Alexander C. Huang

T-cell invigoration to tumour burden ratio associated with anti-PD-1 response

Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.

Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade

Epigenetic profiling suggests that exhausted T cells are a distinct cell linage. The epigenetics of exhaustion During cancer or chronic infection, T cells become dysfunctional, eventually acquiring an “exhausted” phenotype. Immunotherapies aim to reverse this state. Using a mouse model of chronic infection, two studies now show that the epigenetic profile of exhausted T cells differs substantially from those of effector and memory T cells, suggesting that exhausted T cells are a distinct lineage (see the Perspective by Turner and Russ). Sen et al. defined specific functional modules of enhancers that are also conserved in exhausted human T cells. Pauken et al. examined the epigenetic profile of exhausted T cells after immunotherapy. Although there was transcriptional rewiring, the cells never acquired a memory T cell phenotype. Thus, epigenetic regulation may limit the success of immunotherapies. Science, this issue p. 1104, p. 1165; see also p. 1160 Blocking Programmed Death–1 (PD-1) can reinvigorate exhausted CD8 T cells (TEX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX into durable memory T cells (TMEM) is unclear. We found that reinvigoration of TEX in mice by PD-L1 blockade caused minimal memory development. After blockade, reinvigorated TEX became reexhausted if antigen concentration remained high and failed to become TMEM upon antigen clearance. TEX acquired an epigenetic profile distinct from that of effector T cells (TEFF) and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that TEX are a distinct lineage of CD8 T cells. Nevertheless, PD-1 pathway blockade resulted in transcriptional rewiring and reengagement of effector circuitry in the TEX epigenetic landscape. These data indicate that epigenetic fate inflexibility may limit current immunotherapies.

IL-12p70–producing patient DC vaccine elicits Tc1-polarized immunity

BACKGROUND Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients, but dose-limiting toxicities have hindered its incorporation in vaccine formulations. Here, we report on the immunological and clinical outcomes upon vaccination with CD40L/IFN-γ-matured, IL-12p70-producing DCs. METHODS 7 HLA-A*0201+ newly diagnosed stage IV melanoma patients were immunized against the gp100 melanoma antigen using autologous peptide-pulsed, CD40L/IFN-γ-matured DCs. PBMCs were taken weekly for immune monitoring by tetramer analysis and functional assays. CT imaging was performed at baseline, week 9, and week 18 for clinical assessment using RECIST. RESULTS 6 of 7 treated patients developed sustained T cell immunity to all 3 melanoma gp100 antigen-derived peptides. 3 of the 6 immunological responders developed confirmed clinical responses (1 complete remission >4 years, 2 partial response). Importantly, DC vaccine-derived IL-12p70 levels positively correlated with time to progression (P = 0.019, log-rank), as did T-cytotoxic 1 (Tc1) immunity, as assessed by IFN-γ/IL-13 and IFN-γ/IL-5 ratios (P = 0.035 and P = 0.030, respectively, log-rank). In contrast, a pathway-specific defect in IL-12p35 transcription was identified upon CD40L/IFN-γ activation in clinical nonresponder patient DCs, and gp100-specific T cells from these patients displayed a Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ activation protocol corrected the IL-12p70 production defect in DCs derived from clinical nonresponder patients. CONCLUSION These findings underscore the essential role of IL-12p70 in the development of therapeutic type 1 antigen-specific CD8+ T cell immunity in humans with cancer.

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO–CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1–CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.An IL-6/STAT3 signature and memory CD8 T cell subset in preinfusion chimeric antigen receptor–expressing T cells associate with response in patients with high-risk chronic lymphocytic leukemia.

