Combination of OX40 Co-Stimulation, Radiotherapy, and PD-1 Inhibition in a Syngeneic Murine Triple-Negative Breast Cancer Model

Simple Summary This experimental study was designed in order to investigate the efficacy of the triple combination of radiation (SBRT), PD-1 blockade, and OX40 co-stimulation in a syngeneic murine model using 'immunologically cold' triple-negative breast cancer cells. SBRT can induce immunogenic tumor cell deaths and act as an in situ vaccine while OX40 signaling has been shown to improve anticancer immunity combined with PD-1 inhibition via multiple preclinical studies. In our study, triple combination therapy significantly improved primary/abscopal tumor control and reduced lung metastases compared to single or dual therapies. This was found to be through an increased ratio of CD8+ T cells to regulatory T cells and a reduced proportion of exhausted T cells in the tumor microenvironment. Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (alpha-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and alpha-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of alpha-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, alpha-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-gamma, respectively. Together, our results suggest that the combination of alpha-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.