Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs
- 1Charite University Medicine Berlin, Institute of Pathology, Berlin, Germany
Abstract
Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.
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BibTeX
@misc{seitz2026tumorversusnontumor,
title = {{Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs}},
author = {V. Seitz and J. Franzen and K. Gennermann and B. Hirsch and S. Schaper and A. Droege and M. Dreger and N. Genzel and S. Plassmann and D. Bents and J. Gloekler and T. Conrad and C. Loddenkemper and K. Schmelz and M. Morkel and A. Trinks and D. Horst and T. G. Krieger and D. Kainmueller and M. Hummel and S. Hennig and V. Lennerz and S. Elezkurtaj},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.18.750832},
url = {https://www.biorxiv.org/content/10.64898/2026.09.18.750832v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs
AU - V. Seitz
AU - J. Franzen
AU - K. Gennermann
AU - B. Hirsch
AU - S. Schaper
AU - A. Droege
AU - M. Dreger
AU - N. Genzel
AU - S. Plassmann
AU - D. Bents
AU - J. Gloekler
AU - T. Conrad
AU - C. Loddenkemper
AU - K. Schmelz
AU - M. Morkel
AU - A. Trinks
AU - D. Horst
AU - T. G. Krieger
AU - D. Kainmueller
AU - M. Hummel
AU - S. Hennig
AU - V. Lennerz
AU - S. Elezkurtaj
PY - 2026
DA - 2026/09/18
PB - bioRxiv
DO - 10.64898/2026.09.18.750832
UR - https://www.biorxiv.org/content/10.64898/2026.09.18.750832v1
AB - Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.
ER -