Design of Cell-Penetrating Monobodies via Genetic Supercharging and Orthogonal Crosslinking

Domain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high-affinity binders targeting the intracellular proteins implicated in cell signaling. However, it remains challenging to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined strategy combining orthogonal crosslinking mediated by a genetically encoded beta-lactam-lysine (BeLaK) and genetic supercharging to generate cell-penetrating monobodies. When BeLaK was introduced site-specifically to the N-terminal beta-strand of a panel of supercharged monobodies, it enabled efficient interstrand crosslinking with a nearby lysine, generating the rigidified analogs. Compared to the non-crosslinked counterparts, the BeLaK-crosslinked supercharged monobodies exhibited higher thermostability and enhanced cellular uptake at concentrations as low as 40 nM. Most significantly, a +11 charged, orthogonally crosslinked monobody showed significant endosomal escape after endocytosis. The discovery of this stabilized immunoglobin fold should facilitate the design of cell-permeable domain antibodies for targeting intracellular proteins.