A. James Clark School of Engineering
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item LEVERAGING SELF-ASSEMBLY AND BIOPHYSICAL DESIGN TO BUILD NEXT-GENERATION IMMUNOTHERAPIES(2022) Froimchuk, Yevgeniy; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune system has evolved mechanisms to respond not only to specific molecular signals, but also to biophysical cues. Interestingly, research at the interface of biomaterials and immunology has also revealed that the biophysical properties and form of vaccines and immunotherapies impact immunological outcomes. For example, the intermolecular distance between antigen molecules on the surface of nanoparticles can impact formation of T cell receptor clusters that are critical during T cell activation. Despite the importance of biophysical cues in tuning the immune response, the connections between these parameters and immunological outcomes are poorly understood in the context of immunotherapy. Immunotherapies harness an individual’s immune system to battle diseases such as autoimmunity. During autoimmune disease, the immune system malfunctions and mistakenly attacks self-tissue. Immunotherapies can help tailor and guide more effective responses in these settings, as evidenced by recent advances with monoclonal antibodies and adoptive cell therapies. However, despite the transformative gains of immunotherapies for patients, many therapies are not curative, work only for a small subset of patients, and lack specificity in distinguishing between healthy and diseased cells, which can cause severe side effects. To overcome these challenges, experimental strategies are attempting to co-deliver self-antigens and modulatory cues to reprogram dysfunctional responses against self-antigens without hindering normal immune function. These strategies have shown exciting potential in pre-clinical models of autoimmune disease but are unproven in clinical research. Understanding how biophysical features are linked to immunological mechanisms in these settings would add a critical dimension to designing translatable, antigen-specific immunotherapies. Self-assembling materials are a class of biomaterials that spontaneously assemble in aqueous solution. Self-assembling modalities are useful technologies to study the links between biophysical parameters and immune outcomes because they offer precise control and uniformity of the biophysical properties of assembled moieties. Our lab leveraged the benefits of self-assembly to pioneer development of “carrier-free” immunotherapies composed entirely of immune signals. The therapies are composed of self-antigens modified with cationic amino acid residues and anionic, nucleic acid based modulatory cues. These signals are self-assembled into nanostructured complexes via electrostatic interactions. The research in this dissertation utilizes this platform as a tool to understand how tuning the biophysical properties of self-antigens impacts molecular interactions during self-assembly and in turn, how changes in biophysical features are linked to immunological outcomes. Surface plasmon resonance studies revealed that the binding affinity between signals can be tuned by altering overall cationic charge and charge density of self-antigen, and by anchoring the self-antigen with arginine or lysine residues. For example, the binding affinity between signals can be increased by increasing the total cationic charge on the self-antigen, and by anchoring the self-antigen with arginine residues rather than lysine residues. Computational modeling approaches generated insights into how molecular interactions between signals, such as hydrogen bonding, salt-bridges, and hydrophobic interactions, change with different design parameters. In vitro assays revealed that a lower binding affinity between self-assembled signals was associated with greater reduction of inflammatory gene expression in dendritic cells and more differentiation of self-reactive T cells towards regulatory phenotypes that are protective during autoimmunity. Taken all together, these insights help intuit how to use biophysical design to improve modularity of the self-assembly platform to incorporate a range of antigens for distinct disease targets. This granular understanding of nanomaterial-immune interactions contributes to more rational immunotherapy design.Item Leveraging Biomaterial Properties to Reprogram Immune Function in Autoimmunity(2020) Gosselin, Emily A; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Autoimmune diseases occur when immune cells incorrectly identify and attack the body’s tissues as foreign. In Multiple Sclerosis (MS), the immune system targets myelin, the protective layer that insulates nerves. Current MS therapies reduce disease severity without treating the cause, requiring frequent treatments to slow disease progression. Further, existing