CLDN6 antibody or its antigen-binding fragment, or conjugate, with CLDN6; optionally, the anti-CLDN6 antibody or its antigen-binding fragment is detectably labeled; Preferably, the detection is not for diagnostic or therapeutic purposes. A method for inducing cell death in cells expressing CLDN6, the method comprising contacting the cells with an anti-CLDN6 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-3 or a pharmaceutical composition as claimed in claim 8, wherein the cells expressing CLDN6 are tumor cells; Preferably, the tumor is selected from any of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms. An anti-CLDN6 antibody or its antigen-binding fragment as described in any one of claims 1-3, or a drug conjugate as described in claim 5, or a pharmaceutical composition as described in claim 8, wherein the anti-CLDN6 antibody or its antigen-binding fragment, drug conjugate, or pharmaceutical composition is used to treat and / or prevent diseases associated with CLDN6 expression; Preferably, the disease is a tumor; preferably, the anti-CLDN6 antibody or its antigen-binding fragment treats and / or prevents the disease through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis induction, and proliferation inhibition. More preferably, the tumor is a tumor cell that abnormally expresses CLDN6; More preferably, the tumor is selected from any of the following: ovarian cancer, lung cancer, gastric cancer, germ cell and embryonic tumors, testicular cancer, endometrial cancer, uterine cancer, etc., and their metastatic forms.

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