ACE2 protein and IL-6 or TNF alpha antagonist composition and application thereof

An IL-6, alpha antagonist technology, applied in the direction of anti-inflammatory agents, drug combinations, immunoglobulins, etc., can solve the problem that the exact status is not yet clear

Pending Publication Date: 2020-11-03
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 protein and IL-6 or TNF alpha antagonist composition and application thereof
  • ACE2 protein and IL-6 or TNF alpha antagonist composition and application thereof
  • ACE2 protein and IL-6 or TNF alpha antagonist composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Preparation of recombinant polypeptide

[0034] (1) Entrust a gene synthesizer (Suzhou Jinweizhi Company) to optimize the coding nucleotide codons and complete gene synthesis for the composition components and fusion polypeptide amino acid sequences required in this example, and the optimized nucleotide sequences are directly loaded into On the PCDNA3.4 vector, the amino acid sequences encoded by all vectors are described in Table 1. Olamkicept was provided by Ferring Pharmaceuticals, olokizumab was provided by R-Pharm, and adalimumab was purchased from Selleck.

[0035] (2) Entrust the protein manufacturer (Shenzhou Yiqiao Company) to express and purify the components of the composition and the fusion polypeptide 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.Liu S, et al. Clin Immunol.2019 Jun; 203:72-80.) method, using the 293F...

Embodiment 2

[0043] Example 2. Compositions and fusion polypeptides on ACE2-dependent phagocytosis

[0044] The preparation of 293T cells (293-S cells) expressing SARS-CoV-2 spike protein on the cell surface is the same as the literature [LeiC, et al..Nature communications, 2020, 11(1):1-5., preparation of peripheral mononuclear cells , The detection of the phagocytosis ability of peripheral monocytes on 293-S cells is the same as the literature [Klichinsky M, Ruella M, Shestova O, et al. Nature Biotechnology, 2020: 1-7.]. In each treatment group, the representative of the composition of the present invention and the fusion polypeptide is used, and the total concentration of each group of representative treatment is 3 μg / ml, and the results are shown in Table 3:

[0045] Table 3 Phagocytosis of macrophages

[0046]

[0047]

[0048] The results showed that, compared with each component, the composition and the fusion polypeptide significantly increased the immune cell clearance effe...

Embodiment 3

[0049] Example 3 Compositions and fusion polypeptides have anti-inflammatory activity on macrophages

[0050]Raw264.7 macrophages (Cell Bank of Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum (FBS; Gibco Laboratories) at 37°C and 5% CO 2 . Take 1×10 6 Raw 264.7 cells were inoculated into a 96-well plate at a density of 1 cell / mL and cultured overnight; the next day, the above-mentioned medium was replaced with fresh DMEM medium, and the 3 μg / mL various The composition was added to the cells, and a control human IgG (Sigma) was added to the control group. After the cells were incubated with the protein for 30 minutes, LPS (final concentration 1 μg / mL) was added to the medium, and the cells were incubated for another 24 hours before detection experiments.

[0051] 1) NO level test

[0052] Nitric oxide (NO) levels in the above-mentioned Raw 264.7 cell culture medium were measured using the Griess reagent system (Promega, USA). Add...

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Abstract

The invention relates to the technical field of biomedical engineering, provides an ACE2 protein and IL6 or TNF alpha antagonist composition and an application thereof, and particularly an applicationof an ACE2 protein and any one or two of an IL-6 antagonist and a TNF alpha antagonist to preparation of a medicine for treating inflammatory mediator disorder related diseases mediated by ACE2 expression level or function exceptions. The medicine is a medicine composition taking the ACE2 protein and any one or two of the IL6 antagonist and the TNF alpha antagonist as active components, or is a fusion polypeptide formed by the ACE2 protein and any one or two of the IL6 antagonist and the TNF alpha antagonist. The composition or the formed fusion polypeptide can accidentally mediate the ACE2 dependent immune cell clearing effect, reduce the ADE effect of ACE2Ig and reduce the immune cell secretion effect. In an animal model, overexpression release of organ inflammatory mediators can be reduced, organ inflammatory injury can be reduced, the anti-stress capability of organs can be enhanced, and organ injury can be resisted.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to ACE2 protein and variants, IL-6 and / or TNFα antagonist composition, multimer fusion polypeptide and application. Background technique [0002] In animals, when cells or tissues are damaged by bacteria, trauma, toxins, physical or chemical factors (which may be collectively referred to as "inflammatory agents, inflammatory agents"), an inflammatory response occurs. 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, fre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K45/06A61K39/395A61P29/00C12N9/48C07K19/00C12N15/62
CPCA61K38/4813A61K45/06A61K39/3955A61P29/00C12N9/485C12Y304/17023C07K16/248C07K16/241C07K2319/00A61K2300/00
Inventor 胡适傅文燕赵健
Owner PHARCHOICE THERAPEUTICS INC
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