Background Triple negative breast cancer (TNBC) lacks classical targets for hormone and/or antibody therapy, and responses to immune checkpoint inhibitors are rare and generally not sustained. However, the prognostic value of infiltrating CD8 lymphocytes and proven antigenicity of TNBC argue that this disease is amenable to adoptive T cell therapy.

Methods We applied an integrative approach to discover and validate a tumor-restricted intracellular antigen using in silico analyses and laboratory assays such as qRT-PCR and immune-histochemical stainings of large numbers of TNBC- and healthy tissues. We selected immunogenic, non-cross-reactive epitopes derived from it through in silico predictions, immunopeptidomics and in vitro assays. We identified corresponding TCRs and screened them for their specificity using positional amino acid scanning and recognition of tumor- but not healthy cells, as well as for their sensitivity through peptide titrations, killing of patient-derived 3D organoids in vitro and killing of a TNBC cell line in vivo.

Results We identified the target Ropporin (ROPN1), which showed neither gene- nor protein expression in healthy human tissue databases (n=1,709) and over 15 major healthy organs according to qRT-PCR and immune-histochemical (IHC) staining. Notably, this target demonstrated homogenous protein expression in >85% of TNBC patients (n=756 gene expression; n=386 IHC). Epitope predictions and immunopeptidomics using cancer cell lines and tissues enabled identification of 11 HLA-A2-binding epitopes. Epitope-specific T cells were successfully enriched from naïve T cell repertoires for 9 epitopes, which yielded more than 25 clonal TCRs. TCRs directed against 5 epitopes were functionally expressed upon gene transfer into T cells, and TCRs directed against 3 epitopes recognized endogenously processed ROPN1. One TCR (against the FLY-A epitope) demonstrated preferential pairing between the therapeutic TCR alpha and beta chains and harbored a stringent recognition motif according to positional amino acid scanning of the cognate epitope. From an efficacy perspective, this TCR mediated dose-dependent killing of patient-derived TNBC 3D organoids in vitro and a breast cancer cell line in an in vivo murine study. Importantly, in both studies, treatment with FLY-A T cells significantly outperformed the standard of care treatments cisplatin and sacituzumab govitecam.

Conclusions ROPN1 has been identified as a promising target for adoptive T cell therapy for TNBC patients and a specific and active TCR has been selected as the lead candidate for clinical development.

Ethics Approval This study has been approved by the Medical Ethical Committee at Erasmus MC (MEC.02.953, MEC-2020–0090).