An anti-AIBP monoclonal antibody and its application

Abstract

The present invention relates to the technical field of antibody drugs, and in particular to murinized anti-AIBP monoclonal antibodies and applications thereof. The murinized anti-AIBP monoclonal antibodies provided by the present invention can inhibit AIBP protein, inhibit the binding of AIBP protein to human umbilical vein endothelial cells (HUVECs), promote HUVECs proliferation, promote collateral formation and blood flow recovery in lower limb ischemia model mice, and provide a possibility for clinical improvement of collateral circulation in patients with peripheral arterial disease.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to a monoclonal antibody for promoting collateral circulation of peripheral arterial disease, and in particular to a monoclonal antibody that specifically binds to and inhibits the function of AIBP and its application. Background Art

[0002] Peripheral arterial disease

[0003] Peripheral arterial disease (PAD) is mainly lower extremity atherosclerotic occlusive disease (ASO) [1] As the aging of the Chinese population continues to accelerate, the incidence of peripheral arterial disease (PAD) has increased year by year, with an incidence of 19.8% in people over 60 years old and 24.1% in people with diabetes over 50 years old. In addition, about 10% of PAD patients even develop critical limb ischemia (CLI), with clinical manifestations of rest pain (lasting more than 2 weeks), ulcers or gangrene. The quality of life index of CLI patients is similar to that of advanced cancer patients, and their mortality rate exceeds 50% within 5 years after the initial diagnosis. [2] .

[0004] Collateral circulation and critical limb ischemia

[0005] Isner et al. first proposed the concept of therapeutic angiogenesis (TA) in 1999, which is a new therapeutic method that uses cytokines or angioblasts to enter ischemic tissues and transform the local microenvironment to provide an angiogenic environment to promote the formation of capillary networks and the establishment of collateral circulation in ischemic tissues, including angiogenesis, arterial formation and vasculogenesis. In the past 10 years, studies have shown that TA can effectively induce angiogenesis and collateral circulation in CLI tissues, thereby improving blood perfusion in ischemic areas, bringing new ideas and strategies to the treatment of CLI. [2] .

[0006] Arteriogenesis occurs at the arterial level and refers to the expansion of the lumen diameter of existing blood vessels through structural remodeling, forming new collateral arteries with a diameter of 20 to 100 μm and rich in mature tunica media. Studies have shown that the establishment of a collateral artery can increase blood perfusion in the distal ischemic area by 20 to 30 times, which is much better than the increase of the capillary network alone. The effective establishment of new collateral arteries is the main goal of TA. [2] .

[0007] At present, the most extensive research at home and abroad is on VEGF, HIF-1α, hepatocyte growth factor (HGF) and fibroblast growth factor (FGF). Basic research and phase I clinical trial results have confirmed the angiogenesis-promoting effect of direct use of cytokines. However, large-scale controlled studies have shown that it cannot effectively improve the clinical symptoms of CLI patients. [2] .

[0008] AIBP

[0009] Study on the effect of blocking AIBP gene expression in myocardial blood vessels on myocardial microvascular regeneration [3] Reported: Recent studies have found that Apo A-1Binding Protein (AIBP) secreted by peripheral tissues can inhibit angiogenesis. Theoretically, blocking AIBP activity can stimulate vascular regeneration, but there are no reports on the role of AIBP in myocardial microvascular endothelial cells (CMECs) and myocardial tissue. Results In in vitro experiments, blocking the expression of AIBP gene in rat CMECs can promote the regeneration of rat myocardial microvessels; in in vivo animal experiments, blocking the expression of AIBP gene in rat myocardial blood vessels can also significantly promote the regeneration of coronary microvessels after myocardial infarction and improve cardiac function.

[0010] Downregulation of AIBP gene expression promotes angiogenesis in myocardial microvascular endothelial cells [4] This paper reports: The effect of blocking the expression of apolipoprotein AI binding protein (AIBP) on angiogenesis in myocardial microvascular endothelial cells (CMECs) was investigated. Conclusion: Inhibition of AIBP expression can significantly promote the proliferation, migration and tube formation of CMECs.

