{
  "abstract": "Background Pleural mesothelioma (PM) is a highly aggressive malignancy with limited therapeutic options and poor prognosis. 1 Emerging immunotherapeutic approaches, including bispecific T-cell engagers (BiTEs), offer promise by redirecting cytotoxic T cells to target tumour-associated antigens. Protein X, overexpressed in various malignancies including PM, represents a promising immunotherapeutic target.Methods We investigated the efficacy of a novel protein X targeting BiTE construct in five patient-derived PM cell lines (three epithelioid, two biphasic) versus a control BiTE, each in triplicates. The BiTE X construct crosslinks CD3 on T cells with Protein X-expressing tumour cells. Surface expression of Protein X was confirmed via flow cytometry. Allogeneic T cells isolated from healthy donors were co-cultured with PM cells for 72 hours. T cell activation was assessed using antibodies against CD69, CD25, and CD137. Tumour cell cytotoxicity was measured via XTT viability assay, and a dose-response curve established optimal BiTE concentration. Brightfield microscopy visually confirmed mesothelioma cell killing.Results High surface expression of protein X was confirmed across all cell lines. BiTE X induced robust, dose-dependent T cell activation and proliferation. After 72 hours, we observed cell clustering with BiTE X, but not control BiTE, indicating T cell activation. Flow cytometry at 72 hours showed high levels of endogenous T cell activation, ranging between 9%–91% CD25 (T cell activation marker) positivity in a dose-dependent manner. Both CD4+ and CD8+ T cells exhibited BiTE X- induced expression of CD25 and CD137 in all cell lines. Co-culture led to significant tumour cell lysis, with >70% cytotoxicity observed in some lines. No target cell killing or T cell activation occured in control or untreated samples.Conclusion These findings support further evaluation of Protein X-directed BiTEs in unpurified pleural effusion cells, which reflect the tumour microenvironment and heterogeneity of real-world PM patients. Evaluating BiTE responses in this setting may identify subtypes or molecular features linked to therapeutic sensitivity or resistance, informing biomarker development. Ultimately, this work supports the development of personalised BiTE-based immunotherapy which may have the potential to improve clinical outcomes.Reference Kanellakis NI, Asciak R, Hamid MA, et al. Thorax. 2020;75:1004–1008.",
  "authors": [
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, UK",
        "Laboratory of Pleural Translational Research, CAMS Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK"
      ],
      "name": "N Manoharan"
    },
    {
      "affiliations": [
        "Department of Oncology, University of Oxford, Oxford, UK"
      ],
      "name": "P Kok-Ting Wan"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, UK",
        "Medical Research Council Translational Immune Discovery Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK"
      ],
      "name": "T Dong"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, UK",
        "Oxford Centre for Respiratory Medicine, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK",
        "Oxford NIHR Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Oxford, UK"
      ],
      "name": "NM Rahman"
    },
    {
      "affiliations": [
        "Chinese Academy of Medical Sciences Oxford Institute, University of Oxford, Oxford, UK",
        "Laboratory of Pleural Translational Research, CAMS Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK",
        "Oxford Centre for Respiratory Medicine, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK",
        "Oxford NIHR Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Oxford, UK"
      ],
      "name": "N Kanellakis"
    }
  ],
  "title": "S65 Evaluation of protein X-targeting bispecific T cell engager therapy in patient-derived mesothelioma cell cultures",
  "uid": "104e1b6a-88c2-5414-8a33-d6941170d114"
}