therapies cannot differentiate between dysfunctional myelin-reactive inflammatory cells and normal lymphocytes, leaving patients vulnerable to infection. To overcome these limitations, this dissertation investigated biodegradable polymeric microparticles (MPs) co-loaded with myelin peptides and rapamycin, an immunomodulatory signal. Directly injecting these tolerogenic MPs into key immune tissues (e.g. lymph nodes, LNs), induces myelin-specific regulatory immune cells that selectively control myelin-specific inflammation. This work aimed to advance pre-clinical studies and motivate clinical research in two ways: investigating the systemic impact of intra-LN tolerogenic MPs in two MS models, and enhancing MP stability using Chemistry, Manufacturing, and Controls (CMC) considerations. This work showed that across both progressive and relapsing-remitting disease, one tolerogenic intra-LN treatment promoted long-lasting improvements in disease-induced paralysis. Tolerogenic MPs delivered prior to symptom onset promoted tolerance and protected against disease. Treatment at peak disease reversed paralysis and prevented relapse, while treatment during relapse limited disease progression. Strikingly, mice vaccinated against a foreign protein on the same day as intra-LN treatment generated protein-specific T cells and antibodies at similar levels to healthy vaccinated mice, while simultaneously exhibiting significantly reduced paralysis – highlighting the myelin-specific nature of this therapy. While the low dosage requirements of these studies allowed for on-demand preparation, clinical translation requires investigation into manufacturing, preservation, storage, and stability of this immunotherapy. Thus, this dissertation also tested the impact of lyophilization (freeze-drying) and excipients (stabilizing molecules) on MP stability after storage. Lyophilization with low concentrations of excipients significantly improved MP stability and formulation recovery after reconstitution. Storage for 5 months at room temperature did not negatively impact cargo loading, MP size, or biofunctionality. MP formulations with excipients could deactivate inflammatory signaling and restrict myelin-specific immune cell proliferation as well as formulations without excipients. Together, these studies motivate the development of intra-LN delivery of tolerogenic MPs as a potential MS immunotherapy for clinical translation.Item ENGINEERING THE LYMPH NODE MICROENVIRONMENT TO MODULATE ANTIGEN-SPECIFIC T CELL RESPONSE(2019) Gammon, Joshua Marvin; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccines and immunotherapies have provided enormous benefit to human health. However, the development of effective vaccines and immunotherapies for many diseases is hindered by challenges created by the complex pathologies of these targets. For example, in cancer the tumor microenvironment suppresses the function of tumor-specific T cells. In autoimmune diseases, lymphocytes specific for self-antigens attack self-tissue. New technologies providing more sophisticated control over immune response are needed to address these challenges. Lymph nodes (LNs) are tissues where adaptive immune responses develop. Therefore, local delivery of combinations of immune signals is a potential strategy to modulate antigen-specific T cell response for pro-immune or regulatory function. However, application of this idea is hindered since traditional administration routes provide little control over the kinetics, combinations and concentrations with which immune signals are delivered to LNs. Biomaterials have emerged as important tools to overcome these challenges as they provide unique capabilities, including co-delivery, targeting, and controlled release. The research presented here harnesses biomaterials to control immune signals present in LNs to modulate antigen-specific T cell response. In one area, intra-LN injection (i.LN) was used to deposit microparticles (MPs) encapsulating tumor-antigens, adjuvants and immunomodulators to promote tumor-specific central memory T cells. These cells display increased proliferative capacity and resistance to tumor-mediated immunosuppression. MPs encapsulating CpG, an inflammatory adjuvant, and a melanoma antigen potently expanded tumor-specific T cells. MPs delivering low doses of rapamycin – a regulatory immune signal – promoted tumor-specific central memory T cells when co-delivered with the melanoma vaccine. Another important aspect of T cell phenotype which can be modulated for therapeutic benefit is regulatory immune response to control autoimmunity. In this second area, biomaterial-based strategies were used to deliver regulatory immune signals to expand regulatory T cells (TREG) and promote immune tolerance. In one direction, liposomes were designed to deliver regulatory metabolic modulators