[0011] AIBP targets and promoting collateral circulation

[0012] There has been no report on the correlation between free AIBP (sAIBP) in the plasma of PAD patients and the formation of collateral circulation and clinical symptoms. There has also been no report on the correlation between anti-AIBP monoclonal antibodies and the formation of collateral circulation. No therapeutic anti-AIBP monoclonal antibodies have been found to promote the formation of collateral circulation.

[0013] References:

[0014] [1] Wang Shenming, Wang Zhecun. Advances in vascular examination and early treatment of peripheral arterial disease[J]. Chinese Journal of Experimental Surgery, 2020, (Issue 5).

[0015] [2] Gui Liang 1,2 ,Li Yongjun 1 .Research progress of gene therapy angiogenesis in lower limb ischemic diseases[J]. Chinese Journal of Basic and Clinical General Surgery, 2022, (Issue 8).

[0016] [3] Ma Xiaolei. Study on the effect of blocking myocardial vascular AIBP gene expression on myocardial microvascular regeneration[M]. Fourth Military Medical University, 2015.

[0017] [4] Ma Xiaolei, Lü Anlin, Jiang Xiaoyu, et al. Downregulation of AIBP gene expression promotes angiogenesis in myocardial microvascular endothelial cells[J]. Progress in Modern Biomedicine, 2015(22):5. DOI:10.13241 / j.cnki.pmb.2015.22.008. Summary of the invention

[0018] Problems to be solved by the invention:

[0019] In view of the problems existing in the prior art, the present invention provides an anti-AIBP monoclonal antibody that specifically recognizes AIBP, provides the variable region sequence of the monoclonal antibody, provides a nucleotide vector encoding the monoclonal antibody, a host cell, and provides the use of the monoclonal antibody in promoting the formation of collateral circulation and blood flow restoration.

[0020] To achieve the above purpose, the specific technical solution of the present invention is as follows:

[0021] The present invention provides an anti-AIBP monoclonal antibody (MM02), which comprises a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the HCDR1 amino acid sequence is shown in SEQ ID No.1, the HCDR2 amino acid sequence is shown in SEQ ID No.2, and the HCDR3 amino acid sequence is shown in SEQ ID No.3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the LCDR1 amino acid sequence is shown in SEQ ID No.4, the LCDR2 amino acid sequence is shown in SEQ ID No.5, and the LCDR3 amino acid sequence is shown in SEQ ID No.6

[0022] The amino acid sequence of the heavy chain variable region of the anti-AIBP monoclonal antibody is shown in SEQ ID No.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.8.

[0023] The amino acid sequence of the heavy chain of the anti-AIBP monoclonal antibody is shown in SEQ ID No.9, and the amino acid sequence of the light chain is shown in SEQ ID No.10.

[0024] The present invention provides a nucleotide sequence encoding the heavy chain variable region CDR of the anti-AIBP monoclonal antibody MM02.

[0025] The present invention provides a nucleotide sequence encoding the light chain variable region CDR of the anti-AIBP monoclonal antibody MM02.

[0026] Among them, the nucleotide sequences encoding the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-AIBP monoclonal antibody MM02 are shown in SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 13, respectively;

[0027] The nucleotide sequences encoding the light chain variable regions CDR1, CDR2, and CDR3 of the anti-AIBP monoclonal antibody MM02 are shown in SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, respectively.

[0028] Experiments show that the anti-AIBP monoclonal antibody MM02 of the present invention can specifically recognize the AIBP protein and has the best neutralization effect, so the anti-AIBP monoclonal antibody MM02 of the present invention can be used for the detection of AIBP protein.

[0029] The present invention also provides a kit, which includes anti-AIBP monoclonal antibody MM02, and the kit also includes a coating buffer, a washing solution, a blocking solution and / or a color developing solution.

[0030] As certain embodiments of the present invention, anti-AIBP monoclonal antibodies inhibit the binding of AIBP protein to human umbilical vein endothelial cells.

[0031] As certain embodiments of the present invention, anti-AIBP monoclonal antibodies promote proliferation of human umbilical vein endothelial cells.

[0032] As certain embodiments of the present invention, anti-AIBP monoclonal antibodies promote collateral circulation formation and blood flow recovery in lower limb ischemia model mice.

