Anti-claudin antibodies, pharmaceutical compositions thereof, and methods of detection

Abstract

The present application provides an anti-CLDN antibody and a pharmaceutical composition and a detection method thereof, wherein the heavy chain of the antibody comprises one or more than one CDR, the CDR of the heavy chain differs from the CDR sequence of any one of SEQ ID No. 1 to SEQ ID No. 7 or SEQ ID No. 15 to SEQ ID No. 30 by no more than three amino acids, the light chain of the antibody comprises one or more than one CDR, the CDR of the light chain differs from the CDR sequence of any one of SEQ ID No. 8 to SEQ ID No. 14 or SEQ ID No. 31 to SEQ ID No. 46 by no more than three amino acids. The anti-CLDN antibody of the present application has stronger ability to bind to cell lines and tumor tissue cells than IMAB362, and has stronger anti-tumor effect than IMAB362.

Description

[0001] This application is a divisional application of the invention patent application filed on May 16, 2019, with application number 201910410255.8, entitled "Anti-CLDN Antibody and its Pharmaceutical Composition and Detection Method". Technical Field

[0002] This invention relates to an anti-CLDN antibody, a pharmaceutical composition comprising an anti-CLDN antibody, and a method for detecting the presence of CLDN in a biological sample. Background Technology

[0003] Tight junctions (TJs) play a crucial role in intercellular transport and maintain cell polarity by blocking the radial diffusion of membrane proteins and lipids. They also participate in recruiting signaling molecules that regulate cell proliferation, differentiation, and motility. Tight junctions are formed by claudin (CLDN) proteins, a family of over 20 proteins, all containing a four-transmembrane domain and similar amino acid sequences, but exhibiting tissue-specific distribution. Human CLDN genes are distributed in pairs on different chromosomes, suggesting that some CLDN genes originate from gene duplication.

[0004] Most CLDN proteins have a molecular weight ranging from 20 to 34 kDa. The most significant difference lies in the sequence and size of their intracellular C-terminal region. This region contains a PDZ domain binding motif, which allows CLDN proteins to directly interact with cytoplasmic tight junction-related proteins such as ZO-1, ZO-2, ZO-3, and MUPP1. Furthermore, this region contains post-transcriptional modification sites, such as phosphorylation sites, which can influence protein localization and function. MAPK (mitogen-activated protein kinase) or PKC (protein kinase C) can phosphorylate CLDN1, and cAMP (cyclic AMP) induces CLDN5 phosphorylation, both of which promote the barrier function of CLDN proteins. PKA-mediated CLDN16 phosphorylation, on the other hand, enhances magnesium ion transport.

[0005] CLDNs play a crucial role in regulating selective permeation of cellular bypass pathways. CLDN2 and CLDN15 are involved in the formation of cation channels and cation pores, while CLDN4 / 7 / 10 are involved in the formation of anion channels and pores. Claudin protein is highly expressed in some cell lines, thereby affecting transmembrane resistance and permeability. In cultured epidermal-derived cells, CLDN1 / 4 / 5 / 7 can increase transmembrane resistance, while CLDN2 and CLDN10 have the opposite effect.

[0006] Mutations in the CLDN gene are believed to be associated with a variety of diseases. CLDN1 mutations may lead to sclerosing cholangitis and ichthyosis, while CLDN16 and CLDN19 mutations are believed to be associated with hypomagnesemia and hypercalcinuria.

[0007] Differential expression of CLDN proteins is believed to be associated with various cancers. CLDN1 and CLDN7 are downregulated in invasive breast cancer, prostate cancer, and esophageal cancer, while CLDN3 / 4 are upregulated to varying degrees in cervical cancer, colon cancer, esophageal cancer, gastric cancer, and other cancers. Sahin et al. found that in normal tissues, the isoform 2 subtype of CLDN18 (CLDN18.2) is expressed only in post-differentiated epithelial cells of the gastric mucosa, and not in the gastric stem cell region, but abnormally high expression was found in primary gastric cancer and its metastases. High expression of CLDN18.2 has also been reported in pancreatic cancer, esophageal cancer, and lung cancer. Because CLDN18.2 is located on the cell membrane surface, its biological function and characteristics make it an ideal therapeutic target, and monoclonal antibodies targeting this target have emerged in recent years, with Ganymed's IMAB362 (Claudiximab) showing the fastest development. IMAB362 binds to CLDN18.2 on the surface of tumor cells, inducing antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to kill tumor cells. When used in combination with chemotherapy, IMAB362 can also enhance T cell infiltration and upregulate pro-inflammatory factors. Summary of the Invention

[0008] The purpose of this invention is to provide an anti-CLDN antibody, its pharmaceutical composition, and a detection method.

