Application of anti-ADM antibody in tumor blood vessel normalization treatment

Abstract

The invention belongs to the field of biological medicine, and relates to an application of an anti-ADM antibody in tumor blood vessel normalization treatment. The invention provides a novel mouse anti-ADM monoclonal antibody and an application of the novel mouse anti-ADM monoclonal antibody in GBM blood vessel normalization treatment. Specifically, the mouse anti-ADM monoclonal antibody disclosed by the invention is combined with 1-10 amino acid sequences of ADM, although the combination of ADM and a receptor CRLR is not blocked, the biological activity of ADM can be inhibited. The mouse anti-ADM monoclonal antibody provided by the invention can inhibit ADM-mediated tumor vascular endothelial cell proliferation, inhibit interendothelial adhesion connection fracture, and induce capillary structure normalization. Through the implementation of the invention, a new thought can be provided for providing personalized tumor blood vessel targeted therapy for GBM patients with high ADM expression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of an anti-ADM antibody in the treatment of tumor vascular normalization. Background Art

[0002] Glioblastoma multiforme (GBM, WHO grade 4) is a common primary malignant tumor of the central nervous system. Patients have a short survival period and poor prognosis, seriously threatening human health. Abundant neovascularization is one of the most prominent pathological features of GBM, providing necessary nutrients, oxygen, and various growth factors for the rapid growth, high invasiveness, and drug-resistant recurrence of tumors. Traditional anti-angiogenic therapies mainly focus on inhibiting neovascularization and destroying existing tumor blood vessels to limit the blood supply to tumors, thereby "starving" tumor cells. Anti-angiogenic therapy can significantly reduce tumor volume in the short term (Gilbert MR. et al. A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med. 2014, 370(8): 699-708.), but long-term treatment can lead to vascular rarefaction and degeneration, causing extreme hypoxia in tumors and ultimately increasing the drug resistance or invasiveness of tumor cells (Huang M. et al. New insights into antiangiogenic therapy resistance in cancer: Mechanisms and therapeutic aspects. Drug Resist Updat. 2022, 64: 100849.). Vascular normalization therapy mainly focuses on improving abnormally structured and leaky blood vessels within tumors. By reducing vascular tortuosity and dilation, enhancing vascular structural integrity to improve blood perfusion and oxygen supply, enhancing anti-tumor drug delivery, and reducing interstitial fluid pressure, the therapeutic effect can be improved. [3-6]Overall, anti-angiogenic therapy mainly focuses on blocking the formation of new blood vessels, while vascular normalization therapy focuses on improving the structure and function of existing blood vessels. Bevacizumab (BEV), the only currently approved drug for recurrent GBM, reduces the formation of new tumor blood vessels by blocking the binding of vascular endothelial growth factor (VEGF) to its receptor (VEGFR). Although it improves the progression-free survival of patients, it does not improve the overall survival (Sandmann T. et al. Patients With Proneural Glioblastoma May Derive Overall Survival Benefit From the Addition of Bevacizumab to First-Line Radiotherapy and Temozolomide: Retrospective Analysis of the AVAglio Trial. J Clin Oncol. 2015, 33(25):2735-44.). There is still a need to develop new anti-angiogenic drugs or treatment strategies. Rational use of anti-angiogenic drugs can repair the abnormal tumor vascular system before blood vessel regression, normalize the tumor vascular structure, and more effectively transport oxygen and drugs to the tumor parenchyma, thereby enhancing the sensitivity to radiotherapy and chemotherapy. However, although tumor vascular normalization offers hope for improving anti-cancer efficacy, the "window" period of vascular normalization is short (Jain RK. Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med. 2001, 7(9):987-9.). Therefore, developing new treatment strategies targeting the abnormal vascular structure of GBM to induce vascular normalization has clinical significance.

[0003] A large number of tumor-associated macrophages enriched with hypoxia response (hypoxic phenotype TAM) infiltrate around the necrotic area of GBM. Adrenomedullin (ADM), highly secreted by hypoxic phenotype TAM, is a calcitonin superfamily polypeptide composed of 52 amino acids. There are abundant microvessels around the necrosis of GBM, and the microvascular endothelial cells express the ADM receptor CRLR (Calcitonin Receptor Like Receptor). Our study found that ADM can bind to CRLR on endothelial cells, induce AKT phosphorylation, and promote endothelial cell proliferation; at the same time, it induces the phosphorylation of the endothelial cell adhesion junction protein VE-cadherin, promotes the cleavage of VE-cadherin junctions between endothelial cells, and leads to vascular leakage (Wang W. et al. Identification of hypoxic macrophages in glioblastoma with therapeutic potential for vasculature normalization. Cancer Cell. 2024, 42(5): 815-832.).

