Immunogenic monoclonal antibody for treating abdominal aortic aneurysm as well as preparation method and application of immunogenic monoclonal antibody

Abstract

The invention relates to the technical field of biology, in particular to an immunogenic monoclonal antibody for treating abdominal aortic aneurysm as well as a preparation method and application of the immunogenic monoclonal antibody, and the immunogenic monoclonal antibody is a monoclonal antibody obtained by taking an immunogenic peptide fragment specifically acting on a human sphingosine 1 phosphate receptor 2 as an immunogen; wherein the amino acid sequence of the immunogenic peptide fragment is shown as SEQ.ID.NO: 1, the immunogenic monoclonal antibody can be used for preparing an injection preparation for treating abdominal aortic aneurysm, the injection preparation has the advantages of high specificity, lasting effect, good compliance and the like, the defects that JTE-013 is not high in specificity and needs to be administrated for multiple times are overcome, and the immunogenic monoclonal antibody can be used for preparing an injection preparation for treating abdominal aortic aneurysm. And a new thought is provided for treatment of AAA and related vascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to an immunogenic monoclonal antibody for treating abdominal aortic aneurysm, a preparation method thereof, and an application thereof. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a serious vascular disease that poses a major threat to human health. AAA is defined as a permanent local dilation of the abdominal aorta with a diameter ≥ 50% of the normal arterial diameter. Clinically, AAA can also be diagnosed when the abdominal aortic diameter > 3 cm. AAA mostly occurs in the elderly with cardiovascular risk factors (such as smoking, hypertension, and hyperlipidemia), especially in men, and often coexists with atherosclerosis. The Global Burden of Disease Study shows that the mortality rate and disability-adjusted life years caused by AAA have been on the rise in the past 30 years. The latest data shows that 172,427 people die from AAA worldwide every year, far higher than the all-cause mortality rate of 18.2% during the same period. Although our research on the etiology and pathology of AAA has made progress, there is still a lack of effective drug treatment. Clinical trials of antihypertensive drugs, statin drugs, doxycycline drugs, and antiplatelet drugs have not shown significant benefits, and surgical repair remains the only available treatment method for high-risk patients. Therefore, finding new treatment targets and methods is of great significance.

[0003] Sphingosine-1-phosphate (S1P) is an important lipid signaling molecule, which is mainly produced by the catalysis of sphingomyelin on the cell membranes of red blood cells, platelets, and endothelial cells by a variety of enzymes. Sphingosine kinase (SphK), which is the rate-limiting enzyme for S1P synthesis, is divided into SphK1 and SphK2, and SphK1 plays a major role in most cases. Inside the cell, S1P can directly bind to some intracellular targets as a second messenger and regulate the survival, proliferation, differentiation, and apoptosis of cells. Outside the cell, S1P transmits signals by binding to specific receptors. The receptors of S1P are members of the rhodopsin-like GPCR, and 5 subtypes (S1PR1-5) have been identified. Each receptor subtype is expressed in different tissues and cells, so it has different functions. For example, S1PR1 / 2 / 3 are widely distributed in most tissues, especially in the immune, cardiovascular, and central nervous systems, S1PR4 is mainly expressed in the lymphatic system, hematopoietic tissue, and lungs, and S1PR5 is mainly expressed in the brain and spleen. In terms of cardiovascular diseases, S1P / S1PR has been proven to have anti-atherosclerotic, angiogenesis-promoting, ischemia-reperfusion injury-reducing, vascular tone-regulating, and myocardial fibrosis-preventing effects. Therefore, S1P / S1PR may be a powerful candidate target.