[0033] Compared with the prior art, the beneficial effects of this application are:

[0034] The present invention prepares anti-AIBP monoclonal antibodies, and finds that the monoclonal antibodies have high affinity and avidity with AIBP antigens, and have a significant promoting effect on collateral circulation formation and blood flow recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 , the results of the validation of the inhibition of AIBP binding to human umbilical vein endothelial cells by anti-AIBP monoclonal antibodies;

[0036] Figure 2, the results of anti-AIBP monoclonal antibody promoting the proliferation of human umbilical vein endothelial cells;

[0037] Figure 3 , laser Doppler scanning of ischemic lower limb blood perfusion in mice with lower limb ischemia model before and after administration of anti-AIBP monoclonal antibodies and IgG, among which,

[0038] A is the blood perfusion diagram of ischemic lower limbs 1 day before surgery, 0 day after surgery, and 21 days after surgery.

[0039] B is the statistical graph of ischemic lower limb blood perfusion 1 day before surgery, 0 day after surgery, 7 days after surgery, 14 days after surgery, and 21 days after surgery;

[0040] Figure 4 , under confocal fluorescence microscopy, the formation of collateral arteries in the ischemic lower limbs of mice with lower limb ischemia after administration of anti-AIBP monoclonal antibodies and IgG, among which,

[0041] A is the density map of collateral arteries in the ischemic lower limbs 21 days after surgery.

[0042] B is the statistical chart of the number of collateral arteries in the ischemic lower limbs 21 days after surgery.

[0043] *, **, ***, **** represent: P < 0.05, P < 0.01, P < 0.001, P < 0.0001, respectively, indicating that the difference between the two groups is statistically significant. DETAILED DESCRIPTION

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0047] Example 1. Preparation of anti-AIBP monoclonal antibodies

[0048] 1. Immunogen Human AIBP recombinant protein was purchased from Beijing SinoBiological Company, project ID: 22ZN35.

[0049] 2. Animal immunization: The above-mentioned human AIBP recombinant protein was used to immunize 5 AIBP knockout mice (mouse background was C57 / BL strain), with a dose of 50 μg per mouse per immunization. For the first immunization, the human AIBP recombinant protein was mixed with an equal volume of complete Freund's adjuvant to make an emulsion, which was injected subcutaneously at multiple points in the abdomen; at intervals of 2 weeks, the same dose of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant to make an emulsion, which was injected subcutaneously at multiple points in the abdomen. Blood was collected one week after the fourth immunization and the serum titer was determined by ELISA. Mice with qualified titers were waited for fusion. For the fifth immunization, the mice were boosted with immunization (25 μg intravenously + 80 μg intraperitoneally). 3-4 days after the booster immunization, the spleen of the mice was taken for hybridoma fusion.

[0050] 3. Hybridoma cell fusion screening

[0051] According to the serum titer results, the best mouse was selected for hybridoma fusion screening and limiting dilution. The spleen cells of the immunized mice were mixed with SP2 / 0 myeloma cells at a 1:1 ratio and fused by electrofusion to obtain hybridoma cells. The immunogen human AIBP recombinant protein was used as the screening antigen, and the cell supernatant was measured by ELISA. The positive wells that bound to the immunogen were selected as the positive main clones, and subcloning was performed by limiting dilution until a hybridoma cell line that stably secreted monoclonal antibodies was obtained. Three positive hybridoma cells were obtained and the cells were frozen and cultured for antibody production.

[0052] 4. Antibody preparation and purification

[0053] The positive clones obtained by screening were cultured for hybridoma cells, and the supernatant was harvested for protein A purification or hybridoma gene sequencing and recombinant expression to obtain monoclonal antibodies. The titer of AIBP mouse monoclonal hybridoma supernatant was detected by ELISA method, as shown in Table 1.

[0054] Table 1. ELISA method for detecting the titer of AIBP mouse monoclonal hybridoma supernatant

[0055]

[0056] Example 2: Binding performance test of anti-AIBP monoclonal antibody and AIBP protein

[0057] 1. Material information

[0058] name Specifications / Article Number Manufacturer Rabbit Anti-mouse IgG-HRP NA Sinobio AIBP protein 13421-H08H Sinobio Mouse IgG NA Sinobio Goat anti-mouse IgG-HR NA abcam

[0059] 2. Main instruments and equipment

[0060] Instrument Name model factory Multifunctional ELISA reader Spectrammax M5 Molecular Devices

