Anti-Claudin18.2 antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-Claudin18.2 antibody as well as a preparation method and application thereof, and belongs to the technical field of biology. The anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a CDR-H1 as shown in SEQ ID NO.1, a CDR-H2 as shown in SEQ ID NO.2 and a CDR-H3 as shown in SEQ ID NO.3, and the light chain variable region comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises a CDR-L1 as shown in SEQ ID NO. 4, a CDR-L2 as shown in SEQ ID NO. 5, and a CDR-L3 as shown in SEQ ID NO. 6. According to the present invention, the combination of the anti Claudin18.2 antibody and the Claudin18.2 protein molecule has high specificity and high affinity, and can specifically recognize and detect the expression level of Claudin18.2 on cancerous cells. By adopting a rabbit recombinant monoclonal antibody development technology, the Claudin18.2 antibody with high specificity and affinity can be developed and screened out.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an anti-Claudin18.2 antibody, its preparation method, and its application. Background Technology

[0002] Claudin18.2 is a key biomarker in gastric cancer research. A member of the tight junction protein family, it plays a crucial role in maintaining intercellular connections and barrier function. Normally, its expression is limited in healthy tissues, but it is significantly highly expressed in 50%-60% of gastric cancer tissues, exhibiting strong specificity. This characteristic makes it an ideal target for distinguishing cancerous cells from normal cells. Immunohistochemical or molecular detection of Claudin18.2 expression levels can be used to aid in gastric cancer subtyping, especially in diffuse gastric cancer or advanced-stage patients where expression rates are higher.

[0003] Therefore, there is a need to develop an anti-Claudin18.2 antibody with high specificity and affinity. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a highly specific and affinity anti-Claudin18.2 antibody, its preparation method and application.

[0005] The technical solution of the present invention is as follows:

[0006] An anti-Claudin18.2 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.2, and CDR-H3 as shown in SEQ ID NO.3;

[0007] The light chain variable region includes CDR-L1 as shown in SEQ ID NO.4, CDR-L2 as shown in SEQ ID NO.5, and CDR-L3 as shown in SEQ ID NO.6.

[0008] Optionally, the heavy chain variable region includes HFR1 as shown in SEQ ID NO.7, HFR2 as shown in SEQ ID NO.8, HFR3 as shown in SEQ ID NO.9, and HFR4 as shown in SEQ ID NO.10.

[0009] Optionally, the anti-Claudin18.2 antibody includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.11.

[0010] Optionally, the light chain variable region includes LFR1 as shown in SEQ ID NO.12, LFR2 as shown in SEQ ID NO.13, LFR3 as shown in SEQ ID NO.14, and LFR4 as shown in SEQ ID NO.15.

[0011] Optionally, the anti-Claudin18.2 antibody includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.16.

[0012] A nucleic acid, wherein the nucleic acid encodes an anti-Claudin18.2 antibody as described in this invention.

[0013] A recombinant expression vector, wherein the recombinant expression vector contains nucleic acids as described in this invention.

[0014] A recombinant expression cell, wherein the recombinant expression cell comprises a recombinant expression vector as described in this invention.

[0015] A method for preparing an anti-Claudin18.2 antibody according to the present invention, comprising the steps of:

[0016] Recombinant expression cells were obtained by transfecting cells with the recombinant expression vector described in this invention;

[0017] Culture the transfected recombinant expression cells;

[0018] The supernatant was collected and purified to obtain anti-Claudin18.2 antibody.

[0019] A kit for detecting Claudin18.2 protein molecules, wherein the kit comprises an anti-Claudin18.2 antibody as described in this invention.

[0020] Beneficial Effects: This invention provides an anti-Claudin18.2 antibody, its preparation method, and its applications. This anti-Claudin18.2 antibody exhibits high specificity and affinity for the Claudin18.2 protein molecule, enabling it to specifically recognize and detect the expression level of Claudin18.2 on cancerous cells. This invention utilizes rabbit recombinant monoclonal antibody development technology to develop and screen for the aforementioned highly specific and affinity-rich anti-Claudin18.2 antibody. Attached Figure Description

[0021] Figure 1 This is a graph showing the results of antibody-protein immunoblotting.

[0022] Figure 2 This image shows the results of antibody immunohistochemistry staining of normal human gastric tissue.

[0023] Figure 3 This image shows the results of antibody immunohistochemistry staining in human gastric cancer tissue. Detailed Implementation

[0024] This invention provides an anti-Claudin18.2 antibody, its preparation method, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0025] This invention provides an anti-Claudin18.2 antibody, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.2, and CDR-H3 as shown in SEQ ID NO.3;

[0026] The light chain variable region includes CDR-L1 as shown in SEQ ID NO.4, CDR-L2 as shown in SEQ ID NO.5, and CDR-L3 as shown in SEQ ID NO.6.

