Anti-atherosclerosis IgG monoclonal antibody and application thereof

Abstract

The invention provides an anti-atherosclerosis IgG monoclonal antibody, belongs to the technical field of biology, and totally comprises four IgG monoclonal antibodies. The IgG monoclonal antibody can be used for directly inhibiting macrophage phagocytosis of o-LDL (oxidized low-density lipoprotein) and formation of foam cells, so that formation of atherosclerotic plaques is blocked; through phagocytosis mediated by the IgG monoclonal antibody, immune cells can be promoted to phagocytize foam cells, and then fading of atherosclerotic plaques is promoted. Based on the two mechanisms, the IgG monoclonal antibody has the prospect of being developed into a novel medicine for resisting atherosclerosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an anti-atherosclerosis IgG monoclonal antibody and application thereof. BACKGROUND

[0002] Cardiovascular disease is the highest mortality disease in the world, among which atherosclerosis is the root cause of many cardiovascular diseases, including coronary artery disease, myocardial infarction, stroke and peripheral arterial disease. In essence, atherosclerosis is a chronic inflammatory disease, which is triggered by the deposition of foam cells formed by macrophages phagocytosis of ox-LDL in the intima of the artery. A large number of studies have shown that atherosclerosis autoantigens can trigger adaptive immune responses, leading to the production of autoantibodies. Autoantibodies binding to epitopes can weaken atherosclerotic plaque formation, but can also accelerate the development of atherosclerosis, depending on the characteristics of autoantigens and the subtype of antibodies produced. Due to the lack of research on the separation and identification of autoantibodies from atherosclerosis patients, its exact function and role are still unclear.

[0003] The existing atherosclerosis therapy has limitations, and the efficacy of lipid-lowering drugs varies greatly and has obvious side effects, the mechanism of anti-inflammatory drugs is unclear, anti-thrombotic therapy is prone to bleeding and drug resistance, and the restenosis rate after stent surgery is high. The existing scheme does not target the core mechanism of foam macrophages and other mechanisms, and it is difficult to reverse the plaque. Although the existing drugs can reduce 30-40% of cardiovascular events, drug resistance and side effects limit the efficacy, and emerging therapies such as targeting inflammatory pathways (IL-1β inhibitors) and foam cell-specific monoclonal antibodies are becoming a research hotspot.

[0004] Antibody-based therapeutic drugs are a class of biological agents based on antibodies, which play a therapeutic role by targeting specific antigens, and have shown significant advantages in the treatment of various diseases (such as tumors, autoimmune diseases, infectious diseases, etc.). Antibody drugs lead the innovation of disease treatment with the characteristics of precision, efficiency and safety, and with the deep integration of protein engineering and precision medicine, their application prospect will be broader. In summary, traditional treatment methods have been unable to meet the needs of reality, and it is urgent to develop monoclonal antibodies for atherosclerosis for the preparation of new anti-atherosclerosis drugs. SUMMARY

[0005] In order to solve the problem of lack of atherosclerosis monoclonal antibody, the present application aims to provide an anti-atherosclerosis IgG monoclonal antibody and its application. The IgG monoclonal antibody can directly inhibit the phagocytosis of ox-LDL by macrophages and the formation of foam cells, thereby blocking the formation of atherosclerotic plaques; the phagocytosis mediated by the IgG monoclonal antibody can promote the phagocytosis of foam cells by immune cells, thereby promoting the regression of atherosclerotic plaques. Based on the above two mechanisms, the four IgG monoclonal antibodies have the prospect of developing into new anti-atherosclerosis drugs.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: an anti-atherosclerosis IgG monoclonal antibody, comprising four anti-atherosclerosis IgG monoclonal antibodies, respectively named 2E7, 2G3, 3G12 and 3H2, and the four anti-atherosclerosis IgG monoclonal antibodies are composed of heavy chains and light chains.

[0007] Further, the amino acid sequences of the heavy chain variable regions thereof are SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, respectively.

[0008] Further, the amino acid sequences of the light chain variable regions thereof are SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8, respectively.

[0009] Further, the nucleotide sequences of the heavy chain variable regions thereof are SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 and SEQ ID No. 15, respectively.

[0010] Further, the nucleotide sequences of the light chain variable regions thereof are SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 and SEQ ID No. 16, respectively.

