Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.
An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies.
['Kuglstatter|A|A|', 'Stihle|M|M|', 'Neumann|C|C|', 'Müller|C|C|', 'Schaefer|W|W|', 'Klein|C|C|', 'Benz|J|J|', '|||']
D020816:Amino Acid Motifs D018033:Antibodies, Bispecific D001665:Binding Sites D018360:Crystallography, X-Ray D004220:Disulfides D015870:Gene Expression D005822:Genetic Vectors D006031:Glycosylation D057809:HEK293 Cells D006801:Humans D007141:Immunoglobulin Fc Fragments D007074:Immunoglobulin G D008958:Models, Molecular D009154:Mutation D011485:Protein Binding D000072756:Protein Conformation, alpha-Helical D000072757:Protein Conformation, beta-Strand D015202:Protein Engineering D054730:Protein Interaction Domains and Motifs D055503:Protein Multimerization D055550:Protein Stability D011994:Recombinant Proteins
Knob-into-Hole disulfide bond Fc engineering bispecific antibody protein crystallography