Abstract 6121: A novel allogeneic anti-DLL3 CAR-NK cell therapy in treating small cell lung cancer

Small cell lung cancer (SCLC) is an aggressively lethal malignancy with a high unmet medical need. Overexpression of Delta-like ligand 3 (DLL3) has been identified as a poor prognostic factor. Adoptive immunotherapy based on chimeric antigen receptor (CAR) T cells has been developed, however with significant adverse events. To address the critical need for an improved therapeutic intervention, we leveraged the ability of DLL3 targeted peripheral blood derived natural killer (PBNK) allogeneic cells therapy in treating SCLC. We describe preclinical in vitro and in vivo results demonstrating the antitumor efficacy of an off-the-shelf, cryopreserved DLL3 CAR-NK candidate. In addition, we make comprehensive comparison of PB units from different donors to establish criteria of starting materials selection for clinical and commercial supply of CAR-NK cells. PBNK cells were isolated from healthy donors with FcγRIIIA-158 (V/V, V/F) and propagated using feeder cells and cytokine cocktails. Various in vitro assays, including flow cytometry phenotyping, freeze-thaw viability/recovery, short-term and serial killing, cytokine releasing against tumor cells, were used for comprehensive function evaluation of PB units. DLL3 targeted CAR-NK cells were engineered by transducing donor-preselected NK cells with a retroviral vector encoding a DLL3 membrane-proximal epitope targeted scFv, a NK-optimized CAR backbone and a secreted IL15 armor. Activity of CAR-NK cells against target-expression SCLC cells was assessed in vitro and in vivo. PB Donors were selected with excellent NK expansion capacity and freeze/thaw recovery rate, stronger innate cytotoxicity and higher degranulation and intracellular IFN-γ buildup upon co-culturing with different tumor cells. The anti-DLL3 CAR was successfully transduced across all batches of CAR-NK cells derived from selected donors. NK-featured CAR backbone significantly enhanced expansion of NK cells and induced more activated NK phenotype compared to conventional CAR backbone. DLL3 CAR-NK cells was cytotoxic against DLL3+ tumor cells (SHP-77 and NCI-H2171 cells), and DLL3 CAR-NK cells combined with anti-PD-L1 antibodies could further enhance NK cells proliferation in response to tumor cells and could control multiple rounds of tumor cells re-challenge in vitro. Using a cell line derived xenograft mouse model, we showed that a single dose of cryopreserved DLL3 CAR-NK cells delayed tumor growth and prolonged mice survival. Synergistic in vivo antitumor efficacy and enhanced NK cell expansion in peripheral blood were observed when using CAR-NK combined with anti-PD-L1. Collectively, these results demonstrated the favorable manufacturability, potency, and safety of a DLL3 CAR-NK cells product and its potential as a novel allogeneic treatment option for patients with SCLC. Citation Format: Chao Wang, Tingting Liu, Qin Wang, Yanxue Gong, Ling Chen, Fanxiang Gao, Feng Zhou, Zhuoxiao Cao. A novel allogeneic anti-DLL3 CAR-NK cell therapy in treating small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6121.