[0004] Currently, there is no specific S1PR2 antagonist on the market clinically. JTE-013 is the most widely used S1PR2 antagonist, first reported by Japanese scientists in 2001, but it is only used as a tool drug. It inhibits the specific binding of radiolabeled S1P to the cell membrane of Chinese hamster ovary cells stably transfected with human or rat S1PR2, and does not affect the binding of S1P to S1PR1 and S1PR3. However, in recent years, some studies have questioned the specificity of JTE-013, reporting its effects on other S1PRs and its role in non-S1PR-mediated biological functions. For example, in addition to S1PR2, JTE-013 also inhibits S1PR3-mediated cerebrovascular constriction in rodents and S1PR4-mediated calcium mobilization. In addition, JTE-013 can also inhibit the vasoconstriction induced by prostaglandin analogue U46619, endothelin-1, and high potassium chloride (the vasoconstriction induced by high potassium chloride is not S1PR-mediated, but is related to the L-type Ca 2+ channel).

[0005] Key pathological processes related to AAA include inflammatory cell infiltration, cytokine production, matrix metalloproteinase activation, extracellular matrix degradation, phenotypic transformation of vascular smooth muscle cells (VSMCs), VSMC death, neovascularization, and thrombosis. Based on long-term work accumulation and a targeted laboratory platform, we have invented a therapeutic vaccine targeting S1PR2 (National Invention Patent Application No.: 202510089151.7). This vaccine effectively reduces the incidence and severity of AAA in model animals, inhibits abdominal aortic dilation, and protects the structural integrity of the vascular media. Although therapeutic vaccines have the advantages of high target specificity, long-lasting effects (immunization once every 1-3 months can be maintained for several months), high compliance, and low cost, and currently several tumor therapeutic vaccines have been launched on the market, and hypertension vaccines and pulmonary hypertension vaccines have also achieved encouraging results in animal experiments, there is currently no therapeutic vaccine for chronic diseases such as cardiovascular diseases on the market at home and abroad. The main reason is that there is currently no production license standard and system for cardiovascular disease therapeutic vaccines in China, and the transformation and application of the therapeutic vaccine targeting S1PR2 to clinical practice is a long way to go.

[0006] Therefore, it is extremely urgent to provide a drug for chronic diseases such as cardiovascular diseases that can be used clinically. Summary of the Invention

[0007] The object of the present invention is to solve the problem that there is currently no therapeutic vaccine for chronic diseases such as cardiovascular diseases on the market at home and abroad, and there is still a lack of effective drug treatment for abdominal aortic aneurysm. Therefore, an immunogenic monoclonal antibody for treating abdominal aortic aneurysm, a preparation method and an application thereof are provided. The immunogenic monoclonal antibody can be used to prepare an injection preparation for treating abdominal aortic aneurysm. The injection preparation has the advantages of high specificity, long-lasting effect and good compliance, making up for the deficiencies of JTE-013 in weak specificity and multiple administrations, and providing a new idea for the treatment of AAA and related vascular diseases.

[0008] In order to achieve the above object, in the first aspect, the present invention provides an immunogenic monoclonal antibody for treating abdominal aortic aneurysm, and the immunogenic monoclonal antibody is a monoclonal antibody obtained by using an immunogenic peptide segment that specifically acts on human sphingosine 1-phosphate receptor 2 as an immunogen; wherein, the amino acid sequence of the immunogenic peptide segment is shown as SEQ.ID.NO:1.

[0009] In the second aspect, the present invention provides a preparation method of the immunogenic monoclonal antibody as described in the first aspect, and the preparation method includes: immunizing mice with a purified immunogenic peptide segment that specifically acts on human sphingosine 1-phosphate receptor 2 as an immunogen, performing cell fusion after the titer detection reaches the standard, screening and cloning to obtain hybridoma cells capable of secreting monoclonal antibodies, and then obtaining monoclonal antibodies secreted by the hybridoma cell line; wherein, the amino acid sequence of the immunogenic peptide segment is shown as SEQ.ID.NO:1.

[0010] In the third aspect, the present invention provides an injection preparation for treating abdominal aortic aneurysm, and the injection preparation is prepared by using the immunogenic monoclonal antibody described in the first aspect.

[0011] Preferably, the injection preparation is a freeze-dried powder injection or an aqueous solution.

