Dolastatin 10

Therapeutic antibodies represent clinically validated binding interfaces that remain largely unexplored as sources of targeting peptides. Here, we employed a structure-guided approach to extract and optimize an EGFR-binding motif from the anti-EGFR monoclonal antibody necitumumab and transformed it into a peptide-drug conjugate for targeted treatment of EGFR-overexpressed non-small-cell lung cancer (NSCLC). Although necitumumab is an FDA-approved therapeutic for EGFR-positive NSCLC, its clinical effectiveness is constrained by limited antitumor efficacy, inadequate penetration into solid tumors, the development of resistance, and clinically significant on-target toxicities. Like other monoclonal antibodies, necitumumab is a large protein biomolecule whose receptor-binding activity is mediated by a relatively small structural interface, providing an opportunity to re-engineer this functional region into a minimal peptide scaffold. We hypothesized that structure-guided extraction of antibody paratopes could provide a rational route for peptide ligand discovery, retaining receptor specificity while improving tumor penetration and eliminating Fc-driven toxicity. Using the resolved necitumumab Fab-EGFR co-crystal structure, we extracted the primary receptor-contact segment from the antibody paratope by implementing protein-protein interaction analysis and re-engineered it through systematic in silico mutation into an optimized peptide scaffold using MD and MM/GBSA combination protocol. A novel peptide scaffold PE3 (YIYYSFSQDYWP) was identified as the most advantageous carrier, showing favorable affinity toward EGFR (∆G = -66.12 kcal/mol) and subsequently validated to exhibit potent cellular uptake. To evaluate its therapeutic potential, PE3 was converted into a peptide-drug conjugate (PDC) through conjugation with Ahx-DA1, a metabolically stable analogue of dolastatin-10, yielding the conjugate PE3-DA1with IC₅₀ 0.05 ± 0.01 μM against H1975 NSCLC cell line. PE3-DA1 retained potent and selective antiproliferative activity against EGFR-positive cells. In the NSCLC xenograft model, PE3-DA1 induced complete tumor regression without evidence of off-target toxicity. This study demonstrates for the first time that a defined EGFR-binding interface within the necitumumab antibody can be reduced to an optimized peptide scaffold while preserving its targeting function and subsequently exploited as a PDC carrier to achieve selective antitumor activity against EGFR-overexpressing NSCLC.