Preparation method and applications of monoclonal antibody against human CD47

A monoclonal antibody, antibody technology, applied in the direction of anti-animal/human immunoglobulin, application, antibody, etc., can solve the problems of unclear epitope, low affinity, large side effects, etc., to achieve clear epitope identification, The effect of good binding activity

Active Publication Date: 2019-03-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the anti-human CD47 monoclonal antibody currently under development still has the disadvantages of low affinity, large side effects, and unclear targets such as antigenic epitopes

Method used

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  • Preparation method and applications of monoclonal antibody against human CD47
  • Preparation method and applications of monoclonal antibody against human CD47
  • Preparation method and applications of monoclonal antibody against human CD47

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Preparation of Recombinant Human CD47 Extracellular Region Antigen

[0066] Extraction of RNA in MKN-45 cells by Trizol method: 1X10 6 A MKN45 species was placed in a 3cm culture dish. Discard the culture supernatant the next day, rinse with 1xPBS, digest with 1ml Trizol, and centrifuge at 12000×g in a refrigerated high-speed centrifuge for 5 Minutes, carefully take out the centrifuged tube, tilt it slightly and pipette the uppermost liquid into another RNase-free sterilized 1.5ml centrifuge tube, according to the volume ratio of chloroform and Trizol is 1:5 Add chloroform, upside down and fully mix the liquid to make it milky white; after standing at room temperature for 3 minutes, centrifuge in a refrigerated high-speed centrifuge at a speed of 12,000×g for 15 minutes at 4°C, and carefully take out the centrifuged tube. At this time, it can be seen that stratification has appeared in the tube. Add the upper aqueous phase (containing RNA) to another RNase-fr...

Embodiment 2

[0070] Example 2 Immunization of mice with recombinant human CD47 extracellular region antigen

[0071] For the first immunization, the PBS solution containing 50 μg recombinant human CD47 antigen was phacoemulsified with an equal volume of Freund’s complete adjuvant, with a total volume of 200 μl, and Balb / c female mice were injected subcutaneously in multiple points. Five mice were immunized each time. After 3 weeks, the second booster immunization was carried out: 25 μg of antigen per mouse was mixed with an equal volume of Freund’s incomplete adjuvant to make a total volume of 200 μl, phacoemulsification was performed, and subcutaneous injection was performed at multiple points. Two weeks later, the third immunization was carried out: 25 μg of antigen per mouse was mixed with an equal volume of Freund’s incomplete adjuvant to make a total volume of 200 μl for phacoemulsification and multi-point subcutaneous injection. Sera were collected from all mice after three immuniza...

Embodiment 3

[0075] Fusion and screening of embodiment 3 hybridoma cells

[0076]Three days after the shock immunization, the spleen was prepared for cell fusion. Submerge the sacrificed mouse into 70% alcohol and place it on a dissecting board, take out the spleen of the mouse from the upper left side of the abdominal cavity, and place the spleen in a 60mm-diameter petri dish containing 3ml of complete DMEM medium; Transfer the culture dish containing the spleen to the ultra-clean bench, carefully peel off the fat and other tissues on the surface of the spleen with a set of sterile scissors and tweezers; transfer the spleen to a cell filter sieve, and place the sieve in In a 100mm petri dish; use a 3ml syringe with a 26G needle to inject 2ml DMEM into different positions of the spleen; use sterile scissors to cut the spleen filled with DMEM in several places; use a 3ml syringe plug to expel the spleen until only fibrous tissue remains The tissues that remained on the surface of the sieve...

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Abstract

The invention belongs to the field of antibodies, and more particularly relates to a preparation method and applications of a monoclonal antibody against human CD47. The invention discloses the heightvariable region sequence of the monoclonal antibody. The monoclonal antibody against the human CD47 provided by the invention has good binding activity, can effectively recognize the expression of CD47 on the surfaces of tumor cells, and can effectively recognize the extracellular domain recombinant protein of the human CD47 at the protein level. In addition, the antigen epitope identification ofthe monoclonal antibody is clear, thereby being effectively applied to the diagnosis of CD47 target molecules and preparation and development of monoclonal antibody drugs against the human CD47.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the preparation and application of an anti-human CD47 monoclonal antibody. Background technique [0002] CD47 (Cluster of Differentiation 47), also known as integrin-related protein, is widely expressed on the surface of normal cells and is one of the currently known immune checkpoints. As a receptor, CD47 can affect cell-related functions including cell migration, adhesion, proliferation and apoptosis when combined with thrombospondin-1, and can also regulate angiogenesis and inflammation. When it acts as a ligand and binds to signal-regulatory protein-α (SIRPα) on the surface of macrophages, it can transmit inhibitory signals to inhibit the phagocytosis of related cells by macrophages. Aging red blood cells in circulation are more easily cleared by macrophages due to reduced expression of CD47. Studies have shown that CD47 is not only widely expressed in various tissues, but also...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K14/705C12N15/13A61K39/395A61P35/00G01N33/574
CPCA61K2039/505A61P35/00C07K14/70503C07K16/2803C07K2317/24C07K2317/565C07K2317/567C07K2317/73C07K2317/92G01N33/57488G01N2333/70503
Inventor 钱峰俞凯凯孙树洋吴穷张志愿孙磊王军顾子悦胡玉冬
Owner SHANGHAI JIAO TONG UNIV
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