IgM monoclonal antibody for resisting ox-LDL phagocytosis of macrophages and application of IgM monoclonal antibody

Abstract

The invention belongs to the technical field of biology, and provides an IgM monoclonal antibody for resisting ox-LDL phagocytosis of macrophages, and the IgM monoclonal antibody is respectively named as 1B4, 2B4, 2H8 and 4D3. The four IgM monoclonal antibodies have a good inhibition effect on foam cells formed by phagocytosis of ox-LDL by macrophages, and the IgM antibodies injected through tail vein can significantly inhibit formation of atherosclerotic plaques in a mouse body, and can be used for prevention and treatment of atherosclerosis and atherosclerotic cardiovascular and cerebrovascular diseases caused by atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL and their applications. Background Technology

[0002] Atherosclerosis (AS) is a chronic inflammatory vascular disease caused by lipid deposition, and is a major inducing factor for atherosclerotic cardiovascular disease (ASCVD) and stroke. Macrophages, due to excessive phagocytosis of lipids such as oxidized low-density lipoprotein (ox-LDL), are unable to metabolize or excrete them, leading to the formation and death of foam cells. These foam cells then deposit under the vascular endothelium, causing plaque core formation and ultimately resulting in atherosclerotic disease. Current drug treatments mainly focus on lipid-lowering, anti-inflammatory, and antithrombotic therapies, but they can only reduce cardiovascular events by 30-40%.

[0003] Therefore, conducting various drug studies to block cellular phagocytosis of ox-LDL and prevent foam cell formation is one of the keys to the prevention and treatment of atherosclerotic diseases. Summary of the Invention

[0004] The objective of this invention is to screen four IgM monoclonal antibodies that can effectively inhibit macrophage phagocytosis of ox-LDL and foam cell formation through processes such as foam cell vaccine preparation, mouse immunization with the vaccine, and hybridoma screening. The monoclonal antibodies described in this invention are unique, exhibit good inhibitory effects on foam cells formed by macrophage phagocytosis of ox-LDL, and demonstrate good anti-atherosclerotic plaque formation effects in mice, laying the foundation for the subsequent development of antibody drugs for the prevention or treatment of atherosclerosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: an IgM monoclonal antibody against macrophage phagocytosis of ox-LDL, comprising four IgM monoclonal antibodies, named 1B4, 2B4, 2H8, and 4D3, wherein the four IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL are composed of heavy chains and light chains.

[0006] Furthermore, the amino acid sequences of the heavy chain variable region are SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7.

[0007] Furthermore, 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.

[0008] Furthermore, the nucleotide sequences of the heavy chain variable region are SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 and SEQ ID No. 15.

[0009] Furthermore, the nucleotide sequences of the variable region of the light chain are SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 and SEQ ID No. 16.

[0010] Furthermore, the IgM monoclonal antibody against macrophage phagocytosis of ox-LDL has at least one of the following characteristics:

[0011] The heavy chain type is μ;

[0012] The light chain type is κ;

[0013] The monoclonal antibody is a murine antibody.

[0014] Furthermore, it can be used for research on the clinical treatment of atherosclerotic diseases.

[0015] Furthermore, it can be used in conjunction with other drugs to improve the treatment efficacy of atherosclerotic diseases.

[0016] Furthermore, it has applications in the prevention of atherosclerotic diseases and related diseases.

[0017] The beneficial effects of this invention are:

[0018] 1. This IgM monoclonal antibody against macrophage phagocytosis of ox-LDL and its application have a good inhibitory effect on foam cells formed by macrophage phagocytosis of ox-LDL. Furthermore, tail vein injection of the IgM antibody can significantly inhibit the formation of atherosclerotic plaques in mice. It can be used for the prevention and treatment of atherosclerosis and the atherosclerotic cardiovascular and cerebrovascular diseases it causes.

