Preparation of a strain of anti-sema7a functional monoclonal antibody 10A6 and its anti-inflammatory effect in SLE

Abstract

The application provides a preparation of a strain of anti-sema7A functional monoclonal antibody 10A6 and an anti-inflammatory effect of the monoclonal antibody 10A6 in SLE, wherein the CDR-H1 of the heavy chain variable region of the monoclonal antibody 10A6 is an amino acid sequence shown in SEQ ID No. 1, the CDR-H2 of the heavy chain variable region is an amino acid sequence shown in SEQ ID No. 2, and the CDR-H3 of the heavy chain variable region is an amino acid sequence shown in SEQ ID No. 3; the CDR-L1 of the light chain variable region of the monoclonal antibody 10A6 is an amino acid sequence shown in SEQ ID No. 4, the CDR-L2 of the light chain variable region is an amino acid sequence shown in SEQ ID No. 5, and the CDR-L3 of the light chain variable region is an amino acid sequence shown in SEQ ID No. 6. The monoclonal antibody 10A6 can effectively inhibit the expression up-regulation of IL-1beta, TNF-alpha and IL-6 caused by Sema7A recombinant protein; the anti-sema7A monoclonal antibody 10A6 can effectively inhibit the macrophage inflammatory response induced by Sema7A, continuously and effectively reduce the serum anti-double-stranded DNA antibody of a lupus mouse. The monoclonal antibody 10A6 can be used for preparing a pharmaceutical composition for preventing and / or treating systemic lupus erythematosus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, more particularly, to a preparation of a strain of anti-sema7A functional monoclonal antibody 10A6 and its anti-inflammatory effect in SLE. BACKGROUND

[0002] Systemic Lupus Erythematosus (SLE) is a common clinical autoimmune disease, which is characterized by immune system attacking self-tissues, and can involve joints, skin, brain, lungs, kidneys and blood vessels, and cause extensive inflammation and tissue damage. Controlling inflammation is one of the main treatment strategies for SLE. The use of corticosteroids and immunosuppressive agents has greatly improved the prognosis of SLE. However, their side effects cannot be ignored, which limits their clinical application, especially for children with SLE. Therefore, the development of new drugs with effective anti-inflammatory effect and less side effects has become an important demand in the field of SLE treatment.

[0003] Macrophages, as important inflammatory cells, play a key role in the development of SLE. They not only directly produce pro-inflammatory cytokines such as IL-12 and TNF-α, which aggravate SLE tissue inflammation, but also regulate T and B cell immune responses, induce excessive Th1 / Th17 immune responses, and promote the production of pathological anti-dsDNA antibodies, which aggravate tissue inflammation and damage. How to control macrophage inflammation has become the focus and emerging direction of new treatment strategies for SLE.

[0004] The prior art has obtained monoclonal antibodies for different proteins of the human body to treat systemic lupus erythematosus. A Chinese invention patent with publication number CN1503806A discloses a 16 / 6id antibody peptide for treating systemic lupus erythematosus, which relates to a synthetic peptide based on the complementarity determining region of human monoclonal anti-DNA antibodies to regulate the immune system of systemic lupus erythematosus. A Chinese invention patent with publication number CN101155831A discloses an anti-interferon alpha monoclonal antibody and a method of using the same, which controls the activation of MxA promoter or antiviral activity by selectively directly neutralizing the biological activity of human interferon IFN alpha protein subtypes A, 2, B2, C, F, G, H2, I, J1, K, 4a, 4b and WA, but does not significantly neutralize the biological activity of IFN alpha protein subtype D, thereby achieving treatment for lupus. However, there are many proteins or polypeptides that affect the immune system of systemic lupus erythematosus patients, and there is no report on which protein can be used as an antigen to prepare more effective monoclonal antibodies. Therefore, it is extremely necessary to find a B target protein that affects SLE lupus and prepare and identify a therapeutic monoclonal antibody that can effectively alleviate SLE lupus, which is expected to provide another new SLE treatment strategy, and has very important scientific significance and clinical application value.

