Protease‐Responsive Condensation of Engineered Proteins Drives Tumor‐Targeted T‐Cell Activation

ABSTRACT

Liquid‐liquid phase separation (LLPS) drives the formation of biomolecular condensates, which are fundamental to cell signaling. Extracellular phase separation is emerging as a potent strategy for cell‐specific signaling modulation; however, its application is limited by the lack of control over on‐demand condensate formation. Here, we engineered a masked extracellular phase‐separation platform (Condenstim) enabling tumor microenvironment‐specific condensate formation. To achieve this, we first developed an LLPS‐based system that clusters CD3 and CD28 receptors to assemble synthetic immunological synapses for potent T‐cell activation. By employing a protease‐cleavable linker for site‐specific PEGylation, we sterically shielded the protein building blocks, rendering the platform biologically dormant during systemic circulation. Within the tumor microenvironment, localized proteases cleave the linker, thereby restoring LLPS in situ. This modular design ensures spatially confined and efficient T‐cell activation, leading to robust tumor regression in murine models, both as a monotherapy and in combination with PD‐1 blockade. Our findings establish a generalizable strategy to control extracellular LLPS for both fundamental studies and therapeutic applications.