Anti-VEGF antibody and application thereof in preparation of glioblastoma therapeutic agent

Abstract

The invention relates to an anti-VEGF antibody, a heavy chain variable region sequence is shown as SEQ ID NO: 1, and a light chain variable region sequence is shown as SEQ ID NO: 2. According to the present invention, the monoclonal antibody capable of specifically binding to VEGF is prepared, the binding of VEGF and its receptor can be inhibited after the monoclonal antibody is bound to VEGF, the downstream signal transduction pathway of VEGF is blocked, and the animal test results show that the antibody can inhibit the growth of glioblastoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an anti-VEGF antibody and an application thereof in preparing a glioblastoma therapeutic agent. Background Art

[0002] Glioblastoma (GBM) is a highly aggressive malignant tumor of the central nervous system, commonly found in middle-aged and elderly individuals. GBM originates from astrocytes within the brain. These tumor cells are highly proliferative and invasive, rapidly infiltrating surrounding normal brain tissue to form ill-defined lesions.

[0003] GBM treatment faces numerous challenges. Firstly, its high heterogeneity and invasive nature make complete treatment difficult; secondly, the presence of the blood-brain barrier limits the effectiveness of some drugs. However, in recent years, with the continuous advancement of medical research, new treatments and drugs, such as immunotherapy and targeted therapy, have been explored and developed, offering a glimmer of hope for GBM treatment.

[0004] VEGF is a key angiogenic factor overexpressed in GBM. It strongly stimulates the proliferation and migration of vascular endothelial cells, thereby promoting angiogenesis. This provides oxygen and nutrients for tumor growth and invasion, enabling rapid proliferation and spread. Therefore, anti-VEGF therapy is an important treatment for GBM. Commonly used anti-VEGF antibodies include bevacizumab.

[0005] However, there are still few effective antibodies against VEGF, and more anti-VEGF antibodies need to be developed for the preparation of anti-tumor drugs. Summary of the Invention

[0006] To solve the above problems, the present invention provides an anti-VEGF antibody, wherein the heavy chain variable region sequence is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 2.

[0007] The present invention also provides the use of the anti-VEGF antibody in preparing a VEGF protein detection agent.

[0008] The present invention also provides a nucleic acid encoding the above anti-VEGF antibody.

[0009] The present invention also provides an expression vector, which comprises the above nucleic acid and a promoter for initiating transcription of the nucleic acid.

[0010] In a specific embodiment, the expression vector is an adenovirus expression vector.

[0011] The present invention also provides the use of the above-mentioned anti-VEGF antibody, nucleic acid or expression vector in the preparation of a glioblastoma therapeutic agent.

[0012] The present invention prepares a monoclonal antibody that can specifically bind to VEGF. After binding to VEGF, the monoclonal antibody can inhibit the binding of VEGF to its receptor and block the downstream signal transduction pathway of VEGF. Animal experiments have shown that the antibody can inhibit the growth of glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Mouse antiserum titer test.

[0014] Figure 2 This is the effect of monoclonal antibody V1-15 on the binding of VEGF to its receptor.

[0015] Figure 3 The effect of cloned antibody V1-15 on glioblastoma. DETAILED DESCRIPTION

[0016] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0017] 1. Mouse Immunization and Antiserum Production Human recombinant VEGF protein (VEGF 189 , amino acid sequence as shown in SEQ ID NO: 3) was cross-linked with keyhole limpet hemocyanin (KLH) to obtain an immunogen.

[0018] 6-8 week old Balb / c mice were primed with an emulsified mixture of 50 μg of immunogen and 50 μl of complete Freund's adjuvant. Every three weeks, booster immunizations were administered with 25 μg of immunogen and 50 μl of incomplete Freund's adjuvant. Starting with the second booster immunization, blood was collected for antiserum titer determination one week after immunization. When the antiserum titer reached the required level, the spleens of the mice were harvested for hybridoma electrofusion.