[0005] Semaphorin 7A (Sema7A) is a glycosylated phosphatidylinositol (GPI) anchored membrane protein, which can also exist as a soluble protein after proteolytic cleavage; Sema7A is expressed in a variety of immune and non-immune cells. There is no monoclonal antibody prepared against Semaphorin 7A (Sema7A) to treat systemic lupus erythematosus. SUMMARY

[0006] The purpose of the present application is to solve the above technical problems, and to provide a monoclonal antibody 10A6 against Semaphorin 7A, which can treat systemic lupus erythematosus.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A sema7A monoclonal antibody 10A6, wherein a CDR-H1 of a heavy chain variable region of the monoclonal antibody 10A6 is an amino acid sequence shown in SEQ ID No. 1, a CDR-H2 of the heavy chain variable region is an amino acid sequence shown in SEQ ID No. 2, and a CDR-H3 of the heavy chain variable region is an amino acid sequence shown in SEQ ID No. 3; and wherein a CDR-L1 of a light chain variable region of the monoclonal antibody 10A6 is an amino acid sequence shown in SEQ ID No. 4, a CDR-L2 of the light chain variable region is an amino acid sequence shown in SEQ ID No. 5, and a CDR-L3 of the light chain variable region is an amino acid sequence shown in SEQ ID No. 6.

[0009] Further, the heavy chain variable region is an amino acid sequence shown in SEQ ID No. 7.

[0010] Further, the light chain variable region is an amino acid sequence shown in SEQ ID No. 8.

[0011] Further, the monoclonal antibody 10A6 is of IgM subtype, and the light chains are all kappa chains.

[0012] The preparation method of the above sema7A monoclonal antibody is as follows: immunizing an animal with a sema7A protein, isolating a single B lymphocyte from the animal, performing myeloma cell fusion, and screening to obtain a sema7A monoclonal antibody; wherein the sema7A protein is a common amino acid sequence 185-321 (Q9QUR8.1) selected from human (CAJ55404.1) and mouse Sema7A amino acid sequences (Q9QUR8.1), and the common amino acid sequence is a sequence shown in SEQ ID No. 9.

[0013] The present application finds that the sema7A monoclonal antibody 10A6 can effectively inhibit the up-regulation of IL-1β, TNF-α and IL-6 expression caused by a Sema7A recombinant protein, and has no significant effect on the expression of IL-10; the anti-sema7A monoclonal antibody 10A6 can effectively inhibit the macrophage inflammatory response induced by Sema7A, and continuously and effectively reduce the serum anti-double-stranded DNA antibody and kidney damage of a lupus mouse.

[0014] Since the monoclonal antibody 10A6 can effectively inhibit the macrophage inflammatory response induced by Sema7A, and continuously and effectively reduce the serum anti-double-stranded DNA antibody and kidney damage of a lupus mouse, the present application claims to protect a nucleic acid encoding the above sema7A monoclonal antibody 10A6.

[0015] The present application claims to protect an expression vector comprising the above nucleic acid, and capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0016] The present application claims a host cell comprising and capable of expressing the above-mentioned vector and producing the above-mentioned sema7A monoclonal antibody 10A6.

[0017] The present application claims a pharmaceutical composition for preventing and / or treating systemic lupus erythematosus, comprising the above-mentioned sema7A monoclonal antibody 10A6 and a medically acceptable carrier or adjuvant.

[0018] The present application claims the use of the above-mentioned sema7A monoclonal antibody 10A6 in the preparation of a medicine for preventing and / or treating systemic lupus erythematosus.

[0019] The beneficial effects of the present application are as follows:

[0020] (1) The present application finds that the sema7A monoclonal antibody 10A6 can effectively inhibit the up-regulation of IL-1β, TNF-α, IL-6 expression caused by Sema7A recombinant protein, and has no significant effect on IL-10 expression; the anti-sema7A monoclonal antibody 10A6 can efficiently inhibit Sema7A-induced macrophage inflammatory response, and continuously and effectively reduce the serum anti-double-stranded DNA antibody of lupus mice. The sema7A monoclonal antibody 3F12 has no significant effect on the expression of inflammatory factors and SLE inflammatory response.

[0021] (2) The sema7A monoclonal antibody 10A6 of the present application can be used for the preparation of a pharmaceutical composition for preventing and / or treating systemic lupus erythematosus, comprising the above-mentioned sema7A monoclonal antibody 10A6 and a medically acceptable carrier or adjuvant. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the SDS-PAGE diagram of the sema7A antibodies 3F12, 10A6 of the present application.

[0023] Figure 2 is the ELISA detection of the sema7A antibodies 3F12, 10A6 monoclonal antibodies of the present application.

[0024] Figure 3 Binding activity determination of the sema7A antibodies 3F12, 10A6 of the present application.