[0012] In the above technical solution, the present invention uses human sphingosine 1-phosphate receptor 2 (S1PR2) as a target to design an immunogenic monoclonal antibody, and the monoclonal antibody can effectively inhibit the dilation of the abdominal aorta in mice and effectively inhibit the progression of AAA. Compared with vaccines and chemically synthesized drugs, monoclonal antibodies have the remarkable characteristics of high treatment specificity, short onset time and good patient compliance.

[0013] The present invention uses an immunogenic peptide segment that specifically acts on S1PR2 as an immunogen to induce an immune response in BALB / C mice, thereby generating specific antibodies in the mice, and then producing an immunogenic monoclonal antibody for treating abdominal aortic aneurysm through the preparation of hybridoma cells and subcloning. The immunogenic monoclonal antibody can effectively inhibit the dilation of the abdominal aorta and the progression of AAA.

[0014] Moreover, compared with therapeutic vaccines, the immunogenic monoclonal antibodies of the present invention have more mature preparation techniques and related systems, and the dosage is easier to control, showing extremely strong prospects for clinical translation.

[0015] Other features and advantages of the present invention will be described in detail in the following specific implementation section. Brief Description of the Drawings

[0016] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the following specific implementation, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0017] Figure 1 is the flowchart of AAA modeling by injecting male C57BL / 6J mice with anti-SPR-009 monoclonal antibody and JTE-013 in Example 4;

[0018] Figure 2 shows the changes in blood pressure and body weight of male C57BL / 6J mice in each group in Example 4;

[0019] Figure 3 In Example 4, A is the gross picture of the abdominal aorta of different groups, B is the ultrasound picture of the abdominal aorta of different groups, C is the statistical chart of the maximum gross diameter of the abdominal aorta of different groups, and D is the statistical chart of the maximum ultrasound diameter of the abdominal aorta of different groups.

[0020] Figure 4 In Example 4, A is the HE and EVG staining pictures of the abdominal aorta of different groups; B is the statistical chart of the elastin degradation score of the abdominal aorta of different groups. Detailed Description of the Invention

[0021] The following provides a detailed description of the specific implementation of the present invention. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0022] The endpoints and any values disclosed in the ranges in this article are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0023] In a first aspect, the present invention provides an immunogenic monoclonal antibody for treating abdominal aortic aneurysm, and the immunogenic monoclonal antibody is a monoclonal antibody obtained by using an immunogenic peptide segment that specifically acts on human sphingosine-1-phosphate receptor 2 as an immunogen; wherein, the amino acid sequence of the immunogenic peptide segment is shown as SEQ.ID.NO:1.

[0024] SEQ.ID.NO:1

[0025] TPVQWFARE

[0026] The present invention uses S1PR2 as a target to design an immunogenic monoclonal antibody, and this therapeutic monoclonal antibody can effectively inhibit the dilation of the abdominal aorta in mice and inhibit the progression of AAA. Compared with vaccines and chemically synthesized drugs, monoclonal antibodies have significant characteristics such as high therapeutic specificity, short onset time, and good patient compliance.

[0027] In a second aspect, the present invention provides a preparation method of the immunogenic monoclonal antibody as described in the first aspect, and the preparation method includes: immunizing mice with a purified immunogenic peptide segment that specifically acts on human sphingosine-1-phosphate receptor 2 as an immunogen, performing cell fusion after the titer detection reaches the standard, screening and cloning to obtain hybridoma cells capable of secreting monoclonal antibodies, and then obtaining monoclonal antibodies secreted by the hybridoma cell line; wherein, the amino acid sequence of the immunogenic peptide segment is shown as SEQ.ID.NO:1.

[0028] SEQ.ID.NO:1

[0029] TPVQWFARE

[0030] The present invention uses an immunogenic peptide segment that specifically acts on S1PR2 as an immunogen to induce an immune response in BALB / C mice, thereby generating specific antibodies in the mice, and then produces an immunogenic monoclonal antibody for treating abdominal aortic aneurysm through the preparation of hybridoma cells and subcloning. This immunogenic monoclonal antibody can effectively inhibit the dilation of the abdominal aorta and inhibit the progression of AAA.

