ENGINEERED ACTIVITY SENSORS FOR PREDICTIVE IMMUNE MONITORING

Immunotherapies are transforming the treatment of immunological disorders for patients with intractable diseases, for instance through the activation of anti-tumor immunity or the suppression of host reactivity against organ transplants. However, modest response rates and treatment resistance remain clinical barriers, driving efforts to improve response monitoring to better guide clinical decision-making. Most current standards to assess immunotherapy responses rely on evaluation of disease burden by either the core biopsy (e.g., to detect transplant rejection) or radiographic imaging (e.g., to assess tumor regression), yet these approaches primarily focus on morphological features downstream of the immune response. There remains a need for early on-treatment biomarkers to identify patients that may benefit from treatment continuation, alleviate the risks of immune-mediated toxicity, and provide opportunities to treat resistant patients with alternative therapies. Biomarkers of T cell immunity have the potential to monitor the onset of therapeutic responses as elevation of T cell activity in the tumor microenvironment drives tumor control, and suppression of host T cell reactivity towards donor cells promotes transplant tolerance. Proteases are important mediators of immunity and diseases, providing an opportunity to predict responses to immunotherapy early on-treatment. Of note, T cell killing occurs via the classic perforin and granzyme-mediated pathway – the latter of which comprises a family of potent serine proteases – while proteases like matrix-degrading and inflammatory proteases are implicated in major disease hallmarks such as angiogenesis and inflammation. In this thesis, I engineer activity sensors of T cell immunity for two important clinical problems: detecting transplant rejection and monitoring tumor responses during immunotherapy. These sensors monitor the activity of proteases during T cell responses and produce a remote readout in urine. I first develop activity-based nanosensors monitoring granzyme B (GzmB) as noninvasive biomarkers of T cell-mediated acute transplant rejection. Using a skin graft mouse model of organ transplantation, I demonstrate that GzmB nanosensors detect the onset of rejection and indicate allograft failure in recipients treated with subtherapeutic immunosuppression. Then, to noninvasively assess response and resistance to cancer immunotherapy, I design ImmuNe Sensors for monItorinG cHeckpoint blockade Therapy (INSIGHT) by conjugating activity sensors to checkpoint antibodies (e.g., αPD1). In tumor models of immune checkpoint blockade (ICB) response, I show that αPD1-GzmB sensor conjugates retain therapeutic efficacy while producing increased urine signals indicative of early on-treatment responses. Additionally, a multiplexed INSIGHT library sensing tumor and immune proteases enables the development of machine learning classifiers based on urinary outputs to accurately stratify two mechanisms of ICB resistance. This thesis motivates the development of in vivo immune monitoring technologies to maximize the precision and benefit of immunotherapy.Ph.D

Frontiers of Cancer Diagnostics: From Photoacoustic Chemical Imaging to Cellular Morphodynamics

While terrific progress has been made over the last century, cancer continues to be a prevalent, lethal disease and is responsible for millions of deaths each year. The advent of personalized medicine has brought great strides in the treatment of cancer, as clinicians are able to select therapeutic courses that have been tailored to patients specific set of biomarkers. This selection, in principle, maximizes the chances of cancer remission while minimizing overall patient harm. In this spirit, we have focused on developing diagnostic techniques for two separate cancer biomarkers: tumor potassium concentration, and cell morphology. We first developed an ionophore-based potassium sensing nanoparticle. The sensor works on the principle of Donnan exclusion in which the overall charge of the carrier remains constant. The hydrophobic interior of the nanoparticle holds a pH-sensitive dye and a potassium ionophore. As the potassium concentrations rise, the ionophore chelates potassium from the solution which results in a proton being removed from the pH dye to maintain charge neutrality. The deprotonation event can be calibrated for quantitative measurement and this sensor was developed for use in diverse imaging modes, which include UV-VIS absorption, fluorescence, and photoacoustics. At physiological pH and in the presence of interfering ions, we were able to quantitatively measure potassium concentrations using each of these readouts. We modified the potassium sensor to enable in vivo measurements of potassium. This formulation makes use of a solvatochromic dye that transitions from the particle's interior to its surface as potassium is chelated, and thus avoids inherent pH-cross sensitivity. Using photoacoustic chemical imaging, we are able to quantitatively measure the potassium concentration in the tumor microenvironment. As predicted, it was shown that the TME is hyperkalemic, having a potassium concentration of 29mM. The results of the in vivo photoacoustic analysis were verified with ICP-MS measurements of TME potassium. Finally, we combined cell magneto-rotation and machine learning to develop a technique to measure the metastatic potential of a cancer cell population. This technique aims at avoiding the use of expensive and difficult to produce biological labels. By magnetically activating cells, we are able to suspend them in an oscillating magnetic field where they are free to explore their morphological shape space. By collecting fluorescence images of these cells, we are able to train a classifier to recognize cells of a given type. A proof of concept for the technique is provided here, where MCF-7 and MDA-MB-231 cells, both breast cancer but of different metastatic potential, were classified. A random forest classifier trained on cell images was able to correctly identify the cell type with 86.9% accuracy.PHDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163147/1/folzja_1.pd

