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Anti-CD47 monoclonal antibody and use thereof

An antibody and antigen technology, applied in the direction of antibodies, medical preparations with non-active ingredients, and anti-animal/human immunoglobulins

Active Publication Date: 2021-03-05
AKESO BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current studies believe that tumor cells have a mechanism to escape macrophage phagocytosis. During the growth of tumor cells, specific proteins such as calreticulin will be formed on the surface, exposing the tumor identity to attract macrophage phagocytosis, but at the same time, macrophages with SIRPα Tumor cells with high expression of CD47 inhibit the phagocytosis of macrophages due to the activation of CD47-SIRPα pathway, and macrophages are misjudged as normal cells, causing tumor cells to escape the phagocytosis of macrophages (CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Jaiswal S, Jamieson C H M, Pang W W, et al. Cell, 2009, 138(3) 271-285)

Method used

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  • Anti-CD47 monoclonal antibody and use thereof
  • Anti-CD47 monoclonal antibody and use thereof
  • Anti-CD47 monoclonal antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199]Example 1: Preparation of antibody 6f7 of anti-human CD47

[0200]1. Preparation of hybridoma cell line 6f7

[0201]Preparation of hybridoma cell line 6F7 antigen CD47 IGV TEV-HIS (including Genbankid: NP 942088.1) Location: 19-141 CD47 Ripe peptide and TEV (amino acid sequence as enlyfqg, SEQ IDNO: 74) -HIS label The fusion protein, synthesized by Zhongshan Kangfang Biomedical Co., Ltd.) and 3T3-CD47 cells (NiH / 3T3, manufacturers: ATCC, Item No .: CRL-1658, the above-mentioned human CD47 ripe peptide into the cell on the basis of NiH / 3T3 , Construct 3T3-CD47 stabilization expression system). The splenocytes of immunization were fused to mouse myeloma cells, and hybridoma cells were made by CD47 IGV TEV-HIS, 3T3-CD47 cells as an antigen, and the hybridoma cells were screened to the ELISA method. Hybridoma cells for antibodies specifically binding to CD47. The obtained hybridoma cells were screened by competing ELISA (SirpαECD) (where sirpαecd represents the extracellular region ...

Embodiment 2

[0205]Example 2: Sequence analysis of antibody 6f7 of anti-CD47

[0206]The mRNA was extracted from the LT012 cell line cultured in Example 1 in accordance with the method of cultured cell bacteria total RNA extraction kit (TIANGEN, ITD DP430).

[0207]Follow INVITROGENIII FIRST-STRAND SYNTHESIS SYSTEM for RT-PCR kit specification synthesis cDNA and PCR amplification was performed.

[0208]The PCR amplification product was carried out directly to TA clones, and the specific operation is performed with the PEASIY-T1 Cloning Kit (Transgenct101) kit.

[0209]The product of the TA clone is sequenced directly, and the sequencing results are as follows:

[0210]The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the length is 351 bp.

[0211]Its encoded amino acid sequence, such as SEQ ID NO: 2, with a length of 117AA, wherein the sequence of the heavy chain CDR1 is as shown in SEQ ID NO: 5, and the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 6, the heavy ...

Embodiment 3

[0214]Example 3: Light chain and heavy chain design and preparation of humanized antibody 6F7 H1L1 (HG4), 6F7 H2L2 (Hg4) and 6F7 H3L3 (hg4) of anti-human CD47

[0215]1. A anti-human CD47 humanized antibody 6F7 H1L1 (HG4), 6F7H2L2 (HG4) and 6F7 H3L3 (HG4) light chain and heavy chain design

[0216]Three-dimensional crystal structure according to human CD47 protein (Hage T, Reinemer P, Sebald W.Crystals of A1: 1 Complex Between Human Intergeukin-4 and The Extracellular Domain of ItSreceptor Alpha Chain.eur J Biochem. 1998; 258 (2): 831 -6.) and the sequence of the antibody 6f7 obtained in Example 2, by the computer analog antibody model, according to the model design mutation, the variable region sequence of the antibody 6F7H1, 6F7 H2L2 and 6F7 H3L3 (antibody constant region sequence, database from NCBI) Heavy chain constant zone sequence is IgGamma-4 CHAIN ​​C Region, Accession: P01861.1; Light chain constant region is Ig Kappa Chain Cregion, Accession: P01834).

[0217]The variable region s...

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Abstract

The invention relates to an anti-CD47 monoclonal antibody and use thereof. The anti-CD47 monoclonal antibody is secreted from a hybridoma cell strain with an accession number of CCTCC NO: C2018135.

Description

Technical field[0001]The present invention relates to autoimmune disease therapy and molecular immunological fields. Including an antibody against CD47, comprising a pharmaceutical composition thereof and a use thereof. Specifically, the present invention relates to a monoclonal antibody against CD47.Background technique[0002]CD47 is also known as Integrin Associated Protein (IAP). CD47 is 5 transmembrane proteins, and the molecular weight is around 50 kDa, which belongs to the immunoglobulin superfamily. The extracellular N end is an IGV domain, which is coupled to αvβ3 (CD51 / CD61) and αIib [beta] 3 (CD41 / CD61) integrin. CD47 relates to a variety of physiological functions, such as cell metastasis, T cells and dendritic cells (DENDRITIC Cell, DC) activation, axial sporting.[0003]CD47 expressed in all cell types including red blood cells while expressing high expression on tumor cells. The CD47 has two ligands to signal regulatory protein alpha (SIGNAL Regulator Protein], SIRPα)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00C12N5/20C12N15/85C12N5/10A61K39/395A61P35/00
CPCC07K16/2803C12N15/85A61P35/00C07K2317/565C07K2317/567C07K2317/24C07K2319/21A61K2039/505C07K2317/53C07K2317/76C07K2317/92A61K39/395C07K19/00C07K16/28A61K47/68C12N5/10C07K2317/31G01N2333/70596A61K35/13C07K2317/14
Inventor 张鹏李百勇夏瑜王忠民
Owner AKESO BIOPHARMA
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