[0004] Therefore, developing monoclonal antibodies targeting ADM to block the biological activity of ADM and induce vascular structural normalization is of clinical significance. Currently, there is a monoclonal antibody against ADM(1-21) (Publication No.: CN 110167962A), which can increase the half-life of ADM in serum, blood, and plasma and is used to treat congestion in patients with heart failure or kidney disease. The monoclonal antibody targeting ADM(1-10) of the present invention, although it does not prevent ADM from binding to the receptor CRLR, can inhibit the phosphorylation of downstream AKT and VE-cadherin in human brain microvascular endothelial cells (HBMEC) mediated by ADM, ultimately reducing tumor endothelial cell proliferation and the cleavage of adhesion junctions between endothelial cells, inhibiting tumor microvascular neovascularization and inducing vascular structural normalization, and will provide a new strategy for the treatment of GBM vascular normalization. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a novel murine anti-ADM monoclonal antibody and its application in the treatment of GBM vascular normalization. Specifically, the murine anti-ADM monoclonal antibody disclosed in the present invention binds to the 1-10 amino acid sequence of ADM. Although it does not block the binding of ADM to the receptor CRLR, it can inhibit the biological activity of ADM. The murine anti-ADM monoclonal antibody of the present invention can inhibit the proliferation of tumor vascular endothelial cells mediated by ADM, while inhibiting the disruption of endothelial intercellular adhesion junctions and inducing the normalization of microvascular structure. Through the implementation of the present invention, a new idea can be provided for personalized tumor vascular targeted therapy for GBM patients with high ADM expression.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a monoclonal antibody or antigen-binding fragment that specifically binds to ADM. The heavy chain variable region of the antibody or antigen-binding fragment includes:

[0008] CDR1: GYTFTEYTMH (SEQ ID NO:1);

[0009] CDR2: GINPNNGLTK (SEQ ID NO:2);

[0010] CDR3: RDYFARRFAPPDN (SEQ ID NO:3);

[0011] The light chain variable region of the antibody or antigen-binding fragment includes:

[0012] CDR1: KASENVGTYVS (SEQ ID NO:4);

[0013] CDR2: GASNRYT (SEQ ID NO:5);

[0014] CDR3: GQSYSYPLT (SEQ ID NO:6).

[0015] As a specific embodiment, the monoclonal antibody or antigen-binding fragment that specifically binds to ADM includes a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0016] As a specific embodiment, the monoclonal antibody or antigen-binding fragment that specifically binds to ADM has a heavy chain amino acid sequence as shown in SEQ ID NO:9 and a light chain amino acid sequence as shown in SEQ ID NO:10. The heavy chain is of IgG1 type and the light chain is of Kappa type.

[0017] The present invention also provides nucleic acids encoding the antibody or antigen-binding fragment thereof. As a specific embodiment, the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID NO:11, and the nucleic acid encoding the light chain variable region is as shown in SEQ ID NO:12.

[0018] As an embodiment, the nucleic acid encoding the heavy chain is as shown in SEQ ID NO:13, and the nucleic acid encoding the light chain is as shown in SEQ ID NO:14.

[0019] The present invention also provides a vector containing the above nucleic acid. As a specific embodiment, the vector is the pcDNA3.4 vector.

[0020] The present invention also provides a host cell containing the above vector. As a specific embodiment, the host cell can be Expi CHO cells.

[0021] The present invention also provides the use of the above monoclonal antibody or antigen-binding fragment specifically binding to ADM in the preparation of an ADM blocking reagent or a therapeutic drug for glioblastoma vascular normalization.

