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Human-mouse chimeric monoclonal antibody against human platelet membrane glycoprotein Ib alpha and applications of human-mouse chimeric monoclonal antibody

A monoclonal antibody and membrane glycoprotein technology, applied in the biological field, can solve the problems of destroying the therapeutic effect, weakening, allergies, etc.

Inactive Publication Date: 2012-07-11
苏州元德维康生物科技有限公司
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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
[0008] The dissertation "Humanization of Anti-Platelet Membrane Glycoprotein Ibα Monoclonal Antibody and Research on Active Small Molecular Compounds Based on the Structure of Membrane Glycoprotein Ibα" published by Dr. Yang Jianfeng disclosed an anti-platelet membrane glycoprotein Ibα monoclonal antibody, but the The antibody only recognizes human platelet membrane glycoprotein Ibα, and has no cross-reaction with common model animal platelets such as beagle dogs and macaques, which limits various druggability evaluation studies for this antibody
[0009] In addition, mouse-derived antibodies may induce human anti-mouse immune responses in humans, which may weaken or destroy its therapeutic effect, and even cause allergic reactions or hypersensitivity reactions in patients, thus limiting the use in patients. application
In addition, in the treatment of thromboembolic disease, often need to continue medication, which increases the probability of these immune reactions

Method used

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  • Human-mouse chimeric monoclonal antibody against human platelet membrane glycoprotein Ib alpha and applications of human-mouse chimeric monoclonal antibody
  • Human-mouse chimeric monoclonal antibody against human platelet membrane glycoprotein Ib alpha and applications of human-mouse chimeric monoclonal antibody
  • Human-mouse chimeric monoclonal antibody against human platelet membrane glycoprotein Ib alpha and applications of human-mouse chimeric monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of a chimeric antibody eukaryotic expression plasmid

[0054] 1. Construction of cDNA for the heavy and light chain variable regions of monoclonal antibodies

[0055] Cloning of the variable region sequence of the antibody in the anti-human platelet glycoprotein GPIbα human-mouse chimeric antibody SZ-138 cell line, using 5'RACE (Rapid Amplification of cDNA Ends) technology to secrete anti-human platelets The variable region sequence of the functional antibody was cloned from the hybridoma cell line SZ-2A of GPIbα mouse monoclonal antibody. The steps can be briefly summarized as follows: synthesize the first strand of cDNA by an antisense gene-specific primer (GSP1). After the first strand of cDNA is purified, use terminal deoxynucleotidy transferase (TdT) in the cDNA. Add a synthetic homopolynucleotide anchor sequence to the 3'end. A second nested gene-specific primer (GSP2) and an anchor primer that can anneal to the homopolynucleotide tail are use...

Embodiment 2

[0083] Example 2: Chimeric antibody expression and screening

[0084] material:

[0085] -dhfr-deficient CHO cells were purchased from the Shanghai Institute of Cell Research, Chinese Academy of Sciences, and were monoclonal and serum-free before transfection

[0086] -Calcium transfection kit (Invitrogen, Cat.No.K2780-01)

[0087] -MTX (Sigma company, Cat.No.M9929)

[0088] 1. Transfection of cell lines, using calcium phosphate method for transfection.

[0089] The cells are passaged 24h before transfection, and the transfection can be carried out when the cell density reaches 50%-60% of the bottom. The transfection method is strictly in accordance with the method of the kit. The chimeric antibody eukaryotic expression plasmid DNA obtained in Example 1 was digested and linearized with PvuI. Cultivate the cells under standard growth conditions for 24 hours, take the supernatant for ELISA to detect transient expression; after confirming that the transfected cells can secrete and expres...

Embodiment 3

[0103] Example 3: Purification of chimeric antibodies and antibody Fab fragments

[0104] 1. Purification of chimeric antibody

[0105] The chimeric antibody was purified by protein A affinity chromatography. The specific steps are: (1) Wash the protein A affinity chromatography column with 3-5 column volumes of water. (2) 20mM phosphate buffer, pH7.0 equilibrated protein A affinity chromatography column. (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 Figure 4 (The left side is the non-reducing gel electrophoresis of the full-length SZ 138IgG antibody, and the right side is the reducing gel electrophoresis image). After purification, the purity of the chimeric antibody was determined by HPLC to be 94.4%.

[0106] 2. Purification of chimeric antibody Fab fragment

[0107] The chimeric antibody is digested with papain to obt...

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Abstract

The invention belongs to the biotechnology field and particularly relates to a chimeric monoclonal antibody which is capable of being combined with high specificity and high affinity of platelet membrane glycoprotein and has an antithrombotic bioactivity and applications of the chimeric monoclonal antibody. The human-mouse chimeric monoclonal antibody against human platelet membrane glycoprotein Ib alpha is characterized by comprising a mouse heavy chain variable region and a mouse light chain variable region, wherein amino acid sequence of the mouse heavy chain variable region is represented as SEQ ID No.1, and amino acid sequence of the mouse light chain variable region is represented as SEQ ID No.2. Immune reactivity of antibody molecules is mainly produced by a constant region, so that the chimeric antibody containing a human constant region is not easy to generate an anti-mouse immune reaction in human bodies. The monoclonal antibody against platelet glycoprotein (GP) Ib alpha is capable of being combined with high specificity and high affinity of human and rhesus monkey platelet membrane glycoprotein Ib alpha (GP Ib alpha), and the antithrombotic effect is achieved.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a chimeric monoclonal antibody with high specificity and high affinity binding to platelet membrane glycoprotein and antithrombotic biological activity and its application. 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 serious diseases that endanger human health. Platelets play a key role in the formation of thrombus, and platelet aggregation is a necessary process for thrombosis. Under normal physiological conditions, thrombus can prevent blood cells from leaking out of the blood vessels. However, under certain disease conditions, thrombus can inhibit or completely prevent blood flow and cause cellular necrosis. [0003] Platelet aggregation that occurs in atherosclerotic plaques and subse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N5/10A61K39/395A61K49/00A61K51/10A61P7/02
Inventor 阮长耿赵益明季顺东杨剑锋江淼沈飞
Owner 苏州元德维康生物科技有限公司
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