Treatment with doxorubicin and PD-1/CTLA-4 blockade improves T cell activation and anti-tumor efficacy in MCA-205 murine fibrosarcoma

Abstract

Abstract Immune checkpoint inhibitors (ICIs) against PD-1 and CTLA-4 produce responses in about 20% of adult soft tissue sarcomas (STSs), however, most patients do not respond, potentially from poor T cell activation. Standard chemotherapy for STSs includes the anthracycline doxorubicin (DOX) which has been shown in preclinical cancer models to induce immunogenic cell death (ICD) via tumor production of type I interferons (IFN). We hypothesized that induction of ICD and IFN release by doxorubicin would enhance T cell activation, infiltration, and boost anti-tumor efficacy when combined with dual anti-PD-1/CTLA-4 blockade in the MCA-205 murine fibrosarcoma model. MCA-205 tumors were generated in C57BL/6 mice and were treated with DOX, anti-PD1 (RMP1–14), anti-CTLA4 (9D9 mouse IgG2b) or a mouse surrogate of botensilimab, an anti-CTLA4 (9D9 mouse IgG2b.DLE) antibody with enhanced binding to FcγR, or combination DOX plus anti-PD-1/CTLA-4. We found reduction of tumor growth and improved survival (median survival not reached) with DOX/PD-1/CTLA-4 compared to DOX (median survival 29.5 days). This correlated with a 2-fold increased influx of TILs as measured by flow cytometry and immunohistochemistry, with a marked increase in CD8:CD4 T cell ratio with increased expression of activation markers (PD-1, 4–1BB and TIM3). TCR Vβ clonotyping revealed a shift in clonotype frequency with DOX/PD-1/CTLA-4 relative to other treatment groups. Pathway analysis from bulk RNA sequencing demonstrated upregulation of T cell activation in DOX/PD-1/CTLA-4 tumors compared to control tumors. Further work will correlate these findings with human STS patients receiving DOX plus CTLA-4/PD-1 blockade in an ongoing clinical trial (NCT04028063). This work was supported by grants from the University of Colorado Cancer Center (P30CA046934) and the University of Colorado School of Medicine.