Anti-CLDN182 antibody and in-vitro diagnostic kit

Abstract

The invention relates to an anti-CLDN18.2 antibody and an in-vitro diagnostic kit of the anti-CLDN18.2 antibody.

Description

Technical Field

[0001] The present invention relates to an anti-CLDN18.2 antibody and an in vitro diagnostic kit containing the anti-CLDN18.2 antibody. Background Art

[0002] Gastric cancer is one of the major malignant tumors that threaten life and health. Data show that in 2020, there were 479,000 new cases of gastric cancer and 374,000 deaths in China. From 2003 to 2015, the 5-year relative survival rate of gastric cancer in China increased from 27.4% to 35.1%, but it is still significantly lower than that in some other countries / regions. (Guidelines for Screening, Early Diagnosis and Treatment of Gastric Cancer in China (2022), National Cancer Center of China, http: / / chinancpcn.org.cn). These statistics clearly show that the limited treatment options for gastric cancer are seriously insufficient to meet medical conditions.

[0003] Antibodies are a relatively new class of targeted therapeutic compounds that have been widely used in the treatment of various cancers. Antibody targeted therapy has the potential to have higher specificity and lower side effects than many traditional non-antibody types of tumor treatments. Claudin 18.2 was recently discovered as an antibody target for the treatment of gastric cancer and esophageal cancer (J Hematol Oncol. 2017(1):105). It is also a target for the development of antibody drugs for pancreatic cancer. Currently, there are multiple anti-Claudin 18.2 monoclonal antibodies under clinical development, such as Zolbetuximab (IMAB362), ASKB589, etc.

[0004] Claudin is an important molecule that constitutes tight junctions. Tight junctions determine the permeability of epithelial cells and also play a role in blocking the diffusion of proteins and lipids on the cell membrane surface. Claudin18 belongs to the Claudins protein family and is encoded by the Claudin18 gene in the human body. The human Claudin18 gene has two different exons 1. After transcription, it undergoes alternative splicing and ultimately generates two protein subtypes, Claudin18.1 and Claudin18.2, which have different sequences only at the N-terminus. The expression of Claudin18.1 and Claudin18.2 in the human body is tissue-specific. Claudin18.1 is mainly expressed in lung tissue and is not expressed in gastric tissue or gastric cancer. As a highly specific cell surface molecule, Claudin18.2 is only expressed on differentiated epithelial cells on the gastric mucosa in normal tissues. Most primary gastric cancers and their metastatic cancer types express Claudin18.2 molecules. Due to the high specificity of Claudin18.2 expression in normal tissues and its activated expression in various cancers, Claudin18.2 has become a very potential target for epithelial tumors.

[0005] In order to accurately benefit the gastric cancer or gastroesophageal junction cancer population that expresses Claudin18.2, diagnostic screening of Claudin 18.2-positive patients is required before using therapeutic anti-Claudin 18.2 monoclonal antibody treatment. Therefore, the cell tight junction protein (Claudin) 18.2 antibody reagent (immunohistochemistry method) was developed as an original companion diagnostic reagent for the drug. When performing clinical companion diagnostic testing, gastric cancer or gastroesophageal junction cancer pathological tissues are usually paraffin-embedded and sectioned, and then immunohistochemical staining of Claudin 18.2 is performed. Summary of the invention

[0006] In a first aspect, the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; the anti-CLDN18.2 antibody comprises: (1) an antibody heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, (2) an antibody heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, (3) an antibody heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, (4) an antibody light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, (5) an antibody light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and (6) an antibody light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0007] In one embodiment of the present invention, the anti-CLDN18.2 antibody comprises: an amino acid sequence as shown in SEQ ID NO: 7, or an antibody heavy chain variable region having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 7, and an amino acid sequence as shown in SEQ ID NO: 8, or an antibody light chain variable region having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 8.

[0008] In one embodiment of the present invention, the anti-CLDN18.2 antibody is the DS-3 antibody described in the present invention.