[0022] First, through the human brain microvascular endothelial cell (HBMEC) proliferation experiment, the present invention found that the mouse anti-ADM monoclonal antibody of the present invention significantly inhibits the promoting effect of human recombinant protein ADM on HBMEC proliferation; the present invention found that the mouse anti-ADM monoclonal antibody exerts its effect by inhibiting the activation of the AKT signaling pathway mediated by human recombinant protein ADM; secondly, through the cell immunofluorescence experiment, the present invention found that the mouse anti-ADM monoclonal antibody of the present invention significantly restores the reduction of the membrane expression of the adhesion junction protein VE-cadherin in HBMEC induced by human recombinant protein ADM. Therefore, the mouse anti-ADM monoclonal antibody of the present invention can be used as an ADM blocking reagent for the preparation of a therapeutic drug for glioblastoma vascular normalization.

[0023] The present invention also provides a therapeutic drug for glioblastoma vascular normalization, and the active ingredient in the drug includes the above monoclonal antibody or antigen-binding fragment specifically binding to ADM.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) In the HBMEC proliferation experiment, the mouse anti-ADM monoclonal antibody of the present invention significantly inhibits the promoting effect of human recombinant protein ADM on HBMEC proliferation.

[0026] (2) The mouse anti-ADM monoclonal antibody of the present invention can inhibit the activation of the AKT signaling pathway mediated by human recombinant protein ADM.

[0027] (3) In the cellular immunofluorescence assay, the murine anti-ADM monoclonal antibody of the present invention significantly restored the reduced membrane expression of the adhesion junction protein VE-cadherin in human recombinant protein ADM-induced HBMECs. <>

[0028] Generally speaking, the murine anti-ADM monoclonal antibody of the present invention can be used as an ADM blocking reagent to inhibit GBM microvascular angiogenesis and normalize blood vessel structure, thereby achieving a therapeutic effect.

[0029] Other advantages, objectives, and features of the present invention will be described to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the examination and research of the following text, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail preferably in conjunction with the accompanying drawings, where:

[0031] Figure 1 Detection of the binding force between the ADM monoclonal antibody and the antigen ADM(1-10)-BSA; A: ELISA test of the affinity between the ADM monoclonal antibodies - B077 / -A022 / -A033 / -B195 and the antigen ADM-(1-10)-BSA; B: EC50 results of the ADM monoclonal antibodies - B077 / -A022 / -A033 / -B195.

[0032] Figure 2 Inhibitory effect of the ADM monoclonal antibody on the proliferation of human brain microvascular endothelial cells induced by human recombinant protein ADM; A: The proliferation ability of HBMECs in the murine anti-ADM monoclonal antibody - B077 group was greatly reduced; B: There was no significant difference in the proliferation ability of HBMECs in the murine anti-ADM monoclonal antibody - A022 group.

[0033] Figure 3 Inhibitory effect of the murine anti-ADM monoclonal antibody - B077 on the adhesion of human brain microvascular endothelial cells induced by human recombinant protein ADM

[0034] Inhibitory effect on the cleavage of the adhesion junction protein VE-cadherin.

[0035] Figure 4 Inhibitory effect of the murine anti-ADM monoclonal antibody - B077 on the activation of AKT and VE-cadherin mediated by human recombinant protein ADM in human brain microvascular endothelial cells

[0036] Inhibitory effect. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Example 1. Preparation of ADM monoclonal antibody

[0039] 1. Antigen preparation

[0040] Synthesis of ADM(1-10): According to the amino acid sequence MKLVSVALMY of ADM(1-10), the polypeptide was synthesized by chemical synthesis method. Then ADM-(1-10)-BSA and ADM-(1-10)-KLH were synthesized by solid-phase synthesis method. The crude products were purified by liquid chromatography for many times, and the molecular weight and amino acid composition of the purified products were determined by mass spectrometry and amino acid analysis techniques.

[0041] 2. Construction and screening of mouse immune antibody library

[0042] I. Mouse immunization

[0043] ADM-(1-10)-BSA and ADM-(1-10)-KLH were used for mouse immunization. Specifically, ADM-(1-10)-KLH was used for the first immunization, and ADM-(1-10)-BSA was used for the second immunization to immunize the mice crosswise in turn; the intraperitoneal and subcutaneous multi-point injection methods were adopted during immunization. The antigen amount for each mouse in the first immunization and the fifth immunization was 100 μg emulsified with 100 μL of Freund's complete adjuvant; the antigen amount for each mouse in the remaining immunizations was 50 μg emulsified with 100 μL of Freund's incomplete adjuvant. Immunization was carried out once every two weeks. After the third immunization, the immune titer was detected by ELISA. The serum titer greater than 40,000 was considered qualified for immunization.

