Browsing by Subject "Immunotherapy"

Now showing 1 - 20 of 22
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    CD16xCD33 bispecific killer cell engager (BiKE) activates natural killer (NK) cells from myelodysplastic syndrome (MDS) patients against primary MDS and myeloid-derived suppressor cell (MDSC) CD33-positive targets
    (2014-11) Gleason, Michelle Kathleen
    Myelodysplastic syndromes (MDS) are stem cell disorders that can progress to acute myeloid leukemia (AML). While hematopoietic cell transplantation (HCT) can be curative, additional therapies are needed for a disease that disproportionally afflicts the elderly. We tested the ability of a CD16xCD33 bispecific killer cell engager (BiKE) to induce natural killer (NK) cell function from 67 MDS patients. Compared to age-matched normal controls, CD7+ lymphocytes, NK cells, and CD16 expression were markedly decreased in MDS patients. Despite this, reverse-antibody dependent cell-mediated cytotoxicity (R-ADCC) assays showed potent degranulation and cytokine production when resting MDS-NK cells were triggered with an agonistic CD16 mAb. Blood and marrow MDS-NK cells treated with BiKE significantly enhanced degranulation, TNF-alpha and IFN-gamma production against HL-60 and endogenous CD33+ MDS targets. MDS patients had a significantly increased proportion of immunosuppressive CD33+ myeloid derived suppressor cells (MDSC) that negatively correlated with MDS lymphocyte populations and CD16 loss on NK cells. Treatment with the CD16xCD33 BiKE successfully reversed MDSC immunosuppression of NK cells and induced MDSC target cell lysis. Lastly, the BiKE induced optimal MDS-NK cell function irrespective of disease stage. Our data suggest that the CD16xCD33 BiKE functions against both CD33+ MDS and MDSC targets and may be therapeutically beneficial for MDS patients.
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    Cd4 T Cell Activation And Ox40 Agonist Immunotherapy In Tuberculosis
    (2023-09) Gress, Abigail
    After Mycobacterium tuberculosis (Mtb) infection millions of effector T cells traffic to the lungs, but relatively few find antigen and become activated. We used an antigen receptor reporter mouse (Nur77-GFP) infected with Mtb to distinguish recently activated CD4 T cells from others in the lungs. Recently activated Nur77-GFPHI cells more often expressed protective markers and were enriched for expanded TCR clonotypes. Nur77-GFPHI cells differentially expressed co-stimulatory genes including Tnfrsf4/OX40 and were functionally more protective than Nur77-GFPLO. Nur77-GFPLO cells more often expressed markers of terminal exhaustion, cytotoxicity, and the trafficking receptor S1pr5, associated with vascular localization. A short course of immunotherapy with an agonist monoclonal antibody targeting OX40+ cells during early infection transiently expanded CD4 T cell numbers and shifted their phenotype towards parenchymal protective cells, which decreased the lung bacterial burden and extended host survival, offering an additive benefit to antibiotics. We have newly identified OX40 as a conserved marker of recently activated CD4 T cells at the infection site and a target for immunotherapy in tuberculosis.
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    Characterizing the migration and therapeutic potential of CD8 tumor-infiltrating lymphocytes
    (2023-06) Gavil, Noah
    Beginning in the 19th century, immunologists slowly uncovered the mechanisms ofcancer immunosurveillance, ultimately identifying thymic-dependent “T” lymphocytes (T cells), and not antibodies, as the primary mediators of cancer cell elimination and control. T cell surveillance is performed by diverse subsets. After antigen encounter, T cells differentiate, adopting many phenotypic fates. In settings of acute infection, T cells diversify into ‘effector’ and long-lived ‘memory’ states. While some memory T cells circulate throughout the body, others remain resident, patrolling tissues locally. The migration and function of memory T cells has been well studied, providing a systemic view of immune surveillance for microbial pathogens. In settings of chronic antigen, such as cancer, antigen-specific T cells diverge from the memory program, existing along a spectrum of differentiation and exhibiting restrained functional capabilities. While the early stages of cancer immunosurveillance mirror immune responses to microbial pathogens, the T cell surveillance of progressive malignant tumors is poorly understood. Many studies describe the heterogeneity of tumor-infiltrating T cells (TILs). Generally, the density of CD8+ TILs correlates with improved prognosis, but the density of CD8+ TILs with resident-memory (TRM) phenotypes better predicts patient outcomes and responsiveness to immunotherapies. These TRM-like cells may directly control tumor growth, but their migration properties have not been directly studied, leaving their direct function unknown. Importantly, newer data shows that many CD8+ TILs are bystanders, specific for microbial pathogens, not tumors. Reactivation of these bystander T cells with cognate peptide can orchestrate potent anti-tumor immune responses. In this thesis, I study the migration properties of tumor-specific and virus-specific CD8+ T cell subsets. Distinct resident populations of CD8+ TILs exist, differing based on the presence or absence of chronic antigen. Resident CD8+ TILs do not recapitulate the resident T cell programs of healthy tissues. I also investigated the anti-tumor immune mechanisms initiated by antiviral CD8+ TILs reactivation. Cytokine production and innate immune mechanisms were the predominant source of tumor killing. Taken together, T cell immunosurveillance is characterized by the coexistence of T cell subsets that represent lineages associated with chronic antigen exposure and memory T cells, which possess potent therapeutic potential (e.g. TRM).
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    Developing a Mouse Model of Metastatic Osteosarcoma for Immunotherapy
    (2022-12) Sonam
    Osteosarcoma is a primary bone malignancy that primarily affects children and young adults. The 5-year survival rate for patients with localized osteosarcoma is 60-70% and only 20% for patients who present metastasis at the diagnosis. The standard of care treatment has not improved the prognosis in over 30 years and new approaches addressing tumor resistance and immune evasion are required. The long-term goals of this project are to address two objectives: i) To study the potential benefits and risks of immunotherapeutic drugs eBAT and ONIx on syngeneic mouse models of metastatic osteosarcoma, and ii) To study their mechanism of action. K7M2 is a well-characterized model of spontaneous pulmonary metastasis, but intravenous injection of these cells did not result in lung colonization, at any dosage, in the mice in our study. We inferred from the data that the tumor cells were rejected by the immune-competent host. To study the mechanism of action of ONIx, we have made progress in optimizing the protocol for the phagocytosis assay, and further analysis with a different target cell line is planned. Assays to verify the mechanism of action of eBAT are being planned. Overall, this project provided significant learnings on objective planning and designing of the experiments and provided opportunities to acquire expertise in several new methodologies.
