A kind of anti-complement c5 molecule humanized single-chain antibody and its application

A single-chain antibody and anti-complement technology, applied in the field of peptides, can solve the problems of difficulty in obtaining highly specific anti-C5 antibodies, controversy and limitations of eculizumab efficacy, achieve excellent antigen binding activity, improve survival rate, and reduce symptoms Improved effect

Active Publication Date: 2021-09-07
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the clinical efficacy of eculizumab in the treatment of hemolytic uremic syndrome is still controversial.
At present, there are still some technical difficulties in the inhibition of complement activation by anti-C5 antibodies, such as the difficulty in obtaining highly specific anti-C5 antibodies, and the immunogenicity of mouse monoclonal antibodies or chimeric antibodies prepared by existing technologies to humans , and it is difficult for antibody molecules to reach some lesion tissues due to the excessive molecular weight and the hindrance of its spatial conformation
These technical issues limit the practical application of inhibition of complement activation by anti-C5 antibodies to varying degrees

Method used

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  • A kind of anti-complement c5 molecule humanized single-chain antibody and its application
  • A kind of anti-complement c5 molecule humanized single-chain antibody and its application
  • A kind of anti-complement c5 molecule humanized single-chain antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, preparation of anti-C5 single chain antibody

[0028] Use the following method to screen anti-C5 single-chain antibody, and the specific method includes 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. This application is incorporated by reference, and the disclosure content of CN109575132A is incorporated into the specification of this application as a part of this application.

[0030] 1.2 Screening of recombinant phage antibody: Coat polyethylene culture dish with C5 antigen (Complement Technology, Inc; product number: A120Lot#, Accession#P06684), and incubate the supernatant containing recombinant phage 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 growth phase ...

Embodiment 2

[0038] Example 2, Kinetic analysis of the interaction between anti-C5 single chain antibody and C5 ligand

[0039] The kinetic analysis of the interaction between anti-C5 single chain antibody and C5 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] protein name Sodium acetate pH fixed channel antigen 6.0 / 5.5 / 5...

Embodiment 3

[0064] Example 3. In vitro experiment of inhibiting complement activation by anti-C5 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-derived single-chain antibody against complement C5 molecule. The light chain and heavy chain of the antibody have unique CDR regions, have excellent antigen-binding activity, and have an equilibrium dissociation constant K D (m) up to 2.74×10 ‑11 , in the treatment of MRL / lpr lupus erythematosus mice, the anti-C5 single-chain antibody provided by the present invention can significantly improve the survival rate of mice, and the proteinuria, glomerular integral, interstitial inflammation, and vasculitis of the treatment group Symptoms such as crescent and necrosis are obviously improved, showing that the anti-C5 single-chain antibody provided by the invention has excellent application prospects in the preparation of drugs for the treatment of 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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/85C12N5/10A61K39/395A61P37/02
CPCA61K2039/505A61P37/02C07K16/18C07K2317/24C07K2317/56C07K2317/565C07K2317/567
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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