[0019] 2. The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL and its application: The amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody and the nucleotide sequences encoding the heavy and light chain variable regions of the monoclonal antibody were successfully obtained. Homology analysis results showed that the sequences in this invention are unique. Attached Figure Description

[0020] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This invention provides a schematic diagram of macrophage uptake of ox-LDL stained with Oil Red staining for an embodiment of the invention;

[0022] Figure 2 This invention provides schematic diagrams illustrating the inhibition of macrophage phagocytosis by four types of hybridoma cell supernatants using ox-LDL Oil Red staining.

[0023] Figure 3 A schematic diagram of antibody purification using Protein L affinity chromatography column is provided for embodiments of the present invention to illustrate the formation of ascites from hybridoma cells 1B4 and 4D3.

[0024] Figure 4 Western blot analysis of the heavy chains of four IgM antibodies using HRP-conjugated goat anti-mouse IgM antibodies is provided for embodiments of the present invention.

[0025] Figure 5 A schematic diagram of the agarose gel electrophoresis verification results of PCR amplification of the variable regions of the 1B4 and 2B4 heavy and light chains is provided for embodiments of the present invention.

[0026] Figure 6 This invention provides a schematic diagram of the agarose gel electrophoresis verification results of PCR amplification gel recovery for 1B4 and 2B4 heavy and light chains.

[0027] Figure 7 This invention provides a schematic diagram of fluorescence detection results for 1B4 and 4D3 monoclonal antibodies against macrophage phagocytosis of Dio-LDL;

[0028] Figure 8 The schematic diagram of the inhibitory titers of four IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL in this embodiment of the invention;

[0029] Figure 9 A percentage graph of the variable region of four IgM monoclonal antibody sequences relative to the Ig BLAST and IMGT reference sequences is provided for embodiments of the present invention. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] like Figure 1-9 As shown, the present invention has the following specific embodiments.

[0034] Example 1

[0035] 1. Vaccine preparation

[0036] Mice were euthanized by intraperitoneal injection of sodium pentobarbital. The peritoneum was then disinfected by immersion in 75% alcohol for 10 minutes. The abdominal skin was cut open in a biosafety cabinet to expose the peritoneum. 5 mL of 1640 culture medium was injected into the peritoneum. After massaging the abdomen for several minutes, the peritoneal fluid was aspirated into cell culture dishes and placed in a CO2 incubator for 1 hour to allow peritoneal macrophages to adhere. Contaminating cells were washed away with PBS, and the dishes were cultured in complete culture medium for 6-8 hours. Lipopolysaccharide (LPS) was then added to induce macrophage differentiation into the M1 type. Finally, ox-LDL was added, and the cells were incubated for 24 hours. Cells were stained with Oil Red O to assess their ox-LDL phagocytosis activity. Figure 1 As shown.

[0037] 2. Preparation of monoclonal antibodies against macrophage phagocytosis of ox-LDL

[0038] Foam cells formed by macrophages engulfing ox-LDL were sonicated and mixed with adjuvant to immunize 6-week-old BALB / c mice. For the first immunization, the antigen was emulsified and mixed with complete Freund's adjuvant and administered subcutaneously at multiple sites. After the first immunization, booster immunizations were given every two weeks, using the antigen emulsified and mixed with incomplete Freund's complete adjuvant for subcutaneous multi-site immunization, for two booster immunizations. One week after the third immunization, blood was collected from the tail vein to detect the immunization effect. For shock immunization, the antigen was thoroughly mixed with sterile PBS and injected intraperitoneally 3 days before cell fusion, and the spleen lymphocytes of mice were counted three days later for later use.

[0039] Logarithmic growth phase mouse myeloma cells (SP2 / 0) were counted, and myeloma cells were fused with spleen 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 for hybridoma cell selection experiments. The hybridoma cell supernatant was mixed with 50 μg / ml ox-LDL and then added to culture dishes for lipid phagocytosis for 24 hours. Finally, the cells were stained with Oil Red O to assess ox-LDL phagocytosis, and the inhibitory effect of the hybridoma cell culture supernatant on ox-LDL uptake was observed. Figure 2 As shown. Cells containing positive clones were cloned using the limiting dilution method, resulting in four hybridoma cell lines that stably secreted IgM monoclonal antibodies against macrophages' phagocytosis of ox-LDL. These four hybridoma cell lines were named 1B4, 2B4, 2H8, and 4D3.

