EndoS2 Mutants for Antibody Glycan Remodeling With Low Hydrolysis
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Solution Overview
Problem
Existing endoglycosidases, such as EndoS and EndoS2, exhibit significant hydrolytic activity, limiting their effectiveness in transferring a broad range of N-glycans to therapeutic antibodies, and require large enzyme quantities for efficient glycoengineering, leading to high production costs and labor-intensive purification processes.
Innovation Solution
Development of EndoS2 mutants with specific mutations, such as T138D, T138E, and others, that reduce hydrolytic activity and enhance transglycosylation efficiency, allowing for the transfer of diverse N-glycans like high mannose, hybrid, and complex types to therapeutic antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type endoglycosidases (EndoS or EndoS2) are used for glycan transfer, then the enzyme can catalyze the reaction, but the significant hydrolytic activity limits effectiveness and requires large enzyme quantities
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the endoglycosidase sequence to alter the enzyme's catalytic properties. Mutations at positions such as 182, 226, 227, 228, or 138 change the enzyme's behavior from predominantly hydrolytic to predominantly transglycosylating, thereby resolving the contradiction between effectiveness and harmful hydrolytic activity
Solution Approach 2:
The patent converts the harmful hydrolytic activity into a beneficial transglycosylation activity through mutagenesis. The mutated enzyme retains catalytic capability but redirects it toward productive glycan transfer rather than destructive hydrolysis, turning the enzyme's inherent reactivity from a liability into an asset
2Productivity
If large quantities of enzyme are used to overcome hydrolytic activity, then transglycosylation can proceed, but production costs increase and purification becomes labor-intensive
Solution Approach 1:
By changing the enzymatic parameters through mutagenesis, the patent creates enzymes with enhanced transglycosylation activity and reduced hydrolytic activity. This allows efficient glycoengineering reactions using smaller enzyme quantities, directly reducing production costs and simplifying purification processes while maintaining high productivity
3Adaptability or versatility
If wild-type endoglycosidases are used, then the enzyme shows broad substrate activity, but the hydrolytic activity prevents homogenous glycan composition
Solution Approach 1:
The patent changes the catalytic parameters of the enzyme through targeted mutagenesis, creating variants that favor transglycosylation over hydrolysis. This allows the enzyme to work with diverse glycan substrates (maintaining versatility) while producing homogeneous glycan compositions on therapeutic antibodies (improving precision)
Solution Approach 2:
The patent converts the problematic hydrolytic activity that prevents homogeneity into beneficial transglycosylation activity that promotes homogeneity. The mutated enzymes transfer glycans efficiently without cleaving them, enabling precise control over final glycan composition while retaining broad substrate acceptance
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
The EndoS2 mutants enable the production of homogeneously glycosylated therapeutic antibodies with improved effector functions and pharmacological properties, reducing the need for excessive enzyme use and simplifying the production process.
Implementation Method 1
selected mutants of endoglycosidase S2 (EndoS2) from Streptococcus Pyogenes that display improved transglycosylation activities
Implementation Method 2
EndoS2 mutants enable the production of homogeneously glycosylated therapeutic antibodies
Implementation Method 3
reduced hydrolyzing activities for the synthesis of glycoproteins or glycopeptides
Data Source
Figure 1
Figure 2
Figure 3(A)~3(B)
AI summary
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 the wild-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 oligosaccharide is a glycan oxazoline.