Modular dimerization of nanobodies via a bioorthogonal proximity-driven reaction

In recent years, nanobodies, the smallest-known functional antibodies, have gained considerable attention for their unique characteristics, including small size, high antigen-binding affinity, remarkable stability under harsh conditions, and cost-effective production, making them ideal candidates for a wide range of biomedical applications.1 Bispecific nanobodies represent a promising frontier in therapeutic development, with multiple candidates currently in clinical trials, due to their ability to target distinct epitopes increasing their functional affinity via avidity effects.2 Considering both in vivo and in vitro approaches, achieving nanobody heterodimerization with precise structural control and high functionality remains a significant challenge due to risk of molecular heterogeneity and unwanted immunogenicity.3 To address this challenge, we employed a recently discovered proximity-induced ligation between a 1,4 diketone (DOP) and an α-nucleophile (α-Nu), which proceeds without the need of external stimuli, with high specificity and without off-target reactivity.5,6 Its effectiveness was also confirmed in complex reaction media, such as cell lysates, allowing its application to macrobiomolecule ligation. To achieve the desired proximity for nanobody dimerization, each protein is site-selectively conjugated6 to a peptide nucleic acid (PNA) strand bearing one of two complementary reactive moieties (Fig.1). The PNA acts both as a molecular ruler, spacing the nanobodies at programmable distance, and as a molecular barcode, guiding the recognition between complementary strands and enabling the bioorthogonal reaction. Here, we will present preliminary results regarding this novel chemical strategy for inducing selective, covalent, and “on-demand” heterodimerization of nanobodies.