RESUMEN
The plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the Fab region of the monoclonal antibody produced in the plant; thus, it is necessary to compare the antigen affinity of this antibody with that of the prototype. In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana, and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells. Two forms of plant ofatumumab (with or without HDEL-tag) were generated and their production yields were compared. The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL-tag. The specificity of the target recognition of plant-derived ofatumumab was confirmed by mCherry-CD20-expressing HEK cells via immuno-staining, and the capping of CD20 after ofatumumab binding was also confirmed using Ramos B cells. In the functional equivalence tests, the binding affinities and complement-dependent cell cytotoxicity efficacy of plant-ofatumumab-HDEL and plant-ofatumumab without HDEL were significantly reduced compared to those of CHO-derived ofatumumab. Therefore, we suggest that although ofatumumab is not a good candidate as a template for plant-derived monoclonal antibodies because of its decreased affinity when produced in plants, it is an interesting target to study the differences between post-translational modifications in mammals and plants.
Asunto(s)
Anticuerpos Monoclonales Humanizados/genética , Fragmentos Fab de Inmunoglobulinas/química , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Antígenos CD20/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptosis , Linfocitos B , Células CHO , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cricetulus , Citotoxicidad Inmunológica/efectos de los fármacos , Células HEK293 , Humanos , Conformación Proteica , Rituximab/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0191075.].
RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Linfocitos B/citología , Muerte Celular , Nicotiana/genética , Animales , Células CHO , Cricetinae , Cricetulus , Epítopos/inmunología , Citometría de Flujo , Hojas de la Planta/metabolismoRESUMEN
A simple and multitopic ligand, pyrimidine-5-carboxylate (pmc), has been used to obtain a series of metal-organic frameworks (MOFs) based on Co(2+), Cd(2+), and Cu(2+). The networks possess well-defined topologies of body-centered-cubic, rutile, and interpenetrated NbO structures, respectively. Among those, [Cu(pmc)(2)] possesses a permanent porosity resulting from straight one-dimensional channels of 5.5 Å free passages. Unexpectedly, this porous MOF displays a highly selective sorption behavior for CO(2), and the sorptions of N(2), Ar, O(2), H(2), and CH(4) at two different temperatures are found to be negligible. The results of diffraction and spectroscopic analyses exclude framework dynamics or incomplete evacuation as the origin of the gas-sorption selectivity.