Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins

一种糖蛋白、突变体的技术，应用在糖蛋白合成领域，能够解决不清楚Endo-S2转糖基活性等问题，达到延长体内半衰期、提高靶向能力、小免疫原性的效果

Active Publication Date: 2018-12-21
UNIV OF MARYLAND
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, it is unclear whether Endo-S2 has transglycosylation activity and, if so, whether Endo-S2 glycoside synthase can be generated by site-directed mutagenesis

Method used

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  • Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins
  • Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins
  • Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins

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Embodiment

[0115] Generation of Endo-S2 glycosynthase mutants and their use for glycosylation remodeling of the intact monoclonal antibody rituximab

[0116] Glycoside synthases have previously been shown to be responsible for promoting during hydrolysis oxazoline Site-directed mutagenesis of key asparagine (Asn) in the GH85 family or aspartic acid (Asp) residue in the GH18 family of ionic intermediates formed by several GH85 endoglycosidases (ENGases), described GH85 endoglycosidases include EndoA, EndoM, EndoD and GH18 endoglycosidase EndoS. [36,38] Endo-S2 is an endoglycosidase belonging to glycoside hydrolase family 18 (GH18) [33], which is in the same GH family as EndoS, EndoF1, EndoF2, and EndoF3, which were recently confirmed to have transglycosylation activity middle. Based on the hypothesis that EndoS2-catalyzed hydrolysis also involves the formation of oxazoline A substrate-assisted mechanism of ionic intermediates proceeds, as shown by other GH18 endoglycosidases such...

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Abstract

The present invention provides for recombinant Endo-S2 mutants (named Endo-S2 glycosynthases) that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sugar chain is added to a fucosylated or nonfucosylated GlcNAc-IgG acceptor. As such, the present invention allows for the synthesis and remodeling of therapeutic antibodies thereby providing for certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and / or ability to deliver a therapeutic agent.

Description

[0001] Government Rights in This Invention [0002] This invention was made with government support under Grant Nos. R01 GM096973 and R01 GM080374 awarded by the National Institutes of Health. The government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application claims priority to co-pending US Provisional Application No. 62 / 279,087, filed January 15, 2016, the contents of which are hereby incorporated by reference for all purposes. technical field [0005] The present invention relates to glycoprotein synthesis and more specifically to the use of the recombinant mutant Endo S2, which is the endo-β-N-acetylamino group from strain NZ131 of Streptococcus pyogenes serotype M49 Glucosidase, with transglycosylation activity and limited hydrolytic activity, thereby providing efficient glycosylation remodeling of antibodies. Background technique [0006] Monoclonal antibodies (mAbs) represent a major class of therapeutic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C12P21/00A61K47/50
CPCC07K16/2887C07K16/32C12Y302/01096C12N9/2402C07K2317/41A61K47/549C12P21/005A61P31/18A61P35/00A61P43/00
Inventor 王来曦杨强李铁正童新
Owner UNIV OF MARYLAND
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