Site-directed multivalent conjugation of antibodies to ubiquitinated payloads

Antibody conjugates are the foundation of a wide range of diagnostic and therapeutic applications. Although many antibody-conjugation techniques are robust and efficient, obtaining homogeneous multimeric conjugation products remains challenging. Here we report a modular and versatile technique for the site-directed multivalent conjugation of antibodies via the small-protein ubiquitin. Specifically, multiple ubiquitin fusions with antibodies, antibody fragments, nanobodies, peptides or small molecules such as fluorescent dyes can be conjugated to antibodies and nanobodies within 30 min. The technique, which we named 'ubi-tagging', allowed us to efficiently generate a bispecific T-cell engager as well as nanobodies conjugated to dendritic-cell-targeted antigens that led to potent T-cell responses. Using both recombinant ubi-tagged proteins and synthetic ubiquitin derivatives allows for the iterative, site-directed and multivalent conjugation of antibodies and nanobodies to a plethora of molecular moieties.