to bias T cells. In a parallel direction, MPs encapsulating rapamycin and islet self-antigens were designed to promote tolerance in T1D. i.LN delivery of MPs expanded islet-specific TREG and inhibited disease in a mouse model of T1D. Together this work demonstrates potent and modular strategies to therapeutically modulate T cell response.Item PRUSSIAN BLUE NANOIMMUNOTHERAPIES FOR NEUROBLASTOMA(2019) Cano-Mejia, Juliana; Fernandes, Rohan; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of cancer-related deaths. Despite improvements in diagnosis and surgical techniques, neuroblastoma remains challenging to treat due to the heterogeneity of the tumor, low neoantigen expression, immunosuppressive tumor environment, and high recurrence rate. We have therefore engineered a nanoimmunotherapy that combines the advantages of nanotechnology and immunotherapy to combat the aforementioned challenges in treating neuroblastoma. Specifically, our ensemble comprises of Prussian blue nanoparticles (PBNPs) biofunctionalized with the immune adjuvant CpG-oligodeoxynucleotide (CpG). We utilize PBNPs for photothermal therapy (PTT), which ablates tumor cells and releases tumor antigens and adjuvants that increase tumor immunogenicity. Additionally, the PBNPs are biofunctionalized with CpG (CpG-PBNPs) to serve as a depot for local delivery of exogenous immune adjuvants that play an important role in breaking tolerance to tumor antigens and improving tumor antigen presentation. We hypothesize that this approach of targeting tumor cells, antigen presenting cells, and T cells, may hold the key in converting a non-responsive “cold” tumor such as neuroblastoma into a responsive “hot” tumor, leading to better treatments. We first describe the synthesis and characterization of CpG-PBNPs using a facile layer-by-layer coating scheme. The resultant nanoparticles exhibit monodisperse size distributions, multiday stability, and are not cytotoxic. The strong, intrinsic absorption of PBNPs in the CpG-PBNPs is leveraged to administer PTT (CpG-PBNP-PTT) that triggers immunogenic tumor cell death releasing tumor antigens, which increases tumor antigenicity. Simultaneously, the CpG coating functions as an exogenous adjuvant that complements the endogenous adjuvants released by the CpG-PBNP-PTT (e.g. ATP, calreticulin, and HMGB1), increasing adjuvanticity. When administered in a murine model of neuroblastoma, CpG-PBNP-PTT results in complete tumor regression in a significantly higher proportion (70%) of treated animals relative to controls. Further, the long-term surviving, CpG-PBNP-PTT-treated animals reject tumor rechallenge suggesting that our nanoimmunotherapy generates immunological memory. When we treat a synchronous model of neuroblastoma, 50% of nanoimmunotherapy-treated mice show complete eradication of both tumors compared to controls, which showed no survival efficacy. Our findings show the importance of simultaneous cytotoxicity, antigenicity, and adjuvanticity in generating robust and persistent antitumor immune responses. The strategies described in this dissertation encompass novel examples of nanoimmunotherapies to be applied in the clinic for the treatment of neuroblastoma.Item Design of Self-Assembling Nanostructures to Promote Immune Tolerance(2018) Hess, Krystina; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In autoimmune diseases, which affect more than 23 million Americans, the immune system mistakenly attacks healthy tissue. This occurs when the process that normally controls self-reactive inflammatory cells (i.e. tolerance) fails. In multiple sclerosis (MS), the myelin sheath, which insulates nerves, is recognized as a foreign antigen. Demyelination by immune cells results in serious symptoms of neurodegeneration. Current treatments for MS are not curative, but rather manage symptoms by broadly suppressing the immune system, leaving patients unable to fight infection. New therapies that are more specific and effective could greatly improve the quality of life for patients. Biomaterials offer specific advantages for generating antigen-specific tolerance, such as cargo protection, targeted delivery, and controlled release of signals. Additionally, recent reports demonstrate that materials themselves can be intrinsically immunogenic. Two promising biomaterials-based strategies for combating autoimmunity involve: 1) delivery of self-antigen with a regulatory molecule or 2) delivery of self-antigen alone. Aim 1 of this dissertation focuses on the first strategy, creating a novel delivery system for myelin peptide and GpG, an immunomodulatory oligonucleotide. This approach involves electrostatic self-assembly of the two immune signals, eliminating the need for a carrier that could exacerbate inflammation, while still offering attractive features of biomaterials, such as co-delivery. The goal is for