Preparation of monoclonal antibody 8a12 against sema7a and its therapeutic effect on lupus nephritis

Abstract

The application provides a preparation of a sema7A monoclonal antibody 8A12 and a treatment effect of the sema7A monoclonal antibody 8A12 on lupus nephritis, wherein the CDR-H1 of the heavy chain variable region of the monoclonal antibody 8A12 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 8A12 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 8A12 can effectively inhibit the expression up-regulation of IL-1beta, TNF-alpha and IL-6 caused by Sema7A recombinant protein, can effectively inhibit the macrophage inflammatory response induced by Sema7A, and can continuously and effectively reduce serum anti-double-stranded DNA antibodies and kidney damage of lupus mice. The monoclonal antibody 8A12 can be used for preparing a pharmaceutical composition for preventing and / or treating systemic lupus erythematosus and / or lupus nephritis thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, more particularly, to a preparation of anti-sema7A monoclonal antibody 8A12 and its therapeutic effect on lupus nephritis. BACKGROUND

[0002] Systemic Lupus Erythematosus (SLE) is a complex and diverse clinical manifestation of a fatal autoimmune disease, which is different from general inflammation, and its pathological manifestations are mainly the appearance of extensive inflammation in multiple organs and tissues throughout the body. Therefore, the SLE activity score is largely dependent on the degree of inflammation in each organ. Systemic lupus erythenlatosus nephritis (SLEN) is one of the end-stage diseases of SLE patients. IgG autoantibodies against double-stranded DNA are deposited in the glomerular basement membrane, a large number of inflammatory factors are released, and inflammatory cells are infiltrated, which are the main causes of lupus pathogenesis and death.

[0003] At present, for severe lupus nephritis, anti-inflammatory and immunosuppression are mainly used. Generally, the SLE treatment regimen includes corticosteroids, non-steroidal immunosuppressive drugs, anti-malarial drugs and non-steroidal anti-inflammatory drugs. These drugs destroy the integrity of all immune effector responses, rather than specifically targeting the pathogenesis of SLE, and have moderate to severe side effects. Therefore, it is necessary to develop more new drugs with small side effects and effectiveness, which become the focus of research and development for the treatment of lupus nephritis and other SLE tissue lesions.

[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 target protein that affects SLE lupus nephritis and to prepare and identify a therapeutic monoclonal antibody that can effectively alleviate SLE lupus nephritis, 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 report on preparing monoclonal antibodies against Semaphorin 7A (Sema7A) to treat systemic lupus erythematosus, especially lupus nephritis. SUMMARY

[0006] The purpose of the present application is to solve the above technical problems, and to provide a monoclonal antibody 8A12 against Semaphorin 7A, which can treat systemic lupus erythematosus and lupus nephritis.

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

[0008] A sema7A monoclonal antibody 8A12, wherein a CDR-H1 of a heavy chain variable region of the monoclonal antibody 8A12 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 8A12 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 8A12 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 the 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 8A12 can effectively inhibit the up-regulation of IL-1β, TNF-α and IL-6 expression caused by the Sema7A recombinant protein, and has no significant effect on the expression of IL-10; the anti-sema7A monoclonal antibody 8A12 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 lupus mice.

[0014] Since the monoclonal antibody 8A12 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 lupus mice, the present application claims to protect a nucleic acid encoding the above sema7A monoclonal antibody 8A12.

[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 8A12.

[0017] The present application claims a pharmaceutical composition for preventing and / or treating systemic lupus erythematosus and / or its lupus nephritis, comprising the above-mentioned sema7A monoclonal antibody 8A12 and a medically acceptable carrier or adjuvant.

[0018] The present application claims the use of the above-mentioned sema7A monoclonal antibody 8A12 in the preparation of a medicine for preventing and / or treating systemic lupus erythematosus and / or its lupus nephritis.

