Abstract B63: The utility and mechanistic basis of CTLA-4 and PD-1 immunotherapeutic checkpoint inhibitors in enhancing tumor-specific adaptive responses to effectively treat triple-negative breast cancer
Authors: Crosby Erika , Wei Jun-Ping , Yang Xiao-Yi , Lei Gangjun , Wang Tao , Lyerly H Kim , Hartman Zachary C. 
Published: May 2016

While most breast cancers driven by estrogen-mediated signaling (ER+) or HER2 expression (HER2+) can be effectively treated by agents targeting these pathways, recent deep sequencing efforts have failed to identify widespread targetable drivers in Triple Negative Breast Cancers (TNBCs). Despite a lack of unifying drivers, our lab and others have uncovered that these cancers are highly inflammatory and associated with higher numbers of infiltrating immune cells (including CD8+ T-cells and Foxp3+ T-regulatory cells) , as well as expressing immuno-suppressive molecules (such as PD-L1). We hypothesized that despite TNBC molecular heterogeneity, the intrinsic inflammatory and immuno-suppressive features of these cancers could make them highly susceptible to checkpoint inhibitor-based immunotherapy. To test this hypothesis, we explored the utility and mechanistic basis of both PD-1 and CTLA-4 inhibition in generating tumor-specific immunity in an established murine model of TNBC.

To test this hypothesis, we utilized a murine TNBC line (E0771) engineered with an OVA antigen to track and quantify tumor-specific immune responses. Consistent with patient samples, we found that TNBC tumors from this model contained significant numbers of T-cell infiltrates, a large proportion of which were CTLA-4+ PD-1+ Foxp3 T-regulatory cells (~66% of CD4+ TILs). We further found that TNBC tumor cells expressed surface PD-L1, which was significantly enhanced by INF-gamma stimulation. While these tumor-bearing mice exhibited systemic T-cell and B-cell immunity to tumor-specific antigens, established TNBC tumors were highly immuno-suppressive and resistant to antigen-specific T-cell attack in vivo. Having established a clinically applicable model of immuno-suppressive TNBC in an immuno-competent setting, we next tested the efficacy of CTLA-4 and PD-1 targeting antibodies, both alone and in combination. We found that delivery of CTLA-4 targeting antibodies elicited a significant anti-tumor response that was mediated by ADCC suppression of Foxp3+ T-regulatory cells in the tumor microenvironment; as well as partially by blockade of CTLA-4 function, which together allowed for more potent tumor-specific T-cell immunity in treated animals. Administration of PD-1 antibodies also inhibited TNBC tumor growth through blockade of PD-L1/PD-1 signaling in T-cells in the TNBC tumor microenvironment. When these therapies were combined, we found that they greatly enhanced anti-tumor responses through significant reduction in Foxp3+ T-regulatory cells, as well as through significantly induction of tumor-specific T-cell responses, due to tandem non-redundant CTLA-4 and PD-1 blockade. Surprisingly, we found that these responses were individually heterogeneous, despite the use of a single cell type in an inbred strain of mouse under identical conditions. Notably, these responses strongly correlated with the strength of the adaptive tumor-specific immune response suggesting that individual microenvironment differences impact the establishment of robust anti-tumor immunity in TNBC. Collectively, our study demonstrates the efficacy of a combined checkpoint inhibitor approach utilizing CTLA-4 and PD-1 in TNBC and suggests that the clinical use of these approved therapies may vary based on individual microenvironmental perturbations that influence tumor immunity.

Citation Format: Erika Crosby, Jun-Ping Wei, Xiao-Yi Yang, Gangjun Lei, Tao Wang, H Kim Lyerly, Zachary C. Hartman. The utility and mechanistic basis of CTLA-4 and PD-1 immunotherapeutic checkpoint inhibitors in enhancing tumor-specific adaptive responses to effectively treat triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B63.