嵌合抗原受体 T 细胞 (CAR-T) 疗法通过靶向 CD19 在治疗晚期 B 细胞恶性肿瘤方面显示出显着疗效，但抗原阴性复发和鼠源抗体引发的免疫反应仍然是重大挑战，因此需要开发新型人源化疗法多靶点 CAR-T 疗法。在这里，我们设计了第二代基于 4-1BB-CD3ze 的 CAR 构建体，其中包含人源化 CD19 单链可变片段 (scFv) 和重链 (VHH) 上的 BAFFR 单变量结构域（也称为纳米抗体）。由此产生的具有不同结构的 CAR-T 细胞在体外和体内进行了功能比较。我们发现，最佳的串联和双顺反子（BI）结构保留了各自的抗原结合能力，并且在靶细胞刺激时均表现出特异性激活。同时，BI CAR-T细胞（BI CAR）比单靶点CAR-T细胞表现出更强的肿瘤杀伤能力以及更好的白细胞介素2和肿瘤坏死因子-α分泌能力。此外，BI CAR 在重复抗原刺激后表现出较少的耗竭表型，并在小鼠异种移植模型中表现出更有效和持久的抗肿瘤作用。总体而言，我们开发了一种基于 scFv 和 VHH 组合的新型人源化 CD19/BAFFR 双顺反子 CAR (BI CAR)，其在体外和体内均显示出有效且持续的抗肿瘤能力，包括针对具有 CD19 或 BAFFR 缺陷的肿瘤。© 2024英国血液学会和约翰·威利

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating advanced B-cell malignancies by targeting CD19, but antigen-negative relapses and immune responses triggered by murine-derived antibodies remain significant challenges, necessitating the development of novel humanized multitarget CAR-T therapies. Here, we engineered a second-generation 4-1BB-CD3ζ-based CAR construct incorporating humanized CD19 single-chain variable fragments (scFvs) and BAFFR single-variable domains on heavy chains (VHHs), also known as nanobodies. The resultant CAR-T cells, with different constructs, were functionally compared both in vitro and in vivo. We found that the optimal tandem and bicistronic (BI) structures retained respective antigen-binding abilities, and both demonstrated specific activation when stimulated with target cells. At the same time, BI CAR-T cells (BI CARs) exhibited stronger tumour-killing ability and better secretion of interleukin-2 and tumour necrosis factor-alpha than single-target CAR-T cells. Additionally, BI CARs showed less exhaustion phenotype upon repeated antigen stimulation and demonstrated more potent and persistent antitumor effects in mouse xenograft models. Overall, we developed a novel humanized CD19/BAFFR bicistronic CAR (BI CAR) based on a combination of scFv and VHH, which showed potent and sustained antitumor ability both in vitro and in vivo, including against tumours with CD19 or BAFFR deficiencies.© 2024 British Society for Haematology and John Wiley & Sons Ltd.