LRRC15 monoclonal antibody and application thereof

Abstract

The invention belongs to the fields of immunity, tumor treatment and biological detection, and provides an LRRC15 monoclonal antibody and application thereof, and the amino acid sequence of a heavy chain CDR1 of the monoclonal antibody is SEQ ID NO.1; the amino acid sequence of the heavy chain CDR2 is SEQ ID NO. 2; the amino acid sequence of the heavy chain CDR3 is SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is SEQ ID NO.6; the amino acid sequence of the light chain CDR2 is SEQ ID NO.7; and the amino acid sequence of the light chain CDR3 is SEQ ID NO.8. The antibody shows a remarkable inhibition effect on breast cancer, can be used for accurate and specific detection of LRRC15, and provides a more reliable tool for diagnosis and treatment of related diseases.

Description

Technical Field

[0001] This application pertains to the fields of immunology, tumor therapy, and biological detection. Specifically, this application provides an LRRC15 monoclonal antibody and its applications. Background Technology

[0002] LRRC15 (Leucine-rich repeat-containing protein 15) is a type I transmembrane protein rich in leucine repeats and belongs to the LRR protein family. Its structure includes a signal peptide, 15 leucine repeats (LRRs), a transmembrane domain, and a short cytoplasmic domain. LRRC15 plays an important role in extracellular matrix (ECM) interactions, cell adhesion, and signal transduction. Studies have shown that LRRC15 activates the focal adhesion kinase signaling pathway by interacting with fibronectin and β1 integrin, promoting tumor cell migration and invasion.

[0003] In vivo distribution: LRRC15 is expressed in various tissues, particularly in the brain, placenta, and tumor-associated fibroblasts. LRRC15 is highly expressed in various solid tumors, especially in tumor-associated fibroblasts (CAFs) and mesenchymal-derived tumor cells. For example, in sarcomas, glioblastomas, and melanomas, high expression of LRRC15 is closely associated with tumor invasiveness and metastasis. Furthermore, under the regulation of the TGFβ signaling pathway, LRRC15 participates in the formation of the immunosuppressive tumor microenvironment (TME), making it an important target for cancer therapy.

[0004] Significant progress has been made in the study of LRRC15 in both oncology and non-oncology diseases: High expression of LRRC15 in the stromal region of lung adenocarcinoma was significantly associated with improved 5-year survival, suggesting that it may affect clinical outcomes by regulating immune cell function. In breast and ovarian cancer, LRRC15 promotes the migration and invasion of triple-negative breast cancer cells through the Wnt / β-catenin signaling pathway and promotes ovarian cancer metastasis through the integrin signaling pathway. The antibody-drug conjugate ABBV-085, an ADC targeting LRRC15, has shown significant anti-tumor activity in preclinical models, particularly in LRRC15-positive stromal and tumor cells. In rheumatoid arthritis (RA), LRRC15 has been identified by machine learning algorithms as a potential immunodiagnostic biomarker for RA, and its expression is closely related to the infiltration of M1 macrophages, with an area under the curve (AUC) of 0.964, demonstrating strong diagnostic capability. In osteoarthritis and osteogenic differentiation, LRRC15 plays an important role in cartilage damage and osteogenic differentiation, and may affect disease progression by regulating the release of pro-inflammatory cytokines.

[0005] Currently, LRRC15 detection mainly relies on immunological methods, including immunohistochemistry (IHC), flow cytometry, and ELISA. IHC is suitable for analyzing the localization and expression levels of LRRC15 in tissue samples, but its quantitative capabilities are limited. Flow cytometry is suitable for rapid detection of LRRC15 on cell surfaces, but it requires sophisticated sample processing. ELISA is suitable for quantitative detection of LRRC15 in serum or cell culture supernatants, but it cannot provide spatial distribution information. The limitations of existing methods include insufficient sensitivity and specificity, operational complexity, and limitations in quantitative analysis, necessitating the development of more efficient detection technologies. Summary of the Invention

[0006] This application aims to develop a highly sensitive and specific LRRC15 monoclonal antibody and optimize its detection method. By combining novel antibody engineering technologies (such as recombinant antibody technology) and multimodal detection platforms (such as multiplex immunofluorescence technology), the technical solution of this application will significantly improve the accuracy and practicality of LRRC15 detection, providing a more reliable tool for disease diagnosis and treatment. This will contribute to the advancement of LRRC15 detection and cancer diagnostic technologies.

