Self-Immolative Nanobody-Cysteine Residue Modification for Controlled Immunodrug Delivery

Applications of antibody-drug conjugates are rapidly growing, however, arduous fabrication of antibodies and impairment of highly potent drugs by covalent fixation to the protein is urging for alternatives to these conventional strategies. Here, a procedure on genetically engineered single domain antibodies, so-called nanobodies, is demonstrated for their site-specific reversible bioconjugation using self-immolative linkers (SILs). Straight-forward fluorescent labelling at their C-terminal cysteine can be reversed under reductive conditions due to its disulfide-containing SIL. Flow cytometry and microscopy images demonstrate cellular uptake and confirm the integrity of the nanobodies' biological affinity notwithstanding being modified. Following this strategy, a potent small molecular immunomodulator can be installed and its stimulatory effect on a cellular level is boosted in vitro compared to non-degradable alternatives. Furthermore, this protocol is extended to further therapeutically relevant representatives of nanobodies, underlining the versatility of this reversible reductive-responsive bioconjugation for a broad field of applications.