Use of an anti-ADM antibody in the treatment of tumor vascular normalization

Abstract

The application belongs to the field of biological medicine, and relates to application of an anti-ADM antibody in tumor blood vessel normalization treatment. The application provides a novel mouse anti-ADM monoclonal antibody and application thereof in GBM blood vessel normalization treatment. Specifically, the mouse anti-ADM monoclonal antibody disclosed in the application can inhibit the biological activity of ADM by combining with the 1-10 amino acid sequence of ADM, although the combination does not block the combination of ADM and the receptor CRLR. The mouse anti-ADM monoclonal antibody of the application can inhibit the tumor blood vessel endothelial cell proliferation mediated by ADM, and simultaneously inhibit the breakage of intercellular adhesion connection of endothelium, and induce the normalization of microvessel structure. Through implementation of the application, a new idea can be provided for personalized tumor blood vessel targeted treatment of GBM patients with high expression of ADM.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of an anti-ADM antibody in tumor angiogenesis normalization therapy. Background Technology

[0002] Glioblastoma (GBM, WHO grade 4) is a common primary malignant tumor of the central nervous system, characterized by short survival and poor prognosis, posing a serious threat to human health. Abundant angiogenesis is one of the most prominent pathological features of GBM, providing essential nutrients, oxygen, and various growth factors for rapid tumor growth, high invasiveness, and drug resistance / relapse. Traditional anti-angiogenic therapy mainly focuses on inhibiting angiogenesis and destroying existing tumor blood vessels, restricting the tumor's blood supply, 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 atrophy and degeneration, causing extreme tumor hypoxia, ultimately resulting in increased drug resistance or invasiveness of tumor cells (Huang M. et al. New insights into anti-angiogenic therapy resistance in cancer: Mechanisms and therapeutic aspects. Drug Resist Updat. 2022, 64: 100849.). Vascular normalization therapy mainly focuses on improving abnormal and leaking blood vessels within the tumor. 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 pressure, the therapeutic effect is improved. [3-6]In general, anti-angiogenic therapy 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), currently the only approved drug for recurrent GBM, reduces tumor angiogenesis by blocking the binding of vascular endothelial growth factor (VEGF) to its receptor (VEGFR). Although it improves progression-free survival, it does not improve overall survival (Sandmann T. et al. Patients With Proneural Glioblastoma May Derive Overall Survival BenefitFrom the Addition of Bevacizumab to First-Line Radiotherapy and Temozolomide: Retrospective Analysis of the AVAglio Trial. J Clin Oncol. 2015, 33(25):2735-44.), and new anti-angiogenic drugs or treatment strategies still need to be developed. The rational use of anti-angiogenic drugs can repair abnormal tumor vascular systems before angiogenesis occurs, normalizing tumor vascular structure and more effectively transporting oxygen and drugs to the tumor parenchyma, thereby improving the sensitivity to radiotherapy and chemotherapy. However, although tumor vascular normalization offers hope for improving anti-cancer efficacy, the "window" for 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 therapeutic strategies that target GBM vascular structural abnormalities to induce vascular normalization is of clinical significance.

[0003] The necrotic area of ​​GBM is surrounded by a large number of tumor-associated macrophages enriched with hypoxia response (hypoxic phenotype TAM). Hypoxic phenotype TAM highly secretes adrenomedullin (ADM), a calcitonin superfamily polypeptide composed of 52 amino acids. There are abundant microvessels around GBM necrosis, and 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 phosphorylation of VE-cadherin, an endothelial cell adhesion junction protein, promoting the rupture of VE-cadherin junctions between endothelial cells, leading 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 its biological activity and induce vascular normalization is clinically significant. Currently, there are monoclonal antibodies against ADM (1-21) (application publication number: CN 110167962A), which can increase the half-life of ADM in serum, blood, and plasma, and are used to treat congestion in patients with heart failure or kidney disease. While the monoclonal antibody targeting ADM (1-10) of this invention does not prevent ADM from binding to the receptor CRLR, it can inhibit ADM-mediated phosphorylation of downstream AKT and VE-cadherin in human brain microvascular cells (HBMEC), ultimately reducing tumor endothelial cell proliferation and endothelial adhesion junction disruption, inhibiting tumor microangiogenesis, and inducing vascular normalization, providing a new strategy for the treatment of GBM vascular normalization. Summary of the Invention

