Browsing by Subject "Protein engineering"
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Item Open Access Antibody-mediated Immunotherapy of Brain Tumors(2017) Gedeon, Patrick ChristopherConventional 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.
Item Open Access Bioorthogonal Functionalization of Elastin-like Polypeptides(2019) Costa, SimoneRecombinant technology has given us the powerful ability to imagine and create novel biological entities, from potent therapeutics to functionally active materials. By harnessing nature’s building blocks and reconfiguring these components, recombinant engineering unlocks the potential to tailor drug specificity and pharmacokinetics, rationally design biomaterials, understand and define protein structure, and probe cellular function with molecular precision. These technological feats are made possible with a few simple biological ingredients: nucleotides, sugars, and amino acids. These components, exquisitely crafted by evolution, are individually combined in useful ratios and precise sequences in living systems to synthesize DNA, RNA, polysaccharides, and proteins. These macromolecules collectively support organismal structure and function and give rise to the incredible diversity in Charles Darwin’s “great tree” of life. However, the seemingly infinite potential for new materials built from these components is, in fact, limited. The chemical identity of these building blocks – with a particular focus herein on the twenty naturally-occurring amino acids – limits the scope and functionality of the recombinant materials we can produce. In order to functionalize these products, to fundamentally change their chemical identity while preserving their biological functionality, we require the finesse of bioorthogonal chemistries and modification techniques.
Bioorthogonal reactions modify biological materials within living systems without perturbing function, much as two orthogonal lines can extend in different directions and intersect only at a single point. That point of intersection can be precisely defined through recombinant technology and gives us access to new classes of biomaterials. The term “bioorthogonal”, coined by Carolyn Bertozzi, importantly defines these unique chemistries, which inertly co-exist with biology until the exact moment when the desired reactions are initiated, to enhance – and even transform – biological systems.
Bioorthogonal modification of proteins will, by definition, require expansion of the biochemical toolbox; there are a variety of techniques used to achieve this goal. In these studies, we explore the use of genetic code expansion for incorporation of unnatural amino acids. This technology permits co-translational incorporation of amino acids with unique and non-canonical R-groups directly into the polypeptide backbone of a protein or biopolymer. These residues introduce unique chemical reactivity for further functionalization with desired moieties or chemical transformation.
We have used this technology to develop novel therapeutic and material platforms comprised of a unique biopolymer, elastin-like polypeptide (ELP). This thermally responsive biopolymer is easily recombinantly synthesized, though more biochemically complex ELPs require successful bioorthogonal modification. We designed the unnatural amino acid-containing ELPs necessary to enable our strategies for developing three distinct biomaterial platforms: 1) photoreactive ELPs which can generate stable hydrogel particles spanning four orders of magnitude in size; 2) a universal strategy for drug-loaded, targeted ELP nanoparticles by incorporation of a unique site for drug attachment; 3) a sustained-release therapeutic for treatment of brain tumors combining proteins of distinct cellular origin.
We have combined existing tools, technologies, and materials to generate these novel platforms with utility in biomaterials, drug delivery, and cancer therapeutics. The optimizations performed in developing each of these systems will inform future studies with similar goals; similarly, the reactions and strategies employed will contribute to furthering our understanding of the full potential of these important bioorthogonal chemistries.
