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Genetically engineered monoclonal antibody specifically binding to a-beta oligomer

A technology of monoclonal antibodies and oligomers, which is applied in the direction of antibodies, drug combinations, and microbial-based methods, can solve the problems of three-dimensional epitopes that have not been explored clearly, and achieve inhibition of fibrosis aggregation, broad application prospects, Active effect

Active Publication Date: 2011-12-28
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The present invention solves the technical problem that existing antibodies bind to A-beta monomers, oligomers and filaments at the same time, and uses phage display technology to successfully screen out antibodies that specifically bind to A-beta oligomers but do not bind to A-beta oligomers. beta monomer and filament-bound monoclonal antibody
Its three-dimensional epitope has not been explored clearly so far

Method used

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  • Genetically engineered monoclonal antibody specifically binding to a-beta oligomer
  • Genetically engineered monoclonal antibody specifically binding to a-beta oligomer
  • Genetically engineered monoclonal antibody specifically binding to a-beta oligomer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A-be

[0049] Example 1 Preparation of A-beta oligomer

[0050] A-beta monomer (purchased from American Peptide Company in the United States) was dissolved in HFIP (hexafluoroisopropanol, purchased from Sigma Company) to prepare a solution with a concentration of 1 mg / ml. Ultrasound in a water bath at room temperature for 10 minutes, dispensed into a 1.5ml centrifuge tube, placed in a ventilating cupboard to completely volatilize HFIP, and stored at -20°C for later use.

[0051] After the above-treated A-beta was equilibrated at room temperature for 10 minutes, DMSO (dimethyl sulfoxide, purchased from Sigma) was added to fully dissolve the A-beta, and the final concentration was 1 mg / ml. A certain amount of A-beta was added to the pH 7.4 PBS buffer to make the A-beta concentration 10μM. After the A-beta solution was incubated at 37°C for 12 hours, it was centrifuged at 14000 rpm for 20 minutes, and the bottom sediment was discarded to obtain the supernatant containing A-beta oligomers. ...

Embodiment 2

[0052] Example 2 Screening of positive clones

[0053] Dilute the A-beta oligomer obtained in Example 1 with coating buffer (PBS, pH=7.4) to 10-100 μg / mL, add 4 mL to the immunotube, and coat overnight at 4°C. Discard the supernatant and quickly wash the tube 3 times with PBS. Fill the immunotube with 3% BSA and close it vertically at room temperature for 2 hours. Discard the supernatant and quickly wash the tube 3 times with PBS. The phage antibody library (purchased from MRC Center in the United Kingdom) was suspended in 4 mL of 3% BSA and added to the immunotube, incubated upside down at room temperature for 1 hour, and then incubated vertically for 1 hour. Wash 10 times with 0.1% Twenn-20 in PBS (20 washes for the second round of screening and thereafter). After the PBS was sucked dry, 500 μL of trypsin-PBS solution was added to elute the phage, and the phage was incubated upside down at room temperature for 10 min. Add 250 μL of the eluted phage to 1.75 mL of Escherichia...

Embodiment 3

[0060] Example 3 Identification of antibodies

[0061] Abeta monomers, oligomers and fibrous bodies (Abeta monomers incubated at 37°C for more than 4 days and verified by atomic force microscopy) were respectively spotted on NC (nitrocellulose) membrane 3μL. After the membrane was blocked with 5% BSA, scFv W20 was added to incubate for 1 hour, and the membrane was washed 3 times with PBS for 5 minutes each. Then, 1:5000 diluted Protein A (purchased from Santa cruz, USA) was added to incubate for 1 hour, and the membrane was washed 3 times with PBST (Tween-20 concentration of 0.1%) for 5 minutes each time, and DAB was used for color development. Only clones that show spots at the A-beta oligomers, but not spots in the A-beta monomers and fibrous bodies are the desired clones.

[0062] The above-mentioned positive clones are sequenced and identified, and the clones conforming to the basic structure of the antibody in the antibody library are complete single-chain genetically enginee...

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Abstract

The present invention relates to the technical field of genetically engineered antibodies, and provides a monoclonal antibody whose heavy chain variable region contains the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEO ID NO.3, and the light chain variable region The region contains the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The present invention also specifically provides a humanized single-chain antibody produced by a genetically engineered strain of Escherichia coli with a deposit number of CGMCC No. 2821, and its amino acid sequence is shown in SEQ ID NO.7. The antibody of the invention can specifically bind to the A-beta oligomer, effectively inhibit the fibrosis aggregation of A-beta, and obviously reduce the cytotoxic effect of A-beta on cells. The invention also relates to a pharmaceutical composition containing the antibody. The antibody of the invention has strong activity, good specificity, is easy to prepare, and has broad experimental application and clinical application prospects.

Description

Technical field [0001] The present invention belongs to the technical field of genetically engineered antibodies, and specifically relates to genetically engineered monoclonal antibodies that specifically bind to A-beta oligomers. The present invention also relates to a method for preparing the monoclonal antibodies and pharmaceutical compositions containing the monoclonal antibodies. Background technique [0002] Studies have shown that Alzheimer's Disease (AD), commonly known as senile dementia, is composed of non-toxic β-amyloid monomer molecules (β-Amyloid, A-beta 40 / 42 hereinafter referred to as A-beta, also Can be written as Aβ) Aggregate to form toxic oligomers, which are mainly neurodegenerative diseases characterized by memory decline and senile plaque formation in the brain (Selkoe et al., Science 1997, 275, 630-631; Koo et al., PNAS) 1996, 9989-9990). Medical statistics show that 5 to 6% of people over 60 in my country and European and American countries suffer from A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577A61K39/395A61P25/28C07K16/18C12R1/19C12N1/21
Inventor 刘瑞田王小平
Owner TSINGHUA UNIV
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