[0005] In view of this, the purpose of this 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 this invention, while not blocking the binding of ADM to its receptor CRLR by binding to the 1-10 amino acid sequence of ADM, can inhibit the biological activity of ADM. The murine anti-ADM monoclonal antibody of this invention can inhibit ADM-mediated tumor vascular endothelial cell proliferation, while simultaneously inhibiting the disruption of interendothelial adhesion junctions and inducing microvascular structure normalization. Through the implementation of this invention, a new approach can be provided for personalized tumor vascular targeted therapy for GBM patients with high ADM expression.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a monoclonal antibody or antigen-binding fragment that specifically binds to ADM, wherein 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 variable region of the light chain 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] In one 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] In one specific embodiment, the monoclonal antibody or antigen-binding fragment that specifically binds to ADM has the amino acid sequence of the heavy chain as shown in SEQ ID NO:9 and the amino acid sequence of the light chain as shown in SEQ ID NO:10. The heavy chain is of type IgG1 and the light chain is of type Kappa.

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

[0018] In one implementation, the nucleic acid encoding the heavy chain is shown in SEQ ID NO:13, and the nucleic acid encoding the light chain is shown in SEQ ID NO:14.

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

[0020] The present invention also provides a host cell containing the above-described vector. As one specific embodiment, the host cell may be an Expi CHO cell.

[0021] This invention also provides the application of the above-mentioned monoclonal antibody or antigen-binding fragment that specifically binds to ADM in the preparation of ADM blocking agents or glioblastoma vascular normalization therapy drugs.

[0022] First, through human brain microvascular endothelial cell (HBMEC) proliferation experiments, this invention found that the mouse anti-ADM monoclonal antibody significantly inhibited the promoting effect of human recombinant protein ADM on HBMEC proliferation. This invention also 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. Second, through cell immunofluorescence experiments, this invention found that the mouse anti-ADM monoclonal antibody significantly reversed the reduction in membrane expression of the adhesion junction protein VE-cadherin induced by human recombinant protein ADM in HBMEC. Therefore, the mouse anti-ADM monoclonal antibody of this invention can be used as an ADM blocking agent in the preparation of drugs for the treatment of glioblastoma vascular normalization.

[0023] The present invention also provides a glioblastoma vascular normalization therapy, wherein the active ingredient in the drug includes the aforementioned monoclonal antibody or antigen-binding fragment that specifically binds to ADM.

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

[0025] (1) In the HBMEC proliferation experiment, the mouse anti-ADM monoclonal antibody of the present invention significantly inhibited 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 cell immunofluorescence experiment, the mouse anti-ADM monoclonal antibody of the present invention significantly restored the membrane expression of VE-cadherin, the adhesion linker protein of HBMEC induced by human recombinant protein ADM.

[0028] In summary, the mouse anti-ADM monoclonal antibody of the present invention can be used as an ADM blocking agent to inhibit GBM microvascular angiogenesis and vascular structure normalization, thereby achieving a therapeutic effect.

[0029] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0031] Figure 1 For the binding affinity detection of ADM monoclonal antibody to antigen ADM(1-10)-BSA; A: ELISA test of the affinity of ADM monoclonal antibody-B077 / -A022 / -A033 / -B195 to antigen ADM-(1-10)-BSA; B: EC50 result of ADM monoclonal antibody-B077 / -A022 / -A033 / -B195.

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

[0033] Figure 3 Mouse anti-ADM monoclonal antibody-B077 induces adhesion of human brain microvascular endothelial cells to recombinant human protein ADM.

[0034] Inhibitory effect of VE-cadherin cleavage.

[0035] Figure 4 The mouse anti-ADM monoclonal antibody-B077 targets the human recombinant protein ADM-mediated AKT in human brain microvascular endothelial cells.