[0031] In a third aspect, the present invention provides an injection preparation for treating abdominal aortic aneurysm, and the injection preparation is prepared by using the immunogenic monoclonal antibody described in the first aspect.

[0032] In a preferred embodiment of the present invention, in order to make the injection preparation easier to store, the injection preparation is a freeze-dried powder injection or an aqueous solution.

[0033] The following further clarifies the main content of the present invention in conjunction with specific embodiments with reference to the drawings for those skilled in the art to understand. However, the content of the present invention is not limited to the following embodiments.

[0034] The immunogenic peptide specifically acting on human sphingosine 1-phosphate receptor 2 hereinafter referred to as the SPR-009 immunogenic peptide, and the monoclonal antibody prepared using the same hereinafter referred to as the anti-SPR-009 monoclonal antibody.

[0035] Example 1

[0036] This example is used to illustrate the preparation of the immunogenic peptide against the extracellular amino acid sequence of human S1PR2:

[0037] According to the spatial conformation, bioinformatics and pharmacological characteristics of S1PR2, an immunogenic peptide against the extracellular amino acid sequence of human S1PR2 was designed, named SPR-009, which is the SPR-009 immunogenic peptide. The specific amino acid sequence is shown in SEQ ID No.1.

[0038] SEQ ID No.1:

[0039] TPVQWFARE,

[0040] The above-mentioned SPR-009 peptide was synthesized by solid-phase synthesis (entrusted to Shanghai Gil Biochemical Co., Ltd. for synthesis and quality inspection). The purity of the synthesized peptide was analyzed by high-performance liquid chromatography. The purity of the SPR-009 peptide was detected to be over 98%. The obtained SPR-009 peptide was freeze-dried, aliquoted and placed in a cryotube, and stored at -80°C for later use.

[0041] Example 2

[0042] This example is used to illustrate the preparation of the monoclonal antibody specifically acting on human S1PR2:

[0043] The SPR-009 immunogenic peptide of Example 1 was conjugated with an adjuvant and used to immunize BALB / C mice. By preparing hybridoma cells and subcloning, a monoclonal antibody specifically acting on human S1PR2 was produced, named anti-SPR-009. The specific process is as follows:

[0044] (1) Six-week-old male BALB / C mice were selected and immunized by injection on the 0th day, 21st day, 35th day, and 49th day, with 50 μg of SPR-009 polypeptide each time. The SPR-009 polypeptide was emulsified with an adjuvant (Freund's complete adjuvant or Freund's incomplete adjuvant) and injected subcutaneously or intraperitoneally to enhance the immune response. After immunization, the tail vein blood or orbital venous plexus blood of the mice was collected regularly, the serum was separated, and the antibody titer of anti-SPR-009 polypeptide in the serum was detected. The results are shown in Table 1.

[0045] Table 1 OD values of anti-SPR-009 polypeptide antibodies in sera of different mice

[0046] Dilution ratio Mouse No. 1 Mouse No. 2 Mouse No. 3 Mouse No. 4 Mouse No. 5 1:1k 1.5629 2.3690 1.1770 2.4225 1.3151 1:3k 1.0340 1.7405 0.6776 1.6245 0.6345 1:9k 0.6158 1.2200 0.3491 0.8473 0.2725 1:27k 0.2813 0.6059 0.1703 0.3783 0.1363 1:81k 0.1265 0.2623 0.0942 0.1597 0.0761 1:243k 0.0770 0.1277 0.0643 0.1006 0.0553 1:729k 0.0592 0.0792 0.0595 0.0725 0.0749 BLANK 0.0565 0.0600 0.0542 0.0613 0.0614

[0047] (2) On the 54th day, the mouse (No. 2#) with the highest antibody titer was selected for spleen lymphocyte isolation. After the mouse was anesthetized, the spleen was removed under sterile conditions, and a single-cell suspension was obtained through mechanical grinding and treatment with erythrocyte lysate. The isolated spleen lymphocytes were mixed with the mouse myeloma cell line SP2 / 0 in a certain proportion, and cell fusion was carried out under the mediation of polyethylene glycol. The fused cell suspension was inoculated into hypoxanthine-aminopterin-thymidine selective medium to screen out hybridoma cells.