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    The Divergent Role of Co-receptors and Cytokines in the Immune Response to Cancer and Self
    (2020-08) Tucker, Christopher
    The mammalian immune system has evolved over millennia to respond to countless biologic factors and environmental cues. This exquisite system has been shaped over time, to destroy hostile invaders all while sparing healthy surrounding tissue. The immune system is vital to survival, a fact that is readily apparent when immune components are mutated in human disease or mouse models. When immune components are lost, this balance can be distorted in both directions. Alterations to pro-inflammatory molecules, such as Tumor Necrosis Factor alpha (TNFα) can increase infection and cancer risk. Conversely, mutations in immunoregulatory pathways such as programmed death-1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been associated with increased autoimmune disease incidence. This balance is uniquely apparent in the PD-1 pathway and CTLA4 pathways. Blockade of the PD-1 pathway can elicit potent anti-tumor responses, as evidenced by monoclonal antibody therapy Nivolumab and Pembrolizumab response rates, but also can unleash off-tissue pathology such as type one diabetes (T1D). T1D in these cancer patients is rapid and severe. This is perhaps not surprising, as Non Obese Diabetic (NOD) mice treated with anti-PD-1 or anti-PD-L1 therapy rapidly succumb to fulminant T1D. In common, CTLA4 blockade has been shown to halt tumor growth and even cure patients of metastatic cancer, however it has also unleashed potent autoimmune responses in the gut, brain, heart and eyes. Being able to better dissect these pathways, determine who will respond poorly to therapy and how these therapies can be maximized for tumor destruction, are key questions to be answered by the field of immunology. Complementary to the idea of blocking an inhibitory signal, positive signals are also used to invigorate the immune system. OX40, a TNF receptor family member, has been shown to be integral in provide co-stimulation to effector and naïve T-cells, and agonism through this receptor can cause potent anti-tumor effects. Interconnected and also important, the cytokine IL-12 has been shown to induce profound changes in T-cells, driving cytolytic T-cell programming, higher cytokine production, better tumor trafficking, proliferation and survival in the tumor environment while preventing tumor induced T-cell exhaustion. Like PD-1 and CTLA4, both OX40 and IL-12 are associated with their own unique set of autoimmune conditions. The focus of this chapter and this thesis as whole will be on these two immunoregulatory pathways, PD-1 and CTLA-4 as well as two immunostimulatory pathways OX40 and IL-12 and their relative importance in autoimmunity and cancer immunity.
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    Engineering a CD19-Based Bispecific Molecule for CAR T Cell Therapy
    (2018-10) Schrack, Ian
    Cancer is a profoundly devastating disease both globally and within the United States. Current standards of care for treating cancer often includes surgical resection, chemotherapy, and radiation, each of which associates with its own set of adversities. An emerging class of treatment, immunotherapy, aims to utilize a patient’s own functional immune system as the therapeutic agent. Adoptive T cell therapy, but more specifically, chimeric antigen receptor (CAR) expressing T cell transfer, has had notable clinical success particularly against hematological malignancies. Chimeric antigen receptors are synthetic immunoreceptors which can redirect T cells towards varying tumor associated antigens, and, as a living cell, have the aptitude to develop sustainable memory and anti-tumor efficacy. However, conventional CAR T cells lack clinical modularity afforded by other treatments because, once transfused into a patient, the modified immune cell cannot be further altered. This nuance has resulted in several adverse side-effects which can be lethal to a subset of patients. Several resolutions have been posed to solve these reported complications, one of which is genetic encoding CAR specificity towards a secondary, bispecific molecule. This split-CAR approach has the propensity to improve antigen specificity, resolve antigen loss, afford dose-able T cell activation, and more. However, while many bispecific molecules have been developed, many lack both tunability and developability, both of which are important for the complexities and ever-changing nature of cancer. To meet this demand for engineered ligands, several high-throughput ligand selection methods have been developed for discovering ligands with a desired specificity. Furthermore, the associated CAR T cells may have poor aptitude for activation and expansion due to insufficient antigen availability. Conversely, conventional anti-CD19 CAR T cells can harness both healthy or malignant CD19-positive B cells for activation and expansion and thus have an abundance of available antigen. To these points, we utilized yeast-surface display and directed evolution as a pipeline for developing an CD19-based bispecific molecule capable of harnessing the proliferative aptitude of anti-CD19 CAR T cells to target antigens conventionally associated with solid tumors. Human CD19 was evolved for improved structural integrity through conformational selections using anti-CD19 monoclonal antibodies. Improved mutants were sequenced and provided input for designing a stably expressing, generation 2 CD19 library (termed Frame2). The second-generation diversity applied experimentally determined, beneficial mutations in multi-site fashion to drive the enhanced CD19 framework towards a higher stability and/or functionality. The Frame2 CD19 library was constructed as several fusion constructs containing either an anti-EGFR fibronectin domain or an anti-HER2 scFv in both N-terminal and C-terminal orientations and selectively evolved with anti-CD19 antibodies and the ligand-respective antigen. A set of functional bispecific CD19-ligand fusions were successfully developed. In theory, because the anti-CD19 antibodies used for fusion development have an identical binding domain to several anti-CD19 CAR constructs, these fusions should be detectable by CD19-targetted CAR T cells. Moreover, if the ligand domain also retains specificity, the CD19-ligand bispecific molecule should be capable of redirecting anti-CD19 CAR T cells to EGFR or HER2 expressing tumors.
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    Engineering human pluripotent stem cells for enhanced lymphocyte development and function
    (2012-10) Knorr, David Arthur
    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide accessible, genetically tractable and homogenous starting cell populations to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and non-malignant hematological diseases. Our group has previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells. hESCs and iPSCs, which can be reliably engineered in vitro, provide an important model system to study human lymphocyte development and produce enhanced cell-based therapies with potential to serve as a "universal" source of anti-tumor lymphocytes for novel clinical therapies. My studies have focused on the generation of NK cells from hESCs and iPSCs, their function both in vitro and in vivo against a variety of different tumor types, and modification of these cells with genetic constructs to enhance their anti-tumor capabilities.
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    Enhancing the efficacy of a nicotine vaccine
    (2013-03) Cornish, Katherine E.
