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Anti-VEGFR (vascular endothelial growth factor receptor) 2 monoclonal antibody and application thereof

A monoclonal antibody and amino acid technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc., can solve problems that hinder the development of antibody drugs

Active Publication Date: 2017-06-27
CHANGCHUN GENESCIENCE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the early stage of the development of antibody drugs, mouse-derived monoclonal antibodies played a major role in promoting the research of human diseases, but because they were derived from mice, they caused severe immune reactions in patients, which seriously hindered the development of antibody drugs

Method used

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  • Anti-VEGFR (vascular endothelial growth factor receptor) 2 monoclonal antibody and application thereof
  • Anti-VEGFR (vascular endothelial growth factor receptor) 2 monoclonal antibody and application thereof
  • Anti-VEGFR (vascular endothelial growth factor receptor) 2 monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Positive antibody (PCAb) gene synthesis, expression vector construction and preparation of antibody

[0063] 1. Referring to the patent CN 1345334A, select an antibody with good biological activity as the positive antibody (PCAb). The amino acid sequence of PCAb is as follows:

[0064] PCAbH, as shown in SEQ ID NO: 13:

[0065] QVKLQQSGAELVGSGASVKLSCTTSGFNIKDFYMHWVKQRPEQGLEWIGWIDPENGDSDYAPKFQGKATMTADSSSNTAYLQLSSLTSEDTAVYYCNAYYGDYEGYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0066] PCAbL, as shown in SEQ ID NO: 14:

[0067] DIELTQSPAIMSASSPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR...

Embodiment 2

[0071] Example 2: Construction of natural human single chain antibody phage display library

[0072] 1. Construction of phagemid vector

[0073] Select pCANTAB5E as the phage display vector, and carry out vector transformation according to the needs of cloning and phage display. The transformation results are as follows: figure 2. The SfiI-NcoI-XhoI+Linker+NheI-NotI sequence (SED ID NO: 15) was gene-synthesized, then digested with SfiI and NotI, and ligated with the pCANTAB5E vector to obtain the transformed vector pCANTAB5E-SF.

[0074] 2. PBMC isolation and mRNA extraction

[0075] Aseptically extract fresh peripheral blood from healthy volunteers, use lymphocyte separation medium (GE) to separate the lymphocytes therein, and use Invitrogen’s reagent (15596-026) extracts 100×10 6 The total RNA of each cell, the result is as follows image 3 shown.

[0076] 3. Antibody library primer design, synthesis and RT-PCR

[0077] According to the antibody gene sequence inform...

Embodiment 3

[0090] Example 3: Phage display and screening of human antibody library

[0091] 1. Phage display and panning of antibody library

[0092] Inoculate 880ml of 2YT-AG medium (containing 100μg / ml ampicillin and 2% glucose) with 100 times the library capacity of the above-mentioned human VH and VL single-chain antibody library, culture at 37°C and 200rpm until OD600=0.5-0.6, add Helper phage with a cell density of 100 times, infect for 1.5 hours, collect the bacteria by centrifugation, resuspend the cells in 400ml 2YT-AK medium (containing 100μg / ml ampicillin and 75μg / ml kanamycin), and culture overnight at 30°C and 200rpm .

[0093] Centrifuge the culture from the previous step at 10,000g at 4°C for 20min, collect the supernatant and add 1 / 4 volume of PEG / NaCl, mix well, and let it stand on ice for 1h; centrifuge at 12,000g at 4°C for 25min, discard the supernatant, and place the centrifuge tube upside down on the plate Remove the liquid on the paper; resuspend the phage pellet...

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Abstract

The invention belongs to the field of antibody drug, and discloses a full human-derived anti-human VEGFR2 monoclonal antibody screened by a phage antibody base technology and prepared by a gene engineering method, and further discloses a carrier including polynucleotide for encoding the monoclonal antibody, a host cell and an application. Through combining with VEGFR2, the monoclonal antibody stops the combination of VEGF with VEGR2, and does not induce receptor dimerization, cause the following tyrosine residue phosphorylation in intracellular tyrosine kinase structure and activate downstream signal access including activation of phospholipase C and increase of calcium ion concentration in the cell and others; the antibody drug triggers endothelial proliferation, survival, cytoskeleton rearrangement, cell migration, gene expression and others, and finally stops the vascular proliferation caused by combination of VEGF and VEGFR2 and induction of dimerization. The monoclonal antibody can be used for treating diseases caused by tumor angiogenesis.

Description

[0001] This application claims the priority of the Chinese patent application with the application number 201710060867.X and the invention title "anti-VEGFR2 monoclonal antibody and its application" filed with the China Patent Office on January 25, 2017, the entire contents of which are hereby incorporated by reference In this application. technical field [0002] The invention belongs to the field of antibody drugs, and in particular relates to a fully human anti-VEGFR2 monoclonal antibody and applications thereof. Background technique [0003] Angiogenesis refers to the sprouting or division of existing blood vessels (capillaries and venules) to generate new blood vessels, which play an important role in many physiological processes, such as embryogenesis, wound healing, inflammatory response, and menstruation. At the same time, angiogenesis also plays an important role in some pathological processes, such as uncontrolled angiogenesis is the main reason for tumor prolifera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61K2039/505C07K16/2863C07K2317/21C07K2317/24C07K2317/56C07K2317/92
Inventor 冯晓金磊王涛
Owner CHANGCHUN GENESCIENCE PHARM CO LTD
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