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Humanized targeted complement inhibitor protein double-mutant mCR2-mDAF and application thereof

A technology of inhibitors and complement receptors, applied in the field of fusion proteins, can solve problems such as slow process, and achieve the effects of improved inhibition efficiency, excellent anti-adhesion, and good ligand binding ability

Active Publication Date: 2019-10-15
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subsequently, iC3b is digested by serum proteases into membrane-bound fragments C3dg and C3d, but this process is relatively slow

Method used

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  • Humanized targeted complement inhibitor protein double-mutant mCR2-mDAF and application thereof
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  • Humanized targeted complement inhibitor protein double-mutant mCR2-mDAF and application thereof

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Experimental program
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Embodiment 1

[0037] Preparation of embodiment 1.mCR2-mDAF, mCR2-DAF, CR2-DAF fusion protein

[0038] 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-DAF mAb 1H4 and 1A10, mouse anti-human CR2 mAb 171, anti-goat erythrocyte IgM and all secondary antibodies were purchased from Sigma.

[0039] 2. Method

[0040] 2.1 The preparation of rabbit anti-CHO cell membrane and human DAF 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.

[0041] 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 DAF. The mutation sites of mCR2 mutant include: ...

Embodiment 2

[0048] Example 2. Kinetic analysis of interaction between CR2 fusion protein and C3 ligand

[0049] 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 ev...

Embodiment 3

[0053] Embodiment 3. Complement lysis experiment

[0054] 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 complement inhibitor DAF mutant, a fusion protein thereof with a complement receptor 2 mutant and use of the fusion protein in preparation of medicines treating autoimmune diseases. The DAF mutant is a molecular modification obtained by computer modeling and amino acid substitution. The fusion protein of the DAF mutant and the complement receptor 2 mutant has higher ligand binding and dissociation rates than wild-sequence fusion protein thereof, and has better ligand binding force. Biological distribution experiments prove that the fusion protein can rapidly aggregatein the arthritis position after entering a rheumatoid arthritis mouse model, and has a significant anti-adhesion / anti-inflammatory targeted inhibitory effect. In treatment for MRL / lpr lupus erythematosus mice, the fusion protein can significantly increase the survival rate of the mice, and symptoms such as proteinuria, glomerular score, interstitial inflammation, vasculitis and crescent / necrosisin the mice in the treatment group are 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. Cell or tissue destruction can also be caused indirectly by complement activation and its deposition on target structures. Complement activation products that mediate tissue damage are produced at various points in the complement pathway. Inappropriate complement activation on host tissues play...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/12C07K19/00C12N15/62A61K38/17A61K47/64A61P37/02A61P19/02A61P29/00
CPCA61K38/00A61P19/02A61P29/00A61P37/02C07K14/705C07K14/70596C07K2319/00
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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