[0009] The present invention adopts the following technical solution: An anti-CLDN antibody, characterized in that it comprises a heavy chain and a light chain: The heavy chain of the antibody contains one or more CDRs, and the CDR of the heavy chain is approximately three amino acids different from the CDR sequence of any one of SEQ ID No. 1 to SEQ ID No. 7 or SEQ ID No. 15 to SEQ ID No. 30. The light chain of the antibody contains one or more CDRs, and the CDR of the light chain is approximately three amino acids different from the CDR sequence of any one of SEQ ID No. 8 to SEQ ID No. 14 or SEQ ID No. 31 to SEQ ID No. 46.

[0010] Furthermore, the anti-CLDN antibody of the present invention also has the following feature: the heavy chain of the antibody is selected from any one of SEQ ID No. 1 to No. 7.

[0011] Furthermore, the anti-CLDN antibody of the present invention also has the following feature: the light chain of the antibody is selected from any one of SEQ ID No. 8 to SEQ ID No. 14 or SEQ ID No. 31 to SEQ ID No. 46.

[0012] Furthermore, the anti-CLDN antibody of the present invention also has the following feature: the heavy chain of the antibody is selected from any one of SEQ ID No. 15 to SEQ ID No. 30.

[0013] Furthermore, the anti-CLDN antibody of the present invention also has the following feature: the light chain of the antibody is selected from any one of SEQ ID No. 31 to SEQ ID No. 46.

[0014] Furthermore, the anti-CLDN antibody of the present invention also has the following characteristic: wherein the combination of the heavy chain and light chain of the antibody is: SEQ ID No.1 and SEQ ID No.8, SEQ ID No.2 and SEQ ID No.9, SEQ ID No.3 and SEQ IDNo.10, SEQ ID No. 4 and SEQ ID No. 11, SEQ ID No. 5 and SEQ ID No. 12, SEQ ID No. 6 and SEQ ID No. 13, SEQ ID No. 7 and SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 31, SEQ ID No. 16 and SEQ ID No. 32, SEQ ID No. 17 and SEQ ID No. 33, SEQ ID No. 18 and SEQ ID No. 34, SEQ ID No. 19 and SEQ ID No. 35, SEQ ID No. 20 and SEQ ID No. 36, SEQ ID No. 21 and SEQ ID No. 37, SEQ ID No. 22 and SEQ ID No. 38, SEQ ID No. 23 and SEQ ID No. 39, SEQ ID No. 24 and SEQ ID No. 40, SEQ ID No. 25 and SEQ ID No. 41, SEQ ID No. 26 and SEQ ID No. 42, SEQ ID No. 27 and SEQ ID No. 43, SEQ ID No. 28 and SEQ ID No. 44, SEQ ID No. 29 and SEQ ID No. 45, SEQ ID No. 30 and SEQ ID No. 46.

[0015] The present invention also provides a polynucleotide encoding an antibody as described above.

[0016] The present invention also provides a pharmaceutical composition comprising the antibody described in any one of the above claims.

[0017] The present invention also provides the use of any of the antibodies described above in the preparation of antitumor drugs.

[0018] The present invention also provides a method for detecting the presence of CLDN in a biological sample, characterized by comprising: administering an antibody to the biological sample as described in any one of the preceding claims, wherein the antibody has a detectable marker; and detecting the presence of the detectable marker, or detecting the content of the detectable marker.

[0019] Beneficial effects of the invention The anti-CLDN antibody of this invention has a stronger ability to bind to cells than IMAB362. Furthermore, the antibody of this invention exhibits better tumor growth inhibition effects in in vivo animal studies compared to IMAB362. Attached Figure Description

[0020] Figure 1a This refers to the binding ability of the control antibody QP024025 to CHOS cells. Figure 1b This refers to the binding ability of the hybridoma-selected antibody QP188189 to CHOS cells. Figure 1c The binding ability of the hybridoma-selected antibody QP190191 to CHOS cells. Figure 1d The binding ability of the hybridoma-selected antibody QP192193 to CHOS cells. Figure 1e The binding ability of the hybridoma-selected antibody QP196198 to CHOS cells. Figure 1fThis refers to the binding ability of the hybridoma-selected antibody QP199200 to CHOS cells. Figure 1g The binding ability of the hybridoma-selected antibody QP201202 to CHOS cells. Figure 1h This refers to the binding ability of the hybridoma-selected antibody QP207208 to CHOS cells. Figure 2a This refers to the binding ability of the phage-selected antibody QP10731074 to CHOS cells.

