Development of a novel CAR T-cell therapy targeting thymic carcinoma

Introduction: Thymic carcinoma is a rare, highly aggressive mediastinal malignancy with limited effective therapies and considerable toxicity associated with immune checkpoint inhibitors. Although CAR T-cell therapy has revolutionized the treatment landscape for hematologic malignancies, its application to solid tumors, including thymic epithelial tumors, remains challenging due to disease rarity, a paucity of validated surface targets, and the risk of on-target, off-tumor autoimmune toxicity. In this study, we sought to address these barriers by characterizing antigen expression profiles across thymic carcinoma-relevant cell lines, engineering novel CAR constructs against prioritized candidate antigens, and evaluating antigen-specific cytotoxicity. These efforts aim to inform the development of next-generation cellular therapies tailored to the unique biology of this aggressive and understudied tumor type. Methods: Surface expression of TROP-2, c-KIT, and CD70 was quantified on candidate cell lines using flow cytometry. CAR T-cells were produced from healthy donor PBMCs via lentiviral transduction and expanded ex vivo. CAR expression was confirmed by detection of Fab fragments on CD3⁺ T cells. Antigen-specific cytotoxicity was evaluated in vitro using co-culture assays with selected antigen-positive cell lines, and target cell killing was quantified by 7-AAD staining and flow cytometric analysis. Results: Flow cytometry demonstrated robust surface expression of TROP-2 (83%), c-KIT (88%), and CD70 (99%) across the T1889, Kasumi, and U266 cell lines, respectively. Lentiviral transduction yielded CAR⁺ T cell populations of 48.2% (TROP-2), 24.7% (c-KIT), and 47.5% (CD70). All three constructs exhibited potent antigen-specific cytotoxicity substantially exceeding GFP⁺ CD3⁺ control T cells, confirming selective recognition and killing of target cells. These findings provide some of the first in vitro evidence supporting TROP-2, c-KIT, and CD70 as viable immunotherapeutic targets for thymic carcinoma and demonstrate effective CAR expression and selective cytotoxicity. This work establishes a foundation for rational multi-antigen CAR T cell design in a historically understudied solid tumor. Ongoing studies include FACS-based CAR enrichment, characterization of exhaustion markers, development of a dual-antigen synNotch CAR to mitigate off-tumor toxicity, and in vivo validation in murine models to support future clinical translation.