[0036] Inhibition of VE-cadherin activation. Detailed Implementation

[0037] The following specific examples illustrate the implementation 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 embodiments, and 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): The polypeptide was synthesized chemically according to the amino acid sequence MKLVSVALMY of ADM(1-10). Then, ADM-(1-10)-BSA and ADM-(1-10)-KLH were synthesized using solid-phase synthesis. The crude products were purified multiple times by liquid chromatography, and the molecular weight and amino acid composition of the purified products were determined by mass spectrometry and amino acid analysis.

[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 immunization in mice. Specifically, mice were cross-immunized sequentially with ADM-(1-10)-KLH for the first immunization and ADM-(1-10)-BSA for the second immunization. Immunization was performed via multiple intraperitoneal and subcutaneous injections. For the first and fifth immunizations, each mouse received 100 μg of antigen emulsified with 100 μL of Freund's complete adjuvant. For subsequent immunizations, each mouse received 50 μg of antigen emulsified with 100 μL of Freund's incomplete adjuvant. Immunization was performed every two weeks. Immunization titers were measured by ELISA after the third immunization. A serum titer greater than 40,000 was considered a successful immunization.

[0044] II. Construction of Mouse Immune Antibody Library

[0045] The construction and screening method of mouse immune antibody libraries was based on Example 4 of Chinese Invention Patent Application CN117050165A. RNA was extracted from spleen cells of immunized mice and reverse transcribed into cDNA to obtain all antibody nucleotide sequences. The light and heavy chain genes were amplified using molecular cloning technology. The antibody genes were recombined using in vitro ligation. The vector and Fab gene sequences were digested with enzymes to construct the antibody nucleotide sequences into a phage display vector. The constructed vector was then electroporated into *E. coli* to obtain two mouse-derived immune phage display libraries. The library volumes of the two Fab libraries were determined using the dilution plating method. The correct insertion rate of the antibody genes was verified by single-clone sequencing analysis. The sequenced antibody sequences were further analyzed using the NCBI website to check whether the inserted antibody sequences were mouse antibodies, examining the CDR3 of the light and heavy chains, and confirming the number of specific sequences. Based on the sequence analysis results of the two antibody libraries, it was determined that all inserted antibody sequences were mouse-derived antibodies.

[0046] III. 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 immunoassay tubes, and the tubes were coated overnight at 4°C. The next day, the coating solution was discarded, and the tubes were blocked with PBS containing 5% skim milk powder for 2 hours. After rinsing with PBS, the prepared phages were added and incubated for 2 hours. Rinsing was performed to remove non-specifically bound phages, and then 0.8 mL of PBS containing 0.5% skim milk powder was added to the immunoassay tubes. EDTA trypsin digestion solution was used to elute phages that specifically bind to the target antigen. The eluted phages were then used to infect logarithmic-phase Escherichia coli SS320 (Lucigen, 60512-1), which was incubated at 37°C for 30 min, then cultured at 220 rpm for 1 h. VSCM13 helper phages were then added and incubated for 30 min, followed by another 1 h of culture at 220 rpm. The culture was then centrifuged and transferred to C+ / K+ 2×YT medium, and cultured overnight at 30°C and 220 rpm. The phages were prepared on the second day and used in the second round of screening, and this process was repeated. The enrichment levels of the different output sets obtained from the first, second, and third rounds were detected by ELISA. The enriched output sets were then screened by ELISA to identify positive clones that specifically bind. The positive clones were then sequenced and the sequences were compared using IgBlast to obtain molecules with unique sequences. The molecules with unique sequences were then subjected to diversity analysis to remove sequences containing glycosylation modification sites, free cysteine ​​residues, and identical CDR regions. The remaining molecules were then constructed to their full length.

[0048] 3. Purification of target antibody

[0049] Preparation of target antibody: The gene sequence of the target antibody was constructed into the pcDNA3.4 vector, and the antibody was transiently expressed using Expi CHO cells. 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 greater than 95%). The obtained target antibody is the ADM monoclonal antibody.

