{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Charite University Medicine Berlin, Institute of Pathology, Berlin, Germany"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 23,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "V. Seitz"
    },
    {
      "name": "J. Franzen"
    },
    {
      "name": "K. Gennermann"
    },
    {
      "name": "B. Hirsch"
    },
    {
      "name": "S. Schaper"
    },
    {
      "name": "A. Droege"
    },
    {
      "name": "M. Dreger"
    },
    {
      "name": "N. Genzel"
    },
    {
      "name": "S. Plassmann"
    },
    {
      "name": "D. Bents"
    },
    {
      "name": "J. Gloekler"
    },
    {
      "name": "T. Conrad"
    },
    {
      "name": "C. Loddenkemper"
    },
    {
      "name": "K. Schmelz"
    },
    {
      "name": "M. Morkel"
    },
    {
      "name": "A. Trinks"
    },
    {
      "name": "D. Horst"
    },
    {
      "name": "T. G. Krieger"
    },
    {
      "name": "D. Kainmueller"
    },
    {
      "name": "M. Hummel"
    },
    {
      "name": "S. Hennig"
    },
    {
      "name": "V. Lennerz"
    },
    {
      "name": "S. Elezkurtaj"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/asxe5r54ebh9z.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.18.750832",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.18.750832v1.full",
  "id": "asxe5r54ebh9z",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.18.750832v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "immunology"
    ],
    [
      "openalex",
      "2730|Cancer Immunotherapy and Biomarkers"
    ]
  ],
  "openalex": "W7214206248",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.18.750832v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-18",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/asxe5r54ebh9z.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:24:23Z",
      "native_id": "10.64898/2026.09.18.750832",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.18.750832v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:immunology",
      "subfield": "immunology"
    }
  ],
  "title": "Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs",
  "title_text": "Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs",
  "type": "preprint",
  "updated_at": "2026-10-30T12:24:23Z",
  "url": "https://research.draconitic.com/paper/asxe5r54ebh9z",
  "venue": "bioRxiv",
  "versions": []
}