[0027] CDR-H1, CDR-H2, and CDR-H3 refer to the three complementary determinant regions contained in the variable region of the heavy chain; CDR-L1, CDR-L2, and CDR-L3 refer to the three complementary determinant regions contained in the variable region of the light chain.

[0028] Western blotting confirmed that the anti-Claudin18.2 antibody has high specificity in binding to the Claudin18.2 protein molecule, and that the anti-Claudin18.2 antibody can specifically recognize and detect the expression level of the Claudin18.2 protein molecule.

[0029] It is important to emphasize that antibodies suitable for Western blotting need to accurately distinguish the target protein from complex protein mixtures (such as cell lysates), thus requiring extremely high specificity. Furthermore, whether the molecular weight of the detected band matches the expectation most directly demonstrates specificity. Non-specific bands typically have different molecular weights, while immunohistochemistry or immunofluorescence may show non-specific binding to other target proteins but with the same colorimetric localization.

[0030] Furthermore, the heavy chain variable region includes HFR1 as shown in SEQ ID NO.7, HFR2 as shown in SEQ ID NO.8, HFR3 as shown in SEQ ID NO.9, and HFR4 as shown in SEQ ID NO.10.

[0031] HFR1, HFR2, HFR3, and HFR4 refer to the four skeletal regions contained in the heavy chain variable region.

[0032] Furthermore, the anti-Claudin18.2 antibody comprises a heavy chain constant region (Tail), the amino acid sequence of which is shown in SEQ ID NO.11.

[0033] Furthermore, the light chain variable region includes LFR1 as shown in SEQ ID NO.12, LFR2 as shown in SEQ ID NO.13, LFR3 as shown in SEQ ID NO.14, and LFR4 as shown in SEQ ID NO.15.

[0034] LFR1, LFR2, LFR3, and LFR4 refer to the four skeletal regions contained in the variable region of the light chain.

[0035] Furthermore, the anti-Claudin18.2 antibody comprises a light chain constant region (Tail), the amino acid sequence of which is shown in SEQ ID NO.16.

[0036] Furthermore, the heavy chain of the anti-Claudin18.2 antibody is arranged in the following order from one end to the other: HFR1, CDR-H1, HFR2, CDR-H2, HFR3, CDR-H3, HFR4 and the heavy chain constant region (Tail).

[0037] Furthermore, the light chains of the anti-Claudin18.2 antibody are arranged in the following order from one end to the other: LFR1, CDR-L1, LFR2, CDR-L2, LFR3, CDR-L3, LFR4, and the light chain constant region (Tail).

[0038] Furthermore, the anti-Claudin18.2 antibody is an anti-Claudin18.2 monoclonal antibody, and more specifically, an anti-Claudin18.2 rabbit recombinant monoclonal antibody.

[0039] This invention provides a nucleic acid, wherein the nucleic acid encodes an anti-Claudin18.2 antibody as described in this invention.

[0040] This invention provides a recombinant expression vector containing the nucleic acid as described in this invention.

[0041] This invention provides a recombinant expression cell, wherein the recombinant expression cell comprises the recombinant expression vector as described in this invention.

[0042] This invention provides a method for preparing the anti-Claudin18.2 antibody, comprising the following steps:

[0043] Recombinant expression cells were obtained by transfecting cells with the recombinant expression vector described in the embodiments of the present invention;

[0044] Culture the transfected recombinant expression cells;

[0045] The supernatant was collected and purified to obtain anti-Claudin18.2 antibody.

[0046] Specifically, recombinant human Claudin18.2 full-length protein (aa1-261) was used as an immunogen to immunize experimental rabbits. Peripheral blood mononuclear cells (PBMCs) were isolated from the rabbits, labeled, and screened. Genetic information of B cells expressing Claudin18.2 antibody was obtained, amplified, and recombined. Selected cells with suitable genetic information were loaded into a vector and transfected into cells. The antibody secreted by the recombinant expression cells was purified to obtain anti-Claudin18.2 antibody.

[0047] The embodiments of the present invention employ rabbit recombinant monoclonal antibody development technology, which can develop and screen anti-Claudin18.2 antibodies with high specificity and affinity.

[0048] This invention provides a kit for detecting Claudin18.2 protein molecules, wherein the kit includes an anti-Claudin18.2 antibody as described in this invention.

[0049] The present invention will be further described below through specific embodiments.

