# Engineering antibody effector functions by Glycan Remodeling Yeast Display

> **NIH NIH R21** · EMORY UNIVERSITY · 2022 · $233,950

## Abstract

Antibodies constitute a growing class of drugs that are being administered for the treatment of an increasing
range of human diseases, including but not limited to autoimmunity, infection and cancer. While engineering
antibodies to recognize virtually any antigen has become technologically straightforward, engineering antibodies
to induce distinct immune signals, or effector functions, which direct the killing of cells in vivo, remains technically
challenging. This latter property is carried out by the Fc region of antibodies and the difficulty in engineering
antibody Fc regions is due to the presence of a conserved N-linked glycan attached to Asn297 in clinically-
relevant IgG antibodies. The next generation of immunotherapeutic antibodies, as well as our abilities to identify
and better understand antibody-mediated killing mechanisms, depends on our ability to engineer IgG Fc domains
to bind with altered affinities and specificities to Fc γ receptors (FcγRs), including both activating and inhibitory
receptors, and complement in order to customize antibody-mediated effector functions. The major barrier to Fc
engineering is that there are currently no methods by which to perform directed evolution (i.e., combinatorial
mutagenesis and selection) of glycoproteins, such as Fc domains, while maintaining and/or controlling the glycan
chemistry required for their interactions with FcγRs and complement. We combined two established technologies
– chemoenzymatic synthesis of glycoproteins (i.e., the use of glycosylation-modifying enzymes and chemical
synthesis of glycans) and traditional yeast display directed evolution – to create a novel method for engineering
glycosylated Fc domains that we call Glycan Remodeling Yeast Display, or GRYD. In GRYD, a library of IgG Fc
domain proteins is displayed on the yeast cell surface, where they are decorated with the high mannose glycans
that yeast naturally produce. We then use chemoenzymatic synthesis to remodel the Asn297-linked glycans,
while still on the yeast cell surface, to complex type glycans, representative of those on human antibodies.
Finally, using a fluorescently-labeled FcγR tetramer, we select yeast cells expressing Fc domain variants with
higher FcγR binding by fluorescence-activated cell sorting (FACS). By introducing a chemoenzymatic synthesis
step to remodel the Fc glycans on the yeast cell surface, we not only produce a library of properly glycosylated
Fc domain variants from which to select for desirable properties, but we maintain the link between the Fc domain
genes and the proteins that they encode in the same cell – the key requirement of directed evolution. Antibodies
created using the GRYD technology could constitute a novel set of tools that the immunological community can
use to manipulate and evaluate the in vivo antibody-mediated killing mechanisms of the entire catalog of
antibodies, both currently available and to be developed in the future. Immunotherapeutic antibodies that have
b...

## Key facts

- **NIH application ID:** 10494252
- **Project number:** 5R21AI156376-02
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** ERIC JOHN SUNDBERG
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $233,950
- **Award type:** 5
- **Project period:** 2021-09-23 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10494252

## Citation

> US National Institutes of Health, RePORTER application 10494252, Engineering antibody effector functions by Glycan Remodeling Yeast Display (5R21AI156376-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10494252. Licensed CC0.

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