Influence of onboard, tethered IL-12 on potency of the Tmod NOT gate and selectivity.

2562 Background: To realize the full therapeutic potential of engineered immune cells in solid tumors, high potency must be coupled with absolute selectivity. While synthetic NOT logic gates, such as the LIR-1 NOT gate (Tmod) system, effectively address non-specific cytotoxicity, overcoming the suppressive tumor microenvironment (TME) requires auxiliary stimulation. This study focused on Interleukin-12 (IL-12)—a potent pro-inflammatory cytokine—as an “armoring” strategy to bridge this gap. Methods: We designed an antigen-inducible, membrane-tethered IL-12 system to boost antigen-specific Tmod activity. The performance of this inducible IL-12 construct was evaluated across various long-term in vitro and in vivo assays to measure Tmod T cell exhaustion (specifically the upregulation of PD-1 and downregulation of CD62L) and its ability to mitigate the immunosuppressive effects of TGFβ, a primary barrier in the solid tumor microenvironment. We further assessed the reversibility of IL-12 expression upon antigen clearance and monitored for IL-12 shedding both in vitro and in vivo. Results: The inducible IL-12 construct boosted antigen-specific Tmod activity, prevented T cell exhaustion, and effectively mitigated the immunosuppressive effects of TGFβ that typically hamper CAR-T persistence. Crucially, this potency boost did not “override” the LIR-1 blocker; the IL-12-enhanced cells remained selective, sparing normal cells. Furthermore, data showed that the expression of membrane-tethered IL-12 is reversible once the antigen is cleared. Minimal IL-12 shedding was observed, suggesting the pro-inflammatory signal remained localized to the immunological synapse. Conclusions: The antigen-inducible membrane-tethered IL-12 system enhances Tmod potency while maintaining a high degree of selectivity and an acceptable safety profile for clinical translation. An Investigational New Drug (IND) application for MSLN-targeted Tmod boosted by antigen-inducible membrane-tethered IL-12 has been approved by the FDA, and a Phase 1 clinical trial is currently ongoing.