[0025] Figure 4 is the effect of RAW264.7 cells on the production of IL-1β (A), TNF-α (B), IL-6 (C), IL-10 (D) in the experimental grouping of Example 3.

[0026] Figure 5The sema7A antibody 10A6 of the present application can effectively reduce serum anti-inflammatory factors IL-1β (A), TNF-α (B), IL-6 (C), IL-10 (D) of SLE-like mice.

[0027] Figure 6 The sema7A antibody 10A6 of the present application can effectively reduce serum anti-inflammatory factors IL-1β (A), TNF-α (B), IL-6 (C), IL-10 (D) of SLE-like mice. DETAILED DESCRIPTION

[0028] In order to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0029] Example 1: Obtaining of anti-sema7A antibody 10A6

[0030] 1. Antigen preparation, the specific steps are as follows:

[0031] The common amino acid sequence 185-321 (Q9QUR8.1) was selected from the human (CAJ55404.1) and mouse Sema7A amino acid sequences (Q9QUR8.1) searched from NCBI, the common amino acid sequence 185-321 is shown as SEQ ID No. 9, wherein the nucleotide sequence encoding the common amino acid sequence 185-321 is SEQ ID No. 10, and the sequence optimization, DNA synthesis and construction of the prokaryotic expression plasmid pET30a-Sema7A were performed by GenScript Biotech Co., Ltd.

[0032] The pET30a-Sema7A was transformed into BL21 competent cells, screened with solid LB medium containing ampicillin, and single colonies were picked and extracted for plasmid, double enzyme digestion and sequencing to verify that the sequence of the target gene was correct. The obtained strain was stored in a culture solution containing 25% glycerol at-80℃.

[0033] The above strain was cultured at 37°C to OD600 of about 0.6-0.8, 0.1 mM IPTG expression inducer was added, and induction was performed overnight at 16°C. The bacterial cells were disrupted by ultrasonic, and the supernatant and precipitate were separated. SDS-PAGE detection was performed, and it was found that the recombinant protein Sema7A mainly existed in the bacterial cell precipitate. The precipitate was dissolved with 8M urea, mixed with Ni Sepharose 6FF Beads (GE Healthcare), and combined at 4°C overnight. After centrifugation, the supernatant was discarded, and the beads were washed 2-3 times. Sema7A protein solution was obtained by elution with an imidazole gradient eluent, which was used as an antigen for subsequent preparation of monoclonal antibodies.

[0034] 2. Preparation and purification of monoclonal antibodies

[0035] (1) Immunization of animals

[0036] Six 6-8 week old SFP level female Balb / c mice were immunized with the purified Sema7A protein as an antigen. 60 μg of Sema7A protein was mixed with 300 μg of MnJ(β) adjuvant in equal volume and vortexed to mix, and then subcutaneous multi-point immunization of the mice was performed. The mice were subcutaneously immunized for a total of 3 times, with an interval of 2 weeks each time. Seven days after the last immunization, serum antibody titer determination was performed, and the mouse with the highest titer was selected for abdominal impact, i.e., intraperitoneal injection of Sema7A protein antigen (80 μg per mouse) mixed with MnJ(β) adjuvant. Three days later, the mouse spleen was taken for cell fusion.

[0037] (2) Preparation of mouse peritoneal cells

[0038] Before cell fusion for 24 h, feeder cells were prepared. The mouse was sacrificed by dislocation, and 75% alcohol was used for disinfection. The abdomen was cut open, torn open, and the abdominal wall was lifted with forceps. Seven milliliters of HAT 1640 complete culture solution were injected into the abdominal cavity. The abdomen was massaged, and about 6 ml of liquid was withdrawn. The liquid was diluted with 100 ml of HAT 1640 culture solution to mix, and the feeder layer cells were plated in a 96-well plate at 100 μl per well.

[0039] (3) PEG cell fusion

[0040] The mouse was sacrificed by dislocation, and the spleen was taken to prepare a single cell suspension. After breaking the red, the cells were resuspended by centrifugation, and cell counting was performed. The myeloma cells SP2 / 0 were mixed with the above spleen cells at a ratio of 1:4, and 1 ml of 50% (w / v) PEG-1450 was added for 1 min. The reaction was terminated by dilution with basic DMEM medium. Centrifugation was performed at 1000 rpm for 7 min, the supernatant was discarded, and the cells were resuspended in HAT complete culture medium containing 20% fetal bovine serum at 37°C in a 5% CO2 cell incubator for 1 h. Subsequently, the cells were plated at 2×10 7Seeds were placed into pre-prepared feeder cell culture plates and incubated at 37°C with 5% CO2 for two weeks. Observation was then conducted to check for the appearance of positive clones.

