John Ohlfest - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Glioblastoma Multiforme (GBM) is a lethal brain tumor that typically causes death within two years after conventional therapies consisting of surgery, radiation, and chemotherapy. Recent studies have shown that rare CD133+ brain tumor stem cells (BTSCs) are the likely cause of therapy resistance and brain tumor recurrence. Therefore, novel therapies that target BTSCs should be developed. BTSCs have been found to rely on a perivascular niche to sustain self-renewal, which makes anti- angiogenic therapy a rationale adjuvant to combine with BTSC-targeted cytotoxic therapy. Recent clinical data demonstrates that dendritic cell vaccines can elicit a tumor- reactive immune response in GBM patients, but this has typically not been curative. We hypothesize that the combination of anti-angiogenic gene therapy with a BTSC-targeted dendritic cell vaccine will provide synergistic and superior anti-tumor effects to eradicate murine GBM. To test this hypothesis this project entails the translational development of a novel nonviral vector called Sleeping Beauty (SB) for anti-angiogenic gene transfer, and an innovative BTSC-enriched dendritic cell vaccine for immunotherapy against BTSCs. In Specific Aim 1, we will determine the effects of SB-mediated anti-angiogenic gene therapy on BTSC survival and potency in vitro and in vivo. In Specific Aim 2, the anti-tumor efficacy of a BTSC-enriched, lysate-pulsed dendritic cell vaccine will be compared to a parental cell, lysate-pulsed vaccine in mice bearing intracranial gliomas. In Specific Aim 3, the most effective anti-angiogenic gene therapy will be combined with the most effective immunotherapy in glioma-bearing mice to determine if synergistic anti- tumor efficacy is achieved. This project has high impact potential because we may identify an effective and scalable anti-angiogenic therapy, an immunotherapy capable of killing glioma cells responsible for tumor renewal, and assess the efficacy of the combination of these therapies. Together these studies will provide information regarding the feasibility of using these novel approaches for treating patients with GBM. Statement of Relevance Glioblastoma is a fatal brain tumor that kills nearly 13,000 people every year in the United States alone. In this project we will develop and test a combined gene therapy / immune therapy for the treatment of glioblastoma in mice. The project has high impact potential because it may identify a new treatment approach for glioblastoma patients. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is a disease of the entire brain. Even complete surgical resection of the tumor-bearing hemisphere inevitably leads to recurrence and has been abandoned. Nonetheless, the majority of clinical trials employing small molecule drugs have focused their measurements of efficacy (both clinical outcome and biomarkers) on the bulk tumor mass that can be surgically removed. This is done in spite of mounting evidence that suggests the inevitable relapse and lethality of GBM is due to a failure to effectively target invasive glioma cells. Brain tumor cells overexpress protective active efflux transport systems, including p-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). The blood-brain barrier (BBB) in the tumor core is "leaky", allowing systemic drug delivery, but glioma cells infiltrate normal brain structures centimeters away from the margin of surgical resection where the BBB is intact and has functional efflux transport systems. Molecularly- targeted anti-tumor agents such as tyrosine kinase inhibitors (TKIs, e.g., imatinib, erlotinib, dasatinib) have their efficacy limited by sequential barriers to delivery to the actual target, including barriers to macroscopic distribution (active efflux at the BBB) and barriers to microscopic delivery (active efflux from invasive glioma cell). Therefore, drug delivery strategies that (1) improve the delivery of selected "molecularly-targeted" chemotherapeutic agents through the BBB, and (2) improve the intracellular drug accumulation in invasive glioma cells, will significantly enhance the efficacy of molecularly-targeted therapy. Our central hypothesis is that invasive glioma cells can be targeted through specific inhibition of active efflux at the level of both the BBB and the invasive tumor cell leading to improved efficacy of molecularly-targeted tyrosine kinase inhibitors. We propose three specific aims to test this hypothesis. Aim 1 will characterize strategies to improve TKI delivery and efficacy in both human and mouse primary glioma cell lines. Aim 2 will determine the influence of active efflux, and optimize strategies to overcome efflux, on TKI efficacy in a novel spontaneous mouse model of glioma that grows invasively. Aim 3 will determine the influence of inhibiting active efflux transport using a novel prodrug targeting Pgp and BCRP on the efficacy of TKIs in a primary human glioma xenograft model that grows invasively relative to traditional xenografts. Completion of these aims will indicate if active efflux transport at the BBB, the invasive glioma cell barrier, or both is an important mechanism that can influence the efficacy of molecularly-targeted therapy by limiting drug delivery to the invasive glioma cell. If successful, this information should be readily translatable to clinical trials and lead to eventual improvement in the treatment of gliomas, and other tumors of the central nervous system. PUBLIC HEALTH RELEVANCE: Currently, there are no effective treatments for malignant brain tumors. This represents a significant unmet medical need, and if active drug efflux limits drug delivery to the brain leading to therapeutic failure, then improved delivery of an effective agent is possible through inhibiting drug efflux. Improving drug delivery could lead to improved patient outcomes, including longer progression free survival and possible cure.