[0007] On one hand, this application provides a monoclonal antibody of LRRC15, wherein the amino acid sequence of the heavy chain CDR1 is SEQ ID NO.1; the amino acid sequence of the heavy chain CDR2 is SEQ ID NO.2; the amino acid sequence of the heavy chain CDR3 is SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is SEQ ID NO.6; the amino acid sequence of the light chain CDR2 is SEQ ID NO.7; and the amino acid sequence of the light chain CDR3 is SEQ ID NO.8.

[0008] Furthermore, the heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.4, and / or the light chain variable region sequence of the monoclonal antibody is SEQ ID NO.9.

[0009] On the other hand, this application provides the use of the above-mentioned monoclonal antibody in the preparation of a drug for treating cancer.

[0010] Furthermore, the cancer in question is breast cancer.

[0011] On the other hand, this application provides the use of the above-mentioned monoclonal antibody in the preparation of reagents for detecting human LRRC15.

[0012] Furthermore, the reagent used to detect LRRC15 is a tumor diagnostic reagent.

[0013] Furthermore, the reagent is an ELISA reagent, a quantitative immunoPCR reagent, a colloidal gold detection reagent, or a chemiluminescent immunoassay reagent.

[0014] On the other hand, this application provides a non-diagnostic method for detecting LRRC15, which uses the aforementioned monoclonal antibody.

[0015] On the other hand, this application provides a nucleic acid encoding the above-mentioned monoclonal antibody, the nucleotide sequence of which includes SEQ ID NO.5 and SEQ ID NO.10.

[0016] On the other hand, this application provides a vector containing the aforementioned nucleic acid.

[0017] On the other hand, this application provides a host cell containing the aforementioned vector.

[0018] On the other hand, this application provides a method for producing the above-mentioned monoclonal antibody, the method including the step of culturing the above-mentioned host cells.

[0019] The host cell can be any suitable yeast, Escherichia coli, Bacillus subtilis, or cell-free expression system. Those skilled in the art can also select a suitable commercial vector based on the host and perform insertion operations according to the instructions.

[0020] When the antibody CDR region or variable region is known, those skilled in the art can routinely design the backbone region and other steps, and design the corresponding gene sequence according to the host preference.

[0021] The beneficial effects of the present invention include at least the following: (1) The present invention prepared a monoclonal antibody that can specifically recognize human LRRC15.

[0022] (2) The LRRC15 monoclonal antibody and its preparation method provided by the present invention have good specificity, high efficiency, accuracy, high sensitivity and low cost. They have great application value in actual production and provide material support for the diagnosis, monitoring and early warning of diseases such as cancer. Attached Figure Description

[0023] Figure 1 The SDS-PAGE results for recombinant human LRRC15 protein expressed in Example 1 are shown. The lanes from left to right are: marker, LRRC15 first wash, LRRC15 first wash, LRRC15 second wash, LRRC15 second wash, LRRC15 third wash, and LRRC15 third wash. The molecular weight of LRRC15 is approximately 60 kDa.

[0024] Figure 2 Results of Western blotting detection of endogenous LRRC15 protein.

[0025] Figure 3Results of Western blotting detection of endogenous LRRC15 in mouse cells.

[0026] Figure 4 The results are from a sensitivity ELISA assay for the LRRC15 D-5-2B-1 antibody.

[0027] Figure 5 Tumor growth curves in mice treated with LRRC15 D-5-2B-1 antibody.