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

[0020] (1) The present application finds that the sema7A monoclonal antibody 8A12 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 8A12 can efficiently inhibit Sema7A-induced macrophage inflammatory response, and continuously and effectively reduce serum anti-double-stranded DNA antibody and kidney damage in lupus mice. The sema7A monoclonal antibody 3F12 has no significant effect on the up-regulation of IL-1β, TNF-α, IL-6, IL-10 expression caused by Sema7A recombinant protein, and has no significant effect on lupus mice.

[0021] (2) The sema7A monoclonal antibody 8A12 of the present application can be used to prepare a pharmaceutical composition for preventing and / or treating systemic lupus erythematosus and / or its lupus nephritis, comprising the above-mentioned sema7A monoclonal antibody 8A12 and a medically acceptable carrier or adjuvant. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the SDS-PAGE diagram of the sema7A antibodies 3F12, 8A12 of the present application.

[0023] Figure 2 is the ELISA detection of the sema7A antibodies 3F12, 8A12 monoclonal antibodies of the present application.

[0024] Figure 3 Binding activity determination of the sema7A antibodies 3F12, 8A12 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 5 are self-DNA-induced lupus nephritis mouse kidney local Sema7A expression and physiological indicators;

[0027] Figure 6 The sema7A antibody 8A12 of the present application can effectively alleviate the condition of lupus nephritis mice. DETAILED DESCRIPTION

[0028] In order to make the person skilled in the art better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the 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 the person skilled in the art without creative labor should belong to the protection scope of the present application. Embodiment 1 Obtaining of anti-sema7A antibody 8A12

[0029] 1. Preparation of antigen, the specific steps are as follows:

[0030] The common amino acid sequence 185-321 (Q9QUR8.1) is selected from the human (CAJ55404.1) and mouse Sema7A amino acid sequences (Q9QUR8.1) found from NCBI, wherein the common amino acid sequence 185-321 is shown as SEQ ID No. 9, and the nucleotide sequence encoding the common amino acid sequence 185-321 is SEQ ID No. 10. The sequence optimization, DNA synthesis and construction of the prokaryotic expression plasmid pET30a-Sema7A are performed by Kecheng Biotechnology Co., Ltd.

[0031] The pET30a-Sema7A is transformed into BL21 competent cells, screened with solid LB medium containing ampicillin, and single colonies are picked and extracted for plasmid, double enzyme digestion and sequencing to verify that the sequence of the target gene is correct. The obtained strain is stored in a culture solution containing 25% glycerol at-80℃.

[0032] The above strain is cultured at 37℃ to about 0.6-0.8 OD600, 0.1mM IPTG expression inducer is added, and induction is performed overnight at 16℃. The bacterial cells are broken by ultrasonic, the supernatant and the precipitate are separated, and SDS-PAGE detection is performed. It is found that the recombinant protein Sema7A mainly exists in the bacterial cell precipitate. After the precipitate is dissolved with 8M urea, it is mixed with Ni Sepharose 6FF Beads (GE Healthcare), combined overnight at 4℃, centrifuged to discard the supernatant, and the beads are washed 2-3 times; the sema7A protein solution is obtained by elution with imidazole gradient eluent, which is used as the antigen for subsequent preparation of monoclonal antibody.

[0033] 2. Preparation and purification of monoclonal antibody

[0034] (1) Immunizing animals

[0035] Six 6-8 week old SFP grade female Balb / c mice were immunized with the purified Sema7A protein as antigen. 60 μg of Sema7A protein was mixed with 300 μg of MnJ(β) adjuvant in equal volume and vortexed to mix. The mice were then subcutaneously immunized with multiple points. The mice were subcutaneously immunized with multiple points for a total of 3 times, with 2 weeks interval between each time. Seven days after the last immunization, the serum antibody titer was determined, and the mouse with the highest titer was selected for intraperitoneal challenge, i.e. intraperitoneal injection of Sema7A protein antigen (80 μg per mouse) mixed with MnJ(β) adjuvant. Three days later, the spleen of the mouse was taken for cell fusion.

