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Targeting specificity complement system inhibitor and preparation method and application thereof

A complementary and targeted technology, applied in the fields of biotechnology and pharmaceuticals, can solve the problems of inability to obtain complete remission and unsatisfactory treatment with eculizumab

Active Publication Date: 2014-12-24
SHANGHAI COMGEN BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, theoretically, after the application of eculizumab, the condition of PNH patients still cannot be completely relieved, because the C3 level of the complement cascade is still activated, and C3b / iC3b is produced and deposited on the surface of blood cells, so that these blood cells are replaced by monocytes. Phagocytosis, eventually causing extravascular hemolysis (Risitano AM et al., Blood, 2009; 113:4094-4100), which is why eculizumab treatment is not very satisfactory

Method used

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  • Targeting specificity complement system inhibitor and preparation method and application thereof
  • Targeting specificity complement system inhibitor and preparation method and application thereof
  • Targeting specificity complement system inhibitor and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction, eukaryotic expression and purification of CRIg-fH and CRIg-L-fH protein expression vectors

[0051] 1. Instruments and materials:

[0052] Mastercycler pro-Eppendorf PCR instrument (Eppendorf, Germany), DK-8D electric heating constant temperature water tank (Shanghai Jinghong Experimental Equipment Co., Ltd.), IQ350 gel imaging system (GE Healthcare, USA), CO 2 Cell incubator (Thermo Scientific, USA), FR-980A bioelectrophoresis image analysis system (Furi Technology), BioRAD Mini protein Tera system (BioRAD, USA), NanoVue RNA / DNA concentration / purity detector (IKA, Germany)

[0053] 2. Experimental method:

[0054] 2.1 Gene cloning and vector construction

[0055] Trizol method (invitrogen) was used to extract total RNA from human lymphoma U937 cells, which was reverse-transcribed into cDNA (promega reverse transcription kit), and the extracellular domain of CRIg gene (G19-K137) was amplified by PCR; human liver cancer cell line was extracted by Trizol m...

Embodiment 2

[0090] Example 2 Kinetic Analysis and Affinity Determination of the Interaction of CRIg-fH and CRIg-L-fH with C3 Activation and Degradation Fragments

[0091] 1. Instruments and materials:

[0092] Biacore T200 (GE Healthcare), Series S Sensor Chip NTA, CRIg-L-fH protein solution, C3 activated and degraded protein components (C3b, iC3b, C3c, C3d, Complement Technology), HBS-N solution (GE Healthcare)

[0093] 2. Experimental method

[0094] The expressed and purified CRIg-L-fH was diluted to 0.2ug / ml with HBS-N running buffer and coupled to the NTA chip as a ligand. C3fragments were diluted in a series of concentration gradients (as shown in the figure) as the flow analyte, and applied Biacore T200 instrument was used to measure the kinetic curve and analyzed by Biacore T200Software v2.02. Affinity constants, dissociation constants and equilibrium dissociation constants are shown in Table 1.

[0095] Table 1

[0096]

[0097] 3. The experimental results show that CRIg-L...

Embodiment 3

[0099] CRIg-L-fH protects PNH erythrocytes from hemolysis induced by the alternative complement pathway and the classical pathway

[0100] 1. Instruments and materials:

[0101] Erythrocytes from seven PNH patients, normal human serum, CVF (1mg / ml, comptech company), anti-human erythrocyte polyclonal antibody (Rockland), Bio-Tek synergy HT multifunctional microplate reader (Labsystems, Finland), Minispin desktop high-speed centrifuge (Eppendorf, Germany), DK-8D electric heating constant temperature water tank (Shanghai Jinghong Experimental Equipment Co., Ltd.)

[0102] 2. Experimental method:

[0103] 2.1 Inhibitory effect of CRIg-L-fH on PNH erythrocyte hemolysis induced by complement replacement pathway

[0104] Red blood cells were collected from seven PNH patients, washed three times with PBS (5000rpm, centrifuged for 3 minutes), and made into 4% RBC solution; 200ul reaction system, 25ul of 4% RBC (final concentration 1%) was added, and CRIg-L-fH protein solution doubled ...

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Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a targeting specificity complement system inhibitor and the design, preparation and clinical application of the targeting specificity complement system inhibitor. CRIg which can be combined with fragments C3b and / or iC3b which are formed after a complement component C3 is activated to generate targeting effect is directly connected with another component with a complement inhibiting effect, for example factor H or is indirectly connected by virtue of a flexible peptide fragment (Gly4Ser)3 and the like to prepare a fusion protein by methods such as genetic engineering, thus achieving the effect of inhibiting complement activation in a targeting manner. The medicine can be applied to treatment and prevention of a plurality of human diseases which are related to abnormal complement activation.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and in particular relates to the design, preparation and clinical application of a target-specific complement inhibitor. Background technique [0002] The complement system is the main component of innate immunity and an important regulator of acquired immunity. It plays an important role in immune surveillance. It can not only clear the invading pathogenic microorganisms and host cell fragments, but also coordinate the entire immune and inflammatory process (Ricklin D et al., Nat Immunol 2010;11:785-797; Morgan BP et al., Immunology letters 2005;97:171-179). Complement can be activated through the classical pathway, the alternative pathway and the lectin pathway, and its physiological functions are mainly carried out through the products formed after activation, including the deposition of C3b / iC3b on the surface of the attacked cell membrane, and the recruitment of immune effector cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/17A61K47/48A61P7/00
CPCA61K38/00A61K45/06A61P1/04A61P7/00A61P7/06A61P9/10A61P11/06A61P17/00A61P17/06A61P19/00A61P21/04A61P25/00A61P25/16A61P25/28A61P29/00A61P37/00A61P37/02A61P37/08C07K14/4703C07K14/70503C07K14/70596C07K2319/00A61K38/1709A61K38/177
Inventor 胡维国乔倩张鑫
Owner SHANGHAI COMGEN BIO PHARMA CO LTD
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