Advances in nanobody multimerization and multispecificity: from in vivo assembly to in vitro production.

Abstract

NANOBODIES® (Nbs) have emerged as valuable tools across therapeutic, diagnostic, and industrial applications owing to their small size and consequent ability to bind unique epitopes inaccessible to conventional antibodies. While Nbs retrieved from immune libraries normally possess sufficient affinity and specificity for their cognate antigens in the practical use case, their multimerization will often increase functional affinity via avidity effects. Therefore, to rescue binding affinity and broaden targeting specificities, recent efforts have focused on conjugating multiple Nb clones - of identical or unique antigen cognates - together. In vivo and in vitro approaches, including flexible linkers, antibody domains, self-assembling coiled coils, chemical conjugation, and self-clustering hydrophobic sequences, have been employed to produce multivalent and multispecific Nb constructs. Examples of successful Nb multimerization are diverse, ranging from immunoassaying reagents to virus-neutralizing moieties. This review aims to recapitulate the in vivo and in vitro modalities to produce multivalent and multispecific Nbs while highlighting the applications, advantages, and drawbacks tied to each method.