{
  "abstract": "Background Breast cancer (BC) is the most common cancer among women worldwide and remains a major therapeutic challenge, particularly in advanced, relapsed, or refractory cases. Although CAR T cell therapy has shown success in hematologic malignancies, its application to solid tumors such as BC is limited by physical barriers, an immunosuppressive tumor microenvironment, high manufacturing costs, and adverse events including cytokine release syndrome.Methods To address these challenges, we developed Dual BATs—T cells pre-armed with two bispecific protein engagers (BIPEs) ( figure 1). One BIPE targets folate receptor alpha (FRα), frequently overexpressed in BC, and CD3 to promote T cell activation. The second targets PD-L1 and CD28 to deliver costimulatory signaling and counteract immunosuppression from PD-L1. BIPEs were produced in HEK293T cells via lentiviral transduction and harvested to arm human primary T cells. BIPE binding was confirmed by flow cytometry, and phenotypic analysis of Dual BATs was performed. Antitumor activity was assessed using fluorescence-based cytotoxicity assays against BC cells co-expressing FRα and PD-L1.Results Both BIPEs were successfully produced from HEK293T cells and demonstrated high binding affinity to their respective targets. Dual BATs were produced with favorable T cell phenotype and increased expression of anti-apoptotic protein. Compared to single-armed T cells, Dual BATs showed enhanced antigen-specific cytotoxicity, indicating robust functional activity through CD3 and CD28 co-engagement.Conclusions We successfully developed Dual BATs by co-arming human T cells with FRαxCD3 and PD-L1xCD28 BIPEs. This dual-targeting strategy promoted full T cell activation and selective cytotoxicity against BC cells, highlighting its potential as a novel immunotherapy for solid tumors such as BC. These findings support further in vivo studies to assess antitumor efficacy, persistence, and safety, with the goal of advancing Dual BATs toward clinical translation.Acknowledgements The authors are affiliated with the Thailand Hub of Talents in Cancer Immunotherapy (TTCI), supported by the National Research Council of Thailand (grant number N34E670096).Ethics Approval This work was performed in accordance to ethical guideline under the approval from the Siriraj Institutional Review Board (SIRB) of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA no. Si 078/2025).Consent All donors provided written inform consent prior to data collection.Abstract 336 Figure 1Concept of dual bispecific protein engager-armed T cell (Dual BAT) production",
  "authors": [
    {
      "affiliations": [
        "Mahidol University, Bangkok-noi, Bangkok, Thailand"
      ],
      "name": "Piriya Luangwattananun"
    },
    {
      "affiliations": [
        "Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok, Thailand"
      ],
      "name": "Thanawitch Sangkheereeput"
    },
    {
      "affiliations": [
        "Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok, Thailand"
      ],
      "name": "Nunghathai Sawasdee"
    },
    {
      "affiliations": [
        "Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok, Thailand"
      ],
      "name": "Kamonlapat Supimon"
    },
    {
      "affiliations": [
        "Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok, Thailand"
      ],
      "name": "Kwanpirom Suwanchiwasiri"
    },
    {
      "affiliations": [
        "Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok, Thailand"
      ],
      "name": "Pa-thai Yenchitsomanus"
    }
  ],
  "title": "336 Dual bispecific protein engager-armed T cells targeting FRα and PD-L1 enhance cytotoxicity against breast cancer cells",
  "uid": "2f862a39-59d7-54e0-bdc5-12f118e38099"
}