[0021] Figure 2b This refers to the binding ability of the phage-selected antibody QP10791080 to CHOS cells.

[0022] Figure 2c This refers to the binding ability of the phage-selected antibody QP10851086 to CHOS cells.

[0023] Figure 2d This refers to the binding ability of the phage-selected antibody QP10911092 to CHOS cells.

[0024] Figure 2e This refers to the binding ability of the phage-selected antibody QP10971098 to CHOS cells.

[0025] Figure 2f This refers to the binding ability of the phage-selected antibody QP10991100 to CHOS cells.

[0026] Figure 3a This refers to the binding ability of the phage-selected antibody QP11051106 to CHOS cells.

[0027] Figure 3b This refers to the binding ability of the phage-selected antibody QP11071108 to CHOS cells.

[0028] Figure 3c This refers to the binding ability of the phage-selected antibody QP11091110 to CHOS cells.

[0029] Figure 3d It refers to the binding ability of the phage-selected antibody QP11111112 to CHOS cells.

[0030] Figure 3e It refers to the binding ability of the phage-selected antibody QP11131114 to CHOS cells.

[0031] Figure 3fThis refers to the binding ability of the phage-selected antibody QP11151116 to CHOS cells.

[0032] Figure 4a This refers to the binding ability of the phage-selected antibody QP11171118 to CHOS cells.

[0033] Figure 4b This refers to the binding ability of the phage-selected antibody QP11031104 to CHOS cells.

[0034] Figure 4c This refers to the binding ability of the phage-selected antibody QP10451046 to CHOS cells.

[0035] Figure 4d This refers to the binding ability of the phage-selected antibody QP10471048 to CHOS cells.

[0036] Figure 5a This is the binding ability of the control antibody QP024025 to different 293T transiently transfected cell lines.

[0037] Figure 5b This refers to the binding ability of the hybridoma-selected antibody QP188189 to different 293T transiently transfected cell lines. Figure 5c This refers to the binding ability of the hybridoma-selected antibody QP190191 to different 293T transiently transfected cell lines. Figure 5d It is the binding ability of the antibody QP192193, selected from hybridoma screening, to different 293T transiently transfected cell lines.

[0038] Figure 5e This refers to the binding ability of the hybridoma-selected antibody QP196198 to different 293T transiently transfected cell lines.

[0039] Figure 5f It is the binding ability of the antibody QP199200, selected from hybridoma screening, to different 293T transiently transfected cell lines.

[0040] Figure 5g This refers to the binding ability of the hybridoma-selected antibody QP201202 to different 293T transiently transfected cell lines.

[0041] Figure 5h It is the binding ability of the antibody QP207208, selected from hybridoma screening, to different 293T transiently transfected cell lines.

[0042] Figure 6a It is the binding ability of the phage-selected antibody QP10451046 to different 293T transiently transfected cell lines.

[0043] Figure 6b It is the binding ability of the phage-selected antibody QP10711072 to different 293T transiently transfected cell lines.

[0044] Figure 6c It is the binding ability of the phage-selected antibody QP10731074 to different 293T transiently transfected cell lines.

[0045] Figure 6d It is the binding ability of the phage-selected antibody QP10851086 to different 293T transiently transfected cell lines.

[0046] Figure 6e It is the binding ability of the phage-selected antibody QP10911092 to different 293T transiently transfected cell lines.

[0047] Figure 6f It is the binding ability of the phage-selected antibody QP10991100 to different 293T transiently transfected cell lines.

[0048] Figure 6g It is the binding ability of the phage-selected antibody QP11031104 to different 293T transiently transfected cell lines.

[0049] Figure 6h It is the binding ability of the phage-selected antibody QP11051106 to different 293T transiently transfected cell lines.

[0050] Figure 7a It is the binding ability of the phage-selected antibody QP11071108 to different 293T transiently transfected cell lines.

[0051] Figure 7b It is the binding ability of the phage-selected antibody QP11091110 to different 293T transiently transfected cell lines.

[0052] Figure 7c It is the binding ability of the phage-selected antibody QP11111112 to different 293T transiently transfected cell lines.

[0053] Figure 7d It is the binding ability of the phage-selected antibody QP11131114 to different 293T transiently transfected cell lines.

[0054] Figure 7e It is the binding ability of the phage-selected antibody QP11151116 to different 293T transiently transfected cell lines.

[0055] Figure 7f It is the binding ability of the phage-selected antibody QP11171118 to different 293T transiently transfected cell lines.

[0056] Figure 8a This is the curve of the control group IMAB362 in the experiment of the binding ability of the control antibody to the gastric cancer PDX model GA0006 tumor cells.

