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Human target complement inhibitor protein mcr2-fh and its application

A complement inhibitor and fusion protein technology, applied in the field of fusion proteins, can solve the problems of increased protection rate in animal protection experiments, and the inhibition effect is not as good as in vitro inhibition experiments, etc., and achieves excellent anti-adhesion/anti-inflammatory targeted inhibition effect, crescent / Necrosis improvement, excellent application prospects

Active Publication Date: 2022-07-22
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some technical problems in the inhibitory activity of CR2 and fH fusion protein. For example, the inhibitory effect in vivo is not as good as that in vitro, and the protection rate of animal protection experiments needs to be further improved.
Problems arising from the prior art suggest that altered spatial conformation obtained by sequence variation of fusion proteins may be a means of promoting full activity of complement inhibitors

Method used

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  • Human target complement inhibitor protein mcr2-fh and its application
  • Human target complement inhibitor protein mcr2-fh and its application
  • Human target complement inhibitor protein mcr2-fh and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Preparation of CR2 mutant (mCR2)-fH fusion protein

[0034] 1 Materials The expression vector was pEE14.1 (Lonza biologics); CHO cells were used for protein expression, and the culture medium was DMEM containing 10% fetal bovine serum, purchased from Invitrogen Company. Mouse anti-fH mAbs 1H4 and 1A10, mouse anti-human CR2 mAb171, anti-goat erythrocyte IgM and all secondary antibodies were purchased from Sigma.

[0035] 2 methods

[0036] 2.1 Preparation of antiserum of rabbit anti-CHO cell membrane and human fH according to the literature Harlow, E., and Lane, D. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York, USA. 1988:726. method to obtain.

[0037] 2.2 Construction of expression recombinant and protein expression The cDNA structure gene is formed by connecting the four N-terminal SCR units encoding CR2 and the sequence encoding the extracellular region of fH. The complement inhibitor sequence is the 322 ami...

Embodiment 2

[0044] Example 2. Kinetic analysis of interaction between CR2 fusion protein and C3 ligand

[0045]Kinetic analysis of the interaction of the CR2 fusion protein with biotin-labeled C3dg (C3dg-biotin) was detected with a Surface Cytoplasmic Resonance (SPR) detection system (BIAcore 3000 instrument). Human C3dg-biotin (Guthridge, J.M., et al. Structural studies insolution of the recombinant N-terminal pair of short consensus / complementrepeat domains of complement receptor type 2 (CR2 / CD21) and interactions with its ligand C3dg. Biochemistry. 2001, 40(20): 5931–5941.) were injected onto the BIAcore streptavidin sensor chip at a rate of 2 μL / min for 20 min, and the buffer was 0.5× PBS (pH 7.4) (containing 0.5 g / L Tween20). The SPR signal acquired from the captured C3dg yielded BIAcore response units (range 250 to 500). The group without fusion protein was used as a control. After washing with 0.5×PBST (0.5g / LTween20) at 25°C at a flow rate of 25μL / min, the affinity of the CR2 ...

Embodiment 3

[0049] Example 3. Complement lysis assay

[0050] To determine the inhibitory activity against complement, 60%-80% confluent CHO cells were split with EDTA, washed twice with DMEM, and then resuspended in DMEM to a final concentration of 10 6 cells / mL. 100 mL / L rabbit anti-CHO cell membrane antiserum was added to the cell suspension, and the cells were sensitized for 30 min at 4°C. Antiserum was then discarded and cells were resuspended in NHS diluted in DMEM in a final volume of 50 μL or 100 μL. The cells were incubated at 37°C for 60 min, and finally the cell viability was measured by placental blue staining exclusion method (both live and dead cells were counted). The recombinant fusion protein was diluted with DEME and added to NHS and then to the CHO cell suspension. The final concentration was based on the lysis of control CHO cells in which 100 g / L NHS resulted in approximately 90% antibody sensitization. The complement-mediated inhibition of erythrocyte lysis assay...

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Abstract

The invention discloses a fusion protein of a complement receptor 2 variant and a complement inhibitor fH and an application of the fusion protein in the preparation of a drug for treating autoimmune diseases. The complement receptor 2 variant is a molecular modification obtained after computer modeling and amino acid substitution, and has higher ligand binding and dissociation rates and better ligand binding capacity than its wild-type sequence. The biological distribution experiment proves that the fusion protein provided by the present invention can rapidly and highly aggregate in the arthritis site after entering the rheumatoid arthritis mouse model, and has significant anti-adhesion / anti-inflammatory targeting inhibition effect. In the treatment of MRL / lpr lupus erythematosus mice, the fusion protein can significantly improve the survival rate of mice. Symptoms such as necrosis were significantly improved.

Description

technical field [0001] The invention discloses a fusion protein, which belongs to the technical field of polypeptides. Background technique [0002] The complement system is composed of more than 30 kinds of soluble protein molecules, which are part of the natural immune system. The complement system can be activated through three relatively independent and interconnected pathways, thereby exerting various biological effects such as opsonophagocytosis, lysis of cells, mediation of inflammation, immune regulation, and clearance of immune complexes, including enhanced phagocytosis and enhanced phagocytosis. Cell chemotaxis; increase vascular permeability; neutralize virus; cell lysis; regulation of immune response, etc. Complement activation and its deposition on target structures can also indirectly cause cell or tissue destruction. Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K47/64A61K38/17A61P37/02A61P19/02A61P29/00
CPCC07K14/705C12N15/85C12N5/0682A61K47/6425A61K38/1709A61P37/02A61P19/02A61P29/00C07K2319/00C12N2510/02
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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