John H. Sampson, M.D., Ph.D. - US grants

(Adapted from the applicant's abstract): The brain is the most frequent site of crippling and incurable human disease, and malignant primary brain tumors alone are more common than Hodgkin's disease, and cause more deaths than cancer of the bladder or kidney, leukemia, or melanoma. Conventional therapy for malignant brain tumors is ineffective and incapacitating, and represents the most expensive medical therapy per quality- adjusted life-year saved currently provided in the U.S. At the investigators institution, direct injection of (131)I-labeled, operationally-specific, monoclonal antibodies (MAbs) into brain tumor resection cavities delivers extremely high radiation doses to tumor cells around the resection cavity and has produced promising results in Phase II clinical trials. However, these MAbs diffuse only short distances beyond the cavity. Therefore, most of the radiation extending beyond the cavity is not specifically targeted to tumor cells and the radiation dose delivered beyond the cavity declines exponentially from the cavity interface. As a result, tumor cells that are known to infiltrate the brain for significant distances beyond the cavity are subopitimally treated and lethal tumors always recur within 2cm of the radiated resection cavity. Continuous microinfusion is a promising technique that allows homogeneous delivery of even large molecular weight molecules at high concentrations throughout large areas of the brain. Although this technique may enhance the delivery of (131)I-labeled MAbs and other therapeutic agents to diffusely infiltrating malignant brain tumors and reduce recurrence rates, the parameters that govern this technique and its limitations have not been defined. One of the major goals of this proposal is to define these parameters. In addition, this proposal is designed to investigate whether targeted radiotherapy might be improved through the use of human chimeric MAbs with increased biostability and the use of high linear energy transfer radioisotopes, such as (211)At, with greater relative biological effectiveness. The hypothesis to be tested in this proposal is that continuous microinfusion will widely deliver operationally tumor-specific MAbs conjugated to (131)I or the alpha-emitter (211)At such that they will be specific and potent therapeutic agents against malignant brain tumors with major reductions in toxicity to normal brain over conventional whole brain radiotherapies.

DESCRIPTION (provided by applicant): Despite aggressive surgical resections, high-dose radiation therapy, and chemotherapy delivered at toxic doses, the vast majority of patients with malignant brain tumors survive less than one year making conventional therapy for malignant brain tumors the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. Moreover, the failure of these treatment modalities to be tumor-specific at the molecular level, results in inevitable damage to surrounding normal brain that incapacitates patients treated with these traditional modalities. The inherent specificity of immunologic recognition offers the prospect of targeting malignant cells more precisely. Within our program, direct injection of 131-I-labeled, operationally-specific, monoclonal antibodies (MAbs) into brain tumor resection cavities delivers extremely high radiation doses to tumor cells around the resection cavity and has produced promising results in Phase II clinical trials. These MAbs diffuse only short distances beyond the cavity, however. Therefore, most of the radiation extending beyond the cavity is not specifically targeted to tumor cells and the radiation dose delivered beyond the cavity declines exponentially from the cavity interface. As a result tumor cells that are known to infiltrate the brain for significant distances beyond the cavity are sub-optimally treated and lethal tumors always recur within 2 cm of the radiated re section cavity. Continuous microinfusion is a promising technique that allows homogeneous delivery of even large molecular weight molecules at high concentrations throughout large areas of the brain. Although this technique may enhance the delivery of 131-I-labeled MAbs and other therapeutic agents to diffusely infiltrating malignant brain tumors and reduce recurrence rates, the parameters that govern this technique and its limitations have not been defined. One of the major goals of this proposal is to define these parameters. In addition, this proposal is designed to investigate whether targeted radiotherapy might be improved through the use of human chimeric MAbs with increased biostability and the use of high linear energy transfer radioisotopes, such as 211-At, with greater relative biological effectiveness.The hypothesis to be tested in this proposal is that continuous microinfusion will widely deliver operationally tumor-specific monoclonal antibodies conjugated to 131-I or the alpha-emitter 211-At such that they will be specific and potent therapeutic agents against malignant brain tumors with major reductions in toxicity to normal brain.

DESCRIPTION (provided by applicant): Despite aggressive surgical resections, high-dose radiation therapy, and toxic chemotherapy, the vast majority of patients with malignant brain tumors survive less than one year making conventional therapy for malignant brain tumors the most expensive therapy per quality-adjusted life-year saved currently provided. Moreover, the failure of these treatment modalities to be tumor-specific at the molecular level, results in inevitable damage to surrounding normal brain that incapacitates patients treated with these traditional modalities. The inherent specificity of immunologic recognition offers the prospect of targeting malignant cells more precisely. Several studies have documented the exceptional ability of dendritic cells (DCs) to activate the immune system and produce encouraging human antitumor responses. The epidermal growth factor mutation, EGFRvIII, found on the majority of malignant gliomas, represents a tumor-specific target for such an approach. Our preclinical results demonstrate that DCs loaded with a KLH conjugate of an EGFRvIII peptide induce potent humoral and cell-mediated immune responses. Although anti-EGFRvIII, DC-based immunotherapy will allow antigen-specific immune responses to be clearly monitored and potentially optimized, we believe that human antitumor responses will likely be enhanced and the spectrum and utility of this paradigm expanded by targeting additional antigens. However, existing techniques for identifying potential targets are labor intensive, do not systematically assess the entire neoplastic genome, and do not assess the potential risk of autoimmunity posed by targeting these antigens. Serial analysis of gene expression (SAGE) is a contemporary approach to gene expression analysis that allows rapid identification of genes that are over-expressed in neoplastic cells. SAGE databased mining and rapid expression screening has allowed our group to identify a large number of genes uniquely expressed in malignant gliomas that may function as specific tumor antigens. To select those with immunologic relevance and those that are unlikely to induce autoimmune reactivity, we have developed a unique system based on the loading of autologous DCs with genes or gene fragments. Using this technique, we have demonstrated that DCs loaded with tumor-specific RNAs can specifically activate autologous T cells without activating autoreactive T cells. The hypothesis to be tested in this project is that malignant gliomas can be selectively targeted for therapeutic immunotherapy without the induction of autoimmunity using DCs loaded with the tumor-specific EGFRvIII and other additional genes found by SAGE to be uniquely expressed by malignant gliomas.

Malignant gliomas (MGs) are univerally fatal, and effective therapy is limited by collateral damage to normal tissue. Immunotherapy directed against tumor-specific antigens may allow neoplastic cells to be targeted more precisely, and our dendritic cell (DC)-based vaccinations targeting of a mutated tumor-specific epidermal growth factor receptor have produced immunologic and radiographic responses in patients with MGs. The discovery that MGs, but not surrounding normal brain, serve as a refuge for Cytomegalovirus (CMV) reactivation provides an unparalleled opportunity to subvert, as a tumor-specific antigen, the highly immunogenic CMV protein, pp65. Despite the numerous advantages of targeting CMV antigens in MGs with DC-based vaccines, a number of factors clearly limit ant/tumor immune responses in these patients. Innovative complementary strategies that eliminate CD25+ regulatory T cells or block cytotoxic 3; lymphocyte antigen-4-induced T cell tolerance may enhance such immune responses, but the indiscriminate application of these potent adjuvants carries the risk of inducing autoimmune encephalomyelitis. In order to understand the limitations and risks of targeting CMV antigens in MGs, we have developed a novel murine astrocytoma cell line that supports infection with murine CMV and is tumorigenic in syngeneic mice. Our preliminary murine studies demonstrate that these tumors in the brain can be targeted with RNA-loaded DCs. We have also shown that DCs from patients with MGs that are loaded with pp65mRNA, induce interferon-gamma, production from CD4+ and CDS+ T-cells in an antigen-specific manner and incite T-cells to kill malignant astrocytes infected with human CMV. Interestingly, we have also found that CMV-specific T-cells preferentially accumulate at the tumor site in patients with MGs. We believe that our murine model system and the complementary human studies proposed will allow selection and translation of the most effective strategies for targeting CMV-associated antigens in patients with MGs, without the induction of autoimmunity. In this project, we will use the murine model, in combination with in vitro human studies to evaluate the safety of, and to gain a better understanding of the mechanisms involved in the therapeutic targeting of CMV-associated proteins in malignant gliomas. The results will then be used to rationally design and conduct a clinical CMV-targeted clinical trial.

Malignant gliomas (MGs) are univerally fatal, and effective therapy is limited by collateral damage to normal tissue. Immunotherapy directed against tumor-specific antigens may allow neoplastic cells to be targeted more precisely, and our dendritic cell (DC)-based vaccinations targeting of a mutated tumor-specific epidermal growth factor receptor have produced immunologic and radiographic responses in patients with MGs. The discovery that MGs, but not surrounding normal brain, serve as a refuge for Cytomegalovirus (CMV) reactivation provides an unparalleled opportunity to subvert, as a tumor-specific antigen, the highly immunogenic CMV protein, pp65. Despite the numerous advantages of targeting CMV antigens in MGs with DC-based vaccines, a number of factors clearly limit ant/tumor immune responses in these patients. Innovative complementary strategies that eliminate CD25+ regulatory T cells or block cytotoxic 3; lymphocyte antigen-4-induced T cell tolerance may enhance such immune responses, but the indiscriminate application of these potent adjuvants carries the risk of inducing autoimmune encephalomyelitis. In order to understand the limitations and risks of targeting CMV antigens in MGs, we have developed a novel murine astrocytoma cell line that supports infection with murine CMV and is tumorigenic in syngeneic mice. Our preliminary murine studies demonstrate that these tumors in the brain can be targeted with RNA-loaded DCs. We have also shown that DCs from patients with MGs that are loaded with pp65mRNA, induce interferon-gamma, production from CD4+ and CDS+ T-cells in an antigen-specific manner and incite T-cells to kill malignant astrocytes infected with human CMV. Interestingly, we have also found that CMV-specific T-cells preferentially accumulate at the tumor site in patients with MGs. We believe that our murine model system and the complementary human studies proposed will allow selection and translation of the most effective strategies for targeting CMV-associated antigens in patients with MGs, without the induction of autoimmunity. In this project, we will use the murine model, in combination with in vitro human studies to evaluate the safety of, and to gain a better understanding of the mechanisms involved in the therapeutic targeting of CMV-associated proteins in malignant gliomas. The results will then be used to rationally design and conduct a clinical CMV-targeted clinical trial.

