Antibody–Drug Conjugates Prepared with Peptide Asparaginyl Ligases Achieve Strong In Vitro and In Vivo Antitumor Activity

Abstract Antibody–drug conjugates (ADC) are a rapidly expanding class of biopharmaceuticals for cancer-targeted therapy, with 15 ADCs approved for clinical use and more than a hundred candidates in clinical development. ADCs comprise a tumor-specific monoclonal antibody to which a cytotoxic payload is attached via a linker. Approved ADCs are heterogeneous products encompassing a range of drug-to-antibody ratios (DAR) due to the chemical methods employed for conjugation. Achieving precise DAR control is expected to improve product homogeneity, pharmacokinetics, safety, and clinical efficacy. In this study, we developed a rapid and efficient two-component bioconjugation system comprising a recombinant peptide asparaginyl ligase (PAL) and a human glutaminyl cyclase (hQC) to conjugate anti-HER2 antibodies. Trastuzumab and disitamab were conjugated with single or branched payloads, yielding ADCs with DAR values ranging from 2 to 8. These ADCs demonstrated cytotoxicity comparable with their chemically conjugated counterparts, trastuzumab deruxtecan (DXd, ENHERTU) and disitamab vedotin (Aidixi), against HER2-expressing breast cancer cell lines and Patient-Derived Xenograft (PDX) models. Notably, SGZ026, a PAL-conjugated trastuzumab DXd with a DAR of 3.9, exhibited better in vivo mouse plasma stability compared with ENHERTU (DAR 8). Despite carrying approximately half the cytotoxic payload per antibody, a single dose of SGZ026 effectively inhibited tumor growth in breast cancer PDX models with high or intermediate HER2 expression, achieving efficacy comparable with ENHERTU. These findings highlight a novel and robust enzyme-based conjugation technology for developing homogeneous ADCs with an improved therapeutic window.