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

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 (MPRA), we identified CHS-selective synthetic promoters and engineered a Boolean AND-gate circuit that ensures high-fidelity expression of a surface T-cell engager (STE) exclusively within malignant cells.

To further resolve the immunosuppressive TME, we identified macrophage migration inhibitory factor (MIF) as a blockade target. Single-cell RNA sequencing in human-PBMC-reconstituted CHS-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.