DESCRIPTION (provided by applicant): A subset of cells in glioblastoma multiforme (GBM) has been identified that enjoy a unique capacity to regenerate tumors. These brain tumor stem cells (BTSC) can be segregated by the neural stem cell marker, CD133, and are widely believed to be the cells responsible for resistance to conventional therapies. An effective means of specifically eliminating these cells may reduce the need for intensive and non-specific conventional therapy and lower the risk of tumor recurrence. EGFRvIII is a tumor-specific mutation found on BTSC. We have successfully targeted EGFRvIII using a peptide vaccine that allowed rapid translation to an ongoing Phase III trial. EGFRvIII expression is heterogeneous, however, and the recurrence of EGFRvIII-negative tumors suggests that BTSC can rely on other oncogenic pathways. While our data suggests that targeting tumor-specific mutations in BTSC may be important, few highly-conserved tumor-specific mutations like EGFRvIII will be identified and antigen defined vaccine approaches will ultimately be limited. Dendritic cells (DCs) loaded with amplified total tumor RNA is an innovative strategy to induce cellular and humoral antitumor immune responses. Although CD133(+) BTSC are a minority subpopulation of GBM that cannot be reliably isolated or propagated in sufficient quantities to serve as an antigen source for human vaccination protocols, we have been able to reproducibly amplify the RNA content from as few as 500 sorted CD133(+) tumor cells to generate RNA libraries sufficient for clinical scale DC-based vaccination. In order to focus the immunologic response on antigens preferentially or uniquely expressed within BTSC and limit the potential for autoimmune reactivity against shared antigens expressed in normal cells, we will evaluate approaches to enrich for antigens preferentially or uniquely expressed in BTSC by using full length cDNA affinity based substractive hybridization or an innovative strategy that leverages the ability of the DNA mismatch binding protein, MutS, to isolate cDNAs that contain tumor-specific mutations. These various preparations will be evaluated for differential toxicity and efficacy in an inbred transgenic murine malignant astrocytoma model, in which a subpopulation of CD133(+) tumor cells with BTSC qualities have been identified and CD8(+) and CD4(+) epitopes have been found. If efficacy is seen, the least toxic strategy will be translated into a Phase I study within the context of our existing clinical trial platform. PUBLIC HEALTH RELEVANCE: Treatment for malignant primary brain tumors, which are the most common cause of death among children and account for more deaths in adults than melanoma, currently represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. A subset of malignant primary brain tumor cells (BTSCs), called brain tumor stem cells, enjoy a unique capacity to regenerate tumors and to resist conventional therapies. In this proposal we will see if targeting antigens preferentially or uniquely expressed by BTSCs will enhance the efficacy and reduce toxicity of immunotherapy.

[unreadable] DESCRIPTION (provided by applicant): The immune system has the potential to eliminate altered neoplastic cells with incredible specificity. A consistent in-frame deletion in the extra-cellular domain of the epidermal growth factor receptor (EGFRvIII) represents a truly tumor-specific target amenable to immunotherapeutic attack. Our multi-institutional Phase II study demonstrated that vaccination with an EGFRvIII-specific peptide in patients with newly-diagnosed glioblastoma multiforme (GBM) induces potent T- and B-cell immunity, produces nearly complete radiographic responses in all patients with residual tumor, and universally eliminates EGFRvIII-expressing cells. Recurrent tumors, however, continue to express wild-type EGFR suggesting that the immune response is specific, but productive intra-molecular cross-priming against other potential tumor-associated antigens is incomplete. We believe that productive extension of such secondary immune responses is hindered by the presence of regulatory T-cells (TRegs). We have recently shown that TRegs are disproportionately represented within the peripheral blood and tumors of patients with GBM and serve to induce a state of profound, but reversible, immunosuppression. TRegs are characterized by constitutive expression of the high affinity interleukin (IL)-2 receptor (IL-2R1)(CD25) and are uniquely dependent on IL- 2R1 signaling for their function and survival. Using our spontaneous murine glioma model, we have demonstrated that treatment with an antibody that blocks IL-2R1 signaling functionally inactivates and eliminates TRegs without inducing autoimmune toxicity. Our pre-clinical studies have shown that these unarmed IL-2R1-specific antibodies when given in vivo to mice during recovery from lymphopenia induced by therapeutic temozolomide (TMZ) are capable of not only functionally inactivating TRegs, but also dramatically enhance vaccine-induced immune responses. Daclizumab, an existing, humanized, unarmed IL-2R1-specific antibody, functions identically to the antibody used for TReg inactivation studies in mice. We hypothesize that daclizumab therapy during the recovery from therapeutic TMZ-induced lymphopenia in patients with newly-diagnosed GBM will inhibit the functional recovery of TRegs, enhance immune responses against an EGFRvIII-targeted vaccine, and promote productive cross-priming without the induction of deleterious autoimmunity. Because NK cells also express CD25 and may be potent activators or inhibitors of innate and antigen-specific immune responses, the effect of daclizumab on NK cells will also be assessed. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Primary malignant brain tumors, like glioblastoma (GBM), remain universally fatal. Like most neoplasms, they develop through the acquisition of multiple genetic alterations that lead to a heterogeneous deregulation of cell signaling pathways. Despite this complexity, recent advances in gene expression technology have been successful at providing more accurate prognostic information to patients. Still, they have little impact on patient care because they do not alter treatment choice. Innovative approaches pioneered by our group, however, offer an opportunity to identify more elaborate structure in the patterns of gene expression in these tumors by extrapolating the findings obtained with defined cell culture manipulations in vitro, such as activation of a given cell signaling pathway, to the complexity of human cancers in vivo. The resulting "gene signatures" can be thought of as a "fingerprint" shared between experimental cell cultures and patient tumors. To the degree to which they are shared, we believe these signatures have the potential to be used as guides for directing the use of targeted therapeutic agents to treat human cancers. In support of this hypothesis, we have recently shown that these gene signatures accurately predict oncogenic pathway activation and response to targeted therapeutics in various murine and human tumors. The advantage of targeting therapy to susceptible tumors is well illustrated by the examples of trastuzumab for HER2-expressing breast cancer and imatinib for Philadelphia chromosome chronic myeloid leukemia. Similarly, support for the basic concept that genetic analysis can inform targeted therapy in GBM has recently been provided in two retrospective studies - one demonstrating that O6-methylguanine-DNA methyltransferase promoter methylation can inform the use of temozolomide chemotherapy in GBM and the other identifying a significant association between clinical response to epidermal growth factor receptor (EGFR) inhibitors in patients and tumors that co-express PTEN and EGFRvIII. These studies provide evidence that molecular analysis could be used to select patients with GBM that are more likely to respond to a given therapy. These observations combined with the dismal results of studies using various single agents in GBM, suggests that the complexity and heterogeneity of GBM will need to be matched with an equally complex therapeutic combination. This is not much different than in other biologic systems, for example AIDS, leukemia, or bacterial infections, where the potency of combinatorial therapy has been evident in many early and dramatic treatment successes. Therefore, our OVERALL GOAL in this proposal is to enhance the efficacy of targeted combinatorial therapeutics for patients with brain tumors. PUBLIC HEALTH RELEVANCE: Brain tumors remain the most common cause of cancer death among children and account for more deaths in adults than melanoma, and treatment for these tumors represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. Innovative analysis of genetic "fingerprints" in these and other tumors may allow therapy to be enhanced by matching specific susceptibilities of the tumor to targeted therapeutic agents. This proposal tests this "personalized" medicine approach in human tumors grown in mice as a prelude to human clinical studies.