[0036] (2) Preparation of mouse intraperitoneal cells

[0037] Before cell fusion for 24 h, the feeder cells were prepared. The mouse was sacrificed by dislocation, and was sterilized by 75% alcohol immersion. The abdomen was cut open, and the abdominal wall was lifted with forceps for intraperitoneal injection of 7 ml of HAT 1640 complete culture medium. The abdomen was massaged, and about 6 ml of liquid was withdrawn. The liquid was diluted with 100 ml of HAT 1640 culture medium to mix, and the feeder layer cells were plated in a 96-well plate at 100 μl per well.

[0038] (3) PEG cell fusion

[0039] The mouse was sacrificed by dislocation, and the spleen was taken to prepare a single cell suspension. After red blood cell lysis, the cells were centrifuged and resuspended. 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. The cells were centrifuged at 1000 rpm for 7 min, and the supernatant was discarded. The cells were resuspended in HAT complete culture medium containing 20% fetal bovine serum, and were placed in a 37°C, 5% CO2 cell incubator for 1 h. Then the cells were plated at 4 x 10 4 / well in a pre-prepared feeder cell culture plate, and were cultured at 5% CO2, 37°C for two weeks. Whether there were positive clones was observed.

[0040] (4) Screening of positive hybridoma cells and establishment of cell lines

[0041] When the single-hole hybridoma cells grow to about 10,000 cells or more, the supernatant sema7A specific antibody production is detected by ELISA method, and positive hybridoma cells are screened: the enzyme-labeled plate is coated with Sema7A recombinant protein, and after blocking, the cell culture supernatant (100 μl / well) is added, and incubated at 37°C for 1.5 h. After PBST washing, the secondary antibody is added, and incubated at 37°C for 1 h. After PBST washing, the color developing solution is added for color development for 10 min and terminated. The OD value at 450 nm wavelength is detected by an enzyme-labeled instrument. The clones with OD value greater than 2.1 times of the negative value are selected for the next round of testing.

[0042] The wells with high positive values (OD450>2.0) in the fusion plate are selected for limited dilution, and the number of single clone wells is counted as 60% of each plate for subcloning. The single clone wells with higher positive values are selected for limited dilution each time, and ELISA detection can be performed after 5-7 days of subcloning each time, until the single clone cell strain that can stably secrete positive antibodies is finally screened out for expansion culture.

[0043] The cell strain that stably secretes positive antibodies screened in the subcloning stage is expanded and cultured, and the supernatant is collected. The antibody production stability and specificity are verified by ELISA gradient dilution and western blot. The cell is expanded and stored, that is, the hybridoma cell strain.

[0044] (5) Large-scale preparation and purification of monoclonal antibodies

[0045] The cell strains 3F12 and 8A12 obtained above are expanded and cultured with hybridoma cell special SFM, and the cell culture supernatant is collected. The antibody is purified by thiophilic affinity chromatography. First, ammonium sulfate is added to the cell supernatant to 0.8 M, then a thiophilic affinity chromatography column is prepared, the column is balanced with buffer 1 (20 mM sodium phosphate, 0.8 M ammonium sulfate), and then the cell culture supernatant filtered by 0.45 μm filter membrane is passed through the column, then 15 CV of buffer 1 is used for washing, and then 20 mM sodium phosphate solution is used for elution. After dialysis, it is ready for use.

[0046] Example 2 Identification of anti-sema7A monoclonal antibody

[0047] 1. Experimental method

[0048] (1) Molecular weight and purity determination of monoclonal antibodies 3F12 and 8A12

[0049] The Sema7A monoclonal antibodies obtained in Example 1 are subjected to SDS-PAGE electrophoresis, and the molecular weight and purity of the separated and purified 3F12 and 8A12 are determined.

[0050] (2) Type and light chain type of monoclonal antibodies 3F12 and 8A12

[0051] The antibody subtype of the Sema7A monoclonal antibody was identified by using IsoStripTM mouse monoclonal antibody subtype identification kit.

[0052] (3) Anti-sema7A monoclonal antibody 3F12, 8A12 binding activity detection

[0053] The recombinant Sema7A protein was coated on a plate, and the purified antibodies 3F12 and 8A12 were added in a dilution of 300 μg / mL, and incubated at 37°C for 1 h; then 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.

