Human anti-vascular endothelial cell growth factor antibody and application thereof

An antibody, whole antibody technology, applied in the direction of anti-growth factor immunoglobulin, application, antibody, etc.

Active Publication Date: 2013-04-03
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as far as the current development status is concerned, there is still a certain gap between the successful cases of antibody library technology and transgenic mice

Method used

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  • Human anti-vascular endothelial cell growth factor antibody and application thereof
  • Human anti-vascular endothelial cell growth factor antibody and application thereof
  • Human anti-vascular endothelial cell growth factor antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Large-scale screening of large-capacity phage antibody library

[0057] 1. Materials and methods:

[0058] 1. Materials: A large-capacity fully synthetic phage single-chain antibody library was constructed by the Academy of Military Medical Sciences of the Chinese People's Liberation Army (ZL200910091261.8), with a library capacity of 1.35×10 10 . The antigen for screening the antibody library was recombinant VEGF165 expressed by HEK293T cells (concentration: 1 mg / ML), the strain was XLI-Blue (Stratagene, USA); the phage used was M13KO7 (Invitrogen, USA). The eukaryotic expression vectors pABG1, pABκ and pABL are preserved in this laboratory, and the vector information can be found in ( Figure 7A , Figure 7B , Figure 7C ), mammalian HEK293T cells were purchased from Invitrogen.

[0059] 2. Method

[0060] 2.1 Preparation of recombinant protein antigen VEGF165:

[0061] The VEGF165 gene (gifted by Dr. Minglei Shi from the Institute of Bioengineering, ...

Embodiment 2

[0081] Example 2 Computer-aided design of in vitro affinity maturation of anti-VEGF antibody VA6

[0082] 1. Experimental method

[0083] 1.1 Construction of 3D structure model of VA6-VEGF complex

[0084] 1.1.1 Database search for amino acid sequences

[0085] Using the variable region sequences of the light and heavy chains of antibody VA6 as probes, in the latest protein structure database (PDB), the Fasta3.0 program was used to search for sequences, and found highly homologous to the light and heavy chains of VA6, respectively. Reference protein, the homology between the reference protein sequence and the antibody sequence obtained by the search is more than 40%, and the homology between the complex and ICZ8\VEGF complex crystallization reaches 94% after structural superposition, which meets the requirements for obtaining a high-precision structural model .

[0086] 1.1.2 Homology modeling of 3D structures

[0087] Homology modeling was carried out using the program mo...

Embodiment 3

[0107] Example 3 Construction and screening of VA6-9B light chain Fab recombinant phage antibody library

[0108] 1. Materials and methods

[0109] 1. Construction of Phage Fab Antibody Library

[0110] In order to obtain mutant antibodies with higher affinity, the heavy chain of VA6-9B was selected as the object, and the Vκ light chain gene library was constructed through the chain exchange strategy, which was combined with the heavy chain of VA6-9B to construct the light chain mutation Antibody library H9B-Vκ. References for light chain gene library Vκ gene design (Knappik A, Ge LM, Honegger A, et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 2000, 296: 57- 86; Dewey's doctoral dissertation: Academy of Military Medical Sciences, 2006), the framework gene sequence of Vκ is shown in SEQ ID No.35. The amino acid sequences of its CDR1 region and CDR2 region are RAS...

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Abstract

The invention provides a human anti-vascular endothelial cell growth factor (VEGF) antibody as well as a coding gene and an application thereof. According to the invention, by virtue of genetic engineering means and a phage surface display technology, an anti-VEGF genetic engineering single-chain antibody is screened from a complete synthesis single-chain human antibody library, a variable region gene sequence of the anti-VEGF genetic engineering single-chain antibody is obtained, amino acids in a CDR (complementary determining region) of the anti-VEGF genetic engineering single-chain antibody are transformed by virtue of bioinformatics means, and finally a series of high-affinity anti-VEGF antibodies are obtained by constructing and screening mutation libraries and utilizing a chain exchange method, wherein affinity of the anti-VEGF antibodies with human VEGF is 10<-9>-10<-11>M. According to the invention, identification on immunocompetence and biological activity of the antibody is completed, the anti-VEGF antibody is verified to have the characteristic of being antagonistic to combination of VEGF and VEGFR2 and can effectively inhibit proliferation of vascular endothelial cells induced by VEGF. The invention provides a new specific candidate antibody molecule for resisting tumour and senile macular degeneration targeted by VEGF in future.

Description

technical field [0001] The present invention relates to the preparation and application of human genetic engineering antibody for treatment, which is mainly specific to new blood vessels, especially VEGF, which is a key factor in the formation of new blood vessels in malignant tumors and retinopathy, and has the ability to inhibit tumors and other diseases related to the formation of new blood vessels The biological activity of VEGF in the medium, so as to achieve the therapeutic or diagnostic genetic engineering antibody for inhibiting the growth and metastasis of malignant tumors and various intraocular neovascular lesions. Background technique [0002] Neovascularization is implicated in the pathogenesis of a variety of conditions, including solid tumors, proliferative retinopathy, age-related macular degeneration, arthritis, and psoriasis. In solid tumors, angiogenesis enables tumor cells to obtain a favorable growth environment and the ability to self-proliferate. A la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N15/63C12N1/21C12N1/19C12N5/10A61K39/395A61P35/00G01N33/68C12N15/85C07K19/00
Inventor 孙志伟王双曾大地常红艳张畅仇玮祎孙九如范志和杨涛范铁炯
Owner SHANGHAI SERUM BIOTECH
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