Browsing by Subject "Immunotherapy"

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.

Dendritic cells (DC) have been the key elements in developing cancer vaccines to induce potent T cell responses to eradicate tumors. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous primary DC have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer to endogenous DC from currently available Ag-delivering vehicles needs to be further improved. Here, I aim to develop an alternative cellular vaccine platform that can circumvent the aforementioned problems. I reason that classical Ly-6Chi monocytes (i.e. monocytes hereafter) can be a promising candidate to be loaded with tumor Ag and induce effective T cell responses. With advantages including easy-purification from human peripheral blood, monocytes evidently can present antigens directly via in vivo differentiation into bona fide DC or indirectly via antigen transfer to lymphoid resident DC to induce strong Th1 or cytotoxic T lymphocyte (CTL) responses. However, whether monocytes exploit favorably direct or indirect pathway to present the same Ag they are carrying to trigger effective immune responses remains unclear. Furthermore, how exactly monocytes or monocyte-derived cells transfer antigens to lymphoid resident DC has yet to be elucidated. I hypothesized that Ag-loaded monocytes can induce strong anti-tumor immunity and began the research by investigating the immune responses that can be induced by Ag-loaded monocytes. I then went on to determine the mechanisms that mediate monocyte-induced immune responses and evaluate anti-tumor efficacy of this monocyte vaccine.

In the first part of this study, I characterized the immune responses induced by Ag-loaded monocytes. By using negative selection via magnetic-activated cell sorting (MACS) columns, I was able to purify monocytes from bone marrow (BM) cells and determined that these monocytes could be successfully loaded with Ag in the forms of proteins, peptides and mRNA. I found that intravenously (IV) injected Ag-loaded monocytes induced robust Ag-specific CD4+ and CD8+ T cell responses in mice without triggering antibody responses. This vaccine activity of Ag-loaded monocytes appeared to be dose-dependent and required live monocytes with no need of ex vivo stimulation. I found that Ag-specific CD8+ T cells induced by Ag-loaded monocytes were functionally more robust than those induced by protein Ag emulsified in a traditional adjuvant CFA.

In the second part of this study, I investigated how IV injected Ag-loaded monocytes stimulate T cell responses. I identified that the spleen is the primary immune niche for Ag-loaded monocytes to induce T cell responses. I found that Ag-loaded monocytes mainly retain in the spleen where they begin to differentiate into phenotypic DC. Surprisingly, major histocompatibility complex (MHC)-deficient monocytes maintain full capacity to stimulate T cell responses, suggesting that Ag-loaded monocytes do not present Ag by themselves. I determined that endogenous splenic DC is absolutely required for monocyte-induced T cell responses. Therefore, Ag-loaded monocytes induce T cell responses indirectly via transferring Ag to splenic DC even they do differentiate into phenotypic DC in the spleen. I elucidated that this monocyte-to-DC Ag transfer occurs via gap junctions for CD8+ T cell responses and via macrophages for CD4+ T cell responses.

In the final part of this study, I demonstrated that IV injected Ag-loaded monocytes have robust anti-tumor efficacy targeting both model and validated tumor Ag in prophylactic, memory and therapeutic murine SQ melanoma models. The anti-tumor efficacy is superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Furthermore, I demonstrated that Ag-loaded monocytes have a clear anti-tumor efficacy in an intracranial glioblastoma (GBM) model targeting against mutant isocitrate dehydrogenase 1-R132H (mIDH1-R132H), a validated tumor Ag of GBM.

In conclusion, IV injection of unactivated Ag-loaded monocytes without adjuvants induces highly efficacious anti-tumor T cell responses via dual independent and efficient Ag transfer pathways to splenic DC. These findings revise the paradigm that monocytes have to be activated ex vivo to achieve optimal vaccine efficacy and reveal unappreciated cell-associated Ag acquiring pathways of splenic DCs that can be specifically manipulated for future vaccine design in the treatment of human cancers.

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

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.