Epigenomic-Guided Mass Cytometry Profiling Reveals Disease-Specific Features of Exhausted CD8 T Cells

SUMMARY Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic‐ and epigenetic‐guided mass cytometry approach to define core exhaustion‐specific genes and disease‐induced changes in Tex cells in HIV and human cancer. Single‐cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co‐expression patterns. An exhaustion severity metric was developed and integrated with high‐dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer. Graphical Abstract Figure. No Caption available. HighlightsUnbiased identification of unique Tex genes using transcriptomics and epigenomicsHigh‐dimensional CyTOF profiling of human Tex gene products reveals heterogeneityIdentification of key disease‐relevant Tex cell populations in HIV and lung cancerDevelopment of exhaustion metrics applicable to human immune monitoring &NA; Exhausted T (Tex) cells have poor function in chronic infections and cancer but can be therapeutically re‐invigorated. Bengsch et al. use genes modified epigenetically during exhaustion and high‐dimensional CyTOF profiling to define Tex cell heterogeneity in humans with HIV or lung cancer and link Tex cell features to disease progression and response to immunotherapy.

Feasibility of monitoring advanced melanoma patients using cell-free DNA from plasma

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell‐free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra‐deep sequencing for a 61‐gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next‐generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%.

A phase I trial of pembrolizumab with hypofractionated radiotherapy in patients with metastatic solid tumours

BackgroundWe conducted a phase I trial evaluating pembrolizumab+hypofractionated radiotherapy (HFRT) for patients with metastatic cancers.MethodsThere were two strata (12 patients each): (i) NSCLC/melanoma progressing on prior anti-PD-1 therapy, (ii) other cancer types; anti-PD-1-naive. Patients received 6 cycles of pembrolizumab, starting 1 week before HFRT. Patients had ≥2 lesions; only one was irradiated (8 Gy × 3 for first half; 17 Gy × 1 for second half in each stratum) and the other(s) followed for response.ResultsOf the 24 patients, 20 (83%) had treatment-related adverse events (AEs) (all grade 1 or 2). There were eight grade 3 AEs, none treatment related. There were no dose-limiting toxicities or grade 4/5 AEs. Stratum 1: two patients (of 12) with progression on prior PD-1 blockade experienced prolonged responses (9.2 and 28.1 months). Stratum 2: one patient experienced a complete response and two had prolonged stable disease (7.4 and 7.0 months). Immune profiling demonstrated that anti-PD-1 therapy and radiation induced a consistent increase in the proliferation marker Ki67 in PD-1-expressing CD8 T cells.ConclusionsHFRT was well tolerated with pembrolizumab, and in some patients with metastatic NSCLC or melanoma, it reinvigorated a systemic response despite previous progression on anti-PD-1 therapy. Clinical Trial Registration: NCT02303990 (www.clinicaltrials.gov).

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 B104: Impact of PD-1 blockade on epigenetic and transcriptional reprogramming of exhausted T cells

Blocking PD-1 can reinvigorate exhausted CD8 T cells and improve control of chronic infections and cancer. One potential advantage of this immunotherapy is durable protection if immune memory can be established. It is unclear, however, whether blocking PD-1 can reprogram exhausted CD8 T cells into effector or durable memory CD8 T cells. Here, we found reinvigoration of exhausted T cells by PD-L1 blockade caused re-acquisition of some features of effector T cells, but minimal memory development. Indeed, after checkpoint blockade, reinvigorated T cells became re-exhausted if antigen remained high, and failed to become memory T cells upon antigen clearance. Exhausted T cells acquired an epigenetic profile distinct from effector and memory T cells that was minimally altered by blocking PD-L1. Nevertheless, modest enhancer changes resulted in transcriptional network rewiring that may provide opportunities to enhance checkpoint blockade. These data indicate that epigenetic fate inflexibility may limit current immunotherapies and suggest that improving (re)differentiation to memory following PD-1 pathway blockade could enhance clinical outcomes. Citation Format: Kristen E. Pauken, Morgan A. Sammons, Pamela M. Odorizzi, Sasi K. Manne, Jernej Godec, Omar Khan, Debattama Sen, Makoto Kurachi, R. Anthony Barnitz, Bertram Bengsch, Alexander C. Huang, Jason M. Schenkel, Golnaz Vahedi, W. Nicholas Haining, Shelley L. Berger, E. John Wherry. Impact of PD-1 blockade on epigenetic and transcriptional reprogramming of exhausted T cells [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 B104.

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.