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Antibody targeted complement inhibitor with anti-inflammatory action

A technology of inhibitor and complement, applied in the field of gene products, can solve problems such as infection

Inactive Publication Date: 2009-12-16
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although such systemic complement inhibitors improve the inflammatory response, since the complement system is an important part of the body's immune defense mechanism, systemic complement inhibition can also cause potential side effects such as infection

Method used

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  • Antibody targeted complement inhibitor with anti-inflammatory action
  • Antibody targeted complement inhibitor with anti-inflammatory action
  • Antibody targeted complement inhibitor with anti-inflammatory action

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Acquisition of anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-CD59 and construction of its eukaryotic expression vector

[0059] 1. Obtaining anti-P-selectin single chain antibody

[0060] Use the following method to screen anti-P-selectin single-chain antibody, and the specific method includes the following steps:

[0061] 1. Cell culture and identification: the hybridoma cells secreting P-selectin antibody (constructed with reference to the hybridoma cell and monoclonal antibody experimental technology, Xu Tinggui, 1982.) were cultured in complete RPMI1640 medium containing 15% bovine serum middle. Incubator with 5% CO 2 A mixed gas with a humidity of 98%. The specificity and titer of the monoclonal antibody in the culture supernatant were identified by ELISA method, and the antibody titer reached 1:3200 with obvious species specificity. .

[0062] 2. Isolation and purification of mRNA: use rapid preparation and purification ...

Embodiment 2

[0077] Example 2. Screening of cell lines with high expression of anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-CD59 and purification of anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-CD59

[0078] 1. Screening of cell lines with high expression of anti-P-selectin single chain antibody targeting complement inhibitor ScFv-CD59

[0079] In order to obtain higher biological protein molecules that are closer to natural proteins in terms of molecular structure, physical and chemical properties and biological functions, the recombinant eukaryotic expression plasmid pEE14.1 / ScFv-CD59 obtained in Example 1 was transfected into Chinese hamsters using liposomes Ovarian cells in CHO. After 24 hours of transfection, the medium was sucked off, and 10 mL of fresh selective medium DMEM+10% FCS+25 μm MSX was added. at 5% CO 2 The mixed gas was cultivated in a 37°C incubator with a humidity of 98%. After 2 weeks, clones of about 1-2 mm ...

Embodiment 3

[0082] Embodiment 3, in vivo and in vitro biological activity identification and animal experiments

[0083] 1. Complement lysis inhibition experiment

[0084] 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 9 / L. 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 fusion protein ScFv-CD59 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...

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Abstract

The invention discloses an anti-P-selectin single-chain antibody targeted complement inhibitor ScFv-CD59 and application thereof. The invention aims to provide an anti-P-selectin single-chain antibody targeted complement inhibitor ScFv-CD59 with specific targeting and the application thereof in preparing medicaments for treating systemic inflammations. The anti-P-selectin single-chain antibody targeted complement inhibitor ScFv-CD59 is a fusion protein obtained by connecting the amino terminal of the anti-P-selectin single-chain antibody ScFv with the complement inhibitor CD59 by using a connecting peptide. The experiment proves that the ScFv-CD59 can obviously improve the efficiency of inhibiting the complement-mediated cell cracking, can achieve high-degree accumulation at the immune injury part, can obviously inhibit the occurrence and degeneration of inflammations and can obviously reduce the side effect of infection caused by the complement inhibitor. Therefore, the ScFv-CD59 can be used as an active ingredient to be prepared into a novel gene engineering targeted protein medicament with a targeted P-selectin complement inhibitor for treating inflammations. The invention plays an important role in the pharmaceutical field and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of gene products, in particular to an anti-P-selectin single-chain antibody targeting complement inhibitor ScFv-CD59 with specific targeting and its application in the preparation of medicines for treating body inflammation. Background technique [0002] Inflammation is the most common and complex course of human disease, which can occur anywhere in the body and in any tissue. In many diseases such as tumors, cardiovascular and cerebrovascular diseases, diabetes, autoimmune diseases and trauma, there are different degrees of inflammatory reactions. Recent studies have found that when inflammation is severe or chronic, it can also lead to a variety of diseases. Prevention and treatment of inflammation and related syndromes are always thorny problems in the field of medicine. Although various inflammations have their particularities, from the point of view of local pathological changes, they all have a lot in common, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A61K39/395A61K48/00A61P29/00
Inventor 宋宏彬张传福贾雷立周育森徐元勇王勇刘雪林靳连群孙岩松黄留玉
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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