[0044] II. Construction of mouse immune antibody library

[0045] The construction and screening method of mouse immune antibody library are with reference to Example 4 of Chinese invention patent application CN117050165A. The RNA of mouse spleen cells after immunization is extracted, reverse transcribed into cDNA and all antibody nucleotide sequences are obtained. Light and heavy chain genes are obtained by molecular cloning technology amplification. The antibody genes are recombined by the method of in vitro connection. The vector and Fab gene sequence are respectively digested. The antibody nucleotide sequence is constructed into phage display vector. The constructed vector is further transformed into Escherichia coli by electricity to obtain two mouse immune phage display libraries. The storage capacity of two Fab libraries is measured by dilution point plate method. The correct insertion rate of antibody gene is verified by monoclonal sequencing analysis. The antibody sequence after sequencing is further analyzed by NCBI website. It is checked whether the inserted antibody sequence is mouse antibody. The CDR3 of its light and heavy chain is checked and the number of specific sequences is confirmed. According to the sequence analysis results of 2 antibody libraries, it is judged that the inserted antibody sequence is all mouse antibody.

[0046] 3. Screening of Mouse Immune Antibody Library

[0047] In the first round of screening, 4 mL of 30 μg / mL ADM-(1-10)-BSA or ADM-(1-10)-KLH was added to the immunotube and coated overnight at 4°C. The next day, the coating solution was discarded and PBS containing 5% skim milk powder was added to block for 2 h. After rinsing with PBS, the prepared phage was added and incubated for 2 h. After rinsing to remove non-specifically bound phage, 0.8 mL of 0.5% EDTA trypsin digestion solution was used to elute phages that specifically bound to the target antigen; the eluted phages were then used to infect logarithmic phase Escherichia coli SS320 (Lucigen, 60512-1), allowed to rest at 37°C for 30 minutes, and then cultured at 220 rpm for 1 hour. VSCM13 helper phage was then added and allowed to rest for 30 minutes, and cultured at 220 rpm for another hour; the culture was centrifuged and replaced with C+ / K+2×YT medium, and cultured overnight at 30°C and 220 rpm. The next day, phages were prepared and used in the second round of screening, and the process was repeated. The different output sets obtained from the first, second, and third rounds were tested for enrichment by ELISA. The enriched output sets were then screened by ELISA to screen for specifically binding positive clones. The positive clones were sequenced and analyzed. The sequences were aligned by IgBlast to obtain molecules with unique sequences. These molecules were then subjected to diversity analysis, and sequences containing glycosylation modification sites, free cysteines, and identical CDR regions were removed. The remaining molecules were then constructed in their full length.

[0048] 3. Purification of target antibody

[0049] Preparation of the target antibody: The gene sequence of the target antibody was constructed into the pcDNA3.4 vector, and Expi CHO cells were used for transient expression of the antibody. After 7 days of expression, the antibody was purified. The purity of the antibody was detected by SDS-PAGE and size exclusion chromatography (SEC) (antibody purity > 95%), and the obtained target antibody was the ADM monoclonal antibody.

[0050] Example 2. Detection of antigen-antibody affinity ranking

[0051] To screen the binding ability of the candidate antibody (the target antibody obtained in Example 1) to the detection antigen ADM(1-10)-BSA, and to obtain the antibody with the best affinity for subsequent experiments, the following antigen-antibody affinity ranking detection was carried out.

[0052] 1. Coating the plate: The concentration of the antigen ADM(1-10)-BSA was 2 μg / mL, and 30 μL per well was added to a 96-well ELISA plate and left to stand overnight at 4°C.

[0053] 2. Blocking: Wash the plate 3 times with PBST, and add the blocking solution (PBS with 5% skim milk powder) to block at room temperature for 2 h.

[0054] 3. Incubation of the ADM monoclonal antibody: Wash the plate 3 times with PBST, dilute the antibody with PBS containing 1% skim milk powder, add 30 μL per well, and incubate at room temperature for 60 min.

[0055] 4. Incubation of the secondary antibody: Wash the plate 3 times with PBST, add the secondary antibody Goat-Anti-human-IgG-Fc-HRP, and incubate at room temperature for 50 min.

[0056] 5. Color development: Wash the plate 3 times with PBST, and add 30 μL of TMB to each well.