Breast cancer outcomes globally are dependent on access to advanced operating room technology and radiation therapy facilities. In low-income countries, 90% of patients cannot access either radiation or surgery due to a lack of infrastructure, medical specialists, and funds. Therefore, there is a dire need for effective, resource-appropriate technology to improve cancer care in the absence of radiation and surgery, particularly for breast cancer which is the most common cancer in women globally.Breast cancer is a disease with a significant disease burden in both low- and high-resource settings. In both settings, breast cancer is fundamentally treated based on the degree of spread. Non-metastatic, focal tumors are treated with "local" therapy with or without additional "locoregional" therapy based on the degree of local invasiveness. When invasive tumors are found, treatment must include "systemic" immuno- or chemo-therapy as there is a presumed presence of circulating tumor cells. Some treatments, like radiation, occasionally incite an "abscopal effect" whereby tumor death in situ exposes tumor-associated antigens (TAAs), eliciting systemic, immune-mediated destruction of distant tumors; however, this mechanism remains elusive. An alternative "local" cancer therapy, ablation, involves focal destruction of tissue using a small instrument delivered under the skin with image guidance. Various ablation modalities (cryotherapy and radiofrequency ablation) have been observed producing the aforementioned abscopal effect because the necrotic/apoptotic tumor milieu remains in situ, activating tumor-specific cytotoxic T cells. Ablation with ethanol is particularly suited for low-resource settings as it can be performed with only a needle and syringe and may be guided with minimal imaging (ultrasound). Ablation with ethanol has been extensively used for hepatocellular carcinomas, and even though it is fast and effective, the injection of liquid ethanol into a dense tumor is difficult to control. Currently, ethanol ablation often requires multiple treatment sessions for residual or recurrent tumors. Here I utilized the phase-changing formulation of ethanol and the polymer ethylcellulose to increase coverage of a target ablation zone and produce a greater anti-tumor response. Previously, our lab has shown that ethyl cellulose-ethanol (ECE) ablation could more efficiently ablate superficial hamster cheek-pouch tumors than pure ethanol. However, a treatment strategy for breast cancer in low-HDI settings must address invasive disease which previous work with ECE had yet to address. In low-HDI settings, where there is less access to diagnostic services, many patients present with advanced disease. Even in high-HDI settings, current treatment options fall short for patients with recurrent or metastatic breast cancer. The goal of the dissertation was to develop a low-cost, easily accessible method for treating invasive breast cancers. To achieve this goal, I attempted to produce a reliable abscopal response using ECE ablation and other easily accessible drugs. I first optimized ECE ablation for use in a mouse breast cancer model, finding the maximum tolerable dose, and optimizing target tissue necrosis by modulating ethylcellulose concentration. I then characterized the local and systemic immune response to ECE ablation in several tumor models to identify a strategy for improving anti-tumor responses. To enhance the likelihood of an abscopal effect after ECE, I then utilized cyclophosphamide (CP) and buffer therapy to reverse tumor microenvironment (TME) hostility. Oral sodium bicarbonate buffer therapy (bicarb) reduces tumor acidosis and has been shown to increase cytotoxic T lymphocyte (CTL) infiltration into tumors and decrease CTL anergy. CP, a widely accessible chemotherapy, has immunomodulatory effects when used at low, non-curative doses, specifically depleting pro-tumor regulatory T cells. I demonstrated that an anti-tumor response after ECE ablation is more likely in a tumor primed with sodium bicarbonate and low-dose CP. I will refer to this combination treatment as ECE + CP + bicarb. To optimize the treatment and demonstrate efficacy, small animal tumor models were utilized to determine in vivo anti-cancer responses. Both non-metastatic and metastatic models were utilized to determine both the local and systemic response to the new therapeutic ECE + CP + bicarb to understand for which types of breast cancer this therapy was appropriate. Aim 1) Maximize breast tumor necrosis using ethanol ECE injections. First, I optimized ECE delivery to increase target tissue necrosis while minimizing adverse events and tumor growth. I used various dosing schedules to determine the maximum tolerable ECE dose in murine 67NR flank tumors, which is 6 mL/kg or 150 µL for a 25 g mouse. The concentration of ethylcellulose in ECE was modulated to determine the role of the phase-changing polymer on the target tissue ablation. I found that 6% ethylcellulose produced the most tumor necrosis and injectate retention at the injection site, thus 6% ECE was selected as the optimal concentration for these non-metastatic 67NR tumors. I also demonstrated that compared to ethanol alone, ECE improves the ablation zone's compactness and decreases local adverse events due to ethanol leakage. Using Raman spectroscopy through ex vivo tissue, I found that ECE slows ethanol diffusion through 67NR tumors compared to pure ethanol alone. Finally, I demonstrated that ECE improves long-term survival compared to an injection of the same volume of pure ethanol in murine tumors. While I developed a method of ECE that able to reduce primary tumor growth in a non-metastatic model, the local and systemic effect of ECE was still unknown. Aim 2) To characterize the local and distant immune response to ECE. To develop a therapy capable of treating invasive breast cancer, our goal was to create a systemic anti-tumor immune response initiated by tumor ablation. However, the immune response to ECE ablation had yet to be characterized. By comparing an injection of ECE to an injection of the same volume of saline in a mouse tumor model, the effect of ECE could be monitored. In this aim I demonstrated that ECE increases tumor-infiltrating lymphocytes in several models, including chemically-induced and cell-line derived tumors. Additionally, in mice lacking CD8+ T cells, the anti-tumor response of ECE was significantly reduced when compared to immunocompetent mice, suggesting reliance on CD8+ T cell immunity. In the metastatic 4T1 model, ECE increased splenic populations of activated CD8+ T cells and decreased the number of splenic CD11b+Ly6G+Ly6C+ neutrophils. Finally, I discovered that after a single ECE injection, the number of metastases were decreased compared to saline injections and standard of care treatment: surgical excision. Local ECE ablation was found to produce local and systemic immunomodulation favoring an anti-tumor immune phenotype; however, most primary tumors never completely regressed. Therefore, the readily-accessible, low-cost agents CP and bicarb were implemented to further enhance the anti-tumor immune response following ECE ablation. Aim 3) Enhancing ECE with readily-accessible, low-cost immunomodulatory agents. In Aim 2 the immune response to ECE ablation was characterized, however, it was not strong enough to cure animals with invasive TNBC. I hypothesized that ECE ablation was insufficient to cure malignant TNBC due to the highly immunosuppressive TME. Two methods for reducing TME immunosuppression were employed: low-dose CP and oral bicarb therapy. A single low-dose of CP was utilized to deplete Tregs before ablation. Bicarb was ingested by mice for the duration of the study to decrease tumor acidosis and increase the infiltration of anti-tumor T cells into the TME. TNBC cell lines with a range of natural immunogenicity were utilized to test the efficacy of ECE + CP + bicarb including 4T1, 67NR and EO771. The combination of ECE + CP + bicarb eradicated a majority of tumors, eliminating primary tumors and metastatic disease for most animals. Furthermore, the anti-tumor response was found to have a CD8+ T cell-dependent manner in EO771 tumors. In all three cell lines, mice cured with ECE + CP + Bicarb experienced a reduced tumor growth rate when re-challenged with a tumor. When surgery was used instead of ECE ablation, the antimetastatic effect was reduced implying that the in situ necrosis left by ECE ablation is crucial for the systemic anti-tumor response. In summary, I successfully created a novel anti-cancer therapeutic using ECE + CP + bicarb that is effective in aggressive TNBC tumors. The work in these Aims laid a foundation for the use of ECE ablation in breast tumors. A safe and effective dosing strategy was identified in small animal models, as well as methods for boosting the anti-tumor response to ECE ablation. An anti-tumor response to ECE ablation was identified along with the antimetastatic properties of local ECE ablation. These findings provoke many new research questions about the interplay of acidosis, wound healing, inflammation, and necrosis in the TIME and how they affect systemic disease progression. ECE ablation still requires much more investigation to reach the ultimate goal of impacting patient outcomes. For example, the mechanism for the anti-tumor and anti-metastatic response has yet to be fully elucidated. The work here suggests that CD8 T cells are implicated in the therapeutic response; however, the impact of ECE ablation on other crucial players in the TIME (myeloid cell populations, tumor metabolism, hypoxia, and the extracellular matrix) are largely unknown. Additionally, since the therapeutic power of ECE + CP + bicarb does not rely on specific tumor biomarkers, ECE + CP + bicarb could be effective in other tumor types. Specifically, we are interested in using ECE for cervical cancer which disproportionately affects low-HDI settings resulting in significant mortality globally. Another strategic use of the immunomodulatory effect of ECE is in combination with immunotherapies. ECE ablation induces a local inflammatory response and releases necrotic tumor debris that may increase the strength of the response to checkpoint inhibitors. Future research is needed to assess these new combinations.