Item Embargo GRIP Display: A One-Pot Library Display Platform for the Directed Evolution of Proteins(2023) Goldenshtein, VictoriaLibrary display technologies have enabled the development of peptides with affinity for a given substrate. Such affinity-capture reagents have driven progress in many fields, from basic biochemistry to neuropharmacology. A major limitation in the development of neuro-pharmaceuticals has been an inability to examine how the behavioral effects of drugs are mediated by each of the distinct yet intermingled cell types in any given brain region. DART (Drugs Acutely Restricted by Tethering) is the first method to overcome this technical barrier, enabling the delivery of therapeutics to a precise genetically defined neuronal cell type. At the core of DART’s specificity is a capture of a chemical Rx-HTL (HaloTag Ligand conjugated to a drug) by a genetically encoded HTP (HaloTag protein), creating an artificial dosing window. Although the technology has already revealed novel neurobiological insights, a narrow dosing window currently limits DART to neurobiological questions where dose can be tightly controlled, such as via intracranial infusion over a small brain volume. Our goal is to adapt the principles of directed evolution and library display to improve the dosing window of DART and enable its brain-wide delivery. Moreover, using the same principles, we aim to develop an orthogonal DART pair for multiplexed delivery of any combination of drugs to two distinct cell types. The underlying principle of a library display tool is a physical linkage between phenotype (a protein) and genotype (its corresponding nucleotide sequence). This conjugated mRNA, encoding the displayed protein, serves as a unique identifier for each variant. Over the past three decades, several display systems have been developed, each with a unique set of limitations. Typically, there is a tradeoff between the stability of this linkage and the number of unique variants (library size). Thus, no existing platform offers the desired trifecta of linkage stability, library size, and product yield. This work introduces a novel in vitro protein display technology called GRIP Display (Gluing RNA to Its Protein) that permits the generation and simultaneous screening of vast protein libraries (~10^14 variants) against a target of interest, with minimal genetic cross-talk, significant selection enrichment, and one-step simple experimental protocol. Here, we demonstrate 1) the development of GRIP Display and its utility in the optimization of a large binding tunnel of HTP to enhance the covalent capture of its chemical ligand; 2) the development of high-affinity orthogonal HTP/HTL pairs with minimal cross-reactivity; 3) a rational design of a novel peptide/RNA interaction to promote the avidity of binding and create a “single read” display technology GRIP.2. GRIP Display represents a valuable resource for the protein engineering community, and can substantially advance the range of neurobiological questions amenable to DART.
Item Open Access Integrating Protein Engineering and Genomics for Cancer Therapy(2018) Manzari, Mandana TaghizadehWe have developed a broadly applicable platform that harnesses the power of protein engineering and genetic screening to produce efficacious protein-drug combinations for cancer therapy. For proof-of-concept, we implemented this strategy to engineer targeted pro-apoptotic drug combinations that overcome cancer resistance to protein agonists of death receptor 5 (DR5), a key upregulated marker in colorectal cancer (CRC). Over the past decade, various DR5 agonists have shown poor clinical efficacy, including both engineered antibodies and TRAIL, the natural ligand for this receptor. Comprehensive studies suggest that there are three major obstacles to success of these agents: 1) potency, 2) delivery, and 3) resistance.
We have systematically addressed these challenges by engineering a sustained-release formulation of a highly potent, hexavalent death receptor 5 agonist (DRA), and administering the agonist as a sustained release depot, in combination with rationally nominated targeted drugs that overcome intrinsic resistance to DRAs. To address the need for sustained delivery of therapeutic proteins, we developed injectable depots of DRAs recombinantly fused to thermally responsive elastin-like polypeptide (ELP) biopolymers. The bioactive ELP-DRA fusions undergo temperature-driven phase transition upon subcutaneous injection in vivo, resulting in the formation of a gel-like depot suitable for sustained drug delivery. A single 30 mg/kg injection of the gel-like ELP-DRA depot induced significant tumor regression in Colo205 mouse xenografts. To pinpoint the genetic drivers of CRC resistance to the DRA, we used a gain-of-function ORF screen and a CRISPR/Cas9 knockout screen. The screens identified genes that confer sensitivity to the DRA in resistant CRC cell lines. Over twenty small molecule drugs targeting pathways and proteins identified from the screens were then tested in combination with the DRA to identify highly synergistic combinations using cytotoxicity assays. Clonogenic, time-to-progression, and cell viability assays showed that pharmacological blockade of XIAP, Bcl-XL, and CDK4/6 strongly enhances antitumor activity of DRA in established human CRC cell lines and patient-derived CRC cells. In vivo tumor regression studies demonstrated the potent anti-tumor efficacy of combining inhibitors of XIAP and Bcl-XL with the sustained release formulation of ELP-DRA.
By addressing both delivery and resistance issues with our protein engineering and genomics platform, we have overcome the key obstacles to DRA translation as a successful drug in the clinic. Our rational approach elegantly provides optimal protein-small molecule drug combinations that elicit a robust anticancer response, exhibit minimal toxicity, and combat drug resistance.
Item Open Access Molecular Bioengineering: From Protein Stability to Population Suicide(2010) Marguet, Philippe RobertDriven by the development of new technologies and an ever expanding knowledge base of molecular and cellular function, Biology is rapidly gaining the potential to develop into a veritable engineering discipline - the so-called `era of synthetic biology' is upon us. Designing biological systems is advantageous because the engineer can leverage existing capacity for self-replication, elaborate chemistry, and dynamic information processing. On the other hand these functions are complex, highly intertwined, and in most cases, remain incompletely understood. Brazenly designing within these systems, despite large gaps in understanding, engenders understanding because the design process itself highlights gaps and discredits false assumptions.
