While small molecule inhibitors have made an impact in human diffuse large B-cell lymphoma (DLBCL), immunotherapy, specifically using anti-CD20 based approaches, has had the most profound effect on treatment strategies and long-term outcomes. Despite this progress, up to 40% of patients will ultimately succumb to their disease. Furthermore, even when treatment is successful, cytotoxic chemotherapy carries a high risk of long- term morbidities that impact quality of life. There is now substantial data that specific genetic changes and pathway aberrations are conserved across dogs and people with DLBCL supporting the notion that the canine disease can be used as a relevant spontaneous large animal model of the human counterpart.

Toward this end, the purpose of this proposal is to use dogs with spontaneous nave DLBCL to rapidly evaluate rational small molecule/immunotherapy combination approaches, with the ultimate goal of identifying the most effective combination to move forward in human patients with DLBCL. Specifically, we hypothesize that optimal combinations of anti-CD20, anti- PD1, XPO1 inhibition, NAMPT/PAK4 and PI3Kdelta inhibition will have better outcomes than doxorubicin based chemotherapy, resulting in a “chemo-free” blueprint for future human trials. Using an adaptive mini-pilot trial approach, those combinations deemed antagonistic and/or associated with unacceptable adverse events can be rapidly removed from consideration, while those with clear therapeutic promise can be most effectively studied in the front-line setting and enhanced. The data generated from this proposal will create a framework for effectively leveraging information gained from integrated immunotherapeutic trials in canine patients with DLBCL to develop chemo-free strategies that ultimately improve human outcomes.

Spontaneous canine osteosarcoma (OS) is a well-defined large animal model of human OS, exhibiting similar clinical presentation and molecular aberrations. Despite best efforts, progress in the prevention and treatment of metastatic disease has essentially stalled for the past 3 decades; 30% of people and 90% of dogs still die of tumor spread, primarily to the lungs. This is particularly evident with respect to immune checkpoint inhibitors that do not induce the dramatic disease regressions typically observed in other cancers such as melanoma and lung cancer. One particularly daunting challenge for immunotherapy-based therapeutics in OS relates to the permissiveness of the tumor microenvironment (TME) for inducing anti-tumor immune responses. Our data suggests that a relatively low mutational load combined with a dampened overall immune response in OS may contribute to the lack of response to current treatment approaches. We propose that targeting the immune suppressive TME in OS is essential to generating potent and durable anti- tumor immunity. To accomplish this, it may be necessary to simultaneously modulate several elements in the TME, including Tregs, MDSCs, M2 macrophages and overexpressed inhibitory checkpoint molecules. In support of this, we have generated a body of data demonstrating immunological activity of multiple therapeutics, including repurposed drugs with good safety records (losartan, oclacitinib), small molecule inhibitors with established PK/PD in dogs (toceranib, RV1001, reparixin, JHU-292) and antibodies specific for checkpoint molecules (anti- PD1). However, the exact combinations that are most effective against metastatic OS have not yet been identified, and this is a major goal of this proposal. We hypothesize that an adaptive pilot trial design can be used to rapidly screen TME-targeting immunotherapy drug combinations in dogs with macroscopic chemotherapy-resistant metastatic OS and that this information can be refined to assess activity of the most active approach against microscopic metastases in a subsequent adjuvant trial. The data generated from this proposal will create a blueprint for future immunotherapy studies in people with OS by eliminating approaches deemed inactive and generating a set of clinical biomarkers to guide treatment.

