Anti-human CD47 monoclonal antibody

A monoclonal antibody, expression vector technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-tumor drugs, etc.

Active Publication Date: 2020-12-08
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, CD47 antibodies have been reported to cause platelet aggregation and hemagglutination of red blood cells

Method used

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  • Anti-human CD47 monoclonal antibody
  • Anti-human CD47 monoclonal antibody
  • Anti-human CD47 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The acquisition of embodiment 1 hybridoma cell strain

[0063] a. Antigen preparation

[0064] (1) Obtain the target gene

[0065] In this embodiment, the gene sequence of CD47 (NCBI sequence number XP005247966.1, numbered as SEQ ID NO: 25 in the present invention) and its signal is searched from NCBI, and enzyme cutting sites AvrII and BstZ17I are added at both ends, kozak sequence, connecting peptide murine antibody-mFc sequence (NCBI sequence number AAK53870.1, numbered as SEQ ID NO: 26 in the present invention); or adding enzyme cutting sites AvrII, BstZ17I, kozak sequence, -his at both ends Tag, wherein His-tag is a short peptide composed of 6 histidines. The sequence was sent to Shanghai Bioengineering Co., Ltd. for sequence optimization and synthesis. The amino acid sequence design is shown in SEQID NO:17,18. The target gene encoding this protein is synthesized by gene synthesis technology, and its nucleotide sequence is shown in SEQ ID NO:19,20.

[0066] (2)...

Embodiment 2

[0094] Example 2 Preparation and Purification of Antibodies

[0095] The cell lines 3D8, 3D5, 1C6, and 3E7 obtained in Example 1 were recovered, subcultured and expanded, and the cell culture supernatant was collected. Antibodies were purified by Protein A affinity chromatography. First prepare the protein A affinity column. After equilibrating the column with PBS, pass the cell culture supernatant that was centrifuged and filtered through a 0.4um filter through the column, then wash with PBS until the OD value is close to zero, and use 50mmol / L glycine at pH7.5- Elute with hydrochloric acid buffer solution, collect the eluate in the peak area, and set aside after dialysis.

Embodiment 3

[0096] Example 3 SDS-PAGE detects target protein molecular weight and expression

[0097] The size and purity of the target protein were detected by SDS-PAGE reduction electrophoresis. Electrophoresis was performed according to the method of the fourth part of the Chinese Pharmacopoeia 2015 edition, and the grayscale scanning of the electropherogram was performed to identify the molecular weight and expression level of the monoclonal antibody. According to electrophoresis results figure 1 As shown, the light chain is about 25KD, the heavy chain is about 50KD, and the protein purity is greater than 95%.

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Abstract

The invention belongs to the field of biology, and discloses a mouse anti-human CD47 monoclonal antibody and a hybridoma cell strain secreting the monoclonal antibody. Specifically, the hybridoma cellstrain secreting the monoclonal antibody in the invention is 3D8. The antibody in the invention comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is SEQ ID NO:1; and the amino acid sequence of the light chain variable region is SEQ ID NO:2. The antibody provided by the invention is high in affinity, can promote phagocytic action of macrophages on tumour cells by blocking human SIRP alpha and human CD47 signals, and plays an important role in the aspect of human tumour treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-human CD47 monoclonal antibody. Background technique [0002] Cluster of differentiation 47 (CD47), also known as integrin-associated protein (IAP), is a transmembrane glycoprotein widely expressed on the cell surface and belongs to the immunoglobulin superfamily. [0003] The molecular weight of CD47 is between 47-55KD, and its structure includes an amino-terminal extracellular variable region, a transmembrane region composed of 3-5 highly hydrophobic transmembrane segments and a hydrophilic carboxy-terminal cytoplasmic tail region . It interacts with various ligands such as integrins, SIRP-α (signal regulatory protein alpha), SIRPγ and thrombospondin. [0004] Anti-CD47 monoclonal antibody treatment of tumors is related to multiple mechanisms. First, the anti-CD47 monoclonal antibody causes tumor cells to be phagocytized by blocking the binding of CD47 on tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/20C12N15/70C12N1/21C07K14/705A61K39/395A61P35/00C12R1/91
CPCC07K16/2803C07K14/70503C12N15/70A61P35/00A61K2039/505
Inventor 赵丽丽王玲玲刘忠
Owner LUNAN PHARMA GROUP CORPORATION
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