[0057] Figure 8b This is the curve of QP190191 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0058] Figure 8c This is the curve of QP192193 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0059] Figure 8d This is the curve of QP201202 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0060] Figure 8e This is the curve of QP207208 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0061] Figure 8f This is the curve of QP11091110 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0062] Figure 8g This is the curve of QP11131114 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0063] Figure 8h This is the curve of QP11151116 in the experiment of antibody binding ability with gastric cancer PDX model GA0006 tumor cells.

[0064] Figure 9 This is a pharmacodynamic test of the antibody against the gastric cancer PDX model GA0006.

[0065] Figure 10A This is the tumor growth curve after grouping in the PBS group (negative control).

[0066] Figure 10B This is the tumor growth curve after grouping in the QP192193 group.

[0067] Figure 10C This is the tumor growth curve after grouping in the QP207208 group.

[0068] Figure 10D This is the tumor growth curve after grouping for the QP11151116 treatment group.

[0069] Figure 10E This is the tumor growth curve after grouping the control antibody IMAB362 group.

[0070] Figure 11 The weight of tumor D31 in mice in each group containing the antibodies of this invention and the control antibody is [the weight of tumor D31].

[0071] Figure 12 These are real-life images of the tumor volume in each experimental group.

[0072] Figure 13 These are the weight curves for each group of mice.

[0073] Figure 14 These are the curves showing the rate of change in body weight for each group of mice. Detailed Implementation

[0074] The specific embodiments of the present invention will be described below with reference to the accompanying drawings.

[0075] The antibodies of this invention include, but are not limited to, human antibodies. The various antibody screening methods provided in this invention typically prioritize the more convenient hybridoma screening method; however, the antigen for this target (claudin18.2) is difficult to obtain, therefore, phage library screening is also employed. The sequence of the anti-claudin18.2 antibody obtained in this embodiment is shown below: The antibody used for hybridoma screening has a heavy chain variable region (VH) CDR, as shown in the underlined portion of the sequence. QP189 QVQLQQSGAELVKPGASVKLSCKASGYTFT SYGIN WVRQRPEQGLEWIG WLFPGDGTIKYNENFKG KATLTTDRSSSAAYMQLSRLTSEDSAVYFCAR GGYYGNAMDY WGQGTSVTVSS QP191 EVKLVESGGGLVKPGGSLKLSCAASGFTFS NYAMS WVRQTPEKRLEWVA SIISGGRTYYLDSEKG RFTISRDNARNNLYLQMSSLRSEDTAMYYCTR IYYGNSFDY WGQGTTLTVSS QD193 QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIYPGNGDTTYNGKFKG QATLTADKSSSTVYMQLSSLTSEDSAVYFCAR FVKGNAMDYWGQGTSVTVSS >QD198 QVQLKESGPGLVAPSQSLSITCTVSGFSLT IYGVH WVRQPPGRGLEWLG VIWAGGSTNYNSALMS RLSISKDNSKSQVFLKVNSLQTDDTAMYYCAR DYYYGSGFDY WGQGTTLTVSS >QD200 DVQLVESGGGLVQPGGSRKLSCAASGFTFS SFGMH WVRQAPEKGLEWVA YISSGSNSIYYVDTVKG RFTISRDNPKNTLFLQMTSLKSEDTAMYYCAR NAYYGNSFDY WGQGTTLTVSS >QD202 EVQLQQSGPELVKPGASVKMSCKASGYTFT NYFVH WVKQKPGQGLEWIG YINPYNDDTKYNEKFKG KATLTSDKSSSTAYMDLSSLTSEDSAVYYC LSLRFFAY WGQGTLVTVSA >QD208 EVQLQQSGPELVKPGASVKMSCKASGYTFT SYIMH WVKQKPGQGLEWIG YINPYNDGTKYNEKFKG KATLTSDKSSSTVYMELSSLTSEDSAVYCCAR LGFTTRNAMDY WGQGTSVTVSS Light chain sequence: Wherein, the CDRs of the light chain variable region (VL) are shown in the underlined portions of the sequence.

