Nanobody assemblies with fully flexible topology enabled by genetically encoded tetrazine amino acids

Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups-site-specifically inserted by genetic code expansion at Nb surface sites-are compatible with Nb folding and function, enabling Nb assembly at any desired point. Using two anti-SARS-CoV-2 Nbs with viral neutralization ability, we created Nb homo- and heterodimers with improved properties compared with conventionally linked Nb homodimers, which, in the case of our tetrazine-conjugated trimer, translated into enhanced viral neutralization. Thus, this tetrazine-based approach is a generally applicable strategy that greatly increases the accessible range of Nb assembly topologies, and thereby adds the optimization of topology as an effective avenue to generate Nb assemblies with improved efficacy.