Citations

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 Background: Phase 3 clinical results demonstrate that nab-paclitaxel (ABX) exhibits single agent activity in patients with metastatic melanoma relative to dacarbazine. Additionally, phase 2 clinical data suggest that ABX combined with bevacizumab (BEV) with/without carboplatin exhibits greater clinical efficacy than ABX alone. We hypothesized that the latter may be due to a pharmacokinetic, non-covalent interaction between bevacizumab (BEV) and nab-paclitaxel (ABX) allowing for greater localization of ABX to sites of VEGF overproduction (metastases). Herein we present pre-clinical data describing the utility of experimentally created ABX/BEV complex nanoparticles used to deliver ABX to VEGF producing cancer targets. Methods and Results: To test whether BEV binds to ABX we employed flow cytometry and discovered that the BEV does bind to ABX while preserving the ability of BEV to bind its ligand, VEGF. To measure binding affinity we used Biolayer Interferometry (BLItz) to measure association and dissociation constants. Results of BLItz experiments demonstrate that the binding affinity of BEV to ABX is pH and temperature dependent. The size and stability of the nanoparticle formed by binding BEV and ABX was determined using light refraction (Mastersizer) and Brownian motion (Nanosight). Results of these experiments suggest that a range of nanoparticles can be produced and stability of the BEV particle is increased relative to ABX particles alone. Finally the tumor efficacy of the BEV/ABX complex relative to ABX and BEV as well as sequential administration of the two drugs was tested in a xenograft model of A375 human melanoma cell line. Pharmacokinetic studies in these mice suggest that more ABX is deposited in the tumor site when treated with the complexes relative to ABX alone. Conclusions: The therapeutic antibody BEV has the capability to bind to the chemotherapy drug ABX while still binding its ligand, VEGF. The resulting nanoparticle is more stable than the original ABX particle. In a preclinical model of human A375 xenografts in nude mice, AB160 treatment offers significantly improved antitumor activity beyond that of ABX, BEV or ABX+BEV with no additional toxicity. Initial results indicate that the improved efficacy of the ABX/BEV nanoparticle relative to ABX alone is primarily due to the ability of the anti-VEGF antibody (BEV) to target the chemotherapy agent to the tumor where local VEGF concentration is very high. Clinical translation of the safety of a 160nm AB particle (AB160) in patients with metastatic melanoma is in progress. Citation Format: Wendy K. Nevala, Svetomir N. Markovic. Targeted nano-immune conjugates to melanoma: Preclinical testing of bevacizumab targeted nab-paclitaxel. [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 B77.

Abstract Background: The metastatic advancement of melanoma is largely dependent on its ability to evade immunity and gain access to the lymphatics. Characterization of tumor-draining, sentinel lymph nodes in patients has identified the nodes to express an immunosuppressed, Th2-dominant immune profile. Importantly, this profile is observed in sentinel lymph nodes even prior to evidence of metastasis supporting a model in which melanoma-derived factors prime tumor-draining, sentinel lymph nodes for tumor progression. Recent work in our lab has characterized the cellular interactions and the contribution of soluble factors in facilitating this immunosuppressive immune profile in fresh sentinel lymph nodes of patients; however, an appreciation for the role of subcellular counterparts remains to be explored. In this study, we considered a novel role for melanoma-derived, membrane-bound nanoparticles as drivers in polarizing immunity towards an immunosuppressive, tumor-promoting state. Methods and Results: Utilizing recent technology advances in Nanoparticle Tracking Analysis (NTA), characterization of melanoma derived vesicles from human melanoma cell lines showed a size distribution of particles ranging from 30nm to 1um, representing both exosomes and microvesicles. Following hypoxic culture, vesicle production was amplified, suggesting the increasingly hypoxic tumor microenvironment may trigger vesicle secretion. To evaluate the immune regulating potential of melanoma-derived vesicles, proteomic analysis of vesicle cargo by mass spectrometry identified over 100 proteins including known immune regulators Galectin 3, MHC I, ICAM1 and MIF. Functional assessment revealed that dendritic cells matured in vitro in the presence of melanoma-derived vesicles have significantly reduced surface marker expression of co-stimulatory markers CD80 and CD86 compared to controls. In addition to an activation defect, microarray analysis of dendritic cells matured in the presence of melanoma vesicles displayed a unique RNA expression profile. Pathway analysis of the expression arrays identified numerous overlapping signaling pathways involving proteins present in the melanoma-derived vesicles by mass spectrometry suggesting tumor-derived vesicle cargo has the ability to modulate dendritic cell function. Conclusion: Taken together, this study demonstrates a critical function for melanoma-derived vesicles in driving immunosuppression by characterizing their generation, protein cargo and immune regulating properties. The enrichment of melanoma-derived vesicles for immune mediators and ability of the vesicles to suppress dendritic cell function suggests a mechanism by which regional immunosuppression precedes nodal involvement and may foster an environment for metastasis to occur. Moving forward, we are currently translating these findings into fresh sentinel lymph nodes obtained from patients in order to elucidate the role of melanoma-derived vesicles in metastasis. Understanding this mechanism may yield valuable insight as we seek to develop efficacious immunotherapies targeting nodal metastasis. Citation Format: Rachel Goldenstein, Wendy Nevala, Svetomir N. Markovic. Melanoma-derived nanovesicles as drivers of immunosuppression. [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 A70.

