VEGF (Vascular Endothelial Growth Factor) resistant antibody and application thereof

A technology of human antibody and variable region, which is applied in the field of biotechnology and immunology to achieve the effects of growth inhibition, strong antibody antigen binding ability, and inhibition of proliferation of vascular endothelial cells

Inactive Publication Date: 2014-04-23
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human SCDGF exhibits mitogenic activity on ThllTh2 cells in vitro, although its function in the human body is unclear, but one of the possible functions is to play a mitotic role on special types of spinal cord cells in embryonic development, while its effect on blood vessels The role of endothelial cells has not been reported

Method used

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  • VEGF (Vascular Endothelial Growth Factor) resistant antibody and application thereof
  • VEGF (Vascular Endothelial Growth Factor) resistant antibody and application thereof
  • VEGF (Vascular Endothelial Growth Factor) resistant antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Antibody Screening

[0030] In order to avoid the bias of individual immune background and ensure the diversity of the antibody library as much as possible, the peripheral blood of 162 healthy adults and 10 neonatal umbilical cord blood lymphocytes were separated by lymphocyte separation medium, and a total of 2×10 lymphocytes were collected. 9 cells. Total RNA was extracted by Trizol method, reverse transcribed into cDNA, and the variable region genes of different antibody subtypes were amplified by conventional PCR. According to the pDF information of the antibody library vector, the restriction site BssH II, Nhe I and the connecting peptide with the Loxp511 sequence were introduced, and then spliced ​​into ScFv (single-chain antibody: VL-Linker (containing the Loxp511 sequence)-VH, above) by overlapping PCR method BssH II and Nhe I sites) were introduced downstream respectively (for specific methods, see "Biological Library Technology", edited by Shao Ning...

Embodiment 2

[0033] Example 2 Antibody expression and purification

[0034] The obtained 4D3 clone variable region gene was cloned into the eukaryotic expression vector containing the human IgG constant region gene to construct the whole antibody expression vector pCMV-163, its physical map is as follows figure 1 shown. The whole antibody is called FD006 antibody. The obtained eukaryotic expression vector was transfected into CHO cells by liposome-mediated method, and ELISA test (using goat anti-human IgG and horseradish enzyme-labeled goat anti-human IgG to perform double-sandwich ELISA method to detect the supernatant Antibody content (with non-transfected supernatant as negative control and pure human IgG as standard), detect antibody expression in culture supernatant, and screen to obtain monoclonal cell lines with higher expression.

[0035] The results of ELISA experiments showed that the antibody was expressed in the expression supernatant, and the expression level was about 15.3±...

Embodiment 3

[0036] Example 3 Antibody Affinity Determination

[0037] Antibody association and dissociation constants were determined using Fortibio.

[0038] Using Octet RED (Fortebio, USA) instrument, the affinity of anti-VEGF antibody was determined. Antibody concentration diluted in PBS was 10 μg / mL, coated with AHC sensor; PBS was used as control, diluted VEGF concentration was 1 μg / mL, 3 μg / mL, 10 μg / mL, and the binding and dissociation curves of the interaction between anti-VEGF antibody and VEGF were determined; Globe Fitting fits the curve and calculates the binding constant, dissociation constant and affinity constant of the interaction between the two. The results are shown in Table 1, the FD006 antibody affinity is 3.11×10 -10 , slightly higher than Avastin's 6.50×10 -10 .

[0039] Table 1. Affinity analysis of the interaction between Avastin antibody and FD006 antibody and VEGF

[0040]

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PUM

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Abstract

The invention discloses a VEGF (Vascular Endothelial Growth Factor) resistant antibody belonging to the fields of biotechnology and immunology and application of the VEGF resistant antibody. Amino acid sequences of three complementary determining regions (CDR) of heavy chain variable regions of the antibody are respectively shown as SED ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; the amino acid sequences of the three complementary determining regions of light chain variable regions of the antibody are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. According to the VEGF resistant humanized antibody, specific targeting combines with VEGF antigens, and an antibody affinity is 3.11*10<-10>, the binding capacity of the antibody and the antigens is high; the VEGF resistant humanized antibody has a good biological activity and can inhibit cell proliferation of vascular endothelial cells, obviously inhibit tumor growth, and effectively inhibit the growth of corneal neovascularization, and can be used for preparing targeted medicines of specific targeting VEGF antigens or angiogenesis inhibiting medicines.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology, and relates to an anti-VEGF antibody and its application, in particular to an anti-VEGF human antibody and its application. technical background [0002] Angiogenesis plays an important role in the normal growth and development of the human body, such as fetal growth and development, wound healing, female menstruation, splicing of severed limbs, etc. However, abnormal new blood vessels in some abnormal physiological processes can lead to specific diseases, such as cancer, rheumatoid arthritis, degenerative arthritis, diabetic retinopathy, etc. For example, angiogenesis in tumor tissue can promote malignant development, invasion, and metastasis of tumors. Therefore, targeting VEGF to regulate angiogenesis has become an important strategy for the treatment of this type of disease. In 1971, Folkman proposed that tumor growth and metastasis could be inhibited by blocking angiogenesis in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13A61K39/395A61P35/00A61P9/10
Inventor 吕明冯健男乔春霞林周杨静王群黎燕沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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