[0076] >QD188 DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKSYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYFC QNDYFYPYT FGGGTKLEIK >QD190 DIVMTQSPSSQTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISNMQAEDLAVYYC QNDYSYPFT FGSGTKLEIK QD192 DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQNPGQPPKMLIY WASTRES GVPDRFTGSGSGIDFSLTISSVQAEDLALYYC QNAYSYPFT FGSGTKLEIK QD196 DIVMTQSPSSLSVSAGEKVTMSC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFTLTISSVRAEDLAVYYC QNDHYYPFT FGSGTKLEIK QD199 DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTVFTLTISSVQAEDLAVYFC QNNYYYPLT FGAGTKLELK QD201 DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQAPKLLIY WASTRES GVPDRFIGSGSGTDFTLTISHVQAEDLAVYFC QNDYSYPLT FGAGTNLELK QD207 DIVMTQSPSSLSVSAGEKVTMNC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDHSYP FTFGSGTKLEIK Antibodies screened from phage libraries have heavy chain variable region (VH) CDRs as shown in the underlined portion of the sequence. QD1045 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG IINPSGGSTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR DYAFTGFDY WGQGTLVTVSS >QD1047 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG IINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR SSAYGTYSMDY WGQGTLVTVSS >QD1073 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTGVYYCAR GSGSWFGPYFDY WGQGTTVTVSS >QD1079 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR TDGATPFDY WGQGTTVTVSS >QD1085 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR RSYYGTGAFDY WGQGTTVTVSS >QD1091 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR SLGYFSGLAFDY WGQGTTVTVSS >QD1097 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GYNWSFGMDY WGQGTTVTVSS >QD1099 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR AGYFPRSLDY WGQGTTVTVSS >QD1103 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GYSWYWLFGFDY WGQGTTVTVSS >QD1105 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GGDWGGYMDY WGQGTTVTVSS >QD1107 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DYYYYFWFDY WGQGTLVTVSS >QD1109 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DYYYYYWFDY WGQGTLVTVSS >QD1111 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GYYYYFWFDY WGQGTLVTVSS >QD1113 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SAAYYYFWFDY WGQGTLVTVSS >QD1115 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSYYYYFWYDY WGQGTLVTVSS >QD1117 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK AIDYYTFDY WGQGTLVTVSS Light chain sequence: Wherein, the CDRs of the light chain variable region (VL) are shown as the underlined parts in the sequence.

[0077] >QD1046 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALMTPT FGQGTKVEIK >QD1048 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQDLWPRT FGQGTKVEIK >QD1074 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQAAQSPT FGQGTKVEIK >QD1080 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALNTPPT FGQGTKVEIK >QD1086 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVTDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALMTPT FGQGTKVEIK >QD1092 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQGRQFPT FGQGTKVEIK >QD1098 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQGLNTFT FGQGTKVEIK >QD1100 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQWDT FGQGTKVEIK >QD1104 DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTGT FGQGTKVEIK >QD1106 DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYTTPFT FGQGTKVEIK QD1108 DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYST FGQGTKVEIK >QD1110 DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYSSYSPT FGQGTKVEIK >QD1112 DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTKFTLTISSLQPDDFATYYC QQYSTYPLT FGQGTKVEIK >QD1114 DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYLSYPPT FGQGTKVEIK >QD1116 DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYPLT FGQGTKVEIK >QD1118 DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYSTYPLT FGQGTKVEVK Antibody binding ability experiment: Experiment 1: FACS detection of antibody binding ability to tumor cell lines a) Plant cells in 1.96-well plates, 2 × 10⁶ cells per well.5 Centrifuge each cell at 1000 rpm for 5 minutes, wash the cells once with 1×PBS, and remove the supernatant. b) Block the cells with 3% BSA-PBS solution and incubate at 4°C for 60 minutes; c) Dilute the antibody to be tested with blocking buffer to 5 μg / ml, add it to the wells, and incubate at 4°C for 60 minutes; d) Remove the antibody, and wash the cells three times with 220ul of 1×PBS per well; e) Add 50 μL of PE-anti-human FC (1:200 dilution) secondary antibody to each well and incubate at 4°C in the dark for 40 minutes; f) Remove the antibody and wash each well with 1×PBS four times; g) On-machine testing.