[0050] Example 2. Antigen-antibody affinity ranking detection

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

[0052] 1. Plate preparation: The concentration of antigen ADM(1-10)-BSA is 2 μg / mL. Add 30 μL / well to each well of a 96-well ELISA plate and incubate overnight at 4°C.

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

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

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

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

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

[0058] The results are as follows Figure 1 As shown, ELISA experiments revealed that the ADM monoclonal antibody-B077 / -A022 obtained in Example 1 exhibited greater affinity activity for the antigen ADM(1-10)-BSA, while the ADM monoclonal antibody-A033 / -B195 showed weaker affinity activity. More preferably, the ADM monoclonal antibody-B077 had the lowest EC50 value, at 0.005987 μg / mL.

[0059] For 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 shown in SEQ ID NO:11, and the nucleic acid encoding the light chain variable region is shown in SEQ ID NO:12. Specifically, for the ADM monoclonal antibody-B077, the amino acid sequence of its heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of its light chain is shown in SEQ ID NO:10; the heavy chain is of the IgG1 type, and the light chain is of the Kappa type; the nucleic acid encoding the heavy chain is shown in SEQ ID NO:13, and the nucleic acid encoding the light chain is shown in SEQ ID NO:14. Example 3. Mouse anti-ADM monoclonal antibody significantly inhibited the promoting effect of human recombinant protein ADM on the proliferation of HBMECs.

[0060] 1. HBMEC culture: HBMECs (purchased from Cell Systems, #ACBRI376) were cultured in endothelial cell medium (supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001).

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

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

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

[0064] 5. Count the cells from step 3 and add them to the four groups of culture media from step 4. Mix thoroughly and seed 1200 cells / well into a 96-well plate. Prepare the four groups of culture media from step 4 every 2 days and replenish the medium at 50 μL / well.

[0065] 6. On day 6, CCK-8 was added, and the OD values ​​of each group were measured to compare the proliferation capacity of HBMEC in each group.

[0066] This embodiment uses a cell proliferation assay to detect the proliferation of HBMEC cells. The results are as follows: Figure 2 As shown, compared with the control group, the addition of human recombinant protein ADM (purchased from Bachem) promoted HBMEC proliferation. When human recombinant protein ADM and mouse anti-ADM monoclonal antibody-B077 / -A022 were added simultaneously, the proliferation capacity of HBMEC in the mouse anti-ADM monoclonal antibody-B077 group was significantly reduced, while the proliferation capacity of HBMEC in the mouse anti-ADM monoclonal antibody-A022 group was not reduced. The addition of mouse anti-ADM monoclonal antibody-B077 / -A022 did not affect HBMEC proliferation. This indicates that mouse anti-ADM monoclonal antibody-B077, as a blocking antibody, can effectively inhibit the promoting effect of ADM on HBMEC proliferation.

[0067] Example 4. Mouse anti-ADM monoclonal antibody-B077 significantly reversed the breakage of HBMEC adhesion junctions induced by human recombinant protein ADM.

[0068] 1. HBMEC culture: Same as in Example 3;

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

[0070] 3. HBMEC cells were seeded at 25,000 cells / well in confocal dishes and cultured for 48 hours to obtain a fused monolayer with good cell-to-cell contact.

[0071] 4. After starving HBMEC cells for 16 h with endothelial cell culture medium (without FBS and endothelial cell growth additives, purchased from Sciencell, Endothelial Cell Medium, #1001), cells were treated with human recombinant protein ADM and / or mouse anti-ADM monoclonal antibody-B077 for 30 min (cells were divided 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). The supernatant was discarded, cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with VE-cadherin antibody and DAPI. Cells were imaged using a Zeiss LSM900 confocal microscope. VE-cadherin expression and membrane / cytoplasmic distribution in HBMEC cells of each group were compared and quantitatively analyzed.

