title: Production of an active anti-CD20-hIL-2 immunocytokine in Nicotiana benthamiana.
abstract: 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.
authors: Laboratory of Biotechnology, ENEA Research Center Casaccia, Rome, Italy.
authors: Carla
authors: C
authors: Marusic
authors: Laboratory of Radiation Biology and Biomedicine, ENEA Research Center Casaccia, Rome, Italy.
authors: Flavia
authors: F
authors: Novelli
authors: Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Napoli, Italy.
authors: Anna M
authors: AM
authors: Salzano
authors: Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, Napoli, Italy.
authors: Andrea
authors: A
authors: Scaloni
authors: Laboratory of Biotechnology, ENEA Research Center Casaccia, Rome, Italy.
authors: Eugenio
authors: E
authors: Benvenuto
authors: Laboratory of Radiation Biology and Biomedicine, ENEA Research Center Casaccia, Rome, Italy.
authors: Claudio
authors: C
authors: Pioli
authors: Laboratory of Biotechnology, ENEA Research Center Casaccia, Rome, Italy.
authors: Marcello
authors: M
authors: Donini
mesh_terms: D060054:Agrobacterium / Q000378:metabolism; D000595:Amino Acid Sequence; D000920:Antibody-Dependent Cell Cytotoxicity; D018951:Antigens, CD20 / Q000276:immunology*; D015153:Blotting, Western; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis*; D010936:Plant Extracts / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D046549:Plantibodies / Q000737:chemistry / Q000302:isolation & purification; D030821:Plants, Genetically Modified; D011485:Protein Binding; D015202:Protein Engineering; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000235:genetics*
chemical_list: D018951:Antigens, CD20; D007376:Interleukin-2; D010936:Plant Extracts; D046549:Plantibodies
keywords: antibody-dependent cell-mediated cytotoxicity; human interleukin-2; immunocytokine; molecular farming; non-Hodgkin lymphomas