Browsing by Author "Sampson, John H"

Glioblastoma (GBM) is a uniformly lethal cancer with an overall survival of less than 15 months. Aggressive standard of care therapies fail to eradicate these tumors and are non-specific, resulting in incapacitating toxicities. In contrast to such therapies, by virtue of exploiting the inherent specificity and vigilance of the immune system, immunotherapy provides an exquisitely precise approach for safe and effective tumor treatment. Specifically, peptide vaccines offer a promising strategy for inducing potent cytotoxic glioma-specific immune responses. However, they are limited by various mechanisms of glioma-mediated immunosuppression, including low/dysfunctional antigen-presentation, an increased fraction of regulatory T cells, T cell inhibitory pathways, and cytokine dysregulation. Such challenges can be overcome by the combined use of immunomodulatory adjuvants to improve the setting in which T cells recognize and respond to glioma antigens. To this end, a clinically-relevant high-affinity human anti-human CD27 immunomodulatory antibody (αhCD27) that induces potent antitumor T cell responses through engagement of the CD27 T cell costimulatory pathway was recently developed. This antibody is efficacious as a monotherapy in preclinical tumor models and has given rise to significant clinical responses in early phase trials. Given the preliminary success of monotherapy αhCD27 in inducing endogenous antitumor immunity, the overall goal of this dissertation research was to develop a peptide vaccine platform that employs αhCD27 as a vaccine adjuvant for its translation as a novel brain tumor immunotherapeutic.

Chapter 1 provides an overview of brain tumor immunotherapy, including the evolution of the field to date, various genres of treatment modalities, and ongoing progress and challenges. Chapter 2 discusses the approach of T cell immunomodulation, an emerging field in cancer treatment, including the clinical development of various FDA-approved antibodies and their relevance to brain tumors, synergy with current brain tumor standard of care, and emerging immunomodulatory targets. Chapter 3 provides the rationale for targeting the CD27 costimulatory molecule in particular and includes preliminary data that serves as the basis for the preclinical development of αhCD27 as an immunotherapy for brain tumors. Chapter 4 shows the systematic approach for optimizing αhCD27 as a vaccine adjuvant in a murine model of intracranial melanoma alongside a vaccine targeting a model tumor antigen. Lastly, Chapter 5 explores the use of αhCD27 to combat tumor-mediated immunosuppression, an important aspect of its adjuvant activity and the basis for two upcoming phase I clinical trials for malignant glioma.

This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix.

In summary, this work 1) identifies costimulatory T cell immunomodulation as a promising strategy for brain tumor immunotherapy, 2) explores and optimizes the potential for an agonist CD27 to enhance the tumor immune response when combined with a vaccine, 3) has opened up a new line of investigation into the role of CD27 in tumor-mediated immunosuppression, and 4) provides future prospects of utilizing an agonist CD27 antibody as a vaccine adjuvant for the treatment of brain tumors. Together, these studies hold great promise to improve the clinical outlook for brain tumor patients.

Conventional therapy for malignant glioma (MG) fails to specifically target tumor cells. In contrast, immunotherapy offers an exquisitely precise approach, and substantial evidence indicates that if appropriately redirected, T cells can eradicate large, well-established tumors. Even the latest generation of redirected T cell therapies are limited, however, in that they require a centralized manufacturing infrastructure with heavily trained laboratory personnel to genetically modify each patient’s own T cells, use viral transduction which poses uncertain risks, are limited to the initial subset of T cells manipulated and infused, and still face uncertainty as to the optimal T cell phenotype to infuse. This dissertation reports the rational development and clinical translation of a fully-human, bispecific antibody (hEGFRvIII-CD3 bi-scFv) that overcomes these limitations through a recombinant antibody approach that effectively redirects any human T cell to lyse MG cells expressing a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII).

Chapters one, two and three provide an overview of T cell based immunotherapy of cancer and advances in antibody engineering. Also included is a discussion of the current standard-of-care therapy for MG, other immunotherapeutic approaches for MG, and relevant targets and their therapeutic potential for the treatment of MG.