    Tobacco addiction is the leading cause of preventable death worldwide. Many people continue to smoke cigarettes despite clear detrimental health effects. Available smoking cessation therapies are only partially effective, making new treatment approaches necessary to increase smoking cessation rates. Immunization against nicotine features a different mechanism of action than currently available medications. As a pharmacokinetic antagonist, immunization against nicotine alters distribution, metabolism, and clearance of nicotine to attenuate nicotine-induced behavior in animal models. Nicotine vaccines in clinical trials show efficacy but are limited by the modest and highly variable nicotine-specific antibody (NicAb) concentrations produced. This thesis focuses on ways to improve efficacy of a nicotine vaccine by combining it with additional forms of immunotherapy. The first aim of this thesis examined the effects of supplementing vaccination against nicotine with individualized doses of Nic311, a nicotine-specific monoclonal antibody. Compared to either immunotherapy alone, combining active and passive immunization produced greater alterations in nicotine pharmacokinetics and nicotine-induced behavior using a locomotor activity model. Only small doses of Nic311 were necessary to supplement vaccine-generated NicAb concentrations to a previously effective threshold. This decreased cost and use of typically expensive monoclonal antibodies, potentially increasing viability of this approach in a clinical setting. The second aim of this thesis examined the effects of concurrent administration of two immunologically distinct nicotine immunogens in a bivalent vaccine over a range of vaccine formulations and immunization conditions. Immunogens were co-administered in a bivalent vaccine without compromising immunogenicity of either immunogen when delivered subcutaneously in alum, but not when delivered intraperitoneally in Freund's adjuvant. When combined in alum, immune responses elicited by the two immunogens were largely independent of one another. This suggests that subjects who responded poorly to one immunogen may have responded better to the second, immunologically distinct immunogen in the bivalent vaccine. These results indicate that the bivalent vaccine strategy is a feasible way to increase antibody concentrations above what can be achieved using one immunogen alone, but integrity of the response is highly dependent on vaccine formulation and administration conditions.
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    The function and utility of self-specific CD8 T cells
    (2018-02) Nelson, Christine
    Self-specific CD8 T cells have the potential to provide great benefit, but also to cause great harm. This thesis research seeks to uncover the mechanisms controlling CD8 T cell tolerance to self and tumor antigens. We show that the induction of CD8 T cell tolerance is dependent on inhibitory receptor signaling, but the maintenance of tolerance is not. Thus, only newly primed self-specific CD8 T cell are amenable to immune checkpoint blockade activation. We establish CD8 T cell tolerance as an active state of differentiation that is dependent on suppressed antigen-sensing. We demonstrate that robust stimulation with self-antigen and inflammation induces avidity maturation of the self-specific CD8 T cell repertoire, which serves to renew susceptibility to immune checkpoint and aid in anti-tumor responses. We validate that endogenous self-specific CD8 T cells exist within the immune repertoire and can expanded by similar vaccination methods. Expanded self-specific CD8 T cells generate tissue resident memory in non-lymphoid tissues and secondary lymphoid organs, that are phenotypically distinct from non-self-specific populations. Finally, we show that self-specific CD8 T cells synergize with tumor neo-antigen specific CD8 T cells in the treatment and prevention of cancer. This data provides significant insight into the requirements for the induction of self and anti-tumor T cell responses.
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    A heroin/morphine vaccine: mechanism of action and extending its use to other abused opioids
    (2013-07) Raleigh, Michael Dennis
    Heroin is more widely used than any other illicit opioid and mortality rates among heroin users are an average of 13 times higher than the general population. Intravenous heroin use is associated with crime, social disruption, and transmission of blood-born pathogens such as human immunodeficiency virus and hepatitis C. Effective pharmacotherapies are available to treat heroin abuse but have been largely unsuccessful because they require frequent dosing, have a high abuse potential, or have low compliance. Vaccines against heroin and its metabolites (e.g. morphine) are being considered as a complementary treatment for heroin abuse because they are long-acting, selective, have no abuse potential, and may benefit those unwilling to take the current pharmacotherapies. Vaccination with morphine-conjugate vaccines can elicit a strong immune response that reduces the behavioral effects of heroin in animals, presumably by morphine-specific antibodies binding opioids in blood and reducing their distribution to brain. This thesis explores the use of M-KLH, a morphine hapten conjugated to keyhole limpet hemocyanin (KLH) using a tetraglycine linker and mixed with either Freund's or alum adjuvant for increasing the immune response. Morphine vaccines present many challenges that make translation to clinical use difficult. Heroin is sequentially metabolized to its active intermediates 6-monoacetylmorphine (6-MAM), morphine, and then to morphine-6-glucuronide (in vivo and ex vivo). Heroin enters brain and is rapidly converted to 6-MAM, which is presumed to mediate most of heroin's early effects. With regard to the mechanism of action of morphine vaccines, it is unclear whether the antibodies they generate must bind heroin, its downstream metabolites, or both to prevent opioid distribution from plasma to brain and reduce heroin's behavioral effects. However, because analytical assays to measure heroin and metabolite concentrations in tissues have used a wide range of conditions and varying degrees of stability have been reported, studying the effect of vaccination on heroin distribution is not straightforward. In addition, heroin and metabolite distribution after i.v. heroin administration, the most common route of abuse by humans, has not been well characterized in non-vaccinated rodents. Finally, blockade of heroin by vaccination may not prevent the abuse of structurally distinct opioids. The overall goal of this thesis was to better understand the mechanism of action of morphine vaccines and to extend their use to other abused opioids. The specific aims were to stabilize heroin in blood and brain tissues for subsequent pharmacokinetic studies, study distribution of heroin and its metabolites in non-vaccinated and vaccinated rats, explore the effects of vaccination on heroin-induced behaviors, and determine if vaccine efficacy is retained when combined with a vaccine targeting oxycodone, another commonly abused opioid. These aims were explored using clinically relevant drug doses. Heroin and metabolite degradation was significantly reduced by 1) the addition of ice-cold sodium fluoride (a general esterase inhibitor) and formate buffer (pH 3.0) in heroin-spiked tissues, 2) rapid removal of red blood cells via centrifugation, and 3) drying opioids after extraction from tissues prior to measuring their levels. Using these conditions heroin and its metabolites