[0048] (3) The hybridoma cells were inoculated into 96-well plates and cultured in an incubator at 37 °C and 5% CO2. The antibody titer in the culture supernatant was detected regularly to screen out antibody-positive hybridoma cells.

[0049] (4) The antibody-positive hybridoma cells screened out were monoclonalized. The cells were inoculated into 96-well plates by the limited dilution method to ensure that each well contained only one cell. After amplification culture, the monoclonal cell lines were injected into the abdominal cavity of BALB / C mice. One week later, the mouse ascites was collected, cell debris was removed by centrifugation, and the monoclonal antibody in the ascites was purified by protein A / G affinity chromatography. Among them, the subtypes of the monoclonal antibody obtained are shown in Table 2.

[0050] Table 2 Subtypes of anti-SPR-009 monoclonal antibody

[0051] Anti-SPR-009 monoclonal antibody IgG1 2.3909 IgG2a 0.1550 IgG2b 0.0986 IgG3 0.1381 IgM 1.2322 IgA 0.1372 κ 0.4165 λ 0.0777

[0052] (5) To verify the specific binding ability of the anti-SPR-009 monoclonal antibody to the SPR-009 polypeptide, the antibody affinity was detected using a solid-phase plate coated with the SPR-009 polypeptide. The detection results are shown in Table 3.

[0053] Table 3 OD values of antibodies in the serum of mice immunized with anti-SPR-009 monoclonal antibody

[0054] Dilution proportion 1:1k 1:3k 1:9k 1:27k 1:81k 1:243k 1:729k Blank OD value 2.9573 2.3100 2.0113 1.5560 0.8761 0.4285 0.2088 0.0681

[0055] Example 3

[0056] This example is used to illustrate the establishment of an abdominal aortic aneurysm (AAA) model:

[0057] Six-week-old male C57BL6 / J mice were taken. On the 0th day, they were locally incubated with porcine pancreatic elastase (Sigma-Aldrich, E1250) to induce AAA in the mice. The control group (Control group) was treated with an equal amount of 0.9% saline. The AAA group, anti-SPR-009 group, and JTE-013 group were subjected to the same modeling.

[0058] (1) Anesthesia and skin preparation: The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 ml / kg. The mice were fixed in the supine position on a thermostatic operating pad with the temperature adjusted to 37°C. The chest and abdomen were exposed, and the abdominal skin was prepared. The area was disinfected three times with iodophor cotton balls.

[0059] (2) Exposure of the abdominal aorta: A 2.5-cm longitudinal incision was made on the skin along the midline of the lower abdomen with surgical scissors. The underlying muscles were gently lifted, and a 2.5-cm longitudinal incision was made along the white line to enter the abdominal cavity. The intestines and stomach were moved to the right side of the mouse with a wet cotton swab (ideally, this would expose the lower renal segment of the aorta. If the aorta was difficult to locate, the right kidney and the right renal artery could be used to identify the aorta because the anatomical position of the right kidney is slightly lower than that of the left kidney). The connective tissue covering the abdominal aorta and inferior vena cava was gently removed with forceps (the abdominal aorta and inferior vena cava are located within the same vascular sheath, and gentle actions were taken to avoid damaging the two blood vessels). The muscles behind the abdominal aorta and inferior vena cava were gently dissected with forceps (the tip of the forceps should enter the back of the sheath horizontally to create a hole in the fascia connecting the sheath and the underlying muscles. Once the hole was made, the forceps were slowly released to expand the size of the hole), thereby fully exposing the lower renal segment of the abdominal aorta.

