Fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and preparation method as well as application thereof

一种单克隆抗体、全人源的技术，应用在生物领域，能够解决亲和力不高、未能达到全人源化等问题

Active Publication Date: 2011-08-31
YUEHAI BIOPHARM SHAOXING LTD SHAOXING CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Bevacizumab is a humanized antibody, which retains the mouse CDR region and a small amount of mouse residues in the FR region, and has not yet achieved full humanization, and there is a problem of low affinity

Method used

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  • Fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and preparation method as well as application thereof
  • Fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and preparation method as well as application thereof
  • Fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and preparation method as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0019] Example Preparation of Antibody

[0020] (1) Cloning of human antibody light and heavy chain constant region genes

[0021] Lymphocyte separation fluid (product of Dingguo Biotechnology Development Co., Ltd.) was used to separate healthy human lymphocytes, and total RNA was extracted with Trizol reagent (product of Invitrogen Company). Research, 1982, 10: 4071-4079) respectively designed primers using RT-PCR reaction to amplify the antibody heavy chain and light chain constant region genes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (promega company product). After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO: 1 and SEQ ID NO: 2 show the heavy chain constant region (C H ) nucleotide sequence and amino acid sequence. SEQ ID NO: 3 and SEQ ID NO: 4 show the light chain constant region (C L ) nucleotide sequence and amino acid sequence. The correct clone in this...

experiment example 1

[0034] Experimental Example 1. VEGF Antibody Affinity Determination

[0035] The affinity constant of VEGF antibody was detected using Biacore T100 (Biacore AB, Uppsala, Sweden). VEGF165 (product of R&D Company) was covalently bonded to the CM5 biosensor chip (GE Healthcare) through amino groups, and the fully human antibody 11A7, Bevacizumab, and human antibody 6A6 (according to Chinese patent application number 02111093.X application date 2002 Prepared by the method disclosed in the invention title "Humanized anti-vascular endothelial growth factor monoclonal antibody and its preparation method and pharmaceutical composition" on February 30) and negative control antibody (Rituximab commercially available product) (in PBS / 0.05% TWEEN-20 (ICIAmericas) (detergent) solution was made into different concentrations (2-fold ratio concentration dilution) and passed through the chip at a flow rate of 50 μl / min. After each check, residual antibody was eluted from the immobilized ligan...

experiment example 2

[0038] Experimental example 2. VEGF antibody inhibits HUVEC cell proliferation experiment

[0039] Experimental procedure: take HUVEC cells (Cascade Biologics) in a good growth state, and adjust the cell concentration to 2.5×10 4 / ml, seeded in 96-well cell culture plate, 200μl / well, at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, replace the serum-free medium, continue culturing for 72 hours, add VEGF antibodies with different concentration gradients, and use the anti-CD20 antibody Rituximab as a negative control, take 3 parallel wells for each concentration, incubate at 37°C for 1 hour, add the final concentration 25ng / ml of VEGF165 (R&D) was cultured for 24 hours, then 10 μl [3H].TdR (18.5 kBq / well) was added, and incubated in a 37°C incubator for 7 hours. 3H] liquid scintillation counter for determination. Such as figure 1 shown.

[0040] The results showed that the negative control antibody (Rituximab) could not effectively inhibit VEGF-induced HUVEC c...

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Abstract

The invention relates to the field of biotechnology, in particular to a fully human anti-VEGF (Vascular Endothelial Growth Factor) monoclonal antibody and a preparation method as well as application thereof. In the invention, a large-capacity natural human phage antibody library is constructed; a fully human anti-VEGF antibody 11A7 is screened from the library; an amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID NO: 6; and an amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID NO: 8. The invention also discloses the preparation method of the 11A7 antibody, a nucleotide sequence encoding the 11A7 antibody, a nucleic acid sequence-containing expression vector and a host cell. Compared with a humanized antibody bevacizumab, the 11A7 antibody has higher affinity of the antibody and higher capacity of suppressing proliferation of tumor cells and can be used for obviously suppressing growth of tumors and preparing anti-tumor medicaments.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, the invention discloses a fully human monoclonal antibody, its preparation method and application. Background technique [0002] Tumors, especially malignant tumors, are diseases that seriously endanger human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend. The curative effect of malignant tumors is poor, the rate of late metastasis is high, and the prognosis is often poor. Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve. [0003] The growth of tumor has two distinct stages, that is, from the slow growth period without blood v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00A61P1/00
CPCC07K2317/92C07K2317/21C07K16/22A61K2039/505C07K2317/76C07K2317/73A61P1/00A61P35/00
Inventor 王淑蕙李川阚颖仝昕
Owner YUEHAI BIOPHARM SHAOXING LTD SHAOXING CITY
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