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.
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Data availability
Data are available upon reasonable request from the corresponding authors. Publicly available datasets used in this study include Km Plotter https://kmplot.com/, GSE16152927, and GSE21061628.
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Acknowledgements
The authors acknowledge support by the Biotechnology and Biological Sciences Research Council (BB/T008709/1, BB/Y002180/1), Breast Cancer Now (147; KCL-BCN-Q3), the British Skin Foundation (006/R/22), Cancer Research UK City of London Centre (C7893/A31530), Worldwide Cancer Research (24-0087). This research was supported by the King’s Health Partners Centre for Translational Medicine. The views expressed are those of the author(s) and not necessarily those of King’s Health Partners. We thank the Nikon Imaging Centre at King’s College London for help with light microscopy. The authors acknowledge the Advanced Cytometry Platform team, located in the R&D Department, Guy’s and St Thomas’ NHS Foundation Trust, Guy's Hospital, London SE1 9RT. We thank Roman Laddach for his guidance on the analysis of the transcriptomic data.
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S.N.K. and D.E.T. designed and conceived the study. B.E., Y.L., A.M.C., A.C., and S.N.K. developed the methodology. B.E., Y.L., A.M.C., K.S., N.Ł., P.R.C., K.M.I., B.N.L., E.S., J.T., T.B., N.W., M.G., L.C.G.F.P., A.M., S.C., A.J., P.G., V.C., J.R.B., D.E.T., and S.N.K. acquired the data or helped with the data analysis. B.E., Y.L., A.C., A.M.C., S.N.K., and D.E.T. discussed and interpreted the data, and wrote and edited the manuscript. A.M.C., S.I., A.G., P.G., S.T., V.C., J.R.B., A.N.J.T., A.C., D.E.T., and S.N.K. were responsible for supervising data acquisition and for securing funding. S.N.K. and D.E.T. led and oversaw the study. All authors reviewed the manuscript and approved the submitted version. All authors agree to all manuscript contents, the author list and its order, and the author contribution statements.
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S.N.K. is the founder and shareholder of Epsilogen Ltd. S.N.K., A.M.C., and A.C. declare patents on antibodies for cancer. D.E.T. is a co-founder of Pheon Therapeutics Ltd. The authors declare no pending patents. The other authors do not declare any competing interests.
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Esapa, B., Liu, Y., Chenoweth, A.M. et al. An antibody-drug conjugate designed through clone and isotype selection restricts the growth of CSPG4-expressing triple-negative breast cancer. npj Precis. Onc. (2026). https://doi.org/10.1038/s41698-026-01341-0
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DOI: https://doi.org/10.1038/s41698-026-01341-0