This proposal seeks to build on our extensive preliminary data in canine immunotherapy trials to demonstrate that novel methods of exogenous cytokine delivery in combination with autologous natural killer (NK) cells in first-in-dog clinical trials represents a potentially high impact approach to optimize non-T cell based immunotherapies. This work is targeted to dogs with typically lethal, naturally occurring osteosarcoma (OSA) and melanoma, and the findings are expected to have important relevance for the design and translation of innovative immunotherapy approaches in humans. Inhaled (IH) IL-15 offers the advantages of local delivery of this immune-stimulatory cytokine, while limiting systemic exposure and thus potential toxicity. Super-agonist IL- 15 offers increased half-life and greater anti-tumor effects. Therefore, each of these cytokines is anticipated to alter the risk/benefit ratio in favor of their use. To accomplish these objectives, we propose the following three specific aims: 1) Targeting gross pulmonary metastases (OSA and melanoma) with first-in-dog delivery of inhaled (IH) human IL-15 and super-agonist IL-15; 2) Phase II trial of IH IL-15 and autologous NK cells to treat gross OSA and melanoma pulmonary metastases, and; 3) Targeting micro-metastatic disease using super- agonist IL-15 in primary OSA. These studies are designed to evaluate novel treatment combinations for advanced pulmonary metastases in the setting of gross disease, but also will determine the impact of this approach as first-line therapy when combined with standard-of-care treatments. Moreover, this work seeks to elucidate the cellular and molecular mechanisms that mediate an anti-tumor response (or non-response) in dogs with naturally occurring cancers. Our proposed canine trials and bio-marker studies represent an ideal strategy to inform and facilitate the rapid translation of novel, potentially high impact immune therapies to human patients with aggressive cancers.

Malignant gliomas are the most common primary brain tumors in humans, accounting for 30% of all primary central nervous system (CNS) tumors in adults. [2] With few major advances in decades, there has been no significant reduction in mortality and only a modest improvement in median survival. Dogs and humans develop sporadic benign and malignant brain tumors at about the same rate and with similar histopathology. Dogs are often euthanized due to the cost of care. We propose a multi-institutional consortium to test an innovative combinatorial immunotherapeutic approach in dogs that spontaneously and sporadically develop malignant glial brain tumors that resemble in most important aspects, high-grade malignant gliomas in humans. Our approach will inject a clinical grade oncolytic herpes simplex virus (HSV) M032 that expresses human interleukin-12 (IL-12) into the resected tumor bed, creating a potent local inflammatory response and an antigen-rich tumor cell debris field. We will conduct longitudinal safety, survival and correlative biology evaluations as a means of assessing the dog as an appropriate and informative model for design and implementation of clinical studies in humans with high-grade malignant brain tumors. To extend these findings, we propose, in subsequent studies, to combine the HSV immunotherapy approach with molecules that block the action of innate checkpoint inhibitor(s). These studies are designed to address key issues of safety and efficacy of combinatorial immunotherapy in the dog that can be translated to humans with malignant gliomas. Recently, objective responses and long-term survivals have been observed in human glioma patients who have received oncolytic virotherapy.  New approaches to utilize checkpoint inhibitors promises improved efficacy, but the ability to develop more effective combinatorial approaches has been slow, in large part due to lack of a faithful model of spontaneous glioblastoma multiforme (GBM) in immunocompetent hosts. Thus, the spontaneously occurring canine brain tumor represents an ideal opportunity to improve the lives of people and pets through comparative oncology and genomics using the One Medicine approach to research and direct clinical application.

There is a growing body of evidence that spontaneous cancers in dogs represent attractive translational models. Intracranial neoplasia occurs frequently in dogs with a reported prevalence from 0.15 to 4.5% compared to 18.2 cases per 100,000 human. Astrocytoma or glioma account for 20-36% of primary brain tumors in dogs and 25% in humans. Brachycephalic breeds such as Boxers, French and English bulldogs, and Boston terriers have a significantly increased risk of developing gliomas. Primary canine brain tumors have similar histologic classification as those reported by the World Health Organization for human brain tumors. Similar to that in humans, the prognosis for dogs with brain tumors in general is poor regardless of therapeutic intervention. However, much less is known about canine glioma treatment outcomes because only a small number of studies with few dogs have been reported.

There is little information about median survival time for dogs with glioma that received any type of treatment, but estimates of days to 2 or 3 months are often given to owners. The clinical similarities between dogs and humans suggest that dogs may represent an outstanding model for testing targeted therapies; both dogs and humans might benefit from these studies. Herein, we are proposing a multi-pronged immunotherapeutic approach to improve efficacy and survival times. We hypothesize that combination immunotherapy in a canine glioma model will enhance efficacy and accelerate successful translation into phase I human trials for GBM. The objective is to use pet dogs with spontaneous GBM to demonstrate the safety and efficacy of combination immunotherapy. We propose two Specific Aims: 1. Determine the safety and efficacy of immune checkpoint blockade in spontaneous canine GBM in combination with standard of care, and 2. Assess the efficacy of immune-mediated gene therapy in combination with CD200 blockade to enhance anti-glioma immunotherapy.