Nanobody-Based DLL3 CAR T-Cell Therapy for Small Cell Lung Cancer

Introduction: Small-cell lung cancer (SCLC) is aggressive with poor prognosis, and current immunotherapies have limited efficacy. Delta-like ligand 3 (DLL3), expressed in ∼80% of SCLC with restricted normal-tissue expression, is a promising CAR T target. We developed nanobody-based, autologous DLL3 CAR T cells armed with a dominant-negative TGFβ receptor II (DNR) to resist TGFβ-driven immunosuppression and enhance antitumor activity. Methods: Candidate DLL3 binders were derived from a camelid-immunized phage display library and engineered into second-generation CARs. Constructs were tested in vitro for specific lysis with target cell lines. Lead binders were screened for cross-reactivity against Notch ligands. The DLL3 epitope was mapped, and membrane-proximal binders targeting EGF3-6 were prioritized. Off-target interactions were assessed using a membrane proteome array. Multiple CARs—with varying binders/biparatopic combinations, co-stimulation domains, and hinges—were compared in short-term and repeated antigen-stimulation assays across cells with varying DLL3 levels in vitro. Lead CAR T cells with or without DNR were compared in vitro and in vivo. Top constructs advanced to testing in multiple CDX and PDX models to evaluate antitumor efficacy and safety. Manufacturing was compared between a 3-day FasTCAR and an 8-day conventional process. Results: Three VHH binders demonstrated high DLL3 specificity, with no cross-reactivity to Notch family ligands and no off-target binding, and similar affinity for mouse DLL3. The lead biparatopic CAR construct, B2, demonstrated superior cytotoxicity and durability in short-term and repeated antigen-stimulation assays. When armored with DNR, B2-DNR CAR T cells showed augmented persistence versus the unarmored counterparts. In vivo safety assessments showed no body-weight loss in CAR T-treated mice across multiple dose levels, suggesting a wide therapeutic index. T cells were present in the mouse pituitary pars intermedia, where DLL3-positive cells are enriched, but their DLL3 expression was largely intracellular rather than on the cell surface. B2 did not show on-target, off-tumor toxicity. B2-DNR CAR T cells rapidly cleared established tumor xenografts with high and low DLL3 expression in immunodeficient mice, correlating with robust intratumoral CD3+ T-cell infiltration. In a TGFβ-rich PDX model, B2-DNR CAR T cells outperformed the unarmored counterpart, supporting the functional benefit of DNR. The 3-day FasTCAR manufacturing process demonstrated greater efficacy and durability than the 8-day process. Conclusions: The camelid nanobody-derived, DLL3-targeted CAR T-cell therapy manufactured by a 3-day FasTCAR process demonstrates the potential to elicit deep and durable antitumor responses with a favorable safety profile. Preclinical data for the lead candidate, B2-DNR, support further clinical development for the treatment of SCLC. Citation Format: Qi Dong, Wenjie Yin, Xiangling Dai, Tao Wang, Guangyao Zhu, Manli Yin, Yu Zou, Yu Yang, Di Wu, Mark Cobbold, Lianjun Shen. Nanobody-based, DLL3-directed FAST-CAR T-cell therapy for small cell lung cancer (SCLC) [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 1526.