Rational Design of a Therapeutic Nanobody for the Direct Inhibition of the KRAS Oncoprotein

Abstract The KRAS oncoprotein remains a therapeutic challenge due to the limitations of current covalent inhibitors. This work presents the rational design of PIAKRASv2-Nb, a 100% humanized nanobody generated via the Protein Interaction Architect (PIA) method, which binds the DEYDPTIEDS epitope in the Switch I region of KRAS with exceptional affinity (ipTM = 0.78). Unlike classical approaches, the VHH scaffold of PIA-KRASv2-Nb emerged intrinsically humanized (VH3 family, Hu-mAb score = 1.0), eliminating the need for a posteriori engineering. This study demonstrates that the PIA method can generate therapeutically optimal nanobodies ab initio, combining high affinity, intrinsic humanization, and conformational reproducibility. The results position PIA-KRASv2-Nb (seed 72) as a leading candidate for the direct inhibition of KRAS.