Abstract / Summary
Abstract Background Antibody–drug conjugates (ADCs) have transformed the treatment of breast cancer (BC), yet their efficacy remains constrained by heterogeneous target expression and the emergence of resistance. We hypothesized that simultaneous targeting of complementary tumor-associated antigens could broaden tumor-cell coverage and overcome antigen-driven therapeutic escape. Methods We investigated combinations of the HER2-directed ADC trastuzumab deruxtecan (T-DXd) with the TROP2-directed ADCs datopotamab deruxtecan (Dato-DXd) or sacituzumab govitecan (S-Gov) in three BC cell lines using matrix-based drug interaction analyses. To define the biological rationale for dual targeting, we characterized HER2 and TROP2 expression and coexpression across 13,664 primary BC tumors using bulk transcriptomic data and at single-cell resolution in 206,347 cancer cells from 112 primary BCs. Protein-level expression and spatial organization were further assessed by multiplex immunofluorescence in eight invasive BC specimens. Results T-DXd combined with either Dato-DXd or S-Gov demonstrated synergistic antiproliferative activity across all BC cell lines, including at clinically achievable concentrations. In primary tumors, HER2 and TROP2 expression was complementary, with 47% of tumors expressing at least one target compared with 34% for TROP2 alone. Single-cell analysis revealed marked intratumoral heterogeneity, with HER2-only, TROP2-only, double-positive, and double-negative cancer-cell populations frequently coexisting within individual tumors. Dual targeting increased the proportion of theoretically targetable cancer cells by a median of 13% across individual tumors, with substantial interpatient variability. Multiplex immunofluorescence confirmed these distinct cellular phenotypes at the protein level and revealed heterogeneous spatial organization, including enriched niches and intermixed heterotypic patterns. Conclusions HER2 and TROP2 represent complementary therapeutic vulnerabilities within heterogeneous BC ecosystems. Dual HER2/TROP2 targeting may increase both the breadth of tumor-cell coverage and, in double-positive cells, the intensity of ADC-mediated payload delivery. These findings provide a rationale for biomarker-driven ADC combinations based on quantitative single-cell target complementarity and spatial organization rather than conventional binary tumor-level classification. Prospective clinical studies will be required to determine whether this approach can translate into improved therapeutic efficacy and overcome antigen-driven resistance.