[0011] Further, the anti-atherosclerosis IgG monoclonal antibody has at least one of the following characteristics:

[0012] The heavy chain type is IgG;

[0013] The light chain type is kappa;

[0014] The monoclonal antibody is a murine antibody.

[0015] Further, it is used for researching the clinical treatment of atherosclerosis disease.

[0016] Further, it is used for improving the therapeutic effect of other drugs in treating atherosclerosis disease.

[0017] Further, as the application in the field of preventing the formation of atherosclerosis and related diseases caused thereby.

[0018] The beneficial effects of the present application are:

[0019] 1. The anti-atherosclerosis IgG monoclonal antibody and its application can directly inhibit the phagocytosis of ox-LDL by macrophages and the formation of foam cells, thereby blocking the formation of atherosclerotic plaques. In addition, the IgG monoclonal antibody can promote the phagocytosis of foam cells by immune cells, thereby promoting the regression of atherosclerotic plaques, thereby reducing the symptoms of atherosclerosis. Therefore, based on the above two mechanisms, the four IgG monoclonal antibodies have the potential to be developed into new drugs for treating atherosclerosis.

[0020] 2. The anti-atherosclerosis IgG monoclonal antibody and its application successfully obtained the amino acid sequence of the heavy chain and light chain variable region of the monoclonal antibody and the nucleotide sequence encoding the heavy chain and light chain variable region of the monoclonal antibody. Homology analysis results show that the sequence in the present application has uniqueness. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings based on these drawings without creative labor.

[0022] Figure 1 The SDS-PAGE antibody purification electrophoresis chart provided for the embodiments of the present application;

[0023] Figure 2 The result chart of Western blot technology for identifying the type of monoclonal antibody provided for the embodiments of the present application;

[0024] Figure 3 The cell immunofluorescence staining result chart of the ox-LDL inhibition effect provided for the embodiments of the present application;

[0025] Figure 4 The half maximal inhibitory concentration (IC50) value result chart of the ox-LDL inhibition effect provided for the embodiments of the present application;

[0026] Figure 5 The IgG monoclonal antibody heavy chain agarose gel electrophoresis verification result chart provided for the embodiments of the present application;

[0027] Figure 6This is a graph showing the verification results of IgG monoclonal antibody light chain agarose gel electrophoresis provided in an embodiment of the present invention;

[0028] Figure 7 This is a homology comparison diagram of the heavy and light chain nucleotide sequences of the IgG monoclonal antibody provided in an embodiment of the present invention. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0030] Throughout this specification, unless otherwise specified, the terminology used herein should be understood to have the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0032] like Figures 1-7 As shown, the present invention has the following specific embodiments.

[0033] Example 1

[0034] This embodiment prepares a monoclonal antibody against atherosclerosis, including the following steps:

[0035] 1. Preparation of foam cells containing atherosclerotic antigens

[0036] At a dosage of 1 mL / mouse, 0.029 g / mL of thioglycolate solution was injected intraperitoneally into 8-week-old Balb / c mice. 48 h later, the mice were sacrificed and dissected. 5 mL of RPMI 1640 medium was injected into the peritoneum and the cells were allowed to stand for 5-10 min to extract peritoneal macrophages. The cell-containing medium was aspirated, and the cells were transferred to cell culture dishes and incubated in a CO2 incubator for 1 h until cell adhesion occurred. The medium was discarded, and the cells were incubated for another 6 h in 5 mL of RPMI 1640 medium containing 10% fetal bovine serum and 1% P / S. Lipopolysaccharide (LPS) was added to the culture dishes at a concentration of 100 ng / mL, and ox-LDL at a concentration of 50 μg / mL. The peritoneal macrophages were incubated in a CO2 incubator for another 24 h, and the medium was discarded to obtain foam cells.

[0037] 2. Preparation of anti-atherosclerotic monoclonal antibodies

[0038] Freund's incomplete adjuvant was added to a culture dish at a dosage of 100 μL per mouse. Foam cells were then scraped off using a cell scraper to prepare the antigen. Six- to eight-week-old SPF-grade Balb / c mice were used for immunization. Each mouse received 100 μL of the antigen, with 75 μL administered intraperitoneally and 25 μL subcutaneously in the back. Immunization was repeated every month.

[0039] Boost immunization of mice: 100 μL of antigen per mouse; three days after completion, mice were sacrificed, and mouse spleen lymphocyte suspensions were prepared and counted.

