装配型 DLL3 双表位复合纳米抗体 CAR T 细胞的临床前评估

Introduction Delta-like ligand 3 (DLL3) is markedly and selectively expressed in small cell lung cancer (SCLC) and other high-grade neuroendocrine tumors with minimal expression in normal tissues, making it an attractive target for cellular immunotherapy. DLL3-directed CAR approaches in solid tumors have shown limited durability, underscoring the need for improved potency. To address these challenges, we developed DLL3 bi-epitope compound nanobody CAR T cells. We incorporate IL-18 armoring to enhance effector cell survival and function. Herein we report the feasibility of an armored DLL3 bi-epitope cCAR T construct. Experimental Procedures Alpacas were immunized with recombinant human DLL3 extracellular domain protein. Peripheral blood mononuclear cell (PBMC) mRNA was extracted and converted into a VHH (nanobody) to generate a library. The library was panned against DLL3 antigen to enrich for high-affinity binders, which were identified through ELISA screening and cellular binding analysis by flow cytometry. VHH clone sequencing were used to generate our cCAR. The DLL3 bi-epitope cCAR T is comprised of two unique and independently functioning CAR units targeting different DLL3 epitopes reducing risk of tumor evasion due to antigenic variation. A DLL3-targeting nanobody pair was used to generate DLL3 bi-epitope cCAR. Human T cells were activated and transduced to produce CAR T cells. CAR expression was assessed by flow cytometry. Cytotoxicity was evaluated using SHP-77 (endogenous DLL3⁺) and REH-DLL3xp (overexpressing DLL3) targets. NSG mice received CAR T cells to assess antitumor activity. Results The bi-epitope DLL3 cCAR T construct demonstrates potent cytotoxicity lysing target cells in vitro and in vivo. At various effector-to-target ratios, the cCAR was observed to have strong killing activity and specificity against the primary tumor cell line and artificially DLL3- expressing cell line. The cCAR also demonstrated strong potency in SHP-77 xenograft mouse models. Conclusions Our bi-epitope armored DLL3 cCAR T therapy demonstrated exceptional efficacy in vitro and in vivo. These results support future investigation of our cCAR in patients with SCLC and/or DLL3 expressing neuroendocrine tumors.