Chapter four details the rational development of a fully-human, anti-human bispecific antibody, hEGFRvIII-CD3 bi-scFv, for immunotherapy of MG. By generating a panel of fully human bispecific single chain variable fragments (bi-scFvs) and testing their specificity through successive stages of screening and refinement, a highly-expressed and easily purified construct with high-affinity to both CD3 and EGFRvIII target antigens was obtained (hEGFRvIII-CD3 bi-scFv). In vitro, hEGFRvIII-CD3 bi-scFv re-directed naïve human T cells to upregulate cell surface activation markers, secrete pro-inflammatory cytokines, and proliferate in response to antigen-bearing targets. Each of these anti-tumor effects were robust and occurred exclusively in the presence of target antigen, illustrating the specificity of the approach. Using MG cell lines expressing EGFRvIII and patient derived MG with endogenous drivers and levels of EGFRvIII expression, bispecific antibody induced specific lysis was assessed. In each case, hEGFRvIII-CD3 bi-scFv was both potent and antigen-specific, mediating significant target-specific lysis at exceedingly low antibody concentrations. Tumor growth and survival was assessed in xenogenic subcutaneous and orthotopic models of human MG, respectively. In both these models, well-engrafted, patient-derived MG was effectively treated. Intravenous administration of hEGFRvIII-CD3 bi-scFv resulted in significant regression of tumor burden in the subcutaneous models and significantly extended survival in the orthotopic models.

Chapter five discusses challenges associated with intratumoral heterogeneity and details two mechanisms by which bispecific antibodies like hEGFRvIII-CD3 bi-scFv can induce epitope spreading, or an immunological response against tumor antigens other than those initially targeted. These mechanisms include: 1) re-activation of pre-existing T cell clones that have specificity for the tumor but fail to mount an immune response prior to bispecific antibody induced stimulation and 2) tumor cell death that results in release of tumor antigens and subsequent antigen uptake, processing and presentation by antigen presenting cells (APCs) leading to a secondary immune response. The chapter concludes with a discussion of a novel class of recombinant antibody molecules developed as part of this dissertation work, Bispecific Activators of Myeloid Cells (BAMs), that function to enhance phagocytosis and antigen presentation. BAM molecules may be useful in conjunction with other immunotherapeutic modalities to induce epitope spreading and combat intratumoral heterogeneity.

Chapter six describes research examining hEGFRvIII-CD3 bi-scFv in a unique human CD3 transgenic murine model. These studies have furthered the rationale for continued clinical translation of hEGFRvIII-CD3 bi-scFv as a safe and effective therapy for MG and have led to the discovery of a novel mechanism of drug delivery to brain tumors. The transgenic murine model was advantageous given that the CD3 binding portion of the fully-human bispecific antibody binds only to human CD3. Accordingly, the model provides a platform where the same molecule to be advanced to human studies can be tested pre-clinically in a pharmacologically responsive, fully-immunocompetent, syngeneic, murine glioma model. In vitro, hEGFRvIII-CD3 bi-scFv induced potent human CD3 transgenic T cell activation, pro-inflammatory cytokine secretion and proliferation exclusively in the presence of the highly-invasive and aggressive murine glioma, CT-2A, bearing EGFRvIII antigen (CT-2A-EGFRvIII). hEGFRvIII-CD3 bi-scFv mediated significant lysis of CT-2A-EGFRvIII at exceedingly low antibody concentrations. In vivo, hEGFRvIII-CD3 bi-scFv significantly reduced tumor growth in human CD3 transgenic mice with well-established, subcutaneous tumors and extended survival of human CD3 transgenic mice with well-established, orthotopic, MG. In the orthotopic setting, adoptive transfer of pre-activated human CD3 transgenic T cells significantly increased efficacy compared to human CD3 transgenic mice treated with hEGFRvIII-CD3 bi-scFv alone.

