Novel recombinant bifunctional fusion protein as well as preparation and application thereof

A fusion protein and dual-function technology, which can be applied in the direction of drug combination, fusion polypeptide, peptide/protein components, etc., can solve the problem of insufficient CD47 affinity and activity

Active Publication Date: 2016-11-23
IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The binding activity of SIRPα-Fc and SIRPαD1-Fc to CD47 has been reported before (Lee WY et al, 2007), but their affinity activity with the target CD47 is insufficient

Method used

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  • Novel recombinant bifunctional fusion protein as well as preparation and application thereof
  • Novel recombinant bifunctional fusion protein as well as preparation and application thereof
  • Novel recombinant bifunctional fusion protein as well as preparation and application thereof

Examples

Experimental program
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Embodiment 1

[0074] Experimental materials and methods

[0075] 1. Construction of SIRPα-Fc and SIRPαD1-Fc expression vectors

[0076] The expression vector used was pIg-Tail (R&D Systems). Use primer 1 (SEQ ID No.: 9) and primer 2 (SEQ ID No.: 10), primer 1 (SEQ ID No.: 9) and primer 3 (SEQ ID No.: 11) respectively the outer membrane of SIRPα The end and SIRPαD1 gene coding sequence from THP-1 ( TIB-202 TM ) cells, and the PCR products were respectively cloned into the HindIII / EcoRI sites of the improved pIg-Tail vector to form pSIRPα-Fc and pSIRPαD1-Fc vectors.

[0077] 2. Construction of HY03M and HY03MM expression vectors

[0078] The SIRPαD1 gene sequence carrying the N89A mutant was sent to Nanjing GenScript Synthesis (Project No.: 7009323-1), and the synthesized gene was cloned into the HindIII / EcoRI site of the pIg-Tail vector to form the HY03M expression vector. Using primer 4 (SEQ ID No.: 12) and primer 5 (SEQ ID No.: 13), through site-directed mutagenesis PCR, the gene seq...

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Abstract

The invention discloses a genetic recombinant fusion protein. Tumors can be eliminated through two mechanisms, one mechanism refers to blockage of inhibitory signals for tumor-cell-induced macrophage, and the other mechanism refers to direct activation of the phagocytosis of the macrophage. The protein (SIRPalphaD1-Fc (signal-regulatory proteins alpha D1-Fc)) is formed by connecting human SIRPalphaD1 (a first Ig sample region of an outer membrane end of SIRPalpha) with an Fc segment of human IgG1. The invention provides a nucleotide molecule encoding the recombinant bifunctional fusion protein, an expression vector expressing the protein, a method for preparing the protein and a method for treating CD47 overexpression-related diseases. The protein belongs to a homodimer and has the molecular weight being 90 kDa.

Description

technical field [0001] The invention relates to a recombinant bifunctional fusion protein and its preparation method and application, especially its application in tumor treatment. Background technique [0002] In order to accelerate their own growth, tumor cells often "invent" some ways to escape the host's immune surveillance of tumor cells. The known pathways include three: 1) Evasion from the immune surveillance of T lymphocytes. Tumor cells often highly express the membrane proteins PD-L1 and PD-L2, and PD-L1 / PD-L2 can bind to PD-1 on the surface of T cell membranes to induce T cell apoptosis. 2) Evasion of immune surveillance by natural killer (NK) cells. There is a protein NKG2D on the cell membrane of NK cells that can activate NK cells. When NKG2D combines with MICA / MICB on the tumor cell membrane, it can activate NK cells to kill tumor cells. However, tumor cells themselves have a mechanism that can promote the shedding of MICA / MICB from the cell membrane, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10A61K39/395A61P35/00A61P35/02A61P29/00A61P19/02A61P1/00A61P11/06A61P37/08
CPCA61K39/395C07K19/00C12N5/10C12N15/62C12N15/63A61P11/06A61P19/02A61P35/02A61P1/00A61P35/00A61K38/00C07K14/4702C07K2319/30C07K2319/74
Inventor 刘利娟景德强王华
Owner IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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