[0050] Example 1

[0051] This embodiment provides a method for preparing anti-Claudin18.2 antibody, which includes the following steps: Rabbits are immunized by injection with recombinant human Claudin18.2 full-length protein (aa1-261, whose amino acid sequence is shown in SEQ ID NO.17). Peripheral blood mononuclear cells (PBMCs) are isolated from the rabbits, B cells in the PBMCs are labeled and screened, and the genetic information of B cells expressing Claudin18.2 antibody is obtained, amplified, and recombinantly processed. The genetic information of cells meeting the requirements is loaded into a vector and transfected into cells. The antibody secreted by the recombinant expression cells is purified to obtain anti-Claudin18.2 antibody.

[0052] The specific steps for preparing anti-Claudin18.2 antibody are as follows:

[0053] 1. Synthesize recombinant human Claudin18.2 full-length protein (aa1-261) as an immunogen.

[0054] 2. Immunogen and adjuvant were mixed in equal volumes and then injected subcutaneously into the groin of rabbits for immunization. A total of four immunizations were administered to two rabbits. For the first immunization, Freund's complete adjuvant was used to mix the immunogen with the immunogen; subsequent immunizations used Freund's incomplete adjuvant mixed with the immunogen.

[0055] 3. One week after the fourth immunization, blood was collected from the experimental rabbits and serum was separated. A 96-well ELISA plate was coated with 1 μg / mL Claudin 18.2 full-length protein, and the serum titer was detected using an indirect enzyme-linked immunosorbent assay (iELISA). The rabbit serum was serially diluted six times (1:200) in two fold increments, with the final OD value at 1:64000 being recorded. 450 Serum with a reading higher than 0.5 is considered acceptable.

[0056] 4. Anticoagulated blood was collected from rabbits with qualified serum values, and peripheral blood mononuclear cells (PBMCs) were separated and collected using lymphocyte separation fluid.

[0057] 5. After the isolated lymphocytes were incubated with biotin-crosslinked recombinant human Claudin18.2 full-length protein, an adsorption column filled with avidin magnetic beads and a magnetic rack were added to adsorb and wash away impurities before collecting the specific lymphocytes.

[0058] 6. Perform limiting dilutions at 5 cells / well and seed lymphocytes into 96-well plates for culture.

[0059] 7.3 days later, 50 μL of supernatant was collected and coated with 1 μg / mL Claudin 18.2 full-length protein in a 96-well microplate. Serum titers were detected using an indirect enzyme-linked immunosorbent assay (iELISA). OD was collected. 450 Serum with a reading higher than 0.5 is considered acceptable.

[0060] 8. Lyse lymphocytes from qualified serum wells to extract total RNA. Use the mRNA as a template and reverse transcribe it into cDNA using primers and reverse transcriptase. Then, use the cDNA as a template for PCR amplification to obtain the H and L chain genes of positive B-cell monoclonal antibodies.

[0061] 9. The H and L chain genes were ligated into a vector and transformed into E. coli. The plasmid was extracted and transformed into 293 cells for expression to obtain a rabbit recombinant monoclonal antibody against Claudin18.2.

[0062] The final selected heavy chain amino acid sequence of the anti-Claudin18.2 rabbit recombinant monoclonal antibody is shown in SEQ ID NO.18, and the light chain amino acid sequence is shown in SEQ ID NO.19. Specifically, the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6.

[0063] Example 2

[0064] This embodiment uses Western blotting (WB) to verify the specificity of the antibody prepared in Example 1:

[0065] The binding specificity of the anti-Claudin18.2 rabbit recombinant monoclonal antibody was detected using Western blotting. The basic principle is to stain protein, cell, or biological tissue samples treated with gel electrophoresis with specific antibodies. Information on the expression of specific proteins in the analyzed cells or tissues is obtained by analyzing the location and depth of the staining.

[0066] The detailed experimental procedure was performed according to the conventional Western blotting method for proteins. The sample concentration was 60 ng / ml (where the sample refers to the antibody prepared in Example 1), and the loading amount was 0.2 μg / lane (where the sample refers to the Claudin18.2 recombinant protein in the lysate). The secondary antibody was HRP-labeled goat anti-rabbit IgG (H+L), and the secondary antibody dilution was 1:10000. After chemiluminescence (ECL) substrate development, the sample was exposed and photographed in a chemiluminescence imaging system.

[0067] Western blot analysis was performed on the anti-Claudin18.2 monoclonal antibody and the lysate of recombinant Claudin18.2 protein. The results are shown in the figure below. Figure 1 ,from Figure 1 The result was positive, and the positive band was around 28 kDa, consistent with the theoretical molecular weight. This indicates that the anti-Claudin18.2 monoclonal antibody is specific for the recombinant Claudin18.2 protein.