immune cells to encounter both signals simultaneously, biasing the response towards tolerance. This work represents the first studies using self-assembled materials to target toll-like receptor signaling, recently shown to be implicated in many autoimmune diseases. Aim 2 of this dissertation is based on the second strategy above, which relies on evidence that changing the trafficking and processing of a self-antigen can impact the development of inflammation or tolerance. Quantum dots, NPs that are intrinsically fluorescent and rapidly drain to lymph nodes, can be decorated with a large and controllable number of myelin peptides. These key features of QDs were exploited to reveal that parameters of self-antigen display (i.e. dose, density) impact biodistribution and immune cell uptake, and are directly correlated to the level of tolerance induced. Together, the described nanotechnologies offer opportunities to probe important questions towards the design of antigen-specific therapies.Item Engineering biomaterials to promote systemic, antigen-specific tolerance(2017) Tostanoski, Lisa Hoban; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In autoimmune diseases, such as multiple sclerosis (MS) and type 1 diabetes, the immune system incorrectly identifies and attacks “self” molecules. Existing therapies have provided important benefits, but are limited by off-target effects, reduced efficacy as disease progresses, and lack of cure potential, necessitating frequent, life-long dosing. An exciting strategy being explored is the design of vaccine-like therapies that selectively reprogram immune responses to self-molecules. This approach could, for example, control the attack of myelin – the protective coating around neurons – that occurs during MS, without leaving patients immunocompromised. However, the realization of this idea has proven difficult; once injected, conventional approaches do not provide control over the combinations, concentrations, and kinetics of signals that reach key tissues that orchestrate immune responses, such as lymph nodes (LNs). Biomaterials have emerged as a promising strategy to confront this challenge, offering features including co-delivery of cargos and controlled release kinetics. The research in this dissertation harnesses biomaterials to develop novel strategies to promote effective, yet selective control of autoimmunity, termed antigen-specific tolerance. In the first aim, direct injection was used to deposit degradable microparticles in LNs, enabling local controlled release of combinations of myelin peptide and Rapamycin, a drug shown to promote regulatory immune function. This work demonstrates the potency of intra-LN delivery in mouse models of MS, as a single dose of co-loaded microparticles permanently reversed disease-induced paralysis in a myelin-specific manner. The results also support this approach as a platform to study the link between local LN signaling and resultant responses in non-treated tissues and sites of disease during autoimmunity. In the second aim, myelin peptide and GpG, a regulatory ligand of an inflammatory pathway overactive in mouse models and patients with autoimmunity, were self-assembled. This approach generated microcapsules that mimic attractive features of conventional biomaterials, but eliminate synthetic carrier components that can complicate rational design and, due to intrinsic inflammatory properties, might exacerbate autoimmunity. These materials promoted tolerance in mouse cells, mouse models of MS, and samples from human MS patients. Together, these strategies could offer novel, modular approaches to combat autoimmune diseases and inform design criteria for future therapies.Item Harnessing Degradable Materials to Study and Engineer Lymph Node Function(2017) Andorko, James; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccines have benefited global health by controlling or eradicating multiple previously fatal diseases. While many early vaccines were efficacious, sophisticated new vaccines and immunotherapies need to address current challenges in the field, including diseases that avoid immune detection or lack strong molecular targets for the immune system. Overcoming these hurdles requires strategies to specifically control the magnitude and type of immune response generated. Biomaterials offer attractive features to achieve this goal, including protection of encapsulated signals, controlled release of cargos, and tunable features for cell targeting. Intriguingly, recent research reveals many common biomaterials activate the immune system, even without other signals. This intrinsic activation results, at least in part, from biomaterial physicochemical features that mimic pathogens and other foreign materials. Surprisingly, although degradable materials are being intensely studied as vaccines carriers, little