[0009] In one embodiment of the present invention, the anti-CLDN18.2 antibody comprises: an amino acid sequence as shown in SEQ ID NO: 9, or an antibody heavy chain having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 9, and an amino acid sequence as shown in SEQ ID NO: 10, or an antibody light chain having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 10.

[0010] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0011] The third aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid described in the second aspect of the present invention.

[0012] In a fourth aspect of the present invention, an in vitro diagnostic kit is provided, comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; the anti-CLDN18.2 antibody comprises: (1) an antibody heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, (2) an antibody heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, (3) an antibody heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, (4) an antibody light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, (5) an antibody light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and (6) an antibody light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 6.

[0013] In one embodiment of the present invention, the anti-CLDN18.2 antibody comprises: an amino acid sequence as shown in SEQ ID NO: 7, or an antibody heavy chain variable region having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 7, and an amino acid sequence as shown in SEQ ID NO: 8, or an antibody light chain variable region having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 8.

[0014] In one embodiment of the present invention, the anti-CLDN18.2 antibody comprises: an amino acid sequence as shown in SEQ ID NO: 9, or an antibody heavy chain having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 9, and an amino acid sequence as shown in SEQ ID NO: 10, or an antibody light chain having a similarity of not less than 90% (e.g., not less than 90%, or not less than 95%, or not less than 98%, or not less than 99%) to the amino acid sequence as shown in SEQ ID NO: 10.

[0015] In one embodiment of the present invention, the anti-CLDN18.2 antibody or antigen-binding fragment thereof is coupled to at least one detectable marker.

[0016] The fifth aspect of the present invention provides a method for detecting a sample using the in vitro diagnostic kit described in the fifth aspect of the present invention.

[0017] In one embodiment of the present invention, a method for detecting a sample comprises: (i) fixing the sample on a solid support; (ii) contacting the sample with the anti-CLDN18.2 antibody or its antigen-binding fragment included in the in vitro diagnostic kit according to the fifth aspect of the present invention; (iii) contacting the anti-CLDN18.2 antibody or its antigen-binding fragment with a detectable marker; (iv) determining the binding level of the CLDN18.2 antibody or its antigen-binding fragment to the sample; the detectable marker is used to label the anti-CLDN18.2 antibody or its antigen-binding fragment.

[0018] One embodiment of the present invention provides a method for detecting a sample, comprising: (i) fixing the sample on a solid support; (ii) contacting the sample with the anti-CLDN18.2 antibody or antigen-binding fragment thereof included in the in vitro diagnostic kit according to the fifth aspect of the present invention; (iii) determining the binding level of the antibody or antigen-binding fragment thereof to the sample by a detectable marker. The anti-CLDN18.2 antibody or antigen-binding fragment thereof is coupled to at least one detectable marker.

[0019] In one embodiment of the present invention, the detectable marker is horseradish peroxidase.

[0020] In one embodiment of the present invention, the sample is a tissue section.

[0021] In one embodiment of the present invention, the tissue is selected from the group consisting of pancreas, esophagus, ovary, lung, pancreas, stomach, bronchus, breast or ear, nose and throat.

[0022] In one embodiment of the present invention, the solid support is a glass slide.

[0023] A person skilled in the art can easily determine the CDR sequence based on the sequence of the heavy chain or light chain variable region. To annotate the CDRs of the antibody variable region, it is necessary to select an antibody numbering scheme and a definition scheme. The numbering schemes include, but are not limited to, IMGT, Chothia, Kabat, Martin (extended Chothia); the definition schemes include, but are not limited to, Chothia, Kabat, IMGT, and Contact.

[0024] Sequence alignment, usually one sequence is used as a control sequence to compare the test sequence. In the present invention, the term "similarity" of a sequence refers to the proportion of identical amino acids between the test sequence and the target sequence in the entire sequence (a relatively macroscopic description). In amino acid sequence alignment, similarity also includes, in addition to identical residues, whether the two residues at corresponding positions have similar properties, such as the size, charge, hydrophilicity, etc. of the side chain group. The method of arranging sequences for comparison is common knowledge in the art.

