Time-of-day of first checkpoint inhibitor dose influences clinical outcomes and immune responses in hepatocellular carcinoma

Background Although immune checkpoint inhibitors (ICIs) have long half-lives, preclinical and early clinical studies across multiple tumor types suggest that the time-of-day of ICI infusion may influence therapeutic efficacy by aligning initial drug exposure with circadian peaks in T-cell responsiveness. However, the impact of initial drug infusion timing on outcomes has not been established. The immunologic basis of improved outcomes in early-day infusions and its clinical relevance in hepatocellular carcinoma (HCC) also remain unknown. Methods We prospectively followed patients with advanced/metastatic HCC receiving ICI therapy at Johns Hopkins from 2021-2025, classifying them into the morning group (first infusion before 12:00) or the afternoon group (first infusion after 12:00). We assessed clinical outcomes and compared immunologic responses from baseline to early-on-treatment (month 1 or month 2) by profiling 28 immune cell clusters in peripheral blood mononuclear cells using Cytometry by Time-of-Flight (CyTOF) and 39 plasma cytokines using a Luminex multiplex assay. Results Our cohort included 85 patients, 40 of whom received their first infusion in the morning. There were no statistically significant differences in baseline demographic or clinical characteristics between patients initiating therapy in the morning versus afternoon. The morning group had superior progression-free survival (multivariable HR 0.49, 95% CI 0.29-0.82, adjusted p<0.01) and higher odds of objective treatment response (multivariable OR 3.26, 95% CI 1.08-10.90, adjusted p<0.05), with no significant increase in immune-related adverse events. The timing of subsequent infusions after the first dose had no impact on survival outcomes. Immunologic responses diverged after the initial dose, with morning-treated patients showing reduced IL-6 levels (Wilcoxon rank-sum, p<0.01) and greater expansion of cytotoxic central memory CD8+ T-cells (Wilcoxon rank-sum, p=0.01) and cytotoxic effector CD8+ T-cells (Wilcoxon rank-sum, p<0.05) compared to afternoon-treated patients. Conclusions Morning first-dose infusion of ICIs in HCC was associated with improved clinical outcomes and distinct immune responses, including reduced IL-6 signaling and expansion of cytotoxic central memory and effector CD8+ T cells. These findings suggest that the timing of the initial infusion can imprint an immunologic program that shapes subsequent anti-tumor immunity, providing a mechanistic rationale for strategically scheduling ICI administration.