[0054] 2. Experimental results

[0055] (1) The molecular weight and purity determination results of the monoclonal antibodies 3F12 and 8A12 are shown in Table 1, and the SDS-PAGE patterns of the purified anti-sema7A monoclonal antibodies 3F12 and 8A12 are shown in Table 2, with molecular weights of about 70 kDa and 25 kDa, respectively, which are consistent with the molecular weights of antibody heavy and light chains, and the purity is more than 90%. Figure 1 Figure 1 (2) The heavy chain type and light chain type determination results of the monoclonal antibodies 3F12 and 8A12 are shown in Table 3, and the anti-Sema7A monoclonal antibodies 3F12 and 8A12 in the present application are IgM subtype, and the light chains are κ chains.

[0056] (3) The anti-sema7A monoclonal antibody 8A12 binding activity detection results are shown in Table 4, and the EC50 of the anti-Sema7A monoclonal antibody 8A12 in the present application is 19.18 μg / ml. The EC50 of the anti-Sema7A monoclonal antibody 3F12 is 18.79 μg / ml. Figure 2

[0057] The sequences of the heavy chain and light chain of 8A12 are shown in Table 1:

[0058]

[0059] Note: The heavy chain variable region CDR1 represents CDR-H1; CDR2 represents CDR-H2; CDR3 represents CDR-H3; the light variable region CDR1 represents CDR-L1; the light variable region CDR2 represents CDR-L2; and the light variable region CDR3 represents CDR-L3; V(D)J-IMGT represents the amino acid sequence of the heavy chain or light chain.

[0060] (3) The anti-sema7A monoclonal antibody 8A12 binding activity detection results are shown in Table 4, and the EC50 of the anti-Sema7A monoclonal antibody 8A12 in the present application is 19.18 μg / ml. The EC50 of the anti-Sema7A monoclonal antibody 3F12 is 18.79 μg / ml. Figure 3

[0061] ​​​Example 3 Anti-sema7A monoclonal antibody 8A12 on macrophage inflammatory response

[0062] 1. Experimental method

[0063] T cells can interact with macrophage surface receptors through surface Sema7A, and then regulate macrophage immune response. In the present application, we evaluated the effect of anti-sema7A monoclonal antibodies 3F12 and 8A12 on the inflammatory response of macrophages induced by Sema 7A protein. The specific experimental method is as follows:

[0064] The experimental groups were incubated with macrophages for 6h with PBS (NC), 5 μg / ml Sema7A recombinant protein (Pr), 5 μg / ml 8A12 monoclonal antibody, and a mixture of 5 μg / ml Sema7A recombinant protein and 8A12 monoclonal antibody; 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. Cell RNA was extracted, reverse transcribed according to the instructions of the whole kit, and the effect of 3F12 and 8A12 antibodies on the ability of sema7A to induce IL-1β, TNF-α, IL-6, and IL-10 production in RAW264.7 cells was detected by Real-time PCR.

[0065] 2. Experimental results

[0066] The experimental results are shown in Figure 4 The experimental results show that Sema7A recombinant protein can significantly up-regulate the mRNA levels of IL-1β Figure 4 A), TNF-α Figure 4 B), IL-6 Figure 4 C), and IL-10 Figure 4 D) in RAW264.7 cells. The 8A12 monoclonal antibody can effectively inhibit the up-regulation of IL-1β, TNF-α, and IL-6 expression caused by Sema7A recombinant protein, but has no significant effect on IL-10 expression. The 3F12 monoclonal antibody has no significant effect on the up-regulation of IL-1β, TNF-α, IL-6, and IL-10 expression caused by Sema7A recombinant protein.