[0025] In the present invention, the terms "Claudin 18", "claudin 18" or "CLDN18" are interchangeable terms; the terms "Claudin 18.1", "claudin 18.1", "CLDN18.1" or "CLDN18_1" are interchangeable terms; the terms "Claudin 18.2", "claudin 18.2", "CLDN18.2" or "CLDN18_2" are interchangeable terms.

[0026] In the present invention, the term "in vitro diagnostic reagents" or "in vitro diagnostic kits" refers to reagents, kits, calibrators, quality control products and other products used for in vitro detection of human samples in the process of disease prediction, prevention, diagnosis, treatment monitoring, prognosis observation and health status evaluation. They can be used alone or in combination with instruments, instruments, equipment or systems.

[0027] In the present invention, the term "comprising" or "including (comprising)" may be open, semi-closed or closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0028] In the present invention, "mass volume percentage" or "% (w / v)" or "% w / v" is a way of expressing mass volume concentration, which means the grams of solute contained in every 100 ml of solution. For example, 20% (w / v) means that 20 g of solute is contained in every 100 ml of solution.

[0029] The present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, and an in vitro diagnostic kit containing the anti-CLDN18.2 antibody or the antigen-binding fragment thereof; the antibody or the antigen-binding fragment thereof has high binding activity with the CLDN18.2 protein; the in vitro diagnostic kit containing the antibody or the antigen-binding fragment thereof is easy to operate, has high detection sensitivity, and has good storage stability. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0031] Example 1

[0032] Female Balb / c mice aged 6-8 weeks were immunized with human Claudin18 protein. Blood was collected one week after three immunizations. The serum titer was measured by indirect ELISA. The titer was qualified by 1000-fold diluted serum OD-blank>0.5. The mouse sera with qualified titer were subjected to immunohistochemistry detection, and the histochemically superior mice were selected for shock immunization. The mouse spleen was collected 3-4 days after shock immunization for hybridoma fusion.

[0033] The hybridoma cell culture supernatant was subjected to binding detection by indirect ELISA to screen positive cells. After culturing each positive cell obtained from the screening, the supernatant was taken for ELISA and IHC detection, and ELISA and IHC double-positive cells were selected and subjected to 1-2 rounds of limiting dilution again until stable positive cells were obtained. The obtained stable positive cells were used to further prepare and purify anti-CLDN18.2 monoclonal antibodies to obtain DS-3.

[0034] After sequencing, the antibody DS-3 has an antibody heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and an antibody light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; the antibody DS-3 has an antibody heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and an antibody light chain with an amino acid sequence as shown in SEQ ID NO: 10; its CDR information is as follows (the numbering scheme is based on Kabat, and the annotation scheme is based on Kabat):

[0035] Heavy chain CDR1 SEQ ID NO: 1 Wlq Heavy chain CDR2 SEQ ID NO: 2 EIDPNNGRTNFNEKFKR Heavy chain CDR3 SEQ ID NO: 3 ARSSLAY Light chain CDR1 SEQ ID NO: 4 KSSQSLLDSDGKTYLH Light chain CDR2 SEQ ID NO: 5 LVSKLDS Light chain CDR3 SEQ ID NO: 6 QTfP

[0036] SEQ ID NO: 7

[0037]

[0038] SEQ ID NO: 8

[0039]

[0040] Once the amino acid sequence of antibody DS-3 is known, genetic engineering technology can be further used to construct a plasmid, and the constructed plasmid is then transfected / transformed into a suitable host cell (including but not limited to CHO cells, E. coli) to express antibody DS-3.

[0041] Example 2 DS-3 antibody binding activity assay

[0042] The activity of DS-3 antibody was detected by antigen-antibody binding experiment, coated with 5μg / ml CLDN18.2 capture protein, set the maximum detection concentration of DS-3 antibody to 1.2ug / ml, and used 3-fold gradient dilution; the secondary antibody used goat anti-mouse IgG, with a dilution ratio of 1: 1500. The experiment showed that the binding activity EC50 of DS-3 and CLDN18.2 protein was 3.59ng / ml.

