CTC-Informed Pipeline for Personalized CAR-NK Cell Therapy in Pancreatic Cancer

Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to treatment resistance heterogeneity and an immunosuppressive microenvironment, necessitating personalized therapeutics. CAR-NK cell therapy shows promise but faces challenges including target identification in heterogeneous tumors and rapid exhaustion within the TME. Cytokine-armed CAR-NK cells that self-secrete immune-activating cytokines may overcome these barriers. Circulating tumor cells (CTCs) offer dynamic insights into tumor biology, including mutation and protein profiles, enabling personalized CAR target identification. We present a pipeline integrating CTC-informed antigen selection with cytokine-armed CAR-NK therapy, leveraging minimally invasive CTC sampling and allogeneic CAR-NK potential for rapid, adaptive personalized immunotherapy. Method & Result: Using label-free CTC isolation, we performed multi-omics analysis and identified mucin4 (MUC4) and mesothelin (MSLN), two highly prevalent PDAC antigens, in CTCs across patients (n=13; 6 metastatic, 7 localized). MUC4+CTCs were exclusively detected in 67% of metastatic patients, whereas 92% of all patients exhibited MSLN+CTCs, revealing the potential complementary coverage and enabling patient stratification for dual MUC4&MSLN CAR-NK treatment. We generated anti-MUC4 and anti-MSLN CAR-NK cells showing robust cytotoxicity against target-matched PDAC cell lines.To enhance efficacy, we tested IL-12 (for NK activation) and IL-15 (for NK proliferation) armed CAR-NK cells individually and combined. Anti-tumor efficiency improved in all cytokine-armed CAR-NK cells in short-term assays with no advantage of dual versus single cytokine arming. However, synergistically armed CAR-NK cells displayed drastically higher tumor-specific interferon-γ secretion post 18-hour exposure (4208 pg/mL vs 244 pg/mL for tumor-stimulated vs resting NK) compared to single cytokines (1819 & 252 pg/mL for IL-12 & IL-15), indicating sustained activity. In serial killing assays with repeated 72-hour tumor exposures at 2:1 NK: tumor ratio, unarmed CAR-NK lost efficacy by round 2, IL-15 by round 3, IL-12 by round 4, while synergistically armed CAR-NK sustained cytotoxicity through six rounds (16 days). Suggesting cytokine synergy is not only more physiologically relevant than solo-cytokine, but also more effective in immunotherapy activation. Summary: Our platform enables adaptive precision immunotherapy, leveraging minimally invasive CTC sampling to identify patient-specific target for rapid CAR-NK administration, while synergistic cytokine arming ensures durable activity against hostile PDAC TME. This real-time molecular monitoring allows dynamic therapeutic adjustments as tumor profiles evolve. Citation Format: Yin Zou, Mubin Tarannum, Shamileh Fouladdel, Yu Zhang, Nicole Peterson, Vaibhav Sahai, Deepak Nagrath, Rizwan Romee, Sunitha Nagrath. Circulating tumor cell (CTC) informed pipeline for rapid personalization of synergistic cytokine-armed CAR-NK cell therapy in PDAC [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 1313.