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SIRP alpha fusion protein, preparation method and application thereof

A fusion protein and sequence technology, applied in the field of fusion proteins, can solve the problems of effectiveness, safety and production cost limitations, and achieve the effects of reducing production costs, improving phagocytosis, and enhancing affinity

Active Publication Date: 2018-09-04
上海高菲生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, the current molecular design of SIRPα still has limitations in effectiveness, safety and production cost.

Method used

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  • SIRP alpha fusion protein, preparation method and application thereof
  • SIRP alpha fusion protein, preparation method and application thereof
  • SIRP alpha fusion protein, preparation method and application thereof

Examples

Experimental program
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Effect test

experiment example 1

[0040] Experimental Example 1 Preparation of SIRPα fusion protein of the present invention

[0041] like figure 1 Shown is a schematic structural diagram of the SIRPα fusion protein of the present invention. SIRPα-Fc and SIRPα-Fc-Fc fusion proteins are composed of human IgG1 Fc region (SEQ ID NO. 2), human IgG4 Fc region (SEQ ID NO. 3) , human IgG1 Fc-Fc or human IgG4 Fc-Fc fused with the N-terminal V domain (variable region domain) of human SIRPα (SEQ ID NO. 1) to obtain a fusion protein.

[0042] Among them, the same human SIRPα domain is connected to the human IgG4 Fc region containing the hinge stabilizing S288P mutation that prevents the formation of disulfide bonds in the chain, and SIRPα-Fc (Fc is human IgG4 Fc) and SIRPα-Fc-Fc (Fc It is human IgG4 Fc-Fc) fusion protein, and its sequences are respectively shown in SEQ ID NO. 8 and SEQ ID NO. 9.

[0043] All fusion proteins described above were produced by overlapping PCR using standard molecular biology techniques to ...

experiment example 2

[0058] Experimental example 2 The binding effect of SIRPα-lgG4-Fc fusion protein of the present invention on CD47

[0059] (1) Western blot experiment of binding of SIRPα-lgG4-Fc fusion protein of the present invention to CD47

[0060] Mix the above purified fusion protein with different concentrations (equal ratio increase, 0.1nM, 1nM, 10nM, 100nM) with purified CD47 protein (10ng, Abcam) and protein A sepharose beads in PBS solution, 4 degrees Overnight, the immune complexes were pelleted by centrifugation and run on SDS-PAGE gel electrophoresis. Finally, CD47 protein levels were quantified by Western blot assay. Wherein, the amount of CD47 added was directly used as a control.

[0061] like figure 2 As shown, it is the western blotting figure (IP, Immunoprecipitation, immunoprecipitation experiment; WB, Western Blotting, immunoblotting experiment) of the SIRPα-lgG4-Fc fusion protein of the present invention that carries out IP-WB experiment, it can be seen that with the...

experiment example 3

[0067] Experimental example 3 Induction of the phagocytosis effect of the SIRPα-lgG4-Fc fusion protein of the present invention in vitro

[0068] (1) Preparation of M1 macrophages

[0069] Human M1 macrophages were prepared from monocytes obtained from normal healthy human donors (human peripheral blood mononuclear cells were provided by StemCelltechnologies). Monocytes were differentiated into macrophages by culturing in special differentiation medium (supplied by StemCell technologies) supplemented with M-CSF (Macrophage Colony-Stimulating Factor) (20 ng / mL), Specific steps are as follows:

[0070] Monocytes were differentiated into macrophages by culturing in RPMI 1640-based differentiation medium for 6-10 days. The medium was supplemented with the following components: 10% heat-inactivated human AB serum, 1% GlutaMax, 1% penicillin and streptomycin (purchased from GIBCO LifeTechnologies).

[0071] One day before the measurement of phagocytosis, IFN-γ (Interferon-γ, γ-in...

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Abstract

The invention discloses an SIRP alpha (signal regulatory protein alpha) fusion protein, a preparation method and application thereof. The fusion protein comprises a V structural domain of SIRP alpha and an Fc segment with effector function; the V structural domain comprises an amino acid sequence SEQ ID No.1; and the Fc segment comprises a constant region of human body IgG1 or IgG4 antibody. The fusion protein provided by the invention enhances the affinity of SIRP alpha and CD47, the half-life in vivo, and the Fc segment mediated effector function, and can strongly induce the phagocytosis ofmacrophages to a variety of tumor cells.

Description

technical field [0001] The present invention relates to a fusion protein, in particular to a SIRPα fusion protein and its preparation method and application. Background technique [0002] Evasion of the immune system is one of the hallmarks of tumors. In recent years, using the body's original immune response to fight against tumors has gradually become a new treatment strategy. According to the chronological order of the application of different mechanisms of therapy, it mainly includes non-specific immune stimulation, immune checkpoint monoclonal antibody, adoptive cell reinfusion, monoclonal T Cell receptor therapy, CD47 monoclonal antibody, tumor vaccine, etc., are among the most promising therapies both in the experimental stage and in clinical application. Normally, macrophages can infiltrate the tumor body and their antitumor effects can be exploited to benefit the patient. [0003] CD47, also known as Integrin Associated Protein (IAP), is a member of the immunoglob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/17A61K47/42A61P35/00
CPCA61K38/00A61P35/00C07K14/4702C07K2319/30
Inventor 武向卫孙建成于志恒
Owner 上海高菲生物科技有限公司
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