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Human single-chain antibody of anticomplement C3 molecule and application for humanized single-chain antibody

A single-chain antibody and anti-complement technology, applied in the field of polypeptides, can solve the problems of inability to obtain inhibitory effect, incomplete elimination of the body's immune defense, and space conformational hindrance, and achieve crescent/necrosis improvement, excellent antigen binding activity, The effect of excellent application prospects

Active Publication Date: 2020-05-19
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The selective inhibition of the complement activation pathway can eliminate the pathological damage while not completely eliminating the immune defense of the body, but it cannot achieve an ideal inhibitory effect on the three complement activation pathways that are interrelated as described above
Inhibition of complement activation through anti-C3 antibodies still has some technical difficulties, such as the difficulty in obtaining highly specific anti-C3 antibodies, and the immunogenicity of mouse monoclonal antibodies or chimeric antibodies prepared by prior art to humans , it is difficult for antibody molecules to reach some hidden lesions due to the excessive molecular weight and the hindrance of the formed space conformation
These technical issues limit the practical application of inhibition of complement activation by anti-C3 antibodies to varying degrees

Method used

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  • Human single-chain antibody of anticomplement C3 molecule and application for humanized single-chain antibody
  • Human single-chain antibody of anticomplement C3 molecule and application for humanized single-chain antibody
  • Human single-chain antibody of anticomplement C3 molecule and application for humanized single-chain antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the preparation of anti-C3 single-chain antibody

[0028] Screen and prepare anti-C3 single-chain antibody with the following method, specifically comprising the following steps:

[0029] 1.1 Construction and expression of single-chain antibody phage expression library Refer to Example 1 of Chinese invention patent application CN109575132A, and the disclosure content of CN109575132A is cited in this application as a part of this application and incorporated into the description of this application.

[0030] 1.2 Screening of recombinant phage antibodies: A polyethylene culture dish was coated with C3 antigen (Complement Technology, Inc; product number: A113, sequence accession number K02765), and the supernatant containing recombinant phage was incubated with the culture dish at 37°C for 2 hours. Wash the plate 20 times with PBS, then wash the plate 20 times with PBST (PBS containing 0.05% Tween 20), discard the PBST. Add 10 mL of TG1 cells in logarithmic ...

Embodiment 2

[0038] Example 2, Kinetic analysis of the interaction between anti-C3 single chain antibody and C3 ligand

[0039] The kinetic analysis of the interaction between anti-C3 single chain antibody and C3 ligand was detected by surface plasmon resonance (SPR) detection system.

[0040] 2.1 Experimental instruments and reagents

[0041] Instrument: Reichert2SPR (Reichert Company), chip: SAM chip (macromolecular detection), (ReichertInc Company, PART NO: 13206061).

[0042] Reagents: 1xPBST 500ml (filtered, 0.22uM membrane filter), EDC (preparation for current use), NHS (preparation for current use), 1MpH8.5 ethanolamine (5-10ml), 10mM pH2.0 HCl (5-10ml) , 10 mM pH 2.0 Glycine (5-10 ml).

[0043] 2.2. Experimental steps

[0044] 2.2.1 Pre-enrichment

[0045] 2.2.1.1 Dilute the protein with different pH sodium acetate to 10 μg / mL, 200 μL.

[0046] Table 1. Sodium acetate pH selection table

[0047]

[0048] 2.2.1.1 Inject protein in a certain channel, 25μL / min, 2min.

[0049] ...

Embodiment 3

[0064] Example 3. In vitro experiment of inhibiting complement activation by anti-C3 single chain antibody

[0065] 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 single chain antibody 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 sensitizatio...

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Abstract

The invention discloses a human single-chain antibody of an anticomplement C3 molecule. The antibody has a light chain and a heavy chain with unique CDR regions and excellent antigen binding activity;an equilibrium dissociation constant KD(m) reaches 2.06 * 10<-11>; and in treatment of MRL / lpr lupus erythematosus mice, the anti-C3 single-chain antibody provided by the invention can obviously enhance the survival rate of mice; the symptoms like proteinuria, glomerular integration, interstitial inflammation, vasculitis, crescent / necrosis in a treatment group are obviously improved; and the anti-C3 single-chain antibody provided by the invention is shown to achieve excellent application prospect in preparation of drugs for treating autoimmune diseases.

Description

technical field [0001] The invention discloses a polypeptide, more specifically, the invention discloses an antibody. 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. While complement activation provides a valuable first line of defense against potential pathogens, complem...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/85C12N5/10A61K39/395A61P37/02A61P19/02A61P29/00
CPCA61K2039/505A61P19/02A61P29/00A61P37/02C07K16/18C07K2317/565C07K2317/622C07K2317/92C12N15/85
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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