# Development of streamlined one-pot chemoenzymatic glycan remodeling systems for antibody conjugation

> **NIH NIH R44** · GLYCOT THERAPEUTICS, LLC · 2024 · $758,302

## Abstract

Project abstract/summary
In the proposed research, GlycoT will expand its cutting-edge chemoenzymatic platform by further optimizing
and streamlining a one-pot single-zyme glycan-specific conjugation technology, that can be applied to a wide
variety of application. Conjugation of various functional molecules to antibodies are frequently used for a wide
variety of applications within the life science sectors, such as fluorescent labeled antibodies for the detection and
imaging, antibody-drug conjugate (ADC) for cancer therapy, LYTAC for targeted degradation. The most
exemplified application of antibody conjugation is the development of ADC therapeutics. For approved 15 ADCs
and others in clinical trials, the payloads have been mainly conjugated to antibody by non-specific random linkage
to either cysteine or lysine, resulting in heterogenous ADC regioisomers, with varied antigen affinity, aggregation
potential, serum half-life, and other limitation. As a result, site-specific ADCs with improved pharmacokinetics,
and enhanced therapeutic index have been developed. Among different approached to generate site-specific
ADCs, remodeling of Fc-glycan on the conserved Asn-297 position to generate Fc-glycan specific ADC is
particularly attractive. The use of the galactosyltransferase (GalT) mutants capable of accommodating modified
UDP-Gal derivatives as the donor substrates has enabled the incorporation of a selected tag at the Fc glycans
for subsequent site-specific conjugation with modified cytotoxic agents. This technology route has been adopted
by several clinical stage companies. However, the GalT mutant is not very efficient and can only transfer azide
or keto based small Gal-GDP derivative. In contrast, another endoglycosidase-based one-pot transglycosylation
method has overcome such limitations. This convergent approach combined two key enzymatic steps,
deglycosylation of the antibody, and subsequent attachment of a tagged disaccharide, in the same reaction
system, with a single endoglycosidase like EndoS2 from S. pyogenes. This one-pot platform has been
exclusively licensed by GlycoT. The core enzyme EndoS2 is highly efficient and can transfer azido disaccharide
modified with single or multiple extended linkers, as demonstrated in the proof-of-concept studies. In the
proposed Phase 2 R&D, GlycoT will streamline this amazing bioconjugation technology by pursuing the following
five specific aims: Aim 1. Scale-up and optimization of the synthesis of disaccharides oxazolines, Aim 2.
Combability of single-enzyme transglycosylation with engineered human IgG, Aim 3. Design and evaluate
EndoS2 fusion enzyme to further simplify the glycoengineering process, Aim 4. Streamline the whole process
to synthesize glycan-specific ADC, Aim 5. In vivo evaluation of glycan-specific ADCs. With the successful
execution of this R&D plan, for the academic researchers, we can provide convenient, highly efficient, and robust
labeling kits for site-specific conjugation of...

## Key facts

- **NIH application ID:** 10822116
- **Project number:** 1R44GM153003-01
- **Recipient organization:** GLYCOT THERAPEUTICS, LLC
- **Principal Investigator:** Guanghui Zong
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $758,302
- **Award type:** 1
- **Project period:** 2024-03-01 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10822116

## Citation

> US National Institutes of Health, RePORTER application 10822116, Development of streamlined one-pot chemoenzymatic glycan remodeling systems for antibody conjugation (1R44GM153003-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10822116. Licensed CC0.

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