Evaluation of nanobodies engineered with extra-cysteines for site-specific functionalization

Antibody labeling represents a critical step for the preparation of application-specific immunoreagents because the process can affect the binder functionality and result in a highly heterogeneous sample. Therefore, site-specific derivatization methods are preferable, and the addition of a cysteine is a common solution because its thiol group enables the reaction with several commercially available reagents. We first showed that the addition of an extra cysteine did not decrease the yield and functionality of nanobodies, indicating that it did not interfere significantly with the natural disulfide bond present in such macromolecules. The major limitation of single amino acid labeling is that the signal might be weak and compromise the sensitivity. Consequently, we evaluated the possibility of modifying a nanobody by introducing multiple cysteines into its sequence. The resulting variants were assessed for their labeling homogeneity and performance as reagents for flow and mass cytometry. The experimental data indicated that multiple cysteines favored the formation of dimers and resulted in multiple degrees of functionalization. However, the worst consequence was that their efficiency as reagents was significantly lower than that of nanobodies possessing a single extra cysteine.