[0078] Experiment 2: FACS detection of antibody binding ability to PDX tissue tumor cells a) Prepare one gentleMACS™ C Tube and 3ml of digestion solution for each tumor. The digestion solution should be prepared according to the TumorDissociation Kit (miltenyibiotech, 130-096-730) instructions and used immediately after preparation. b) Sacrifice the mice, remove the tumors using clean tools, wash with 1×PBS, and remove blood vessels, fat, fascia, and other tissues attached to the tumor surface. Each digestive tube can digest no more than 0.8g of tumor tissue to ensure complete digestion of the tumor tissue by the digestive solution; c) Place the tumor tissue into the wells of a 6-well plate, add digestion solution, and use clean forceps and a scalpel to cut the tumor tissue into pieces approximately 1 mm in size. 3 Small pieces; d) Place the tissue block into the gentleMACS™ C Tube, wash the well plate with residual digestion solution and transfer it together into the digestion tube, then place it on ice; e) Invert the digestion tube and place it into the gentleMACS automated tissue processor. Select program 37_c_m_TDK_1 for digestion. After the program is complete, remove the digestion tube and collect cells and remaining tissue by brief centrifugation at 300×g. f) Discard the supernatant, resuspend the cells and tissues with 1×PBS, add the cell suspension to a cell filter screen, place it in a 50ml centrifuge tube, and use 10ml PBS to pass the suspension through the sieve to obtain a single-cell suspension. g) Centrifuge at 300×g for 5 minutes, discard the supernatant, resuspend in 5ml PBS, count cells and adjust cell concentration to 1×10⁻⁶ cells per sample. 6 One cell; h) Add 100 μL of 1 µg / ml Mouse BD Fc Block (CAT#553141) PBS solution to each sample to resuspend the cells, add 20 μL of human FcR Blocking Reagent, mix well, and incubate at room temperature in the dark for 10 minutes. i) Add 5ug / ml of primary antibody and incubate at 4°C in the dark for 60 minutes; j) Add 2 ml of FACS wash buffer, gently resuspend the cells, centrifuge at 300×g for 5 minutes, discard the supernatant, and repeat twice. k) Add 100 μL of FACS wash buffer containing PE-labeled human IgG Fc secondary antibody and dye, and incubate the cells in the dark for 60 minutes. l) Add 2 ml of FACS wash buffer, gently resuspend the cells, centrifuge at 300×g for 5 minutes, discard the supernatant, and repeat twice. m) Resuspend cells in 200ul FACS wash buffer and perform analysis.

[0079] In the experimental diagram, "hybridoma clone in CHOS system" and "phage clone in CHOS system" refer to the binding ability of antibodies selected from hybridoma and phage screening to CHOS cells, respectively. In the diagram, CHO18.2: CHOS-CLDN18.2 stable transfected cells, CHO18.1: CHOS-CLDN18.1 stable transfected cells, and CHOS: CHOS transfected with empty vector.

[0080] CHOS is a cell line obtained by immortalizing hamster ovarian cells. FACS (flow cytometry fluorescence sorting technology) was used to detect the binding ability of antibodies to the cell line.

[0081] Figures 1a to 1h The binding ability of the antibodies screened from hybridomas to CHOS cells was demonstrated.

[0082] Figures 2a to 2f , Figures 3a to 3f as well as Figures 4a to 4d The binding ability of the antibodies selected by phage screening to CHOS cells was demonstrated.

[0083] Experimental objective: To verify the binding ability of the antibody to claudin18.1 (non-specific binding) and claudin18.2 in the CHOS cell line.

[0084] In the attached diagram of the CHOS cell lines, each panel consists of a combination of FACS detection results from three CHOS cell lines using one test antibody, where: 1. Red curve ③ indicates the binding ability of the antibody to be tested to the CHOS cell line (CHOS) transfected with empty vector, i.e., the negative control; 2. Blue curve ② indicates the binding ability of the test antibody to the CHOS cell line (CHO18.1) transfected with claudin18.1, i.e., the detection of non-specific binding; 3. The yellow curve ① indicates the binding ability of the antibody to be tested to the CHOS cell line (CHO18.2) transfected with claudin18.2, that is, the binding ability of the target protein.

[0085] Figures 5a to 5h This demonstrates the binding ability of the antibodies screened from hybridomas to 293T cells.

[0086] Figures 6a to 6h as well as Figures 7a to 7f The binding ability of the antibodies selected by phage screening to CHOS cells was demonstrated.

[0087] In the figure, 293T-QD012: 293T-CLDN18.1 transiently transfected cells 293T-QD010: 293T-CLDN18.2 transiently transfected cells Experimental objective: To verify the binding ability of the antibody to claudin18.1 (non-specific binding) and claudin18.2 in the 293-T cell line.

[0088] 293T-QD210: 293T-CLDN18.2-18.1ECD1 transiently transfected cells; the first extracellular domain of CLDN18.2 was replaced with the first extracellular domain of CLDN18.1. 293T-QD211: 293T-CLDN18.1-18.2ECD1 transiently transfected cells; the first extracellular domain of CLDN18.1 was replaced with the first extracellular domain of CLDN18.2. Experimental objective: To verify in the 293-T cell line that the antibody binding region is encoded by the exon-1 domain of claudin18.2.

[0089] Similarly, "hybridom clone in 293T" and "phage clone in 293T" refer to the binding ability of antibodies selected from hybridomas and phages to different 293T transiently transfected cell lines, respectively.