were stabilized in tissues for subsequent distribution studies. In non-vaccinated rats 6-MAM was the predominant metabolite in brain as early as one minute after administration of 0.26 mg/kg i.v. heroin, which is consistent with previous studies that suggest that 6-MAM mediates heroin's early behavioral effects. Vaccination with a morphine-conjugate vaccine (M-KLH) led to a reduction of 6-MAM and morphine, but not heroin, distribution to brain after heroin administration, suggesting that morphine vaccines reduce accumulation of 6-MAM in brain. The mechanism by which this occurs is likely through antibody binding of 6-MAM in plasma to prevent its distribution to brain and is consistent with very high plasma 6-MAM concentrations in vaccinated rats after i.v. heroin or 6-MAM administration. Vaccination with M-KLH led to a reduction of heroin-induced anti-nociception and locomotor activity and remained effective for up to 16 days after repeated dosing suggesting that heroin vaccines may have long-lasting efficacy. These results are consistent with findings from the distribution studies and support the hypothesis that morphine vaccines function by retaining 6-MAM in plasma and prevent its accumulation in brain. To determine whether opioid vaccines could be combined without reducing individual vaccine efficacy and prevent heroin addicts from abusing structurally distinct opioids, rats were vaccinated with M-KLH, an oxycodone-conjugate vaccine (Oxy-KLH), or the bivalent vaccine (both M-KLH and Oxy-KLH). Total morphine- and oxycodone-specific antibody titers were significantly increased in rats that received the bivalent vaccine compared to rats that received individual vaccines. Concurrent i.v. administration of 6-MAM and oxycodone in M-KLH vaccinated rats led to increased 6-MAM retention in plasma and reduced 6-MAM distribution in brain. A similar effect on oxycodone distribution was seen in Oxy-KLH vaccinated rats. There was a trend towards greater efficacy in altering both 6-MAM and oxycodone distribution in the bivalent group compared to individual vaccine groups. These data suggest that combining opioid vaccines will retain, and possibly enhance, individual vaccine efficacy and might be a viable option to prevent addicts from abusing structurally distinct opioids. These findings contribute to the understanding of how morphine vaccines elicit their effects on heroin-induced behaviors and suggest that morphine vaccines, alone or in combination with other pharmacotherapies, may benefit those seeking treatment for heroin addiction.
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    Immune-based tumor regression induced by Fc-OX40L in a murine model of glioma
    (2012-05) Murphy, Katherine Anne
    Glioblastoma multiforme, the most aggressive form of glioma, is a lethal brain tumor with a dismal median survival of 15-19 months. Immunotherapy is a promising adjuvant therapy for malignant brain tumors. Lack of adequate, endogenous antigen presentation and the immune suppressive nature of the tumor microenvironment present significant hurdles that need to be overcome in order to mount an effective immune response, capable of tumor elimination. Vaccination with tumor-derived antigen has sought to overcome inadequate antigen presentation, yet has shown to been inefficient at complete tumor elimination. Functionally unresponsiveness, anergy, can be induced in immune cells when antigen recognition occurs without proper costimulation. To test if additional costimulation was necessary to achieve a functional immune response, costimulatory molecules 41BBL, CD80, GITRL and OX40L, fused to the Fc portion of human immunoglobulin, were tested in combination with tumor vaccine. Vaccine/Fc-OX40L yielded the most potent response resulting in complete tumor regression in the majority of animals and the generation of immunological memory capable of rapidly clearing tumor upon tumor rechallenge. Additionally, combining vaccine/Fc-OX40L with the standard of care chemotherapy resulted in regression of 100% of glioma tumors, however 80% of these animals developed fatal secondary lymphoid malignancy. These data demonstrate Fc-OX40L has incredibly potent activity against experimental gliomas relative to the other molecules tested. In addition, it reveals a potential hazard in combining mutagenic chemotherapeutics with immunotherapy. Lymphocytes isolated from vaccine/Fc-OX40L treated animals had enhanced cytolytic activity and increased proliferative capacity, compared to controls. Additional analysis demonstrated a CD4 T cell, NK, and B cell dependent, and CD8 T cell independent mechanism of action. The presence of tumor reactive antibody in the serum of treated mice and deposition of antibody at the tumor site, combined with the observation that loss of the Fc receptor negatively affected the ability of animals to control tumor growth generated interest in antibody-mediated mechanisms. Tumor infiltration of perforin expressing NK, NKT, and neutrophil-like cells in vaccine/Fc-OX40L treated animals may provide a possible mechanism of tumor killing. Further understanding the necessary effector cells and mechanisms involved in tumor elimination will aid in the design of future immunotherapy approaches.
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    Mechanisms of Action for Ad5-TRAIL/CpG Immunotherapy for the Treatment of Renal Cell Carcinoma
    (2014-06) James, Britnie Ryan
    Renal cell carcinoma (RCC) affects ~65,000 people in the U.S. annually. About 30% of RCC patients have multiple metastases at diagnosis, and an equal percentage will develop metastatic tumor recurrence after nephrectomy. Metastatic RCC is incurable, with a median survival time of only 18 months. Immune-based therapy for RCC provides the potential for long-lived protection against reoccurrence. However, even the most successful immunotherapy-based clinical trials only show objective response rates in <50% of the patients. Many factors may account for this limited clinical success, including pre-clinical use of young, normal weight ("lean") animals lacking immunomodulatory co-morbidities present in many cancer patients. Obesity is one of the main risk factors and co-morbidities for RCC. The reasons for this are likely complex and multifactorial, but generalized immune suppression during obesity may contribute to these findings. Due to the negative effects of obesity on the immune system, studies are needed to provide a framework from which novel immunotherapies can be developed for patients with metastatic RCC that is complicated by such co-morbidities. In a subcutaneous RCC model we have demonstrated that Ad5-TRAIL/CpG immunotherapy could eradicate local tumors. However, the mechanisms by which this therapy worked in a metastatic model and the negative effects obesity may exert were not known. Using a model of metastatic RCC we found that lean mice required CD8αDC and pDC to mount an antitumor CD8+ T cell response capable of clearing tumors, following Ad5-TRAIL/CpG treatment. Mice complicated with diet-induced obesity (DIO) presented with immune dysregulations in both the DC and CD8+ T cell compartments. Additionally, Ad5-TRAIL/CpG therapy modulated the immunosuppressive MDSC population in lean mice, but not in DIO mice. These data correlated with the inability of DIO mice to respond to Ad5-TRAIL/CpG therapy and they ultimately succumbed to tumor burden. The research presented here highlights the immunosuppression evident in the obese environment and demonstrate the importance of examining co-morbidities, such as obesity, in pre-clinical studies for novel therapies.
