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A completely humanized anti-VEGFR-2 monoclonal antibody and a preparing method thereof

A VEGFR-2, monoclonal antibody technology, applied in the field of bioengineering, can solve the problems of difficult assembly, small molecules, high tumor penetration, achieve good biochemical and biological activities, effective anti-angiogenesis effect.

Active Publication Date: 2016-06-08
SICHUAN KELUN BIOTECH BIOPHARMACEUTICAL CO LTD
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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: ScFv has the advantages of small molecule and high tumor penetration, while 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 easier to synthesize and better tolerated

Method used

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  • A completely humanized anti-VEGFR-2 monoclonal antibody and a preparing method thereof
  • A completely humanized anti-VEGFR-2 monoclonal antibody and a preparing method thereof
  • A completely humanized anti-VEGFR-2 monoclonal antibody and a preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction and Screening and Separation of Antibody Library

[0062] 1. Construction of antibody library

[0063] The single-chain antibody library used in the present invention is a human natural antibody library, and its main construction process is:

[0064] (1) Isolate B lymphocytes from human peripheral blood or spleen, lymph nodes and other tissues, Trizol extracts total mRNA and reverse transcribes it into cDNA;

[0065] (2) According to the human immunoglobulin gene sequence library (such as Kabatdatabase, V-base, IMGT, etc.), design a set of primers for antibody heavy chain and light chain with degenerate sequences to amplify different VH and The VL gene fragment is spliced ​​into a full-length scFv single-chain antibody through the linker region;

[0066] The base sequences of the primers used are as follows:

[0067] V H back (amplification heavy chain variable region VH primer)

[0068] HuVH1B / 7A-BACK5'-CAGRTGCAGCTGGTGCARTCTGG-3'

[0069] H...

Embodiment 2

[0112] Example 2 Construction and expression of high-expression engineered cell lines

[0113] 1. Antibody cloning and transfection

[0114] (1) The single-chain antibody with the screened variable region sequence consisting of SEQIDNO: 1-6 is cloned into the FC fusion eukaryotic expression vector Pcantab-5E or pFUSEIgG1-FC to express the full-length antibody vector by conventional gene cloning method middle. The correct insertion of the antibody gene was identified by enzyme digestion and sequencing, followed by transfection and antibody secretory expression.

[0115] (2) 24 hours before transfection, digest 1×10 5 / mL cell density HEK293T / 17 cells were seeded in 6-well plates and cultured overnight. Take two test tubes containing 200ul serum-free medium, add 1ugDNA working solution, mix well, incubate at room temperature for 5 minutes, quickly add 2ulPEI (polyethyleneimine polymer) dilution solution to DNA dilution solution, vortex immediately for 15 seconds, Incubate at...

Embodiment 3

[0132] Identification of Biochemical and Biological Functions of Example 3 Antibodies

[0133]1. Antibody affinity detection

[0134] The affinity constants of the target antibody samples of the three clones (A139, A140, A141) were detected using BiacoreT100 (BiacoreAB, Uppsala, Sweden). The reference product VEGF165 (product of R&D Company) is covalently bonded to the CM5 biosensor chip (GE Healthcare) through the amino group, and the RU response value of the antibody to be tested is measured, and the appropriate antibody concentration is determined according to the result, usually 100-200RU is appropriate . Then test the concentration of antigen VEGFR2, determine the range of antigen concentration and exposure time. Finally, the kinetic parameters and affinity of the test antibody were determined using the kinetics / affinity method. The results are shown in Table 1.

[0135]

[0136] Table 1 Affinity test results

[0137] It can be seen from the table that different a...

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Abstract

The invention discloses a completely humanized anti-VEGFR-2 monoclonal antibody, and discloses a method of screening by adopting a phage antibody library technique and preparing the novel anti-VEGFR-2 monoclonal antibody or a segment thereof by utilizing a genetic engineering manner. The antibody can be a univalent antibody or a bivalent antibody. The antibody can interdict VEGF-A, VEGF-C, VEGF-D and VEGF-E simultaneously so that the antibody has more effective anti-angiogenesis effects. The antibody can be applied for treating diseases caused by tumor angiogenesis. The diseases comprise, but not limited to, gastric cancer, adenocarcinoma of esophagogastric junction, non-small cell lung cancer, metastatic non-small cell lung cancer, hepatocellular carcinoma, metastatic hepatocellular carcinoma, HER2-negtive metastatic breast cancer, metastatic colorectal cancer, metastatic melanoma, metastatic renal cell carcinoma, glioblastoma, ovarian cancer, prostate cancer and solid tumor.

Description

technical field [0001] The invention relates to a novel fully humanized anti-VEGFR-2 monoclonal antibody and its preparation method and application, belonging to the technical field of bioengineering. Background technique [0002] Tumor formation needs the support of surrounding normal matrix, blood components and lymphatic vessels. The formation of new blood vessels is necessary for tumor growth, invasion and metastasis. Studies have shown that VEGF and its receptor family play a very important role in this process . The identified VEGF receptors are VEGFR-1 (Flt-1, fms-like tyrosine kinase), VEGFR-2 (also known as KDR / Flk-1, kinase-inserted chimeric receptor, fetal liver kinase-1) , VEGFR-3 (Flt-4), neuropilin-1 (neuropilin-1), neuropilin-2. Human VEGF receptor 2 (VEGFR-2), also known as KDR (kinaseinsertdomain-containingreceptor, KDR) is the main signal transduction receptor of VEGF, which participates in the migration, proliferation and survival of neovascular endothel...

Claims

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Application Information

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IPC IPC(8): C07K16/30A61K39/395A61P35/00
Inventor 薛彤彤李明雄杨秋艳刘立平闫岩王利春王晶翼
Owner SICHUAN KELUN BIOTECH BIOPHARMACEUTICAL CO LTD
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