[0090] The experimental objectives in 293T are the same as in the CHO cell line. The meanings of the curves and their representations are explained below.

[0091] 1. Red curve ⑤ indicates the binding ability of the test antibody to the 293T cell line transiently transfected with empty vector, i.e., the negative control; 2. Blue curve ④ indicates the binding of the test antibody to the 293T cell line (293T-QD010) transiently transfected with claudin18.2, detecting the binding ability of the target protein; 3. Orange curve ③ indicates the binding of the test antibody to the 293T cell line (293T-QD012) transiently transfected with claudin18.1, detecting non-specific binding; 4. Dark green curve ② indicates the binding of the test antibody to the 293T cell line (293T-QD211) transiently transfected with claudin18.1-18.2ECD1 (the first extracellular domain of CLDN18.1 is replaced with the first extracellular domain of 18.2, which is the region where the designed antibody binding site is located), verifying the domain where the antibody binding site is located; 5. The light green curve ① indicates the binding of the test antibody to the 293T cell line (293T-QD210) transiently transfected with claudin18.2-18.1ECD1 (the first extracellular domain of CLDN18.2 was replaced with the first extracellular domain of 18.1, the former being the region where the designed antibody binding site is located), verifying the necessity of this domain for antibody binding.

[0092] Figures 8a to 8h The experiment demonstrated the binding ability of the antibody to the GA0006 gastric cancer PDX model tumor cells.

[0093] Table 1: Binding ratio of antibody to GA0006 gastric cancer PDX model tumor cells The above experimental results show that the antibody provided by the present invention has better binding ability to proteins and lower non-specific binding.

[0094] In other embodiments, the present invention also provides a polynucleotide encoding an antibody as described above. When provided in the form of DNA, these polynucleotides encoding the antibodies provided by the present invention may contain non-coding sequences that will be removed during subsequent transcription and editing, or may contain only sequences encoding the antibodies corresponding to those provided in the embodiments of the present invention and sequences necessary for protein expression.

[0095] The present invention also provides a pharmaceutical composition comprising any one of the antibodies described above. The pharmaceutical composition provided by the present invention may comprise only one or at least two of the antibodies provided in the embodiments.

[0096] Those skilled in the art should understand that pharmaceutical compositions also include pharmaceutically acceptable excipients, and conventional excipients required for the preparation of powders or tablets and other dosage forms should be included as components to be added during the pharmaceutical manufacturing process.

[0097] The present invention also provides a method for detecting the presence of CLDN in biological samples, characterized in that it includes: The steps of administering an antibody to a biological sample as described in any of the above-mentioned steps, wherein the antibody has a detectable biomarker, and detecting the presence of the detectable biomarker or detecting the amount of the detectable biomarker.

[0098] Animal efficacy trials a) Tumor tissue was collected from GA0006 tumor-bearing mice, a gastric cancer xenograft model, and cut into tumor blocks with a diameter of 2-3 mm. These blocks were then subcutaneously implanted into the right anterior scapula of Balb / c nude mice. b) When the average tumor volume in Balb / c nude mice reaches approximately 100 mm 3 Mice were randomly divided into groups of six. The weight of all animals was measured, and tumor volume was measured using calipers. Grouping was based on tumor volume, ensuring similarity in tumor volume between groups. The coefficient of variation (CV) of tumor volume within each group was calculated using the formula CV = SD / MTV × 100%, and should be less than 40%. Randomization was performed using StudyDirector™. Grouping was designated Day 0, and medication was initiated on that day. Detailed administration methods, dosages, and routes are shown in the table below.

[0099] Table 2: Dosing parameters for animal efficacy studies The administration volume was 10 μL / g. The meanings of the antibody numbers in Tables 1 and 2, such as QP190191, indicate a combination of a heavy chain and a light chain.

[0100] c) After drug administration, mouse body weight and tumor volume were measured twice weekly. Tumor volume calculation formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (where a represents the major axis and b represents the minor axis). The experiment was terminated one week after the last administration, mice were sacrificed, tumors were collected, weighed, and photographed. The following analytical methods were used for data analysis: The relative tumor proliferation rate, T / C (%), is the percentage of tumor volume or weight in the treatment group and the control group at a certain time point. The calculation formula is: T / C % = TRTV / CRTV × 100% (TRTV: average RTV in the treatment group; CRTV: average RTV in the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment). The relative tumor inhibition rate, TGI (%), is calculated as follows: TGI% = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively).

