Endo-S Mutants for Homogeneous Antibody Glycoforms

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Solution Overview

Problem

Current methods for glycoprotein glycosylation remodeling, particularly for monoclonal antibodies, face challenges in achieving consistent and homogeneous glycoforms due to limitations in enzymatic deglycosylation and reglycosylation processes, especially with endoglycosidases like EndoA and EndoM, which struggle with core-fucosylated IgG-Fc domains, and lack the ability to transfer intact complex type N-glycans.

Innovation Solution

Development of recombinant Endo-S mutants with reduced hydrolysis activity and enhanced transglycosylation efficiency, specifically the D233Q and D233A mutants, which can efficiently transfer oligosaccharides to both core-fucosylated and nonfucosylated GlcNAc-IgG acceptors, enabling the synthesis of homogeneous glycoforms of IgG antibodies and Fc fragments with predetermined oligosaccharide moieties.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If endoglycosidases like EndoA and EndoM are used for deglycosylation and reglycosylation, then glycosylation remodeling can be performed, but the enzymes struggle with core-fucosylated IgG-Fc domains and cannot transfer intact complex type N-glycans

Engineering Contradiction:

Improveability to process core-fucosylated IgG-Fc domainsVSAvoidconsistency and homogeneity of glycoforms

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent introduces a novel endoglycosidase enzyme with modified catalytic parameters that enable efficient processing of core-fucosylated IgG-Fc domains. This enzyme exhibits enhanced ability to cleave fucosylated N-glycans and transfer intact complex type N-glycans, overcoming the limitations of conventional endoglycosidases and achieving consistent homogeneous glycoforms

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The glycosylation remodeling process is divided into distinct stages: deglycosylation of the Fc domain, followed by reglycosylation with desired N-glycan structures. This segmentation allows independent optimization of each step, enabling the enzyme to first remove problematic fucosylated glycans and then install homogeneous complex type N-glycans

Inventive Principle:

Principle #1Segmentation

2Manufacturing precision

If conventional enzymatic methods are used for glycosylation remodeling, then some glycoforms can be produced, but consistent and homogeneous glycoforms are difficult to achieve

Engineering Contradiction:

Improvehomogeneity of glycoformsVSAvoidsimplicity of enzymatic process

Core Design Contradiction:

Manufacturing precisionVSEase of manufacture

Solution Approach 1:

The patent employs a specifically designed endoglycosidase enzyme as an intermediary catalyst that mediates the transfer of intact complex type N-glycans to the Fc domain. This enzymatic mediator ensures high precision in glycoform production by catalyzing the formation of homogeneous glycosylated products without requiring complex chemical synthesis procedures

Inventive Principle:

Principle #24Intermediary (Mediator)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

These mutants allow for the production of antibodies with prolonged in vivo half-life, reduced immunogenicity, enhanced targeting ability, and improved therapeutic efficacy by achieving specific glycoforms that were previously difficult to obtain, such as fully sialylated IVIG for anti-inflammatory activity and nonfucosylated glycoforms for enhanced ADCC activity.

Implementation Method 1

Endo-S, an Endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, that possesses transglycosylation activity and limited hydrolyzing activity thereby providing for efficient glycosylation remodeling of antibody-Fc domain

Methodology Applied

Scientific EffectTransglycosylation: Chemical Bonding

Implementation Method 2

Endo-S, an Endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, that possesses transglycosylation activity and limited hydrolyzing activity

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Data Source

PatentUS11643450B2Chemoenzymatic glycoengineering of antibodies and Fc fragments thereof

Publication Date: 2023.05.09 UNIV OF MARYLAND

AI summary

The present invention provides for recombinant Endo-S mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sialylated oxazoline or synthetic oligosaccharide oxazoline is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor. Such recombinant Endo-S mutants are useful for efficient glycosylation remodeling of IgG1-Fc domain to provide different antibody glycoforms carrying structurally well-defined Fc N-glycans.