Production of an active anti‐CD20‐hIL‐2 immunocytokine in Nicotiana benthamiana. Issue 1 (16th April 2015)

• Record Type: Journal Article
• Authors: Marusic, Carla; Novelli, Flavia; Salzano, Anna M.; Scaloni, Andrea; Benvenuto, Eugenio; Pioli, Claudio; Donini, Marcello
• Is Part Of: Plant biotechnology journal. Volume 14:Issue 1(2016)
• Page Start: 240
• Page End: 251
• Publication Date: 2015-04-16
• Subjects: molecular farming -- immunocytokine -- human interleukin‐2 -- antibody‐dependent cell‐mediated cytotoxicity -- non‐Hodgkin lymphomas; Plant biotechnology -- Periodicals; Plant genetic engineering -- Periodicals; 630.272
• DOI: 10.1111/pbi.12378
• Languages: English
• ISSNs: 1467-7644

Abstract

Summary: Anti‐CD20 murine or chimeric antibodies (Abs) have been used to treat non‐Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti‐CD20 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 mAb therapy is the use of anti‐CD20 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‐CD20‐human interleukin‐2 (hIL‐2) immunocytokine (2B8‐Fc‐hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana . The scFv‐Fc‐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. 2B8‐Fc‐hIL2 and the cognate 2B8‐Fc antibody, devoid of hIL‐2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody‐dependent cell‐mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8‐Fc‐hIL2, 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 NHLs treatment.