Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core alpha 1,3-fucose and beta 1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFc(Delta XF), has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFc(Delta XF), but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFc(Delta XF) treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFc(Delta XF) before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFc(Delta XF) was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.