# Development of streamlined chemoenzymatic glycan remodeling systems for antibodies and other important glycoproteins

> **NIH NIH R44** · GLYCOT THERAPEUTICS, LLC · 2021 · $774,934

## Abstract

Project abstract/summary
The objectives of this proposal are to develop efficient and streamlined cheomoenzymatic systems for glycan
remodeling of antibodies and other important glycoproteins, through enzyme immobilization, scalable glycan
production, and kits development. Such immobilized enzymes, when combined with activated glycan library as
kits, can help general academic and industrial users, particularly non-specialists, to prepare glycan-defined
glycoproteins for structural and functional studies. The streamlined approach can also be applied in scale-up
preparation of homogenous glycoproteins with therapeutic potential. Protein glycosylation is one of the most
ubiquitous posttranslational modifications. It profoundly affects a protein’s properties such as folding, in vivo
stability, immunogenicity, and pharmacokinetics, and also directly participate in many important biological
processes, including cell adhesion, cancer progression, host-pathogen interactions, and immune responses.
A major issue in glycoprotein studies is the structural heterogeneity and the difficulty in isolating
homogeneous glycoforms for detailed structural and functional studies. Although significant progresses have
been made for producing glycan-defined glycoprotein such as total chemical synthesis and biosynthetic pathway
engineering in different host expression system, the pure glycoforms that can be achieved are still quite limited.
To address this challenge, a novel chemoenzymatic method to produce homogeneous glycoprotein and
glycopeptides has been developed recently. This convergent approach consists of two key steps: deglcosylation
of glycoproteins with an endoglycosidase and subsequent attachment of a desired activated N-glycan to the
protein-GlcNAc acceptor by a novel glycosynthase. This SBIR II application is built on the success of the Phase
I study (1R43GM134816-01), where we have successfully finished the immobilization of endoglycosidase S2
(EndoS2) from S. pyogenes and its corresponding glycosynthase mutant EndoS2-D184M. The EndoS2-D184M
glycosynthase was immobilized by a site-oriented approach which almost fully retains the activity of enzyme. We
have demonstrated that the immobilized enzymes are highly efficient for glycan remodeling of therapeutic
antibodies. In this Phase II research, we propose to pursue the following three specific aims. Aim 1 is to expand
this immobilization strategy to other glycosidases/glycosynthases to establish a tool box of enzymes for glycan-
remodeling. Aim 2 is optimize and scale up our production protocol for different oxazolines and significantly
lower their production cost. Aim 3 is to develop glycan remodeling kits and establish scalable glycan remodeling
protocols.

## Key facts

- **NIH application ID:** 10324865
- **Project number:** 2R44GM134816-03
- **Recipient organization:** GLYCOT THERAPEUTICS, LLC
- **Principal Investigator:** Qiang Yang
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $774,934
- **Award type:** 2
- **Project period:** 2019-08-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10324865

## Citation

> US National Institutes of Health, RePORTER application 10324865, Development of streamlined chemoenzymatic glycan remodeling systems for antibodies and other important glycoproteins (2R44GM134816-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10324865. Licensed CC0.

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