The adaptive immune system plays a crucial role in combating tumors through immunosurveillance and responding to immunotherapies. However, many studies investigating the anti-tumor immune response focus primarily on tumor-infiltrating lymphocytes (TILs), which often lack specificity for the antigenic epitopes presented on tumor cells, making them less relevant for effective anti-tumor immunity. To gain insights into novel immunotherapeutic targets and biomarkers of response, it is essential to characterize the phenotypic features and dysfunctional mechanisms of tumor-specific T cell populations. This requires the use of tumor models that express known neoantigens in order to study tumor-specific T cell responses in vivo. While transplant models with known neoantigen expression are widely used, there is a limitation in the availability of autochthonous tumor models where the tumor coevolves with the immune system. In this dissertation, various approaches to studying the tumor-specific immune response in the autochthonous setting are presented and discussed. One such approach that has been developed involves combining CRISPR/Cas9 and sleeping beauty transposase technology to create an autochthonous orthotopic murine sarcoma model. This model incorporates key genetic elements such as oncogenic KrasG12D, functionally impaired p53, and the expression of known MHCI and MHCII sarcoma neoantigens. By utilizing MHC tetramer flow cytometry, a tumor-specific immune response in the peripheral blood was identified as early as 10 days after tumor induction, leading to effective tumor clearance. Interestingly, when CD8 and CD4 T cells were co-depleted, tumors developed at a high penetrance. However, depleting either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These findings indicate that both CD8 and CD4 T cells can independently contribute to immunosurveillance and participate in the clearance of sarcomas expressing MHCI and MHCII neoantigens. Understanding the tumor-specific immune response in autochthonous models is crucial for uncovering new targets for immunotherapy and identifying biomarkers of response. The development of the autochthonous orthotopic murine sarcoma model described in this dissertation provides a valuable tool for investigating the mechanisms and characteristics of tumor-specific T cell responses in an in vivo setting.

Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials.

However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward.

This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.

The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models.

The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.

In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.

Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. The heterogeneity of MB molecular subtypes as well as the failure of standard therapies to treat metastatic or recurrent disease necessitates more potent targeted approaches that minimize collateral toxicity. Immunotherapy presents a promising strategy by specifically targeting cancer cells and to date, there have been few successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting the proliferation and differentiation of progenitor cell populations within the developing cerebellum. To test the developing cerebellum as a source of tumor rejection antigens, we adapted two animal models of MB recapitulating human Sonic Hedgehog (SHH) and Group 3 tumors for immunotherapeutic evaluation. Immunologic characterization of these murine models revealed subtype-specific differences in the tumor microenvironment and a differential response to immune checkpoint blockade. We used total embryonic RNA from the developing mouse cerebellum (P5) to generate antigen-specific T cells and confirmed the immunogenicity of targeting developmentally regulated antigens in vitro. Developmental antigen-specific T cells produced high levels of Th1-type cytokines in response to two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells did not show any cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens conferred a significant survival benefit and long term cures in intracranial treatment models of SHH and Group 3 tumor bearing animals. We additionally tested whether the enrichment of select developmental antigens through the exclusion of normal brain transcripts would potentiate antitumor responses in both animal models. Finally, we evaluated the relevance of targeting fetal antigens across human MB subtypes. Our studies demonstrate that developmental antigens can safely target multiple MB subtypes and can be further refined to preferentially target individual subgroups. Further studies targeting immunogenic developmental antigens and leveraging this strategy with specific immune modulatory interventions represent a novel approach at utilizing patient molecular classification information to mediate safe and effective immunotherapy.