This led to the hypothesis that activated T cells, known to cross the blood-brain barrier (BBB) to perform routine immunosurveillance of the central nervous system (CNS), may bind to hEGFRvIII-CD3 bi-scFv intravascularly, via its CD3 receptor, and carry or “hitchhike” the large CD3 binding macromolecule to tumors located behind the BBB. Indeed, studies have revealed that adoptive transfer of activated T cells significantly increases the biodistribution of intravenously administered hEGFRvIII-CD3 bi-scFv to orthotopic glioma. Furthermore, blocking T cell extravasation, using natalizumab, for example, a drug used clinically to prevent the migration of T cells to the CNS in patients with multiple sclerosis, completely abrogates the increase in efficacy observed with the adoptive transfer of activated T cells. This newly uncovered hitchhiking mechanism of drug delivery to the CNS provides an important tool to enhance the immunotherapy of brain tumors and has potentially far-reaching consequences for the treatment of other CNS disorders, such as Alzheimer’s or Parkinson’s disease, where issues regarding drug delivery to the CNS are relevant. To begin to study this mechanism of drug delivery in disorders where the blood-brain barrier is intact, we have developed a novel transgenic murine model that expresses EGFRvIII at very low levels within neurons in the brain and have demonstrated that intravenously administered EGFRvIII-targeted recombinant antibody can accumulate in the CNS parenchyma, even in the presence of an intact BBB.

On the basis of these results, a series of clinical research development activities were conducted that have led to the initiation of a clinical study to test the hitchhiking mechanism of drug delivery in patients and ultimately to translate hEGFRvIII-CD3 bi-scFv therapy as a safe and effective treatment for patients with MG. These activities have resulted in a foundation in pre-clinical toxicology, clinical grade biologic manufacturing, clinical protocol development, and regulatory processes necessary to safely translate hEGFRvIII-CD3 bi-scFv therapy to the clinic.

This has involved conducing an extended single-dose toxicity study of hEGFRvIII-CD3 bi-scFv in animals to support studies in humans, the results of which are detailed in chapter seven. To assess for toxicity, human CD3 transgenic mice were administered hEGFRvIII-CD3 bi-scFv or vehicle as a control. Animals were observed for 14 days post-dosing with an interim necropsy on day two. Endpoints evaluated included clinical sings, body weights, feed consumption, clinical chemistries, hematology, urinalysis, and histopathology. There were no clinical observations, evidence of experimental autoimmune encephalomyelitis (EAE), or change in body weight or feed consumption noted during the study that would be associated with toxicity. Furthermore, no statistical difference was observed between drug- and control-receiving cohorts in hematological parameters or urinalysis and no pathological findings related to EGFRvIII-CD3 bi-scFv administration were observed. Statistical differences were observed between drug-treated and control-treated cohorts for some of the clinical chemistries assessed, such as hematocrit, calcium and phosphorus among the female, 14-day analysis cohorts.

To produce hEGFRvIII-CD3 bi-scFv and autologous activated T cells to be administered to patients for clinical study, chemistry, manufacturing and control protocols for the production of clinical grade hEGFRvIII-CD3 bi-scFv and autologous activated T cells were developed and implemented. The data presented in chapter eight describe optimized manufacturing processes and rationale for the selection and implementation of in-process and release analytical methods. This work includes the generation of a stable Chinese hamster ovary (CHO) cell line that expresses high levels of hEGFRvIII-CD3 bi-scFv, the generation and certification of a current Good Manufacturing Practice (cGMP) master cell bank (MCB), optimization and scale up of upstream and downstream manufacturing procedures, and development of standard operating procedures (SOPs) for the manufacture and assessment of clinical grade hEGFRvIII-CD3 bi-scFv and autologous activated T cells. Together, these have allowed for the production of clinical grade antibody and autologous patient derived cells within Duke University Medical Center. The production of recombinant antibodies for use in the clinic is a complex endeavor often performed in industry with teams of highly skilled scientists who test and optimize manufacturing protocols using a large, well-established manufacturing infrastructure. The successful production of clinical grade recombinant antibody at an academic center, therefore, represents a significant achievement and would likely be of interest to other academic-based researchers and clinicians embarking on similar clinical endeavors.