Seeinstructions): Project 2. Enhancing Glioma Immunotherapy with Temodar-lnduced Lymphopenia and a Multivalent Vaccine. John H. Sampson, M.D., Ph.D., M.H.Sc., Project Leader Despite the demonstrated capacity for the immune system to eliminate tumor cells with exquisite precision in preclinical models, active immunotherapy against human neoplasms has met with relatively few clinical successes. However, our vaccines targeting the tumor-specific epidermal growth factor receptor mutation, EGFRvlll, when given during recovery from temozolomide (TMZ)-induced lymphopenia have produced strong humoral and CD8+ and CD4+ T-cell responses that are accompanied by radiographic responses in all patients with residual disease and a median survival that exceeds 32 months. Vaccination eliminates EGFRvlll-expressing tumor cells. This illustrates the effectiveness of immunologic targeting, but EGFRvlll-negative tumor recurrence in this setting underscores the need to develop an effective therapy that addresses the heterogeneity of antigen expression in GBM. We believe that the potent and specific immune responses generated against EGFRvlll in our studies were potentiated by TMZ. The homeostatic proliferation that occurs after TMZ-induced lymphodepletion reduces the threshold for lymphocyte activation and proliferation. Our murine models have demonstrated that vaccine responses can be dramatically enhanced in TMZ-pretreated mice in a dose-dependent manner and dose-intensified TMZ regimens in patients with GBM have resulted in dramatically enhanced cellular and humoral responses to vaccination. These results have highlighted the potential to leverage the recovery from TMZ-induced lymphopenia as a potentially novel mechanism to enhance active immunotherapy against GBM. In an attempt to broaden the response seen when targeting EGFRvlll in this context, we propose to evaluate total tumor-RNA loaded dendritic cell vaccines. Mechanistic understanding of the effects of TMZ on the induction and maintenance of antitumor immunity will be explored in relevant murine astrocytoma models in order to rationally develop enhanced immunotherapeutic treatments for patients with GBM. RELEVANCE (See instructions): Treatment for malignant primary brain tumors is ineffective and represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the USA. The only major side effect of temozolomide, the only successful chemotherapeutic for this disease, is bone marrow suppression. We believe this suppression may dramatically enhance antitumor immunity and will investigate this possibility.

DESCRIPTION (provided by applicant): Program Description: The brain is the most frequent site of crippling and incurable human disease. Although basic discoveries in the neurosciences are being made at an unprecedented pace, these discoveries cannot be leveraged to reduce the burden of human disease unless they are responsibly translated into human studies and critically evaluated in the context of clinical practice. While many residency training programs in Neurology or Neurosurgery provide training in the basic sciences, the later demands of clinical practice rarely permit these specialists to excel in basic science research. Neurologist and Neurosurgeons, however, have unique access to the human brain and spinal cord and to patients with neurologic disease. As such, they are ideally positioned to translate basic science discoveries into the clinical arena concurrent with their clinical practice. Formal training in the principles, costs, and responsible conduct of translational and clinical research is lacking in most medical school curricula and is non-existent in traditional residency training programs, however. Thus, there is a clear need to enhance the interest and capability of Neurologists and Neurosurgeons in training to proceed on to academic careers as clinician-scientists who will fill the need to translate basic discoveries into novel treatments designed to reduce the burden of neurological disease. The overall goal of this translational and clinical research training program is to ensure that a diverse group of residents and fellows in the clinical neurosciences become highly-trained clinician-scientists with sufficient knowledge of clinical investigation principles and regulations to become competent, responsible, and independently-funded investigators capable of translating basic discoveries into clinical practice. The program proposed here will provide a unique and rigorous, but proven, approach to engage and educate physicians focused within the neurosciences. The program will integrate didactic training within the context of the Clinical Research Training Program, a formal degree program with a thesis requirement within the School of Medicine, and mentorship by a multidisciplinary faculty with significant experience in translational and clinical research and training. Emphasis will be placed on critical interpretation of the literature, statistical methodologies, and mechanisms of funding. The Program Director and an External Advisory Board will review program and trainee performance quarterly, as well as review applicants and mentors. Trainees will be recruited during and after residency training from a local, national, and international pool. Public Health Relevance: The brain is the most frequent site of crippling and incurable human disease. Important basic discoveries in the neurosciences are not being translated into clinical practice where they could reduce the burden of human neurologic disease because Neurologists and Neurosurgeons lack the requisite skills to conduct high-quality and responsible clinical and translational research. This proposal describes a program to enhance the interest and capability of Neurologists and Neurosurgeons as clinician-scientists who will fill the need to translate basic discoveries into novel treatments for human neurologic diseases.

DESCRIPTION (provided by applicant): The most common malignant primary brain tumor, glioblastoma (GBM), remains uniformly fatal despite surgical resection, incapacitating radiation therapy, and myelodepleting temozolomide (TMZ) chemotherapy. Adjuvant immunotherapy promises to induce robust tumor-specific immune responses that eliminate neoplastic cells with unparalleled specificity, but TMZ-induced lymphopenia would be expected to curtail the induction and persistence of productive antitumor immune responses. However, following periods of lymphopenia, such as those induced by TMZ, there is a homeostatic proliferation of remaining lymphocytes. Thus, T-cells that predominate during this recovery period have a competitive advantage and may become disproportionately over-represented in the recovering lymphocyte population. We and others have identified this as an opportunity to enhance the preferential expansion and maintenance of ex vivo expanded and adoptively transferred anti-tumor T-cells. While high-dose IL-2 has been utilized in most human clinical trials of adoptive immunotherapy for maintenance and expansion of transferred lymphocytes, recent studies in murine models have demonstrated capacity to achieve T cell maintenance and anti-tumor efficacy using in vivo vaccination in the absence of exogenous cytokine supplementation. Given the documented neurotoxicity of IL-2 in patients with GBM, the capacity to facilitate in vivo engraftment of tumor-specific lymphocytes using concomitant vaccination is of paramount interest. Furthermore, our preliminary data demonstrate that vaccines given during the recovery from TMZ-induced lymphopenia result in dramatically enhanced humoral responses and antigen- specific T-cell frequencies in mice and humans. Although our preliminary data uses T-cells from mice with transgenic T-cell receptors to evaluate the combination of vaccine and adoptive immunotherapy in the context of TMZ, such approaches are not easily translated into human studies. Dendritic cells (DCs) loaded with the total antigenic content of tumor cells in the form of RNA (TTRNA), however, provide an innovative strategy that we and others have safely and successfully to expand tumor-specific lymphocytes against a broad repertoire of tumor antigens. Our OVERALL GOAL then is to evaluate the combination of adoptive cellular therapy and TTRNA-loaded DC vaccines during recovery from serial lymphodepletion with TMZ in a murine brain tumor model prior to clinical studies in humans. PUBLIC HEALTH RELEVANCE: Treatment for brain tumors represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States, and brain tumors remain the most common cause of cancer death among children and account for more deaths in adults than melanoma. Vaccines are an attractive adjuvant approach to therapy for these tumors, but are thwarted by the chemotherapy used for these tumors that kills lymphocytes that might respond to these vaccines. This effect of the chemotherapy, however, actually helps maintain anti-tumor lymphocytes infused immediately after the chemotherapy so we will evaluate the combined efficacy of vaccines and transferred antitumor lymphocytes in this proposal.

DESCRIPTION (provided by applicant): The most common malignant primary brain tumor, glioblastoma (GBM), remains uniformly fatal despite surgical resection and incapacitating radiation therapy. Temozolomide (TMZ) chemotherapy has shown a survival benefit in patients with GBM, but median survival remains <15 months. Immunotherapy represents a promising additional approach, but the profound lymphopenia induced by TMZ would curtail the induction of vaccine-induced antitumor immune responses. Recently, however, non-myeloablative lymphodepletion, such as that produced by TMZ, has emerged as a potent adjuvant to adoptive T-cell immunotherapy. Following periods of lymphopenia there is a homeostatic proliferation of lymphocytes designed to recover normal lymphocyte counts. Thus, T-cells that predominate during this recovery period, including those anti-tumor T-cells provided through adoptive transfer, become disproportionately represented in the recovering lymphocyte population. Unfortunately, immunosuppressive regulatory T-cells (TRegs) also undergo homeostatic proliferation after lymphodepletion. Moreover, attempts at eliminating TRegs from adoptively transferred cells are thwarted because these immunosuppressive cells are re-generated de novo from the transferred cells. While antibodies specific for the high-affinity interleukin (IL)-2 receptor alpha (IL-2R1) (CD25) have been shown to abrogate TReg function in animal models, effector T-cell functions are also inhibited. Effector T-cells may not always be dependent on IL-2 signaling, however. During homeostatic proliferation, effector T-cells enjoy a reduced activation threshold and can differentiate into effector memory T-cells directly driven by surges in cytokines that share receptors with IL-2 such as IL-7 and IL-15. Signaling through these cytokines, then, may uniquely bypass the need for IL-2 signaling in effector T-cells in this context. If so, lymphodepletion may provide a unique context wherein adoptively transferred effector T-cells may not require IL-2 signaling and may not be susceptible to inhibition by IL-2R blocking antibodies while TRegs may remain susceptible to this mode of inhibition because of their unique dependence on IL-2 specifically. Consistent with this, our preliminary studies have shown that unarmed IL-2R1-specific antibodies given to mice during recovery from transient lymphopenia, while capable of functionally inactivating TRegs actually dramatically enhance effector T-cell responses. In this proposal, we will test the HYPOTHESIS that during hematopoietic recovery from treatment-induced lymphopenia the generation of de novo TRegs from adoptively transferred anti- tumor T-cells can be selectively inhibited by anti-IL2R1 antibodies leading to enhanced antitumor immunity without induction of limiting autoimmunity. If correct, the translatability of this approach is high because daclizumab, a commercially-available, humanized IL-2R1-specific antibody, functions identically to the antibody used for TReg inactivation studies in mice.

