Coffey, Matt C.

2562 Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal cancer with limited immunotherapeutic options. Pelareorep (pela) is an intravenously delivered unmodified reovirus containing a double stranded RNA genome that has been studied as an immunotherapeutic in multiple cancers including breast, anal, colorectal and pancreatic. We previously reported high tumor response rates in first-line metastatic PDAC patients treated with pela combined with gemcitabine, nab-paclitaxel and atezolizumab. We report here the immunologic effects of pela in a cohort of first-line metastatic PDAC subjects treated with pela plus chemotherapy and atezolizumab and the correlation of these effects with tumor response. Methods: To examine its effects on pancreatic cancer, a phase 1/2 Simon 2-stage platform study (GOBLET) was performed that included patients with first-line locally advanced/metastatic unresectable PDAC. Anti-reovirus T cell activity was assessed by interferon gamma secretion (ELISPOT). Changes in the expression of plasma proteins were analyzed by Olink Response panels. T cell receptor sequencing (TCR-seq; ImmunoSEQ Assay, Adaptive Biotechnologies) was performed on tissue collected prior to the start of therapy and on blood from baseline through 3 treatment cycles to identify TILs expansion. Tumor responses were scored according to the modified RECIST v1.1 criteria. Results: Increases in anti-reovirus T cell activation as determined by ELISPOT after cycle 3 of therapy were observed in 6/8 subjects. Three subjects with maximum responses (> 300 spots) showed >30% decreases in tumor volume. Significant changes in plasma proteins as determined by Olink included PD-L1, CXCL9, CXCL10, CXCL11 and IFN-γ. Pre-treatment tumor tissue was used to identify the TIL clonal populations prior to therapy. Increased TIL clones in the blood pre-treatment was associated with tumor responses. Pela treatment increased the expansion of pre-existing and new TIL clones in the blood after one cycle of treatment. Sustained increases in pre-existing TIL clonal populations in the blood through cycle 3 of therapy were observed in subjects who exhibited reductions in tumor volume. Conclusions: These findings, while preliminary, demonstrate that pela induces not only anti-reovirus T cells but also activates innate and adaptive anti-tumor immunity in PDAC subjects treated with chemotherapy and atezolizumab. Tumor responses are associated with both the presence of TIL clones in the blood prior to treatment and the expansion of pre-existing TILs in the blood on treatment. These findings provide additional insights into the immunologic mechanisms by which pela-based therapy may provide clinical benefit in patients with metastatic PDAC.

Abstract Background Brain tumors are the leading cause of cancer death for pediatric patients. Pelareorep, an immunomodulatory oncolytic reovirus, has intravenous efficacy in preclinical glioma models when preconditioned with GM-CSF (sargramostim). We report a phase I trial with the primary goal of evaluating the safety of sargramostim/pelareorep in pediatric patients with recurrent or refractory high-grade brain tumors and a secondary goal of characterizing immunologic responses. Methods The trial was open to pediatric patients with recurrent or refractory high-grade brain tumors (3 + 3 cohort design). Each cycle included 3 days of subcutaneous sargramostim followed by 2 days of intravenous pelareorep. Laboratory studies and imaging were acquired upon recruitment and periodically thereafter. Results Six patients participated, including three glioblastoma, two diffuse intrinsic pontine glioma, and one medulloblastoma. Two pelareorep dose levels of 3 × 108 and 5 × 108 tissue culture infectious dose 50 (TCID50) were assessed. One patient experienced a dose limiting toxicity of persistent hyponatremia. Common low-grade (1 or 2) adverse events included transient fatigue, hypocalcemia, fever, flu-like symptoms, thrombocytopenia, and leukopenia. High-grade (3 or 4) adverse events included neutropenia, lymphopenia, leukopenia, hypophosphatemia, depressed level of consciousness, and confusion. All patients progressed on therapy after a median of 32.5 days and died a median of 108 days after recruitment. Imaging at progression did not show evidence of pseudoprogression or inflammation. Correlative assays revealed transient but consistent changes in immune cells across patients. Conclusions Sargramostim/pelareorep was administered to pediatric patients with recurrent or refractory high-grade brain tumors. Hyponatremia was the only dose limiting toxicity (DLT), though maximum tolerated dose (MTD) was not determined.

Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)–modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

10524 Background: Reolysin is a Dearing strain, naturally occurring, ubiquitous human reovirus. The PKR (double stranded RNA-activated protein kinase) is inhibited and therefore the virus replicates specifically in transformed cells possessing an activated Ras pathway producing lysis. In vitro and in vivo studies with Reolysin in sarcoma cell lines revealed significant antitumor activity. Methods: This phase II open-label, single agent study was designed to characterize the efficacy and safety of Reolysin given IV every 28 days in patients (pts) with bone or soft tissue sarcoma with lung metastasis using a Simon two-stage design. 38 pts were accrued to the first stage. If 1 or more pts experience clinical benefit (prolonged SD > 6 months, partial or complete response) up to 52 pts could be accrued. The agent will be considered active if 3 or more responses or prolonged SD are observed. Results: Since July 2007, 43 pts age 19–76 (median 49) were enrolled (20 female) and received a total of 141 cycles (range 1–18). All pts had performance status 1 (29 pts) or 0 (14 pts). 38 pts received prior chemotherapy, radiotherapy, biological agents or combinations for their metastatic disease, 15 pts received more than 3 chemotherapy regimens. The sarcoma subtypes included: synovial sarcoma (13 pts), osteosarcoma (7 pts), leiomyosarcoma (7 pts), MFH (5 pts), Ewing/PNET (1 pt), chordoma (1 pt), others (9 pts). Side effects were mild to moderate (grade 1–2) and included constitutional symptoms fever, chills, fatigue. Two pts experienced respiratory side effects (cough and dyspnea) and 2 pts had diarrhea. Hematological side effects included grade 2–3 neutropenia (6 pts) and grade 2 thrombocytopenia (2 pts). One patient experienced grade 2 AST elevation. 33 pts are evaluable for response to date: 14 pts (42%) had SD for 2+ months including 5 pts having SD for more than 6 months. Conclusions: Utilization of single agent reovirus for treatment of sarcoma is a novel and unique therapeutic approach to date. Reolysin is well tolerated and shows promise for the treatment of metastatic sarcoma. Primary efficacy goals have been met. Accrual is ongoing to a total of 52 pts. [Table: see text]

Abstract Antitumor T-cell responses raised by first-line therapies such as chemotherapy, radiation, tumor cell vaccines, and viroimmunotherapy tend to be weak, both quantitatively (low frequency) and qualitatively (low affinity). We show here that T cells that recognize tumor-associated antigens can directly kill tumor cells if used at high effector-to-target ratios. However, when these tumor-reactive T cells were present at suboptimal ratios, direct T-cell–mediated tumor cell killing was reduced and the ability of tumor cells to evolve away from a coapplied therapy (oncolytic or suicide gene therapy) was promoted. This T-cell–mediated increase in therapeutic resistance was associated with C to T transition mutations that are characteristic of APOBEC3 cytosine deaminase activity and was induced through a TNFα and protein kinase C–dependent pathway. Short hairpin RNA inhibition of endogenous APOBEC3 reduced rates of tumor escape from oncolytic virus or suicide gene therapy to those seen in the absence of antitumor T-cell coculture. Conversely, overexpression of human APOBEC3B in tumor cells enhanced escape from suicide gene therapy and oncolytic virus therapy both in vitro and in vivo. Our data suggest that weak affinity or low frequency T-cell responses against tumor antigens may contribute to the ability of tumor cells to evolve away from first-line therapies. We conclude that immunotherapies need to be optimized as early as possible so that, if they do not kill the tumor completely, they do not promote treatment resistance.

Abstract The oncolytic reovirus (RV) has demonstrated clinical efficacy and minimal toxicity in a variety of cancers, including multiple myeloma (MM). MM is a malignancy of plasma cells that is considered treatable but incurable because of the 90% relapse rate that is primarily from drug resistance. The systemic nature of MM and the antitumor immunosuppression by its tumor microenvironment presents an ongoing therapeutic challenge. In the present study, we demonstrate that RV synergizes with the standard-of-care MM drug bortezomib (BTZ) and, importantly, enhances its therapeutic potential in therapy-resistant human MM cell lines in vitro. Using the syngeneic Vk*MYC BTZ-resistant immunocompetent transplantable MM murine model, we also demonstrate that mice harboring BTZ-insensitive MM tumors respond to the RV/BTZ combination treatment in terms of decreased tumor burden and improved overall survival (P < .00001). We demonstrate that BTZ augments RV replication in tumor-associated endothelial cells and myeloma cells, leading to enhanced viral delivery and thereby stimulating cytokine release, immune activity, apoptosis, and reduction of the MM-associated immune suppression. We conclude that combined RV/BTZ is an attractive therapeutic strategy with no safety signals for the treatment of MM.

