Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor–positive Breast Cancer

Abstract Estrogen receptor–positive (ER+) breast cancer is not considered immunogenic and, to date, has been proven resistant to immunotherapy. Endocrine therapy remains the cornerstone of treatment for ER+ breast cancers. However, constitutively activating mutations in the estrogen receptor alpha (ESR1) gene can emerge during treatment, rendering tumors resistant to endocrine therapy. Although these mutations represent a pathway of resistance, they also represent a potential source of neoepitopes that can be targeted by immunotherapy. In this study, we investigated ESR1 mutations as novel targets for breast cancer immunotherapy. Using machine learning algorithms, we identified ESR1-derived peptides predicted to form stable complexes with HLA-A*0201. We then validated the binding affinity and stability of the top predicted peptides through in vitro binding and dissociation assays and showed that these peptides bind HLA-A*0201 with high affinity and stability. Using tetramer assays, we confirmed the presence and expansion potential of antigen-specific CTLs from healthy female donors. Finally, using in vitro cytotoxicity assays, we showed the lysis of peptide-pulsed targets and breast cancer cells expressing common ESR1 mutations by expanded antigen-specific CTLs. Ultimately, we identified five peptides derived from the three most common ESR1 mutations (D538G, Y537S, and E380Q) and their associated wild-type peptides, which were the most immunogenic. Overall, these data confirm the immunogenicity of epitopes derived from ESR1 and highlight the potential of these peptides to be targeted by novel immunotherapy strategies. Significance: Estrogen receptor (ESR1) mutations have emerged as a key factor in endocrine therapy resistance. We identified and validated five novel, immunogenic ESR1-derived peptides that could be targeted through vaccine-based immunotherapy.