Novel Ligands for IFNg derived from Streptococcal Protein G scaffold


Jawid Ahmad1, Jijngjin Li2, Lada Biedermannová2, Milan Kuchař2, Hana Šípová3, Alena Semerádtová4, Jiří Černý2, Hana Petroková2, Pavel Mikulecký2, Jiří Polínek2, Ondřej Staněk1, Jiří Vondrášek2, Jiří Homola3, Jan Malý4, Radim Osička1, Peter Šebo1,2, and Petr Malý2


1Institute of Microbiology AS CR, v.v.i. and 2Institute of Biotechnology AS CR, v.v.i., Vídeňská 1083, 142 20 Prague; 3Institute of Photonics and Electronics AS CR, v.v.i., Chaberská 57, 182 51, Prague; 4Faculty of Science, Jan Evangelista Purkyně University, České Mládeže 8, 400 96 Ústí nad Labem, Czech Republic


Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and as next generation protein therapeutics. Here we describe high-affinity binders for human interferon gamma (hIFNγ) that were derived from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. On the basis of computational interaction energy mapping, solvent accessibility assessment and in silico alanine scanning analysis, 11 residues from the albumin-binding surface of ABD were selected for randomization, thus generating a combinatorial scaffold library of a theoretical complexity of 1014. Upon two independent campaigns of in vitro ribosome display selection of such scaffolds, high affinity recombinant ligands were obtained that exhibited Kd values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ and in vitro competition for hIFNγ binding between pairs of individual selected ligands or with the human IFNγ receptor 1 (hIFNγR1), revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which was different from the site of hIFNγR1 binding. The novel hIFNγ ligands thus represent a proof of concept for development of novel diagnostic reagents derived from the ABD scaffold.