[0040] Logarithmic growth phase mouse myeloma cells (SP2 / 0) were counted, and myeloma cells were fused with splenic lymphocytes at a ratio of 1:5. The fused cell suspension was added to 96-well plates and cultured at 37°C with 5% CO2. After cell clones appeared, the cell supernatant was collected, and antibody levels were detected by Oil Red staining. Positive clones were selected. Cells containing positive clones were cloned using limiting dilution, resulting in the establishment of four stable hybridoma cell lines secreting anti-atherosclerotic monoclonal antibodies. The four anti-atherosclerotic monoclonal antibodies were named 2E7, 2G3, 3G12, and 3H2, respectively.

[0041] The amino acid sequences of the heavy chain variable regions of the four anti-atherosclerotic monoclonal antibodies 2E7, 2G3, 3G12 and 3H2 are SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, respectively, and the amino acid sequences of the light chain variable regions are SEQ ID No.2, SEQ ID No.4, SEQ ID No.6 and SEQ ID No.8, respectively.

[0042] Of the four anti-atherosclerotic monoclonal antibodies, the nucleotide sequences encoding the heavy chain variable region are SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15, and the nucleotide sequences encoding the light chain variable region are SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No. 16.

[0043] 3. Ascites expansion and antibody purification

[0044] Using an in vivo induction method, sterile liquid paraffin was injected intraperitoneally into 8-week-old Balb / c mice at a dosage of 300 μL / mouse. One week later, hybridoma cells were injected intraperitoneally. 6 Two weeks later, ascites fluid was collected from each animal, and the antibody was purified using a Protein L affinity chromatography column or a Protein G affinity chromatography column to obtain an anti-atherosclerotic monoclonal antibody.

[0045] Example 2

[0046] This embodiment analyzes and detects four types of anti-atherosclerotic IgG monoclonal antibodies.

[0047] 1. Western blot identification

[0048] Western blot was used to identify the types of anti-atherosclerotic monoclonal antibodies. The purified antibodies were subjected to SDS-PAGE electrophoresis, and their molecular weight was estimated. The results are as follows: Figure 1 As shown in the figure. The sample was then transferred to an Immobilon-PVDF membrane and blocked. HRP-labeled goat anti-mouse IgG (10 μg / mL) was used as a secondary antibody for detection. Finally, the membrane was placed in DAB solution for color development, and four antibodies were identified as IgG types. The results are shown in the figure. Figure 2 As shown.

[0049] 2. Indirect ELISA method detection

[0050] The binding affinity of anti-atherosclerotic IgG monoclonal antibodies to antigens was detected using an indirect ELISA method. Foam cells were collected and sonicated, and the protein concentration in the cell lysate was determined using the Bradford method. Samples were prepared by coating ox-LDL (10 μg / mL), cell lysate (20 μg / mL), and IgG monoclonal antibody diluted to 20 μg / mL. The detection antibody was HRP-labeled anti-human IgG diluted 1:10000. Finally, the samples were developed using TMB substrate, and absorbance (OD) values ​​were read at 450 nm. Results showed that the 3H2 monoclonal antibody exhibited moderate binding affinity to both foam cells and macrophages. The binding affinity of the 2E7, 2G3, and 3G12 monoclonal antibodies to ox-LDL antigen, foam cells, or normal macrophages showed no significant difference.

[0051] Example 3

[0052] This embodiment examines the effect of anti-atherosclerotic IgG monoclonal antibody on inhibiting macrophage phagocytosis of ox-LDL.

[0053] Mouse peritoneal macrophages were used at 10 5Cells were seeded per well in 96-well plates and stimulated with lipopolysaccharide (LPS) at a final concentration of 200 ng / mL for 2 hours. Different concentrations (0.02–0.5 mg / mL) of IgG monoclonal antibody were mixed with 30 μg / mL Dio-ox-LDL and added to the 96-well plates for lipid phagocytosis for 4 hours. Then, 100 μL of 0.5 μg / mL DAPI working solution was added, and staining was performed for 5 minutes. The inhibitory effect of IgG monoclonal antibody on ox-LDL phagocytosis was observed using fluorescence microscopy. Randomly selected fields of view were used for observation. The results are shown below. Figure 3 As shown.

