Abstract

Chimeric antigen receptor-engineered T (CAR T) cell therapy has achieved remarkable clinical efficacy against hematological malignancies but exhibits limited therapeutic outcomes in solid tumors, where immune checkpoint signaling is highly active. Here, we demonstrate that the second-generation CD28-CD3{zeta} (28Z) CAR undergoes liquid-liquid phase separation with the Src-family kinase LCK to assemble a signaling condensate crucial for CAR activation. PD-1, but not LAG3 or CTLA4, disrupts these CAR-LCK condensates through competitive binding to CAR rather than through its canonical phosphotase-dependent inhibitory pathway. Incorporation of a CD3{varepsilon} cytoplasmic module into the CAR introduces additional LCK interactions that reinforce CAR-LCK condensation, thus limited PD-1 incorporation and stabilize signalosome assembly. Consequently, the CD3{varepsilon}-engineered CAR (E-CAR) resists PD-1-mediated condensates disassembly, preserves immunological synapse organization, and sustains proximal signaling following PD-L1 engagement. E-CAR T cells are therefore resistant to PD-1-mediated functional suppression and maintain potent antitumor activity against PD-L1-positive solid tumors. Together, these findings identify disruption of CAR signaling condensates as a previously unrecognized mechanism of PD-1-mediated inhibition and establish CD3{varepsilon} modular signalosome engineering as a rational engineering strategy to overcome immune checkpoint suppression.