Multimerization and Conjugation to Antiviral and Cytotoxic Payloads Enhance the Activity of Virus-Neutralizing Nanobodies In Vitro.

The emergence and rapid evolution of novel viral pathogens have challenged conventional antibody-based therapeutics, driving the need for versatile strategies with robust and broadly neutralizing antiviral potential. Nanobodies, compact antibody fragments derived from camelid heavy-chain-only antibodies, represent a promising platform for engineering therapeutics with enhanced antiviral capabilities. We explored targeted modifications of three high-affinity SARS-CoV-2 spike-binding nanobodies raised against the Wuhan variant (1H3, 2F8, and 1D10) through oligomerization and conjugation with cytotoxic and antiviral payloads. We evaluated the impact of these modifications on neutralizing or antiviral activities against the Omicron variant (B.1.1.529 BA.1). In addition to increasing molecular weight, which could extend the serum half-life, PEG-mediated tetramerization of the nanobodies enhanced their affinity, and Fc fusion increased neutralizing potency. Importantly, Sortase A-mediated conjugation of nanobodies with ARVI11, a 1,3-diaza-2-oxophenoxazine derivative exhibiting both anti-SARS-CoV-2 activity and cytotoxicity toward infected host cells, significantly enhanced antiviral efficacy, with the effect being dependent on the type of linker used.