[0040] Among them, monoclonal antibody 1B4 has the amino acid sequence of its heavy chain variable region as SEQ ID No. 1 and its light chain variable region as SEQ ID No. 2; monoclonal antibody 2B4 has the amino acid sequence of its heavy chain variable region as SEQ ID No. 3 and its light chain variable region as SEQ ID No. 4; monoclonal antibody 2H8 has the amino acid sequence of its heavy chain variable region as SEQ ID No. 5 and its light chain variable region as SEQ ID No. 6; and monoclonal antibody 4D3 has the amino acid sequence of its heavy chain variable region as SEQ ID No. 7 and its light chain variable region as SEQ ID No. 8.

[0041] The four IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL have the following nucleotide sequences: 1B4, encoding the heavy chain variable region (SEQ ID No. 9) and the light chain variable region (SEQ ID No. 10); 2B4, encoding the heavy chain variable region (SEQ ID No. 11) and the light chain variable region (SEQ ID No. 12); 2H8, encoding the heavy chain variable region (SEQ ID No. 13) and the light chain variable region (SEQ ID No. 14); and 4D3, encoding the heavy chain variable region (SEQ ID No. 15) and the light chain variable region (SEQ ID No. 16).

[0042] 3. Expansion of ascites and antibody purification

[0043] Using an in vivo induction method, 0.3 mL of sterile liquid paraffin was injected intraperitoneally into each 8-week-old Balb / c mouse. Seven days later, approximately 1.0 × 10⁻⁶ hybridoma cells were injected intraperitoneally. 6 Ascites fluid was collected from each individual animal after 10 days. Antibodies were purified using a Protein L affinity chromatography column to obtain IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL. Electrophoresis images of purified antibodies 1B4 and 4D3 are shown below. Figure 3 As shown. Western blot analysis of the heavy chains of four IgM antibody strains against horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM antibodies is performed. Figure 4 As shown.

[0044] Example 2

[0045] This embodiment detects the function of four IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL.

[0046] To observe the inhibitory effect of IgM monoclonal antibody on lipid phagocytosis, 1.0 × 10⁶ cells were seeded in a 96-well culture plate. 5 Peritoneal macrophages at a specific cell density were cultured for 8 hours, then transferred to serum-free RPMI 1640 medium and cultured for another 8 hours. After stimulating cells with LPS for 2 hours, a mixture of monoclonal antibodies at different concentrations (0.02–0.5 mg / ml) and 30 μg / ml fluorescently labeled oxidized low-density lipoprotein (Dio-ox-LDL) was added to RPMI 1640 medium for lipid phagocytosis. Next, DAPI staining was performed at room temperature for 5 minutes. After washing with PBS, internalized Dio-ox-LDL was visualized by Dio fluorescence, and quantitative analysis was performed using Image Pro Plus software to calculate the IC50. 50 Value, such as Figure 5 , 6 As shown.

[0047] Example 3

[0048] This embodiment provides the cloning process of light and heavy chains of an IgM monoclonal antibody against macrophage phagocytosis of ox-LDL, including:

[0049] 1. Hybridoma cell culture and total RNA extraction: Hybridoma cells were cultured in KD-Advance complete medium at 37°C and 5% CO2. Approximately 1.0–5.0 × 10⁶ cells were extracted. 6 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Novizan).

[0050] 2. Synthesis of the first strand of cDNA: cDNA was synthesized using a reverse transcription kit (purchased from Novizan).

[0051] 3. Gene amplification: Design universal upstream and downstream primers for both the light and heavy chains, and perform amplification using semi-nested PCR, such as... Figure 7 As shown, Figure 7 In the left figure, lane M: Marker; lane 1: heavy chain amplification of 2B4 antibody heavy chain using heavy chain primer set; lane 2: heavy chain amplification of 1B4 antibody heavy chain using heavy chain primer set; Figure 7 In the right figure, lane M: Marker; lane 1: amplification of the light chain of 2B4 antibody using the light chain primer set; lane 2: amplification of the light chain of 1B4 antibody using the light chain primer set.