Over half of all cancer patients receive radiation therapy, and it contributes to over 40% of cancer cures. Within the last decade, cancer immunotherapy has become a pillar of cancer therapy, along with surgery, chemotherapy, and radiation therapy. The most commonly used type of immunotherapy is immune checkpoint blockade, which can increase immune cell activity by blocking inhibitory signals. Preclinical studies with transplanted tumors demonstrate high cure rates with either immune checkpoint blockade, radiotherapy, or combination treatment. These studies have led to hundreds of clinical trials testing checkpoint blockade and radiotherapy alone or in combination with other therapies, but emerging results are disappointing. My thesis work seeks to develop novel mouse models of cancer that recapitulate human disease, understand mechanisms of tumor resistance to radiation and immunotherapy, and identify novel immunologic targets that can enhance patient responses to radiation therapy.

Using complex genetically engineered mouse models of cancer, we investigated the contributions of the tumor microenvironment to therapeutic resistance. First, we explored the role of neutrophils in mediating radiation response. We showed that elevated neutrophil levels were associated with poor local control and survival in cervical cancer patients treated with definitive chemoradiation. Furthermore, in a genetically engineered mouse model of sarcoma, we demonstrated that genetic and antibody-mediated depletion of neutrophils increases radiosensitivity and decreases a mitogen-activated protein kinase transcriptional program. These results demonstrate that neutrophils promote tumor resistance to radiotherapy.

In complementary work using novel genetically engineered mouse models of sarcoma, we found that cells with high tumor mutational burden transplanted into syngeneic mice were cured by immune checkpoint blockade and radiation therapy, but the identical treatment failed in autochthonous sarcomas. To understand the mechanisms by which primary tumors were resistant to tumor cure by radiation and immunotherapy, we generated a single cell atlas of tumor-infiltrating immune cells from transplant and primary sarcomas treated with radiation and immunotherapy, which revealed marked differences in their immune landscapes. We found that radiation therapy remodeled myeloid cell phenotypes in primary and transplant sarcomas, but only transplant tumors were enriched for the effector CD8+ T cells that mediate response to combination therapy. In contrast, mice with autochthonous sarcomas demonstrated tumor-specific tolerance. These results indicate that radiation and immunotherapy cooperate to promote immunity within the tumor microenvironment, but identify immune tolerance in autochthonous tumors that must be overcome for this promising combination treatment to cure cancers that co-evolve with the immune system.