DESCRIPTION (provided by applicant): Malignant brain tumors are the leading cause of cancer-related death in people under age 35. Novel therapies that selectively target residual migratory glioma cells after surgery are urgently needed. Therapies that will be effective against these extremely heterogeneous tumors should have broad molecular targets. Immunotherapy using autologous tumor cell lysate vaccines targeting multiple tumor antigens is a promising approach being tested in numerous clinical trials. However, complete tumor regression is rarely achieved. The effect of cell culture conditions during vaccine production on the immune response and clinical outcome is poorly understood. Moreover, although tumor-reactive T cells may be correlated to prolonged survival in select patients, the functional heterogeneity of responding T cells and their relation to antibody response and clinical outcome is inadequately defined. We discovered that the oxygen concentration used to culture tumor cells during vaccine production flips an immunologic switch, dictating whether cytotoxic T lymphocytes (CTL) or antibody production dominate in the resulting immune response. Glioma cells grown in physiologic brain oxygen (5% O2) have enhanced adjuvant properties, are enriched for a cancer stem cell phenotype, and increase expression of known immunogenic glioma antigens relative to cells grown in the conventional oxygen level (20% O2). Furthermore, glioma-bearing mice vaccinated with glioma lysates prepared from 5% O2 cultures mixed with CpG oligodeoxynucleotides (ODN) as an adjuvant exhibit a three- fold increase in tumor infiltrating T cells and significantly increased survival relative to mice vaccinated with lysates from 20% O2 cultures. We propose to elucidate the effect that oxygen concentration used during vaccine production has on the immune response and clinical response in an innovative large animal model of glioma. We have established that pet dogs with spontaneous gliomas represent an outstanding animal model in which surgery, steroids, and postoperative chemotherapy can be given similar to human patients, lending great translational relevance to our findings. In specific aim 1, we will determine the difference in survival between dogs with glioma treated by surgery, chemotherapy, and vaccination with glioma lysate / CpG ODN prepared from cells cultured in 5% or 20% O2. Tumor burden and toxicity will be determined similar to human patients. In specific aim 2, the frequency of "polyfunctional" CTLs that secrete multiple effector cytokines and degranulate to kill tumor cells will be determined and correlated with tumor-reactive antibody response and survival. The deliverables of this study will be: i) new knowledge relating oxygen tension used in vaccine production to T and B cell responses and patient survival, ii) validation of immune monitoring assays useful in predicting which patients may respond to immunotherapy, iii) efficacy and safety data in the only useful large animal model of glioma in the world to justify accelerated clinical trial design in human glioma patients. PUBLIC HEALTH RELEVANCE: Glioma is an aggressive brain tumor that is very difficult to treat. Vaccines have been tested in glioma patients with suboptimal results. We have developed a novel vaccine with increased efficacy in mouse models. In this project we will determine if vaccination can increase the survival of pet dogs with glioma as a prelude to human clinical trials.

DESCRIPTION (provided by applicant): There are currently no comprehensive or satisfactory explanations for how tumors evade the immune response. Much of our knowledge about tumor immunoevasion is limited to individual mechanisms that have been therapeutically targeted with single drugs. However, tumors evolve multiple, redundant strategies to circumvent immune surveillance. This plasticity has contributed to the modest efficacy of cancer immunotherapies that only target one/several pathway(s). The challenge we face is: to comprehensively decipher the mechanisms tumors use to evade immune surveillance. Specifically, how can we determine which of these may be redundant and which may be ideal drug targets (e.g., non-redundant mechanisms)? Similarly, which pathways crucial for tumor immunoevasion might synergize and require several drugs to achieve effective immunotherapy? In order to address this challenge we will establish assays that intentionally select for mutations that allow a tumor to escape an immune response. Using the power of immunologic selective pressure, we will indentify mechanisms that work alone and in combination to allow a tumor to escape immunity. Our central hypothesis is that tumors evolve immunosuppressive gene expression patterns specific to their microenvironment to evade immune attack. We further hypothesize that those changes in gene expression can be rapidly indentified by functional genomics approaches. We have previously used transposon-based mutagenesis to indentify mutations that initiate tumors. Transposons are mobile genetic elements that can be designed to cause mutations that are rapidly identifiable. Herein we will customize transposon-based mutagenesis to identify genes that allow a tumor to thrive despite the presence of high numbers of tumoricidal T cells. Our specific aim is to identify and validate genes that promote escape from immune surveillance in gliomas, an