[0061] 3. Test steps

[0062] 3.1 Coating: Coat AIBP protein at 4°C overnight, with a coating concentration of 100 μg / mL, 100 μL / well;

[0063] 3.2 Blocking: Wash the plate 3 times with PBST, pat dry, add 3% BSA / PBST solution, and incubate at room temperature for 2 hours;

[0064] 3.3 Sample dilution and addition:

[0065] Anti-AIBP mouse monoclonal antibody (MM02) was diluted to 1 μg / mL with 1% BSA / PBST, and then diluted 4-fold to a total of 8 concentrations. The plate was washed 3 times with PBST and patted dry. 100 μL of the above sample was added to each well of the ELISA plate and incubated at room temperature for 2 hours;

[0066] 3.4 Add secondary antibody for detection:

[0067] Wash the plate 4 times with PBST and pat dry. Dilute goat anti-mouse IgG-HRP with 1% BSA / PBST at 1:5000, add 100 μL / well to the corresponding wells of the plate, and incubate at room temperature for 1-2 hours;

[0068] 3.5 Color development: Wash the plate three times with PBST and pat dry. Add 100 μL / well of TMB substrate to the ELISA plate and incubate at room temperature in the dark for about 20 minutes.

[0069] 3.6 Termination: Add 100 μL 1M H3PO4 to each well to terminate the reaction;

[0070] 3.7 Reading the plate: After terminating the color development reaction, read the measurement value at a wavelength of 450nm and record the OD value.

[0071] 4. Test results

[0072] The results are shown in Table 2. The anti-AIBP mouse monoclonal antibody MM02 can specifically bind to AIBP with a stronger affinity than MM05 and MM06.

[0073] Table 2. Comparison of binding affinity between AIBP monoclonal antibodies MM02, MM05 and MM06 and AIBP

[0074] AIBP-mAb ID OD-AIBP-mAb OD-Blank MM02 5A9C5A5 1.893 1.325 MM05 4E1F2E5F8 0.905 0.603 MM06 6H9E5E4H8 0.744 0.652

[0075] Example 3: Verification of the inhibition of AIBP protein binding to human umbilical vein endothelial cells (HUVECs) by anti-AIBP monoclonal antibodies

[0076] 1. Material information

[0077]

[0078]

[0079] 2. Main instruments and equipment

[0080] Instrument Name model factory Multifunctional ELISA reader Spectrammax M5 Molecular Devices

[0081] 3. Test steps

[0082] 3.1 Couple AIBP protein to fluorescence: Mix Cy3-NHS fluorescent group (1 mM) with AIBP recombinant protein (500 ng), stir overnight at 4°C, purify the mixture using a 10KD ultrafiltration tube, and adjust the AIBP mixture to 100 ng / uL.

[0083] 3.2 Cell seeding: HUVECs were seeded into 96-well plates at a density of 5000 cells per well and cultured in a cell culture incubator for 4 hours to allow them to adhere to the plate.

[0084] 3.3 Pretreatment of monoclonal antibodies: Anti-AIBP monoclonal antibodies were combined with AIBP recombinant protein (1 μg / mL antibody corresponding to 100 ng / mL AIBP recombinant protein) in a water bath at 37 degrees Celsius for 30 minutes.

[0085] 3.4 Cell treatment: Incubate HUVECs with blank control (medium control), AIBP-Cy3 (100 ng / mL) / IgG, and AIBP-Cy3 / anti-AIBP monoclonal antibody at 4°C for 2 hours. After incubation, wash the plate three times with PBS to remove excess unbound complexes.

[0086] 3.5 Plate reading: Use a multifunctional microplate reader to read the fluorescence value of each well.

[0087] 4. Test results and conclusions:

[0088] Results: Figure 1 As shown, compared with the blank control, AIBP + The relative fluorescence intensity of the group was significantly increased, P < 0.01; + Compared with the AIBP group + MM02 + The relative fluorescence intensity of the group was significantly decreased, P < 0.01; + Compared with the AIBP group + HDL + MM05 + Group and AIBP + HDL + MM06 + There was no significant difference in relative fluorescence intensity among the groups.

[0089] Conclusion: Monoclonal antibody MM02 significantly inhibits the binding of AIBP to human umbilical vein endothelial cells; monoclonal antibodies MM05 and MM05 do not inhibit the binding of AIBP to human umbilical vein endothelial cells.

