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The Macrophage Migration Inhibitory Factor (MIF) is an inflammatory cytokine that is overexpressed in a number of cancer types, with increased MIF expression often correlating with tumor aggressiveness and poor patient outcomes. In this study, we aimed to better understand the link between primary tumor expression of MIF and increased tumor growth. Using the MMTV-PyMT murine model of breast cancer, we observed that elevated MIF expression promoted tumor appearance and growth. Supporting this, we confirmed our previous observation that higher MIF expression supported tumor growth in the 4T1 murine model of breast cancer. We subsequently discovered that loss of MIF expression in 4T1 cells led to decreased cell numbers and increased apoptosis in vitro under reduced serum culture conditions. We hypothesized that this increase in cell death would promote detection by the host immune system in vivo, which could explain the observed impairment in tumor growth. Supporting this, we demonstrated that loss of MIF expression in the primary tumor led to an increased abundance of intra-tumoral IFNgamma-producing CD4+ and CD8+ T cells, and that depletion of T cells from mice bearing MIF-deficient tumors restored growth to the level of MIF-expressing tumors. Furthermore, we found that MIF depletion from the tumor cells resulted in greater numbers of activated intra-tumoral dendritic cells (DCs). Lastly, we demonstrated that loss of MIF expression led to a robust induction of a specialized form of cell death, immunogenic cell death (ICD), in vitro. Together, our data suggests a model in which MIF expression in the primary tumor dampens the anti-tumor immune response, promoting tumor growth.

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Summary T cells use highly diverse receptors (TCRs) to identify tumor cells presenting neoantigens arising from genetic mutations and establish anti-tumor activity. Immunotherapy harnessing neoantigen-specific T cells to target tumors has emerged as a promising clinical approach. To assess whether a comprehensive peripheral mononuclear blood cell analysis predicts responses to a personalized neoantigen cancer vaccine combined with anti-PD-1 therapy, we characterize the TCR repertoires and T and B cell frequencies in 21 patients with metastatic melanoma who received this regimen. TCR-α/β-chain sequencing reveals that prolonged progression-free survival (PFS) is strongly associated with increased clonal baseline TCR repertoires and longitudinal repertoire stability. Furthermore, the frequencies of antigen-experienced T and B cells in the peripheral blood correlate with repertoire characteristics. Analysis of these baseline immune features enables prediction of PFS following treatment. This method offers a pragmatic clinical approach to assess patients’ immune state and to direct therapeutic decision making.

Background: Neoantigens arise from DNA mutations in cancer cells and are important targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides designed based on a patient’s neoantigen and HLA profile that is directed at inducing tumor-specific T cell responses to neoantigens. Here, we report relationships between baseline tumor characteristics, immune response and clinical outcome for NT-001, a Phase 1b study of NEO-PV-01 + adjuvant in combination with nivolumab in either first or later line therapy for patients with metastatic melanoma, bladder and non-small cell lung cancer (NCT02897765). This analysis is focused on the melanoma cohort. Methods: Serial blood and tumor biopsies were scheduled for collection: i) prior to treatment, ii) after 12 weeks of nivolumab monotherapy and iii) after completion of NEO-PV-01 vaccination. Baseline features and immune responses in tumor cells were characterized by immunohistochemistry for multiple immune and tumor markers, gene expression, whole exome and TCR sequencing. Antigen-specific responses by IFNγ ELISpot, intracellular cytokine staining, multi-parameter surface and functional phenotyping by FACS and the presence of cytolytic properties were monitored in serial peripheral blood samples. Durability of immune responses up to 104 weeks post start of treatment will be measured. Results: A cohort of 10 melanoma patients demonstrated CD4 and CD8 T cell responses against 56% of vaccine peptides that were detected primarily in the post-vaccination samples as measured by IFNγ ELISpot. Analysis of additional patients is ongoing. T cell responses were neoantigen-specific for most peptides tested (86%; 12/14). Vaccine-induced immune responses were durable in patients who reached the week 52 treatment timepoint. Most T cell responses were polyfunctional, as evident by secretion of multiple cytokines, exhibited a memory and effector memory phenotype and were cytolytic. Epitope spreading, defined as post-vaccination T cell responses to neoantigens not included in the vaccine, was observed in multiple melanoma patients analyzed and evaluated for association with post vaccine clinical responses. Further, multi-platform assessments of immune and molecular responses including gene expression and TCR repertoire analysis demonstrate extensive responses in patients continuing study past 52 weeks. Additional correlates of clinical outcomes with molecular and immunologic responses will be presented. Conclusions: Treatment with NEO-PV-01 + adjuvant in combination with nivolumab induced broad de novo neoantigen-specific immune responses in metastatic melanoma. Immune responses were specific and correlations with clinical outcomes will be discussed. Citation Format: Siwen Hu-Lieskovan, Patrick A. Ott, Aung Naing, Rana H. Besada, Samantha J. Gates, Victoria R. Kohler, Riley R. Curran, Meghan E. Bushway, Julian Scherer, Kristen N. Balogh, Tracey E. Sciuto, Ying S. Ting, Michael S. Rooney, Dewi Harjanto, Zhengping Huang, Yuting Huang, Yvonne Ware, April Lamb, Lisa D. Cleary, Melissa A. Moles, Richard B. Gaynor, Matthew J. Goldstein, Les H. Brail, Joel Greshock, Lakshmi Srinivasan. The personalized vaccine, NEO-PV-01 with anti-PD1, induces neoantigen-specific de novo immune responses in patients with advanced metastatic melanoma: Association with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 942.