Studying SARS-CoV-2 interactions using phage-displayed receptor binding domain as a model protein.

SARS-CoV-2 receptor binding domain (RBD) mediates viral entry into human cells through its interaction with angiotensin converting enzyme 2 (ACE2). Most neutralizing antibodies elicited by infection or vaccination target this domain. Such a functional relevance, together with large RBD sequence variability arising during viral spreading, point to the need of exploring the complex landscape of interactions between RBD-derived variants, ACE2 and antibodies. The current work was aimed at developing a simple platform to do so. Biologically active and antigenic Wuhan-Hu-1 RBD, as well as mutated RBD variants found in nature, were successfully displayed on filamentous phages. Mutational scanning confirmed the global plasticity of the receptor binding motif within RBD, highlighted residues playing a critical role in receptor binding, and identified mutations strengthening the interaction. The ability of vaccine-induced antibodies to inhibit ACE2 binding of many mutated RBD variants, albeit at different extents, was shown. Amino acid replacements which could compromise such inhibitory potential were underscored. The expansion of our approach could be the starting point for a large-scale phage-based exploration of diversity within RBD of SARS-CoV-2 and related coronaviruses, useful to understand structure-function relationships, to engineer RBD proteins, and to anticipate changes to watch during viral evolution.

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.