[0067] Example 4 Anti-sema7A monoclonal antibody in vivo treatment of lupus nephritis

[0068] 1. Experimental method

[0069] Lupus nephritis-like mouse model establishment and physiological index determination: extract BALB / c mouse activated lymphocyte-derived self-DNA (Self-DNA) and normal-DNA mixed with Freund's adjuvant, subcutaneously immunize 6-8 week old female mice of the same strain every other week (100 μg DNA per mouse per time, 6 mice per group), a total of three times, which can effectively establish a lupus nephritis-like mouse model. The established mouse model is determined by the following physiological indexes, and the determination method is as follows:

[0070] During the modeling process, serum and urine samples were collected every other week for detection of anti-dsDNA autoantibodies in serum and urine protein content. After 12 weeks of first immunization, the mouse kidney paraffin section was detected by H&E for kidney pathological condition; after 12 weeks of first immunization, the mouse kidney frozen section was detected by immunofluorescence for IgG class immune complex deposition.

[0071] Anti-dsDNA autoantibody detection: enzyme-labeled plate was treated with 0.5% protamine sulfate, 100 μl / well, 37°C incubation for 1 h; washed with PBST, 3 times, and dried; bovine thymus DNA was used as coating antigen, 50 μg / mL, 100 μL / well, 37°C incubation for 2 h, and 4°C overnight; washed with PBST for 3 times, and dried; add 1×Diluent blocking, 100 μL / well, 37°C incubation for 1 h; washed with PBST for 3 times, and dried; dilute the serum to be tested with 1×Diluent according to 1:100, 100 μL / well, 37°C incubation for 1 h; washed with PBST for 3 times, and dried; add goat anti-mouse IgG-HRP (1:1000), 100 μL / well, 37°C incubation for 1 h; washed with PBST for 4 times, and dried; add TMB color developing liquid, 100 μL / well, 37°C incubation for 15 min; add ELISA stop solution, 50 μL / well, and measure OD 450 .

[0072] Urine protein detection: according to the operation steps of urine protein quantitative test kit (CBB method), mix 2 μL ddH2O, 2 μL sample, 2 μL protein standard (563 mg / L) and CBB application liquid thoroughly, stand for 5 min, and detect OD value at 595 nm wavelength by enzyme-labeled instrument.

[0073] Kidney paraffin sections and H&E staining: Mouse kidneys were fixed with 4% paraformaldehyde for 48 h; dehydrated using an automated dehydrator, embedded in paraffin, sectioned, and dried at 60 °C for 1 h; sections were soaked in xylene for 7 min, repeated 3 times; then soaked in anhydrous ethanol, 95% ethanol, and 80% ethanol for 2 min, and rinsed briefly with water; stained with hematoxylin for 5 min, rinsed briefly with water, rinsed briefly with 1% hydrochloric acid-ethanol, and rinsed again with water; bluing was performed in a 55 °C water bath for 5 min, rinsed briefly with water, stained with eosin for 10 s, and rinsed again with water; then rinsed briefly with 80% ethanol, 95% ethanol, and anhydrous ethanol; after drying, the sections were mounted with neutral resin; observed and photographed under an upright fluorescence microscope.

[0074] Kidney cryosection and immunofluorescence: Mice were anesthetized; cardiac perfusion was performed on mice with PBS and paraformaldehyde; kidneys were soaked in paraformaldehyde for 6-12 h; dehydration was performed with 30% sucrose; mouse kidneys were embedded in OTC, flash-frozen at -20℃, and cut into 5 μm thin sections; sections were fixed with acetone at room temperature for 10 min; washed with PBS, 5 min × 3 times; anti-Mouse IgG-Alexa Fluor 488 (1:100 dilution) was added, and incubated overnight at 4℃; washed with PBS, 5 min × 3 times; and examined under a microscope after mounting.

[0075] (2) Therapeutic effect of anti-sema7A monoclonal antibody 8A12

[0076] Six to eight-week-old female BALB / c mice were randomly divided into groups of six. They were injected subcutaneously at multiple points on the back every two weeks for a total of three times. Within one week of the last immunization, 50 μg of sema7A monoclonal antibody 8A12 was administered via the tail vein, and SLE-related physiological indicators were continuously monitored.