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 Interleukin 9 (IL-9), the signature Th9 cytokine, is associated with T regulatory cell function, parasite expulsion and allergic airway inflammation. Because IL-9 can promote a tolerogenic/immunosuppressive environment, we used IL-9 knock-out (IL-9ko) mice to estimate the role of endogenous IL-9 in the biology of two aggressive breast cancers, TUBO and 4T1, and CT26 a colon carcinoma. Merely eliminating endogenous IL-9 enabled efficient sensitization of host T cells leading to early rejection of growing tumors. Duplicate tumor challenges simply grew progressively in wildtype mice. Interestingly, tumor rejection occurred without additional vaccines or immunomodulatory therapies. The IL-9ko mice acquired immunologic memory, which actively protected from residual disease and tumor rechallenges. The ability of a normally lethal tumor challenge to stimulate an enduring therapeutic response in IL-9ko mice was linked to the activation of CD8+ T cells, as IL-9ko mice failed to be cured if either CD8+ or CD4+ T cells were depleted. Importantly, T cells from tumor-rejecting IL-9ko mice retained effector competence in wildtype mice, and treatments with neutralizing anti-IL-9 significantly slowed tumor progression in wildtype mice. IL-9 inhibits the immune system's ability to prime effective immunological memory after exposure to growing tumor; it therefore functions as a checkpoint inhibitor of adaptive immunity. Optimization of IL-9 neutralization may play a unique role in immunotherapy. Citation Format: Dominique B. Hoelzinger, Ana Lucia Dominguez, Peter A. Cohen, Sandra J. Gendler. Checkpoint inhibition of adaptive immunity by IL-9 can be disrupted to achieve rapid T cell sensitization and rejection of progressive tumor challenges. [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 A44.

Abstract Introduction: Galectin-9 (Gal-9) is a carbohydrate-binding protein that interacts with T-cell immunoglobulin mucin 3 (TIM3) to modulate immune homeostasis. The Gal-9/TIM3 interaction down regulates activated T cells which promotes tumor growth, much like program death 1 (PD-1) and its ligand (PD-L1). Still, the effect Gal-9 has on the immune environment of melanoma patients is weakly understood. This study focuses on the role of Gal-9 in immunoregulation of healthy persons and metastatic melanoma patients. Methods: Soluble Gal-9 levels were determined from plasma collected from patients and healthy persons using standard ELISA methods. Proliferation and apoptosis of cells in the presence and absence of Gal-9 was determined by mixed lymphocyte reactions and Tunnel staining. Supernatants from these reactions were collected and run using a cytokine bead assay to determine changes in immune parameters with and without Gal-9. Results: Soluble Gal-9 was significantly higher in metastatic melanoma patients compared to healthy controls (3929 vs. 877 pg/mL, p=0.0003 respectively). In the presence of Gal-9, the proliferative effect of an allostimulus could be overridden until the level of responder cells to stimulator cells was 4:1. Using an apoptosis assay, Gal-9 was found to increase the level of cellular apoptosis; however, apoptosis was decreased as the allostimulus increased. Healthy cells incubated with Gal-9 were found to increase secretion of growth factors (EGF and FGF-2), chemokines (CXCL1, CXCL8, CCL2 and CCL3), T helper 1 cytokines (IFN-g and IL-7) which have shown tumor promoting affects and T helper 2 cytokines (IL-13 and IL-4). Conclusions: Gal-9 may potentially play a role in suppressing anti-tumor immunity. This suggests the use of a Gal-9 neutralizing antibody for therapeutic benefit in melanoma. Citation Format: Elizabeth Ann L. Enninga, Wendy K. Nevala, Svetomir N. Markovic. Galectin-9 has tumor promoting properties that may be targetable in metastatic melanoma. [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 A41.

