Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain

Abstract

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (K_D = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Keywords: EndoS2; human Fc gamma receptor IIIa (FcγIIIa); human immunoglobulin 1 (hIgG1); truncated N-glycan.

Figure 1

N-glycan processing in yeasts and mammals shares a common starting point. However, the end products are substantially different with yeasts capable of extensive mannosylation. The dashed line indicates the site of endoglycosidase (endo) cleavage; EndoS and EndoS2 cleave IgG1 N-glycans. OST—oligosaccharyltransferase; dol-dP—dolichol diphosphate.

Figure 2

EBY100-(GPD) EndoS2 is a dual-function yeast strain that can display glycoengineered Fc on the yeast surface for protein engineering or produce glycoengineered antibodies.

Figure 3

IgG1 Fc display and processing by the EndoS2 yeast strains and the respective controls (pYD1-Fc and pYD1-Fc/T299A). Flow cytometry for IgG1 Fc using an anti-IgG1 antibody (A–D) and GFP-FcγRIIIa binding (F–I). Boundaries for the highlighted boxes were drawn based on negative controls (not shown). (E,J) Western blots showing mobility of the Aga2p-IgG1 Fc fusions. T299A is an aglycosylated IgG1 Fc variant which shows reduced binding to GFP-FcγRIIIa.

Figure 4

Purification of IgG1 Fc and Rituximab expressed from EBY100-(GPD)EndoS2. IgG1 Fc purified using Protein A chromatography (A) and size-exclusion chromatography (B). Rituximab purified using Protein A chromatography (C) and size-exclusion chromatography (D). The largest peak in D at 140 mL is Rituximab. Arrows indicate the Rituximab heavy and light chain. (A,C) are 12% reducing SDS-PAGE gels.

Figure 5

Identification of oligomannose N-glycans on secreted IgG Fc. Concanavalin A purification of Fc derived from the (A) EBY100 and the (B) EBY100-(GPD) EndoS2 strains. “S” is the starting material before loading on the column, “FT” is the flow-through unbound fraction, “W” wash and “E” elutions.

Figure 6

Yeast-expressed Rituximab composition and receptor binding. (A) Total ion current from ESI-MS spectra of trypsinized Rituximab expressed from the EBY100-(GPD) EndoS2 strain summed from 11–15 min; doubly charged ions corresponding to oligomannose N-glycoforms were not observed. Isobaric ions were not distinguished, thus each cartoon represents multiple possible configurations. (B) ESI-MS/MS of the (1) GlcNAc glycopeptide from panel A showing a single GlcNAc residue at N297. Rituximab displaying either truncated (C) or yeast N-glycans (D) binding to high affinity FcγRIIIa (V158) variant as determined by surface plasmon resonance.

Figure 7

Binding of yeast-expressed Rituximab to Raji B cells. (A) Schematic diagram of the detection strategy. Flow cytometry analysis of Raji B cells incubated with the two detection antibodies shown in panel A, with or without commercially sourced Rituximab (B) or EBY100-(GPD) EndoS2 expressed Rituximab(C).