A logic-gated synthetic circuit coupled with microenvironmental reprogramming overcomes immune exclusion in antigen-poor chondrosarcoma.

Abstract

Chondrosarcoma remains refractory to immunotherapy due to a paucity of targetable antigens and a suppressive tumor microenvironment (TME). We developed a modular strategy integrating tumor-intrinsic genetic logic with rational TME reprogramming to overcome these barriers. Using a 57,715-element massively parallel reporter assay, we identified chondrosarcoma-selective synthetic promoters and engineered a Boolean AND-gate circuit that ensures high-fidelity expression of a surface T cell engager exclusively within malignant cells. To further address the immunosuppressive TME, we identified macrophage migration inhibitory factor (MIF) as a target for blockade. Single-cell RNA sequencing in human PBMC-reconstituted chondrosarcoma xenograft models revealed that the circuit initiates T cell activation, while MIF blockade prevents exhaustion by promoting proliferative T cells and M1-polarized macrophages. Local administration induced a systemic abscopal effect, regressing distant, untreated tumors. Collectively, these findings provide a programmable blueprint for converting immunologically cold, antigen-poor malignancies into systemically immune-responsive states.