[0077] 2. Experimental Results

[0078] (1) The results of the index measurement of the model mice are as follows: Figure 5 As shown, by Figure 5 It was found that Sema7A expression was significantly elevated in the mouse lupus nephritis model. Specific analysis is as follows: High titers of anti-double-stranded DNA antibodies persisted in the serum of the model mice. Figure 5 A), high levels of proteinuria ( Figure 5 B), in the model mice, the glomeruli were enlarged, structurally disordered, and showed extensive infiltration of inflammatory cells. Figure 5 C), a large amount of immune complex deposition was observed in the kidneys. Figure 5 D). Meanwhile, real-time PCR was used to detect the expression of sema7A protein in various organs of lupus mice, and it was found that the expression of this gene was most significantly upregulated in the kidneys, by more than 10-fold.

[0079] (2) The therapeutic effect of anti-sema7A monoclonal antibody 8A12, such as Figure 6As shown, the anti-sema7A monoclonal antibody 8A12 consistently and effectively reduced serum anti-double-stranded DNA antibodies and kidney damage in lupus mice. Inflammatory macrophages are key pathogenic cells driving the development of lupus nephritis. Combined with our in vitro experimental results, 8A12 antibody can inhibit Sema7A-induced macrophage inflammatory response. Therefore, during the lupus mouse modeling process, we administered 8A12 monoclonal antibody (50 μg / mouse) intravenously one week after the last DNA immunization, and continuously monitored serum anti-double-stranded DNA antibody levels and kidney damage. Specific analysis is as follows: Compared with the control group, 8A12 monoclonal antibody effectively reduced the production of anti-dsDNA antibodies (…). Figure 6 A) Reduce the production of protein in urine ( Figure 6 B) significantly reduces glomerular inflammatory proliferation and inflammatory infiltration. Figure 6 C) Simultaneously, IgG deposition in the glomerular basement membrane was significantly reduced, indicating that the anti-Sema7A monoclonal antibody 8A12 could significantly alleviate lupus nephritis in mice. However, 3F12 and IgM isotype monoclonal antibodies had no effect on the above physiological indicators.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the solutions. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention based on the understanding of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An anti-sema7A monoclonal antibody 8A12, characterized in that, The amino acid sequence of CDR-H1 in the heavy chain variable region of the monoclonal antibody 8A12 is shown in SEQ ID No. 1, the amino acid sequence of CDR-H2 in the heavy chain variable region is shown in SEQ ID No. 2, and the amino acid sequence of CDR-H3 in the heavy chain variable region is shown in SEQ ID No. 3; the amino acid sequence of CDR-L1 in the light chain variable region of the monoclonal antibody 8A12 is shown in SEQ ID No. 4, the amino acid sequence of CDR-L2 in the light chain variable region is shown in SEQ ID No. 5, and the amino acid sequence of CDR-L3 in the light chain variable region is shown in SEQ ID No.

6.

2. The anti-sema7A monoclonal antibody 8A12 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

7.

3. The anti-sema7A monoclonal antibody 8A12 according to claim 1, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID No.

8.

4. The anti-sema7A monoclonal antibody 8A12 according to claim 1, characterized in that, The monoclonal antibody 8A12 is an IgM subtype, and all light chains are κ chains.

5. A nucleic acid encoding the anti-sema7A monoclonal antibody 8A12 as described in claim 1.

6. An expression carrier, characterized in that, It comprises the nucleic acid of claim 5 and is capable of expressing the nucleic acid in a eukaryotic host cell.

7. A host cell, characterized in that, It contains and can express the vector of claim 6, and produces the anti-sema7A monoclonal antibody 8A12 of any one of claims 1-4.

8. A pharmaceutical composition for treating lupus nephritis, characterized in that, It includes the anti-sema7A monoclonal antibody 8A12 as described in any one of claims 1-4 and a medically acceptable carrier or excipient.

9. The use of the anti-sema7A monoclonal antibody 8A12 according to any one of claims 1-4 in the preparation of a drug for treating lupus nephritis.

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