Optimized pipeline for generating highly potent neutralizing Affitins: Application to SARS-CoV-2 spike protein

Viral infections remain a major health threat, causing approximately three million deaths annually. While most antiviral drugs are chemical molecules targeting viral replication or entry, protein-based therapies such as monoclonal antibodies (mAbs) offer high neutralizing activities and immune system engagement. As alternatives or complements to mAbs, Affitins - small (7 kDa) highly stable artificial affinity proteins derived from hyperthermophilic archaea - have emerged as a promising scaffold. Selected from high-diversity libraries (∼10¹² variants), they are easy to engineer and cost-effective to produce. Here, we developed a pipeline using ribosome display to generate Affitins in different multimerization formats targeting the SARS-CoV-2 spike protein. The most potent constructs, engineered as dimers or trimers (17-27 kDa), demonstrated potent neutralization across several SARS-CoV-2 variants, with IC₅₀ values as low as 39 pM, high affinity (22.8 pM) and high thermal stability (84.7 °C). We also developed the first hexameric Affitins (∼106 kDa), by Fc-mediated dimerization of trimers, which achieved an excellent neutralization efficacy (IC₅₀ = 0.8 pM) ranking them among the most potent B1.351 SARS-Cov-2 neutralizing proteins. These results position Affitins as versatile and promising antiviral agents, capable of forming high-valency constructs with smaller size than antibodies, expanding our arsenal against viral threats.