The advent of immune checkpoint blockade as a new strategy for immunotherapy has changed the outlook for many aggressive cancers. Although complete tumor eradication is attainable in some cases, durable clinical responses are observed only in a small fraction of patients, underlining urgent need for improvement. We previously showed that RON, a receptor tyrosine kinase expressed in macrophages, suppresses antitumor immune responses, and facilitates progression and metastasis of breast cancer. Here, we investigated the molecular changes that occur downstream of RON activation in macrophages, and whether inhibition of RON can cooperate with checkpoint immunotherapy to eradicate tumors. Activation of RON by its ligand, MSP, altered the gene expression profile of macrophages drastically and upregulated surface levels of CD80 and PD-L1, ligands for T-cell checkpoint receptors CTLA-4 and PD-1. Genetic deletion or pharmacological inhibition of RON in combination with anti-CTLA-4, but not with anti-PD-1, resulted in improved clinical responses against orthotopically transplanted tumors compared to single-agent treatment groups, resulting in complete tumor eradication in 46% of the animals. Positive responses to therapy were associated with higher levels of T-cell activation markers and tumor-infiltrating lymphocytes. Importantly, co-inhibition of RON and anti-CTLA-4 was also effective in clearing metastatic breast cancer cells in lungs, resulting in clinical responses in nearly 60% of the mice. These findings suggest that RON inhibition can be a novel approach to potentiate responses to checkpoint immunotherapy in breast cancer.

Cancer immunotherapy continues to make headway as a treatment for advanced stage tumors, revealing an urgent need to understand the fundamentals of anti-tumor immune responses. Noteworthy is a scarcity of data pertaining to the breadth and specificity of tumor-specific T cell responses in metastatic breast cancer. Autochthonous transgenic models of breast cancer display spontaneous metastasis in the FVB/NJ mouse strain, yet a lack of knowledge regarding tumor-bound MHC/peptide immune epitopes in this mouse model limits the characterization of tumor-specific T cell responses, and the mechanisms that regulate T cell responses in the metastatic setting. We recently generated the NetH2pan prediction tool for murine class I MHC ligands by building an FVB/NJ H-2q ligand database and combining it with public information from six other murine MHC alleles. Here, we deployed NetH2pan in combination with an advanced proteomics workflow to identify immunogenic T cell epitopes in the MMTV-PyMT transgenic model for metastatic breast cancer. Five unique MHC I/PyMT epitopes were identified. These tumor-specific epitopes were confirmed to be presented by the class I MHC of primary MMTV-PyMT tumors and their T cell immunogenicity was validated. Vaccination using a DNA construct encoding a truncated PyMT protein generated CD8 + T cell responses to these MHC class I/peptide complexes and prevented tumor development. In sum, we have established an MHCligand discovery pipeline in FVB/NJ mice, identified and tracked H-2D q /PyMT neoantigen-specific T cells, and developed a vaccine that prevents tumor development in this metastatic model of breast cancer.

Metastasis is the cause of death for nearly all types of cancer, including breast cancer. An exciting new area of research in metastatic breast cancer centers on immune therapy. Although new immune checkpoint blockade therapies have provided benefit for a fraction of patients tested so far, the majority of patients still do not respond to these drugs. A better understanding of how the immune system can be harnessed against metastatic breast cancer is required in order to improve patient outcomes in this area. We previously discovered that macrophage Ron receptor tyrosine kinase promotes breast cancer metastasis by inhibiting CD8+ cytotoxic T lymphocyte activity. We hypothesized that dual blockade of Ron activity and existing immune checkpoint molecules would unleash a more effective CD8+ T cell response to control or eliminate metastatic breast cancer. Our strategy would simultaneously disable tumor-mediated immune evasion mechanisms on both the innate and adaptive immune systems. To test the potential of combination therapy, the Ron inhibitor BMS-777607 and/or the immune checkpoint blocking agents anti-PD1 or anti-CTLA4, were administered in our mouse mammary tumor model to examine the effects on breast tumor progression. To examine Ron-specific effects of BMS-777607, we also examined the effect of genetic deletion of host Ron signaling activity in combination with immunotherapy. The anti-tumor immune response was comprehensively examined by multi-color flow cytometry and immunohistochemical staining for tumor-infiltrating lymphocytes (TILs). Our data suggest that the combination of Ron inhibition with immune checkpoint blockade may be an effective therapy in breast cancer. Citation Format: Shu Chin Alicia Lai, Atakan Ekiz, Alana Welm. Ron kinase inhibition to improve immunotherapy for breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A30.