Here we cover results from design projects that span several scales of complexity. First we describe the adaptation and experimental validation of protein functional assays on minute amounts of material. This work enables the application of cell-free protein expression tools in a high-throughput protein engineering pipeline, dramatically increasing turnaround time and reducing costs. The parts production pipeline can provide new building blocks for synthetic biology efforts with unprecedented speed. Tools to streamline the transition from the in vitro pipeline to conventional cloning were also developed. Next we detail an effort to expand the scope of a cysteine reactivity assay for generating information-rich datasets on protein stability and unfolding kinetics. We go on to demonstrate how the degree of site-specific local unfolding can also be determined by this method. This knowledge will be critical to understanding how proteins behave in the cellular context, particularly with regards to covalent modification reactions. Finally, we present results from an effort to engineer bacterial cell suicide in a population-dependent manner, and show how an underappreciated facet of plasmid physiology can produce complex oscillatory dynamics. This work is a prime example of engineering towards understanding.
Item Open Access Protein Engineering for Biosensor Development(2008-11-24) Miklos, AleksandrBiosensors incorporating proteins as molecular recognition elements for analytes are used in clinical diagnostics, as biological research tools, and to detect chemical threats and pollutants. This work describes the application of protein engineering techniques to address three aspects in the design of protein-based biosensors; the transduction of binding into an observable, the manipulation of affinities, and the diversification of specificities. The periplasmic glucose-binding protein from the hyperthermophile Thermotoga maritima (tmGBP) was fused with green fluorescent protein variants to construct a fluorescent ratiometric sensor that is sufficiently robust to detect glucose up to 67°C. Ligand-binding affinities of tmGBP were changed by altering a C-terminal helical domain that tunes ligand binding affinity through conformational coupling effects. This method was extended to the Escherichia coli arabinose-binding protein. Computational design techniques were used to diversify the specificity of the E. coli maltose-binding protein (ecMBP) to bind ibuprofen, a non-steroidal antiinflammatory drug. These designs ranged in affinity from 0.24 to 0.8 mM and function as reagentless fluorescent sensors. The ligand affinities of ecMBP are tuned by complex interactions that control conformational coupling. These experiments demonstrate that long-range conformational effects as well as molecular recognition interactions need to be considered in the design of high-affinity receptors.
Item Open Access Sortase as a Tool in Biotechnology and Medicine(2016) Bellucci, JosephWe have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.
We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.
We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells
Item Open Access The Immunoengineering Toolbox: A Set of Thermoresponsive Biopolymers for Sustained Delivery of Cancer Immunotherapies(2022) Kelly, GarrettTherapeutic cancer vaccines have the potential to revolutionize cancer treatment by providing systemic control of both local and metastatic malignancies. However, stimulating antitumor immune responses in patients with cancer has proven difficult and the success rate associated with cancer vaccines is low. Therefore, there is an urgent need to develop novel cancer vaccine strategies to overcome immunosuppression and produce robust anticancer immunity. Addressing this problem will require the development of new tools to achieve improved localized control of the microenvironment in which cancer antigens are present. Motivated by this rationale, we developed a toolbox of sustained-release immunostimulatory fusions to create cancer vaccines that provide tunable spatiotemporal control of immunostimulatory signals. The backbone of these immunostimulatory fusions is a set of protein biopolymers, elastin-like polypeptides (ELPs), that can undergo a thermally triggered phase transition to form a depot upon injection in vivo for localized and sustained delivery of their payload. To create the toolbox, a set of ELPs were covalently fused to the cytokines, granulocyte-macrophage colony-stimulatory factor (GM-CSF), and interleukin-12 (IL-12), the antigenic peptides SVYFFDWL and SIINFEKL, and the immune adjuvant fibronectin III extra domain A (Fn3EDA). The CpG oligodeoxynucleotide (ODN) adjuvant was bound to an ELP with an oligolysine tail by electrostatic complexation. This toolbox of immunostimulatory, depot-forming ELP fusions was used to develop two new cancer vaccines to improve anticancer immunity: 1) an intratumoral (i.t.) in situ vaccine, consisting of a previously developed ELP-Iodine-131 (131I-ELP) radioisotope conjugate for localized radiotherapy combined with the ELP fusion to locally deliver CpG ODN and 2) a subcutaneous (s.c.) subunit vaccine, consisting of ELP fusions to provide sustained release of an antigenic peptide, GM-CSF, and CpG. Characterization studies demonstrated the depot-forming phase behavior and immunostimulatory activity for each fusion in the toolbox, the ability of the ELP-GM-CSF fusion to recruit antigen-presenting cells (APCs) in vivo, and the ability of an ELP-oligolysine fusion to prolong retention and enhance the activity of electrostatically complexed CpG. Furthermore, we demonstrated that an in situ i.t. depot vaccine improves the local and systemic control of 4T1 mammary carcinoma, leading to a synergistic improvement in survival. Finally, we demonstrated that an optimized s.c. depot vaccine augments CD8 T cell response to both a model antigen and a cancer neoantigen and provides protection from melanoma in a tumor challenge experiment. Altogether, these studies establish a versatile delivery platform to spatiotemporally control immune signaling that advances the development of cancer vaccines.
