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Single-chain antibody of human source anti-alexin C3d molecules and application thereof

A single-chain antibody and anti-complement technology, which is applied in the field of peptides, can solve the problems of limitations, difficulty in obtaining highly specific anti-C3d antibodies, and difficulty in reaching the lesion tissue in the spatial conformation of antibody molecules, so as to achieve good therapeutic effect and excellent anti-adhesion/anti-complement Inflammation targeting inhibitory effect, crescent/necrosis improvement effect

Active Publication Date: 2019-04-05
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some technical difficulties in inhibiting the combination of C3d and CR2 through anti-C3d antibodies, such as the difficulty in obtaining highly specific anti-C3d antibodies, and the effects of mouse monoclonal antibodies or chimeric antibodies prepared by prior art on humans. Immunogenicity, and it is difficult for antibody molecules to reach some lesion tissues due to the hindrance of their spatial conformation
These technical issues limit the practical application of inhibition of complement activation by anti-C3d antibodies to varying degrees

Method used

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  • Single-chain antibody of human source anti-alexin C3d molecules and application thereof
  • Single-chain antibody of human source anti-alexin C3d molecules and application thereof
  • Single-chain antibody of human source anti-alexin C3d molecules and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, preparation of anti-C3d single chain antibody

[0032] Use the following method to screen anti-C3d single-chain antibody, and the specific method includes the following steps:

[0033] 1.1 Preparation of cDNA

[0034] 20 ml of peripheral blood from 50 healthy individuals were collected, and mononuclear cells were separated with lymphocyte separation medium (Tianjin Blood Research Institute). Total RNA was extracted from isolated human peripheral blood lymphocytes with Trizol reagent (Invitrogen), and then mixed in the same ratio. The cDNA was reverse transcribed using a cDNA reverse transcription kit (Takara Company). The above steps were carried out according to the instructions provided by the manufacturer.

[0035] 1.2 Amplification of antibody light chain and heavy chain variable region genes: Using PCR method, using the cDNA synthesized by reverse transcription as a template, add primers for amplifying the light chain and heavy chain variable regio...

Embodiment 2

[0056] Embodiment 2, ELISA detection antibody binding activity

[0057] ScFv affinity was detected by non-competitive enzyme immunoassay. Specific steps: coat the ELISA plate according to the concentration of 2, 1, 0.5, 0.25 μg / ml C3d, add 2-fold serial dilution of ScFv, the secondary antibody is HRP / anti-ScFv antibody, after the color development is terminated, the microplate reader measures at 450nm single wavelength Optical density value (D). The Curve Expert 1.4 software was used to calculate the corresponding antibody concentration when the D value reached 1 / 2D under different antigen dilutions based on the D value and the antibody concentration [Ab]. According to the formula: Ka=(n-1) / 2×(n[Ab']-[Ab]), where [Ab'] and [Ab] represent two different concentrations of [Ag'] and [Ag'] and Under [Ag], the corresponding antibody concentration when the D value reaches 1 / 2D, n represents the dilution factor between the antigen [Ag'] and [Ag]. The result is: ScFv affinity consta...

Embodiment 3

[0058] Example 3. Biodistribution experiment of anti-C3d single chain antibody

[0059] Anti-C3d single-chain antibody ScFv was labeled by lodogen method 125 I. In the EP tube coated with 200 μg lodogen, add 150 μl of 50 mmLo / L PBS (pH7.4) prepared now, 100 μl (100 μg) of ScFv dissolved in 1 mg BSA), Na 125 I solution 15μl (185MBq), at room temperature, shake the marked tube intermittently for 15min. The SEP-PAK C18 column was washed with 5 mL each of methanol, double distilled water and 0.1% trifluoroacetic acid (TFA) to activate it; the labeled mixture was put on the column and washed with 0.1% TFA; 1.5 mL of eluent. After freeze-drying, it was diluted with PBS solution containing 1 mg / mL BSA, aliquoted, and stored in a -80°C refrigerator for later use. Male DBA / 1J mice were subcutaneously injected with 0.1mL collagen II and complete Freund's adjuvant (both purchased from Sigma, USA) at the base of the tail, and boosted once on the 21st day to establish a mouse model of r...

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Abstract

The invention discloses a single-chain antibody of human source anti-alexin C3d molecules. A light chain and a heavy chain of the antibody have a unique CDR region; excellent antigen binding activityis realized; the affinity constant reaches 1.22*10<-7> mol / L. Biological distribution experiments prove that the anti-C3d single-chain antibody provided by the invention can be highly gathered in thearthritis positions after entering a mouse model with rheumatoid arthritis; the arthritis serious degree of the three ScFv treatment groups is obviously lower than that of a PBS group; the healing degree has the obvious dose dependency relationship; the result shows that the excellent anti-adhesion / anti-inflammatory targeted inhibition effects are achieved. In the treatment process on MRL / lpr mice with lupus erythematosus, the anti-C3d single-chain antibody provided by the invention can obviously improve the survival rate of the mice; the symptoms of proteinuria, glomerular score, interstitial inflammation, vasculitis and crescent / necrosis and the like in the treatment group are obviously relieved. The result shows that the anti C3d single-chain antibody provided by the invention has excellent application prospects in the preparation of medicine for treating autoimmune diseases.

Description

technical field [0001] The invention discloses a polypeptide, more specifically, the invention discloses an antibody. background technology [0002] The complement system is composed of more than 30 kinds of soluble protein molecules and is 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. Cell or tissue destruction can also be caused indirectly by complement activation and its deposition on ta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/85C12N5/10A61K39/395A61P37/02A61P19/02
CPCA61K2039/505A61P19/02A61P37/02C07K16/18C07K2317/56C07K2317/622C07K2317/92C12N2510/00
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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