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

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R43GM134816-01
Agency Tracking Number: R43GM134816
Amount: $300,000.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: 300
Solicitation Number: PA16-157
Solicitation Year: 2016
Award Year: 2019
Award Start Date (Proposal Award Date): 2019-08-01
Award End Date (Contract End Date): 2021-07-31
Small Business Information
Ellicott City, MD 21042-4921
United States
DUNS: 079908008
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (301) 405-1848
Business Contact
Phone: (410) 707-3183
Research Institution

Project abstract/summary
This proposal responds to the NIH PA-16-157 “New Technologies for the Glycosciences (R43/R44)”. We
aim to develop efficient and streamlined cheomoenzymatic systems for glycan remodeling of antibodies and
other important glycoproteins, through optimizing enzyme immobilization and simplifying the reaction procedures.
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 a number of 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 in multiple
approaches to prepare glycan-defined glycoprotein (such as total chemical synthesis and glycosylation through
glycosylation 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. In the proposed study,
GlycoT Therapeutics attempts to streamline and simplify this enzymatic glycan remodeling method through
immobilizing the enzymes and developing easy-to-use kits and protocols, which are particularly useful for non-
specialists. Immobilization will offer multiple valuable benefits: 1) stabilizing the enzyme for sustained activity,
repeated use, and easy storage; 2) removing the protein purification steps for protein-GlcNAc acceptor and final
products, thus greatly simplifying the process; 3) eliminating the potential problem of wild type enzyme
contamination from the deglycosylation step. To achieve these objectives, we propose to pursue the following
two specific aims in Phase I study. Aim 1 is to immobilize key enzymes for glycan remodeling of antibodies,
which include: the endoglycosidase S2 (Endo-S2) from Streptococcus pyogenes, the Endo-S2 glycosynthase
mutant (D184M), and an α1,6-fucosidase for defucosylation. Different methods of immobilization will be
investigated. Aim 2 is to develop reaction kits and simply protocols for streamlined application of the enzymatic
glycan remodeling of antibodies. In prospective phase II research, the study will expand to other
endoglycosidases, to cover other glycoproteins/glycopeptides, for both academic and industrial applications.Project NarrativeThis proposal research aims to develop efficient and streamlined cheomoenzymatic systems for glycan
remodeling of antibodies and other important glycoproteins.

* Information listed above is at the time of submission. *

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