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ACE2 immune fusion protein and application thereof

A fusion protein, Z1-Z2 technology, applied in the direction of fusion polypeptide, peptide/protein component, hybrid peptide, etc., can solve the problem that the exact position is not yet clear

Active Publication Date: 2021-02-19
PHARCHOICE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some ACE2-based recombinant protein drug candidates have shown some therapeutic effects in animal models of acute inflammatory injury studies, but the exact status of these candidate drugs in the treatment of related diseases has not yet been clarified

Method used

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  • ACE2 immune fusion protein and application thereof
  • ACE2 immune fusion protein and application thereof
  • ACE2 immune fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Preparation of recombinant polypeptide

[0044] (1) Entrust a gene synthesizer (Suzhou Jinweizhi Company) to optimize the coding nucleotide codon and complete gene synthesis for the amino acid sequence of the immune fusion protein required in this example, and the optimized nucleotide sequence is directly loaded into the PCDNA3.4 vector Above, the amino acid sequences encoded by all vectors are described in Table 1.

[0045] (2) Entrust the protein manufacturer (Shenzhou Yiqiao Company) to express and purify the immune fusion protein for this example. Using literature Finck B K. Science, 265.; Mihara M et al.. Journal of Clinical Investigation.2000; 106:91-101; Yu X, et al. Nature Immunology.2009; 10:48-57.LiuS, et al. Clin Immunol.2019Jun; 203:72-80.) method, using 293F system for transient transfection expression technology for protein expression, and then using peptide M, SSL7 and ion exchange methods to obtain a large number of recombinant peptides, SDS-...

Embodiment 2

[0058] Example 2. Neutralizing effect of immune fusion protein on virus invasion

[0059] Firstly, the recombinant virus system was used to detect the virus invasion of Vero E6 cells mediated by the recombinant SARS-CoV-2S protein of the immune fusion protein. This method is a routine detection method in this field. Refer to the non-patent literature [Lei C, et al..Naturecommunications, 2020 ,11(1):1-5.]. The results are shown in Table 4:

[0060] Table 4 Virus infection

[0061] Group (drug concentration, 5μg / ml) Recombinant virus invasion (compared to background fluorescence intensity multiples) SD P value (compared to positive control group) Blank control (no virus) 1.204 0.213 Positive control (virus only) 3622.65 201.66 rhACE2 1208.37 28.68 Control IgG 3854.327 350.180 Control IgA 4143.753 203.011 ACE2-IgG 0.927 0.497 P＜0.05 ACE2-IgA1 1.113 0.932 P＜0.05 ACE2-IgA2 1.115 0.755 P＜0.05 S...

Embodiment 3

[0063] Example 3. Immune fusion protein mediates phagocytosis of virus particles by immune cells

[0064]For further experimental research on the evaluation of peripheral blood mononuclear cells (PBMC) phagocytosis of virus particles, the method of detecting cell phagocytosis of virus particles by flow cytometry and the calculation of phagocytosis fraction can refer to non-patent literature [Gach J S, et al. al..PLoS pathogens,2017,13(12):e1006793.], the anti-S protein antibody carrying FITC was used as a detection method to detect the recombinant virus. It is a routine technique in the field to prepare recombinant virus systems and detect intracellular proteins by flow cytometry, and reference can be made to non-patent literature [Fu W, etal. Naturecommunications, 2019, 10(1): 1-12.]. The results are shown in Table 5:

[0065] Table 5 Phagocytosis of SARS-CoV-2 recombinant virus particles

[0066]

[0067] The results show that, compared with the IgG type fusion protein,...

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Abstract

The invention relates to the technical field of biomedical engineering, and provides ACE2 immune fusion protein and application thereof. A structural general formula of the ACE2 immune fusion proteinis Z1-Z2, wherein Z1 is ACE2 or a functional variant or fragment thereof, and Z2 is an IgA heavy chain constant region or a functional variant or fragment thereof. Experiments prove that the ACE2-IgAimmune fusion protein enhances the ACE2 enzyme activity, effectively enhances the anti-immune cell IgA receptor activation, enhances the ACE2-dependent immune cell phagocytosis and clearance effects and lowers the immune cell secretion effect, and ACE2-IgG type fusion protein has no similar effect. Through animal model experiments, the immune fusion protein can reduce overexpression release of organ inflammatory mediators, relieve organ inflammatory injury, enhance organ anti-stress ability and resist organ injury. Through independent application or combination with other related disease drugs, inflammatory mediator related diseases caused by ACE2 down-regulation and / or dysfunction can be effectively treated, and wide clinical application prospects are achieved.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to ACE2 immune fusion protein, its preparation method and application. Background technique [0002] When animal cells or tissues are damaged by bacteria, trauma, toxins, physical or chemical factors (which may be collectively referred to as "inflammatory substances, inflammatory agents"), an inflammatory response will occur. The pathophysiological features of the inflammatory response are regulated by the complex interplay of multiple pro-inflammatory or anti-inflammatory stimuli or mediators synthesized and released by cells. Some known classes of pro-inflammatory and anti-inflammatory stimuli or mediators include cytokines, nitrous oxide, thromboxane, aridine, phospholipid-like platelet activating factor, prostaglandins, elicitors, complement factors, coagulation factors , superantigen, monokine, chemokine, interferon, free radical, protease, arachidonic acid meta...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/48C12N15/62A61K38/48A61K47/68A61P29/00
CPCC12N9/485C12Y304/17023A61K38/48A61K47/6801A61P29/00C07K2319/30C07K2319/00Y02A50/30
Inventor 胡适傅文燕
Owner PHARCHOICE THERAPEUTICS INC
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