[0054] Internalized Dio-ox-LDL was visualized using Dio fluorescence, and then quantitative analysis was performed using Image Pro Plus software to calculate the half-maximal inhibitory concentration (IC50). Results are as follows: Figure 4 As shown, all four IgG monoclonal antibodies significantly inhibited the phagocytosis of ox-LDL by macrophages in a dose-dependent manner.

[0055] Example 4

[0056] This embodiment provides the cloning process for the light and heavy chains of anti-atherosclerotic IgG monoclonal antibodies, including:

[0057] 1. Total RNA was extracted from hybridoma cells using a total RNA extraction kit (purchased from Novizan).

[0058] 2. Synthesis of the first strand of cDNA: cDNA was synthesized using a reverse transcription kit (purchased from Novizan).

[0059] 3. Gene amplification:

[0060] Primers: Mouse antibody variable region specific primers were provided by Anhui General Biotechnology Co., Ltd.

[0061] First-strand PCR was performed using the first strand of cDNA as a template, while second-strand PCR was performed using the first-strand product as a template. Both reaction volumes were 25 μL.

[0062] PCR reaction mixture: Template 2 μL, dNTPs 0.5 μL, 5×SF buffer 5 μL, Primer Forward (10 μM) 1 μL, Primer Reverse (10 μM) 1 μL, Phanta Super-Fidelity DNA Polymerase 0.5 μL

[0063] PCR reaction conditions: 97℃ pre-denaturation for 3 min; followed by 35 cycles (97℃ denaturation for 10 s, 59℃ annealing for 30 s, 72℃ extension for 30 s); final extension at 72℃ for 10 min, and storage at 4℃ after completion.

[0064] 4. Cloning and screening of PCR amplification products

[0065] The PCR products were subjected to 1% agarose gel electrophoresis, and the results are as follows: Figure 5 , Figure 6 As shown, antibody DNA was recovered using a DNA gel recovery kit (Novizan), and the fragment was inserted into the pBM23 vector using the pBM23 Toposmart kit (purchased from Biomed Biotechnology). The vector was then transformed into DH5α competent cells (ampicillin-resistant), and recombinant positive clones were screened and sequenced.

[0066] Example 5

[0067] This embodiment performs a homology analysis.

[0068] After sequencer sequencing of the variable regions, the sequences of each antibody variable region were submitted to the IgBLAST and IMGT / BlastSearch databases (using default parameters and selecting mice as the species for querying sequences) using the NCBI (GenBank+EMBL+DDBJ+PDB) and IMGT databases to determine their percentage of homology with light and heavy chain reference sequences. The results are as follows: Figure 7 As shown, the homology range of the IgG variable region is 91.32% to 98.57%.

[0069] Finally, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0070] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0071] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An anti-atherosclerotic IgG monoclonal antibody, characterized in that, It contains four anti-atherosclerotic IgG monoclonal antibodies, named 2E7, 2G3, 3G12 and 3H2, respectively. The four anti-atherosclerotic IgG monoclonal antibodies are composed of heavy chains and light chains.

2. The anti-atherosclerotic IgG monoclonal antibody according to claim 1, characterized in that: The amino acid sequences of the variable region of the heavy chain are SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7, respectively.

3. The anti-atherosclerotic IgG monoclonal antibody according to claim 1, characterized in that: The amino acid sequences of the variable region of the light chain are SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, respectively.

4. The anti-atherosclerotic IgG monoclonal antibody according to claim 1, characterized in that: The nucleotide sequences of the variable region of the heavy chain are as shown in SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15, respectively.

5. The anti-atherosclerotic IgG monoclonal antibody according to claim 1, characterized in that: The nucleotide sequences of the variable regions of the light chain are as follows: SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No.

16.

6. The anti-atherosclerotic IgG monoclonal antibody according to claim 1, characterized in that: The anti-atherosclerotic IgG monoclonal antibody has at least one of the following characteristics: The heavy chain type is IgG; The light chain type is κ; The monoclonal antibody is a murine antibody.

7. The application of the anti-atherosclerotic IgG monoclonal antibody according to claims 1-6, characterized in that: As a research tool for clinical treatment of atherosclerotic diseases.

8. The application of the anti-atherosclerotic IgG monoclonal antibody according to claims 1-6, characterized in that: It is used in conjunction with other drugs to improve the treatment effect of atherosclerotic diseases.

9. The application of the anti-atherosclerotic IgG monoclonal antibody according to claims 1-6, characterized in that: Its application in the prevention of atherosclerosis and related diseases.

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