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Abstract
Antibody Dependent Cellular Cytotoxicity is an important mechanism for immunotherapeutics. This mechanism involves target cells with surface antigens, antibodies, and immune effector cells such as macrophages and natural killer (NK) cells. The Fc domain of the antibody engages with the Fc γ Receptors (FcγRIIIa) on the cell surface of macrophages or NK cells. This interaction has a strict N-glycan requirement restricting the production of antibodies to mammalian expression platforms. In addition to this, the amino acid composition of Fc also influences its affinity towards the receptor.This work represents two disparate approaches to enhance FcγRIIIa receptor binding. We first combined protein and N-glycan engineering to create a novel glycoengineered Yeast Surface Display (YSD) platform (EBY100-(GPD)-EndoS2) to simultaneously meet both these objectives. This platform could be used for engineering Fc through the creation of YSD libraries and for the production of Rituximab. This yeast expressed Rituximab possessed the desired engineered glycoform (>99%). In addition to this observation, the antibody also displayed properties in terms of Fab binding and receptor engagement similar to commercial antibody. We are currently in the process of validating hits identified using this platform for antibody engineering. In addition to
this, we have also utilized YSD to create library of novel aglycosylated disulphide variants to eliminate the requirement of the N-glycan completely. Our best hits displayed significant receptor-binding affinity with KD 1-3μM.
Another part of this work describes creation of a novel yeast platform to boost protein expression for 15N-labeling of Fc. Isotope labeling has been restricted to either mammalian or prokaryotic expression systems, yeast however combines advantages of both these platforms. We created yeast strain EBY100-Kar2p-T2A-PDI by utilizing a ribosome skip site to simultaneously produce both proteins, possibly lowering E.R stress, and resulting in increased target protein expression. With this platform, we recovered 2.2 mg/L of uniformly 15N-labeled human immunoglobulin (Ig)G1 Fc domain with 90.6% 15N labeling. NMR spectroscopy revealed a high degree of similarity between the yeast and mammalian expressed IgG1 Fc domains. In the future, we could utilize this platform to 15N and 13C label Fc to further understand kinetics and map the binding interface between Fc and the FcγRIIIa.