Inflammatory bowel disease (IBD) is a chronic disorder characterized by persistent inflammation in the gastrointestinal tract. Current therapies for IBD, such as anti-inflammatory or immunosuppressant drugs and anti-cytokine biologics, only temporarily alleviate symptoms and vary widely in effectiveness among patients. Consequently, there exists a critical unmet need for a long-lasting and broadly effective IBD treatment. Natural antibodies against the small molecule epitope phosphorylcholine (PC) are an important component of innate immunity with diverse functions including the clearance of bacterial and autologous targets in a non-inflammatory manner. The cells that produce these antibodies, B1a cells, however, have been shown to be reduced in patients with IBD, with this decrease being associated with a more advanced disease state. It has also been demonstrated that the adoptive transfer of B1a cells in a murine model of IBD results in the increased production of anti-PC antibodies and lessens disease severity. Furthermore, active immunotherapies are an alternative to monoclonal antibody biologics and a promising approach for generating a long-lasting IBD therapy because they exploit the ability of a patient’s own immune system to produce antibodies against a therapeutic target. Building on these concepts, we strove to develop an active immunotherapy consisting of PC as an epitope displayed on self-assembling peptide nanofibers to produce a therapeutic anti-PC antibody response for the treatment of IBD. The first part of this dissertation (Chapter 3) describes the process of designing, determining the therapeutic efficacy of, and gaining mechanistic insights into a nanofiber-based anti-PC active immunotherapy. We began by developing conjugation strategies for attaching PC to Q11 self-assembling peptides to render PC immunogenic. In an effort to find a balance between immunogenicity, stability, and ease of synthesis, we compared phosphoramidite and phosphodiester linkages for PC and concluded that the phosphodiester linkage was critical for PC epitope integrity. We then investigated how altering the multivalency of PC on Q11 nanofibers could further augment the anti-PC antibody response by synthesizing two nanofiber and PC conjugates with either 1 or 1-4 PC copies per Q11 peptide termed PC-Q11 and PCM-Q11, respectively. Intraperitoneal (i.p.) immunization with PCM-Q11 was found to induce a significantly greater anti-PC antibody response than i.p. immunization with PC-Q11. Additionally, PCM-Q11 was more selectively taken up by and able to activate natural antibody-producing B1a cells compared to all other B cells than PC-Q11 or Q11 alone. Further, control over the immune phenotype elicited was achieved via the inclusion of a T-cell epitope and/or CpG adjuvant, with the addition of both greatly augmenting the immune response elicited. We then evaluated the efficacy of immunizations with PCM-Q11/T-cell epitope with or without CpG in several different dextran sodium sulfate (DSS)-induced murine colitis models. We first investigated the ability of these immunizations to prevent severe disease when administered prior to the induction of a 30-day chronic DSS colitis model. Interestingly, immunization with both formulations was protective, significantly improving weight loss, disease severity indices, and colon lengths over unimmunized controls. This efficacy was repeated in a 1 cycle (10-day) DSS colitis model in both male and female mice and not attributed to CpG administration alone. Immunizations against PC also lowered bacterial spread to the spleen due to colon damage, with this effect being more pronounced in female mice. Additionally, we determined the efficacy of PCM-Q11 immunizations in a therapeutic setting where mice received one cycle of DSS colitis followed by three immunizations and then one more cycle of DSS colitis. This showed that immunizations with PCM-Q11 have therapeutic efficacy by significantly improving weight loss, disease activity indices, colon lengths, and bacterial spread to the spleen in this model. Furthermore, we have conducted several other studies to gain mechanistic insight into the observed efficacy of PCM-Q11 immunizations. We found that anti-PC immunization decreases microbiome diversity. We were also able to use flow cytometry to detect IgG and IgM antibodies in serum from mice immunized with PCM-Q11 that bind apoptotic colon epithelial cells in vitro. Additionally, we have shown through passive transfer of PCM-Q11 immunized sera that induced anti-PC antibodies offer some protection against severe DSS-induced colitis. Moreover, through both histological examination of colon damage and immunofluorescence imaging of tight junction proteins, we determined that PCM-Q11 immunizations were not acting by improving barrier function in the colon. Finally, we observed reduced efficacy of PCM-Q11 immunizations in an Il10-/- murine model of colitis. Collectively, this data demonstrates that immunization with PCM-Q11 was both preventative and therapeutic in multiple DSS-induced models of colitis in mice, with considerable efficacy attributed to the induced anti-PC antibody response. In the second part of this dissertation (Chapter 4), peptide nanofibers were modified for use as oral vaccines. While oral delivery offers direct access for eliciting immune responses within the gastrointestinal tract, it poses substantial obstacles for vaccines to overcome including acidic and proteolytic environments, thick mucus barriers, and a limited window for absorption. We therefore focused on two main design improvements to peptide nanofibers: synthesis with protease-resistant D-amino acids and incorporation of muco-penetrative peptide sequences rich in proline, alanine, and serine (PAS). We showed that D-amino acid Q11 was not degraded in simulated gastrointestinal environments in contrast to its L-amino acid counterpart. Additionally, we determined that PASylation enhanced muco-penetration in vitro and accelerated nanofiber transport through the GI tract in vivo. Ultimately, however, we found that oral immunization with PASylated L-amino acid nanofibers with cholera toxin B subunit mucosal adjuvant was the optimal formulation for the generation of both local and systemic immune responses. The primary areas affected in IBD are the distal small intestines and the colon, so the induction of therapeutic antibodies in these tissues is paramount. Thus, we sought to translate the above design principles to the small molecule epitope, PC, to enable oral administration. Oral immunization with PASylated anti-PC formulations was able to generate both local and systemic anti-PC immune responses. We then illustrated that oral vaccination with PASylated PC-bearing nanofibers was effective in both therapeutic and prophylactic models of DSS-induced colitis, observing comparable reductions in disease severity to i.p. anti-PC immunizations, thus, increasing the translatability of our therapy by offering a needle-free formulation. Overall, this data indicates that PASylated supramolecular peptide nanofibers are a promising platform for oral immunization. This dissertation outlines an encouraging first example of an active immunotherapy engaging natural antibody responses against phosphorylcholine as a durable therapy for IBD. More broadly, the strategies developed offer a potentially versatile approach for engaging natural antibody therapies and oral nanofiber peptide vaccines towards a variety of inflammatory and infectious diseases.

