{
  "abstract": "Background Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, and emerging T cell engager (TCE) immunotherapies require predictive preclinical models that capture patient-specific tumour biology and tumour-immune interactions. Although patient-derived organoid (PDO)-immune co-culture systems show promise for evaluating immunotherapy responses, many rely on matrix-embedded cultures that limit scalability and reproducibility. We therefore developed and validated a low-matrix organoid-T cell co-culture platform for functional assessment of TCE activity in patient-derived CRC models. Methods Patient-derived CRC organoids representing diverse molecular and genetic backgrounds were co-cultured with activated allogeneic CD3+ T cells in a suspension low-matrix format. Matrix concentration, medium composition, T cell activation status, and assay duration were optimized. Tumour killing, T cells activation, cytokine secretion, apoptosis, and motility were assessed using flow cytometry, live-cell imaging, cytokine profiling, and immunofluorescence. The platform was evaluated using EGFR- and HER2-targeting bispecific T cell engagers across 21 CRC organoid models and multiple healthy donor-derived T cell populations. Results Optimization identified 1% matrix and a 1:1 organoid: T cell medium that preserved organoid integrity while maintaining T cell viability, activation, and motility. The final workflow enabled reproducible co-culture of 5-day mature organoids with 7-day activated T cells for 72 hours. EGFR-targeting TCEs induced tumour killing, T cell activation, and IFN-{gamma} secretion across donor-organoid combinations. Screening of 21 CRC organoid models revealed substantial inter-patient heterogeneity, with 12 models maintaining [≥]50% baseline viability and supporting functional TCE evaluation. EGFR- and HER2-targeting TCEs produced potent dose-dependent cytotoxicity, with IC50 values ranging from 0.0188 to 54.77 nM across responsive models and up to 75%-85% tumour killing in the most sensitive organoids. These responses were accompanied by increased effector cytokine secretion. Real-time imaging confirmed dynamic T cell engagement and apoptosis-driven organoid destruction following TCE treatment. Conclusions We establish a robust, scalable, human-relevant low-matrix organoid-T cell co-culture platform for functional screening of T cell engagers in patient-derived CRC models. By enabling integrated assessment of tumour killing, immune activation, cytokine responses, and tumour-immune interactions while preserving inter-patient heterogeneity, this system provides a translational framework for immunotherapy development. The platform may support candidate prioritisation, biomarker discovery, patient stratification, and preclinical evaluation of immune-engaging therapeutics.",
  "abstract_shown": true,
  "affiliations": [
    "University of Birmingham, UK"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 16,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "C. M. A. PINNA"
    },
    {
      "name": "K. Rakhimova"
    },
    {
      "name": "J. Zhang"
    },
    {
      "name": "L. Bills"
    },
    {
      "name": "L. Tee"
    },
    {
      "name": "C. Blanc"
    },
    {
      "name": "D. G. Millar"
    },
    {
      "name": "E. Rottoli"
    },
    {
      "name": "O. Huhn"
    },
    {
      "name": "M. Gaspar"
    },
    {
      "name": "N. Lal"
    },
    {
      "name": "R. Lara"
    },
    {
      "name": "K. A. Marley"
    },
    {
      "name": "S. Cemerski"
    },
    {
      "name": "S. Dovedi"
    },
    {
      "name": "A. D. Beggs"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/cs4fy728t4mem.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.21.751493",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.21.751493v1.full",
  "id": "cs4fy728t4mem",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.21.751493v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "cancer biology"
    ],
    [
      "openalex",
      "2730|Cancer Immunotherapy and Biomarkers"
    ]
  ],
  "openalex": "W7214614276",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.21.751493v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-21",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/cs4fy728t4mem.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:24:44Z",
      "native_id": "10.64898/2026.09.21.751493",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.21.751493v1"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "biorxiv:cancer biology",
      "subfield": "oncology"
    }
  ],
  "title": "A low-matrix organoid-T cell co-culture platform for functional evaluation of T cell engagers in patient-derived colorectal cancer organoids",
  "title_text": "A low-matrix organoid-T cell co-culture platform for functional evaluation of T cell engagers in patient-derived colorectal cancer organoids",
  "type": "preprint",
  "updated_at": "2026-10-30T12:24:44Z",
  "url": "https://research.draconitic.com/paper/cs4fy728t4mem",
  "venue": "bioRxiv",
  "versions": []
}