DESCRIPTION (provided by applicant): Recently, temozolomide (TMZ), a myelosuppressive alkylating chemotherapy, has shown a benefit in patients with glioblastoma multiformes (GBM), but median survival is still less than 15 months. Moreover, these conventional chemotherapies lack specificity and result in incapacitating damage to surrounding normal brain and systemic tissues. Immunotherapy may provide an opportunity to eliminate altered neoplastic cells without adding additional toxicity to multi-modality therapy, but the lymphopenia induced by cycles of adjuvant TMZ, now the standard-of-care in patients with GBM, would be predicted to curtail the induction of productive immune responses. However, when given to patients with newly diagnosed glioblastoma multiforme during the recovery from TMZ-induced lymphopenia, the investigators demonstrated that a vaccine targeting a EGFRvIII mutation on GBM induced strong immunologic responses in all vaccinated patients that were accompanied by complete radiographic responses in patients with residual disease and prolonged survival compared to matched historical controls. However, the EGFRvIII mutation is expressed in only about a third of GBMs, limiting the number of patients who may benefit from this therapy and highlighting the need to target more ubiquitously expressed antigens. The recent discovery that greater than 90% of GBMs are associated with tumor-restricted reactivation of human cytomegalovirus (CMV) provides an opportunity to leverage the well characterized viral antigens of CMV as tumor-specific targets. Following a developmental pathway used successfully to advance EGFRvIII specific vaccines to large scale efficacy trials and commercialization, the investigators propose to evaluate the preclinical and clinical advancement of peptide vaccines targeting the immunodominant antigens of CMV. The objectives of this phase I STTR proposal are to finalize the composition of a multi-component peptide vaccine targeting CMV antigens through investigation of the impact of peptide length, hapten conjugation, and the inclusion of class II-restricted helper T cell epitopes on immunologic responses in HLA transgenic mice, and to determine the impact of vaccine dose and timing with respect to administration of TMZ in order to define variables worthy of investigation within the context of early phase clinical trials. PUBLIC HEALTH RELEVANCE: The significance of this research is that it may advance a new therapy for malignant brain tumors as well as provide a strategy for treatment that can be applied to many other cancers. Improved therapy for cancer has significant potential to improve public health and quality of life for patients affected by malignant disease.

PROJECT SUMMARY (See instructions): Project 2: Targeting Immunosuppression Pathways to Enhance Brain Tumor Immunotherapy. John H. Sampson, M.D., Ph.D., M.H.Sc, Project Leader Glioblastoma multiforme (GBM) is the most common of primary malignant brain tumors and despite incapacitating conventional therapy, remains universally fatal. Immunotherapy is an attractive therapeutic alternative, but is limited by the lack of frequent and homogeneously-expressed tumor-specific antigens and a profoundly immunosuppressive host environment. This proposal addresses both of these limitations. The nearly universal presence and homogeneous expression of CMV antigens in GBM, but not normal brain, has now been well-established and provides an unparalleled opportunity to subvert these highly immunogenic viral proteins as tumor-specific targets. Despite the potential immunogenicity of CMV antigens, endogenous immune responses may be limited, at least in part, by an excessive number of immunosuppressive T-cells (TRegs). TRegs are a phenotypically distinct CD4+ CD25 + Foxp3+ population that normally prevent autoimmunity and are uniquely dependent on the high affinity interleukin (lL)-2 receptor (IL-2Ra/CD25) for their function and survival. Monoclonal antibodies (MAbs) that block lL-2Ra have been shown to abrogate Tpeg function in animal models but can also inhibit effective anti-tumor immune responses in mice and humans. Our preliminary data demonstrates that treatment with lL-2Ra blocking MAbs in mice undergoing homeostatic proliferation in response to transient lymphodepletion, as might be seen after therapeutic cycles of chemotherapy, still eliminates Tpegs, but in stark contrast, no longer impairs effector Tcell immune responses. Instead, vaccine-induced immune responses are strongly accentuated. This may be due to homeostatic surges in y receptor cytokines (IL-7 and IL-15) that share receptors with IL-2 and may bypass the need and effects of IL-2 signaling in activated T-cells in this context Preliminary clinical studies show that patients vaccinated with CMV pp65 mRNA transfected dendritic cells and then treated with a commercially-available MAb specific for the human IL-2Ra after therapeutic TMZ chemotherapy, have reduced Tpeg levels, increased pp65-specific CDS* T-cell responses, and progression-free survival interval of 27.4 months. In this proposal, we will test the HYPOTHESIS that during hematopoietic recovery from treatment-induced lymphopenia TRegs can be selectively inhibited by anti-lL2Ra MAbs to enhance anti-tumor immunity without the induction of limiting autoimmunity.

DESCRIPTION (provided by applicant): Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM), the most common of these tumors, is uniformly lethal. Moreover, current therapy is non-specific and produces a median overall survival of only <15 months. In contrast, immunotherapy promises an exquisitely precise approach, and substantial evidence suggests that T cells can eradicate large, well-established tumors in mice and humans even when tumors reside within the brain. Notably, tumor immunotherapy has been recently validated in phase III clinical trials, leading to pivotal approvals by the FDA of at least two prototypic immune-based cancer treatments within the past two years. Bispecific T cell Engagers (BiTEs) represent an emerging class of bispecific antibody that has been shown to effectively redirect T cells against tumor cells. BiTEs promise to overcome many critical barriers that have traditionally limited translation of immunotherapy to the clinic. Separating this platform from other available immunotherapeutic approaches, our preliminary data support that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier (BBB) and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with GBM. To date, BiTEs have not previously targeted tumor-specific antigens, and until our studies, have not yet been tested for their ability to mediate activity against tumors of the central nervous system (CNS). Here, we have designed a BiTE against the EGFRvIII tumor-specific antigen and perform preclinical tests to determine its efficacy against EGFRvIII- expressing GBM. In Aim 1 of this proposal, we will evaluate the risk of toxicity and validate efficacy of the EGFRvIII-specific BiTE in a novel human CD3 transgenic mouse background. The unique model, along with a murine homologue of human EGFRvIII that we have created, will allow us to evaluate the actual construct we plan to take into clinic. Moreover, because the CD3 transgenic mice are heterozygotes and have a normal immune response, this model will allow us to assess the development of secondary autoimmunity and the potential for development of endogenous immune responses against tumor cells that do not express EGFRvIII. In Aim 2, we will explore the possibility that, unlike other macromolecules, BiTEs have the unique ability to penetrate an intact BBB and determine whether this effect can be modulated to improve antitumor efficacy. Finally, in Aim 3, we will conduct early clinical studies to assess biodistribution, early toxicity, and potential efficacy of the EGFRvIII-specific BiTE in patients with EGFRvIII-expressing GBM. Overall, this work has the potential to improve the clinical management of patients with GBM by generating a novel therapeutic.

DESCRIPTION (provided by applicant): Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM) is uniformly lethal, and current therapy is non-specific and produces a median overall survival of <15 months. In contrast, immunotherapy promises an exquisitely precise approach, and substantial evidence suggests that T-cells can eradicate large, well-established tumors in mice and humans even when tumors reside within the brain. Chimeric antigen T-cell receptors (CARs) combine the variable region of an antibody with T-cell signaling moieties to confer T-cell activation with the targeting specificiy of an antibody and are not MHC-restricted. Additionally, co-stimulatory molecules, such as CD28 and 4-1BB, can be added to these constructs to improve T-cell expansion, survival, cytokine secretion, and tumor lysis. Clinical trials utilizing CARs have demonstrated their remarkable potential. However, severe adverse events and even patient deaths have occurred when these CARs have been directed against antigens shared by normal tissues. EGFRvIII is a tumor-specific mutation of the epidermal growth factor receptor that is expressed in GBMs and several other neoplasms. We have previously shown that EGFRvIII can be recognized by highly avid antibodies, so have developed human and murine CAR vectors that specifically recognize EGFRvIII inducing cytokine secretion and in vitro and in vivo tumor lysis. We have also demonstrated that an EGFRvIII-specific peptide (PEPvIII) contains the conformational epitope for EGFRvIII-specific antibodies used in these CARs. Using this peptide, we have shown that these cognate peptides are sufficient antidotes for CARs, suggesting a novel paradigm for reducing the target-specific toxicity of less tumor-specific CARs. Despite their potency, however, CARs still require host conditioning with lymphodepletion for efficacy and are still limited by being susceptible to inhibition by host immunosuppressive factors of which regulatory T-cells (TRegs) have been most frequently implicated. Similarly, while total body irradiation or non-therapeutic chemotherapy has been applied to optimize CAR therapy, it adds additional toxicity without direct anti-tumor efficacy. Our prior experience with TMZ demonstrates that, in addition to having direct clinical benefit in GBM, TMZ can potentiate anti-tumor immune responses directly related to the rebound homeostatic proliferation it induces. To address these issues, in this proposal, we will 1) Evaluate the risk of toxicity, utility of TMZ, and the requirements for efficacy of a tumor-specific, EGFRvIII-targeted CAR in a syngeneic, immunocompetent, orthotopic murine GBM model; 2) Determine if CD3-CD28-4-1BB CAR vectors naturally transfect and activate TRegs and dissect the role of CAR-secreted IL-2 in supporting the growth of intratumoral TRegs and effector T-cells; and 3) Conduct a Phase I clinical trial in TMZ-treated patients with EGFRvIII-expressing GBM to assess CAR safety, kinetics and function.