[0072] This embodiment uses cell immunofluorescence assays to detect the membrane expression of the adhesion junction protein VE-cadherin in HBMECs. The results are as follows: Figure 3 As shown, compared with the control group, human recombinant protein ADM induced a decrease in VE-cadherin membrane expression in HBMECs, while mouse anti-ADM monoclonal antibody-B077 did not affect VE-cadherin expression. When human recombinant protein ADM and mouse anti-ADM monoclonal antibody-B077 were added simultaneously, VE-cadherin membrane expression increased, indicating that mouse anti-ADM monoclonal antibody, as a blocking antibody, can effectively inhibit ADM-induced HBMEC adhesion junction breakage.

[0073] Example 5. Mouse anti-ADM monoclonal antibody-B077 inhibits ADM-mediated AKT and VE-cadherin activation.

[0074] 1. HBMEC culture is the same as in Example 3;

[0075] 2. Take HBMEC cells in good growth condition, discard the culture medium, wash once with PBS, add 1 mL of Accutase enzyme for 3 min digestion, and terminate digestion with endothelial cell culture medium (added with 2% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% endothelial cell growth additive, purchased from Sciencell, Endothelial cell medium, #1001).

[0076] 3. HBMEC cells were seeded at 25,000 cells / well in confocal dishes and cultured for 48 hours to obtain a fused monolayer with good cell-to-cell contact.

[0077] 4. After starving HBMEC cells for 16 h with endothelial cell culture medium (without FBS and endothelial cell growth additives, purchased from Sciencell, Endothelial cell medium, #1001), the cells were treated with human recombinant protein ADM and / or mouse anti-ADM monoclonal antibody-B077 for 15 min (the cells were divided 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). The supernatant was discarded, the cells were washed once with PBS, and the prepared RIPA protein lysis buffer (RIPA: protease / phosphatase inhibitor = 100:1) was added to the cells. The cells were then lysed on ice for 30 min.

[0078] 5. Aspirate the lysis buffer and centrifuge at 15000g for 15 minutes at 4°C;

[0079] 6. Transfer the supernatant to a centrifuge tube and determine 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℃ metal bath for 10 min.

[0081] 8. Take 20 μg of protein and 5 μL of marker and slowly add them to the electrophoresis lane of the electrophoresis tank after removing the comb. Run electrophoresis at a constant voltage of 80V for 120 min.

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

[0083] 10. After electroporation, place the PVDF membrane in the prepared 5% skim milk powder and seal it on a shaker at room temperature for 60 minutes.

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

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

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

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

[0088] 15. Develop using an ECL kit.

[0089] This embodiment uses Western blot to detect the activation of AKT and VE-cadherin in HBMEC, and the results are as follows: Figure 4 As 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 act as a blocking antibody to inhibit ADM-mediated activation of AKT and VE-cadherin 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 embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within 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 includes CDR1, CDR2, and CDR3. Among them, CDR1 is GYTFTEYTMH; CDR2 is GINPNNGLTK; CDR3 stands for RDYFARRFAPPDN; The variable region of the light chain of the antibody or antigen-binding fragment includes CDR1, CDR2, and CDR3. Among them, CDR1 is KASENVGTYVS; CDR2 is GASNRYT; CDR3 is GQSYSYPLT.

2. The monoclonal antibody or antigen binding fragment specifically binding to ADM according to claim 1, characterized in that, The antibody or antigen-binding fragment 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.

3. The monoclonal antibody or antigen-binding fragment specifically binding to ADM according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the antibody or antigen-binding fragment is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is 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 of claim 4, wherein Nucleic acids encoding the heavy chain variable region are shown in SEQ ID NO:11, and nucleic acids encoding the light chain variable region are shown in SEQ ID NO:

12.

6. The nucleic acid of claim 4, wherein The nucleic acid encoding the heavy chain is shown in SEQ ID NO:13, and the nucleic acid encoding the light chain is 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 of claim 7.

9. The use of the monoclonal antibody or antigen-binding fragment that specifically binds to ADM as described in any one of claims 1-3 in the preparation of a glioblastoma vascular normalization therapy.

10. A glioblastoma vascular normalization therapeutic drug, characterized by, The active ingredient in the drug includes a monoclonal antibody or antigen-binding fragment that specifically binds to ADM as described in any one of claims 1-3.

Images