[0043] Example 3DS-3 as an in vitro diagnostic reagent

[0044] Claudin18.2 staining experiment was performed on Leica Bond III automatic immunohistochemistry instrument. The experimental steps are as follows:

[0045] (i) Sample pretreatment: (1) Place the sample slices and quality control slices in an oven at 65±5℃ for 30-60 minutes; (2) Take out the slices and cool them to room temperature;

[0046] (ii) Slice staining experimental steps:

[0047]

[0048] (iii) Cleaning and sealing: After the above staining steps are completed, the sample slides are taken out and dehydrated and sealed using the DAKEWE fully automatic staining and sealing machine.

[0049] Example 4 Interpretation of DS-3 Detection Reagent Staining Results

[0050] The cell staining localization pattern of the CLDN18.2 antibody reagent is the cell membrane and / or cytoplasm; in the staining of tumor tissue samples, only the cell membrane staining of tumor cells is judged, and cytoplasmic staining is not considered. Evaluate the entire sample: All valid tumor cells on the slice should be evaluated, and there should be no less than 100 tumor cells. The staining interpretation standard of the CLDN18.2 antibody reagent in tumor tissue: Only the cell membrane staining of tumor cells is judged, and the tumor cell staining ratio (TC) of each cell membrane staining intensity is calculated separately.

[0051] The calculation formula is as follows:

[0052]

[0053] The staining intensity is divided into 0, 1+, 2+ and 3+, and the staining intensity score is interpreted as follows:

[0054] 0: no target staining in tumor cells;

[0055] 1+: tumor cells showed weak or incomplete cell membrane staining;

[0056] 2+: tumor cells showed moderate intensity of cell membrane staining;

[0057] 3+: tumor cells showed strong cell membrane staining;

[0058] 51 different samples were taken and tested with DS-3 detection reagent and control antibody R detection reagent. Antibody R detection reagent is a commercially available diagnostic reagent for CLDN18.2. The steps of DS-3 detection reagent refer to Example 3, and the steps of antibody R detection reagent refer to the instructions for use attached thereto.

[0059] When the critical value is defined as TC(2+&3+)=40%, that is, TC(2+&3+)≥40%, the result is judged to be positive; the comparison of the test results is shown in Table 1. In Table 1, there are two samples judged to be positive by DS-3 detection reagent and negative by antibody R detection reagent; the sample numbers are B1786-1 and 221208-18. The staining results of these two samples are shown in Table 2.

[0060] For sample B1786-1, TC(2+&3+) of DS-3 detection reagent = 45%, and TC(2+&3+) of antibody R detection reagent = 35%; for sample 221208-18, TC(2+&3+) of DS-3 detection reagent = 50%, and TC(2+&3+) of antibody R detection reagent = 30%; After analysis, DS-3 and antibody R showed similar staining patterns, but DS-3 stained stronger on some tumor cells, which may be due to the different antigenic sites recognized by the two clones, or DS-3 has a higher sensitivity.

[0061] Table 1 Paired test results of DS-3 detection reagent and commercially available antibody R detection reagent (cutoff value is 40%)

[0062]

[0063] Table 2

[0064]

[0065] When the critical value is defined as TC(2+&3+)=75%, that is, TC(2+&3+)≥75%, the result is positive; the comparison of the test results is shown in Table 3. Among them, the specific results of the three samples with differences in DS-3 detection reagents and antibody R detection reagents are shown in Table 4. The staining differences of the three samples are small, or DS-3 has higher sensitivity.