[0090] Example 4: Anti-AIBP monoclonal antibody promotes HUVECs proliferation

[0091] 1. Material information

[0092]

[0093]

[0094] 2. Main instruments and equipment

[0095] name Part Number Manufacturer Inverted fluorescence microscope NA Leica

[0096] 3. Test steps

[0097] 3.1 Cell seeding: HUVECs were seeded into 96-well plates at a density of 5000 cells per well and cultured in a cell culture incubator for 4 hours until they adhered to the plate.

[0098] 3.2 Pretreatment of monoclonal antibodies: Anti-AIBP monoclonal antibodies were combined with AIBP recombinant protein (1 μg / mL antibody corresponding to 100 ng / mL AIBP recombinant protein) in a water bath at 37 degrees Celsius for 30 minutes.

[0099] 3.3 Cell treatment: blank control (medium control), HDL / AIBP, HDL / AIBP (100 ng / mL) / IgG, and HDL / AIBP / anti-AIBP monoclonal antibody were incubated in a cell culture incubator at 37°C and 5% CO2 for 16 hours.

[0100] 3.4 Prepare 2X EdU working solution: Since the EdU working solution is added to the well plate in an equal volume with the culture medium, it needs to be prepared into a 2X working solution. The recommended final concentration of EdU is 10μM (1X). Dilute EdU (10mM) with cell culture medium at 1:500 to obtain 2X EdU working solution (20μM).

[0101] 3.5 Add an equal volume of 2X EdU working solution (20μM) preheated at 37℃ to the 6-well plate, so that the final concentration of EdU in the 6-well plate becomes 1X. For example, if the final concentration is designed to be 10μM and the original culture medium in the 6-well plate is 1mL, then add 1mL of 2X EdU working solution (20μM) to the well plate. If the volume of the culture medium is too large, you can first remove an appropriate amount of culture medium and then add an equal volume of 2X EdU working solution; or you can reduce the volume of the working solution and increase the concentration of EdU so that the final EdU concentration in the culture medium is 10μM, for example, add 220μL 0.1mM EdU to 2mL of culture medium. Replacing all the culture medium may affect cell proliferation, so it is not recommended to replace all the culture medium.

[0102] 3.6 Continue to incubate the cells for 2 hours.

[0103] 3.7 After EdU labeling, remove the culture medium and add 1 mL of fixative solution for 15 minutes at room temperature. Note: For flow cytometry, adherent cells are trypsinized and resuspended in culture medium before fixation.

[0104] 3.8 Remove the fixative solution and wash the cells three times with 1 mL of washing solution per well, each time for 3-5 minutes.

[0105] 3.9 Remove the washing solution, add 1 mL of permeabilization solution (you can use Biotech's strong immunostaining permeabilization solution P0097, immunostaining washing solution P0106, or PBS containing 0.3% Triton X-100) to each well, and incubate at room temperature for 10-15 minutes.

[0106] 3.10 Remove the permeabilization solution and wash the cells 1-2 times with 1 mL of washing solution per well, each time for 3-5 minutes.

[0107] 3.11 Preparation of Click Additive Solution: For C0075S, dissolve one tube of Click Additive with 1.3 mL of deionized water and mix until all dissolved to obtain Click Additive Solution; for C0075L, dissolve one bottle of Click Additive provided in the kit with 10.4 mL of deionized water and mix until all dissolved to obtain Click Additive Solution. After preparation, the solution can be appropriately divided and stored at -20°C.

[0108] 3.12 Add 0.5 mL of Click reaction solution to each well and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample.

[0109] 3.13 Incubate at room temperature in the dark for 30 minutes.

[0110] 3.14 Aspirate the Click reaction solution and wash three times with washing solution, each time for 3-5 minutes.

[0111] 3.15 Add 1 mL of 1X Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 minutes to stain the cell nuclei.

[0112] 3.16 The maximum excitation wavelength of Azide 555 is 555nm, and the maximum emission wavelength is 565nm. High-definition images were taken using a fluorescence microscope.