[0019] Antiserum titers were determined by ELISA. Plates were coated with HPV16 VLP protein at a concentration of 0.2 μg / ml. Serially diluted antiserum or purified antibody (100 μl per well) was added (pre-immune mouse serum was used as a control) to each well. The plates were incubated at 37°C for 1.5 hours and washed twice. A 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) secondary antibody was added to each well. The plates were incubated at 37°C for 1 hour. After washing 4-6 times, 100 μl of TMB substrate was added and incubated at 37°C for 10 minutes. The reaction was terminated with 50 μl of 0.2 M H₂SO₄, and the OD₄450nm was measured. The serum titer for ELISA was determined as the highest dilution at which the OD₄450 was greater than 2.1 times that of the blank control and greater than 0.2.

[0020] The results are as follows Figure 1 As shown, the titer of mouse antiserum is high, indicating that mouse serum contains specific antibodies.

[0021] 2. Screening of Hybridoma Cell Lines The mice were intraperitoneally injected with the immunogen for shock immunization after three immunizations. Three days later, the spleens of the mice were harvested and spleen cell suspensions were prepared for electrofusion with SP2 / O cells.

[0022] After fusion, the hybridoma cells were screened by testing their affinity to the VEGF protein. Among them, a hybridoma cell line V1-15 was selected, which can produce anti-VEGF monoclonal antibodies.

[0023] 3. Monoclonal Antibody Preparation and Sequencing The cell lines were injected into the peritoneal cavity of mice. After approximately seven days, ascites was extracted when the abdomens became distended. Monoclonal antibodies were purified using Protein G affinity purification, and their purity was determined by SDS-PAGE. The results showed that the antibody purity exceeded 90%.

[0024] Total RNA was extracted from cells and reverse transcribed to amplify cDNA of the antibody mRNA for sequencing to obtain the amino acid sequence of the monoclonal antibody V1-15. The heavy chain variable region sequence is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 2.

[0025] The dissociation constant Kd of monoclonal antibody V1-15 and VEGF was determined to be 5.76 nM, indicating that it has a high affinity for VEGF.

[0026] 4. Effect of monoclonal antibody V1-15 on the binding ability of VEGF to receptors Monoclonal antibody V1-15 was mixed with VEGF protein and incubated at room temperature for at least 30 minutes. The antibody-protein mixture was then added to a solid matrix embedded with VEGF receptors and incubated at room temperature for 1 hour. Nonspecifically bound molecules were washed away, and the VEGF content on the solid matrix was measured. VEGF protein without monoclonal antibody V1-15 was used as a control.

[0027] The results are as follows Figure 2 As shown, the binding of monoclonal antibody V1-15 to VEGF greatly reduced the binding of VEGF to its receptor, suggesting that the antibody can block the signaling pathway downstream of VEGF.

[0028] 5. Effects of monoclonal antibody V1-15 on glioblastoma Preparation of nude mouse transplant tumor model: U-87 MG (human astroglioma cells) was prepared into a mixture containing 10 6 A cell suspension containing 10 cells / mL was prepared, and cell viability was determined to be >90% using trypan blue staining. The cells were injected subcutaneously into the right axilla of nude mice. The maximum long diameter (a) and the maximum short diameter perpendicular to it (b) of the tumor were measured every three days, and tumor volume was calculated using the formula V = ab² / 2.

[0029] Nude mice were divided into four groups. The animal treatment group received only tumor cell transplantation. The control treatment group received tumor cell transplantation and local injection of mouse anti-human IgG. Treatment group 1 received local injection of monoclonal antibody V1-15 100 mg / kg daily starting from the first day of tumor cell transplantation. Treatment group 2 received local injection of AAV vector containing the monoclonal antibody V1-15 gene expression cassette on the first day of tumor cell transplantation.

[0030] The results are as follows Figure 3 As shown, the monoclonal antibody V1-15 and its AAV expression vector of the present invention both have an inhibitory effect on tumor growth.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-VEGF antibody, characterized in that The heavy chain variable region sequence is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO:

2.

2. Use of the anti-VEGF antibody according to claim 1 in the preparation of a VEGF protein detection agent.

3. A nucleic acid, characterized in that Encodes the anti-VEGF antibody of claim 1.

4. An expression vector, characterized in that The invention comprises the nucleic acid according to claim 3 and a promoter for initiating transcription of the nucleic acid.

5. The expression vector according to claim 4, characterized in that The expression vector is an adenovirus expression vector.

6. Use of the anti-VEGF antibody according to claim 1, the nucleic acid according to claim 3, or the expression vector according to claim 4 or 5 in the preparation of a therapeutic agent for glioblastoma.

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