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    Myeloid derived suppressor cells in dogs with cancer: phenotype, function and clinical implications
    (2014-06) Goulart, Michelle Rodrigues
    Myeloid-derived suppressor cells comprise phenotypically heterogeneous population of myeloid cells at different stages of differentiation endowed with potent immunosuppressive activity. Abnormal accumulation of MDSC in tumor models and cancer patients produce profound immune suppression, severely impairing T cell antitumor immunity, contributing to angiogenesis, cell invasion and metastasis, and constitute a major hurdle in achieving successful immunebased therapies. Understanding the mechanism that drives MDSC expansion and enhances function in humans and dogs is crucial for the development of efficacious immunotherapy. Studies in dogs with several tumor types, including sarcoma, carcinomas, mast cell tumors and gliomas confirmed MDSC expansion in the peripheral blood of dog cancer patients. MDSC have been identified in dogs using the combination of three-marker phenotype CD11b+CD14-MHCII-cells for granulocytic and CD11b+CD14+MHCII-cells for monocytic subsets. Granulocytic MDSC accumulated in the peripheral blood of dogs with advanced sarcoma, carcinomas and mast cell tumors, co-purified with peripheral blood mononuclear cell (PBMC) fraction and expressed polymorphic mononuclear morphology. This subset of cells showed the ability to efficiently inhibit T cell proliferation and IFN-γ secretion of autologous T cells, as well as allogenic T cells from healthy dogs, and expressed ARG1, iNOS2, TGF-β and IL-10. Monocytic MDSC also demonstrated potent ability to suppress T cell proliferation and preferentially accumulated in the peripheral blood of dogs with glioma. Elevated levels of arginase activity found in the serum of dogs with glioma could potentially be due to the presence of elevated numbers of MDSC. Evaluation of the anti-mouse Gr1 antibody for MDSC staining and identification revealed that does not cross react and therefore is not suitable for canine cells.
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    Overcoming Obstacles to Glioma Immunotherapy
    (2014-04) Litterman, Adam
    Glioma is a type of malignant tumor of the non-neuronal cells of the central nervous system, the glia. These tumors are the most common malignant tumors of the central nervous system. The most aggressive and most prevalent of these, glioblastoma multiforme (GBM) is a deadly disease with a grim prognosis, with median survival at diagnosis of less than a year and a half. Standard treatment with irradiation and the DNA alkylating drug temozolomide yields incremental improvement in survival over irradiation alone but better therapies remain needed. Immune therapies are an emerging class of therapies that have shown great promise in the treatment of hematopoietic malignancies and solid tumors. These therapies harness the capability of the immune system to target and kill large numbers of tumor cells specifically, and it is has been suggested that most or all durable responses to treatment of solid tumors involve generation of an anti-tumor immune response. Several anecdotal reports of dramatic responses in GBM patients after receiving cancer vaccines (a type of immune therapy) suggest that immune therapies for glioma could yield substantial increases in survival of patients with these tumors. However, the overall record of vaccines for the treatment of this disease has been marked by failure, and substantial barriers remain to the implementation of other types of immune therapies in glioma patients. Several mechanisms by which tumors in general, and brain tumors in particular, evade the activity of the immune system have been outlined. These include accumulation of immune suppressive cell types, tumor intrinsic changes that directly suppress the activity of infiltrating immune cells, and brain specific mechanisms of immune privilege. While these mechanisms are doubtless operative in many cases, accumulating evidence from clinical trials of adoptive transfer of T cells demonstrate that the accumulation of sufficient numbers of tumor-specific T lymphocytes at the tumor site can result in an overwhelming anti-tumor immune response and associated durable clinical responses. Therefore, my research over the past several years has focused on clinically relevant mechanisms in glioma patients that present obstacles to the development of a robust T cell mediated anti-tumor immune response. In this thesis, I outline experiments performed to understand and develop strategies for overcoming two obstacles to expanding large numbers of tumor specific cytolytic T lymphocytes in glioma patients: the anti-proliferative effect of the alkylating drug temozolomide on in vivo T cell expansion by cancer vaccination, and the differentiated phenotype of ex vivo expanded T cells for adoptive immunotherapy that is associated with diminished proliferative potential in vivo. A focus in these experiments is the targeting of tumors with T cells that are specific for antigenic determinants derived from tumor-specific mutations. Engineered T cell responses targeting individual patient-specific mutations may someday lead to significant improvements in the efficacy of immune therapy for glioma, and ultimately to improved outcomes for patients with these malignancies.
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    Revolutionizing T cell Therapy for Pancreatic Cancer: Harnessing the Power of T cell Receptor Exchange Mice
    (2023-03) Rollins, Meagan
    Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer characterized by a suppressive tumor microenvironment (TME) including elevated levels of TGF. The adoptive transfer of T cell receptor (TCR) engineered T cells specific to mesothelin (Msln) can effectively target PDA, but efficacy is limited by the suppressive TME that promotes engineered T cell dysfunction. T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin (Msln), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high-affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac-targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. To test the hypothesis that TGFβ is a major driver of engineered T cell dysfunction in PDA, we knocked out Tgfbr2 using CRISPR/Cas9 in in vitro activated Msln-specific TRex cells. The loss of Tgfbr2 signaling in high affinity (1045) Msln-specific TRex T cells drive increases in markers of effector T cells such as Klrg1, Cxcr3, and CD44. When transferred into orthotopic PDA tumor-bearing mice, both Tgbr2-WT and Tgbr2-/- engineered T cells traffic to tumors driven by increased frequency and number of cDC1 and cDC2 dendritic cells. With vaccination, the engineered T cells cause a 10-fold reduction in tumor weight at day 13 post tumor and are highly proliferative. Tumor-infiltrating Tgbr2-/- cells upregulated IFNg, TNFa, and Granzyme b and decreased markers of terminal exhaustion PD-1 and Lag3. Our studies suggest, interfering with TGFβ signaling can alter T cell fate prior to transfer and maintain effector differentiation within the TME promoting cytotoxic Klrg1+ T cells at the expense of PD-1+ exhausted T cells and leading to tumor control.
