title: Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco.
abstract: Complex multimeric proteins such as dimeric and secretory immunoglobulin A (IgA) can be difficult to produce in heterologous systems, although this has been achieved using several platforms including plants. As well as topical mucosal applications, dimeric IgA (dIgA), and secretory IgA (sIgA) can be used in tumor and anti-viral therapy, where their more potent cell-killing properties may increase their efficacy compared to current drugs based on IgG. However, the development of therapeutic IgA formats is hampered by the need to co-express four different polypeptides, and the inability to purify such molecules using conventional protein A or protein G affinity chromatography. The light chain (LC)-specific affinity ligand protein L is a potential alternative, but it only recognizes certain kappa light chain (LC(κ)) subtypes. To overcome these limitations, we have adapted a framework-grafting approach to introduce LCs that bind protein L into any IgA. As a model, we used the chimeric anti-human chorionic gonadotropin (hCG) antibody cPIPP, since this contains a murine LC((κ)) subtype that does not bind protein L. Grafting was achieved by replacing selected framework region 1 (FR1) residues in the cPIPP LC(κ) variable domain with corresponding residues from LC(κ) subtypes that can bind protein L. The grafted antibody variants were successfully purified by protein L affinity chromatography. These modifications affected neither their antigen-binding properties nor the yields achieved by transient expression in tobacco plants. Our results therefore show that LC FR1 grafting can be used as generic strategy for the purification of IgA molecules.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Boes
authors.forename: H
authors.initials: H
authors.lastname: Spiegel
authors.forename: H
authors.initials: H
authors.lastname: Delbrück
authors.forename: R
authors.initials: R
authors.lastname: Fischer
authors.forename: S
authors.initials: S
authors.lastname: Schillberg
authors.forename: M
authors.initials: M
authors.lastname: Sack
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007070:Immunoglobulin A / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013678:Technology, Pharmaceutical / Q000379:methods; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D007070:Immunoglobulin A; D011994:Recombinant Proteins