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Platelet aggregation resistant chimeric monoclonal antibody and/or its fragment

An anti-platelet aggregation and monoclonal antibody technology, applied in the direction of antibodies, hybrid peptides, blood diseases, etc., can solve the problems of increasing the probability of immune response, weakening, and destroying the therapeutic effect, so as to reduce cardiac ischemic events and prevent acute The effect of thrombus formation

Inactive Publication Date: 2007-03-21
上海亚联抗体医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because murine antibodies may induce human anti-mouse immune responses in humans, this response may weaken or destroy its therapeutic effect, and even cause allergic or hypersensitivity reactions in patients, thus limiting its application in patients
In addition, in the treatment of thromboembolic diseases, repeated dosing is often required, which increases the probability of these immune reactions

Method used

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  • Platelet aggregation resistant chimeric monoclonal antibody and/or its fragment
  • Platelet aggregation resistant chimeric monoclonal antibody and/or its fragment
  • Platelet aggregation resistant chimeric monoclonal antibody and/or its fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of Chimeric 813 Antibody Eukaryotic Expression Plasmid

[0042] 1. Construction of heavy chain and light chain variable region cDNA of monoclonal antibody

[0043] Cloning of the variable region sequence of the anti-human platelet glycoprotein IIIa monoclonal antibody hybridoma cell line (R813), using 5'RACE (Rapid Amplification of cDNA Ends, rapid amplification of cDNA ends) technology, from secreted anti-human platelet The variable region sequence of the functional antibody cloned in hybridoma cell line 813 of glycoprotein IIIa murine monoclonal antibody. The steps can be briefly described as follows: an antisense gene-specific primer (GSP1) is used to synthesize the first strand of cDNA, after the first strand of cDNA is purified, terminal deoxynucleotidy transferase (Terminal deoxynucleotidy transferase, TdT) is used to generate A synthetic homopolynucleotide anchor sequence is added to the 3′ end of the DNA. The cDNA is amplified using a se...

Embodiment 2

[0106] Example 2 Expression and Screening of Chimeric 813 Antibody

[0107] Material:

[0108] -dhfr-deficient CHO cells were purchased from Shanghai Cell Institute, Chinese Academy of Sciences

[0109] - Calcium transfection kit (Invitrogen)

[0110] -MTX (Sigma Corporation)

[0111] 1. Cell line transfection, using the calcium phosphate method for transfection.

[0112] Plasmid DNA was linearized with PvuI digestion. The transfection method was strictly operated according to the method of the kit. Cultivate the cells under standard growth conditions for 24 hours, take the supernatant for ELISA to detect the transient expression level; after confirming that the transfected cells can secrete and express antibodies, digest the cells, and use selective medium (excluding H and T) at 1000-8000 Ratio of cells / well Divide the cells into 96-well plates at 37°C, 5% CO 2 down to cultivate.

[0113] 2. Screening of high expression cell lines

[0114] (1) After monoclonal cells a...

Embodiment 3

[0120] Example 3 Chimeric 813 Antibody and Chimeric 813 Antibody F(ab') 2 Fragment purification

[0121] 1. Purification of chimeric 813 antibody

[0122] Chimeric antibodies were purified by protein A affinity chromatography. The specific steps are: (1) washing the protein A affinity chromatography column with 3-5 times column volume of water. (2) Equilibrate protein A affinity chromatography column with 20mM phosphate buffer, pH 7.0. (3) Pump the cell culture supernatant containing the desired purified monoclonal antibody into the protein A affinity chromatography column. (4) Wash the column with 20mM phosphate buffer, pH 7.0, to OD 280 <0.01. (5) Elute with 0.1M citric acid-sodium citrate buffer, pH 3.0, collect the eluted peak, and adjust the pH value of the collected antibody solution to be neutral with 1M Tris-HCl buffer, pH 9.0. (6) Dialysis desalination. After purification, the chimeric 813 antibody was determined by HPLC, and its purity was 94.4%. The electrop...

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PUM

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Abstract

One kind of platelet aggregation resistant chimeric monoclonal antibody and / or its fragment is obtained through connecting the specific antigen binding region of mouse platelet membrane glycoprotein GPIIIa with the constant region of humanized antibody, and the chimeric antibody is digested with pepsin to obtain F(ab')2 fragment. The variable regions of the heavy chain and the light chain in the chimeric antibody have the amino sequences as shown in SEQ ID No.1 and SEQ ID No.2. The chimeric antibody and / or its fragment can avoid or reduce the immune reaction of mouse originated antibody. As the antithrombogenic medicine developed for Asiatic race, the present invention is especially suitable for Asiatic race to prevent and treat thrombus, unstable angina pectoris, etc and to detect thrombus after radionuclide labeling.

Description

technical field [0001] The present invention belongs to the field of biotechnology, in particular, the present invention relates to a chimeric monoclonal antibody or / and fragments thereof that bind to platelet membrane glycoprotein IIIa with high specificity and high affinity and have antithrombotic biological activity . Background technique [0002] Epidemiological studies have shown that with the improvement of living conditions and the aging of the population, the lesions caused or complicated by thromboembolic diseases in recent decades are currently diseases that seriously endanger human health (Circulation 2001104: 2746-2753; Circulation 2001 104:2855-2864). Platelets play a key role in thrombus formation. Platelet aggregation is an essential process for thrombus formation. Normally, clots prevent blood cells from leaking out of blood vessels, but under certain disease conditions, clots can inhibit or completely stop blood flow, leading to cell death. [0003] For ...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/395A61K51/10A61P7/02
Inventor 阮长耿
Owner 上海亚联抗体医药有限公司
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