[0066] Table 3 Paired test results of DS-3 detection reagent and commercially available antibody R detection reagent (cutoff value is 75%)

[0067]

[0068] Table 4

[0069]

[0070] Example 5 DS-3 detection reagent stability

[0071] After the three batches of DS-3 companion diagnostic kits (immunohistochemistry method) produced through research and development and pilot production (0880812071, 0880812072 and 0880812073) passed the inspection, each reagent was divided into appropriate volumes and stored in a 37°C incubator. They were taken out on the 0th day, 3rd day, 7th day and 14th day respectively, and the consecutive sections of the tissue chip were tested once each, and the staining results were compared. The stained sections were interpreted by a professional pathologist and the interpretation results were analyzed.

[0072] The results showed that the localization and intensity of the positive signals of the reagents in the three batches of kits did not change significantly in the four tests within 14 days, and no target positive signals were found in the negative control reagents; in the three batches of kits, the same reagent had no significant difference in the interpretation results of the same tissue in the four tests within 14 days, and ΔTC (≥2+) ≤ 10%; in each test within 14 days, the interpretation results of the ready-to-use CLDN18.2 mouse monoclonal antibody reagents in the three batches of kits for the same tissue had almost no difference, and ΔTC (≥2+) ≤ 5%.

[0073] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately. In addition, it should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-CLDN18.2 antibody or an antigen-binding fragment thereof; the anti-CLDN18.2 antibody comprises: (1) an antibody heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (2) an antibody heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, (3) an antibody heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, (4) an antibody light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (5) an antibody light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, (6) The antibody light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-CLDN18.2 antibodies include: The amino acid sequence shown in SEQ ID NO: 7 or an antibody heavy chain variable region having no less than 90% similarity to the amino acid sequence shown in SEQ ID NO: 7, The amino acid sequence shown in SEQ ID NO: 8 or an antibody light chain variable region having no less than 90% similarity to the amino acid sequence shown in SEQ ID NO:

8.

3. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 3.

5. An in vitro diagnostic kit, characterized in that: The in vitro diagnostic kit comprises an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; the anti-CLDN18.2 antibody comprises: (1) an antibody heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, (2) an antibody heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, (3) an antibody heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3, (4) an antibody light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4, (5) an antibody light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, (6) The antibody light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

6.

6. The in vitro diagnostic kit according to claim 5, characterized in that: The anti-CLDN18.2 antibodies include: The amino acid sequence shown in SEQ ID NO: 7 or an antibody heavy chain variable region having no less than 90% similarity to the amino acid sequence shown in SEQ ID NO: 7, The amino acid sequence shown in SEQ ID NO: 8 or an antibody light chain variable region having no less than 90% similarity to the amino acid sequence shown in SEQ ID NO:

8.

7. The in vitro diagnostic kit according to claim 5 or 6, characterized in that: The anti-CLDN18.2 antibody or antigen-binding fragment thereof is coupled to at least one detectable label.

8. A method for detecting a sample, comprising: (i) fixing the sample on a solid support; (ii) contacting the sample with the anti-CLDN18.2 antibody or antigen-binding fragment thereof included in the in vitro diagnostic kit according to claim 5 or 6; (iii) contacting the anti-CLDN18.2 antibody or antigen-binding fragment thereof with a detectable label; (iv) determining the binding level of the CLDN18.2 antibody or antigen-binding fragment thereof to the sample; The detectable marker is used to label the anti-CLDN18.2 antibody or antigen-binding fragment thereof.

9. A method for detecting a sample, comprising: (i) fixing the sample on a solid support; (ii) contacting the sample with the anti-CLDN18.2 antibody or antigen-binding fragment thereof included in the in vitro diagnostic kit according to claim 7; (iii) determining the level of binding of the antibody or antigen-binding fragment thereof to the sample via the detectable label.

10. The method according to claim 8 or 9, characterized in that: The detectable marker is horseradish peroxidase.

11. The method according to claim 8 or 9, characterized in that: The sample is a tissue section.

12. The method according to claim 11, characterized in that The tissue is selected from the group consisting of pancreas, esophagus, ovary, lung, pancreas, stomach, bronchus, breast, or ear, nose and throat.

13. The method according to claim 8 or 9, wherein the solid support is a glass slide.

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