Discovery of a Novel Tripeptide Linker that Broadens the Therapeutic Window of Auristatin-Based Antibody–Drug Conjugates by Reducing Bone Marrow Toxicity

Vedotin-based antibody-drug conjugates (ADCs) employing the valine-citrulline (Val-Cit) dipeptide linker and monomethyl auristatin E (MMAE) have achieved substantial clinical success but are frequently limited by dose-limiting hematologic toxicities, including neutropenia. These toxicities are hypothesized to arise, in part, from extracellular cleavage of the Val-Cit linker by neutrophil-derived proteases within the bone marrow microenvironment. Here, we describe the discovery and characterization of a tumor-selective tripeptide linker that improves the therapeutic index of MMAE-based ADCs. Using a high-throughput fluorescent peptide library screened against cancer and bone marrow homogenates, we identified peptide motifs preferentially cleaved in cancer lysates. Incorporation of these motifs into MMAE-containing drug linkers revealed a D-amino-acid-containing tripeptide (DLeu-Ala-Glu) that retained intracellular potency while exhibiting reduced susceptibility to neutrophil elastase and proteinase 3. ADCs bearing this linker demonstrated markedly reduced bone marrow toxicity in rats and cynomolgus monkeys relative to vedotin controls, with improved tolerability at higher doses and preserved antitumor efficacy across multiple xenograft models. Mechanistic studies showed that reduced linker proteolysis and increased linker hydrophilicity both contributed to reduced toxicity relative to vedotin. Together, these data demonstrate that rational peptide linker design integrating protease selectivity and physicochemical properties can substantially improve the safety profile of MMAE-based ADCs without compromising efficacy.