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Binding molecules that bind human complement factor C2 and uses thereof

A technology that combines molecules, complement factors, applied in the field of application of inhibitors of complement factors, application in the treatment of human diseases, diseases or disorders, and can solve the problem of increasing the risk of infection

Active Publication Date: 2016-04-13
BROTEIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, inhibition of complement in inflammatory diseases has the inherent disadvantage of increasing the risk of infection

Method used

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  • Binding molecules that bind human complement factor C2 and uses thereof
  • Binding molecules that bind human complement factor C2 and uses thereof
  • Binding molecules that bind human complement factor C2 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Generation of Mouse Anti-Human Complement C2 Antibodies

[0133] (a) Production of recombinant human complement C2

[0134] Recombinant human complement protein C2 with an N-terminal his tag was generated by U-ProteinExpress (Utrecht, The Netherlands). Briefly, cDNA encoding human complement protein C2 (GenBank sequence: NM_000063; see SEQ ID NO. 1) was cloned and subsequently expressed in HEK293 cells. Purify C2 by affinity chromatography and use precast gels System (Invitrogen) SDS-PAGE analysis. Proteins were stained with Coomassie brilliant blue.

[0135] as in figure 1 As shown in (lane 1 ), the C2 preparation showed >95% purity, ie a band with a molecular weight ≈100 kDa was observed, consistent with the expected molecular weight of glycosylated human C2.

[0136] (b) Biochemical characteristics of recombinant human complement C2

[0137]Generated by incubating C2 (100 μL of a 400 μg / mL solution) with plasma-derived activated C1s (100 μL of a 16 ...

Embodiment 2

[0144] Example 2: ELISA for Screening the Inhibitory Activity of Anti-C2 Hybridoma Supernatants

[0145] In the ELISA system, the culture supernatants of hybridomas producing anti-C2 antibodies were initially screened for their inhibitory effect on C2 ( figure 2 ). In this ELISA, agglutinated human IgG is coated in microtiter plates (Greiner-Bio-One) and incubated with diluted fresh serum with or without anti-C2 containing hybridoma The supernatant was pre-incubated. Immobilization of C4 and C3 on the plate, indicative of complement activation, was then measured using biotinylated polyclonal affinity purified anti-C3 and anti-C4 antibodies. Streptavidin-HRP was then used to measure the binding of anti-C3 and anti-C4 to the plate, which was visualized using the 3',5'-tetratmethylbenzidine sequence. Briefly, ELISA plates (Greiner-Bio-One) were coated with 100 μl / well of aggregated IgG at 10 μg / ml in PBS overnight at room temperature. In the ELISA, this step and all subseque...

Embodiment 3

[0147] Example 3: Binding characteristics of purified murine anti-C2 antibodies 13, 32, 35, 60 and 5F2.4

[0148] (a) Binding of monoclonal antibodies anti-C2-13, -32, -35, -60 and -5F2.4 to glycosylated recombinant human C2

[0149] Inhibitory and non-inhibitory anti-C2 mAbs were purified using protein G affinity chromatography (GE Healthcare). IsoQuick for isotyping using mouse monoclonal TM Kit (Sigma), heavy and light chains were typed into isotypic species (Table 1).

[0150] Table 1. Antibody classes for some murine mAbs against human C2

[0151]

[0152]

[0153]Using the procedure as described in Example 1(c) above, by assaying for binding to high (200 ng / well) and low (25 ng / well) glycosylated recombinant C2 (U-protein Express), purified The monoclonal antibodies anti-C2-5F2.4, anti-C2-13, anti-C2-32, anti-C2-35 and anti-C2-60 were further characterized.

[0154] as in Figure 4 Antibodies anti-C2-5F2.4, anti-C2-13, anti-C2-32, anti-C2-35 and anti-C2-60 dos...

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Abstract

The invention relates to means and methods that relate to binding molecules that bind human complement factor C2. Specific binding molecules are described with specific C2 activity inhibiting properties. Such binding molecules are useful in the treatment of symptoms of various human diseases among which there is inflammatory disease, neuro-inflammatory disease or ischemia-reperfusion (I / R) injury. Disease.

Description

technical field [0001] The present invention relates to the field of immunology / biochemistry. The present invention relates to means and methods for inhibiting the activation of the classical and lectin activation pathways of the complement system, and their use in the treatment of human disorders. The present invention relates to inhibitors of complement factors and their use. In particular, the present invention relates to binding molecules that bind to human complement factor C2, and their usefulness in the treatment or prevention of diseases or disorders mediated by complement activation, such as ischemia-reperfusion in antibody-mediated inflammatory diseases and ischemic disorders ( I / R) damage) application. Background technique [0002] The complement system consists of proteins that circulate in the blood. Complement factors circulate as inactive precursor proteins. Activation of the system results in an activation cascade in which one factor activates subsequent ...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395
CPCC07K16/18C07K2317/24C07K2317/565C07K2317/76A61P13/12A61P17/00A61P19/02A61P25/00A61P29/00A61P31/04A61P37/06A61P43/00A61P7/00A61P7/06A61P9/10Y02A50/30C07K16/40
Inventor C·E·哈克C·伊尔迪兹L·波恩P·J·西蒙斯
Owner BROTEIO PHARMA
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