The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L.) is equivalent to obinutuzumab produced in CHO cells

Jin Won Lee; Woon Heo; Jinu Lee; Narae Jin; Sei Mee Yoon; Ki Youl Park; Eun Yu Kim; Woo Taek Kim; Joo Young Kim

doi:10.1371/journal.pone.0191075

The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L.) is equivalent to obinutuzumab produced in CHO cells

Jin Won Lee, Woon Heo, Jinu Lee, Narae Jin, Sei Mee Yoon, Ki Youl Park, Eun Yu Kim, Woo Taek Kim, Joo Young Kim

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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.

Original language	English
Article number	e0191075
Journal	PloS one
Volume	13
Issue number	1
DOIs	https://doi.org/10.1371/journal.pone.0191075
Publication status	Published - 2018 Jan

Bibliographical note

Funding Information:
This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2013M3A9A9050574 to W.T.K. and NFR-2017R1A2B4010319 to J.Y.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2013M3A9A9050574 to W.T.K. and NFR-2017R1A2B4010319 to J.Y.K.

Publisher Copyright:
© 2018 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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title = "The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L.) is equivalent to obinutuzumab produced in CHO cells",

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.",

author = "Lee, {Jin Won} and Woon Heo and Jinu Lee and Narae Jin and Yoon, {Sei Mee} and Park, {Ki Youl} and Kim, {Eun Yu} and Kim, {Woo Taek} and Kim, {Joo Young}",

note = "Funding Information: This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2013M3A9A9050574 to W.T.K. and NFR-2017R1A2B4010319 to J.Y.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2013M3A9A9050574 to W.T.K. and NFR-2017R1A2B4010319 to J.Y.K. Publisher Copyright: {\textcopyright} 2018 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

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AU - Heo, Woon

AU - Lee, Jinu

AU - Jin, Narae

AU - Yoon, Sei Mee

AU - Park, Ki Youl

AU - Kim, Eun Yu

AU - Kim, Woo Taek

AU - Kim, Joo Young

N1 - Funding Information: This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2013M3A9A9050574 to W.T.K. and NFR-2017R1A2B4010319 to J.Y.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2013M3A9A9050574 to W.T.K. and NFR-2017R1A2B4010319 to J.Y.K. Publisher Copyright: © 2018 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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N2 - 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.

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The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L.) is equivalent to obinutuzumab produced in CHO cells

Abstract

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