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Recombinant protein prepared based on SIRP-alpha D1 mutant and application

A recombinant protein and mutant technology, applied in the field of genetic engineering, can solve the problems of not improving the phagocytosis of macrophages to cancer cells, not improving the targeting affinity of cancer cells, etc.

Active Publication Date: 2019-02-05
BETA PHARM SUZHOU LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this patent does not improve the targeting affinity with cancer cells, nor does it functionally improve the phagocytosis of macrophages on cancer cells

Method used

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  • Recombinant protein prepared based on SIRP-alpha D1 mutant and application
  • Recombinant protein prepared based on SIRP-alpha D1 mutant and application
  • Recombinant protein prepared based on SIRP-alpha D1 mutant and application

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Embodiment 1

[0054] The present invention relates to two recombinant proteins, including SIRPαD1 contact surface amino acid point mutation combination coupled with human IgG1-Fc (CD001), and deglycosylation point mutation (N80A) on CD001 to obtain CD002. The above recombinant protein can be obtained through two mechanisms To kill tumor cells, one is to block the SIRPα-CD47 signal to activate the phagocytosis of tumor cells by macrophages, and the other is to combine Fc with the Fc receptor of macrophages to activate the immune function of macrophages. The present invention uses human embryonic kidney cells (293F) to express and purify the recombinant protein, and finds that the mutant has better target binding activity and macrophage ADCP activation function, and has a lower degree of hemolysis to red blood cells.

[0055]In the present invention, CD001 (I31 / K53 / E54 / H56 / S66) is obtained by point mutation of part of the surface amino acids in the binding region of SIRPαD1 and CD47, or the 80...

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Abstract

The invention discloses a recombinant protein prepared based on a SIRP-alpha-D1 mutant and application. A mutant I is obtained on the basis of performing contact surface amino acid multi-point mutation on an outer end IgV area (SIRP-alpha-D1) of a SIRP alpha film, a mutant II is obtained by performing deglycosylation mutation on an amino acid asparaginate (N) at the 80th site on the basis of the mutant I, and a human IgG1-Fc area is coupled for performing fusion expression to obtain the recombinant protein CD001 / CD002. Compared with a wild-type or deglycosylation single-point mutant, these mutants have higher affinity with the recombinant protein CD47 and multiple types of cancer cell lines, and have a better blocking effect on CD47 and SIRPa combination in a protein level and a cell level; the mutants have no blood clotting to a white blood cell (a red blood cell); and in the aspect of function, the mutants are capable of more effectively activating phagocytosis of macrophage to a tumor cell. The CD001 / CD002 has better target spot bonding activity, better target spot blocking effect and stronger immune activation function, and has no side effect to the red blood cell.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant protein prepared based on a SIRP-αD1 mutant and its application. Background technique [0002] CD47 is also known as integrin-associated protein (integrin associated protein, IAP), which is widely expressed on the surface of cells and can bind to signal regulatory protein α (Signal regulatory protein α, SIRPα), thrombospondin-1 (TSP1) and integrin (integrins ) interaction to mediate a series of responses such as apoptosis, proliferation, and immunity. CD47 is a 5-time transmembrane protein with a molecular weight of about 50kDa, belonging to the immunoglobulin superfamily, and the extracellular N-terminal is the IgV domain. CD47 was first identified as a tumor antigen of human ovarian cancer in the 1980s, and then CD47 was found to be expressed in various types of human tumors, including acute myeloid leukemia (AML), chronic myeloid leukemia ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K47/68A61P35/00
CPCA61K47/6801A61P35/00C07K14/70503C07K2319/30
Inventor 王雷黄苏萍尹建河张楠董丽莎赵永浩汤春彭继荣张晓东
Owner BETA PHARM SUZHOU LTD
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