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Human targeting complement inhibitor protein mCR2-fH and application

A complement inhibitor and fusion protein technology, applied in the field of fusion proteins, can solve the problems that the inhibition effect is not as good as in vitro inhibition experiments, the protection rate of animal protection experiments is improved, etc., and achieve excellent anti-adhesion/anti-inflammatory targeted inhibition effect, crescent / Improvement of necrosis and improvement of inhibition efficiency

Active Publication Date: 2019-08-16
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 targeting complement inhibitor protein mCR2-fH and application
  • Human targeting complement inhibitor protein mCR2-fH and application
  • Human targeting complement inhibitor protein mCR2-fH and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1.CR2 mutant (mCR2)-fH fusion protein preparation

[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-fH mAb 1H4 and 1A10, mouse anti-human CR2mAb171, 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 fH 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 is composed of 4 N-terminal SCR units encoding CR2 connected with the sequence encoding the extracellular region of fH. The complement inhibitor sequence is 322 amino acids encodi...

Embodiment 2

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

[0045]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

[0049] Embodiment 3. Complement lysis experiment

[0050] 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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Abstract

The invention discloses a fusion protein of a complement receptor 2 variant and a complement inhibitor fH and application of the fusion protein in the preparation of autoimmune disease treatment drugs. The complement receptor 2 variant is a molecular modified body acquired by computer modeling and amino acid replacement, and has higher ligand binding and dissociation rate than its wild sequence, as well as better ligand binding force. Biological distribution experiments prove that the fusion protein provided herein can aggregate to a high degree at the inflammatory joint part quickly after entering a mouse model with rheumatoid arthritis and has significant anti-adhesion / anti-inflammatory targeted inhibitory effect. In the treatment of MRL / lpr mice with lupus erythematosus, the fusion protein can significantly increase the survival rate of mice and evidently improve the symptoms of the mice in a treatment group, such as proteinuria, glomerular score, interstitial inflammation, vasculitis and crescent / necrosis.

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 destruction can also be caused indirectly by complement activation and its deposition on targ...

Claims

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Application Information

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Patent Type & Authority Applications(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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