Human targeted complement inhibitor protein mCR2-CD59 and application thereof

A fusion protein and complement receptor technology, applied in the field of fusion proteins, can solve the problems of affecting CD59 inhibitory activity, affecting complement inhibitor activity, and low inhibitory activity, and achieve excellent anti-adhesion/anti-inflammatory targeted inhibition effect, Crescent body/necrosis improvement, beneficial therapeutic effect

Active Publication Date: 2019-07-05
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 inhibition of complement activity by the complement receptor 2 and CD59 fusion protein. For example, in comparison with the complement receptor 2 and DAF fusion protein, its inhibitory activity is not high, and CR2 and CD59 complement Different fusion orientations also significantly affected the inhibitory activity of CD59
Problems in the prior art suggest that the sequence and even the spatial conformation of the fusion protein is a key factor affecting the full activity of complement inhibitors

Method used

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  • Human targeted complement inhibitor protein mCR2-CD59 and application thereof
  • Human targeted complement inhibitor protein mCR2-CD59 and application thereof
  • Human targeted complement inhibitor protein mCR2-CD59 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Preparation of mCR2-CD59 fusion protein and CR2-CD59 fusion protein

[0034] 1. Materials The expression vector was pEE14.1 (Lonza biologicals); CHO cells were used for protein expression, and the culture medium was DMEM containing 10% fetal bovine serum, which was purchased from Invitrogen. Mouse anti-CD59 mAb 1H4 and 1A10, mouse anti-human CR2 mAb 171, anti-goat erythrocyte IgM and all secondary antibodies were purchased from Sigma.

[0035] 2 methods

[0036] 2.1 The preparation of rabbit anti-CHO cell membrane and human CD59 antiserum was described in 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 structural gene was formed by connecting four N-terminal SCR units encoding CR2 and the sequence encoding the extracellular region of CD59. The complement inhibitor sequen...

Embodiment 2

[0045] Example 2. Kinetic analysis of interaction between CR2 fusion protein and C3 ligand

[0046]Kinetic analysis of the interaction between CR2 fusion protein and biotin-labeled C3dg (C3dg-biotin) was detected by surface plasmon resonance (SPR) detection system (BIAcore3000 instrument). Human C3dg-biotin (Guthridge, J.M., et al. Structural studies solution of the recombinant N-terminal pair of short consensus / complement repeat 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 speed of 2μL / min for 20min, and the buffer was 0.5× PBS (pH7.4) (containing 0.5g / L Tween20). SPR signals acquired from captured C3dg yielded BIAcore response units (range 250 to 500). The group without fusion protein was used as the control. After washing with 0.5×PBST (0.5g / LTween20) at a flow rate of 25μL / min at 25°C, the affinity of the CR2 fusion protein was eva...

Embodiment 3

[0050] Embodiment 3. Complement lysis experiment

[0051] To measure the inhibitory activity on complement, 60%-80% confluent CHO cells were separated with ethylenediaminetetraacetic acid, washed twice with DMEM, and then resuspended in DMEM to make the final concentration of 10 6 cells / mL. Add 100mL / L rabbit anti-CHO cell membrane antiserum to the cell suspension and react at 4°C for 30min to sensitize the cells. Then the antiserum was discarded, and the cells were resuspended in NHS diluted with DMEM to a final volume of 50 μL or 100 μL. After 60 minutes at 37°C, the cell viability was measured by placenta blue staining and exclusion method (both live and dead cells were counted). The recombinant fusion protein was diluted with DEME and added to NHS first, and then added to CHO cell suspension. The final concentration was based on the control CHO cells lysed by 100 g / L NHS which can cause about 90% antibody sensitization. Complement-mediated inhibition of erythrocyte lys...

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PUM

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Abstract

The invention discloses a fusion protein of a complement receptor 2 mutant and a complement inhibitor CD 59 and application thereof to preparing autoimmune disease treating drugs. The complement receptor 2 mutant is a molecular modified body obtained through computer modelling and amino acid replacement and is higher in ligand binding and dissociation rate and ligand binding force compared with wild sequences. Biological distribution test shows that the fusion protein can be highly concentrated at arthritic parts after entering mouse models with rheumatoid arthritis and achieve significant anti-adhesion/anti-inflammatory targeted inhibition effects; when applied to treating MRL/lpr (Murphy Roths large/lymphoproliferation) mice with systemic lupus erythematosus, the fusion protein can significantly improve the survival rate of the mice, and symptoms of the mice of a treatment group such as proteinuria, glomerular score, interstitial inflammation, vasculitis and crescent glomerulonephritis/necrosis can be significantly improved.

Description

technical field [0001] The invention discloses a fusion protein and belongs to the technical field of polypeptides. Background technique [0002] The complement system is composed of more than 30 kinds of soluble protein molecules and is a part of the natural immune system. Its components include more than 30 kinds of molecules such as complement intrinsic components, various regulatory factors and complement receptors. The complement system can be activated through three relatively independent and interrelated pathways, thereby exerting various biological effects such as opsonizing phagocytosis, lysing cells, mediating inflammation, immune regulation, and clearing immune complexes, including enhancing phagocytosis, enhancing phagocytosis Chemotaxis of cells, increase of vascular permeability, neutralization of viruses, cell lysis, regulation of immune response, etc. Cell or tissue damage can also be caused indirectly by complement activation and its deposition on target st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K47/64A61P19/02A61P37/02A61P29/00
CPCC07K14/70596C07K14/705A61K47/6425A61P19/02A61P37/02A61P29/00C07K2319/33
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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