Abstract
The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.
MeSH terms
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Amino Acid Sequence
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Antibodies, Monoclonal / immunology
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Antibodies, Monoclonal / metabolism*
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Cystatins / pharmacology
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Cysteine Proteinase Inhibitors / pharmacology*
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Humans
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Immunoglobulin G / immunology*
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Immunoglobulin G / metabolism
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Neoplasms / immunology
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Neoplasms / therapy*
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Nicotiana / drug effects
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Nicotiana / genetics
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Nicotiana / immunology
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Nicotiana / metabolism*
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Peptide Hydrolases / chemistry
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Peptide Hydrolases / metabolism
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Plant Leaves / drug effects
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Plant Leaves / genetics
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Plant Leaves / immunology
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Plant Leaves / metabolism
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Plants, Genetically Modified / drug effects
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Plants, Genetically Modified / genetics
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Plants, Genetically Modified / immunology
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Plants, Genetically Modified / metabolism*
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Proteolysis
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Recombinant Proteins / immunology
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Recombinant Proteins / metabolism
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Solanum lycopersicum / metabolism
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alpha 1-Antichymotrypsin / pharmacology
Substances
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Antibodies, Monoclonal
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Cystatins
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Cysteine Proteinase Inhibitors
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Immunoglobulin G
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Recombinant Proteins
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alpha 1-Antichymotrypsin
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Peptide Hydrolases
Grants and funding
This work was supported by a Great Relevance collaborative grant from the Italian Ministry of Foreign Affairs, ‘Direzione Generale per la Promozione del Sistema Paese, Unità per la Cooperazione Scientifica e Tecnologica bilaterale e multilateral’ to EB and MD, and by a Discovery grant from the Natural Science and Engineering Research Council (NSERC) of Canada to DM. PVJ was the recipient of a graduate scholarship from the AgroPhytoSciences NSERC–CREATE network and a BMP graduate scholarship funded by NSERC, the Fonds de Recherche Québec Nature et Technologies and Medicago inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.