BCMA定向的自体嵌合抗原受体T（CAR-T）细胞在复发或难治性多发性骨髓瘤（RRMM）中显示出优异的临床疗效，然而，目前自体CAR-T细胞的制备过程复杂且成本高昂。此外，在多发性骨髓瘤中观察到CD47表达上调，抗CD47抗体在临床试验中显示出显着的效果。因此，我们专注于开发BCMA/CD47定向的通用CAR-T（UCAR-T）细胞来改善这些局限性。在本研究中，我们采用噬菌体展示技术筛选针对BCMA和CD47蛋白的纳米抗体，并确定了表征纳米抗体。此外，我们利用CRISPR/Cas9系统同时破坏T细胞的内源TRAC和B2M基因，产生TCR和HLA双敲除T细胞，开发BCMA/CD47定向的UCAR-T细胞，并检测体外和体外抗肿瘤活性。我们从免疫VHH文库中分别获得了14个针对BCMA和1个针对CD47蛋白的特异性纳米抗体。 BCMA/CD47 定向的 UCAR-T 细胞表现出优异的 CAR 表达 (89.13-98.03%)，并有效杀死原代人 MM 细胞和 MM 细胞系。 BCMA/CD47导向的UCAR-T细胞对MM表现出优异的抗肿瘤活性，并延长了肿瘤移植NCG小鼠体内的存活时间。这项工作表明BCMA/CD47导向的UCAR-T细胞在体外和体外表现出有效的抗MM活性体内，这为开发用于治疗多发性骨髓瘤的新型“现成”细胞免疫疗法提供了潜在策略。© 2024。作者。

BCMA-directed autologous chimeric antigen receptor T (CAR-T) cells have shown excellent clinical efficacy in relapsed or refractory multiple myeloma (RRMM), however, the current preparation process for autologous CAR-T cells is complicated and costly. Moreover, the upregulation of CD47 expression has been observed in multiple myeloma, and anti-CD47 antibodies have shown remarkable results in clinical trials. Therefore, we focus on the development of BCMA/CD47-directed universal CAR-T (UCAR-T) cells to improve these limitations.In this study, we employed phage display technology to screen nanobodies against BCMA and CD47 protein, and determined the characterization of nanobodies. Furthermore, we simultaneously disrupted the endogenous TRAC and B2M genes of T cells using CRISPR/Cas9 system to generate TCR and HLA double knock-out T cells, and developed BCMA/CD47-directed UCAR-T cells and detected the antitumor activity in vitro and in vivo.We obtained fourteen and one specific nanobodies against BCMA and CD47 protein from the immunized VHH library, respectively. BCMA/CD47-directed UCAR-T cells exhibited superior CAR expression (89.13-98.03%), and effectively killing primary human MM cells and MM cell lines. BCMA/CD47-directed UCAR-T cells demonstrated excellent antitumor activity against MM and prolonged the survival of tumor-engrafted NCG mice in vivo.This work demonstrated that BCMA/CD47-directed UCAR-T cells exhibited potent antitumor activity against MM in vitro and in vivo, which provides a potential strategy for the development of a novel "off-the-shelf" cellular immunotherapies for the treatment of multiple myeloma.© 2024. The Author(s).