Item Open Access Traditional and Computational Engineering of Genetically Encoded Indicators and Actuators for Neuroscience Applications(2023) Beck, ConnorThe brain supports numerous complex processes ranging from signal processing and motor control to learning and memory. These processes rely on signal transduction between interconnected networks of neurons that form neural circuits. Understanding how neural circuits function requires non-invasive, genetically specific technologies to both record and manipulate neural activity. Recording neural activity establishes a correlative relationship between the activity and cognitive function, while manipulating neural activity establishes a causal relationship between the activity and behavioral or physiological processes. Genetically encoded protein tools facilitate neuroscience research in both experimental paradigms. Genetically encoded sensors enable optical recording of neural activity across a wide spatiotemporal range. These indicators detect diverse forms of neural activity, including calcium ion flux, membrane voltage potential, and neurotransmitter concentration. Conversely, optogenetic actuators enable targeted, optical excitation or inhibition of neurons upon activation with a specific wavelength of light.
Advancement of genetically encoded tools will allow researchers to access new experimental regimes of neuroscience. Enhancing the fluorescence response and temporal fidelity of genetically encoded sensors improves signal detection fidelity, enabling neuroscientists to access more neurons at once and more precisely analyze neural circuits. Expanding the spectral diversity of genetically encoded tools makes it possible to record from multiple neural populations simultaneously or to optogenetically excite one population with a specific wavelength of light while recording the activity of another in a distinct optical channel. Such multi-channel experiments enable neuroscientists to investigate the influence of the activity of an ensemble of neurons on the activity of another ensemble downstream in a neural circuit or feedback between neural circuits. However, expanding the palette of protein sensors and actuators for such multi-channel experiments has been challenging. Most state-of-the-art genetically encoded sensors fuse cyan-light-sensitive green fluorescent protein to a sensing domain, so the dual channel experiments described above require a complementary sensor or actuator that is sensitive to a spectrally distinct wavelength of light. However, the performance of red fluorescent genetically encoded tools typically lags relative to their green counterparts, and using cyan-light-activated sensors in conjunction with green-light-activated actuators introduces high optical crosstalk. Additionally, the dynamic properties and context-dependent performance of genetically encoded sensors make high-throughput screens of this class of tools labor intensive and time consuming. This constraint on the throughput of screens has limited development efforts to a miniscule fraction of the possible variants of each sensor.
In this dissertation, I expanded the spectral diversity of the tools described above and developed a novel strategy for high throughput evolution of genetically encoded sensors. First, I developed a red fluorescent genetically encoded voltage sensor by engineering the fluorescence resonance energy transfer (FRET) efficiency between a voltage sensitive domain and a red fluorescent protein. This red fluorescent sensor enabled high fidelity recordings of neural activity with sub-millisecond temporal resolution, dual-channel recordings in parallel with green fluorescent sensors, and simultaneous optogenetic excitation and voltage imaging with minimal optical crosstalk. Second, I developed an optogenetic actuator with a blue-shifted activation spectrum by employing this same FRET mechanism. I demonstrated that the activation spectrum of optogenetic tools could be tuned by engineering FRET efficiency between the actuator domain and a compatible fluorescent protein. This straightforward strategy represents a technical step forward for engineering the spectra of optogenetic actuators, which has been difficult to achieve without compromising functionality. Third, I developed a screening method that enabled pooled, high-throughput screens of diverse libraries containing genetically encoded sensor mutants. This method employed both experimental and computational advancements. I used in situ optical mRNA sequencing to determine the sequence of each screened protein variant and machine learning to predict the function of unscreened variants. I expanded the coverage of the possible sequence space by over an order of magnitude compared to traditional directed evolution.