[0052] The first round of PCR was performed using the first strand of cDNA as a template, and the second round of PCR was performed using the product from the first round as a template. The reaction volume for both rounds was 25 μL.

[0053] PCR reaction system: template 2μL, dNTPs 0.5μL, primers (10μM) 2μL, 5×SF buffer 5μL, ddH2O 15μL, DNA polymerase 0.5μL.

[0054] The PCR reaction conditions were as follows: pre-denaturation at 97℃ for 3 min, followed by 35 cycles (denaturation at 97℃ for 10 s, annealing at 59℃ for 30 s, extension at 72℃ for 30 s), extension at 72℃ for 10 min after the cycle, and storage at 4℃.

[0055] 4. Cloning and screening of PCR amplification products

[0056] Heavy and light chain PCR products were subjected to 1% agarose gel electrophoresis, and antibody DNA was recovered using a DNA gel recovery kit (Novizan). Figure 8 As shown, the fragment was inserted into the pBM23 vector using the fourth-generation TOPO cloning kit, transformed into DH5α competent cells (ampicillin resistant), and recombinant positive clones were screened and sequenced.

[0057] Example 4

[0058] Homology analysis

[0059] After sequencing the variable region, the obtained sequences were compared with the Ig BLAST and IMGT databases for homology.

[0060] The sequence alignment results show:

[0061] The variable regions of IgM antibody sequences, including framework regions and complementarity-determining regions (CDRs), showed a percentage of 75.44% to 99.28% compared to the reference sequence. Among these antibodies, the 2H8 antibody had a lower percentage of its complementary region (CDR) amino acid sequence compared to the reference sequence. The percentages of light chain CDR1, CDR2, and CDR3 compared to the reference sequence were 71.40%, 88.90%, and 88.90%, respectively, while the percentages of heavy chain CDR1, CDR2, and CDR3 compared to the reference sequence were 79.20%, 82.40%, and 71.10%, respectively. Sequence comparison of the four isolated IgM monoclonal antibodies revealed that their sequences were not identical. Clones 1B4 and 4D3 showed an 84.55% percentage in the heavy chain CDR, but only a 26.79% percentage in the light chain CDR. Figure 9 As shown. The variable region of the IgM antibody sequence of this invention is not completely identical to the gene sequences encoding various reported monoclonal antibodies, indicating that this invention is unique in terms of gene sequence.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to specific implementations. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An IgM monoclonal antibody against macrophage phagocytosis of ox-LDL, characterized in that, It contains four IgM monoclonal antibodies, named 1B4, 2B4, 2H8, and 4D3, respectively. These four IgM monoclonal antibodies against macrophage phagocytosis of ox-LDL are composed of heavy and light chains.

2. The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to claim 1, characterized in that: The amino acid sequences of the heavy chain variable region are SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.

7.

3. The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to claim 1, characterized in that: The amino acid sequences of the variable region of its light chain are SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No.

8.

4. The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to claim 1, characterized in that: The nucleotide sequences of the variable region of the heavy chain are SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 and SEQ ID No.

15.

5. The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to claim 1, characterized in that: The nucleotide sequences of the variable region of the light chain are SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 and SEQ ID No.

16.

6. The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to claim 1, characterized in that: The IgM monoclonal antibody against macrophage phagocytosis of ox-LDL has at least one of the following characteristics: The heavy chain type is μ; The light chain type is κ; The monoclonal antibody is a murine antibody.

7. The application of the IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to any one of claims 1-6, characterized in that: As a research tool for clinical treatment of atherosclerotic diseases.

8. The application of the IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to any one of 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 IgM monoclonal antibody against macrophage phagocytosis of ox-LDL according to any one of claims 1-6, characterized in that: Its application in the prevention of atherosclerosis and related diseases.

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