VHH-based CAR-T cells targeting DLL3 show high efficacy in small-cell lung cancer models.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by a high relapse rate, limited treatment options, and a poor prognosis. Delta-like ligand 3 (DLL3) has emerged as a promising target for SCLC. Notably, CAR T cells that use the variable domain of heavy-chain-only antibodies (VHH) demonstrate superior efficacy compared to their ScFv counterparts. However, the therapeutic effectiveness of anti-DLL3 VHH-CAR T cells has yet to be fully explored. To leverage the therapeutic potential of VHHs, we first immunized alpacas and screened for anti-DLL3 VHHs using yeast display. Then, 1-B12 and 5 identified the positive clones and compared them based on their affinity, specificity, and cytotoxicity in CAR T cell models. Moreover, 1-B12 was selected as the humanized sequence due to its higher affinity, greater specificity, and stronger cytotoxicity. Finally, the functionality of the four humanized VHH-CAR T cells from the 1-B12 sequence was evaluated through in vitro assays that measured cytokine production and cytotoxicity, followed by in vivo studies to assess their antitumor efficacy. The anti-DLL3 VHHs exhibited strong affinity, specificity, and cytotoxicity in CAR T cell models. Notably, the HM-CAR T cells exhibited robust cytokine secretion and cytotoxic activity against tumor cells. Moreover, these HM-CAR T cells demonstrated significant antitumor efficacy in vivo. This study highlights effective strategies for developing DLL3-specific VHHs and their application in CAR T therapy, which supports their clinical potential as a promising immunotherapeutic approach for cancers that express DLL3.