The overarching purpose of the Biostatistics, Informatics, and Data Coordination Core is to provide expert data analysis and data management to support all studies in the SPORE in Brain Cancer. Core B will ensure not only the timeliness and efficiency, but also the scientific integrity and quality, of the proposed SPORE studies by providing an infrastructure that offers, across studies: (1) expertise and technical support; (2) sophisticated but streamlined analytic, informatics, and data-related methods; (3) tools and procedures to help investigators exploit cutting-edge data and informatics capabilities while employing consistent, high-quality, research practices; and (4) a central access point that coordinates available resources such as institutional datasets. Staffed by biostatistics and informatics experts, database architects, computer scientists, and data managers, this Core builds upon and fortifies successful and longstanding collaborations with Brain SPORE and Duke Cancer Institute (DCI) investigators and our commitment to data standards and quality control. Core B leverages the Preston Robert Tisch Brain Tumor Center, DCI, and Duke University Health System shared resources, to provide SPORE investigators with centralized biostatistical expertise, state-of-the-art technology and database solutions, and access to an enduring coordinated data resource, tailored to meet the needs of brain cancer research. Specific aims addressed by this Core include: (1) To provide biostatistical leadership and expertise in the design, conduct, analysis, and reporting of Brain SPORE studies. Biostatistical collaboration will span all project stages: study design, sample size calculations, randomization, monitoring, analysis and interpretation, and manuscript preparation. (2) To be the SPORE data coordination center by providing services in data acquisition, transfer, integration, quality control, management and sharing for the SPORE in Brain Cancer Projects and Cores. In order to achieve this aim, Core B will ensure that there is a coordinated system for data collection, storage, and standardization; quality assurance and monitoring of data quality; access to tools and software; and promotion of data management best practices throughout the SPORE, including a system of common data management standardized operating procedures (SOPs) and agreed data governance, in order to ensure data integrity, provenance and security and reproducibility study findings. Finally, (3) To develop a longitudinal brain tumor outcomes database (?datamart?) that will serve as an enduring resource for brain cancer research at Duke. Leveraging institutional resources, this datamart will integrate information from Brain SPORE databases and institutional databases including clinical, health services and patient-reported outcomes to create a large clinical and research dataset for current and future analyses.

Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM) is uniformly lethal, and current therapy is non-specific and produces a median overall survival of <15 months. In contrast, immunotherapy promises an exquisitely precise approach, and substantial evidence suggests that T cells can eradicate large, well-established tumors in mice and humans even when tumors reside within the brain. Chimeric antigen receptors (CARs) combine the variable region of an antibody with T-cell signaling moieties to confer T-cell activation with the targeting specificity of an antibody without MHC-restriction. Clinical trials utilizing CARs have demonstrated their remarkable potential. However, severe adverse events and even patient deaths have occurred when these CARs have been directed against antigens shared by normal tissues. EGFRvIII is a tumor-specific mutation of the epidermal growth factor receptor that is expressed in GBMs and several other neoplasms. Our laboratory has developed human and murine CARs for the transduction of T cells targeting the tumor-specific EGFRvIII mutation (EGFRvIII-CARs) for the lysis of EGFRvIII positive GBM. EGFRvIII-CARs should not lead to direct killing of normal tissues as seen with CARs targeting tumor- associated, but not tumor-specific antigens. However, EGFRvIII is heterogeneously expressed and in patients vaccinated with an EGFRvIII-specific peptide vaccine, tumors recur as a result of outgrowth of the EGFRvIII negative tumor cells. Furthermore, patients with GBM are highly immunosuppressed and CARs can be restrained by host immunosuppressive factors present in the GBM microenvironment such as secreted transforming growth factor beta (TGF-?). While the potency of CAR therapy demands tumor-specificity, future CAR development must address tumor heterogeneity and immunosuppression. In Aim 1 we will formally examine potential toxicity of intracerebrally (IC) delivered EGFRvIII-CARs and determine if IC delivered CARs are retained within the brain. In AIM 2, we will examine the impact of EGFRvIII-CAR therapy on tumor heterogeneity through determining if epitope spreading is engendered in host T cells. In AIM 3, we will address immunosuppression though ongoing collaborations in our laboratory with basic scientists that have identified the micro-RNA miR-23a as a key inhibitor of anti-tumor cytotoxic T lymphocyte responses. miR-23a is upregulated by TGF-? and inhibition of miR-23a in T cells subverts TGF-? induced immunosuppression on T cell effector function and dramatically enhances anti-tumor efficacy within murine models. We will investigate whether miR-23a inhibition within EGFRvIII-CAR transduced T cells enhances cytotoxicity and confers resistance to host immunosuppression. This proposal therefore addresses the key issues of tumor-specificity, heterogeneity and immunosuppression and proposes the first in man trial evaluating IC delivery of EGFRvIII- CARs in patients with GBM.

DESCRIPTION (provided by applicant): Malignant primary brain tumors are the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma, the most malignant primary brain tumor, has a median survival of <15 months, and patients with lower grade gliomas progress to the universally lethal tumor types within ten years. Current therapy is incapacitating and limited by non-specific toxicity to systemic tissue or surrounding eloquent brain; however, immunotherapy promises an exquisitely precise approach. We have previously demonstrated that immune responses can be generated specifically against the tumor-specific mutation, EGFRvIII. These were sufficient to eliminate orthotopic gliomas expressing a murine homologue of EGFRvIII and predicted the ability to generate immune responses in humans. In humans with brain tumors, EGFRvIII-specific immune responses were sufficient to consistently eliminate all EGFRvIII-expressing tumors cells without toxicity. Unfortunately, EGFRvIII is heterogeneously expressed and tumors recur as a result of outgrowth of the EGFRvIII negative tumor cells. Recently, using next-generation sequencing, we discovered another highly-conserved and tumor-specific mutation in gliomas at the active site of isocitrate dehydrogenase 1 (IDH1). IDH1 mutations are frequent (>70%) in almost all glioma subtypes, and greater than 90% of IDH1 mutations are IDH1R132H. Although IDH mutations are associated with longer overall survival, IDHR132H status has been occasionally misunderstood to function as an inhibitor of tumor growth. Rather, it denotes a genetically distinct subset of tumors where IDH1R132H generates the onco-metabolite R-2- hydroxyglutarate (R-2HG) which impairs histone and DNA demethylases, prevents cellular differentiation, and promotes tumorigenesis. Recently, small molecule inhibition of IDH1R132H has been shown to reduce tumor cell proliferation; however, it does not induce apoptosis and tumor cells persist in logarithmic growth. Thus small molecule enzyme inhibition may only be partially effective as a therapeutic approach. Preliminary data from our laboratory shows murine responses to vaccination with an IDH1R132H-specific peptide (PEPIDH1M) are both immunogenic and specific. Unlike EGFRvIII, however, the IDH mutation is homogeneously expressed in nearly all tumor cells. The specific aims of this proposal will optimize PEPIDH1M vaccination through adjuvants and host conditioning, assess potential for toxicity and efficacy of the optimal vaccine strategy, and characterize immune presentation and recognition of IDH1R132H in blood samples from patients with IDHR132H-expressing gliomas.

ABSTRACT ? Administrative Core The Administrative Core provides comprehensive administrative and scientific management for all components of the Duke Brain SPORE, including research Projects 1-3, three Shared Resource Cores, and both Developmental Research and Career Enhancement Programs. Programmatic oversight will be provided by the Administrative Core Co-Leads, as well as an Executive Committee consisting of Project and Core Leads and Internal and External Advisory Boards. The functions of the Administrative Core will be carried out through the following Specific Aims: 1) Provide administrative management and oversight; 2) Provide financial management and ensure compliance with fiscal and research policies; 3) Provide oversight of all data operations; 4) To promote integration within the SPORE and the Institution; 5) Maintain and support access to an adequate cancer patient population; and 6) Provide scientific management and evaluate research progress. Through these Aims, the Core ensures the full range of support required to provide administrative management; promote integration, communication, and collaboration; ensure fiscal and regulatory compliance; and oversee data operations, scientific rigor, and research progress. By serving these functions, the Administrative Core allows SPORE investigators to focus on conducting and advancing the translational science supported by the SPORE.

ABSTRACT ? Overall Building on the Duke Brain Tumor Program's longstanding focus on development, refinement, and testing of immunotherapies to treat low-grade gliomas and glioblastoma (GBM), this renewal of the Duke SPORE in Brain Cancer continues work to develop new or improve existing therapies to improve the life of patients with primary malignant brain tumors. To achieve this goal, the Program provides the infrastructure, oversight, and resources to conduct innovative translational research relevant to these treatments (Aim 1). Innovative research proposed includes: 1) studies of potent neoantigen and Cytomegalovirus vaccines in the context of regulatory T cell depletion using a novel approach targeting CD27 to overcome both host immunosuppression and antigenic heterogeneity endemic to GBM (Project 1); 2) studies employing a novel therapeutic strategy to reverse the recently discovered phenomenon of T cell sequestration in patients with GBM and overcome the limitations imposed on immunotherapy by longstanding lymphopenia in this population (Project 2); and 3) studies examining the mechanisms and efficacy of a novel cellular tumor vaccine strategy that uses antigen- loaded monocytes and an endogenous antigen transfer pathway to stimulate potent anti-tumor T cell responses (Project 3). To support this work, the SPORE ensures the availability of expertise through three Shared Resource Cores, all continued from the current award: a Biostatistics and Bioinformatics Core (Core 1), Clinical Trial Operations Core (Core 2), and a Biorepository, Pathology, and Immune Monitoring Core (Core 3) (Aim 2). Research central to the theme of the SPORE is further enhanced by the Program's commitment to seeding developmental research and implementing approaches to grow the research community through its Developmental Research and Career Enhancement Programs (Aim 3). Finally, the Duke SPORE in Brain Cancer continues to participate in and lead inter-SPORE activities to enhance collective impact (Aim 4). Contributions include continuing leadership of an active inter-SPORE collaboration in Immune Monitoring that is working to establish common standards and to harmonize assays, and proposed contributions to the NCI's new Functional Data Commons effort. Taken together, the Duke Brain SPORE is ideally positioned to address and overcome limitations in existing malignant brain tumor therapies and enhance collective research environment to advance shared research community goals.

