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

Abstract 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.