Abstract B011: Onboard, tethered IL-12 boosts potency of the Tmod NOT gate and preserves selectivity

Abstract For immune cells engineered with synthetic constructs to realize their full therapeutic potential, they must meet the demanding dual requirements of high potency and selectivity, thereby overcoming the central challenge of non-specific cytotoxicity. The challenge is particularly acute in solid tumor treatments, where issues such as antigen expression, tissue accessibility, and the tumor microenvironment complicate therapy. Engineering cells with receptors that function as synthetic logic gates offers a way to improve tumor specificity by recognizing combinations of antigens rather than relying on a single marker. Tmod is a synthetic NOT logic gate construct composed of two receptors: a CAR activating receptor that selectively kills tumor cells (activator), and an inhibitory receptor based on LIR-1 that spares normal cells (blocker). To enhance efficacy beyond the immediate effects of receptor activation, other mechanisms can be brought into play. One strategy is to harness cytokine receptors—key contributors to auxiliary stimulation during natural immune responses. We took an approach that has been previously used to boost CAR-T potency that involves engineered onboard cytokines, a method commonly known as “armoring”. However, potency enhancements from onboard cytokines must not compromise the selectivity of the cell therapy product, especially in products engineered with logic gates designed to improve tumor-specific killing. Here, we thoroughly tested membrane-tethered IL-12 with respect to its effects on the potency and selectivity of MSLN-targeted Tmod. We found that an antigen-inducible construct that encodes membrane-tethered IL-12 boosts antigen-dependent activity of Tmod constructs by ∼10-fold in a variety of longer-term in vitro and in vivo assays without overriding the blocker, a key requirement of booster function. Minimal IL-12 shedding was observed both in vitro and in vivo, consistent with an acceptable safety profile of a Tmod product that carries tethered IL-12. Membrane-tethered IL-12 also mitigated the immunosuppressive effect of TGFβ, thought to be a key component of the tumor microenvironment. We conclude that the membrane-tethered IL-12 effectively enhances Tmods cytotoxicity while preserving selectivity. Citation Format: Sara Imboden, Jon Torres, Alexandre Zampieri, Sanam Shafaattalab, Jushen Liang, Richele Bruno, Talar Tokatlian, Alexander Kamb, Jingli A. Zhang. Onboard, tethered IL-12 boosts potency of the Tmod NOT gate and preserves selectivity [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 B011.