Interaction of plant-produced antibodies with neonatal Fc receptor Szymon Stelter, Muriel Bardor and Julian Ma, 27/05/2016

Introduction Neonatal Fc Receptor provides long IgG serum half-life FcRn in antibody-based therapy extends therapeutic/protective effect lower and less frequent dosing reduced cost of antibody therapy Roopenian et al. (2007) Nat Rev Immunol

Introduction Structural interaction of IgG with FcRn FcRn – lgG binding is independent of human glycosylation; Unusual glycoforms on recombinant antibodies might have an indirect impact on the binding - Jefferis, R. (2009) Nat Rev Drug Discov, Some plant-made antibodies are cleared out faster than the variants made in mammalian cells (Rosenberg 2015, Triguero 2011, Ko 2003) Some have similar clearance rate (Hiatt 2014, Rodriguez 2013, Lee 2013, Gasdanska 2012, Khoudi 1999) Does plant glycosylation affect binding of IgG to FcRn? No studies of plant-made IgG interacting with FcRn

Results Binding of different plant antibody glycovariants to FcRn VRC01HEK original VRC01 from HEK cells VRC01WT plant VRC01 VRC01ΔXF plant VRC01 without fucose and xylose VRC01Gal VRC01 ΔXF extended with galactose VRC01aglyco plant VRC01 without Fc glycan

Results Affinity of different plant antibody glycovariants to FcRn VRC01HEK original VRC01 from HEK cells VRC01WT plant VRC01 VRC01ΔXF plant VRC01 without fucose and xylose VRC01Gal VRC01 ΔXF extended with galactose VRC01aglyco plant VRC01 without Fc glycan

Discussion Antibody oxidation affects FcRn bindnig Met252 Met428 we found that substituting a more hydrophobic residue Leu at Met428 position resulted in a mutant that has a stronger binding to FcRn Met252 oxidation impairs FcRn binding more profoundly than Met428 oxidation IgG Fc Gaza-Bulseco (2008) J Chrom B Gao, X., et al. (2015), J Pharm Sci,

Results Oxidation of plant-made VRC01 glycovariants DTLM252ISR in collaboration with Prof. Muriel Bardor, Université de Rouen, France

Results Oxidation of plant-made VRC01 glycovariants Oxidation, not glycosylation, affects binding of plant-made antibodies to FcRn

ACKNOWLEDGEMENTS St George’s University of London, UK Prof. Julian Ma Dr Rachel Allen Dr Matthew Paul Glyco-Mev Laboratory, University of Rouen, France Prof. Muriel Bardor Prof. Patrice Lerouge Jessica Vanier University of Natural Resources and Life Sciences, Vienna Prof. Herta Steinkellner Dr Alexandra De Vitoria Castilho Prof. Friedrich Altmann Daniel Marsch