CD73 Targeted Combination Therapy Reverses PD-1 Inhibitor Resistance in CD73-High PD-L1-Negative NSCLC: Evidence from Pleural Effusion Organoids

Objectives Patients with PD-L1-negative non-small cell lung cancer (NSCLC) often exhibit inherent PD-1 inhibitor resistance, leading to poor monotherapy efficacy and unfavorable clinical outcomes. Notably, no effective stratification biomarkers are currently available to overcome this resistance. Thus, this study aimed to reverse PD-1 inhibitor resistance in PD-L1-negative NSCLC by identifying novel predictive biomarkers and developing effective targeted immunotherapeutic regimens. Methods Pleural effusion-derived organoids (PEOs) were established and validated from driver gene wild-type, PD-L1-negative NSCLC patients. Four treatment groups were set: control, anti-PD-1 monotherapy (Toripalimab), anti-CD73 monotherapy (Uliledlimab), and anti-PD-1 + anti-CD73 combination (Toripalimab + Uliledlimab). ATP assay was used to detect tumor cell viability. Spearman’s rank correlation analysis was applied to evaluate the association between CD73 expression and tumor inhibition rates. Flow cytometry was employed to measure T cell activation markers. Enzyme-linked Immunospot assay was used to detect IFN-γ and TNF-α secretion. Results PEO models recapitulated the biological features of primary pleural effusion. Toripalimab monotherapy showed no antitumor efficacy in PD-L1-negative subgroup regardless of CD73 expression levels, whereas Toripalimab + Uliledlimab combination significantly reversed PD-1 inhibitor resistance in CD73-high PD-L1-negative NSCLC patients. Furthermore, a significant positive correlation was observed between CD73 expression levels and tumor inhibition rates. Conclusion This study validates CD73 as a potential predictive biomarker for stratifying PD-L1-negative NSCLC patients and establishes anti-PD-1 + anti-CD73 combination regimen that effectively reverses PD-1 inhibitor resistance in CD73-high PD-L1-negative NSCLC patients.