Design of Cell-Penetrating Domain Antibodies via a Genetically Encoded β-Lactam Amino Acid

Domain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high-affinity protein binders targeting the intracellular proteins implicated in cell signalling. However, it remains a challenge to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined approach combining orthogonal crosslinking afforded by a genetically encoded β-lactam-lysine (BeLaK) and genetic supercharging to generate cell-penetrating monobodies. When introduced to the N-terminal β-strand of a series of supercharged monobodies, BeLaK enabled efficient inter-strand crosslinking with the neighbouring lysine. Compared to its non-crosslinked counterpart, a BeLaK-crosslinked, +18-charged monobody exhibited enhanced thermostability and greater cellular uptake at 40 nM. Moreover, this structurally rigidified, supercharged monobody inhibited ERK1/2 phosphorylation in KYSE-520 esophageal cancer cell line at sub-micromolar concentration, indicating significant endosomal escape after endocytosis. Together, the discovery of this BeLaK-encoded, rigidified immunoglobin fold should facilitate the design of cell-penetrating monobodies targeting intracellular signalling proteins.