[0041] (4) Screening for positive hybridoma cells and establishing cell lines

[0042] Once the hybridoma cells in each well have grown to approximately 10,000 cells or more, the production of Sema-specific antibodies in the supernatant is detected using ELISA to screen for positive hybridoma cells: The ELISA plate is coated with Sema7A recombinant protein, blocked, and then incubated with cell culture supernatant (100 μl / well) at 37°C for 1.5 h. After washing with PBST, secondary antibody is added, and the plate is incubated at 37°C for 1 h. After washing with PBST, chromogenic buffer is added, and the incubation is stopped after 10 min. The OD value at 450 nm is measured using a microplate reader. Clones with OD values ​​greater than 2.1 times the negative value are selected for the next round of testing.

[0043] Wells with high positive values ​​(OD450>2.0) in the fusion plate were selected for limiting dilution. Subcloning was performed by counting 60% of the single-clone wells per plate. Each time, single-clone wells with high positive values ​​were selected for limiting dilution. Each subcloning was performed ELISA after 5-7 days until a single-clone cell line that could stably secrete positive antibodies was finally selected for expansion culture.

[0044] The cell lines that stably secreted positive antibodies, selected during the subcloning stage, were expanded and cultured. The supernatant was collected, and the stability and specificity of antibody production were verified by serial dilution using ELISA and Western blot. These cells were then expanded and cryopreserved, thus forming hybridoma cell lines.

[0045] (5) Large-scale preparation and purification of monoclonal antibodies

[0046] The obtained cell lines 3F12 and 10A6 were expanded and cultured using SFM (Self-Fluid Mushroom) specifically for hybridoma cells, and the cell culture supernatant was collected. Antibodies were purified using thiophile affinity chromatography. First, ammonium sulfate was added to the cell supernatant to a concentration of 0.8 M. Then, a thiophile affinity chromatography column was prepared, and the column was equilibrated with buffer 1 (20 mM sodium phosphate, 0.8 M ammonium sulfate). The cell culture supernatant filtered through a 0.45 μm filter was then passed through the column, washed with buffer 1, eluted with 20 mM sodium phosphate solution, and dialyzed for later use.

[0047] Example 2: Identification of anti-sema7A monoclonal antibody

[0048] 1. Experimental Methods

[0049] (1) Determination of molecular weight and purity of monoclonal antibodies 3F12 and 10A6

[0050] The Sema7A monoclonal antibody obtained in Example 1 was subjected to SDS-PAGE electrophoresis, and the molecular weight and purity of the separated and purified 3F12 and 10A6 were determined.

[0051] (2) Type and light chain type of monoclonal antibodies 3F12 and 10A63

[0052] The antibody subtype of the Sema7A monoclonal antibody was identified using an IsoStripTM mouse monoclonal antibody subtype identification kit.

[0053] (3) Detection of binding activity of anti-Sema7A monoclonal antibodies 3F12 and 10A63

[0054] The recombinant Sema7A protein was coated as an antigen, and the purified antibodies 3F12 and 10A6 were added in a dilution of 300 μg / mL, followed by incubation at 37°C for 1 h; a commercially available anti-mouse IgM-HRP secondary antibody was added and incubated at 37°C for 1 h; after PBST washing, TMB was added for color development, and after the reaction was stopped, the OD450 value was read by a multifunctional enzyme label instrument, and the EC50 value of the antibody binding to Sema7A was analyzed.

[0055] 2. Experimental results

[0056] (1) The results of the determination of the molecular weight and purity of the monoclonal antibodies 3F12 and 10A6 are shown in Table 1, and the SDS-PAGE patterns of the purified anti-Sema7A monoclonal antibodies 3F12 and 10A6 are shown in Table 2. Figure 1 Figure 1 The molecular weight of the 3F12 and 10A6 is about 70 kDa and 25 kDa, respectively, which is consistent with the molecular weight of the heavy chain and light chain of the antibody, and the purity is more than 90%.

[0057] (2) The results of the determination of the type and light chain type of the monoclonal antibodies 3F12 and 10A6 are shown in Table 3. Figure 2 The anti-Sema7A monoclonal antibodies 3F12 and 10A6 in the present application are of IgM subtype, and the light chains are κ chains.