[0028] Figure 6 Bioluminescence imaging image of mice treated with LRRC15 D-5-2B-1 antibody. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Example 1: Preparation of LRRC15 monoclonal antibody (1) Expression of recombinant human LRRC15 protein in Escherichia coli: A suitable human LRRC15 protein sequence was selected from NCBI, numbered NP_570843.2. The expression vector was synthesized by Suzhou Genewiz Company: plasmid: pET-28a(+); 5' restriction site: Ndel; 3' restriction site: Xhol; expression strain: BL21(DE3).

[0031] Mix 100 ng of plasmid with 20 μL of competent cells and incubate on ice for 30 min. Heat shock for 45 s, incubate on ice for 2 min, then incubate at 37°C, 200 rpm for 1 h. Centrifuge at 2000 rpm and plate a small amount of supernatant onto Kana antibody plates. Incubate overnight at 37°C. The next day, pick cells for expansion culture, induce expression with 1 mM IPTG at 37°C for 4 h. After successful expression, increase the culture volume and perform protein purification. SDS-PAGE is used to detect the protein expression and purification effect. Results are shown below. Figure 1 As shown.

[0032] (2) Mice were rapidly immunized with recombinant human LRRC15 protein to obtain immunized mice. Lymphocytes from the immunized mice were then fused with myeloma cells. Positive well cells were then screened and subcloned. The immunization process in mice is as follows: First immunization: 100 μg of recombinant human LRRC15 protein and complete Freund's adjuvant were mixed completely at a volume ratio of 1:1 and then injected subcutaneously into the hind paw pads of mice. Second immunization: Two weeks after the first immunization, the second immunization was performed. 100 μg of recombinant human LRRC15 protein and incomplete Freund's adjuvant were mixed completely at a volume ratio of 1:1 and injected subcutaneously into the hind paw pads of mice. Third immunization: Two weeks after the completion of the second immunization, the third immunization is performed by intraperitoneal injection of 100 μg of recombinant human LRRC15 protein and PBS buffer at a volume ratio of 1:1. Ten days after the third immunization, periorbital blood was collected from mice for potency testing. Enhanced immunization: Three days before fusion, 100 μg of recombinant human LRRC15 protein and PBS buffer were mixed at a volume ratio of 1:1 and injected into the tail vein of mice.

[0033] The specific procedures for the fusion experiment of lymphocytes and myeloma cells are as follows: Myeloma cells and lymphocytes were mixed at a ratio of 1:5 to obtain a cell mixture. The cell mixture was placed in a 50 ml centrifuge tube, diluted with RPMI-1640 basal medium, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the centrifuge tube was shaken to homogenize the cells. 1 ml of 50% PEG was slowly added, and the reaction was allowed to proceed for 90 seconds. Then, 10 ml of RPMI-1640 medium was added to stop the PEG reaction. The confluent cells were placed in a 37°C water bath and reacted for 10 min. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in RPMI-1640 complete medium. The fused cells were seeded into 96-well plates, 100 μL per well; the cell culture plates were then placed in a CO2 incubator for culture. After 10 days of fusion, cells from the positive wells were selected.

[0034] The specific steps for subcloning are as follows: Pipe the cells from the positive wells, take 10 μL for counting, add PBS buffer to the centrifuge tube according to the limiting dilution method, take 100 μL of cell suspension into the centrifuge tube, pipette well, take 30 μL, add 500 μL of RPMI-1640 complete medium, pipette well, and then inoculate onto semi-solid medium. After 6-8 days, select visible monoclonal cell lines and inoculate them onto 96-well plates, and then screen for positive monoclonal cell lines.

[0035] (3) Screening of strongly positive cell lines obtained after subcloning in step (2) for ascites preparation, and purification of the ascites: The specific procedures for preparing and purifying ascites fluid are as follows: Preparation of mouse ascites: Six-week-old healthy BALB / c female mice were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. One to two weeks later, the mice were injected intraperitoneally with 10 mL of liquid paraffin. 6 After 7-10 days of observation of significant abdominal distension in mice, ascites was collected from hybridoma cells. The ascites was centrifuged at 4000 rpm for 15 minutes at room temperature, and the supernatant was the monoclonal antibody ascites.

