Abstract 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. Citation Format: Byeong-Hyeok Choi, Ga-Ram Hwang, Morgan Flaherty, Vincent M. DeStefano, Masayuki Wada, Kevin Pinz, Jennifer E. Chow, Nabil Hagag, Yu Ma, Luo Jing, Yupo Ma. Preclinical evaluation of armored DLL3 biepitope compound nanobody CAR T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5200.
Choi et al. (Fri,) studied this question.