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BackgroundTriple 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.MethodsWe 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.ResultsWe 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.ConclusionsROPN1 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 ApprovalThis study has been approved by the Medical Ethical Committee at Erasmus MC (MEC.02.953, MEC-2020–0090).

Treatment with adoptively transferred T cells is challenged by limited longevity of therapeutic cells within tumors. To enhance the durability of anti-tumor T cell products, we have created T cell receptors (TCRs) with built-in co-stimulatory molecules. We observed that TCRs coupled to ICOS mediated exceptionally long-term responses including delay of tumor recurrence and cures in a mouse tumor model. TCR:ICOS T cells showed enhanced and antigen-specific production of inflammatory cytokines and resistance to exhaustion. Genetic ablation of ICOS-mediated activation of the PI3K-NFκB pathway neutralized the long-term anti-tumor effects. To translate TCR:ICOS to human T cells, we identified a single amino acid change in the cytosolic tail which was necessary for functional surface expression. Notably, the optimized receptor sustained performance of human T cells upon repeated stimulation across multiple antigen specificities. Collectively, we present a novel and uniformly applicable TCR:ICOS format that supports fitter T cell products for adoptive cell therapy. Newly designed co-stimulatory TCR, with extracellular TCR-V and C domains coupled to CD28 transmembrane domain, and ICOS and CD3ε intracellular domains (in short TCR:ICOS) provides: ➢durable anti-tumor response and T cell persistence in mouse model➢inflammatory T cell phenotype and resistance to T cell exhaustion➢effects via PI3K and NFκB activation➢translation to human T cells upon single amino acid mutation in TCR:ICOS tail➢extension to multiple clinically relevant TCRs while preserving prolonged T cell fitness durable anti-tumor response and T cell persistence in mouse model inflammatory T cell phenotype and resistance to T cell exhaustion effects via PI3K and NFκB activation translation to human T cells upon single amino acid mutation in TCR:ICOS tail extension to multiple clinically relevant TCRs while preserving prolonged T cell fitness

Triple-negative breast cancer (TNBC) shows an urgent need for new therapies. We discovered Ropporin-1 (ROPN1) as a target to treat TNBC with T-cells. ROPN1 showed high and homogenous expression in 90% of primary and metastatic TNBC but not in healthy tissues. HLA-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCRs) from naive repertoires. Following gene introduction into T-cells and stringent selection, we retrieved a highly specific TCR directed against the epitope FLYTYIAKV that did not recognize non-cognate epitopes from alternative source proteins. Notably, this TCR mediated killing of three-dimensional tumoroids in vitro and tumor cells in vivo and outperformed standard-of-care drugs. Finally, the T-cell product expressing this TCR and manufactured using a clinical protocol fulfilled standard safety and efficacy assays. Collectively, we have identified and preclinically validated ROPN1 as a target and anti-ROPN1 TCR T-cells as a treatment for the vast majority of TNBC patients.Competing Interest StatementThis research was conducted according to the requirements of objectivity and integrity standards. DH is listed as inventor for European patent application no. P128827EP00. MK has received research support from BMS, Roche, AstraZeneca, personal fees from AstraZeneca, Daiichi Sankyo, Domain Therapeutics, Alderaan, BMS, MSD, Gilead, Roche outside the submitted work (all paid to the NKI/AVL). RD has received research support from MSD and Bayer, personal fees from Bluebird Bio, Genticel, other support from Pan Cancer T B.V. outside the submitted work (all paid to the Erasmus MC Cancer Institute) and is listed as inventor for European patent applications P130556EP00 and P128827EP00. All other authors (DK, MvB, RW, DR, KK, MT, CYL, ATJ, RF, JM, MdB, SIB, JAD, MK, EHJD, MBS, JWM, RJMA) declare no competing interests.

IntroductionThe treatment landscape for de novo metastatic hormone sensitive prostate cancer (mHSPC) is rapidly evolving. With an abundance of available treatment strategies, selecting the optimal strategy for an individual patient is becoming increasingly challenging. TripleAiM1 aims to evaluate the impact of mHSPC treatments on health-related quality of life (HRQoL) and to provide real-world data insights on diagnostics, treatment strategies, patient subgroups and related healthcare expenditure for mHSPC. The aspirational target of TripleAiM1 is that in the near future, a more tailored therapy can be offered based on the individual patient’s wishes and needs in accordance with the overarching principle of value-based healthcare.Methods and analysisWe describe the TripleAiM1 study design; a nationwide registry comprising a retrospective and prospective cohort of patients with de novo mHSPC. Starting in May 2020, eligible patients are identified, selected and recruited in 14 participating hospitals in the Netherlands. Our hypothesis is that, in a real-world setting, differences in clinically meaningful HRQoL deterioration will be observed for treatment strategies over time. HRQoL data, assessed with patient-reported outcome measures, costs and clinical data will be collected for 24 months.For the retrospective cohort, all patients diagnosed with de novo mHSPC from January 2017 onwards are eligible for inclusion. Patient and tumour characteristics, imaging modalities and treatment patterns will be analysed descriptively to provide a real-world overview.Time-to-event endpoints will be assessed using the Kaplan-Meier method and regression models will be employed to analyse baseline characteristics associated with an increased likelihood of death, progression and HRQoL deterioration. Longitudinal mixed-effects models will be employed to assess change of patient-reported outcome scores from baseline until the end of follow-up.Ethics and disseminationEthical approval was obtained from the Medical Research Ethics Committee, Twente. Study results will be published in peer-reviewed journals.Trial registration numberNL9719.