Chapter nine describes a clinical protocol for a phase 0 study of hEGFRvIII-CD3 bi-scFv in patients with recurrent EGFRvIII-positive glioblastoma (GBM). The protocol details intravenous administration of single doses of radiolabeled hEGFRvIII-CD3 bi-scFv with and without pre-administration of radiolabeled autologous activated T cells in a given patient. This will allow for imaging studies that will reveal the pharmacokinetics of the recombinant antibody both with and without adoptive transfer of autologous activated T cells. Endpoints include an assessment of the: intracerebral tumor localization of 124iodine (I)-labeled hEGFRvIII-CD3 bi-scFv with and without prior administration of 111indium (In)-labeled autologous T cells; percentage of patients with unacceptable toxicity; percentage of patients alive or alive without disease progression six months after study drug infusion; median progression-free survival; 111-In-autologous T cell intracerebral tumor localization; and percentage of patients who are EGFRvIII-positive at recurrence.

Chapter 10 concludes with a discussion of ongoing and anticipated future pre-clinical and clinical research. Together, these data presented in this dissertation have been submitted to the US Food and Drug Administration (FDA) in support of an Investigational New Drug (IND) application permit for clinical studies of hEGFRvIII-CD3 bi-scFv at Duke University Medical Center. This clinical study of the hitchhiking mechanism of drug delivery and the pharmacokinetics of hEGFRvIII-CD3 bi-scFv may have far reaching implications for disorders of the CNS where drug access past the BBB is relevant and will advance our understanding of hEGFRvIII-CD3 bi-scFv therapy in patients, guiding future clinical study of the molecule as a safe and effective form of immunotherapy for patients with EGFRvIII-positive GBM and other cancers.

The most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.

Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both in vitro and in vivo using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials.

In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.

Malignant gliomas are the most common primary brain tumor in adults, with an incidence of five cases per 100,000 persons per year. Grade IV glioblastoma is the most aggressive form and prognosis remains poor despite the current gold-standard first-line treatment – maximal safe resection and combination of radiotherapy with temozolomide chemotherapy – resulting in a median survival of approximately 20 months. Tumor recurrence occurs in virtually all glioblastoma patients, and there currently exists no accepted treatment for these patients. Recent advances in novel directed therapeutics are showing efficacy and have entered clinical trials. This work spans the pre-clinical and clinical development of a bispecific antibody – EGFRvIII:CD3 bi-scFv – for the treatment of malignant gliomas.

Chapter 1 reviews current front-line immunotherapy research in the fields of antibodies, including BiTEs and checkpoint inhibitors, and tumor vaccinations, including peptide and dendritic cell vaccinations. Furthermore, challenges specific to high-grade gliomas as well as opportunities for combination therapies are discussed. Chapter 2 introduces the architecture of the novel bispecific antibody EGFRvIII:CD3 bi-scFv and provides an overview of the molecule’s efficacy in various models. EGFRvIII:CD3 bi-scFv is a truncated antibody with dual specificity. One arm targets the epidermal growth factor receptor mutation variant III (EGFRvIII), a tumor-specific antigen found on glioblastoma. The other arm targets the human CD3 receptor on T cells. As an obligate bispecific antibody, simultaneous binding of both receptors by multiple EGFRvIII:CD3 bi-scFv’s results in the crosslinking of CD3 receptor, activation of T cells, and release of perforin/granzyme which lyses the proximal EGFRvIII-expressing tumor cells. EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma.

Chapter 3 outlines the in-house development of a scalable clinical production process using a WAVE (GE) bioreactor and describes the cGMP-compliant clinical production of EGFRvIII:CD3 bi-scFv. The 250-liter cGMP-production run yielded more than four grams of clinical drug material.

Chapter 4 demonstrates that EGFRvIII:CD3 bi-scFv produced using the cGMP development process is efficacious in both in vitro and in vivo models of glioblastoma. The chapter also describes the approach used to calculate the starting dose for the upcoming first-in-human clinical trial. First-in-human clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach. In order to establish a first-in-human dose, as advised by the FDA for bispecific antibodies, this work uses a MABEL approach to select a safe starting dose for EGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling. Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng EGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a first-in-human clinical study.

Chapter 5 describes the pharmacokinetic properties of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, the chapter first describes the development of a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, was used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. These results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and Phase I studies of EGFRvIII:CD3 bi-scFv.

Finally, Chapter 6 provides an outlook of the future development of cancer therapeutics for treating malignant gliomas.