[0036] Protein A agarose gel medium was loaded into a nickel ion affinity chromatography column. Ascites fluid and PBS were mixed in equal volumes at a 1:1 ratio and the mixture was slowly loaded onto the column. After antibody binding, the sample was eluted with glycine elution buffer, and finally, 10 monoclonal antibodies were obtained through purification.

[0037] (4) Slowly add saturated ammonium sulfate dropwise to half-saturation while stirring at 4℃, and continue stirring for 30 min: Centrifuge at 13000 rpm for 30 min at 4°C, discard the supernatant. Dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4); slowly add saturated ammonium sulfate dropwise to 33% while stirring at 4°C, continue stirring for 30 min, centrifuge at 13000 rpm for 30 min at 4°C, discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4), dialyze overnight at 4°C. After ammonium sulfate precipitation, further purification is performed using a Protein G column. The concentration of the purified antibody is determined using the BCA method. Finally, 10 monoclonal antibodies (including monoclonal antibody D-5-2B-1) were purified, and their specificity, sensitivity, and biological effects were detected.

[0038] Example 2: Screening of monoclonal antibodies that specifically recognize LRRC15 (a) Western Blot verification of antibody specificity (1) Sample preparation Cells that highly express LRRC15 (A549 cells and HeLa cells) were selected as positive controls, and cells that express low or no LRRC15 (HEK293T cells) were selected as negative controls.

[0039] Total protein was extracted using RIPA lysis buffer (containing protease inhibitors), quantified using the BCA method, and experimentally verified.

[0040] (2) SDS-PAGE electrophoresis Sample loading amount: 20 μg protein per well, mixed with 5×SDS loading buffer, and boiled at 100℃ for 5 min.

[0041] Electrophoresis conditions: 10% SDS-PAGE gel, constant voltage of 80 V for 30 min, then adjusted to 120 V until bromophenol blue reaches the bottom of the gel.

[0042] (3) Transfer membrane PVDF membrane pretreatment: methanol activation for 30 s, TBST equilibration for 5 min.

[0043] Wet transfer conditions: constant voltage of 100 V, transfer film at 4℃ for 90 min (LRRC15 molecular weight is about 60kDa).

[0044] (4) Antibody incubation and color development Sealing: 5% skim milk / TBST sealed at room temperature for 1 h.

[0045] Primary antibody incubation: Hybridoma supernatant (purified antibody 1 μg / mL) was incubated overnight at 4°C. LRRC15 Rabbit pAb was added as a control.

[0046] Secondary antibody incubation: HRP-labeled anti-mouse IgG (1:10000) was incubated at room temperature for 1 h.

[0047] ECL color development: using chemiluminescent substrates (such as SuperSignal™ West Pico) for exposure imaging.

[0048] (5) Analysis of Western Blot Results like Figure 2 As shown, the purified antibody D-5-2B-1 has a specific band, which is consistent with the detection position of the purchased Abcam LRRC15 antibody.

[0049] Negative control: No band was detected in LRRC15 negative cells (HEK293T).

[0050] (ii) Endogenous detection Protein was loaded at a rate of 100 μg per lane using lysates from A549 cells, mouse heart tissue, mouse liver tissue, mouse spleen tissue, mouse lung tissue, mouse kidney tissue, mouse brain tissue, mouse intestinal tissue, and mouse stomach tissue, respectively. The Western blotting method described above was used for detection with the D-5-2B-1 antibody, and the plots were obtained. Figure 3 .

[0051] like Figure 3 As shown, the monoclonal antibody D-5-2B-1 against LRRC15 can specifically recognize the LRRC15 antigen in mouse cells.