[0058] The sequences of the heavy chain and light chain of 10A6 are shown in Table 1:

[0059]

[0060] Note: The heavy chain variable region CDR1 represents CDR-H1; CDR2 represents CDR-H2; CDR3 represents CDR-H3; the light chain variable region CDR1 represents CDR-L1; the light chain variable region CDR2 represents CDR-L2; and the light chain variable region CDR3 represents CDR-L3; V(D)J-IMGT represents the amino acid sequence of the heavy chain or light chain.

[0061] (3) The results of the detection of the binding activity of the anti-Sema7A monoclonal antibody 10A63​Figure 3 As shown, the EC50 of anti-Sema7A monoclonal antibody 10A6 in this invention is 11.67 μg / ml, and the EC50 of anti-Sema7A monoclonal antibody 3F12 is 18.79 μg / ml.

[0062] Example 3: Inflammatory Response of Macrophages by Anti-sema7A Monoclonal Antibody 10A6

[0063] 1. Experimental Methods

[0064] T cells can interact with macrophage surface receptors via Sema7A, thereby regulating macrophage immune responses. In this invention, we evaluated the effects of anti-sema7A monoclonal antibodies 3F12 and 10A6 on Sema7A protein-induced macrophage inflammatory responses. The specific experimental methods are as follows:

[0065] The experimental groups were divided into two groups and incubated with macrophages for 6 h each: PBS (NC), 5 μg / ml Sema7A recombinant protein (Pr), 5 μg / ml 10A6 monoclonal antibody, and a mixture of 5 μg / ml Sema7A recombinant protein and 10A6 monoclonal antibody; and 5 μg / ml 3F12 recombinant protein (Pr), 5 μg / ml 3F12 monoclonal antibody, and a mixture of 5 μg / ml Sema7A recombinant protein and 3F12 monoclonal antibody. Cellular RNA was extracted and reverse transcribed according to the instructions of the Full-Gold standard. Real-time PCR was used to detect the effects of 3F12 and 10A6 antibodies on the ability of sema7A to induce the production of IL-1β, TNF-α, IL-6, and IL-10 in RAW264.7 cells.

[0066] 2. Experimental Results

[0067] Experimental results are as follows Figure 4 As shown, the experimental results indicate that the Sema7A recombinant protein can significantly upregulate IL-1β in RAW264.7 cells. Figure 4 A), TNF-α Figure 4 B), IL-6 Figure 4 C), IL-10 Figure 4 D) mRNA levels. The 3F12 monoclonal antibody had no significant effect on the upregulation of IL-1β, TNF-α, IL-6, and IL-10 induced by the Sema7A recombinant protein. The 10A6 monoclonal antibody effectively inhibited the upregulation of IL-1β, TNF-α, and IL-6 induced by the Sema7A recombinant protein, but had no significant effect on IL-10 expression.

[0068] Example 4: Therapeutic effect of anti-sema7A monoclonal antibody on inflammatory damage in SLE mice

[0069] 1. Experimental methods

[0070] Establishment and treatment of lupus nephritis-like mouse model: After mixing the activated lymphocyte-derived self-DNA of BALB / c mice with Freund's adjuvant, 6-8-week-old female mice of the same strain were subcutaneously immunized (100 μg DNA per mouse per time, 6 mice per group) at 0, 2, and 4 weeks, for a total of three times, which can effectively establish a lupus nephritis-like mouse model. One week after the last DNA immunization (i.e., at week 7), 3F12 monoclonal antibody and 10A6 monoclonal antibody (50 μg per mouse) were intravenously administered, and serum was collected at week 12 for detection of inflammatory factor expression. During the modeling process, serum and urine samples were collected every other week to continuously detect the serum anti-dsDNA antibody and urine protein levels of SLE mice to characterize the therapeutic effects of anti-sema7A monoclonal antibodies 3F12 and 10A6. The specific determination steps are as follows:

[0071] Inflammatory factor detection: Coat the enzyme-labeled plate with 1×Capture Antibody, 100 μL / well, 4°C overnight; wash with PBST for 3 times and dry; add 1×Diluent for blocking, 200 μL / well, 37°C for 1 h; dilute the standard (start from 500 pg / mL with a dilution ratio of 1:2); wash with PBST for 3 times and dry; add cell supernatant or diluted standard, 100 μL / well, 37°C for 1 h; wash with PBST for 3 times and dry; add 1×Detection Antibody, 100 μL / well, 37°C for 1 h; wash with PBST for 5 times and dry; add 1×Avidin-HRP or StreAvidin-HRP, 100 μL / well, 37°C for 30 min; wash with PBST for 7 times and dry; add TMB developing solution, 100 μL / well, 37°C for 15 min; add ELISA stop solution, 50 μL / well, and measure OD 450 .

