Evaluation of Antibody-Drug Conjugate Performances Using a Novel HPLC-DAD Method for Tumor-Specific Detection of DM4 and S‑Methyl-DM4

Antibody-drug conjugates (ADCs) are an emerging class of therapeutics that have gained interest in precision medicine for Cancer treatment, combining the targeted delivery capabilities of monoclonal antibodies with the potent cytotoxicity of small-molecule drugs. The goal is to enhance the therapeutic window by maximizing tumor cell killing while minimizing off-target toxicity. Pivotal aims are precise delivery of payload in the tumor site and a robust analytical methodology to quantify the accumulation of it. 1959-sss/DM4, a novel ADC targeting highly glycosylated LGALS3BP, which is a protein implicated in tumor progression and metastasis, was developed. This ADC has shown potent and durable antitumor activity in various preclinical models. A high-performance liquid chromatography (HPLC) method according to ICH guidelines for the simultaneous quantification of DM4 and its primary metabolite, S-methyl-DM4, in tissue matrices was validated. Was employed a patient-derived neuroblastoma cell line (hNB CTRL), which naturally lacks LGALS3BP expression, alongside a counterpart cell line (hNB LGALS3BP) in which LGALS3BP expression was ectopically induced via lentiviral-mediated gene transduction. A second metastatic model using SKNAS neuroblastoma cells, which endogenously express LGALS3BP, was utilized to study the kinetics of the payload. The approach allows for the simultaneous detection of DM4 and S-methyl-DM4, offering a valuable tool for the comparative assessment of ADC performance in preclinical models.