[0052] This method rigorously screened for the highly specific anti-LRRC15 monoclonal antibody D-5-2B-1 using Western blotting, laying the foundation for the development of subsequent diagnostic or therapeutic antibodies.

[0053] (III) Sensitivity detection of monoclonal antibody D-5-2B-1 against LRRC15 An ELISA assay was constructed using LRRC15 eukaryotic antigen as the plating medium and monoclonal antibody D-5-2B-1 as the detection antibody. The specific detection steps are as follows: (1) Coating: LRRC15 eukaryotic protein at concentrations of 20 μg / mL, 2 μg / mL, 200 ng / mL, 20 ng / mL, 2 ng / mL, 200 pg / mL, 20 pg / mL and 2 pg / mL were used as antigens to coat the plates overnight at 4°C; (2) Blocking: Wash 3 times with PBST (containing 0.05% Tween), block with 5% skim milk powder, 200 μL per well, and incubate at 37°C for 1 h; (3) Add antibody: Wash 3 times with PBST, pat dry and add D-5-2B-1 detection antibody, 2ug / mL, 80μL per well; incubate at 37℃ for 1h; (4) Add secondary antibody: Wash 3-5 times with PBST, dilute secondary antibody (HRP-labeled goat anti-mouse) 8000 times with 5% skim milk powder, 50 μL per well, and incubate at 37℃ for 1 h; (5) Wash 5 times with PBST, 3 min each time, pat dry, add colorimetric solution, incubate at room temperature in the dark for 10 min, then stop the incubation and read the OD value with a microplate reader. 450 The value is used to plot the detection curve. Figure 4 ).

[0054] Test results, such as Figure 4 As shown, the ELISA detection sensitivity of the LRRC15 D-5-2B-1 antibody against the LRRC15 antigen is 0.2 ng / mL, binding... Figure 2 , Figure 3 The LRRC15 D-5-2B-1 monoclonal antibody can specifically recognize the LRRC15 antigen in cells, and the test results are consistent with those of commercially available LRRC15 antibodies. Moreover, it has good affinity and specificity.

[0055] Example 3: Sequence and structural analysis of monoclonal antibody D-5-2B-1 The monoclonal antibody D-5-2B-1 was sequenced by Shanghai Bio-Tech Co., Ltd.

[0056] The heavy chain variable region of monoclonal antibody D-5-2B-1 contains three CDR regions, as follows: VHCDR1:DYAMS (SEQ ID NO:1); VHCDR2:TISNGGGYTYYPDSVKG (SEQ ID NO:2); VHCDR3: HDYRIYDTVDY (SEQ ID NO:3); The amino acid sequence of the heavy chain variable region of monoclonal antibody D-5-2B-1: MNLGLSLIFLVLVLKGVQCAVMLVESGGDLVQPGGSLKLSCTASGFTFSDYAMSWIRQTPEMRLEWVATISNGGGYTYYPDSVKGRFTISRDNAENTLSLQMTSLRSEDTAIYYCSRHDYRIYDTVDYWGQGTSVTVSS (SEQID NO.4); Nucleotide sequence: ATGAACCTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGCAGTGATGCTGGTGGAGTCTGGAGGAGACTTGGTGCAGCCTGGAGGGTCCTGAAACTCTCCTGTACAGCCTCTGGATTCACTTTCAGTGACTATGCCATGTCTTGGATTCGCCAGACTCCGGAGATGAGGCTGGAGTGGGTCGCAACCATT AGTAATGGTGGTGGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCCCAGAGACAATGCCGAGAACACCCTGTCCCTTCAAATGACCAGTCTTAGGTCTGAGGACACGGCCATTTATTACTGTTCACGACATGACTATCGTATCTACGATACTGTGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCG (SEQ ID NO.5); The light chain variable region of monoclonal antibody D-5-2B-1 contains three CDR regions, as follows: VLCDR1:RSSKSLLHRNGITYLY(SEQ ID NO:6); VLCDR2: QMSNLAS (SEQ ID NO:7); VLCDR3:AQNLELLT(SEQ ID NO:8); The amino acid sequence of the light chain variable region of monoclonal antibody D-5-2B-1: MRFSAQLLGLLVLWIPGSTADIVMTQAAISNPVTLGTSASISCRSSKSLLHRNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELLTFGAGTKLELK (SEQ ID NO:9); Nucleotide sequence: ATGAGGTTCTCTGCTCAGCTTCTGGGGCTGCTTGTGCTCTGGATCCCTGGATCCACTGCAGATATTGTGATGACGCAGGCTGCAATCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGAAATGGCATCACTTACTTGTATTGGTATCTGCAGAAGCCAGGC CAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGTGGAGGCCGAGGATGTGGGTGTTTATTACTGTGCTCAAAATCTAGAACTTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO:10).

