Efficient Expression of Functionally Active Aflibercept with Designed N-glycans

Abstract

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLI^GnGn and AFLI^Sia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLI^GnGn and AFLI^Sia displayed similar binding potency to VEGF₁₆₅, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

Keywords: Fc fusion; aflibercept; anti VEGF; glycan engineering; glycosylation; plant expression.

Figure 1

Aflibercept produced in N. benthamiana. (A) Western blot analysis of total soluble proteins (TSP) extracted from plants infiltrated with pICH26211α-aflibercept (lane S, ~10 µg of TSP was loaded). (B) SEC profile of protein A-purified aflibercept and location of collected fractions. (C) SDS-PAGE of protein A-purified aflibercept (ELU), and SEC fractions thereof (F1–F3), under non-reducing (left) and reducing (right) conditions; protein loading (ELU, F1, F2, F3): 6.5, 2, 4, 4 µg/lane.

Figure 2

N-glycan composition of aflibercept expressed in N. benthamiana plants deficient in core xylose and fucose [18,24]. Four variants were analyzed by LC-ESI-MS: AFLI^GnGn and AFLI^Sia purified from TSP and IF. Histogram bars represent the relative abundance (%) of glycoforms present at each individual glycosite (GS1–GS5). For detailed information, see Table S1. Nomenclature according to Proglycan [28].

Figure 3

Binding ELISA of aflibercept variants to VEGF₁₆₅. GnGn TSP: AFLI^GnGn isolated from TSP; Sia TSP and Sia IF: AFLI^Sia isolated from TSP and IF, respectively.