Adeno-Associated Virus (AAV) is a gene therapy vector with immense clinical importance. However, its use as a template for homology directed repair has come under greater examination particularly for the generation of site-specific recombined Chimeric antigen receptor or CAR T-cells. This is because traditional CAR T-cells generated from retro- or lentiviral vectors have risks for insertional oncogenesis or exhaustion from tonic signaling due to use of a constitutively active promoter, both problems which AAV directed knockins may overcome. In fact, the use of site-specific knockin CAR T-cells have now entered clinical trials for the treatment of CD19+ blood-borne cancers. While the use of these next-gen AAV generated CARs have excelled for liquid tumors, their use for the treatment of solid tumors has lagged. This is due to poor preclinical modeling for solid-tumor directed CARs which take place in immunocompromised mouse models that do not fully recapitulate the tumor microenvironment known to be problematic for infiltrating lymphocytes. Thus, there is a clear need to evaluate these therapeutics in immunocompetent hosts, however, there exist no known AAV serotype that can effectively target murine T-lymphocytes to generate these site-specific knockins. To ameliorate this problem, we employed a capsid evolution from the AAV6 background to generate a murine T-lymphocyte tropic AAV variant dubbed Ark313. Ark313 is vastly superior to the parent serotype in transducing, gene editing and site-specific knockins in murine T-cells. To characterize how this was happening, we employed a genome wide CRISPR knockout screen in murine primary T-cells to reveal the essential factor for Ark313 transduction to be Qa-2, a non-classical MHC-1b molecule. Due to the restricted tissue expression of the Qa-2 antigen, we injected mice systemically with Ark313 and saw it could transduce up to 25% of spleen resident T-cells including naïve/memory/effector subsets when using a self-complementary transgene. Additionally, Ark313 displayed a liver de-targeted tropism reducing potential off target tissue transduction when employing an ubiquitous promoter. Together we have generated a novel tool for the facile genetic manipulation of murine T-cells both ex and in vivo. We believe Ark313 will be a fundamental reagent to employ when interrogating T-lymphocyte immunotherapeutic questions and for investigating immune basic biology. This work lays the groundwork for the development of human lymphocyte targeting AAVs for generating CARs to combat liquid and solid tumors via systemic dosing.

Background

Methods

Results

Conclusions

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.