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Polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and application thereof

An immune checkpoint, RS-17 technology, applied in the field of peptides that block the binding of CD47 on the surface of tumor cells and its corresponding ligands, can solve the problems of no research reports on small molecule drugs or peptide drugs, and achieve the inhibition of CD47/SIRPα pathway , Eliminate immunosuppression, weak effects of toxic and side effects

Active Publication Date: 2020-04-10
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both drugs belong to the monoclonal antibody class, and there are no research reports on small molecule drugs or peptide drugs.

Method used

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  • Polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and application thereof
  • Polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and application thereof
  • Polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1RS-17 synthetic product quality detection

[0050] Synthetic RS-17 polypeptide, its sequence is: RRYKQDGGWSHWSPWSS-NH 2 . Its relative molecular mass, yield and purity and other property information were measured by mass spectrometry (Mass Spectrometry, MS) and high performance liquid chromatography (High Performance Liquid Chromatography\HPLC).

[0051] The specific chromatographic conditions are:

[0052] Analytical chromatographic column: SinoChrom ODS-BP, 250mm×4.6mm (5μm filler);

[0053] Mobile phase: Phase A is water (100%), phase B is acetonitrile (100%);

[0054] Loading volume: 20μL;

[0055] Flow rate: 1mL / min;

[0056] Detection wavelength: 220nm;

[0057] Elution gradient: see Table 1.

[0058] Table 1 The elution gradient for detecting the modification yield

[0059]

[0060] Its MS report is identical to the HPLC report such as figure 1 , figure 2 show. The results showed that the relative molecular mass of RS-17 was 2120.29g / mo...

Embodiment 2

[0061] Example 2 MST intermolecular interaction analysis between polypeptide RS-17 and CD47 protein

[0062] Microthermophoresis (MST) is a technique for the analysis of biomolecules. Microscale thermophoresis is the directional movement of particles in a microscopic temperature gradient. Relative changes in motion along temperature gradients due to changes in hydration layers caused by changes in biomolecular structure / conformation can be used to determine affinity (K d value).

[0063] In this part of the examples, the polypeptide RS-17 (RRYKQDGGGWSHWSPWSS-NH 2 ) as an object to study the interaction between it and CD47 extracellular segment recombinant protein (Ala18-Glu141) in vitro.

[0064] 1.1 Experimental materials

[0065] Polypeptide RS-17 (RRYKQDGGWSHWSPWSS-NH 2 ); human recombinant protein CD47 (Ala18~Glu141, Cloud-Clone, USA)

[0066] 1.2 Experimental Instruments

[0067] Monolith NT.115MST micro-thermophoresis instrument (German nanotemper company); Capill...

Embodiment 3

[0079] Western blot detected tumor cell lines with high expression of CD47 and lymphocyte cell lines with high expression of SIRPα as positive cells, which were subsequently used to detect the blocking effect of polypeptides on the CD47 / SIRPα pathway.

[0080] 1.1 Experimental materials

[0081] Human liver cancer cell HepG2: Shanghai Chunmai Biotechnology Co., Ltd.; human epidermal squamous cell carcinoma cell SCC-13: human mononuclear lymphoma cell THP-1: Shanghai Chunmai Biotechnology Co., Ltd.; mouse macrophage lymphoma cell RAW264.7: cell culture Bottle of American Thermo Company; cell culture medium: Gibco Company of the United States; rabbit anti-human / mouse CD47 antibody: BioXcell Company of the United States; rabbit anti-human / mouse SIRPα antibody: Wanlei Biotechnology Co., Ltd.; rabbit anti-human / mouse GAPDH antibody: Linktech bio ltd. HRP Goatanti-Rabbit IgG: Lianke Biological Co., Ltd.

[0082] 1.2 Experimental Instruments

[0083] Inverted microscope (Japan OLY...

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Abstract

The invention discloses a polypeptide RS-17 with anti-CD47 immune checkpoint antagonistic activity and an application thereof, and belongs to the field of biomedicines. The polypeptide sequence of thepolypeptide RS-17 is RRYKQDGGWSHWSPWSS-NH2. The polypeptide can specifically recognize CD47 on the surfaces of tumor cells, and has ability of blocking binding of the CD47 and a corresponding ligandof the CD47, so that an autologous signal generated by interaction between the CD47 and SIRPa on macrophage surfaces is blocked. Therefore, an immunosuppressive effect of tumors is eliminated, and theeffect of specifically killing the tumors is achieved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and more specifically relates to a polypeptide capable of blocking the combination of CD47 on the surface of tumor cells and its corresponding ligand and its application. Background technique [0002] CD47 (Cluster of Differentiation 47), also known as Integrin-Associated Protein (IAP), is encoded by the CD47 gene in the human body and is widely expressed as a glycoprotein with five transmembrane domains, which plays an important role in self-signaling effect. It belongs to the immunoglobulin superfamily and can interact with integrin (Integrin), thrombospondin (TSP-1) and signal regulatory protein α (SIRPα), among which the interaction between CD47 and signal regulatory protein α (SIRPα) was studied The most in-depth part. The bidirectional signal transduction mediated by its corresponding receptors results in different intercellular responses, including inhibition of phagocytosis, cell fusio...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P35/00A61P35/02
CPCC07K7/08A61P35/00A61P35/02A61K38/00
Inventor 徐寒梅王新民
Owner CHINA PHARM UNIV