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Full-humanized agonist single-chain antibody resistant to human death receptor 5 and application thereof

A death receptor and single-chain antibody technology, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, anti-animal/human immunoglobulin, etc., can solve the problem of short half-life, etc. Achieve the effect of simple preparation process

Active Publication Date: 2017-02-15
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it is precisely because it has multiple binding receptors that it is prone to drug resistance [Sheridan, J., et al.Science, 1997, 277:818-821; Merino D., et al. Molecular and cellular biology 2006 , 26(19):7046-7055], and its half-life in vivo is short (about 30 minutes) [Herbst RS., et al.Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2010, 28(17): 2839-2846]

Method used

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  • Full-humanized agonist single-chain antibody resistant to human death receptor 5 and application thereof
  • Full-humanized agonist single-chain antibody resistant to human death receptor 5 and application thereof
  • Full-humanized agonist single-chain antibody resistant to human death receptor 5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Expression and purification of human death receptor DR5 protein

[0034] Using the cDNA of the human death receptor DR5 (product of Beijing Yiqiao Shenzhou Co., Ltd.) as a template, PCR amplifies the gene fragment of the extracellular region (1-130aa) of the death receptor DR5, clones it into the expression vector pET21b, and transforms it into Escherichia coli BL21 (DE3) for protein expression. The expression conditions are as follows: inoculate the seed solution in 2×YT-AG medium with 2% inoculum amount and culture with shaking at 37°C until OD 600nm =0.6, add IPTG with a final concentration of 0.5mM, and induce at 20°C for 20h. Centrifuge at 6000rpm at 4°C for 15min to collect the cells; resuspend the cells in PBS and sonicate (sonication for 3s, interval of 3s, 20min in total); centrifuge at 12000rpm at 4°C for 30min to collect the supernatant; pass the supernatant through a nickel ion affinity column (GE company product) and molecular exclusion chromat...

Embodiment 2

[0035] Example 2. Construction of phage human single-chain antibody library

[0036] The construction of the phage human single-chain antibody library was carried out according to the literature [Sblattero D., et al. Nat Biotechnol, 2000, 18: 74-80; Sblattero D., et al. Immunotechnology, 1998, 3: 271-278]. Using the peripheral blood of 105 healthy people and 58 peripheral blood of tumor patients as sources, the peripheral blood lymphocytes producing antibodies were separated by density gradient centrifugation, the total RNA was extracted, and cDNA (product of Treasure Biotech Co., Ltd.) was synthesized by reverse transcription. As a template, 6 heavy chain upstream primers, 4 heavy chain downstream primers, 7 lambda chain upstream primers and 3 lambda chain downstream primers were selected for PCR amplification of the variable region of the human-specific antibody [Sblattero D., et al. Nat Biotechnol, 2000, 18: 74-80; SblatteroD., et al. Immunotechnology, 1998, 3: 271-278], an...

Embodiment 3

[0037] Example 3. Screening of fully human anti-human death receptor DR5 single chain antibody

[0038] Coating buffer (50mM NaHCO 3 , pH9.6) to dilute the death receptor DR5 extracellular domain protein to 20ug / ml, take 2ml and add it to the immune tube, and coat it at room temperature overnight; the next day, discard the supernatant, and wash the immune tube 3 times quickly with PBS; 2% MPBS ( PBS containing 2% skimmed milk), 37°C for blocking for 2h; discard the blocking solution, and wash the immunotube 3 times quickly with PBS; phage antibody library (~10 12 pfu) suspended in 4ml 2% MPBS and added to the immune tube, placed on a rotary shaker and rotated repeatedly for 2h, washed the immune tube 15 times with PBS containing 0.1% Tween-20, and then washed the immune tube 15 times with PBS; add 1ml 100mM triethylamine, rotate and incubate at room temperature for 10min for specific elution; 0.5ml 1M Tris-HCl (pH7.4) buffer is used to quickly neutralize the eluted phage; the...

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Abstract

The invention relates to a single-chain antibody capable of specific binding and capable of activating human death receptor 5 (DR5), and belongs to a full-humanized antibody; the single-chain antibody is obtained by performing quintuple screening in a high-capacity full-humanized phage antibody library by using human death receptor DR5 extracellular protein as a target antigen; the single-chain antibody may be acquired by induced expression in Escherichia coli, nickel ion affinity chromatography and molecular-exclusion chromatographic purification; pharmacodynamic experiments prove that the single-chain antibody is efficient in suppressing the growth of DR5 positive colon cancer cell COLO205 and breast cancer cell MDA-MB-231; the single-chain antibody and other antibodies modified therefrom, including full-length, bispecific or fusion protein forms, may act as drugs for positive tumor targeting therapy of death receptor DR5; the single-chain antibody has the advantages such as low immunogenicity, high specificity and high penetrability and is an ideal targeting tumor therapeutic drug.

Description

technical field [0001] The invention belongs to the field of genetic engineering pharmaceuticals, and relates to the screening, identification, expression and purification of a fully human single-chain antibody against human death receptor 5 (death receptor 5, DR5), and its application in tumor targeting therapy. [0002] Background of the invention [0003] Tumor necrosis factor-related apoptosis-inducing ligand TRAIL (TNF-related apoptosis-inducing ligand, also known as APO2L) is a member of the tumor necrosis factor superfamily, containing 281 amino acids, like other TNF superfamily members, TRAIL is a type II trans Membrane protein, comprising an N-terminal transmembrane domain and a C-terminal extracellular domain [Nagane M., et al. Neuro-oncology 2010, 12(7):687-700]. TRAIL exists only in the membrane-bound form in vivo, but the extracellular region can be expressed in a soluble form through genetic engineering and maintain a certain activity. It is known that TRAIL ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00G01N33/574
CPCA61K2039/505C07K16/28C07K2317/24C07K2317/565C07K2317/622G01N33/57407G01N33/57411G01N33/57415G01N33/57419G01N33/57423G01N33/57434G01N33/57438G01N33/57449
Inventor 谭树华雷高新徐智盼徐梦龙陆陈晨
Owner CHINA PHARM UNIV
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