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Genetically engineered monoclonal antibody specifically binding to a-beta oligomer

A monoclonal antibody and oligomer technology, applied in the direction of antibodies, drug combinations, microorganism-based methods, etc., can solve the problem that the spatial three-dimensional antigen epitope has not been clearly explored, and achieves inhibition of fibrosis aggregation, easy screening, and reduction of Effects of toxic effects

Inactive 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
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Embodiment 1A-be

[0049] The preparation of embodiment 1A-beta oligomer

[0050] A-beta monomer (purchased from American Peptide Company, USA) was dissolved in HFIP (hexafluoroisopropanol, purchased from Sigma Company) to prepare a solution with a concentration of 1 mg / ml. Sonicate in a water bath at room temperature for 10 minutes, dispense into 1.5ml centrifuge tubes, place in a fume hood to allow HFIP to evaporate completely, and store 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 A-beta to a final concentration of 1 mg / ml. A certain amount of A-beta was added to PBS buffer at pH 7.4, so that the concentration of A-beta was 10 μM. After incubating the A-beta solution at 37° C. for 12 hours, centrifuge at 14,000 rpm for 20 minutes, and discard the bottom precipitate to obtain the supernatant containing A-beta oligomers. The formation of A-beta ol...

Embodiment 2

[0052] The screening of embodiment 2 positive clones

[0053] The A-beta oligomer obtained in Example 1 was diluted to 10-100 μg / mL with coating buffer (PBS, pH=7.4), and 4 mL was added to an immunotube, and coated overnight at 4°C. Discard the supernatant and wash the tube 3 times quickly with PBS. The immunotube was filled with 3% BSA and sealed vertically for 2 hours at room temperature. Discard the supernatant and wash the tube 3 times quickly with PBS. The phage antibody library (purchased from the MRC center in the UK) 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 times for the second round of selection and subsequent washes). After the PBS was blotted dry, 500 μL of trypsin-PBS solution was added to elute the phage, and incubated upside down at room temperature for 10 min. Add 250 μL of the eluted phage to 1.75 mL...

Embodiment 3

[0060] The identification of embodiment 3 antibody

[0061] Abeta monomers, oligomers, and fibrils (Abeta monomers were incubated at 37°C for more than 4 days and verified by atomic force microscopy) were spotted on 3 μL of NC (nitrocellulose) membranes. After the membrane was blocked with 5% BSA, scFv WC2 was added to incubate for 1 hour, and the membrane was washed 3 times with PBS, 5 minutes each time. Add 1:5000 diluted Protein A (purchased from Santa Cruz, USA) and incubate for 1 hour, wash the membrane 3 times with PBST (Tween-20 concentration is 0.1%), 5 minutes each time, and develop color with DAB. Only clones showing spots in A-beta oligomers but not in A-beta monomers and fibrils were desired.

[0062] The above positive clones were sequenced and identified, and the clones that conformed to the basic structure of the antibody in the antibody library were complete single-chain genetically engineered antibodies. The sequencing primers are:

[0063] LMB3: 5'-CAG GAA...

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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. 2818, 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 toxic 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 invention belongs to the technical field of genetic engineering antibodies, and specifically relates to a genetic engineering monoclonal antibody specifically combined with A-beta oligomers. The invention also relates to a preparation method of the monoclonal antibody and a pharmaceutical composition containing the monoclonal antibody. 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) aggregated to form toxic oligomers A neurodegenerative disease in the elderly mainly characterized by memory loss and senile plaques in the brain (Selkoe et al., Science 1997, 275, 630-631; Koo et al., PNAS 1996, 9989-9990). Medical statistics show that 5-6% of the elderly over the age of 60 in my country and European and American countries suffer from Alzheimer's ...

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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