[0113] 4. Test results and conclusions:

[0114] Results: Figure 2 As shown, compared with the blank control, AIBP + HDL + The percentage of EDU-positive cells in the group was significantly reduced, P < 0.01; + HDL + Compared with the AIBP group + HDL + MM02 + The percentage of EDU-positive cells in the group was significantly increased, P < 0.01; + HDL + Compared with the AIBP group + HDL + MM05 + Group and AIBP + HDL + MM06 + There was no significant difference in the percentage of EDU-positive cells among the three groups.

[0115] Conclusion: AIBP protein significantly inhibits the proliferation of HUVECs; anti-AIBP monoclonal antibody MM02 can significantly improve the inhibition of HUVECs proliferation; anti-AIBP monoclonal antibodies MM05 and MM06 did not significantly improve the inhibition of HUVECs proliferation.

[0116] Example 5: Anti-AIBP monoclonal antibody promotes collateral circulation formation and blood flow recovery in lower limb ischemia model mice

[0117] 1. Material information

[0118] name Part Number Manufacturer C57 / BL male mice NA Hunan Slake Company Mouse IgG NA Sigma SMA Antibodies NA Sigma CD31 Antibody NA Abcam (UK) Ki67 Cat#16667 Abcam (UK) F4 / 80 Cat#6640 Abcam (UK) FABP4 Cat#13979 Abcam (UK) COUP-TFII Cat#PP-H7147-00 RD(USA) CXCR4 Cat#181020 Abcam (UK) Donkey vs Sheep 647 Cat#A32849 Thermo (USA) Donkey anti-rabbit 488 Cat#A32790 Thermo (USA) Donkey anti-rat 488 Cat#A32766 Thermo (USA)

[0119] 2. Main instruments and equipment

[0120] name Part Number Manufacturer Laser Doppler blood flowmeter NA OMEGAFLO Confocal fluorescence microscopy LSM900 Zeiss

[0121] 3. Test steps

[0122] 3.1 Mouse femoral artery ligation model: 12-week-old male mice were anesthetized with gas anesthetics, and the hair on both lower limbs was removed with depilatory cream. The skin was disinfected with iodine or alcohol. The skin was incised at the groin of the left lower limb, and the femoral artery, femoral vein and femoral nerve were carefully separated. The femoral artery was ligated with non-absorbable sutures, and the skin was sutured and disinfected after ligation. The mice were placed on a warm blanket for recovery.

[0123] 3.2 Mouse administration: AIBP monoclonal antibody (MM02) or isotype mouse IgG was injected into mice via the tail vein at a dose of 100 ug per B6j mouse (25 g) in the femoral artery ligation model at the following time points: 1 day before surgery, the day of surgery (0 day after surgery), 1 day, 2 days, 3 days, 7 days, 10 days, 12 days, and 14 days after surgery.

[0124] 3.3. Laser Doppler scanning: The lower limbs of mice were shaved, and after femoral artery ligation (days -1, 0, 7, 14, and 21), the left and right lower limbs were scanned with an LDI analyzer (Moor Infrared Laser Doppler Imager Instrument, Wilmington, Delaware) to measure blood flow. The animals were placed on a heating pad under 1% isoflurane anesthesia. The blood perfusion level of the ischemic hindlimb and the normal hindlimb was quantified by the average pixel value within the ROI, and the relative change of hindlimb blood flow was expressed by the ratio of blood perfusion detected by laser Doppler on the left (ischemic) and right (normal) sides.

[0125] 3.4 Immunofluorescence of frozen sections: Slides were rinsed twice with PBS and then incubated with PBST (PBS + 0.1% Triton) at room temperature for 10 minutes to permeabilize the tissue. Slides were rinsed twice with PBS, blocked with 3% goat serum and 3% BSA in PBS for 2 hours, and then incubated with primary antibodies diluted in blocking buffer at 4°C overnight. Slides were rinsed with PBS and then incubated with secondary antibodies at room temperature for 1 hour. Slides were washed again with PBST (PBS + 0.01% Triton) and mounted with DAPI. Primary antibodies were directed against CD31 (1:250), SMA-Cy3 (1:300), Ki67 (1:100), F4 / 80 (1:100), FABP4 (1:250), COUP-TFII (1:250), CXCR4 (1:100). Secondary antibodies included donkey anti-sheep 647 (1:500), donkey anti-rabbit 488 (1:500), and donkey anti-mouse 488 (1:500). Images were collected using a confocal fluorescence microscope, and image data were analyzed using ImageJ to investigate the arterial density (collateral circulation generation) in the adductor muscles of the lower limbs after administration of anti-AIBP monoclonal antibodies and IgG in the lower limb ischemia model mice.

