CONICET | Buscador de Institutos y Recursos Humanos

Plant-based platforms have been extensively used for the expression of therapeutically valuable monoclonal antibodies. However, to generate highly active molecules a detailed understanding of biochemical and cell biological processes that affect protein stability and post-translational modifications is required. In this work we aimed to examine vacuolar deposition as a possible strategy for the expression of mAb. The mAb 14D9 was transiently expressed in Nicotiana benthamiana leaves in a (i) secreted form and (ii) as heavy chain (H) fusion to two vacuolar sorting signals1 (H-CtVSS and H-ssVSS). To monitor subcellular localization secretory H and L chains were fused to the red fluorescent protein mCherry. Confocal microscopy showed that sec-L-Cherry expressed alone localized to the apoplastic space, while sec-H-Cherry was retained in the ER. When constructs encoding sec-L-Cherry and H-CtVSS or H-ssVSS were co-infiltrated, red fluorescence was observed in the central vacuole, no ER staining was found. A vacuolar staining pattern was also obtained when H-RFP-CtVSS and sec-L constructs were co-infiltrated. In addition vacuolar versions of 14D9 were purified and subjected to MS analyses to determine the N-glycosylation profile. Three major glycoforms were detected: Man7, Man8 (oligomannosidic structures) as well as fucosylated (F) and xylosylated (X) structures. No glycans that are assigned as vacuolar typical (namely paucimannosidic structures) were detected. In conclusion, this work shows that vacuolar targeted 14D9 accumulates in the central vacuole of leaves, that the mAb is stable and that it can be easily purified. ER typical glycosylation indicates a direct transport from the ER to the vacuole bypassing the Golgi. However, at least some of the antibody molecules that are sorted to the vacuole pass through the trans-Golgi complex as shown by FX containing glycans. These results point to peculiarities in the vacuolar transport mechanisms of plants.