Novel anti-vegfr2 monoclonal antibody and its preparation and application

A monoclonal antibody, a new type of technology, applied in the field of bioengineering, can solve the problems of low yield and difficult assembly

Active Publication Date: 2014-10-08
BEIJING DONGFANG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two forms also have their own characteristics. Fab contains two chains, which has the disadvantages of difficult assembly and low yield; the main problem of scFv is that it is easy to form dimers, but it is easier to synthesize and has good tolerance.

Method used

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  • Novel anti-vegfr2 monoclonal antibody and its preparation and application
  • Novel anti-vegfr2 monoclonal antibody and its preparation and application
  • Novel anti-vegfr2 monoclonal antibody and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1, construction and screening of antibody library

[0070] 1. Construction of antibody library

[0071] The single-chain antibody library used in the present invention is a human natural antibody library, and its main construction process is:

[0072] (1) Isolate B lymphocytes from peripheral blood or spleen, lymph nodes and other tissues, extract mRNA and reverse transcribe it into cDNA;

[0073] (2) Design antibody heavy chain and light chain primer sequences according to existing antibody gene sequence libraries (such as Kabat database, V-base, IMGT, etc.) (heavy chain primer sequences are shown in Table 1; light chain primer sequences are shown in Table 2 shown), different VH and VK gene fragments are amplified by PCR technology, and spliced ​​into a full-length scFv single-chain antibody through the linker region;

[0074] Table 1. Primers for amplifying the heavy chain variable region VH

[0075]

[0076] K=G / T, M=A / C, R=A / G, S=G / C, W=A / T

[0077] T...

Embodiment 2

[0089] Example 2, Cloning and Expression of Antibodies

[0090] 1. Antibody cloning and transfection

[0091] The screened variable region sequence consisting of SEQ ID NO: 1-12 was cloned into the FC fusion eukaryotic expression vector pFUSEIgG1FC or pSNEO to express the full-length antibody by conventional gene cloning method. The correct insertion of the antibody gene was identified by enzyme digestion and sequencing, followed by transfection and antibody secretory expression.

[0092] The day before transfection, digest 1×10 5 The 293 cells / mL cell density were inoculated in 6-well plates and cultured overnight until the confluence of 50-80% was optimal. The culture medium was incubated in a water bath at 37°C for 30 minutes, 200 μL serum-free medium was added to each of the 2 eppendorf test tubes, 1-2 μg DNA and 2 μl or 4 μl PEI (polyethyleneimine polymer) working solution (DNA / PEI = 1:2), mix well and incubate at room temperature for 5 minutes, quickly add the PEI dil...

Embodiment 3

[0098] Example 3, Identification of Biochemical and Biological Functions of Antibodies

[0099] 1. Antibody binding specificity detection

[0100] The binding specificity of the antibodies was determined using the ELISA method described below. Dilute VEGFR2 or other recombinant proteins with coating buffer to a final concentration of 1 μg / ml, 100 μl / well, and incubate overnight at 4°C. Discard the coating solution, wash the wells with PBS three times, and pat dry on a clean paper towel. Add not less than 300 μL / well of blocking solution, and incubate at 37°C for 2 hours. The blocking solution was discarded, and the wells were washed three times with PBST (phosphate buffered saline, 0.05% Tween). Take 300 μL of cell supernatant after transfection for 48 hours, centrifuge at 1000 r / pm for 5 minutes, and take 100 μL / well of supernatant. Incubate at 37°C for 1h. The sample solution was discarded and washed 3 times with PBST. The detection antibody was diluted with blocking s...

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PUM

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Abstract

The invention discloses a monoclonal antibody or sections thereof, which is sieved by utilizing a bacteriophage antibody database technology and prepared by utilizing a gene engineering method, of VEGFR2, and also discloses a carrier containing polynucleotide, which codes the monoclonal antibody or sections thereof, host cells, a method of preparing and purifying, and applications. The monoclonal antibody or sections thereof is used for preparing medicines for curing diseases caused by cancer new-born blood vessels. The diseases contain but not limited to following cancers: non-small cell lung cancer, metastatic non-small-cell lung cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2 negative metastatic breast cancer, metastatic gastric gland cancer, metastatic colorectal cancer, metastatic melanoma, and metastatic renal cell carcinoma.

Description

technical field [0001] The invention relates to a novel anti-VEGFR2 monoclonal antibody and its preparation and use, belonging to the technical field of bioengineering. Background technique [0002] Angiogenesis refers to the sprouting or division of existing blood vessels (capillaries and venules) to generate new blood vessels, under physiological and pathological conditions, such as embryogenesis, female reproductive cycle, inflammatory response, wound healing, tumorigenesis and other processes are undergoing angiogenesis. In 1971, Folkman first established the connection between angiogenesis and tumor growth, and found that if many tumors do not undergo angiogenesis, they can only grow to a size of a few millimeters. If angiogenesis is inhibited, tumor cells are still proliferating at a high speed, but at the same time With rapid apoptosis, neovascularization is in balance between stimulators and antagonists. Inhibiting tumor angiogenesis can inhibit tumor formation and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/63C12N5/10A61K39/395A61P35/00A61P35/04
Inventor 朱晓东谷香果李先钟高燕牛德云蔡清华岳玲白义白先宏
Owner BEIJING DONGFANG BIOTECH
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