DESCRIPTION (provided by applicant): Glioblastoma (GBM) remains uniformly lethal. It is also the most common of the primary malignant brain tumors, which are the most frequent cause of cancer death in children and young adults. In contrast to current therapy which is limited by off-target toxicity, immunotherapy promises an exquisitely precise approach, and substantial evidence indicates that, if appropriately redirected, T cells can eradicate large, well established tumors. We have developed a novel bispecific T cell engager (BiTE) that effectively tethers CD3+ T cells to the surface of tumor cells that express the tumor-specific epidermal growth factor receptor mutation, EGFRvIII. Our first EGFRvIII-CD3 BiTE eradicated well-established EGFRvIIIPOS human GBM in a xenograft model reconstituted with human T cells without evidence of autoimmune toxicity. Based on these data, we developed a developed a EGFRvIII-CD3 BiTE from fully-human antibody segments, increasing clinical safety by drastically reducing the potential for immunogenicity. Because all available antibodies specific for human CD3 do not cross-react with any other species including primates, we have rederived a unique, pharmacologically responsive, immunocompetent, human CD3 transgenic murine model that will allow for direct assessment of the humanized BiTE destine for clinical trial, drastically increasing the validity and translatability of pre-clinical efficacy and toxicity studis. Our overall goal is to translate the BiTE therapeutic platform for safe, effective immunotherapy in patients with EGFRvIII-expressing GBM. In this proposal, we seek to perform Investigational New Drug (IND) required experiments, as the Food and Drug Administration (FDA) has outlined to us in our formal Pre-IND meeting. The in vitro cytoxicity and in vivo efficacy of the lead human construct, shown to bind to both targets, will be validated in Aim 1. Aim 2 will complete the necessary optimization of protocols for current good manufacturing practice (cGMP) production of the lead human construct and will yield a sufficient quantity for IND-enabling studies. Aim 3 will document the activity and pharmacokinetics of the cGMP drug product, producing information critical in determining the first-in-man dose. Formal toxicology and stability testing will be completed in Aim 4, allowing for assessment of any potential off-target activity and guiding manufacturing timelines for clinical trial respectively. The sum total of data generated in this proposal, as requested by the FDA during our Pre-IND meeting, will be used to assemble the necessary documents and file an IND application with the FDA in Aim 5.

? DESCRIPTION (provided by applicant): Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM) is uniformly lethal, and current therapy is non-specific and produces a median overall survival of <15 months. In contrast, immunotherapeutic approaches are exquisitely precise and can eradicate large, well-established tumors in mice and humans even when tumors reside within the immunologically privileged brain. However, immunotherapy is limited by the lack of frequent and homogeneously-expressed tumor-specific antigens. We have previously demonstrated that a peptide vaccine targeting the tumor-specific EGFRvIII mutation (PEPvIII) induces immune responses sufficient to eliminate all EGFRvIII-expressing tumor cells in mice and humans without toxicity. Unfortunately, EGFRvIII is heterogeneously expressed and tumors recur as a result of outgrowth of the EGFRvIII negative tumor cells. However, the nearly universal presence and homogeneous expression of cytomegalovirus (CMV) antigens in GBM, but not normal brain, has now been well-established and provides an unparalleled opportunity to subvert these immunogenic viral proteins as tumor-specific targets. In two consecutive clinical trials from our laboratory using dendritic cells (DCs) targeting CMV pp65 in patients with GBM, specific immunologic responses were induced along with remarkably enhanced progression free survival and overall survival. However, DC vaccination is expensive, time-consuming, and commercialization is challenging. In contrast, peptide vaccines are cost-effective, easier to produce, and easier to commercialize. Leveraging our experience with PEPvIII, we have developed a CMV-specific multi-epitope peptide cocktail (PEP-CMV). PEP-CMV vaccination is immunogenic in HLA-A2 transgenic mice and peripheral blood mononuclear cells from CMV seropositive patients with GBM respond to stimulation with PEP-CMV, indicating PEP-CMV is broadly immunogenic in our patient population. We have already shown with PEPvIII that lymphopenia induced by both standard of care temozolomide (TMZ) and dose intensified TMZ can be leveraged to augment immunogenicity and the impact these regimens have on PEP-CMV immunogenicity will be compared in our proposed trial. Additionally, we will examine a novel immunostimulant, tetanus (Td), as an adjuvant to PEP-CMV. In a recent pilot trial from our laboratory, patients randomized to receive Td as vaccine- site pre-conditioning prior to CMV pp65-loaded DC immunization, experienced significantly enhanced PFS and OS in comparison to the control arm. A Td booster was included for site pre-conditioning during PEP-CMV immunogenicity analysis and was demonstrated to significantly enhance IFN¿ secretion of CMV targeted T cells in HLA-A2 transgenics. Therefore, in this proposal, we will test the HYPOTHESIS that vaccination with PEP-CMV after Td skin conditioning will be a feasible, safe, and immunogenic tumor-specific therapy in patients with newly-diagnosed GBM during TMZ chemotherapy, without antigen escape or toxicity.

ABSTRACT: Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM) is uniformly lethal, and current therapy is non-specific and produces a median overall survival of <15 months. In contrast, immunotherapy promises an exquisitely precise approach, and substantial evidence suggests that T cells can eradicate large, well-established tumors in mice and humans even when tumors reside within the brain. Dendritic cells (DCs) bearing tumor antigen can be delivered as a vaccine and migrate to the draining lymph nodes (DLN) to trigger the formation of potent tumor-specific cytotoxic T lymphocytes (CTLs) capable of eradicating tumor while leaving normal tissue unharmed. However, despite individual cases of remarkable patient responses to antitumor DC vaccination, overall objective responses in early phase clinical trials have remained under 15%. The migration of vaccine-delivered DCs is low (~5%), and preclinical studies have demonstrated that preconditioning the vaccine site with the inflammatory cytokines can increase DC migration to the DLN and proportionately increase the magnitude of the antigen-specific T cell response. We hypothesized that preconditioning the vaccine site with the recall antigens in Tetanus/diphtheria toxoid (Td) would induce inflammation, increase DC migration, and elicit more consistently efficacious antitumor immunity. In a recent study in patients with newly diagnosed GBM published in Nature, we demonstrated that unilaterally preconditioning one vaccine site with Td resulted in increased bilateral DC migration to the DLNs and a significant increase in progression free survival and OS - with three of the six Td treated patients living past 4.5 years. A recapitulative murine model corroborated these findings, demonstrating that Td preconditioning both enhanced systemic DC migration to the DLNs and suppressed tumor growth in an antigen-dependent manner. Examination of both patient and murine sera revealed that the chemokine (C-C motif) ligand 3 (CCL3) was the only cytokine or chemokine significantly upregulated after Td preconditioning. Furthermore, in mice we demonstrated that the systemic increase in DC migration after Td preconditioning is dependent upon CD4+ memory effector T cells (CD4Td?mem) and CCL3. However, recent pilot data from our laboratory indicate that the CD4Td?mem are actually responsible for the production of CCL3, suggesting CCL3 serves as the primary driver of the improved antigen-dependent immunity from Td preconditioning. We hypothesize that in addition to enhancing the migration of DCs to the DLN, that CCL3 directly increases antigen-specific T cell magnitude and functionality as well as immune cell trafficking to tumor. This proposal will mechanistically determine the specific role of CCL3 in DC migration, antigen-specific T cell responses, as well as immune cell trafficking, and will further assess if the antitumor efficacy of DC vaccination can be further enhanced by the use of exogenous CCL3 as a vaccine-enhancing drug.

PROJECT SUMMARY We recently reported in Nature that patients with glioblastoma (GBM) randomized to receive a vaccine against Cytomegalovirus (CMV) major integument protein pp65 using a tetanus/diphtheria (Td) vaccine site preconditioning regimen had a statistically significant increase in progression-free survival (PFS) and overall survival (OS) in a small but randomized, blinded, and controlled trial. Half of the patients treated this way were still alive nearly 5 years later despite only 10% of patients typically surviving past 5 years. We targeted CMV because many different groups, including our own, had shown that CMV antigens (Ags), like the immunodominant pp65, are found in GBM, but not surrounding normal brain; this suggests CMV pp65 could be subverted as a highly immunogenic and often homogeneously expressed target for anti-tumor immunotherapy. In our preliminary study, combined with in-depth mechanistic studies in mice, we demonstrated that preconditioning the vaccination site with Td recall Ags increased DC migration to the draining lymph nodes (DLNs), which predicted PFS and OS. Mechanistic studies in mice revealed that the antitumor efficacy of these vaccines was dependent on the vaccinating Ag being present in the tumor, underscoring pp65 as a target in GBM. Efficacy was also dependent on a Td recall response and high systemic levels of the chemokine (C-C motif) ligand 3 (CCL3), which was the only immune mediator elevated in mice and patients. We believe these data warrant confirmation in our proposed phase II trial with a larger series of patients. This will also allow us to confirm some of the mechanistic findings in human patients. However, systemic immunosuppression mediated in part by elevated levels of regulatory T cells (TRegs) in patients with GBM still likely limits vaccine efficacy. Recently, we and others have demonstrated that a clinical- grade antibody targeting CD27 specifically depletes TRegs in transgenic mice and humans. We have also demonstrated that, unlike clinical approaches targeting CD25 to deplete TRegs, that the anti-CD27 antibody simultaneously increases vaccine-induced immune responses. Moreover, it specifically coordinates CD4+ and CD8+ T cell responses leading to enhanced vaccine-induced immunogenicity and increased survival in mice with established orthotopic glioma. Overall, we hypothesize that Td preconditioning will increase DC migration, systemic CCL3, and OS, and that TRegs will be reduced while CMV vaccine responses are further enhanced when a novel anti-CD27 mAb is added to this regimen.

