LAUSR

Anti‐CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B‐cell malignancies such as non‐Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8‐Fc‐hIL2), obtained by fusing an anti‐CD20 scFv‐Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL‐2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N‐glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody‐dependent cell‐mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N‐glycosylation plant signature (IC‐ΔXF) and the corresponding scFv‐Fc‐ΔXF antibody not fused to the cytokine, were obtained in a glyco‐engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20–35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC‐ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N‐glycosylation profile (IC) and to rituximab. The glyco‐engineered recombinant molecules also exhibited a strongly improved ADCC and complement‐dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv‐Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N‐glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential.