title: An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.
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.
authors.affiliation: Département de phytologie, Université Laval, Québec Quebec, Canada.
authors.forename: Philippe V
authors.initials: PV
authors.lastname: Jutras
authors.affiliation: Laboratory of Biotechnology ENEA Research Center, Casaccia, Rome, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Marusic
authors.affiliation: Laboratory of Biotechnology ENEA Research Center, Casaccia, Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Lonoce
authors.affiliation: Département de phytologie, Université Laval, Québec Quebec, Canada.
authors.forename: Carole
authors.initials: C
authors.lastname: Deflers
authors.affiliation: Département de phytologie, Université Laval, Québec Quebec, Canada.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Goulet
authors.affiliation: Laboratory of Biotechnology ENEA Research Center, Casaccia, Rome, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Benvenuto
authors.affiliation: Département de phytologie, Université Laval, Québec Quebec, Canada.
authors.forename: Dominique
authors.identifier: http://orcid.org/0000-0002-5177-1142
authors.initials: D
authors.lastname: Michaud
authors.affiliation: Laboratory of Biotechnology ENEA Research Center, Casaccia, Rome, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Donini
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D015891:Cystatins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism; D018551:Solanum lycopersicum / Q000378:metabolism; D009369:Neoplasms / Q000276:immunology / Q000628:therapy*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D018515:Plant Leaves / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000514:alpha 1-Antichymotrypsin / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D000514:alpha 1-Antichymotrypsin; D010447:Peptide Hydrolases