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    The Role of Oxygen in the Immunogenicity of brain tumor cell vaccines
    (2013-07) Andersen, Brian Magne
    Tumor cell vaccination is a promising strategy for patients with primary brain tumors. Cell-derived vaccines have been developed and tested in patients with many cancers, resulting in objective responses in a minority of cases. Tumor cells are often harvested from tissue culture, where phenotype can shift in response to many factors. Oxygen affects expression of thousands of genes, yet its impact on immunogenicity in culture was unknown until recently. Compared to atmospheric oxygen levels, physiologic (5%) oxygen increases the adjuvant properties and efficacy of glioma vaccines. These studies determined the impact of oxygen on cell-mediated and humoral immunity with regard to its potential for improving efficacy in brain tumor patients. Immunogenicity and oxygen were first investigated over a broader oxygen range, with 5% oxygen still yielding the greatest extension of survival. With the effort to maximize immunogenicity, primary autologous meningioma cells from pet dogs were cultured in 5% oxygen for vaccine production. Dogs vaccinated with lysate/adjuvant vaccines based on these cells induced robust antibody responses and survived a median of three-fold longer than historic controls. Further studies in mice led to the discovery that glioma cells cultured in 5% oxygen upregulated a "danger" signal, annexin II. Monomeric annexin II enhanced dendritic cell cross presentation, CD8 T cell priming, and extended survival of murine breast carcinoma and plasmacytoma. Enriching for endogenous danger signals by decreasing tissue culture oxygen is a simple means to enhance the immunogenicity of brain tumor cultures.
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    Salmonella enterica Typhimurium as a tumor-targeting immunotherapy vector
    (2015-08) Drees, Jeremy
    Interest in cancer immunotherapy has grown in recent years due to its potential for significant and durable therapeutic responses. Immune checkpoint blockade has emerged as an immunotherapy as a single agent but has even greater appeal when it is used in combination with other immunostimulatory approaches. However, the dosing of checkpoint blockade and its combinatorial use with other immunotherapies has been limited by systemic immune-related adverse side effects. One way to overcome these adverse effects is to deliver the therapeutic agents specifically to the tumor microenvironment. Salmonella enterica Typhimurium (S. Typhimurium) has been studied for cancer therapy due to its genetic manipulability and tumor-targeting propensity, and in this thesis, the potential of S. Typhimurium as a tumor-targeting immunotherapy vector was investigated. Functional antagonistic single chain antibodies (scFvs) against the immune checkpoints CTLA-4 and PD-L1 were isolated from an immunized chicken library and engineered for secretion from S. Typhimurium. The inherent anti-tumor properties and tumor-targeting capability of S. Typhimurium were then tested in transplanted primary and metastatic tumor models as well as a genetically engineered autochthonous BALB-neuT breast cancer model. In each of these models, S. Typhimurium demonstrated native anti-tumor efficacy; however the bacteria did not adequately colonize the autochthonous tumors of the BALB-neuT model. Disruption of tumor vasculature by treating BALB-neuT mice with a vasculature disrupting agent (VDA) improved the colonization of autochthonous tumors over 1000-fold to levels similar to those observed for transplanted tumors. Subsequent comparison of the tumor targeting capability and efficacy of S. Typhimurium engineered to secrete the antagonistic ?PD-L1 (scFv) versus a control strain showed that secretion of the scFv may further improve the colonization of autochthonous tumors, leading to a greater reduction in tumor burden of treated mice. These findings provide a proof of principle for the expression and delivery of functional immunotherapeutic single chain antibodies using S. Typhimurium, demonstrate S. Typhimurium's native tumoricidal activity independent of tumor-targeting, illustrate the importance of clinically representative tumor models when studying bacterial cancer therapy, and demonstrate the potential of VDA treatment to improve bacterial tumor-targeting. Collectively, this work illustrates S. Typhimurium's promise as a tumor-targeting immunotherapy vector.
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    Targeting The Erβ/Her Oncogenic Network In Lung Cancer: Synergistic Antitumor Interaction And Potentiation Of Anti-Pd1 Efficacy
    (2020-04) Almotlak, Abdulaziz
    Lung cancer is the leading cause of cancer related mortality in the United States, accounting for more than 142,000 estimated deaths in 2019. The major subtype of lung cancer is non-small cell lung cancer (NSCLC), which represents 85% of all cases. Despite the advancement in understanding the molecular basis of NSCLC, the 5-year survival rate is less than 20%. The current treatment strategies for advanced stage patients rely on molecularly-targeted therapies, cytotoxic chemotherapy and immunotherapeutic agents. Because of the complexity and heterogeneity of lung tumors, intrinsic and acquired resistance mechanisms ultimately result in failure to respond to these therapies and early relapse. Combinatorial strategy that involves targeting multiple aspects of tumorigenesis may represent a new avenue for therapeutic intervention in lung cancer management. Estrogen signaling has been frequently shown to be an important mediator of lung cancer progression and metastasis. In a non-genomic fashion, ER mutually interacts with human epidermal growth factor receptors (HERs) to promote lung cancer proliferation and growth. Targeting ER signaling with the antiestrogen fulvestrant has shown moderate activities in preclinical models of lung cancer. The reciprocal interaction between ER and EGFR could limit the activity of using anti-ER agent alone. In a phase II clinical trial, combining fulvestrant with the selective EGFR tyrosine kinase inhibitor erlotinib showed an enhanced activity over erlotinib alone and improved the survival outcomes in NSCLC patients. However, the improvement in overall median survival was modest. Recent retrospective clinical analysis demonstrated that genes contained in the prediction analysis of microarray 50 (PAM50) provide prognostic information in high ERβ+ lung cancer patients. The 7-gene model includes c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1, and PgR. In high-risk patients, who tend to have a poor prognosis and short median survival, c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 are overexpressed and PgR is downregulated. Importantly, the 7-gene model described one interacting network that includes ER and HER2/HER3 as the top upstream regulators for the 7-gene panel. In fact, a significant association between ERβ and HER2 expression was found, in which 70.2 % of ERβ-positive cases were positive for HER2 compared to 34.5% of ERβ-negative cases. HER3, when analyzed with HER2, showed also positive association with ERβ. These observations suggest that ERβ and HER2/HER3 pathways define lung tumors with very aggressive biology, indicating that blocking both pathways could be more efficacious than either one of them alone. These observations also could explain why the magnitude of response was modest when fulvestrant was combined with erlotinib, as erlotinib has weak activities against HER2 and HER3. Combining a pan-HER inhibitor such as dacomitinib (inhibits EGFR, HER2 and HER4) with fulvestrant could be more efficacious than either agent alone and could produce a gene signature that predicts better clinical outcomes in NSCLC. Immune