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Human-mouse chimeric monoclonal antibody against human von Willebrand factor a3 region and its preparation method and application

A hemophilia factor and monoclonal antibody technology, applied in the biological field, can solve the problems of increasing the probability of immune response, destroying the treatment effect, and weakening immune response, so as to reduce cardiac ischemic events, prolong bleeding time, and prevent acute The effect of thrombus formation

Active Publication Date: 2019-05-28
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the mouse-derived monoclonal antibody may induce human anti-mouse immune response in the human body, this immune response will not only weaken or destroy the therapeutic effect, but may even cause allergic or hypersensitivity reactions in patients, which greatly limited clinical application
In addition, in the treatment of thromboembolic diseases, repeated administration is usually required, which further increases the probability of the above-mentioned immune response

Method used

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  • Human-mouse chimeric monoclonal antibody against human von Willebrand factor a3 region and its preparation method and application
  • Human-mouse chimeric monoclonal antibody against human von Willebrand factor a3 region and its preparation method and application
  • Human-mouse chimeric monoclonal antibody against human von Willebrand factor a3 region and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of human-mouse chimeric monoclonal antibody eukaryotic expression plasmid.

[0044] 1. Construction of the heavy chain and light chain variable region cDNA of the monoclonal antibody SZ-123:

[0045] Using 5'-RACE technology, the variable region sequences of the heavy and light chains of the functional antibody were cloned from the hybridoma cell line SZ-123 secreting the mouse monoclonal antibody against the A3 region of human vWF, in which the mouse monoclonal antibody SZ was secreted -123 hybridoma cell line mRNA as a template, and design a set of heavy chain and light chain reverse transcription specific primers HGSP1 and KGSP1, its nucleotide sequence is shown in SEQ ID NO:3 and SEQ ID NO:4 respectively Show. The electrophoresis of the 5'-RACE product is as follows figure 1 As shown, the right side is the light chain (L) result, and the left side is the heavy chain (H) result.

[0046]

[0047] According to the corresponding relationsh...

Embodiment 2

[0052] Example 2: Expression, screening, suspension acclimatization and shake flask expression of chimeric monoclonal antibody.

[0053] 1. CHO-S cell electrotransfection method:

[0054] 20 μg of recombinant plasmids (pMH3-MHC-SZ123VH, pMH3-MHC-SZ123VK) were co-transformed into 3×10 6 CHO-S cells, electroporation reaction system: 200 μL, CHO-S cell suspension + 20 μg plasmid + 10 μg salmon sperm DNA, electroporation reaction conditions: 500V, 500us, 4 times.

[0055] 2. Screening of high expression cell lines:

[0056] Spread the electroporated cell suspension in a Petri dish containing 10 mL of medium (DMEM / F12=1:1, containing 10% FBS). 2.4mg / L G418 (Geneticin) was used to screen the cells under pressure, and single clones were selected and transferred to 96-well plates. When the clone grows to cover 80% of the bottom of the well, remove the FBS medium, add D / F medium (expression medium) at a dose of 100 μL / well, and detect the expression after 48 hours. Samples were dil...

Embodiment 3

[0067] Example 3: Purification of chimeric monoclonal antibody.

[0068] The chimeric monoclonal antibody was purified by protein A affinity chromatography, and the specific steps were as follows:

[0069] (1) Wash the protein A affinity chromatography column with 3 to 5 times the column volume of water;

[0070] (2) Equilibrate the protein A affinity chromatography column with 20mM phosphate buffer (PBS) (pH=7.0);

[0071] (3) Filter the cell culture supernatant containing the desired purified monoclonal antibody with a 0.45 μm filter membrane, and pump it into the protein A affinity column that has been equilibrated with PBS;

[0072] (4) Wash the column with 20mM PBS (pH=7.0) until OD 280 <0.01;

[0073] (5 ） Elute the target protein with 0.1M glycine-HCl buffer (pH=3.0), collect the elution peak, and adjust the collected antibody solution with 1M Tris-HCl buffer (pH=9.0) until the pH value is neutral;

[0074] (6) Dialyze and desalt with 10mM PBS (pH=7.2) to obtain pu...

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Abstract

The invention discloses a human-mouse chimeric monoclonal antibody against the A3 region of human von Willebrand factor and its preparation method and application. Specifically, the chimeric monoclonal antibody of the present invention includes a murine heavy chain variable region, a murine light chain variable region, a human heavy chain constant region, and a human light chain constant region; wherein the murine heavy chain variable region As shown in SEQ ID NO: 1, the murine light chain variable region is shown in SEQ ID NO: 2, and the human heavy chain constant region is selected from any one of IgG, IgM, IgA, IgE, IgD Chain constant region, the human light chain constant region is any light chain constant region selected from kappa or lambda. The chimeric monoclonal antibody of the present invention can not only inhibit the combination of vWF and collagen, but also inhibit the aggregation of platelets, has a good antithrombotic effect, is not easy to generate anti-mouse immune response in the human body, and greatly reduces the occurrence of clinical bleeding risk .

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a human-mouse chimeric monoclonal antibody against the A3 region of von Willebrand factor (vWF) with high specificity, high affinity binding properties and high antithrombotic activity and its Preparation methods and applications. Background technique [0002] Epidemiological studies have shown that in recent decades, with the improvement of material life and the aging of the population, the hazards of thrombotic diseases have become increasingly prominent. Under certain pathological conditions, thrombus can inhibit or completely stop blood flow, leading to cellular necrosis and seriously threatening human health. [0003] Platelets play a key role in thrombus formation, and platelet adhesion and aggregation are necessary processes for thrombus formation. Platelet adhesion and aggregation and subsequent thrombus formation in atherosclerotic plaques play an important role in the pat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/36C12N5/20A61K39/395A61P7/02C12R1/91
CPCA61K2039/505C07K16/36C07K2317/24
Inventor 赵益明阮长耿季顺东江淼沈飞
Owner SUZHOU UNIV
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