[0057] 6. Termination and reading: Add 2M termination solution to terminate the reaction and detect the OD value (450 nm).

[0058] The results are as Figure 1 shown. Identified by ELISA experiment, the ADM monoclonal antibodies -B077 / -A022 obtained in Example 1 had stronger affinity activity with the antigen ADM(1-10)-BSA, while the ADM monoclonal antibodies -A033 / -B195 had weaker affinity activity with the antigen. More preferably, the EC50 value of the ADM monoclonal antibody -B077 was the smallest, which was 0.005987 μg / mL.

[0059] For the ADM monoclonal antibody - B077, it includes a heavy chain variable region as shown in SEQ ID NO:7 (the heavy chain variable region includes: CDR1: GYTFTEYTMH (SEQ ID NO:1); CDR2: GINPNNGLTK (SEQ ID NO:2); CDR3: RDYFARRFAPPDN (SEQ ID NO:3)) and a light chain variable region as shown in SEQ ID NO:8 (the light chain variable region includes: CDR1: KASENVGTYVS (SEQ ID NO:4); CDR2: GASNRYT (SEQ ID NO:5); CDR3: GQSYSYPLT (SEQ ID NO:6)); the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID NO:11, and the nucleic acid encoding the light chain variable region is as shown in SEQ ID NO:12. Specifically, for the ADM monoclonal antibody - B077, the amino acid sequence of its heavy chain is as shown in SEQ ID NO:9, and the amino acid sequence of its light chain is as shown in SEQ ID NO:10; the heavy chain is of IgG1 type, and the light chain is of Kappa type; the nucleic acid encoding the heavy chain is as shown in SEQ ID NO:13, and the nucleic acid encoding the light chain is as shown in SEQ ID NO:14. Example 3. Mouse anti - ADM monoclonal antibody significantly inhibits the promoting effect of human recombinant protein ADM on the proliferation of HBMEC

[0060] 1. HBMEC culture: HBMEC (purchased from Cell systems, #ACBRI376) was cultured with endothelial cell medium (added with 2% fetal bovine serum, 1% penicillin - streptomycin double - antibody solution, 1% endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001).

[0061] 2. Take HBMEC with good growth status, discard the medium, wash once with PBS, add 1 mL Accutase enzyme and digest for 3 min, and terminate the digestion with the endothelial cell medium in step 1.

[0062] 3. Discard the supernatant, wash the cells once with 5 mL PBS, and resuspend the cells with 1 mL endothelial cell medium (added with 0.05% fetal bovine serum, 1% penicillin - streptomycin double - antibody solution).

[0063] 4. Prepare 4 groups of culture media using the endothelial cell medium from Step 3: control group (endothelial cell medium), human recombinant protein ADM treatment group (endothelial cell medium + 100 nM human recombinant protein ADM), mouse anti-ADM monoclonal antibody treatment group (endothelial cell medium + 0.625 μg / mL mouse anti-ADM monoclonal antibody), human recombinant protein ADM + mouse anti-ADM monoclonal antibody treatment group (endothelial cell medium + 100 nM human recombinant protein ADM + 0.625 μg / mL mouse anti-ADM monoclonal antibody). Mix the 4 groups of culture media thoroughly in a shaker at 4°C for 40 min; the mouse anti-ADM monoclonal antibody is mouse anti-ADM monoclonal antibody-B077 or mouse anti-ADM monoclonal antibody-A022;

[0064] 5. Count the cells from Step 3, add them to the 4 groups of culture media from Step 4 respectively and mix well, then inoculate them into 96-well plates at 1200 cells / well. Re-prepare the 4 groups of culture media in Step 4 every 2 days and replenish the liquid at 50 μL / well;

[0065] 6. Add CCK-8 on the 6th day, detect the OD values of each group, and compare the proliferation ability of HBMEC in each group.

[0066] In this example, the proliferation of HBMEC was detected by a cell proliferation experiment. The results are as Figure 2 shown. Compared with the control group, the addition of human recombinant protein ADM (purchased from Bachem) could promote the proliferation of HBMEC; when human recombinant protein ADM and mouse anti-ADM monoclonal antibody-B077 / -A022 were added simultaneously, the proliferation ability of HBMEC in the mouse anti-ADM monoclonal antibody-B077 group was greatly reduced, while the proliferation ability of HBMEC in the mouse anti-ADM monoclonal antibody-A022 group was not reduced, and the addition of mouse anti-ADM monoclonal antibody-B077 / -A022 did not affect the proliferation of HBMEC. It shows that mouse anti-ADM monoclonal antibody-B077, as a blocking antibody, can effectively inhibit the promoting effect of ADM on the proliferation of HBMEC.