checkpoint inhibitors have changed the treatment paradigm for several solid tumors, including lung cancer. Antibodies that target programmed death receptor 1(PD1) or its ligand (PD-L1) have proven great efficacy for certain patients with lung cancer. Patients who respond to these agents tend to have durable effects and longer survival outcomes; however, only 20-29% of patients are predicted to respond. Several combination strategies are being evaluated clinically to maximize the efficacy of these agents and improve the response rate. Preclinical evidence found that the ER blocker fulvestrant effectively sensitizes lung cancer cells to T cell and natural killer (NK) cell mediated cytotoxicity effects. In addition, estrogen signaling is a critical mediator for myeloid derived suppressor cells (MDSCs), which are largely known for their immunosuppressive effects. Evidence also demonstrated that EGFR inhibitors possess dual immunomodulatory effects that include upregulation of major histocompatibility complex I and II (MHC I and II), inducing the recruitment of immune cells, and inhibiting other tyrosine kinases essential for immune cells function. Synergy was observed when EGFR TKI and PD1 inhibitor was combined in an- EGFR-mutant model. Altogether, these previous observations encouragingly support the hypothesis that use of triple therapy (fulvestrant, pan-HER TKI and an immune checkpoint blocker) will be a promising treatment approach for lung cancer. Testing this hypothesis is the subject of this dissertation. To evaluate the therapeutic potential of combining ER blocker with pan-HER in NSCLC, we chose fulvestrant, as an antiestrogen, and dacomitinib, as a tyrosine kinase inhibitor that targets EGFR, HER2, and HER4. We first evaluated the efficacy of this combination in three different human NSCLC cell lines and assessed the ability of this treatment approach to produce a gene signature that predicts better clinical outcomes. We utilized three different cell lines that represent three different categories of NSCLC population; EGFR-mutant, KRAS-mutant and EGFR and KRAS wild-type. Next, we investigated the immunomodulatory effects of this combination treatment on macrophages and CD8+ T cells in vitro. We used a novel syngeneic lung cancer model (FVBW-17/FVB-N) to evaluate the therapeutic effectiveness of fulvestrant-dacomitinib in combination with a mouse anti-PD1. Major results The combination of fulvestrant and dacomitinib significantly suppressed NSCLC cell growth in vitro and produced a combination index < 0.5, indicating strong synergy. The combination also showed potent downregulation of HER activity and marked decrease in amphiregulin (AREG) and neuregulin (NRG1-β1) expression. Importantly, the combination, but not single agents, completely reversed the gene signature associated with poor prognosis. C-Myc, MIA, CXXC5, FGFR4, FOXC1 and Grb7 were downregulated and PgR was upregulated following the combination treatment. The combination significantly reduced c-Fos, JunB and pCREB DNA-binding activities. The c-Fos/Ap-1 inhibitor t-5224, but not CBP-CREB inhibitor, was able to partially mimic the effects of the combination in reversing the gene signature. In vivo, the combination treatment demonstrated a robust synergistic antitumor effect in NSCLC cell lines that were engrafted subcutaneously in immunodeficient mice. Tumor regression was observed in the majority of tumors following the combination treatment. A drastic decrease in HER activity and downstream signaling were observed with the combination, along with a significant decrease in AREG and NRG1-β1 expression. In situ proximity ligation assay revealed a significant decrease in the active dimerization of both p-HER2/p-HER3 and p-HER2/p-EGFR dimers following the combination treatment. Additionally, the gene signature was also completely reversed by the combination but not with single agents. In the EGFR mutant model, the survival of mice was improved after treatment discontinuation, tumors that recurred were less aggressive, and two mechanisms of resistance commonly associated with HER TKIs were blocked. To evaluate the immunomodulatory effects of the combination, bone marrow-derived macrophages and CD8+ T cells were treated with the combination. Surprisingly, macrophages lost their phagocytic function and behave more like M2-macrophages by expressing high IL-10, CD206 and PD1 following the combination treatment. Mechanistically, dacomitinib induced downregulation of phospho-Syk, and fulvestrant was not able to overcome this effect. In CD8+ T cells, the combination impaired the function of T cells, induced high PD1 expression, and severally reduced IFN-ϒ and TNF-α production. These debilitating effects were mostly attributed to the downregulation of Src Family kinases activities, as less phospho-SFK Y416 was detected following the combination treatment. In vivo, adding anti-PD1 antibody to the combination treatment enhanced the immune function and improved the antitumor effects. In a sequential approach, where anti-PD1 was administrated after the combination treatment, the average tumor volume was 4-fold less than placebo and this effect was synergistic. In comparison, the triple therapy given concomitantly showed a 2-fold decrease compared to placebo. Sequential triple therapy was also significantly better than concomitant triple therapy. None of the drugs alone show any sign of activities. Interestingly, after one week of administrating fulvestrant and dacomitinib, tumors showed high immune cell infiltration (inflamed tumor microenvironment), with relatively high PD1 expression on CD8+ T cells. In contrast, the concomitant triple therapy showed a significant increase in CD8+ T cells infiltration but with a decrease in PD1 expression, and fewer M2 macrophages. Conclusion and Significance Retrospective analysis of NSCLC patients revealed that the interaction between HER2/HER3 and ERβ contributes to poor outcomes in NSCLC patients with high ERβ expression. Here, we report that targeting ERβ with the antiestrogen fulvestrant, along with targeting multiple HER pathways with the pan-HER TKI dacomitinib produced synergistic antitumor effects in preclinical models of human ERβ+ NSCLC. The robust antitumor effect seen was accompanied by the ability of this combination treatment to produce a gene signature that predicts better clinical outcomes. The 7-gene model could serve as a predictive tool for identifying patients who will more likely respond to the treatment. These data strongly support its clinical use, giving the fact that both drugs are clinically used for cancer patients. Convincing evidence also suggest an immunomodulatory effect of ER signaling and HER pathways. The combination of fulvestrant and dacomitinib suppressed phagocytic macrophages and CD8+ T cell functions and upregulated PD1 expression. These effects were largely contributed by dacomitinib for its inhibitory effects on Syk and Src kinases on immune cells and might limit the clinical utility of the fulvestrant/dacomitinib combination. However, the ability of the combination to turn the TME into an inflamed microenvironment with upregulated PD1 could potentiate the tumor-mediated killing effects by immunotherapy. The synergistic antitumor effect observed with adding anti-PD1 in a sequential manner after the administration of fulvestrant and dacomitinib is highly encouraging. The significance of these observations is that the triple therapy has shown excellent antitumor effects in a highly aggressive tumor model that is unresponsive to immune checkpoint inhibitor alone or fulvestrant-dacomitinib combination. The clinical usefulness of this approach can have therapeutic applications for other solid tumor models, particularly in tumors that are less inflamed and are unlikely to respond to immune checkpoint inhibitors.
