Endoglycosidase mutants for glycoprotein remodeling and methods of using it

Abstract

A mutant of EndoS2 includes one or more mutations in the sequence of a wild-type EndoS2 (SEQ ID NO: 1), wherein the one or more mutations are in a peptide region located within residues 133-143, residues 177-182, residues 184-189, residues 221-231, and / or residues 227-237, wherein the mutant of EndoS2 has a low hydrolyzing activity and a high tranglycosylation activity, as compared to those of thewild-type EndoS2. A method for preparing an engineered glycoprotein using the mutant of EndoS2 includes coupling an activated oligosaccharide to a glycoprotein acceptor. The activated oligosaccharideis a glycan oxazoline.

Description

[0001] Cross-reference to related applications [0002] This application claims priority to US Patent Application Serial No. 62 / 378,806, filed August 24, 2016, the entire contents of which application is incorporated by reference in its entirety. technical field [0003] The present invention relates to a mutant of endoglycosidase S2 (endoglycosidase S2, EndoS2) selected from Streptococcus pyogenes (Streptococcus Pyogenes), which is useful in the synthesis of various well-defined N-glycans (high mannose type ( High mannose type), mixed type (hybrid type) and complex type (complex type) glycoproteins or glycopeptides have improved transglycosylation (tranglycosylation)-activity and reduced hydrolysis activity. In particular, one or more embodiments of the present invention also provide the use of EndoS2 mutants to effectively reshape the glycans of therapeutic antibodies, thereby forming a homogeneous glycan composition in the Fc region to enhance its effector function. functi...

AI Technical Summary

Problems solved by technology

However, the complete conversion of starting materials into products requires a very large amount of enzymes. The production of large-scale enzymes and the purification steps to remove residual enzymes after the reaction are very cumbersome and labor-intensive, which will have a large impact on the overall glycoengineering preparation method. cost

Furthermore, the transglycosylation activity of EndoS mutants is restricted to specific symmetric two-armed complex glycoforms, but not limited to a broad range of high-mannose, mixed, and three- and four-armed complex glycoforms (these Types have additional native modifications such as α-1,3-trehalose of GlcNAc, α-1,2-trehalose of Gal, extended polyacetyllactosamine positions, and asymmetric Sialylated branches (antennae)) make the use of limited

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