[0067] Example 4. Mouse anti-ADM monoclonal antibody-B077 significantly restores the disruption of adherens junctions induced by human recombinant protein ADM in HBMEC

[0068] 1. Culture of HBMEC: The same as in Example 3;

[0069] 2. Take HBMEC with good growth status, discard the culture medium, wash once with PBS, add 1 mL of Accutase enzyme and digest for 3 min, and terminate the digestion with endothelial cell medium (added with 2% fetal bovine serum, 1% penicillin-streptomycin double antibody solution, 1% endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001);

[0070] 3. Seed HBMECs at 25,000 cells / well in a confocal dish and culture for 48 h to obtain a confluent monolayer with good cell - cell contact.

[0071] 4. After starving HBMECs in endothelial cell medium (without FBS and endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001) for 16 h, treat them with human recombinant protein ADM and / or mouse anti - ADM monoclonal antibody - B077 for 30 min (the cells are divided into 4 groups, namely the control group, the 100 nM human recombinant protein ADM treatment group, the 0.625 μg / mL mouse anti - ADM monoclonal antibody - B077 treatment group, and the 100 nM human recombinant protein ADM + 0.625 μg / mL mouse anti - ADM monoclonal antibody - B077 treatment group). Discard the supernatant medium, wash once with PBS, fix with 4% paraformaldehyde for 30 min, and stain with VE - cadherin antibody and DAPI. Image the cells on a Zeiss LSM900 confocal microscope. Compare the expression and membrane / cytoplasm distribution of VE - cadherin in HBMECs of each group and perform quantitative analysis.

[0072] In this example, the membrane expression of the adhesion junction protein VE - cadherin in HBMECs was detected by immunofluorescence assay. The results are as Figure 3 shown. Compared with the control group, human recombinant protein ADM induced a decrease in the membrane expression of VE - cadherin in HBMECs, while mouse anti - ADM monoclonal antibody - B077 did not affect the expression of VE - cadherin. When human recombinant protein ADM and mouse anti - ADM monoclonal antibody - B077 were added simultaneously, the membrane expression of VE - cadherin increased, indicating that mouse anti - ADM monoclonal antibody, as a blocking antibody, can effectively inhibit the disruption of adhesion junctions induced by ADM in HBMECs.

[0073] Example 5. Inhibition of ADM - mediated AKT and VE - cadherin activation by mouse anti - ADM monoclonal antibody - B077

[0074] 1. Culture HBMECs as in Example 3.

[0075] 2. Take HBMECs with good growth status, discard the medium, wash once with PBS, add 1 mL Accutase to digest for 3 min, and terminate the digestion with endothelial cell medium (supplemented with 2% fetal bovine serum, 1% penicillin - streptomycin double - antibody solution, 1% endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001).

[0076] 3. Seed HBMECs at 25,000 cells / well in a confocal dish and culture for 48 h to obtain a confluent monolayer with good cell-cell contact.

[0077] 4. After starving HBMECs in endothelial cell medium (without FBS and endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001) for 16 h, treat them with human recombinant protein ADM and / or mouse anti-ADM monoclonal antibody - B077 for 15 min (divide the cells into 4 groups: control group, 100 nM human recombinant protein ADM treatment group, 0.625 μg / mL mouse anti-ADM monoclonal antibody - B077 treatment group, and 100 nM human recombinant protein ADM + 0.625 μg / mL mouse anti-ADM monoclonal antibody - B077 treatment group). Discard the supernatant, wash once with PBS, add the prepared RIPA protein lysate (RIPA: protease / phosphatase inhibitor = 100:1) to the cells, and place on ice for 30 min for lysis.

[0078] 5. Aspirate the lysate and centrifuge at 15,000 g for 15 min at 4 °C in a centrifuge.

[0079] 6. Pipette the supernatant into a centrifuge tube, measure the protein concentration according to the instructions of the BCA protein concentration assay kit, measure the absorbance at 562 nm, and calculate the protein concentration.

