Triple-negative breast cancer (TNBC) has 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. Human leukocyte antigen-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCR) from naïve 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 noncognate epitopes from alternative source proteins. Notably, this TCR-mediated killing of three-dimensional (3D) 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 patients with TNBC.

Significance: Metastatic TNBC has a dismal prognosis. This study discovers Ropporin-1 as a target for T-cell therapy for most patients. The selected TCR is highly specific and sensitive in advanced models, and preclinical testing shows that the T-cell product expressing this TCR, manufactured according to good manufacturing practice, has favorable safety and potency.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License .
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Supplementary data

Supplementary methods

This file contains supplementary methods and references.

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Supplementary Table 1

Top-ranked ROPN1/B peptides and their scores for immunogenicity, non-cross reactivity, HLA-A2 binding and immunogenicity.

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Supplementary Table 2

ROPN1/B peptides identified with immunopeptidomics.

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Supplementary Table 3

Overview of healthy cell lines.

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Supplementary Figure 1

Expressions of ROPN1/B, NY-ESO1 and MAGE-A4 target antigens in healthy tissues, TNBC and other tumor types.

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Supplementary Figure 2

Retrieval of ROPN1/B-specific T-cells and TCR genes.

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Supplementary Figure 3

FLY-1A TCR T-cells recognize melanoma tumoroids.

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Supplementary Figure 4

FLY-1A TCR T-cells do not recognize ROPN1-negative and/or HLA-A2-negative tumor cells.

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Supplementary Figure 5

Extended phenotypes of intra-tumoral and peripheral T-cells following adoptive transfer of FLY-1A TCR T cells.

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Supplementary Figure 6

FLY-1A TCR T-cells recognize patient-derived melanoma cells.

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Supplementary Figure 7

The extent of recognition of the ROPN1 target by FLY-1A TCR T cells correlates with level of expression.

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Supplementary Figure 8

Stepwise approach from retrieval and identification of TCRs to the functional testing of their sensitivity, specificity and reactivity.

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Supplementary Figure 9

Presence of ROPN1 and FLY-1A TCRβ in metastatic TNBC in relation to endogenous T cell phenotype and anti-PD1 response.

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