AbstractUnderstanding how incompletely cleared primary tumors transition from minimal residual disease (MRD) into treatment-resistant, immune-invisible recurrences has major clinical significance. We show here that this transition is mediated through the subversion of two key elements of innate immunosurveillance. In the first, the role of TNFα changes from an antitumor effector against primary tumors into a growth promoter for MRD. Second, whereas primary tumors induced a natural killer (NK)–mediated cytokine response characterized by low IL6 and elevated IFNγ, PD-L1hi MRD cells promoted the secretion of IL6 but minimal IFNγ, inhibiting both NK-cell and T-cell surveillance. Tumor recurrence was promoted by trauma- or infection-like stimuli inducing VEGF and TNFα, which stimulated the growth of MRD tumors. Finally, therapies that blocked PD-1, TNFα, or NK cells delayed or prevented recurrence. These data show how innate immunosurveillance mechanisms, which control infection and growth of primary tumors, are exploited by recurrent, competent tumors and identify therapeutic targets in patients with MRD known to be at high risk of relapse. Cancer Immunol Res; 5(11); 1029–45. ©2017 AACR.

Abstract We have developed the use of reovirus as a systemically delivered oncolytic agent in both pre-clinical models and in Phase I and II clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, partly because of disruption of the PKR-mediated anti-viral response in malignant cells. In addition however, we have shown that anti-tumor therapy is directly associated with immune activation by virus replication in tumors. The immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, as well as the generation of adaptive anti tumor immune responses as a result of in vivo priming against tumor associated antigens released during that killing. Therefore, to exploit the immune components of reovirus anti-tumor therapy, we tested the combination of oncolytic therapy with systemic checkpoint inhibition. To do this, we used our murine, immune-competent, model and a protocol in which injection of reovirus into subcutaneous (s.c.) B16 melanomas generates moderate therapy following intra-tumoral injection of reovirus into 5d established subcutaneous B16 melanomas growing in C57Bl/6 mice. In this model, provision of systemic anti-PD-1 antibody along with i.t. reovirus, significantly enhanced survival compared to i.t. reovirus alone (p<0.01) and led to >40% of mice being cured long term. Immune analysis suggests that the enhanced therapeutic benefit of reovirus plus checkpoint inhibition is contributed by at least two factors. Thus, blockade of PD-1 significantly enhanced the ability of NK cells to recognize (TNF-α secretion), and kill, reovirus-infected target tumor cells. Second, antiPD-1 antibody led to a significant reduction in Treg activity in reovirus-treated mice, with the overall effect of increasing the adaptive CD8+ anti tumor T cell response. We also showed that timing of the checkpoint inhibitor antibody was significant in balancing toxicity with anti tumor therapy. These data suggest that combination of checkpoint inhibition therapy with reovirus oncolytic/immunotherapy represents a readily translatable method to enhance the therapeutic value of either alone. Citation Format: Shane Zaidi, Kevin Shim, Karishma Rajani, Rosa Diaz, Elizabeth Ilett, Timothy Kottke, Jill Thompson, Peter Selby, Kevin Harrington, Hardev Pandha, Alan Melcher, Matt Coffey, Richard Vile. Combination therapy of reovirus and checkpoint inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B88.