PROJECT SUMMARY ? Project 1 We recently reported in Nature that patients with glioblastoma (GBM) randomized to receive a vaccine against Cytomegalovirus (CMV) major integument protein pp65 using a tetanus/diphtheria (Td) vaccine site preconditioning regimen had a statistically significant increase in progression-free survival (PFS) and overall survival (OS) in a small but randomized, blinded, and controlled trial. Half of the patients treated this way were still alive nearly 5 years later despite only 10% of patients typically surviving past 5 years. We targeted CMV because many different groups, including our own, had shown that CMV antigens (Ags), like the immunodominant pp65, are found in GBM, but not surrounding normal brain; this suggests CMV pp65 could be subverted as a highly immunogenic and often homogeneously expressed target for anti-tumor immunotherapy. In our preliminary study, combined with in-depth mechanistic studies in mice, we demonstrated that preconditioning the vaccination site with Td recall Ags increased DC migration to the draining lymph nodes (DLNs), which predicted PFS and OS. Mechanistic studies in mice revealed that the antitumor efficacy of these vaccines was dependent on the vaccinating Ag being present in the tumor, underscoring pp65 as a target in GBM. Efficacy was also dependent on a Td recall response and high systemic levels of the chemokine (C-C motif) ligand 3 (CCL3), which was the only immune mediator elevated in mice and patients. We believe these data warrant confirmation in our proposed Phase 2 trial with a larger series of patients. This will also allow us to confirm some of the mechanistic findings in human patients. However, systemic immunosuppression mediated in part by elevated levels of regulatory T cells (TRegs) in patients with GBM still likely limits vaccine efficacy. Recently, we and others have demonstrated that a clinical- grade antibody targeting CD27 specifically depletes TRegs in transgenic mice and humans. We have also demonstrated that, unlike clinical approaches targeting CD25 to deplete TRegs, that the anti-CD27 antibody simultaneously increases vaccine-induced immune responses. Moreover, it specifically coordinates CD4+ and CD8+ T cell responses leading to enhanced vaccine-induced immunogenicity and increased survival in mice with established orthotopic glioma. Overall, we hypothesize that Td preconditioning will increase DC migration, systemic CCL3, and OS, and that TRegs will be reduced while CMV vaccine responses are further enhanced when a novel anti-CD27 mAb is added to this regimen.

PROJECT SUMMARY ? Overall Malignant primary brain tumors, like glioblastoma (GBM), are the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Moreover, current therapy is incapacitating and limited by non-specific toxicity. Despite hundreds of clinical trials, few agents have been approved for clinical use, and the tumors addressed in this application remain uniformly lethal. The OVERALL GOAL of this PPG is to develop completely new therapies or to improve existing novel therapeutic approaches through a better understanding of the immunobiology of patient's response to both the tumor and the therapy to achieve prolonged survival in patients with GBM without concomitant toxicity. Within this overall goal, we have focused on eliminating the key barriers that have thus far restricted successful immunotherapy against brain tumors. In the three proposed clinical trials, we will focus on enhancing immunotherapy through more potent platforms, through reducing immunosuppression, through modulating the tumor microenvironment, and through understanding the immune-mediated mechanisms activated by the different platforms. Importantly, this PPG leverages an extraordinary group of senior scientists with a long history of collaboration and successful translational research to accomplish these goals. Project 1, led by John Sampson, will conduct a Phase 2 trial based on his recently published pilot trial demonstrating that preconditioning the vaccine site with tetanus/diphtheria (Td) recall antigens prior to tumor-targeted DC vaccination against Cytomegalovirus (CMV) antigens shown to be re-activated within the tumor dramatically extended OS in patients with GBM. This Phase 2 trial will validate these pilot findings in a larger group of patients. Furthermore, this trial will also incorporate a novel, fully human, clinically approved anti-CD27 mAb that simultaneously reduces immunosuppression and potentiates vaccination through concomitant regulatory T cell depletion and CD27 costimulation. Project 2, led by Michael Gunn, evaluates a completely novel and extraordinarily potent cellular vaccine strategy and examines if monocyte vaccination in humans is safe and will result in robust anti-tumor antigen-specific T cell responses. Project 3, led by Darell Bigner, will conduct a Phase 2 clinical trial based on the promising Phase 1 work with a recombinant oncolytic poliovirus, to elucidate mechanisms by which this therapy generates an anti-tumor immune response, and to examine the synergistic therapeutic combination with the chemotherapeutic lomustine. These projects will be supported by an Administrative Core, as well as three shared resource cores to provide Biostatistics and Bioinformatics resources (Core 1), Clinical Trials and Imaging infrastructure (Core 2), and Correlative Studies and Immune Monitoring expertise (Core 3). While the individual therapies proposed are diverse, our central theme of brain tumor immunotherapy, and our group focus on isolating and addressing the key limitations preventing successful immunotherapy for GBM, creates a highly synergistic and integrated Program that as a unified program will achieve greater results than each project performed in isolation.

ABSTRACT ? Project 1 In brain tumors like glioblastoma (GBM), failures to develop an effective vaccine and achieve immune checkpoint inhibition have been attributed to both the remarkable immunosuppression and extraordinary antigenic intratumoral heterogeneity. A major contributor to immunosuppression in GBM is elevated regulatory T-cells (TRegs) which dramatically suppress T cell effector function and diminish the efficacy of antitumor vaccination. Efforts to deplete TRegs by targeting the interleukin-2 receptor ? (CD25) have been unsuccessful to date, due to cytotoxic effects on effector T cells, which are required to promote antitumor immunity. To overcome this hurdle, Project 1 builds novel preliminary data demonstrating the ability of a clinically available CD27 agonist antibody (?CD27) to simultaneously deplete TRegs and enhance vaccine-induced immune responses. Specifically, the Project tests the hypothesis that class I neoantigens linked to universal class II epitopes will be well-tolerated and rendered more immunogenic by the ability of the clinically available CD27 agonist antibody to deplete TRegs and simultaneously enhance vaccine-induced immune responses in patients with GBM. Aim 1 will evaluate the safety and therapeutic potential of a neoantigen and Cytomegalovirus antigen vaccine in combination with dose-escalating ?CD27 in patients with GBM. Cumulative results will provide critical data on the feasibility and immunogenicity of neoantigen vaccination in patients with GBM to determine if a larger trial is warranted. Aim 2 will determine if ?CD27 simultaneously depletes TRegs and increases vaccine-induced immune responses. It is expected that ?CD27 will reduce TRegs in this patient population while improving vaccine-induced CD8+ and CD4+ T cell responses. If successful, this work will develop a therapeutic strategy for patients with GBM that has enhanced efficacy by addressing the issues of host immunosuppression and intratumoral heterogeneity.

ABSTRACT ? Developmental Research Program The Developmental Research Program (DRP) is a critical component of the Duke SPORE in Brain Cancer, supporting (1) pilot research projects with translational potential relevant to brain tumors and (2) the participation of experienced investigators from other fields to work in brain tumor research. Projects are encouraged to test novel concepts and paradigms that bring new approaches into the field to advance understanding of brain tumor biology and improve brain tumor diagnosis, treatment, and/or prevention. The DRP will support two to three promising projects each year, with the option of renewed funding for one additional year with excellent progress. Projects that have a strong likelihood of either developing into full SPORE projects or projects that can receive funding as independent research projects (e.g., R01s) will be specifically targeted for support. Both individual investigator and collaborative research are eligible. The Specific Aims of this DRP are: 1) To identify and support meritorious basic, translational or clinical research projects that have a high probability of impacting the diagnosis, treatment or prevention of brain tumors; 2) To attract new, outstanding and experienced investigators, currently not working on brain tumors, to pursue brain tumor research; 3) To facilitate collaborative research to test novel concepts and paradigms relevant to brain tumor biology, diagnosis, treatment or prevention, and 4) To evaluate progress on funded projects and facilitate their evolution into full SPORE projects or into independently funded projects. Through these Aims, the DRP helps to ensure success in the Duke Brain SPORE's charge to translate research findings from the bench to the bedside of brain tumor patients.