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    TLR7/8 Agonist Encapsulating Polymeric Nanoparticles for Cancer Immunotherapy
    (2018-10) Kim, Hyunjoon
    The immune system is important for the prevention of cancer and formed the basis of cancer immunotherapy. That is, enhancement of the immune response for the treatment of malignant cancer cells. The field has undergone significant progress to include the use of checkpoint inhibitors, monoclonal antibodies and cytokine therapies. In addition, a cancer vaccine, composed of tumor associated antigens (TAAs) and vaccine adjuvant, is particularly promising. Effective vaccines can mobilize tumor-specific CD8 T cells to kill selectively tumor cells with cytotoxic granules and secrete IFN-ɣ that sensitize tumors to be susceptible to effector immune cells. Additionally, activated CD8 T cells become memory cells and can respond to same TAA-epitopes, which can be effective for long-term protective immunity to inhibit cancer recurrence. Activation of dendritic cells (DCs), which are the main antigen-presenting cells (APCs), is critical for T cell immunity. To an elicit tumor-specific CD8 T cell response, DCs have to process and present TAAs to CD8 T cells through the major histocompatibility complex (MHC) I. Moreover, co-stimulatory signals and pro-inflammatory cytokines are required to stimulate CD 8 T cells. However, CD8 T cell anergy and exhaustion will occur if TAA treatment is not sufficiently immunogenic to trigger DC activation. Therefore, development of immunostimulatory adjuvant that can trigger DC activation can enhance therapeutic efficacy of cancer vaccines. Imidazoquinoline-structured synthetic toll-like receptor (TLR) 7/8 agonists are strong cytokine inducers that can be a potent vaccine adjuvant. TLR7/8 ligation can activate MyD88 signaling pathways and stimulate DCs to upregulate co-stimulatory molecules and secrete pro-inflammatory cytokines and type I interferons. However, TLR7/8 agonists lack prominent efficacy in vivo due to the rapid clearance from the injection site. Following subcutaneous (SC) injection, small molecules enter the systemic circulation via blood capillaries and only small portion can reach the draining lymph nodes. Therefore, our goal was to develop a SC injectable drug carrier that can more efficiently deliver as well as prolong duration of at the site of action of TLR7/8 agonists. In this study, we fabricated poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) containing TLR7/8 agonists. Nanoparticulate delivery of TLR7/8 agonist showed enhanced DC activation and antigen-presentation compared to the soluble form of TLR7/8 agonists. When combined with peptide/tumor cell lysate-based antigens, NPs potentiated the antigen-specific CD8 T cell expansion and increased cytotoxic functions, which resulted in enhanced efficacy in both prophylactic and therapeutic tumor models. To further enhance endo/lysosomal delivery of TLR7/8 agonists in PLGA NPs, we included a sodium bicarbonate-mediated gas-generating system that is acidic pH-responsive. This approach resulted in 33-fold greater amount of TLR7/8 agonists encapsulated within the NPs. More importantly, the PLGA NP immunization elicited a stronger CD8 T cell response compared to conventional PLGA NPs, which in turn, enhanced therapeutic efficacy. As tumor microenvironment is immune suppressive, we examined whether modulation of tumor microenvironment can enhance the therapeutic efficacy of cancer vaccine. We reduced the immune suppressive cells including myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs) by daily oral dosing of a tyrosine kinase inhibitor (TKI), sunitinib. Additionally, we adapted an anti-PD-L1 antibody to block programmed death ligand 1 (PD-L1) expressed on tumor-associated (M2) macrophages and MDSCs that exhaust CD8 T cells, to augment the CD8 T cell activation at the tumor. In our study, combination of sunitinib and PD-L1 blockade significantly decreased the immune suppressive cell population and reduced PD-L1 expression on these cells. We also examined if nanoparticulate delivery of TLR7/8 agonist can potentiate NK cell-mediated cancer immunotherapy through its known effect on TH1 immunity. Antibody-dependent cellular cytotoxicity (ADCC) of monoclonal antibodies was found to be augmented in response to TLR7/8 agonist encapsulating NPs as a vaccine adjuvant. Overall, our studies demonstrate that PLGA NPs broaden the application of TLR7/8 agonists for improved cancer treatment. Moreover, this platform holds promise to enhance the efficacy of cancer vaccines composed of tumor associated antigens (TAAs) and vaccine adjuvant
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    Unraveling the Signaling Pathways of the Cd200 Activation Receptor Family and Their Implications in Regulating Antitumor Response in Glioblastoma
    (2020-05) Ampudia Mesias, Elisabet
    Glioblastoma multiforme (GBM) is the most aggressive and incurable primary brain tumor with a current median overall survival of approximately 14 months. Immune checkpoint-based therapy has demonstrated successes in solid tumors including melanoma and lung cancer increasing overall survival, however, it has not been successful in combating Central Nervous System (CNS) tumors. Our studies seek to establish a successful checkpoint inhibitor-based immunotherapy model for treating GBM, and our central hypothesis is, synthetic ligands modulate CD200 activation receptors (CD200ARs) overriding the inhibitory effect mediated by CD200 binding to CD200IR. The CRISPR/Cas9 system was used to generate different murine raw264.7 macrophages (MØs) cell lines expressing different combinations or a single CD200 receptor. The resultant cell lines were stimulated with the synthetic ligand, and the effects of this binding were studied. The main achievements of this research were to demonstrate that CD200ARs stimulated by synthetic peptide-binding couples with DAP10, and stimulates downstream activation of phosphatidylinositol 3-kinase, Vav1, cJUN, and ERK1/2. Second, CD200ARs form complexes (CD200ARs 2&3) to interact with the peptide ligands to optimize the biological function of macrophages. Third, the signals initiated by CD200ARs/DAP10 induce cytokine secretion and immune activation that results in tumor control. Our research reveals the signaling pathway of the CD200 immune checkpoint that leads to activation rather than suppression of immune cells and improves the response of GBM to vaccine-based immunotherapy.