An antibody-drug conjugate designed through clone and isotype selection restricts the growth of CSPG4-expressing triple-negative breast cancer

Abstract Antibody–drug conjugates (ADCs) demonstrate therapeutic potential, but aggressive triple-negative breast cancers (TNBCs) require precise target selection and antibody optimisation. We identified chondroitin sulfate proteoglycan 4 (CSPG4) expression in neoadjuvant treatment-resistant TNBC to guide ADC development. Three anti-CSPG4 IgG1 antibodies with distinct variable regions (225.28S, 763.74, and 9.2.27) were engineered and compared. 225.28S IgG1 demonstrated the most efficient internalisation and potent cancer cell cytotoxicity when conjugated to the tubulin inhibitor MMAE. To determine the optimal isotype, we generated 225.28S IgG4 and directly compared it with 225.28S IgG1. The IgG1 isotype showed superior internalisation and killing activity as an MMAE-conjugated ADC. Conjugation of 225.28S IgG1 to the topoisomerase inhibitor DXd produced an ADC with a drug-to-antibody ratio (DAR) of 8. This ADC was capable of robust internalisation into cancer cells and tumour cell cytotoxicity in vitro, and significant growth restriction of two CSPG4-expressing TNBC patient-derived xenografts (PDX) implanted orthotopically in mouse mammary fat pads. Unconjugated 225.28S IgG1 also limited TNBC xenograft growth in immunodeficient mice engrafted with human immune cells, confirming Fc-mediated functional activity. These studies identify 225.28S IgG1 as the optimal clone and isotype, supporting a next-generation DXd-conjugated ADC as a promising therapeutic strategy for hard-to-treat CSPG4-expressing TNBC.