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Fusion protein of anti-C3d targeted single-chain antibody and DAF, and application of fusion protein

A fusion protein and single-chain antibody technology, applied in the field of polypeptides, can solve the problems of affecting the targeting effect of anti-C3d antibodies and increasing the molecular weight of proteins, and achieve excellent anti-adhesion/anti-inflammatory targeting inhibition effects, improvement of vasculitis, and high inhibition efficiency effect

Active Publication Date: 2020-03-27
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the single-chain antibody of the anti-C3d antibody in the prior art is still difficult to meet this technical requirement. The fusion of the anti-C3d antibody and different inhibitors will increase the molecular weight of the protein, which will change the spatial structure of the protein after reconstruction, thereby affecting Targeting effect of anti-C3d antibodies

Method used

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  • Fusion protein of anti-C3d targeted single-chain antibody and DAF, and application of fusion protein
  • Fusion protein of anti-C3d targeted single-chain antibody and DAF, and application of fusion protein
  • Fusion protein of anti-C3d targeted single-chain antibody and DAF, and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Preparation of anti-C3d single-chain antibody

[0026] Use the following method to screen anti-C3d single chain antibody, and the specific method includes the following steps:

[0027] 1.1 Preparation of cDNA

[0028]Collect 20 ml of peripheral blood from 50 healthy people, and separate mononuclear cells with lymphocyte separation medium (Tianjin Institute of Blood). Total RNA of cells was extracted from isolated human peripheral blood lymphocytes with Trizol reagent (Invitrogen), and then mixed in the same proportion. cDNA was reverse transcribed using a cDNA reverse transcription kit (Takara). The above steps are carried out according to the instructions provided by the manufacturer.

[0029] 1.2 Amplification of variable region genes of antibody light chain and heavy chain: using PCR method, using the cDNA synthesized by reverse transcription as a template, add primers for amplifying the variable region genes of light chain and heavy chain into the PCR ...

Embodiment 2

[0050] Example 2 Preparation of anti-C3d single chain antibody-DAF (ScFv-DAF) fusion protein

[0051] 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-DAF mAbs 1H4 and 1A10, mouse anti-human CR2 mAb 171, anti-goat erythrocyte IgM and all secondary antibodies were purchased from Sigma.

[0052] 2 methods

[0053] 2.1 Preparation of antiserum of rabbit anti-CHO cell membrane and human DAF 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.

[0054] 2.2 Construction of expression recombinant and protein expression cDNA structure gene is formed by linking the sequence encoding anti-C3d single-chain antibody and the sequence encoding the extracellular region of DAF. The complement inhi...

Embodiment 3

[0074] Example 3. Kinetic analysis of interaction between ScFv-DAF and C3 ligand

[0075] Kinetic analysis of the interaction of ScFv-DAF 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 in 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 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 / L Tween20) at 25°C at a flow rate of 25μL / min, the affinity of ScFv-DAF was evaluated by de...

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Abstract

The invention discloses a single-chain antibody of a human anti-complement C3d molecule, and a fusion protein of the single-chain antibody and a complement inhibitor DAF. The antibody has excellent antigen binding activity. Biological distribution experiments prove that the fusion protein provided by the invention can be rapidly and highly aggregated at an arthritis part after entering a rheumatoid arthritis mouse model, and has an excellent anti-adhesion / anti-inflammation targeting inhibition effect. In treatment of MRL / lpr lupus erythematosus mice, the fusion protein provided by the invention can obviously improve the survival rate of the mice, and symptoms of proteinuria, glomerular score, interstitial inflammation, vasculitis, crescent / necrosis and the like of a treatment group are obviously improved, so that the fusion protein provided by the invention has an excellent application prospect in preparation of medicaments for treating autoimmune diseases.

Description

technical field [0001] The invention relates to an antibody and 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, which are part of the natural immune system. The complement system can be activated through 3 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 co...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K19/00C12N15/62A61K39/395A61K38/17A61P37/02A61P29/00A61P19/02
CPCA61K38/00A61K2039/505A61P19/02A61P29/00A61P37/02C07K14/70596C07K16/18C07K2317/24C07K2317/56C07K2317/565C07K2317/622C07K2317/76C07K2317/92C07K2319/00
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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