[0126] 4. Test results

[0127] 4.1 As Figure 3 A and Figure 3 As shown in B, 7 days, 14 days and 21 days after surgery, the blood perfusion of ischemic lower limbs of mice in the anti-AIBP monoclonal antibody MM02 group was significantly better than that in the IgG group, and the P values ​​were all < 0.01.

[0128] 4.2 As Figure 4 A and Figure 4 As shown in B, 21 days after surgery, the number of collateral arteries in the ischemic lower limbs of mice in the anti-AIBP monoclonal antibody MM02 group was significantly increased compared with that in the IgG group, P < 0.01.

[0129] 5. Test conclusion

[0130] Anti-AIBP monoclonal antibody MM02 promotes blood perfusion and collateral artery formation in ischemic lower limbs of mice with femoral artery ligation model.

[0131] Example 6: Anti-AIBP mouse monoclonal antibody gene sequencing

[0132] Based on the experimental results of immunization, fusion monoclonalization, binding performance testing with AIBP protein, verification of inhibition of AIBP protein binding to human umbilical vein endothelial cells (HUVECs), promotion of HUVECs proliferation, and promotion of collateral circulation formation and blood flow recovery in lower limb ischemia model mice, the MM02 monoclonal antibody cell line was selected for antibody gene sequencing.

[0133] The steps are: extract total RNA from cells and reverse to obtain cDNA. PCR amplification obtains the heavy chain and light chain variable region genes of the antibody, sequencing and bioinformatics analysis of the sequence, and eliminating non-functional antibody genes. The antibodies are named according to the cell clones from which they are derived. The amino acid sequences of the heavy chain and light chain and their variable regions are shown in Table 2

[0134] Table 2. Amino acid sequences of heavy chain variable region and light chain variable region of MM02 monoclonal antibody

[0135]

[0136] The amino acid sequence of the heavy chain variable region of the MM02 monoclonal antibody is shown in SEQ ID NO:7:

[0137] QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCASATQPYGYAMDYWGQGTSVTVSS

[0138] The amino acid sequence of the light chain variable region of the MM02 monoclonal antibody is shown in SEQ ID NO: 8:

[0139] QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK

[0140] The amino acid CDR sequence of the MM02 monoclonal antibody is shown in Table 3.

[0141] Table 3. CDRs and sequence numbers of anti-AIBP mouse monoclonal antibody MM02

[0142]

[0143]

[0144] The CDR sequence of the MM02 monoclonal antibody nucleotide is shown in Table 4.

[0145] Table 4. Nucleotide CDRs and sequence numbers of anti-AIBP mouse monoclonal antibody MM02

[0146]

[0147] The amino acid sequence of the heavy chain of the MM02 monoclonal antibody is shown in SEQ ID NO.9:

[0148] MEWIWIFLFILSGTAGVQSQVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCASATQPYGYAMDYWGQGTSVTVSSESQSFPNVF PLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNKDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVE SGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCT VTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY

[0149] The amino acid sequence of the light chain of the MM02 monoclonal antibody is shown in SEQ ID NO.10:

[0150] MDFQVQIFSFLLISASVILSRGQIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPW TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0151] The present invention prepares and discovers an anti-AIBP monoclonal antibody, which has high affinity and avidity to AIBP antigen and has a significant promoting effect on collateral circulation.

[0152] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An anti-AIBP monoclonal antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3; The light chain variable region includes LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

2. The anti-AIBP monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-AIBP monoclonal antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The anti-AIBP monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain of the anti-AIBP monoclonal antibody is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:

10.

4. Use of the anti-AIBP monoclonal antibody according to any one of claims 1 to 3 in the preparation of a medicament for treating lower limb ischemia.

5. A polynucleotide, characterized in that The nucleotide sequence encodes the anti-AIBP monoclonal antibody according to claim 1.

6. Use of the anti-AIBP monoclonal antibody according to any one of claims 1 to 3 in the preparation of a product for detecting AIBP expression.

7. A kit comprising the anti-AIBP monoclonal antibody according to any one of claims 1 to 3.

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