上皮性卵巢癌是妇科癌症中最致命的一种。针对单一抗原的嵌合抗原受体（CAR）T细胞治疗效果受到上皮性卵巢肿瘤内抗原异质性表达的限制。为克服这一限制，我们描述了一种既具有双重靶向又具有正交细胞毒性模式的工程细胞，用于抵抗卵巢癌细胞高表达的两种肿瘤抗原：细胞表面的Muc16和细胞内的WT1。将Muc16特异性CAR T细胞（4H11）工程化，以分泌一种由TCR仿制抗体（ESK1）构建的双特异性T细胞结合剂（BiTE），该结合剂与HLA-A2分子呈递的WT1衍生的RMFPNAPYL（RMF）表位具有反应性。分泌的ESK1 BiTE招募和重定向其他T细胞到肿瘤细胞上的WT1。我们展示了分泌ESK1 BiTE的4H11 CAR T细胞在体外和小鼠肿瘤模型中，对低表达Muc16的癌细胞表现出增强的抗癌活性，相比于单独应用4H11 CAR T细胞。具有不同特异性的双重正交细胞毒性模式，可靶向表面和细胞内的肿瘤相关抗原，是克服上皮性卵巢癌和其他癌症对CAR T细胞疗法抵抗性的一种有前景的策略。© 2023. 作者及斯普林格诺许使用斯普林格自然出版集团德国公司的专有许可。

Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR T cell therapy in epithelial ovarian cancer and other cancers.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.