Use of the endoglycosidase endos for treating immunoglobulin g mediated diseases

An endoglycosidase, application technology, applied in endocrine system diseases, sexual diseases, metabolic diseases and other directions, can solve problems such as undiscovered

Active Publication Date: 2009-12-16
ハンサバイオファルマアクチボラゲット
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Use of the endoglycosidase endos for treating immunoglobulin g mediated diseases
  • Use of the endoglycosidase endos for treating immunoglobulin g mediated diseases
  • Use of the endoglycosidase endos for treating immunoglobulin g mediated diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1: EndoS efficiently hydrolyzes IgG in human blood

[0115] To be effective as a therapeutic against pathological IgG, EndoS needs to be active at low concentrations in the context of human whole blood. To investigate this, recombinant EndoS (rEndoS) without a signal sequence (ie, having the sequence of SEQ ID NO: 1 ) was produced and purified as previously described (Collin & Olsén, 2001, Infect. Immun. 69:7187-7189). Increasing final concentrations (0, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 μg / ml) of rEndoS were rolled end over end in 500 μl of heparinized human blood from healthy volunteers at 37°C Incubate with rotation for 1 hour. Samples were centrifuged at 720 xg for 10 min at 4°C, after which IgG in plasma was purified using protein G sepharose according to the manufacturer's instructions (GE Healthcare Bjosciences, Uppsala, Sweden). Consistent with previous findings (Collin & Olsén 2001, EMBO J. 20:3046-3055) on the IgG binding proteins Protein H (from...

Embodiment 2

[0118] Example 2: EndoS efficiently hydrolyzes IgG in rabbits

[0119] To further substantiate the utility of EndoS as a therapeutic agent, the IgG glycan hydrolytic activity of EndoS in the circulation of living animals was studied. Swedish loop rabbits weighing approximately 3 kg were injected intravenously with 1 mg of rEndoS, corresponding to an rEndoS:IgG ratio of approximately 1:2000 (assuming that rEndoS is only distributed in the blood). Animals showed no signs of disease. Serum samples were collected at 0, 1, 2, 4, 6, 8 and 12 hours and on days 1, 2, 3, 4, 5, 6, 8 and 10. Serum IgG was analyzed for glycosylation status by SDS-PAGE and lectin blot analysis as described previously for human blood.

[0120] These experiments showed that, prior to injection, the apparent molecular weight of the IgG heavy chain was comparable to that of fully glycosylated intact rabbit IgG ( Image 6 A, staining, 0 hours and IgG). In contrast, there was already a partial shift of the...

Embodiment 3

[0124] Example 3: EndoS is active in rabbits despite antibodies against the EndoS enzyme

[0125] Since EndoS was fully active at the second and third injections, it was intriguing to determine whether this was due to an absent or low level of immune response to the enzyme or to have specific antibodies against EndoS that did not interfere with enzymatic activity. Interested in. Since it is known that healthy individuals and those infected with S. pyogenes have antibodies against EndoS ( This is of particular interest since et al., 2004, J. Infect. Dis. 189:797-804).

[0126] To investigate this, purified rEndoS was separated on 10% SDS-PAGE and electroblotted onto PVDF cut into 1.5 mn strips. Strips were incubated with a 1:500 dilution of all serum samples from the first, second and third injections, followed by incubation with peroxidase-labeled goat anti-rabbit antibody (Pierce). Strips were developed with chemiluminescence as described above for lectin blots.

[012...

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Abstract

The invention provides use of an EndoS polypeptide, or a polynucleotide encoding an EndoS polypeptide, in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated by IgG antibodies.

Description

field of invention [0001] The present invention relates to methods for treating or preventing diseases or conditions mediated by IgG antibodies, such as autoimmune diseases, transplant rejection, post-surgical therapy, and acquired hemophilia. Background of the invention [0002] IgG is a heterotetramer comprising two heavy and two light chains held together by disulfide bonds, forming a three-protein domain separated by a flexible and protease-sensitive hinge region. Two identical Fab portions bind antigen while a separate Fc portion is responsible for effector functions, including binding and activating complement factor Clq and Fc receptors on leukocytes. [0003] In addition to the polypeptide backbone, the Fc portion contains a conserved glycan linked to Asn-297 on each heavy chain. This oligosaccharide has a complex biantennary pattern with core fucose linked to the innermost N-acetylglucosamine (GlcNAc). These glycans are located at C H 2 domain (the second constan...

Claims

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Application Information

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IPC IPC(8): A61K38/47A61K38/16A61P37/00
CPCA61K48/005A61K38/47A61P1/04A61P1/08A61P1/16A61P13/12A61P15/00A61P17/00A61P17/06A61P17/14A61P19/02A61P19/04A61P21/00A61P21/04A61P25/00A61P27/02A61P27/16A61P29/00A61P3/10A61P35/00A61P37/00A61P37/02A61P37/06A61P5/00A61P5/06A61P5/14A61P5/18A61P5/40A61P7/00A61P7/04A61P7/06A61P7/08A61P9/00A61P9/08
Inventor L·博乔克马迪亚斯·科林阿恩·奥尔森R·霍默达K·S·南达库玛乌纳·莎侬
Owner ハンサバイオファルマアクチボラゲット
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