RAD001-Adjuvanted Influenza Vaccine Synergizes With Anti–Programmed Cell Death Protein 1 Therapy to Enhance Antitumor Immunity in Programmed Cell Death Ligand 1–Low Murine Lung Cancer

Abstract

Background: Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have transformed lung cancer therapy, but their efficacy is often limited in tumors with low programmed cell death ligand 1 (PD-L1) expression and immunologically “cold” tumor microenvironments. This study aimed to evaluate the safety and therapeutic efficacy of combining a RAD001-adjuvanted influenza vaccine with PD-1 blockade and to explore its immunological mechanisms in a murine lung cancer model.

Methods: C57BL/6 mice bearing Lewis lung carcinoma (LLC) tumors, characterized by low PD-L1 levels (10.28%), were randomized to receive control, anti-PD-1 monotherapy, or anti-PD-1 combined with either a split influenza vaccine, an MF59-adjuvanted vaccine, or a RAD001-adjuvanted vaccine. Primary endpoints included tumor growth kinetics and survival. Safety was assessed via histopathological analysis of major organs and biochemical analyses. Mechanistic changes were evaluated through immunophenotyping of tumor-infiltrating lymphocytes (TILs) and serum cytokine profiling.

Results: The RAD001-adjuvanted vaccine synergized with anti-PD-1 therapy, resulting in superior tumor growth inhibition compared to all other groups. This synergy was associated with a remodeled tumor microenvironment, showing increased CD8⁺ T-cell infiltration and a higher CD8⁺/regulatory T-cell ratio. Serum analysis revealed a shift toward a Th1-dominant cytokine profile (a trend toward increased interferon-gamma and interleukin-2). Histopathological and biochemical examination confirmed no treatment-related toxicities in the heart, liver, lungs, kidneys, or spleen.

Conclusions: RAD001-adjuvanted influenza vaccination safely enhances the efficacy of anti-PD-1 therapy in PD-L1–low murine lung cancer by promoting a pro-inflammatory immune microenvironment. These findings suggest that strategic vaccine adjuvant selection can broaden the therapeutic reach of immune checkpoint inhibitors.