Human-mouse chimeric monoclonal antibody for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody

A hemophilia factor and monoclonal antibody technology, applied in the biological field, can solve the problems of increasing the probability of immune response, weakening of immune response, and patient's allergic reaction, so as to reduce cardiac ischemic events, prevent the formation of acute thrombosis, The effect of prolonged bleeding time

Active Publication Date: 2016-07-06
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 for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody
  • Human-mouse chimeric monoclonal antibody for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody
  • Human-mouse chimeric monoclonal antibody for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody

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 chain and light chain of the functional antibody were cloned from the hybridoma cell line SZ-123 secreting the mouse monoclonal antibody against the human vWFA3 region, in which the mouse monoclonal antibody SZ- The mRNA of 123 hybridoma cell lines was used as a template, and a set of heavy chain and light chain reverse transcription specific primers HGSP1 and KGSP1 were designed, the nucleotide sequences of which were shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. 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 cor...

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 / LG418 (Geneticin) was used to screen the cells under pressure, and single clones were selected and transferred to a 96-well plate. 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 20mMPBS (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 10mMPBS (pH=7.2) to obtain pure...

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Abstract

The invention discloses a human-mouse chimeric monoclonal antibody for a human von willebrand factor A3 region as well as a preparation method and application of the human-mouse chimeric monoclonal antibody. Specifically, the human-mouse chimeric monoclonal antibody comprises a mouse source heavy chain variable region, a mouse source light chain variable region, a human source heavy chain constant region and a human source light chain constant region, wherein the mouse source heavy chain variable region is as shown in SEQ ID NO:1; the mouse source light chain variable region is as shown in SEQ ID NO:2; the human source heavy chain constant region is selected from any one of heavy chain constant regions of IgG, IgM, IgA, IgE and IgD; the human source light chain constant region is selected from any one of light chain constant regions of kappa or lambda. The human-mouse chimeric monoclonal antibody can inhibit both combination of vWF and collagen and accumulation of thrombocyte, is good in antithrombus effect and is unlikely to have anti-mouse immunoreaction in human bodies, and moreover the probability of clinical bleeding risk can be greatly reduced.

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 Applications(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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