[0080] 7. Add 4×loading buffer to the protein sample at a volume ratio of 3:1, mix well with a pipette tip, and heat in a 95 °C metal bath for 10 min.

[0081] 8. Slowly add 20 μg of protein and 5 μL of marker to the well in the electrophoresis tank after removing the comb, and run electrophoresis at a constant voltage. The initial voltage is 80 V and run for 120 min. <https: / / www.sciencedirect.com / science / article / pii / S

[0082] 9. After electrophoresis, activate the PVDF membrane with methanol for 5 min, soak it in semi-dry transfer buffer (40 mL 5×Transfer Buffer + 40 mL absolute ethanol + 120 mL ddH2O) together with thick filter paper for 5 min, assemble the electrotransfer clip (in the order from positive electrode to negative electrode: thick filter paper, PVDF membrane, electrophoresis gel, thick filter paper), transfer at 25 V and 2.5 A (constant current and limited voltage) for 20 min.

[0083] 10. After electrotransfer, place the PVDF membrane in the prepared 5% non-fat milk powder and block it at room temperature on a shaker for 60 min.

[0084] 11. Gently wash off the milk powder with PBST, prepare the primary antibody solution with antibody diluent according to the instructions, and incubate overnight at 4 °C on a shaker.

[0085] 12. Wash the PVDF membrane 3 times with PBST at room temperature for 10 min each time;

[0086] 13. Incubate with HRP-Rabbit / Mouse secondary antibody (1:5000) on a shaker at room temperature for 60 min;

[0087] 14. Wash the PVDF membrane 3 times with PBST at room temperature for 10 min each time;

[0088] 15. Develop the image using an ECL kit.

[0089] In this example, the activation of AKT and VE-cadherin in HBMEC was detected by Western blot. The results are as Figure 4 shown. Compared with the control group, the addition of human recombinant protein ADM promoted the phosphorylation of AKT and VE-cadherin in HBMEC, while the addition of mouse anti-ADM monoclonal antibody - B077 did not affect the activation of AKT and VE-cadherin. When human recombinant protein ADM and mouse anti-ADM monoclonal antibody - B077 were added simultaneously, the phosphorylation levels of AKT and VE-cadherin in HBMEC decreased, indicating that mouse anti-ADM monoclonal antibody - B077 can be used as a blocking antibody to inhibit the activation of AKT and VE-cadherin mediated by ADM in HBMEC.

[0090] The above results indicate that the ADM monoclonal antibody - B077 of the present invention can be used for the treatment of glioblastoma vascular normalization.

[0091] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment that specifically binds to ADM, characterized in that, The heavy chain variable region of the antibody or antigen-binding fragment comprises: CDR1: GYTFTEYTMH; CDR2: GINPNNGLTK; CDR3: RDYFARRFAPPDN; The light chain variable region of the antibody or antigen-binding fragment comprises: CDR1: KASENVGTYVS; CDR2: GASNRYT; CDR3: GQSYSYPLT.

2. The monoclonal antibody or antigen-binding fragment specifically binding to ADM according to claim 1, wherein The antibody or antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:

8.

3. The monoclonal antibody or antigen-binding fragment specifically binding to ADM according to claim 1, wherein The amino acid sequence of the heavy chain of the antibody or antigen-binding fragment is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

10.

4. A nucleic acid encoding the antibody or antigen-binding fragment according to any one of claims 1-3.

5. The nucleic acid according to claim 4, wherein, The nucleic acid encoding the heavy chain variable region is as shown in SEQ ID NO:11, and the nucleic acid encoding the light chain variable region is as shown in SEQ ID NO:

12.

6. The nucleic acid according to claim 4, wherein The nucleic acid encoding the heavy chain is as shown in SEQ ID NO:13, and the nucleic acid encoding the light chain is as shown in SEQ ID NO:

14.

7. A vector containing the nucleic acid according to any one of claims 4-6.

8. A host cell containing the vector according to claim 7.

9. Use of the monoclonal antibody or antigen-binding fragment specifically binding to ADM according to any one of claims 1-3 in the preparation of an ADM blocking reagent or a therapeutic drug for glioblastoma vascular normalization.

10. A drug for the treatment of glioblastoma vascular normalization, characterized in that, The active ingredient in the drug comprises the monoclonal antibody or antigen-binding fragment specifically binding to ADM according to any one of claims 1-3.

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