T细胞淋巴瘤（TCL）是一组高度异质性的疾病，预后差，5年总生存率低。目前的治疗方案疗效相对较低。在T淋巴细胞中，临床单靶点嵌合抗原受体T细胞（CAR-T细胞）治疗研究需要大量和多次输注，增加了治疗的风险和费用，因此优化靶向治疗是改善总体预后的一种方式。尽管双特异性CAR-T细胞治疗B细胞淋巴瘤具有显著进展，以避免抗原逃脱，但在TCL的应用需要进一步研究。在这里，我们构建了一个羊驼纳米抗体（Nb）噬菌体文库，并分别生成高亲和力和特异性的针对CD30和CD5的Nb。通过多轮筛选，构建了双特异性NbCD30-CD5-CAR T细胞，并在体外和体内验证了其对TCL的优越抗肿瘤效应。我们的研究结果显示，Nb衍生的双特异性CAR-T细胞与单靶点CAR-T细胞和双特异性单链变量片段（scFv）衍生的CAR-T细胞相比，在TCL治疗中显著改善了抗肿瘤疗效。由于Nbs更小且免疫原性较低，基于Nb的双特异性CAR-T细胞的协同作用可能会提高其在未来临床应用中的安全性和疗效。 © 2023作者

T cell lymphoma (TCL) is a highly heterogeneous group of diseases with a poor prognosis and low 5-year overall survival rate. The current therapeutic regimens have relatively low efficacy rates. Clinical studies of single-target chimeric antigen receptor T cell (CAR-T cell) therapy in T lymphocytes require large and multiple infusions, increasing the risks and cost of treatment; therefore, optimizing targeted therapy is a way to improve overall prognosis. Despite significant advances in bispecific CAR-T cell therapy to avoid antigen escape in treatment of B cell lymphoma, applying this strategy to TCL requires further investigation. Here, we constructed an alpaca nanobody (Nb) phage library and generated high-affinity and -specificity Nbs targeting CD30 and CD5, respectively. Based on multiple rounds of screening, bispecific NbCD30-CD5-CAR T cells were constructed, and their superior anti-tumor effect against TCL was validated in vitro and in vivo. Our findings demonstrated that Nb-derived bispecific CAR-T cells significantly improved anti-tumor efficacy in TCL treatment compared with single-target CAR-T cells and bispecific single chain variable fragment (scFv)-derived CAR-T cells. Because Nbs are smaller and less immunogenic, the synergistic effect of Nb-based bispecific CAR-T cells may improve their safety and efficacy in future clinical applications.© 2023 The Authors.