research has investigated how the intrinsic immunogenicity of these materials changes as polymers degrade. The work in this dissertation reveals parameters impacting material intrinsic immunogenicity and exploits this new understanding to test the influence of biomaterial-based vaccines on the function of lymph nodes (LNs), key tissues that coordinate immunity. In the first aim, the immunostimulatory properties of a library of degradable polymers, poly(beta-amino esters) (PBAEs), were investigated in cell and animal models. PBAEs in soluble forms did not activate innate immune cells (e.g., dendritic cells, DCs). When PBAEs were formulated into particles to mimic a common vaccine strategy, DC activation increased in a molecular weight-specific manner. Using intra-lymph node (i.LN.) injection, a novel technique to control the dose, kinetics, and combination of signals in LNs, PBAE intrinsic immunogenicity was confirmed in mice. In the second aim, microparticles encapsulating immune signals were introduced into mice via i.LN. injection and immune responses were quantified in treated LNs, untreated LNs, and in blood. These results elucidated the interplay between local LN rearrangement and systemic antigen-specific responses which ultimately led to prolonged survival in cancer models. By understanding how the properties and administration of biomaterial-based vaccines impact immunity, this dissertation provides information that can help create new design rules for future vaccines that actively direct the immune system toward a desired response.Item Controlled Delivery of a Glutamate Receptor Modulator to Promote Regulatory T cells and Restrain Autoimmunity(2015) Gammon, Joshua Marvin; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Autoimmunity occurs when the immune system incorrectly recognizes and attacks self-molecules. Current therapies involve broad immunosuppressants that are not curative and leave patients immunocompromised. Dendritic cells (DCs) are a target for new therapies because DCs influence the differentiation of immune effector cells. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), a glutamate receptor enhancer, modulates DC cytokine profiles to polarize T cells toward regulatory phenotypes (TREG ) that are protective in multiple sclerosis (MS). However, PHCCC treatment is limited by poor solubility, a short half-life, and toxicity. We hypothesized that controlled delivery of PHCCC from nanoparticles would alter DC function with reduced treatment frequency. PHCCC nanoparticles attenuated DC activation and promoted TREGs while reducing toxicity 30-fold. In mouse models of MS, these particles delayed disease and reduced severity compared to an equivalent dosing schedule of soluble drug. This outcome demonstrates controlled delivery of metabolic modulators can promote tolerance, suggesting a new route to improve autoimmune therapy.Item Biodegradable Prussian blue nanoparticles for photothermal immunotherapy of advanced cancers(2015) Cano-Mejia, Juliana; Fernandes, Rohan; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Multifunctional nanoparticles represent a class of materials with diverse therapy and imaging properties that can be exploited for the treatment of cancers that have significantly progressed or advanced, which are associated with a poor patient prognosis. Here, we describe the use of biodegradable Prussian blue nanoparticles (PBNPs) in combination with anti-CTLA-4 checkpoint blockade immunotherapy for the treatment of advanced cancers. Our nanoparticle synthesis scheme yields PBNPs that possess pH-dependent intratumoral stability and photothermal therapy (PTT) properties, and degrade under mildly alkaline conditions mimicking the blood and lymph. Studies using PBNPs for PTT in a mouse model of neuroblastoma, a hard-to-treat cancer, demonstrate that PTT causes rapid reduction of tumor burden and growth rates, but results in incomplete responses to therapy and tumor relapse. Studies to elucidate the underlying immunological responses demonstrate that PTT causes increased tumor infiltration of lymphocytes and T cells and a systemic activation of T cells against re-exposed tumor cells in a subset of treated mice. PBNP-based PTT in combination with anti-CTLA-4 immunotherapy results in complete tumor regression and long-term survival in 55.5% of neuroblastoma tumor-bearing mice compared to only 12.5% survival in mice treated with anti-CTLA-4 alone and 0% survival both in mice treated with PTT alone, or remaining untreated. Further, all of the combination therapy-treated mice exhibit protection against tumor rechallenge indicating the development of antitumor immunity as a consequence of therapy. Our studies indicate the immense potential of our combination photothermal immunotherapy in improving the prognosis and outlook for patients with advanced cancers.