[0101] The mean tumor volume in the PBS control group mice on day 31 after administration was 911.16 ± 177.81 mm. 3 The mean tumor volume on day 31 after administration of the antibody molecules QP192193 (10 mg / kg), QP207208 (10 mg / kg), and QP11151116 (10 mg / kg) was 580.97 ± 67.97 mm. 3 680.28±193.50 mm 3 , and 722.38±118.07 mm 3 The TGI values ​​were 36.34%, 25.34%, and 20.72%, respectively. The mean tumor volume of the control molecule IMAB362 (10 mg / kg) on ​​day 31 post-drug administration was 661.28 ± 104.49 mm. 3 The TGI was 27.42% (see table below). All three screened molecules inhibited tumor growth to some extent. Although there was no statistically significant difference, QP192193 showed a trend of being superior to the control molecule IMAB362. QP207208 and QP11151116 also showed the same level of tumor growth inhibition ability as IMAB362. Furthermore, the mice did not experience a significant decrease in body weight during administration, indicating that the antibody molecules had no significant toxic side effects on the mice. The results of tumor weight analysis were similar to those of tumor volume analysis. In the PBS control group, the mean tumor weight on day 31 after administration was 722.57 ± 176.32 mg. In the antibody molecule treatment groups (QP192193, QP207208, and QP11151116), the mean tumor weights on day 31 after the end of administration were 455.5 ± 46.42 mg, 391.93 ± 111.15 mg, and 432.03 ± 66.25 mg, respectively, with TGIs of 36.96%, 45.76%, and 40.21%. In the control molecule IMAB362 treatment group, the mean tumor weight on day 31 after administration was 435.78 ± 91 mg, with a TGI of 39.69%. (See table below) Therefore, through PDX animal efficacy trials, we found that the screened antibody molecules have better or the same level of in vivo tumor-inhibiting ability than the control molecule IMAB362.

[0102] Experimental results are as follows Figures 9 to 14 As shown, the results of the efficacy test of the antibody against the gastric cancer PDX model GA0006 are as follows: Figure 9 As shown. Figure 10A This is the tumor growth curve after grouping in the PBS group (negative control). Figure 10B This is the tumor growth curve after grouping in the QP192193 group. Figure 10C This is the tumor growth curve after grouping in the QP207208 group. Figure 10D This is the tumor growth curve after grouping for the QP11151116 treatment group. Figure 10E This is the tumor growth curve after grouping the control antibody IMAB362 group. Figure 11 The weight of tumor D31 in mice in each group containing the antibodies of this invention and the control antibody is [the weight of tumor D31]. Figure 12 These are real-life images of the tumor volume in each experimental group. Figure 13 These are the weight curves for each group of mice. Figure 14 The figures show the rate of change in body weight for each group of mice. The experimental results demonstrate that the invented antibody exhibits better tumor-inhibiting effects than IMAB362 in vivo animal studies.

Claims

1. An anti-CLDN antibody or its antigen-binding fragment, characterized in that, The antibody comprises heavy chains and light chains: The heavy chain of the antibody contains three CDRs, denoted as HCDR1, HCDR2, and HCDR3, respectively. The light chain of the antibody contains three CDRs, denoted as LCDR1, LCDR2, and LCDR3, respectively. Furthermore, the anti-CLDN antibody or its antigen-binding fragment comprises the following CDR combinations: The sequence of HCDR1 is SYWMN, the sequence of HCDR2 is QIYPGNGDTTYNGKFKG, the sequence of HCDR3 is FVKGNAMDY, and the sequence of LCDR1 is KSSQSLLNSGNQKNYLT, the sequence of LCDR2 is WASTRES, and the sequence of LCDR3 is QNAYSYPFT.

2. The anti-CLDN antibody or its antigen-binding fragment as described in claim 1, characterized in that: The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID No. 3; and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID No.

10.

3. The anti-CLDN antibody or its antigen-binding fragment as described in claim 1, characterized in that: The antibody is a human-derived antibody.

4. A polynucleotide encoding an anti-CLDN antibody as described in claim 1 or 2, or an antigen-binding fragment thereof.

5. A pharmaceutical composition comprising the anti-CLDN antibody as described in claim 1 or 2, or an antigen-binding fragment thereof.

6. The use of an anti-CLDN antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, Used to prepare antitumor drugs, wherein the tumor is gastric cancer, pancreatic cancer, esophageal cancer or lung cancer.

7. A method for non-diagnostic detection of the presence of CLDN in biological samples, characterized in that, include: The step of administering an anti-CLDN antibody or its antigen-binding fragment as described in claim 1 or 2 to a biological sample, wherein the antibody or its active fragment has a detectable biomarker. The steps include detecting the presence of the detectable marker or detecting the content of the detectable marker.

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