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...