[0060] (3) Drug incubation: 10 μL of porcine pancreatic elastase was dropped onto a 5 cm × 5 mm gauze with a pipette, and the gauze was wrapped around the abdominal aorta (0.5 cm from the right renal artery). The Control group was treated with an equal amount of 0.9% saline. After 30 minutes, the gauze was removed with forceps.

[0061] (4) Irrigation of the abdominal cavity: The aorta and abdominal cavity were irrigated with 500 μL of 0.9% sterile saline. The remaining saline was absorbed with a 10 cm × 10 cm gauze.

[0062] (5) Suture: The abdominal organs were repositioned, and the muscles and skin were sutured sequentially with 6-0 non-absorbable monofilament sutures. The area was disinfected three times with iodophor.

[0063] (6) Anesthesia recovery: The mice were removed and placed on a heating pad in the cage. The mice were allowed to breathe the indoor air freely, and the general conditions such as body temperature and respiration of the mice were monitored during the recovery period.

[0064] Example 4

[0065] This example was used to illustrate the therapeutic effects of anti-SPR-009 monoclonal antibody and JTE-013 on elastase-induced AAA:

[0066] (1) Experimental grouping:

[0067] Male C57BL / 6J mice at 6 weeks of age were selected. After 1 week of adaptive feeding, they were divided into 4 groups as follows:

[0068] Group 1: Control group (Control, n = 6): Treated with 0.9% saline on day 0 of the experiment according to the above-mentioned modeling method.

[0069] Group 2: AAA model group (AAA, n = 8): Modeled according to the above method on day 0 of the experiment.

[0070] Group 3: anti-SPR-009 monoclonal antibody group (anti-SPR-009, n = 9): Modeled according to the above method on day 0 of the experiment, and anti-SPR-009 monoclonal antibody was injected via the tail vein at doses of 100 μg / animal on days 0, 7, 14, and 21, respectively.

[0071] Group 4: JTE-013 group (JTE-013, n = 9): Modeled according to the above method on day 0 of the experiment, and JTE-013 was injected intraperitoneally at a dose of 5 mg / kg / time, three times a week.

[0072] (2) Blood pressure monitoring:

[0073] The tail artery blood pressure of mice in each group was measured weekly using a non-invasive mouse tail blood pressure monitor BP-2010A (Softron, Japan). The mice were placed in a dark environment and heated at 37 °C for about 15 minutes before blind measurement began. During the measurement, the mice were kept in a quiet and peaceful state. Each mouse was measured about 15 times, and the average value was statistically analyzed. All measurements were completed between 9:00 and 11:00 in the morning. The results are shown in Figure 2 .

[0074] It can be seen from Figure 2 that there was no significant change in systolic blood pressure in all groups during the entire experimental observation period, and there was no significant difference among the groups.

[0075] (3) Ultrasonic detection:

[0076] Echocardiography was performed on day 28. The mice were anesthetized with 1.5% isoflurane at a temperature of 36.5 - 37.5 °C, and images were obtained using a Vevo 3100 high-resolution imaging system (Visualsonics, Canada) equipped with a 30 MHz transducer. All measurement results were analyzed offline by the observer using the Vevo 3100 workstation software.

[0077] (4) At the end of the experiment, all mice were euthanized, the heart and the entire aorta were exposed, perfused with phosphate-buffered saline, the perimembranous tissue was dissected, and then rapidly excised and photographed. The results are shown in Figure 3 .

[0078] It can be seen from Figure 3From the content of the statistical chart of the maximum gross diameter of the abdominal aorta in different groups, the maximum gross diameter of the abdominal aorta in the anti-SPR-009 monoclonal antibody group was 1.40 mm, that in the JTE-013 group was 1.53 mm, and that in the AAA model group was 1.78 mm; from Figure 3 From the content of the statistical chart of the maximum ultrasonic diameter of the abdominal aorta in different groups, the maximum ultrasonic diameter of the abdominal aorta in the anti-SPR-009 monoclonal antibody group was 1.00 mm, that in the JTE-013 group was 1.05 mm, and that in the AAA model group was 1.37 mm.

