Abstract C007: A versatile microRNA-based platform for activation-dependent regulation of armored CAR T cell payloads

Abstract CAR T-cell therapy has transformed the treatment landscape for blood cancers, yet its success in solid tumors is limited by poor trafficking, immunosuppressive tumor microenvironment, and antigen heterogeneity. “Armored” CAR T cells aim to overcome these barriers by delivering immune-boosting payloads, although achieving precise and safe tumor-specific control of such potent functions remains a critical and unresolved challenge. Here, we present a post-transcriptional regulatory platform that leverages endogenous microRNA (miRNA) circuits to control therapeutic transgene expression in CAR T cells. This approach embeds complementary miRNA target sites (miRTS) into payload-encoding transcripts, making their expression dictated by endogenous miRNA abundance. This design exploits miRNAs naturally expressed in resting T cells to enforce repression of payloads under non-activated conditions. Upon CAR-mediated antigen stimulation, these miRNAs are downregulated, enabling targeted de-repression selectively at the tumor site. To identify microRNAs with this activation-coupled behavior, we combined small-RNA sequencing with a functional screen. Transcriptomic profiling of HER2-28Z CAR T cells activated in vitro and of CAR tumor-infiltrating lymphocytes from treated mice revealed 58 microRNAs that are abundant at rest but diminish upon activation. Using an in vivo, library-based functional screen informed by these data, we prioritized miR-29c-3p, miR-32-3p, miR-150-5p, and miR-181a-5p as the most effective regulators. Incorporating the corresponding target sites into the 3’UTR of the transgene within armored CAR-T generated a miRNA-inducible platform that enabled strong transgene repression in resting T cells while activation-specific payload expression in vitro and in various solid tumor models in vivo. In these models, all candidate miRNAs significantly reduced circulating payload levels, with reductions ranging from 53% to 94% compared to constitutive payload expression, while simultaneously permitting intratumoral expression and enhancing tumor control and T-cell persistence. The platform proved highly versatile. Each miRNA candidate provided distinct repression and induction dynamics, and regulatory strength could be further tuned by varying the number or combination of miRTS, with additional sites enhancing repression. This flexibility enables tailoring to transgenes with diverse safety requirements. The system supported varied payloads, including secreted cytokines (IL-12, IL-18) and membrane-bound proteins such as a secondary CAR. It performed consistently across CARs with different antigen specificities and with both 4-1BB and CD28 signaling domains. Importantly, we show that our platform is compatible with CAR T cells derived from cancer patients, supporting its translational feasibility. Collectively, our results define a post-transcriptional, plug-and-play regulatory layer for precise, context-driven, and modular control of armored CAR T-cell therapies—addressing a central barrier to their safe and effective implementation in solid tumors. Citation Format: Nina Barceló-Genestar, Teresa Lobo-Jarné, Paula Barbao, Marta Gimenez, Guillem Colell, Fleur Chapus, Salut Colell, Maria Hernandez, Hugo Calderón, Ariel Galindo-Albarrán, Marco Antonio Mendoza-Parra, Luca Gattinoni, Aleix Prat, Alena Gros, Xavier M. Anguela, Alba Rodriguez-Garcia, Sonia Guedan. A versatile microRNA-based platform for activation-dependent regulation of armored CAR T cell payloads [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C007.