Human blood vessel endothelial growth factor antigen epitope and epitope vaccine thereof

A vascular endothelium and growth factor technology, applied in anti-tumor drugs, antibody medical components, peptide/protein components, etc., can solve the problems of high treatment cost and antibody heterogeneity, and achieve low clinical use and good immunogenicity. , the effect of low clinical treatment cost

Active Publication Date: 2013-02-20
祖泽再生医学科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a novel human VEGF epitope vaccine with potential medical or pharmaceutical value, so as to realize the active immunotherapy of tumors and solve the problem of antibody heterogeneity in the treatment of tumors by anti-human VEGF antibody drugs currently used clinically. Insufficiency, high cost of treatment, etc.

Method used

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  • Human blood vessel endothelial growth factor antigen epitope and epitope vaccine thereof
  • Human blood vessel endothelial growth factor antigen epitope and epitope vaccine thereof
  • Human blood vessel endothelial growth factor antigen epitope and epitope vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Analysis and verification of human VEGF epitope

[0059] Protein epitope analysis methods generally include synthetic peptide verification method, computer-aided program prediction method, and protein three-dimensional structure analysis method, but any method has limitations, and the antigen epitope prediction based on protein three-dimensional structure is accurate. The highest degree. At present, two or more analysis methods are generally combined to predict the epitope of a protein, and finally to determine whether an epitope is a real epitope must be verified by experiments.

[0060] The present invention predicts the most likely human VEGF antigen epitope by using computer-aided program prediction method and protein three-dimensional structure analysis method, and then verifies whether the epitope is the real antigen epitope through experiment.

[0061] First, use the BepiPred (http: / / www.cbs.dtu.dk / services / BepiPred / ) epitope prediction program to init...

Embodiment 2

[0106] Example 2: Construction, expression and purification of human VEGF epitope vaccine expression vector

[0107] The human single domain antibody BT32 / A6 (Journal of Biology Chemistry 2001, 276: 24774-24780) was camelized, that is, the 9th, 10th and 12th amino acids of FR2 of the human single domain antibody BT32 / A6 Respectively replaced by glutamic acid, arginine and glycine, its sequence is shown in SEQ ID NO: 9, (see attached Figure 8 , wherein the double underlined part is the framework region 2 (FR2); the framed part is the amino acid after camelization transformation; the single underlined part is the CDR3 region).

[0108] Replace the appended with human VEGF epitope sequence "QIMRIKPHQGQHIGEM" Figure 8 The "DRLKVEYYDSSGYYVSRFGA" amino acid sequence of the CDR3 region in the middle, thus forming the amino acid sequence of a new protein, its amino acid sequence is shown in SEQ ID NO: 10, (as attached Figure 9 , wherein the double underlined part is the amino aci...

Embodiment 3

[0125] Embodiment 3: Analysis of immune effect of human VEGF epitope vaccine

[0126] Five female Balb / c mice aged 6-8 weeks were immunized with the human VEGF epitope vaccine obtained in Example 2, and the immune effect of the vaccine was observed.

[0127] The dose of immunization was 50 μg per mouse, the site of immunization was the abdominal cavity, and the immunization procedure was on the 0th, 14th, 21st and 35th days. Freund's complete adjuvant was used for the first immunization, Freund's incomplete adjuvant was used for the second and third immunization, and no adjuvant was added for the last immunization. Seven days after the last immunization, blood was collected to test the immune effect of the vaccine.

[0128] The pre-immune serum and post-immune serum of five animals were diluted 10,000 times, and then ELISA method was used to detect their recognition of human VEGF, and the coating concentration of human VEGF was 2 μg / ml. The test results showed that, after 4 ...

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Abstract

The invention relates to an antigen epitope and of a human blood vessel endothelial growth factor, and an epitope vaccine thereof, and belongs to the field of biotechnology. The invention discloses the antigen epitope of the human blood vessel endothelial growth factor, and also discloses the epitope vaccine of the human blood vessel endothelial growth factor built on the basis of a single domain antibody skeleton. The epitope vaccine can effectively induce animals to produce antibodies for the human blood vessel endothelial growth factor, presents relatively good antineoplastic growth effects in the bodies of the animals, and can be used for drugs of solid tumors such as tumors and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antigenic epitope of human vascular endothelial growth factor, and a human vascular endothelial growth factor epitope vaccine designed by using the antigenic epitope, and the epitope vaccine can be used for solid tumors such as liver cancer treat. Background technique [0002] Tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) and its receptor plays an important role in the occurrence and development of tumors. Anti-tumor angiogenesis is one of the important directions in the research and development of anti-tumor drugs. The most successful anti-tumor angiogenesis drug so far is Avastin produced by Roche. Avastin is an antibody drug, and the antigen molecule it recognizes is human VEGF. At present, Avastin has been approved by the US FDA for the treatment of advanced colorectal cancer, recurrent glioma and renal tumors. [0003] Avastin is pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K19/00C12N15/11C12N15/63A61K39/00A61K38/16A61P35/00
Inventor 高建恩罗时伟钱军
Owner 祖泽再生医学科技(北京)有限公司
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