[0057] The monoclonal antibody D-5-2B-1 obtained in this invention has a specific CDR region, which is significantly different from existing reported monoclonal antibodies; and the monoclonal antibody D-5-2B-1 has excellent affinity and specific recognition performance for human LRRC15.

[0058] Example 4: Effects of LRRC15 antibody on breast cancer in vivo MDA-MB-231-luciferase cells (2 × 10⁻⁶) were subcutaneously injected into the mammary fat pads of 12 seven-week-old female nude mice. 6 (Number of tumors). Once the tumor grows to approximately 20 mm in volume... 3 Mice were divided into a control group and an LRRC15 antibody treatment group. The control group received intraperitoneal injections of normal mouse IgG (3 mg / kg) twice weekly; the LRRC15 antibody treatment group received intraperitoneal injections of LRRC15 monoclonal antibody (3 mg / kg) twice weekly. Treatment continued for 4 weeks, and tumor volume was recorded. The tumor volume was calculated using the formula: V = (length × width) / (length × width) 2) / 2. Bioluminescence imaging was used to analyze the metastatic lesions in mice with orthotopic tumors.

[0059] The results are as follows Figure 5 As shown, the tumor volume in mice treated with LRRC15 monoclonal antibody for 24 days was smaller than that in the control group, demonstrating that LRRC15 monoclonal antibody can inhibit the growth of breast cancer cells in vivo. Figure 6 As shown, mice with orthotopic tumors treated with LRRC15 monoclonal antibody for 4 weeks had fewer metastatic lesions than the control group. The experimental results demonstrate that LRRC15 monoclonal antibody can inhibit the metastasis of breast cancer cells in vivo.

Claims

1. An LRRC15 monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is SEQ ID NO.1; the amino acid sequence of the heavy chain CDR2 is SEQ ID NO.2; the amino acid sequence of the heavy chain CDR3 is SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is SEQ ID NO.6; the amino acid sequence of the light chain CDR2 is SEQ ID NO.7; and the amino acid sequence of the light chain CDR3 is SEQ ID NO.

8.

2. The LRRC15 monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The light chain variable region sequence of the monoclonal antibody is SEQ ID NO.

9.

4. The use of the monoclonal antibody according to any one of claims 1-3 in the preparation of a medicament for treating cancer.

5. The application according to claim 4, wherein the cancer is breast cancer.

6. The use of the monoclonal antibody according to any one of claims 1-3 in the preparation of a reagent for detecting human LRRC15.

7. The application according to claim 6, characterized in that, The reagents are ELISA reagents, quantitative immunoPCR, colloidal gold detection reagents, or chemiluminescent immunoassay reagents.

8. A nucleic acid encoding a monoclonal antibody according to any one of claims 1-3, characterized in that, The nucleotide sequence of the nucleic acid includes SEQ ID NO.5 and SEQ ID NO.

10.

9. A carrier, characterized in that, The vector contains the nucleic acid according to claim 8.

10. A method for producing a monoclonal antibody according to any one of claims 1-3, characterized in that, The method includes the step of culturing host cells containing the vector of claim 9.

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