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A fully human monoclonal antibody against complement c5 molecule and its application

A monoclonal antibody and anti-complement technology, applied in the field of peptides, can solve problems such as the controversy over the efficacy of eculizumab, and achieve the improvement of symptoms such as vasculitis and crescent/necrosis, improvement of survival rate, and excellent antigen-binding activity. 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.

Method used

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  • A fully human monoclonal antibody against complement c5 molecule and its application
  • A fully human monoclonal antibody against complement c5 molecule and its application
  • A fully human monoclonal antibody against complement c5 molecule and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Preparation of fully human monoclonal antibody against complement C5 molecule

[0027] 1.1 Construction and expression of phage antibody 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.

[0028] 1.2 Screening of recombinant phage antibody: Coat polyethylene culture dish with C5 antigen (Complement Technology, I 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 and incubate at 37°C for 1 hour. After centrifugation, the supernatant was collected for the next round of screenin...

Embodiment 2

[0059] Example 2. Kinetic analysis of the interaction between anti-complement C5 monoclonal antibody and C5 ligand

[0060] The kinetic analysis of the interaction between anti-complement C5 monoclonal antibody and C5 ligand was detected by surface plasmon resonance (SPR) detection system.

[0061] 2.1 Experimental instruments and reagents

[0062] Instrument: Reichert2SPR (Reichert Company), chip: SAM chip (macromolecular detection), (ReichertInc Company, PART NO: 13206061).

[0063] 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).

[0064] 2.2. Experimental steps

[0065] 2.2.1 Pre-enrichment

[0066] 2.2.1.1 Dilute the protein with different pH sodium acetate to 10 μg / mL, 200 μL.

[0067] Table 1. Sodium acetate pH selection table

[0068] protein name Sodium acetate pH fixed channel ...

Embodiment 3

[0085] Example 3. In vitro experiment of anti-C5 whole antibody inhibiting complement activation

[0086] 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). Monoclonal antibody diluted with DEME was first added to NHS, 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-media...

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Abstract

The invention discloses a fully human monoclonal antibody against complement C5 molecule. The light chain and heavy chain of the antibody have unique CDR regions, so that the antibody has excellent antigen-binding activity. In the cell lysis inhibition experiment , the antibody has a significant effect of inhibiting the lysis of antibody-sensitized CHO cells and erythrocytes. The anti-C5 monoclonal antibody disclosed in the present invention can significantly improve the survival rate of mice in the treatment of MRL / lpr lupus erythematosus mice, and the proteinuria, glomerular integral, interstitial inflammation, vasculitis and new Symptoms such as moon bodies / necrosis are significantly improved, which shows that the monoclonal 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 an antibody 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. While complement activation provides a valuable first line of defense against potential pathogens, complement act...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/10A61K39/395A61P37/02
CPCA61K2039/505A61P37/02C07K16/18C07K2317/565C07K2317/76
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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