[0079] It can be seen that compared with the AAA model group, the dilation of the abdominal aorta in the anti-SPR-009 monoclonal antibody group and the JTE-013 group was significantly reduced (gross: 1.40 mm vs 1.78 mm, P<0.01; 1.53 mm vs 1.78 mm, P<0.05; ultrasound: 1.00 mm vs 1.37 mm, P<0.01; 1.05 mm vs 1.37 mm, P<0.05). Thus, both anti-SPR-009 monoclonal antibody and JTE-013 can effectively inhibit the dilation of the abdominal aorta and the progression of AAA.

[0080] (5) Fix the tissue in 4% paraformaldehyde overnight, then embed it in paraffin and cut continuous sections at intervals of 5 μm. The sections were stained with hematoxylin and eosin (HE) and elastic van Gieson (EVG) to observe the degradation of elastic fibers in the media. The degradation degree of elastic fibers was graded from 1 to 4 (grade 1, the elastic layer was intact and the tissue was good; grade 2, the fiber density decreased and showed a linear shape; grade 3, occasional fiber breaks; grade 4, severe degradation of elastin, and the rupture site was visible), and the results were as Figure 4 shown.

[0081] From Figure 4 the content, it can be known that the elastin score in the AAA model group was 4.00, and the elastin score in the anti-SPR-009 monoclonal antibody group was 2.67. It can be seen that compared with the AAA model group, the elastin score in the anti-SPR-009 monoclonal antibody group was significantly improved (4.00 vs 2.67, P = 0.039), while the elastin score in the JTE-013 group was 3.00, and compared with the AAA model group, the improvement of the elastin score in the JTE-013 group was not significant (4.00 vs 3.00, P = 0.167). It can be known that anti-SPR-009 monoclonal antibody can effectively retain the elastic structure of the media and thus inhibit the progression of AAA.

[0082] As can be seen from the above figures and content, the anti-SPR-009 monoclonal antibody of the present invention has a therapeutic effect on abdominal aortic aneurysm similar to or even better than that of JTE-013, and has the effects of inhibiting abdominal aorta dilation, preserving the elastic structure of the tunica media, and inhibiting the progression of AAA.

[0083] In summary, the present invention uses S1PR2 as a target, and based on immunogenic peptides, pioneeringly screens out a therapeutic monoclonal antibody specific for S1PR2, which can effectively inhibit the dilation of the abdominal aorta in mice, protect the elastic structure of the tunica media, and effectively inhibit the progression of AAA.

[0084] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0085] In addition, it should be noted that, in the case of no conflict, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0086] In addition, any combination can be made between different embodiments of the present invention, as long as it does not violate the idea of the present invention, it should also be regarded as the content disclosed by the present invention.

Claims

1. An immunogenic monoclonal antibody for treating abdominal aortic aneurysm, characterized in that, The immunogenic monoclonal antibody is a monoclonal antibody obtained by using an immunogenic peptide segment that specifically acts on human sphingosine-1-phosphate receptor 2 as an immunogen; wherein, The amino acid sequence of the immunogenic peptide segment is as shown in SEQ.ID.NO:

1.

2. A method for preparing the immunogenic monoclonal antibody according to claim 1, characterized in that, The preparation method includes: immunizing mice with a purified immunogenic peptide segment that specifically acts on human sphingosine-1-phosphate receptor 2 as an immunogen, performing cell fusion after the titer detection reaches the standard, screening and cloning to obtain hybridoma cells capable of secreting monoclonal antibodies, and then obtaining monoclonal antibodies secreted by the hybridoma cell line; wherein, The amino acid sequence of the immunogenic peptide segment is as shown in SEQ.ID.NO:

1.

3. An injection preparation for treating abdominal aortic aneurysm, characterized in that, The injection preparation is prepared by using the immunogenic monoclonal antibody according to claim 1.

4. The injection preparation according to claim 2, characterized in that, The injection preparation is a freeze-dried powder injection or an aqueous solution.

Images