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Anti-VEGF monoclonal antibody and preparation method and application thereof

A monoclonal antibody and variable region technology, applied in the field of biomedicine, can solve the problems of high price, low affinity, and high price of bevacizumab

Active Publication Date: 2019-01-04
SHANGHAI HENLIUS BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the price of bevacizumab is relatively expensive. In the drug bidding and procurement organized by various provinces in China from 2016 to 2017, the lowest winning bid price was more than 5,000 yuan (100mg / bottle). One administration, 3-4 bottles (100mg / bottle) each time, the individual patient needs to spend nearly 30,000 to 40,000 yuan on bevacizumab for only one month
[0006] Therefore, the existing anti-VEGF monoclonal antibodies also have defects such as few types, poor selectivity, low affinity and high price.

Method used

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  • Anti-VEGF monoclonal antibody and preparation method and application thereof
  • Anti-VEGF monoclonal antibody and preparation method and application thereof
  • Anti-VEGF monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Expression and Purification of Human VEGF Extracellular Region Recombinant Protein

[0059] Synthetic human VEGF-Fc fusion gene (synthesized by Nanjing GenScript Biotechnology Co., Ltd.), wherein the VEGF gene sequence is SEQ ID NO: 13, the Fc gene sequence is SEQ ID NO: 14, and the two sequences are connected to form VEGF-Fc For the fusion gene, design the forward and reverse primers as follows:

[0060] 5'-atccgccggcaagccgccaccatgaactttctgctgtcttgg-3' (SEQ ID NO: 15) and

[0061] 5'-tcactacgtatcatttacccggagacaggggagaggctc-3' (SEQ ID NO: 16), used to amplify the gene encoding VEGF-Fc fusion protein, introducing restriction endonuclease sites Ngo MIV and SnaBI at the upstream and downstream of the gene, PCR conditions 95°C for 10s, 60°C for 10s, 72°C for 15s, 35 cycles, and 72°C for 10min. The DNA polymerase was the product of TAKARA.

[0062] The PCR product was recovered by agarose gel (the recovery kit is a product of Omega bio-tek company), purified, and...

Embodiment 2

[0064] Example 2 Construction, expression, purification and identification of anti-VEGF human antibody eukaryotic expression vector

[0065] Antibody heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:7) genes were synthesized by Nanjing GenScript Biotechnology Co., Ltd. Use PCR reaction to amplify the coding DNA sequences of the heavy chain and light chain above, and introduce appropriate enzyme cutting sites at both ends of the gene of the heavy chain and light chain of the antibody. Ngo MIV and Sna BI restriction sites were introduced respectively, and the sequences of the forward and reverse primers were:

[0066] 5'-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3' (SEQ ID NO: 17) and

[0067] 5'-GCTCTACGTATCATTTACCTGGAGACAGGGAGAGGC-3' (SEQ ID NO: 18), Ngo MIV and SnaB I were respectively introduced at the 5' end and 3' end of the antibody light chain variable region gene, and the forward and reverse primer sequences were respectively:

[0068] 5'-ACCTGCCGGCAAGCCGCC...

Embodiment 3

[0073] Example 3 Affinity Identification of Anti-VEGF Human Antibody

[0074]Antibody affinity identification. The VEGF recombinant protein prepared in Example 1 was coated onto an ELISA plate at 2 µg / ml (working volume 30 µL), and left standing at 4°C overnight. Wash 3 times with regular phosphate buffered saline (PBST) containing 0.05% Tween 20. Block with 5% skimmed milk for 1 hour at room temperature. Wash 3 times with PBST, prepare the monoclonal antibody 2VA7 prepared in Example 2 to a concentration of 26.5 µg / ml, and perform 3-fold serial dilutions, a total of 8 gradients, add 30 µl to each well, and let stand at room temperature for 1 hour. Wash 3 times with PBST, add 30 μl 1:4000 dilution of horseradish peroxidase-labeled goat anti-human light chain constant region secondary antibody (Millipore company product), and let stand at room temperature for 1 hour. Wash 4 times with PBST, add TMB for color development, and use 2M H 2 SO 4 Stop the reaction. Read at 450n...

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Abstract

The invention belongs to the field of biomedical antibodies, and specifically discloses an anti-VEGF monoclonal antibody and a preparation method and application thereof. The anti-VEGF monoclonal antibody has favorable bioactivity, is capable of effectively combining VEGF proteins and better enclosing the VEGF proteins to be combined with other acceptors at a protein level and a cell level. The monoclonal antibody can be independently applied to tumor immunotherapy and tumor diagnosis and screening or combined with other anti-tumor drugs to be applied to tumor immunotherapy and tumor diagnosisand screening, and has favorable prospect for preparing drugs for treating tumors and resisting autoimmune diseases.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-VEGF monoclonal antibody and its preparation method and application. Background technique [0002] The formation and development of tumors are closely related to neovascularization. Usually, tumors start from a single abnormal cell, which can remain dormant for a long period of time due to the very close distance to the available capillary bed. Some tumor cells may switch to an angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillaries. These newly formed blood vessels not only promote the continued growth of the primary tumor, but also allow the dissemination and recolonization of metastatic tumor cells. Compared with normal cells in solid tumors, neovascularization gives tumor cells a growth advantage over normal cells. In breast cancer and some other tumors, a correlation between microvessel density and patient survival has been o...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/13C12N15/85C12N5/10C12P21/08A61K39/395A61P35/00A61P37/02A61P31/00A61P37/06
CPCA61K2039/505A61P31/00A61P35/00A61P37/02A61P37/06C07K16/22C12N15/85
Inventor 何虹霖姜伟东
Owner SHANGHAI HENLIUS BIOTECH INC
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