John Wherry

Abstract PR05: Peripheral blood immune profiling of anti-PD-1 therapy in human melanoma reveals a link between T cell re-invigoration and tumor burden that predicts response

Despite the clinical success of PD-1 based therapies in human melanoma patients, the majority of patients do not have durable clinical benefit from anti-PD-1 monotherapy. A major challenge remains identifying which patients will respond to anti-PD-1 therapy and defining the underlying reasons for successful response versus treatment failure. Pre-existing T cell infiltration and/or PD-L1 expression in tumors may predict clinical responses; however, the use of blood-based profiling to understand the immunologic mechanism of PD-1 blockade has been less explored. Here we used detailed immune profiling of peripheral blood from stage IV melanoma patients before and after pembrolizumab (pembro), and identified pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (TEX). Robust induction of Ki67 in this subset of circulating CD8 T cells post-therapy (re-invigoration) occurred in 78% of patients indicating strong, on target immunological effects of PD-1 blockade in most patients studied here. Despite this high immunological response rate, the objective clinical response rate in this cohort was less than 40%. Ki67 in CD8 T cells alone did not predict clinical outcomes and, in fact, higher systemic immune activation at baseline was associated with lower overall survival. Rather, the magnitude of re-invigoration of circulating TEX in relation to pre-treatment tumor burden correlated with clinical response. We identified a TEX re-invigoration to tumor burden ratio which could be used to predict clinical response and overall survival as early as 6 weeks post therapy. Consistent observations were found in a second independent cohort and suggest that clinical failure of PD-1 blockade in many patients may not solely be due to an inability to induce immune re-invigoration but rather, an imbalance between T cell re-invigoration and tumor burden. Thus, by focused profiling of a mechanistically relevant circulating T cell subpopulation calibrated to pre-treatment disease burden, we identify a clinically accessible predictor of response to PD-1 blockade. These findings also provide a framework for dissecting distinct types of treatment failures in melanoma and have implications for stratifying patients into additional immunotherapeutic treatment approaches. Citation Format: Alexander Huang, Michael A. Postow, Robert J. Orlowski, Rosemarie Mick, Bertram Bengsch, Sasi Manne, Wei Xu, Shannon Harmon, Matthew Adamow, Deborah Kuk, Katherine Panangeas, Cristina Carerra, Phillip Wong, Felix Quagliarello, Kristen E. Pauken, Ramin S. Herati, Suzanne McGettigan, Shawn Kothari, Sangeeth M. George, Brandon Wenz, Kurt D9Andrea, Xiaowei Xu, Ravi K. Amaravadi, Giorgos Karakousis, Lynn M. Schuchter, Katherine L. Nathanson, Jedd D. Wolchok, Tara C. Gangadhar, John Wherry. Peripheral blood immune profiling of anti-PD-1 therapy in human melanoma reveals a link between T cell re-invigoration and tumor burden that predicts response [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR05.

Abstract A036: Anti-PD1 therapy and CD8 T cell invigoration in metastatic melanoma

Antibodies targeting the PD-1 pathway can reverse T cell exhaustion resulting in re-invigoration of immune responses. Anti-PD1 therapies such as pembrolizumab have been shown to be efficacious across a broad range of human cancers including melanoma with clinical responses correlating with immune changes in the tumor microenvironment. However, our understanding of the immune mechanism of anti-PD1 therapy in humans remains incomplete. Specifically, there is relatively little information about changes in the differentiation, activation and reversal of exhaustion in peripheral blood CD8 T cells and correlation with clinical outcome. We collected peripheral blood at serial time points before and after pembrolizumab therapy on 39 consecutive patients with Stage IV melanoma and analyzed changes in T cell subsets and differentiation using 16 parameter flow cytometry. Here we show that pembrolizumab treatment results in increases in the peripheral blood CD8/Treg ratio, as well as invigoration of CD8 T cells. Focusing on key CD8 T cell subsets expressing combinations of T-bet, Eomes, PD-1 and other inhibitory receptors has allowed greater focus on populations responding to re-invigoration by pembrolizumab treatment. This invigoration can be demonstrated by upregulation of granzyme B (GzmB) and Ki67 in key CD8 T cell subsets. An early increase in GzmB+Ki67+ cells translates into a later increase in GzmB+ cells suggesting an early wave of proliferation giving rise to a pool of re-invigorated CD8 T cells. T cell subsets that express markers of exhaustion including T-bet, Eomes, and inhibitory receptors may represent populations that are invigorated by anti-PD1 therapy. These changes may also correlate with clinical outcomes and therefore may be useful as a biomarker of response. These results show that T cell responses to pembrolizumab treatment can be tracked in an easily accessible peripheral blood compartment during therapy. The kinetics of immune response in key CD8 T cell subsets may inform us of optimal treatment duration as well as the nature and timing of cancer response to therapy. Citation Format: Alexander Huang, Wei Xu, Shannon Harmon, Felix Quagliarello, Ramin Herati, Kristen Pauken, Bertram Bengsch, Lynn Schuchter, Ravi Amaravadi, Suzanne McGettigan, Tara Gangadhar, John Wherry. Anti-PD1 therapy and CD8 T cell invigoration in metastatic melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A036.

Abstract PR05: Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients

Immune checkpoint inhibitors result in impressive clinical responses but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) on a phase one clinical trial and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Computational analysis of genome-wide and immune profiles of mice revealed resistance was due to T cell exhaustion driven by adaptive resistance and prolonged interferon-gamma exposure, resulting in STAT1-mediated upregulation of PD-L1 on melanoma cells and tumor macrophages. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells, and macrophage depletion and/or PD-L1 blockade reverses T cell exhaustion. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of the T cell receptor (TCR) repertoire. RT with dual checkpoint blockade shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Similar to results from mice, patients on our clinical trial with tumors showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. In contrast, patients with low PD-L1 on melanoma cells or macrophages had markedly improved survival, with the best survival observed among those patients with low PD-L1 on both cell types. Thus, our results suggest that 1) RT can enhance response to anti-CTLA4 when the TCR and/or antigen repertoire are sub-optimal, 2) upregulation of PD-L1 through STAT1-mediated adaptive resistance mechanisms inhibits response to anti-CTLA4-based therapy unless PD-L1/PD-1 is blocked, and 3) the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms. Finally, although PD-L1 was a dominant resistance mechanism in our models, PD-L1-independent resistance mechanisms were also evident. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Joseph Benci, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Steve Hahn, John Wherry, Robert Vonderheide, Andy Minn. Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR05.