[0072] Anti-dsDNA autoantibody detection: the ELISA plate was treated with 0.5% protamine sulfate, 100 μl / well, 37°C for 1 h; washed with PBST, 3 times, and dried; calf thymus DNA was used as a coating antigen, 50 μg / mL, 100 μl / well, 37°C for 2 h, and then 4°C overnight; washed with PBST, 3 times, and dried; 1×Diluent was added for blocking, 100 μl / well, 37°C for 1 h; washed with PBST, 3 times, and dried; the serum to be tested was diluted with 1×Diluent at 1:100, 100 μl / well, 37°C for 1 h; washed with PBST, 3 times, and dried; goat anti-mouse IgG-HRP (1:1000) was added, 100 μl / well, 37°C for 1 h; washed with PBST, 4 times, and dried; TMB color developing solution was added, 100 μl / well, 37°C for 15 min; ELISA stop solution was added, 50 μl / well, and OD was measured 450 .

[0073] Urine protein detection: according to the operation steps of the urine protein quantitative test kit (CBB method), 2 μl of ddH2O, 2 μl of sample, 2 μl of protein standard (563 mg / L), and CBB application liquid were thoroughly mixed, left to stand for 5 min, and then the OD value at 595 nm wavelength was detected by an enzyme-labeled instrument.

[0074] 2. Experimental results

[0075] (1) As shown in Figure 5 , intravenous infusion of 10A6 monoclonal antibody can effectively reduce the serum IL-1β, TNF-α, IL-6, and IL-10 levels of SLE mice, while intravenous infusion of 3F12 monoclonal antibody has no significant effect on the serum cytokines of SLE mice.

[0076] (2) Continuous detection of the serum anti-dsDNA antibody and urine protein levels of SLE mice found that, compared with 3F12 monoclonal antibody and the control, 10A6 monoclonal antibody treatment can effectively reduce the production of anti-dsDNA antibody since the 4th week, and maintain at a lower level over time Figure 6 A). At the same time, the urine protein of the 10A6 monoclonal antibody group mice is also significantly lower than that of the control Figure 6 B) from the 8th to 12th week. It is shown that anti-Sema7A monoclonal antibody 10A6 significantly improves the condition of SLE mice and has a good therapeutic effect.

[0077] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit the solutions. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. An anti-sema7A monoclonal antibody 10A6, characterized in that, The amino acid sequence of CDR-H1 of the heavy chain variable region of the monoclonal antibody 10A6 is shown as SEQ ID No. 1, the amino acid sequence of CDR-H2 of the heavy chain variable region is shown as SEQ ID No. 2, and the amino acid sequence of CDR-H3 of the heavy chain variable region is shown as SEQ ID No. 3; the amino acid sequence of CDR-L1 of the light chain variable region of the monoclonal antibody 10A6 is shown as SEQ ID No. 4, the amino acid sequence of CDR-L2 of the light chain variable region is shown as SEQ ID No. 5, and the amino acid sequence of CDR-L3 of the light chain variable region is shown as SEQ ID No.

6.

2. The anti-sema7A monoclonal antibody 10A6 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown as SEQ ID No.

7.

3. The anti-sema7A monoclonal antibody 10A6 according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown as SEQ ID No.

8.

4. The anti-sema7A monoclonal antibody 10A6 according to claim 1, characterized in that, The monoclonal antibody 10A6 is of IgM subtype, and the light chains are all κ chains.

5. A nucleic acid encoding the anti-sema7A monoclonal antibody 10A6 according to claim 1.

6. An expression vector, characterized by, The nucleic acid according to claim 5 is contained and can express the nucleic acid in a eukaryotic host cell.

7. A host cell, characterized in that, The vector according to claim 6 is contained and can express the anti-sema7A monoclonal antibody 10A6 according to any one of claims 1-4.

8. A pharmaceutical composition for treating lupus nephritis, characterized by, The anti-sema7A monoclonal antibody 10A6 according to any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient are included.

9. Use of the anti-sema7A monoclonal antibody 10A6 according to any one of claims 1-4 in the preparation of a medicament for treating lupus nephritis.

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