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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 problems such as limiting the evaluation of antibody druggability, weakening, and increasing the probability of immune response

Inactive Publication Date: 2014-09-17
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 Chimeric Antibody Eukaryotic Expression Plasmid

[0054] 1. Construction of heavy chain and light chain variable region cDNA of monoclonal antibody

[0055] Cloning of the variable region sequence of the anti-human platelet glycoprotein GPIbα human-mouse chimeric antibody SZ-138 cell line, 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 from the hybridoma cell line SZ-2A of GPIbα 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 second n...

Embodiment 2

[0083] Example 2: Expression and screening of chimeric antibodies

[0084] Material:

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

[0086] - Calcium Transfection Kit (Invitrogen, Cat.No.K2780-01)

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

[0088] 1. Cell line transfection, using the calcium phosphate method for transfection.

[0089] Cells were subcultured 24 hours before transfection, and transfection could be performed when the cell density reached 50%-60% confluence. The transfection method was strictly operated according to the method of the kit. The chimeric antibody eukaryotic expression plasmid DNA obtained in Example 1 was digested with PvuI and linearized. The cells were cultured for 24 hours under standard growth conditions, and the supernatant was taken for ELISA to detect the transient expression level; after con...

Embodiment 3

[0103] Example 3: Purification of Chimeric Antibody and Antibody Fab Fragment

[0104] 1. Purification of chimeric antibodies

[0105] 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 Figure 4 (The left side is the non-reducing gel electrophoresis of the SZ 138 IgG full-length antibody, and the right side is the reducing gel electrophoresis). The purified chimeric antibody was determined by HPLC, and its purity was 94.4%.

[0106] 2. Purification of chimeric antibody Fab fragments

[0107] Chimeric antibodies were digested with papain ...

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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 application thereof. 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. Platelets play a key role in thrombus formation, and platelet aggregation is a necessary process for thrombus formation. Under normal physiological conditions, a thrombus prevents blood cells from leaking out of blood vessels, however, under certain disease conditions, a thrombus can inhibit or completely stop blood flow leading to cellular necrosis. [0003] Platelet aggregation and subsequent thrombus formation in at...

Claims

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Application Information

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Patent Type & Authority Patents(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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