Despite a lack of unifying drivers in Triple-Negative Breast Cancer (TNBC), our lab and others have uncovered that these cancers have elevated expression of inflammatory genes and immunosuppressive molecules (i.e. PD-L1), as well as elevated numbers of infiltrating immune cells (including CD8+ T-cells and Foxp3+ T-regulatory cells) which suggests the therapeutic potential for single and combinations of checkpoint blockade antibodies. While early trials with PD-1 inhibitors have been encouraging for TNBC, only a fraction of treated patients respond to this therapy. To test and define the mechanisms that govern responses, 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. Consistent with patient samples, we found TNBC tumors from our model exhibited elevated PD-1+ expressing CD8+ T-cell infiltrates, Foxp3+ T-regulatory cell infiltrates (~66% of CD4+ TILs), as well as highly elevated tumor cell expression of PD-L1. We also found that while TNBC cells were easily killed by T-cell in vitro, TNBC tumors were highly immuno-suppressive and resistant to antigen-specific T-cell attack in vivo, even after adoptive transfer of up to 5x10E6 tumor-specific T-cells. However, we found that both CTLA-4 and PD-1 antibodies could curtail this immunosuppression to different degrees and through alternate mechanisms. Specifically, we found that CTLA-4 antibody mediated anti-tumor immunity through the elimination and blockade of Foxp3+ T-regulatory cells in the tumor microenvironment, which allow for potent T-cell expansion. Conversely, PD-1 antibodies elicited anti-tumor immunity through blockade of PDL1/PD1 signaling between tumor cells and T-cells in the TNBC tumor microenvironment that allowed for a more modest expansion of individually tailored T-cell specific clones in vivo. Strikingly, the combination of these antibodies and their alternate mechanisms of action resulted in greatly enhanced anti-tumor responses and led to regression of ~80% of tumors. This was accompanied by an augmented infiltration of T-cells into the tumor microenvironment and significantly enhanced systemic tumor-specific T-cell responses, which appear to be emergent properties of dual CTLA-4/PD-1 antibody treatment. However, we found that these treatments did not expand a common tumor-specific T-cell clone, despite adoptive transfer of identical tumor-specific immunodominant T-cells into mice after tumor implantation. Thus, despite our use of a highly homogeneous model utilizing genetically identical mice implanted with an identical tumor line bearing a unique tumor antigen under identical conditions, the tumor-specific T-cell responses were highly unique for each individual tumor. Collectively, our study suggest that dual blockade could be an effective therapeutic clinical strategy against TNBC and further suggest the utility of monitoring systemic immune response and TCR expansion of TILs as the most useful correlates in clinical studies utilizing CTLA-4 and PD-1 antibodies. Citation Format: Hartman ZC, Crosby EJ, Wei J-P, Yang X-Y, Lei G-J, Wang T, Liu C-X, Agarwal P, Morse MS, Lyerly HK. CTLA-4 and PD-1 checkpoint inhibitors enhance individually tailored adaptive anti-tumor immune responses to overcome tumor immunosuppression and effectively treat triple-negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-27.

Background: Immune-based therapy for metastatic breast cancer has had limited success. Strategies to augment adaptive immunity include vaccines targeting genomic amplifications like Human Epidermal Growth Factor Type 2 (HER2), an established driver of malignancy. Using a novel alphaviral vector, we constructed a vaccine encoding a portion of HER2 (VRP-HER2). Methods: In preclinical studies, mice were immunized before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and anti-tumor function were assessed. We then translated this vaccine into a phase I clinical trial in which subjects with advanced HER2-overexpressing breast cancers received VRP-HER2 every 2 weeks for a total of three doses (cohort 1). In cohort 2, subjects received the same dose of VRP-HER2 along with a standard HER2 targeted therapy. Results: VRP-HER2 induced HER2-specific T cell and antibody responses while controlling tumor growth in murine models. Vaccination with VRP-HER2 was well tolerated in both patient cohorts. PFS was modest, while median OS was 50.2 months in cohort 1 and 32.7 months in cohort 2. In cohort 2, there is one partial response and two patients with continued stable disease. Vaccine induced anti-HER2 antibodies and T cells were identified. Increased perforin expression by memory CD8 T cells post vaccination significantly correlated with improved PFS. Conclusions: VRP-HER2 led to an increase in perforin expressing HER2-specific memory CD8 T cells in preclinical and clinical studies, and had profound antitumor effects in murine models. The generation of HER2-specific memory CD8 T cells was significantly correlated with increased PFS in patients. Subsequent studies will seek to enhance T cell activity by combination with anti-PD-1/PD-L1 antibodies. Citation Format: Crosby EJ, Gwin WR, Chang S, Maecker HT, Lubkov V, Snyder JC, Broadwater G, Hyslop T, Osada T, Hobeika AC, Hartman ZC, Morse MA, Lyerly HK. CD8 T cells induced by novel alphaviral vector predict improved progression free survival in advanced HER2+ breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-16.