Production of an active anti‐ CD 20‐ hIL ‐2 immunocytokine in N icotiana benthamiana

Summary Anti‐ CD 20 murine or chimeric antibodies ( A bs) have been used to treat non‐ H odgkin lymphomas ( NHL s) and other diseases characterized by overactive or dysfunctional B cells. Anti‐ CD 20 Abs demonstrated to be effective in inducing regression of B ‐cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of m A b therapy is the use of anti‐ CD 20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL ‐2‐based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti‐ CD 20‐human interleukin‐2 (h IL ‐2) immunocytokine (2 B 8‐ F c‐h IL 2) based on the C 2B8 m A b ( R ituximab) and the resulting ectopic expression in N icotiana benthamiana . The sc F v‐ F c‐engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS ‐ PAGE and gel filtration. Purification yields using protein‐ A affinity chromatography were in the range of 15–20 mg/kg of fresh leaf weight ( FW ). Glycopeptide analysis confirmed the presence of a highly homogeneous plant‐type glycosylation. 2 B 8‐ F c‐ hIL 2 and the cognate 2 B 8‐ F c antibody, devoid of h IL ‐2, were assayed by flow cytometry on D audi cells revealing a CD 20 binding activity comparable to that of R ituximab and were effective in eliciting antibody‐dependent cell‐mediated cytotoxicity of human PBMC versus D audi cells, demonstrating their functional integrity. In 2 B 8‐ F c‐h IL 2, IL ‐2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHL s treatment.