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Death receptor-5 agitated polyvalent antibody and application thereof in preparation of anti-tumor medicines

A technology of death receptors and multivalent antibodies, which is applied in the direction of anti-tumor drugs, anti-animal/human immunoglobulins, antibodies, etc., to achieve the effect of reducing immunogenicity

Active Publication Date: 2013-02-13
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some tumor cells can highly express decoy receptors and become resistant to TRAIL-induced apoptosis [Science, 1997, 277: 818–821; Cell Signal, 2004, 16:139-44]

Method used

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  • Death receptor-5 agitated polyvalent antibody and application thereof in preparation of anti-tumor medicines
  • Death receptor-5 agitated polyvalent antibody and application thereof in preparation of anti-tumor medicines
  • Death receptor-5 agitated polyvalent antibody and application thereof in preparation of anti-tumor medicines

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Preparation of DR5 single-chain antibody

[0028] Routine immunization was performed by emulsifying 100 μl (100 μg) of DR5 antigen (PeproTech) with an equal volume of complete Freund's adjuvant. Spleen of mice was taken, total RNA was extracted, and cDNA was synthesized by reverse transcription. According to the protocol of "Recombinant Antibodies" (Shen Beifen, Science Press), cDNA was used as a template to amplify a complete set of VH and VL genes, and the amplified heavy and light chain products were subjected to overlap extension PCR to amplify ScFv fragments. The ScFv fragment and the carrier PAK100 were digested with SfiI, ligated with T4 ligase, and electrotransformed into E. coli XL1-Blue competent cells to prepare a phage antibody library. Coat 96-well plates with DR5 antigen (100 μg / ml in the first round, 30 μg / ml in the second round, 10 μg / ml in the third round, 1 μg / ml in the fourth round, and 0.5 μg / ml in the fifth round), After blocking with 1%...

Embodiment 2

[0079] Example 2 Construction of Tetravalent Antibody Expression Vector

[0080] Using the DR5 single-chain antibody gene as a template and L1 and H2 as primers, ScFv was amplified by PCR. The reaction conditions were: 95°C for 3 minutes, 95°C for 30 sec, 60°C for 40 sec, and 72°C for 30 sec, 30 cycles. Using P53a and P53b as primers, the P53 fragment was amplified by PCR, and the reaction conditions were: 95°C for 30 sec, 60°C for 40 sec, 72°C for 30 sec, 30 cycles. Using the above product as a template and L1 and P532 as primers, overlap extension PCR was carried out. The reaction conditions were: 95°C for 3 min, 95°C for 30 sec, 60°C for 40 sec, 72°C for 30 sec, 30 cycles. Take the recovered product VL+VH2+P53 of the target gene, and use the restriction endonucleases XhoI and Hind III as a double-digestion system for fragments. Take the empty vector plasmid pcDNA3.1, and cut it with restriction endonucleases XhoI and HindIII. The target gene was recovered by gel cutting, ...

Embodiment 3

[0086] Example 3 Screening of agonistic tetravalent antibodies

[0087] The tetravalent antibody plasmids of each clone were transiently transfected into COS7 cells, and the supernatant was harvested after 48 hours, and the expression of the tetravalent antibody was confirmed by westenblot (results in image 3 ). Collect and prepare Jurkat cell suspension, add to 24-well culture plate, the final concentration of cells is 1×10 5Each well was cultured at 37°C and 5% CO2 for 12 hrs, and 50 μl of COS7 cell culture supernatant was added; negative control wells used RPMI1640 culture medium. at 37°C and 5% CO 2 Incubate for 20 h under certain conditions, and collect all the cells in each well into corresponding centrifuge tubes. After washing the cells with PBS, suspend the cells in 100 μl of AnnExin V binding solution in each well, add 5 μl of AnnExin V-FITC solution and 5 μl of PI solution, mix well, incubate at room temperature in the dark for 20 min, and use binding Make up ...

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Abstract

The invention provides an antibody in an extracellular region of a death receptor-5 of a tumor necrosis factor-related apoptosis inducing ligand. By a death receptor-5 agitated polyvalent antibody, a single-chain antibody of the death receptor-5 forms a polyvalent antibody through an oligomerization structural domain, and the amino acid sequence is shown as SEQIDNO.1. The single-chain antibody of the death receptor-5 forms a tetravalent antibody through a p53 structural domain, and the amino acid sequence is shown as SEQIDNO.2. After multimerization, the antibody can induce apoptosis of tumor cells, and is nontoxic to normal cells. The invention also provides an application of the death receptor-5 agitated polyvalent antibody in preparation of anti-tumor medicines.

Description

technical field [0001] The invention relates to the preparation of death receptor agonistic antibody, in particular to the preparation of death receptor 5 agonistic multivalent antibody and its application in antitumor drugs. Background technique [0002] Tumor necrosis factor-related apoptosis-inducing ligand (Tumor necrosis factor-related apoptosis inducing ligand, TRAIL) is a new member of the TNF superfamily discovered by Willy et al. in 1995. Experiments have confirmed that TRAIL has a killing effect on most cell lines of almost all systemic malignant tumors, but has no killing effect on normal tissues (Nat Med, 1999, 5: 157-63), so tumor therapy targeting TRAIL receptors has become a research hotspot. [0003] The biological effects of TRAIL are mainly produced by binding to the corresponding receptors on the cell membrane. At present, four kinds of TRAIL receptors have been found in the cell membrane: two are death receptors: DR4 (Death receptor-4), DR5 (Death recep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
Inventor 马远方刘峰涛张军季泽俊刘广超李淑莲
Owner HENAN UNIVERSITY
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