ABSTRACT We have previously reported in Nature that patients with newly-diagnosed glioblastoma (GBM) randomized to receiving vaccines against Cytomegalovirus (CMV) using a potent vaccine site preconditioning regimen had a statistically significant increase in progression-free survival (PFS) and overall survival (OS). Half of the patients treated this way were still alive nearly 5 years later despite having no genetic markers predicting long-term survival. These results have been repeated in an additional cohort which showed a median survival of 44.1 months with ~36% of patients alive at 5 years. These results are remarkable because GBM remains uniformly lethal with a median OS of < 21 months despite surgical resection, high dose radiation therapy, chemotherapy, and tumor-treating fields, and only 10% of patients typically live past 5 years. In addition to demonstrating the potential for efficacy, our preliminary clinical and laboratory studies demonstrated that preconditioning the vaccination site with tetanus/diphtheria (Td) recall antigens increased DC migration to the draining lymph nodes (DLNs), which predicted PFS and OS as did the production of polyfunctional, CMV-specific T cells. These T cell responses were enhanced by GM-CSF at the vaccine site and pre-vaccination lymphodepletion with standard of care (SOC) temozolomide (TMZ), but inhibited by subsequent adjuvant doses of TMZ. Moreover, mechanistic studies in mice and humans revealed that efficacy was also dependent on producing high systemic levels of the chemokine (C-C motif) ligand 3 (CCL3). We believe these results warrant confirmation in a larger series of patients. Our Specific Aims are: 1. To conduct a larger Phase 2 trial of CMV pp65-loaded DC vaccination in patients with GBM. Patients with CMV positive, newly diagnosed GBM will receive serial vaccines with CMV pp65-loaded DCs with GM-CSF and Td vaccine site preconditioning. GM-CSF and Td vaccine site preconditioning will be employed because they have been shown to enhance DC migration and increase polyfunctional T cell responses in prior studies. TMZ will be given prior to vaccination to induce homeostatic proliferation of the vaccine-induced T cell responses but not given subsequently to prevent killing of vaccine-induced T cells. 2. To confirm predictors of survival. In our prior studies DC migration to draining lymph nodes, systemic and local CCL3, and CMV pp65-specific polyfunctional T cells predicted PFS and OS. Here we will collect samples to confirm these predictors.

ABSTRACT ? Career Enhancement Program (CEP) The Career Enhancement Program (CEP) plays an integral role in the Duke Brain SPORE by providing seed support for pilot research projects with translational potential from promising new and early stage investigators. Projects that are broadly collaborative both within and external to Duke; that test novel concepts and paradigms that bring new approaches into the field; and that further expand the scope of the brain tumor diagnosis, prevention, and management will be prioritized for support. The Specific Aims of the CEP are: 1) Identify and support meritorious translational projects led by NIH-designated new or early stage investigators that have a high probability of impacting the diagnosis, treatment or prevention of brain tumors; 2) Recruit and retain promising women and minority new and early stage investigators with a career focus in translational neuro-oncology research; 3) Facilitate access to programs for leadership training and a highly successful program for grant writing that includes mentoring of new and early stage investigators to enable such faculty members to achieve independent faculty status with emphasis on neuro-oncology research; and 4) Provide continuing evaluation of progress on funded projects and facilitate their evolution into full SPORE projects or independently funded projects. By developing the next generation of investigators and new lines of investigation that can either replace projects within the SPORE or progress as independent, externally supported research projects, the CEP plays a critical role advancing the Program's goal to overcome limitations in malignant brain tumor therapies.

ABSTRACT ? Overall Building on the Duke Brain Tumor Program's longstanding focus on development, refinement, and testing of immunotherapies to treat low-grade gliomas and glioblastoma (GBM), this renewal of the Duke SPORE in Brain Cancer continues work to develop new or improve existing therapies to improve the life of patients with primary malignant brain tumors. To achieve this goal, the Program provides the infrastructure, oversight, and resources to conduct innovative translational research relevant to these treatments (Aim 1). Innovative research proposed includes: 1) studies of potent neoantigen and Cytomegalovirus vaccines in the context of regulatory T cell depletion using a novel approach targeting CD27 to overcome both host immunosuppression and antigenic heterogeneity endemic to GBM (Project 1); 2) studies employing a novel therapeutic strategy to reverse the recently discovered phenomenon of T cell sequestration in patients with GBM and overcome the limitations imposed on immunotherapy by longstanding lymphopenia in this population (Project 2); and 3) studies examining the mechanisms and efficacy of a novel cellular tumor vaccine strategy that uses antigen- loaded monocytes and an endogenous antigen transfer pathway to stimulate potent anti-tumor T cell responses (Project 3). To support this work, the SPORE ensures the availability of expertise through three Shared Resource Cores, all continued from the current award: a Biostatistics and Bioinformatics Core (Core 1), Clinical Trial Operations Core (Core 2), and a Biorepository, Pathology, and Immune Monitoring Core (Core 3) (Aim 2). Research central to the theme of the SPORE is further enhanced by the Program's commitment to seeding developmental research and implementing approaches to grow the research community through its Developmental Research and Career Enhancement Programs (Aim 3). Finally, the Duke SPORE in Brain Cancer continues to participate in and lead inter-SPORE activities to enhance collective impact (Aim 4). Contributions include continuing leadership of an active inter-SPORE collaboration in Immune Monitoring that is working to establish common standards and to harmonize assays, and proposed contributions to the NCI's new Functional Data Commons effort. Taken together, the Duke Brain SPORE is ideally positioned to address and overcome limitations in existing malignant brain tumor therapies and enhance collective research environment to advance shared research community goals.

ABSTRACT Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has been successful against some liquid tumors, but has failed to cure solid tumors. A key reason for CAR T cell failure against solid tumors is antigen heterogeneity. However, pre-clinical studies of CAR T cells against solid tumors in animal models show some promise; in a brain tumor mouse model of glioblastoma, CAR T cells recognizing the EGFRvIII tumor-specific antigen are successful in eliminating tumor, but only against homogeneous tumor and only when mice first receive lymphodepletive host conditioning (via total body irradiation) prior to CAR T cell infusion. Although lymphodepletive host conditioning provides immunological space for CAR T cell expansion, it is problematic in the context of heterogeneous solid tumors, as it impairs endogenous host immunity which is critical for targeting alternative antigens found within the solid tumor. For that reason, successful CAR T cell treatment against solid heterogeneous tumors will require innovative methods to improve CAR T cell persistence to eliminate the need for host lymphodepletive conditioning, and allow for preservation of host endogenous immunity. To achieve this, we propose to utilize metabolic reprogramming of EGFRvIII CAR T cells. Many studies over the last decade have now clearly demonstrated a link between T cell differentiation, function, and metabolism. A predominantly oxidative metabolism supports T cell surveillance, survival, and memory, whereas a predominantly glycolytic metabolism supports biosynthesis to promote effector T cell proliferation and function, but is associated with decreased longevity. The objectives of this R21 proposal are to (1) utilize metabolic reprogramming of EGFRvIII CAR T cells to improve CAR T cell persistence in vitro and in vivo, and (2) test the ability of modified EGFRvIII CAR T cells delivered in the absence of lymphodepletive host conditioning to preserve the endogenous immune system and improve heterogeneous tumor killing. We hypothesize that methods that increase oxidative metabolism will improve CAR T cell persistence, eliminating the need for lymphodepletive host conditioning, maintaining host endogenous immunity, and ultimately improving heterogeneous tumor killing. To test our hypothesis, we will perform the following specific aims: 1) Identify genetic and pharmacological strategies to modify EGFRvIII CAR T cells for enhanced metabolic fitness to support persistence; and 2) Test if metabolically fit murine EGFRvIII CAR T cells delivered in the absence of lymphodepletive host conditioning preserve endogenous immunity. If successful, these approaches can be partnered in future studies with strategies to enhance endogenous host immunity against heterogeneous tumors and overcome a hostile immunosuppressive tumor environment. This work, while performed in a brain tumor model, would be relevant for CAR T cell therapy against multiple solid tumors.

In brain tumors like glioblastoma (GBM), failures to develop an effective vaccine and achieve immune checkpoint inhibition have been attributed to the extraordinary antigenic intratumoral heterogeneity of this disease. To overcome this, successful immunotherapy for GBM will require antitumor T cells with increased magnitude and functionality (potency) and T cells targeting multiple antigens simultaneously (diversity). We have identified 3 strategies to accomplish these goals. First, we will confirm that conjoining neoantigen major histocompatibility complex class I (MHCI) epitope peptides with the universal tetanus P30 class II epitope markedly increases the potency of T cell responses and unveils T cells responses against MHC I antigens that are otherwise non-immunogenic, resulting in de novo immune responses capable of inducing antitumor efficacy. Second, we will administer P30 in the tumor microenvironment to stimulate P30-specific CD4+ T cell help. Help provided to CD8+ T cells at the tumor during the effector stage has been shown to improve the magnitude and persistence of CD8+ tumor infiltrating lymphocytes. Third, we will engage a novel, clinically-available checkpoint agonist ??CD27) and program cell death protein 1 (PD-1) blockade. Stimulating CD27 on antigen-engaged, CD4+ and CD8+ T cells increases the immunogenicity and memory of low-affinity CD8 epitopes, and improves the survival, effector function, and migratory capacity of activated T cells. However, as CD27 stimulation can cause expression of inhibitory PD-1 on T cells, we will also explore PD-1 blockade as a way of limiting this escape mechanism and further enhancing efficacy. We propose that multi-antigen P30-conjoined class I neoantigen vaccination with the novel checkpoint agonist ?CD27 and PD-1 blockade will increase the potency and diversity of neoantigen-specific CD8+ T cell responses, resulting in improved antitumor efficacy. Thus, despite a low mutational burden in GBM, our strategy should enable potent neoantigen-specific T cell responses against a breadth of targets to engender efficacy against heterogeneous tumor. Our Specific Aims are: 1. To determine if multi-antigen, conjoined neoantigen vaccination improves survival in mice with heterogeneous intracerebral glioma; 2. To determine if the addition of class II antigen at the tumor site improves efficacy in these tumors; 3. To determine if ?CD27, alone or in combination with PD-1 blockade, increases the potency and diversity of tumor-specific T cell responses and antitumor efficacy against heterogeneous tumors.