Glioblastoma (GBM) is the most common and deadly primary malignant brain tumor. Despite an aggressive multimodal standard of care, prognoses and patient quality of life remain exceptionally poor, due in part to the non-specific and toxic nature of conventional treatment options. By contrast, adoptive cell transfer of T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) has emerged as a promising approach to targeting brain tumors, given that T cells have migratory capacity within the brain parenchyma, a mechanism to discriminate between normal and neoplastic tissue, and can develop immunological memory. This work spans the development of an effective CAR T-cell immunotherapy strategy targeting the tumor-specific driver mutation, EGFRvIII, which is expressed exclusively by GBM and other cancers but not normal tissue.

Chapters 1 and 2 provide an overview of GBM and the current clinical standard of care, the role of the immune system as it relates to the development and eradication of cancer, and an introduction to various immunotherapy platforms under active preclinical and clinical investigation. Chapter 3 details the historical context of adoptive T-cell immunotherapy and its evolution to present day, detailing our early proof-of-principle studies that led to the inception of the original research described herein. Data presented in Chapter 4 summarizes our translational objectives in implementing CAR T-cell immunotherapy clinically for patients with newly-diagnosed GBM. Chapter 5 addresses a perennial limitation to the immunotherapy of solid tumors by demonstrating an ability of modified CARs to circumvent intratumoral immunosuppression mediated by regulatory T cells. In Chapter 6, we present data that demonstrate, for the first time, a novel role for host lymphodepletion in cellular immunotherapy delivered directly into the brain. Lastly, Chapter 7 contains concluding remarks on the current state of CAR technology and important future directions.

In summary, our work here demonstrates that CAR T cell immunotherapy 1) has curative potential against highly established, orthotopic and syngeneic murine GBM, 2) can be strategically implemented within the current clinical treatment paradigm for GBM, and 3) can overcome a major mechanism of immunosuppression, demonstrating the versatility of gene-modified T cells for the treatment of malignant brain tumors. Together, these studies have paved way for the rationale design of two phase I clinical trials in patients with newly-diagnosed and recurrent EGFRvIII-positive GBM at Duke University.

The histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens.

Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes.

This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix.

In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.

Glioblastoma (GBM) is the most common and deadly primary malignant brain tumor with a median survival of <20 months. Despite aggressive standard of care therapies, GBM remains lethal. Alternatively, immunotherapy in the form of adoptively transferred T cells expressing chimeric antigen receptors (CARs) has emerged as a promising approach to targeting brain tumors. Preclinically, CARs for GBM-specific epidermal growth factor receptor variant III (EGFRvIII; CARvIII) have been successful combined with total body irradiation (TBI) in treating tumors exclusively expressing EGFRvIII. While effective at the bench, this model does not translate clinically; therefore, next generation immunotherapy aims to enhance CARs to secrete immunomodulatory factors to better treat GBM. This work spans the development and success of this new CAR “armored” to secrete IL-12, a stimulatory cytokine that enhances T cell persistence and function, to treat orthotopic heterogeneous GBM.Chapters 1 and 2 provide an overview of GBM. Detailed in these chapters are the current clinical standard of care, immune privilege of the brain, and various immunotherapies under active preclinical and clinical investigation. Chapter 3 details the history of adoptive T cell therapy in the context of brain tumors. Specifically, it focuses on existing CAR T cell therapies for GBM. Chapter 4 summarizes the next generation of “armored” CARs currently being developed. In Chapter 5 we present the development of a new CAR T therapy and demonstrate, for the first time, its efficacy in treating an in vivo, heterogeneous brain tumor. Chapter 6 summarizes the data gathered from our single-cell sequencing of immune cells collected from CAR-treated, tumor-bearing brains and flow cytometry. Additionally, we briefly evaluated the toxicity of our CAR treatment. In Chapter 7, we evaluate numerous in vivo, immunological depletion models to better understand the mechanism of action of our CAR therapy. To conclude, Chapter 8 contains closing remarks on the current state of CAR T cell therapy and future directions. To summarize, we have engineered a fourth-generation CAR T cell that can cure homogeneous and kill heterogeneous brain tumors in immunocompetent mice without host lymphodepletive preconditioning through reprogramming endogenous immune cells in the tumor microenvironment.

Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME). GBM is universally lethal and remains highly refractory to immunotherapy, including immune checkpoint blockade (ICB). Resistance to ICB is a central issue in GBM and is thought to be primarily driven by tumor-imposed immune dysfunction. Here, however, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a novel driver of ICB resistance. CaMKK2 is highly expressed in myeloid cells and neurons and is associated with worsened survival in patients with GBM. Using CaMKK2-deficient preclinical murine models, we determine that host CaMKK2 expression reduces survival and promotes ICB resistance in a T cell-dependent manner. Single-cell RNA-sequencing, flow cytometric profiling, and immunofluorescence staining of immune cells in the tumor reveal that CaMKK2 expression is associated with several pro-tumor, ICB resistance-associated immune phenotypes. For instance, CaMKK2 promotes terminal exhaustion in CD8+ T cells and reduces the expansion of effector CD4+ T cells, additionally limiting their tumor penetrance and interactions with myeloid cells. CaMKK2 also maintains myeloid cells in an Apolipoprotein E+, disease-associated microglia-like phenotype, which is associated with ICB resistance. Conversely, CaMKK2 deficiency permits the programming of tumor-associated macrophages (TAMs) to a dendritic cell (DC)-like phenotype that is associated with ICB response. Finally, we determine that it is neuronal CaMKK2 expression, specifically, that is required for maintaining the ICB resistance-associated MHC-IIlow TAM phenotype. Our findings reveal CaMKK2 as a novel contributor to ICB resistance, primarily via non-hematopoietic cells, in GBM and additionally newly identify neurons as a critical driver of pro-tumor immune phenotypes within the GBM TME.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Conventional therapies for GBM typically fail to provide lasting antitumor benefits, owing to their inability to specifically eliminate all malignant cells. Immunotherapy is currently being pursued as a strategy to address this unmet need, in light of the cell-specific cytotoxicity an immune response can afford. Of the various immunotherapeutic modalities, cancer vaccines are currently being evaluated as a means to direct the adaptive immune system to target residual GBM cells that remain following standard-of-care treatment. To date, no cancer vaccines have been proven effective against GBM; however, only a few have reached phase III clinical testing. Clinical immunological monitoring data suggests that GBM vaccines are capable of stimulating immune responses reactive to GBM antigens, but whether these responses have an appreciable antitumor effect on GBM is still uncertain. Nevertheless, there have been several promising outcomes in early phase clinical trials, which lend encouragement to this area of study.

In this dissertation, we explore the therapeutic potential of cancer vaccines targeting malignant astrocytoma-specific somatic missense mutations – or neoantigens. This pursuit was inspired by recent data from a phase III clinical trial with a protein vaccine targeting the GBM-specific antigen EGFRvIII, revealing that most recurrent tumors were composed of EGFRvIII-deficient or -suppressed tumor cell variants. This outcome, known as antigen escape, is likely a consequence of the profound heterogeneity of GBM tumors and, altogether, suggests that monovalent immunotherapeutic strategies targeting subclonal GBM antigens are likely insufficient to treat this disease. Conversely, personalized cancer vaccines targeting patient-specific missense mutations have the potential to elicit a multivalent, tumor-specific immune response that may target a broader repertoire of GBM cells.

Chapters 1-4 offer a comprehensive review of GBM, an overview of immunotherapy for malignant brain tumors, and promising vaccines that are currently being explored for the treatment of GBM. In chapter 5, we present a novel method that we have developed for evaluating neoantigen-specific lymphocytes from miniscule amounts of solid tumor tissue, which we believe can aid in immunological monitoring of neoantigen-specific immune responses in the clinic. In chapter 6, we elucidate the mechanism of an efficacious neoantigen vaccine, which led to the development of rationally-designed, neoantigen-targeting, synthetic long peptide vaccines with enhanced immunogenicity and efficacy using a universal helper epitope. In chapter 7, we explore the utility of minigene-transfected dendritic cell (DC) vaccines for targeting neoantigens, in which we reveal several significant limitations of traditional GM-CSF + IL-4-generated DCs. Finally, chapter 8 discusses future prospects for enhancing the therapeutic response by cancer vaccines. Together, this original work provides several encouraging insights for the development and evaluation of personalized cancer vaccines for GBM.