aggressive form of brain cancer. To accomplish this, mutagenic transposons will be mobilized in cultured glioma cells. Mutagenized glioma cells will be implanted into mice with pre-established immunological memory to select for tumor cells that only grow when immune evasion is achieved. In parallel, we will carry out a similar genetic screen in mice with spontaneously arising gliomas that are treated by infusion of tumor-specific T cells. Transposon insertions will be cloned, sequenced and statistically analyzed to identify genes that are repetitively mutated in many tumors that escape immunity. Candidate genes will be functionally validated in tissue culture and animal model experiments to establish cause and affect relationships between their expression and immunoevasion. IMPACT: The identification of integrated pathways that tumors require to escape immune responses in order to thrive, which will ultimately lead to the development of drugs that increase the efficacy of cancer immunotherapy. PUBLIC HEALTH RELEVANCE: Tumors are continuously eliminated in our bodies by a process called "immunosurveillance". When immunosurveillance fails, clinically apparent tumors arise that cause significant morbidity and mortality. Therefore, discovery of the processes that allow a tumor to escape the immune response is vital to improve the effectiveness of immune-based treatments for cancer. Herein we will establish assays that allow for the rapid identification of novel mechanisms by which tumors escape the immune response. Several of these "immune escape" mechanisms will be validated as potential targets for future drug development. The ultimate impact of these studies will be the development of more effective immune-based therapies for the treatment of cancer.

DESCRIPTION (provided by applicant): Malignant brain tumors are the leading cause of cancer-related death in people under the age of 35. Immunotherapy in the form of personalized vaccines has demonstrated immunologic activity and clinical responses in select glioma patients with minimal toxicity. There are numerous ongoing clinical trials that utilize cultured tumor cells as the source of vaccine antigen for the treatment of a wide array of tumors. However, very little is known about how cell culture conditions affect the immune response and ultimate clinical response following vaccination. The consistent condition used is expansion of tumor cells in atmospheric oxygen (20% O2). We have identified the oxygen concentration used in tissue culture as a primary determinant of the immunogenicity of tumor cell vaccines. Our data have led to the attractive central hypothesis that the oxygen tension in a tumor cell culture acts as a master immunologic switch, dictating the type and strength of immune response induced by vaccination. We have reproducibly shown that lysates from glioma cells cultured in 5% O2 prime cytotoxic T lymphocytes (CTLs) with superior effector functions relative to lysate from glioma cells cultured in 20% O2 in human and murine systems. This difference profoundly affects the efficacy of immunotherapy, as shown by significant improvements in survival in murine models of glioma and breast carcinoma. We demonstrated that administration of 5% O2 lysate vaccination caused superior CTL proliferation, cytokine elaboration, tumoricidal function, and trafficking to tumor sites relative to 20% O2 lysate vaccines. Conversely, 20% O2 lysate vaccines enhance antibody responses. Despite reduced tumor-reactive antibody responses, the 5% O2 vaccines require B cells for therapeutic efficacy, revealing a putative role of B cells in CTL priming. Additionally, we have evidence that glioma cells grown in 5% O2 upregulate toll-like receptor (TLR) 2 ligands because TLR2 is required in several systems to distinguish the immunogenicity of 5% and 20% O2 lysates. The goal of this proposal is to elucidate the molecular basis of the "oxygen switch effect" in order to rationally improve the efficacy of tumor cell vaccines. In specific aim 1 we will determine and optimize tumor cell intrinsic changes that modulate immunogenicity. Pharmacologic strategies to increase hypoxia inducible factors (HIF) in tumor cells will be compared to actual hypoxia in cell cultures that will subsequently be assayed for immunogenicity. Additionally, we will test the hypothesis that HIF2a is the molecular switch that induces expression of TLR2 ligands. In specific aim 2, we will test our hypothesis that B cells are required because IgG-tumor lysate complexes trigger Fc receptor-mediated cross presentation of antigens to enhance CTL responses. In specific aim 3, we will determine if the adjuvant effect of 5% O2 lysates is due to expression of Annexin A2, a novel putative TLR2 ligand we recently found to be upregulated in hypoxia. Collectively, this knowledge will facilitate groundbreaking approaches to enhance the efficacy of immunotherapy for glioma and other non-central nervous system tumors. PUBLIC HEALTH RELEVANCE: Glioma is an aggressive brain tumor that is very difficult to treat. Vaccines have been tested in glioma patients with suboptimal results. We have developed a novel vaccine with increased efficacy in mouse models. In this project we will optimize the efficacy of this vaccine and elucidate the mechanism by which it works with the long-term goal improving the efficacy of immunotherapy for brain tumor patients.