Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B‐cell lineage neoplasms

Summary 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.