Abstract Reovirus is a systemically delivered oncolytic agent with evidence of activity in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, as well as activating anti-tumor innate and adaptive immunity. Having previously shown that intravenously delivered reovirus selectively accesses colorectal cancer metastatic to the liver in patients, in this study we explored the potential of systemic reovirus for the treatment of both primary and secondary brain tumors. We first showed that intravenous reovirus can be detected in tumors implanted into the brains of immunocompetent mice. We then tested our current most potent reovirus-based therapy (systemic reovirus plus granulocyte-macrophage colony stimulating factor [GMCSF] in reovirus pre-immune mice), and showed effective therapy in immunocompetent murine models of primary glioma, and of both directly reovirus sensitive, and insensitive, models of melanoma brain metastases. We also found that addition of reovirus/GMCSF to clinical ‘standard of care’ (radiotherapy and temozolamide chemotherapy) significantly enhances survival. In parallel to these pre-clinical experiments, we have initiated an open-label, non-randomized study of intravenous reovirus administered to patients prior to planned surgery for recurrent high grade glioma or metastatic brain tumors, to test whether the data showing access of the agent to tumors in the brains in mice, also applies to humans. In total, 10 patients will be treated with a single infusion of 1x1010 TCID50 of reovirus, of which 9 have completed the study to date. The primary objective is the presence of reovirus in the resected tumors as assessed by immunohistochemistry, RNA in-situ hybridization and retrieval of infectious virions. Early analysis of the first 3 patients, comprising one glioblastoma multiforme, one grade 3 oligodendroglioma and one colorectal brain metastasis, has shown that all 3 resected tumors contained reovirus RNA and protein. There was also evidence for productive reovirus infection in 2 of the tumors. Within all patients to date, the only grade 3-4 adverse reaction has been neutropaenia in 1 patient. Further tissue analysis is ongoing, as is testing of blood samples from these patients to further charactize how reovirus is carried in the blood and protected from neutralizing antibodies. This clinical study shows, for the first time that an oncolytic virus, reovirus, infects and replicates in brain tumors following intravenous administration. Together with pre-clinical data showing the efficacy of systemic reovirus in combination with GMCSF/radiation/temozolamide, these findings support the future development of trials and combination studies using reovirus in patients with high grade gliomas and brain metastases. Citation Format: Adel Jebar, Liz Ilett, Tim Kottke, Emma West, Karen Scott, Simon Thomson, Matt Coffey, Gerard Nuovo, Susan Short, Richard Vile, Alan Melcher. Systemic oncolytic reovirus for the treatment of primary and secondary brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A49.

Abstract We have developed the use of reovirus as a systemically delivered oncolytic agent in both pre-clinical models and in early Phase clinical trials. Reovirus has direct oncolytic activity against many human/murine tumor cells, partly because of disruption of the PKR-mediated anti-viral response in malignant cells. In addition however, we have shown that anti-tumor therapy is directly associated with immune activation by virus replication in tumors. The immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, as well as the generation of adaptive anti tumor immune responses as a result of in vivo priming against tumor associated antigens released during that killing. Therefore, to exploit the immune components of reovirus anti-tumor therapy, we hypothesized that the combination of reovirus therapy with systemic checkpoint inhibition would augment therapeutic efficacy. To test the hypothesis, we used C57Bl/6 mice, an immune-competent murine model, with established subcutaneous (s.c.) B16 melanomas. In this model, intra-tumoral injection of reovirus into s.c. tumors generated moderate therapy. Provision of systemic anti-PD-1 antibody along with i.t. reovirus, significantly enhanced survival compared to i.t. reovirus alone (p<0.01) and led to >40% of mice being cured long term. Immune analysis suggested that the enhanced therapeutic benefit of reovirus plus checkpoint inhibition is contributed by at least two factors. First, blockade of PD-1 significantly enhanced the ability of NK cells to recognize (TNF-α secretion), and kill, reovirus-infected target tumor cells. Second, anti PD-1 antibody led to a significant reduction in Treg activity in reovirus-treated mice, with the overall effect of increasing the adaptive CD8+ anti-tumor T cell response. Furthermore, in vivo depletion studies demonstrated that NK cells had a dramatic effect in reducing the therapeutic efficacy of reovirus plus anti-PD-1 therapy. Overall, the results indicate that combination therapy of reovirus with PD-1 blockade confers significant survival benefit, by augmenting tumor-specific NK responses and specifically attenuating tumor-specific immunosuppression. These data also suggest that combination of PD-1 inhibition therapy with reovirus oncolytic/immunotherapy represents a readily translatable method to enhance the therapeutic efficacy. Citation Format: Karishma Rajani, Christopher Parrish, Kevin Shim, Liz Ilett, Fiona Errington-Mais, Jill Thompson, Tim Kottke, Rosa Maria-Diaz, Peter Selby, Hardev Pandha, Kevin Harrington, Alan Melcher, Matt Coffey, Shane Zaidi, Richard Vile. Combination therapy of reovirus and PD-1 blockade effectively establishes tumor control via innate and adaptive immune responses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1360. doi:10.1158/1538-7445.AM2015-1360