With the increased availability of computational resources, the past decade has seen a rise in the use of computational fluid dynamics (CFD) for medical applications. There has been an increase in the application of CFD to attempt to predict the rupture of intracranial aneurysms, however, while many hemodynamic parameters can be obtained from these computations, to date, no consistent methodology for the prediction of the rupture has been identified. One particular challenge to CFD is that many factors contribute to its accuracy; the mesh resolution and spatial/temporal discretization can alone contribute to a variation in accuracy. This failure to identify the importance of these factors and identify a methodology for the prediction of ruptures has limited the acceptance of CFD among physicians for rupture prediction. The International CFD Rupture Challenge 2013 seeks to comment on the sensitivity of these various CFD assumptions to predict the rupture by undertaking a comparison of the rupture and blood-flow predictions from a wide range of independent participants utilizing a range of CFD approaches. Twenty-six groups from 15 countries took part in the challenge. Participants were provided with surface models of two intracranial aneurysms and asked to carry out the corresponding hemodynamics simulations, free to choose their own mesh, solver, and temporal discretization. They were requested to submit velocity and pressure predictions along the centerline and on specified planes. The first phase of the challenge, described in a separate paper, was aimed at predicting which of the two aneurysms had previously ruptured and where the rupture site was located. The second phase, described in this paper, aims to assess the variability of the solutions and the sensitivity to the modeling assumptions. Participants were free to choose boundary conditions in the first phase, whereas they were prescribed in the second phase but all other CFD modeling parameters were not prescribed. In order to compare the computational results of one representative group with experimental results, steady-flow measurements using particle image velocimetry (PIV) were carried out in a silicone model of one of the provided aneurysms. Approximately 80% of the participating groups generated similar results. Both velocity and pressure computations were in good agreement with each other for cycle-averaged and peak-systolic predictions. Most apparent “outliers” (results that stand out of the collective) were observed to have underestimated velocity levels compared to the majority of solutions, but nevertheless identified comparable flow structures. In only two cases, the results deviate by over 35% from the mean solution of all the participants. Results of steady CFD simulations of the representative group and PIV experiments were in good agreement. The study demonstrated that while a range of numerical schemes, mesh resolution, and solvers was used, similar flow predictions were observed in the majority of cases. To further validate the computational results, it is suggested that time-dependent measurements should be conducted in the future. However, it is recognized that this study does not include the biological aspects of the aneurysm, which needs to be considered to be able to more precisely identify the specific rupture risk of an intracranial aneurysm.

ABSTRACT This article describes research criteria and probability methodology for the diagnosis of prodromal PD. Prodromal disease refers to the stage wherein early symptoms or signs of PD neurodegeneration are present, but classic clinical diagnosis based on fully evolved motor parkinsonism is not yet possible. Given the lack of clear neuroprotective/disease‐modifying therapy for prodromal PD, these criteria were developed for research purposes only. The criteria are based upon the likelihood of prodromal disease being present with probable prodromal PD defined as ≥80% certainty. Certainty estimates rely upon calculation of an individual's risk of having prodromal PD, using a Bayesian naïve classifier. In this methodology, a previous probability of prodromal disease is delineated based upon age. Then, the probability of prodromal PD is calculated by adding diagnostic information, expressed as likelihood ratios. This diagnostic information combines estimates of background risk (from environmental risk factors and genetic findings) and results of diagnostic marker testing. In order to be included, diagnostic markers had to have prospective evidence documenting ability to predict clinical PD. They include motor and nonmotor clinical symptoms, clinical signs, and ancillary diagnostic tests. These criteria represent a first step in the formal delineation of early stages of PD and will require constant updating as more information becomes available. © 2015 International Parkinson and Movement Disorder Society