Abstract
Plants have attracted attention as bio-drug production platforms because of their economical and safety benefits. The preliminary efficacy of ZMapp, a cocktail of antibodies produced in N. benthamiana (Nicotiana benthamiana L.), suggested plants may serve as a platform for antibody production. However, because the amino acid sequences of the Fab fragment are diverse and differences in post-transcriptional processes between animals and plants remain to be elucidated, it is necessary to confirm functional equivalence of plant-produced antibodies to the original antibody. In this study, Obinutuzumab, a third generation anti-CD20 antibody, was produced in N. benthamiana leaves (plant-obinutuzumab) and compared to the original antibody produced in glyco-engineered Chinese hamster ovary (CHO) cells (CHO-obinutuzumab). Two forms (with or without an HDEL tag) were generated and antibody yields were compared. The HDEL-tagged form was more highly expressed than the non-HDEL-tagged form which was cleaved in the N-terminus. To determine the equivalence in functions of the Fab region between the two forms, we compared the CD20 binding affinities and direct binding induced cell death of a CD20-positive B cells. Both forms showed similar CD20 binding affinities and direct cell death of B cell. The results suggested that plant-obinutuzumab was equivalent to CHO-obinutuzumab in CD20 binding, cell aggregation, and direct cell death via binding. Therefore, our findings suggest that Obinutuzumab is a promising biosimilar candidate that can be produced efficiently in plants.
Highlights
A recent breakthrough in cancer treatment employs immunotherapy with monoclonal antibodies that bind to a highly expressed target on cancer cells[1, 2]
In order for the heavy and light chains to assemble in the endoplasmic reticulum (ER), the localisation signal sequence from the ER protein luminal binding protein (BiP) was attached to the N-terminal region of the light and heavy chains
Of the 16 different anti-CD20 antibodies clinically available at present[32], Obinutuzumab is recognized as superior based on low minimal residual disease (MRD) and increased progression-free survival[2]
Summary
A recent breakthrough in cancer treatment employs immunotherapy with monoclonal antibodies that bind to a highly expressed target on cancer cells[1, 2]. If the Fab function of Obinutuzumab produced in N. benthamiana leaves can be demonstrated as equivalent to that produced in CHO cells, this result would indicate that the protein sequence of the Fab portion of Obinutuzumab is not altered by plant-specific post-transcriptional processes. If this is the case, it would be very easy to produce Obinutuzumab in a variety of transgenic plants or via highly productive expression systems that have solved the issue of plant-specific glycosylation
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