[0068] Example 3

[0069] This embodiment uses immunohistochemistry (IHC) for detection and verification:

[0070] Immunohistochemistry is a technique that uses chromogenically labeled specific antibodies to qualitatively, locally, and quantitatively identify corresponding antigens in situ through antigen-antibody reactions and histochemical colorimetric reactions within tissue cells. The detection sensitivity, i.e., affinity, of the anti-Claudin18.2 rabbit monoclonal antibody was verified using conventional immunohistochemical detection methods.

[0071] The specific steps are as follows:

[0072] 1. Prepare a paraffin block, cut it into 3μm thick sections, and attach them to a glass slide to prepare a test sample.

[0073] 2. Bake the tissue sections at 60℃ for 1 hour.

[0074] 3. Immerse the slices in the dewaxing solution for 30 minutes to perform the dewaxing process.

[0075] 4. After dewaxing, the tissue sections were immersed in anhydrous ethanol, 95% ethanol, and 75% ethanol for 10 minutes each for hydration treatment.

[0076] 5. After hydration, the tissue sections were subjected to high-temperature retrieval of antigens in a Tris EDTA solution at pH 9.0.

[0077] 6. After antigen repair, the tissue sections were blocked in 3% H2O2 for 10 min.

[0078] 7. Add 0.1 μg / mL of the anti-Claudin18.2 rabbit monoclonal antibody prepared in Example 1 at room temperature and incubate for 1 h.

[0079] 8. Clean, add 100 μL of goat anti-rabbit secondary antibody and incubate at room temperature for 1 h.

[0080] 9. Clean, add 100μL DAB colorimetric solution and develop for 2 minutes, then stop.

[0081] 10. Counterstain with hematoxylin for 5 minutes.

[0082] 11. After staining, the tissue sections were immersed in 75% ethanol, 95% ethanol, anhydrous ethanol, and dewaxing solution for 2 minutes each, and then mounted.

[0083] After mounting, the anti-Claudin18.2 rabbit monoclonal antibody could be observed under a microscope in normal human gastric tissue. Figure 2 ), human gastric cancer tissue ( Figure 3 The staining of Claudin18.2 protein in the sample is highly sensitive and has no nonspecific staining.

[0084] In summary, this invention provides an anti-Claudin18.2 antibody, its preparation method, and its applications. This anti-Claudin18.2 antibody exhibits high specificity and affinity for the Claudin18.2 protein molecule, enabling it to specifically recognize and detect the expression level of Claudin18.2 on cancerous cells. This invention utilizes rabbit recombinant monoclonal antibody development technology to develop and screen for the aforementioned highly specific and affinity-rich anti-Claudin18.2 antibody.

[0085] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An anti-Claudin18.2 antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region includes CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.2, and CDR-H3 as shown in SEQ ID NO.3; The light chain variable region includes CDR-L1 as shown in SEQ ID NO.4, CDR-L2 as shown in SEQ ID NO.5, and CDR-L3 as shown in SEQ ID NO.

6.

2. The anti-Claudin18.2 antibody according to claim 1, characterized in that, The heavy chain variable region includes HFR1 as shown in SEQ ID NO.7, HFR2 as shown in SEQ ID NO.8, HFR3 as shown in SEQ ID NO.9, and HFR4 as shown in SEQ ID NO.

10.

3. The anti-Claudin18.2 antibody according to claim 1 or 2, characterized in that, The anti-Claudin18.2 antibody contains a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.

11.

4. The anti-Claudin18.2 antibody according to claim 1, characterized in that, The light chain variable region includes LFR1 as shown in SEQ ID NO.12, LFR2 as shown in SEQ ID NO.13, LFR3 as shown in SEQ ID NO.14, and LFR4 as shown in SEQ ID NO.

15.

5. The anti-Claudin18.2 antibody according to claim 1 or 4, characterized in that, The anti-Claudin18.2 antibody contains a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.

16.

6. A nucleic acid, characterized in that, The nucleic acid encodes the anti-Claudin18.2 antibody as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in claim 6.

8. A recombinant expression cell, characterized in that, The recombinant expression cell comprises the recombinant expression vector as described in claim 7.

9. A method for preparing the anti-Claudin18.2 antibody according to any one of claims 1-5, characterized in that, Including the following steps: Recombinant expression cells were obtained by transfecting cells with the recombinant expression vector described in claim 7; Culture the transfected recombinant expression cells; The supernatant was collected and purified to obtain anti-Claudin18.2 antibody.

10. A kit for detecting Claudin18.2 protein molecules, characterized in that, The kit includes the anti-Claudin18.2 antibody as described in any one of claims 1-5.

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