Nanomedicine-based Immunochemotherapy for the MR Imaging and Treatment of Triple Negative Breast Cancer

Cancer is known to be a detrimental disease and it accounts for many deaths every year. In women Triple negative breast cancer (TNBC) known to be a very aggressive type of tumor. Current treatment approaches are abortive and have many side effects. With the recent advances in nanotechnology and immunotherapy for cancer treatment, we have designed new nanomedicine that combines both the aspects of cancer treatment. In this work, we have developed an anti-PD-L1-conjugated, Doxo-SS-Gd MR imaging agent encapsulating iron oxide nanoparticles (IONPs) to form IONP-Doxo-SS-Gd-PD-L1 nanomedicine for the targeted MR imaging and treatment of TNBC. The anti-PD-L1 acts as a checkpoint inhibitor in the PD-1 & PD-L1 interaction, helping in generating an immune response against the cancer cells. Furthermore, the Gd-DTPA functionalized doxorubicin prodrug (Doxo-SS-Gd), is known as a chemotherapeutic drug and a strong T1 MR agent, which would provide bright T1 MR contrast for the imaging of tumor. To assess the therapeutic potential of the designed nanomedicine in treating cancer, various cell bases experiments were carried out. The bright and dark MR contrast of the IONP-Doxo-SS-Gd- PD-L1 nanomedicine was also evaluated using clinical MRI instrument (B = 9.3T). Therefore, the developed immunochemotherapeutic-nanomedicine provides combination approaches for the synergistic (immunotherapy and chemotherapy) treatment of TNBC and further has MR imaging functionality for diagnosis and treatment monitoring

Magneto-plasmonic nanoparticle platform for detection of rare cells and cell therapy

textMagnetic and plasmonic properties combined in a single nanostructure provide a synergy that is advantageous in a number of biomedical applications, such as contrast enhancement in multimodal imaging, simultaneous capture and detection of circulating tumor cells, and photothermal therapy of cancer. These applications have stimulated significant interest in development of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared region and a strong magnetic moment. In this dissertation, we addressed this need to create a novel immunotargeted magneto-plasmonic nanoparticle platform. The nanostructures were synthetized by self-assembly of primary 6 nm iron oxide core-gold shell particles, resulting in densely packed spherical nanoclusters. The close proximity of the primary particles in the nanoclusters generates a greatly improved response to an external magnetic field and strong near-infrared plasmon resonances. A procedure for antibody conjugation and PEGylation to the hybrid nanoparticles was developed for biomedical applications which require molecular and biocompatible targeting. Furthermore, we presented two biomedical applications based on the immunotargeted hybrid nanoparticle platform, including circulating tumor cell (CTC) detection and cell-based immunotherapy of cancer. In the CTC detection assays, rare cancer cells were specifically targeted by antibody-conjugated nanoparticles and efficiently separated from normal blood cells by a magnetic force in a microfluidic chamber. The experiments in whole blood showed capture efficiency greater than 90% for a variety of cancers. We also explored photoacoustic imaging to detect nanoparticle-labeled CTCs in whole blood. The results showed excellent sensitivity to delineate the distribution of hybrid nanoparticles on the cancer cells. Thus, these works paves the way for a novel CTC detection approach which utilizes immunotargeted magneto-plasmonic nanoclusters for a simultaneous magnetic capture and photoacoustic detection of CTCs. In another application, we introduced a novel approach to label cytotoxic T cells using the magnetic nanoparticles with an expectation to enhance T cell recruitment in tumor under external magnetic stimulus. A series of in vitro experiments demonstrated highly controllable manipulation of labeled T cells. Thus, these results highlight the promise of using our nanoparticle platform as a multifunctional probe to manipulate and track immune cells in vivo and further improve the efficacy of cell-based cancer immunotherapy.Biomedical Engineerin

Fluorescent-based nanosensors for selective detection of a wide range of biological macromolecules: A comprehensive review

Thanks to their unique attributes, such as good sensitivity, selectivity, high surface-to-volume ratio, and versatile optical and electronic properties, fluorescent-based bioprobes have been used to create highly sensitive nano -biosensors to detect various biological and chemical agents. These sensors are superior to other analytical instrumentation techniques like gas chromatography, high-performance liquid chromatography, and capillary electrophoresis for being biodegradable, eco-friendly, and more economical, operational, and cost-effective. Moreover, several reports have also highlighted their application in the early detection of biomarkers associ-ated with drug-induced organ damage such as liver, kidney, or lungs. In the present work, we comprehensively overviewed the electrochemical sensors that employ nanomaterials (nanoparticles/colloids or quantum dots, carbon dots, or nanoscaled metal-organic frameworks, etc.) to detect a variety of biological macromolecules based on fluorescent emission spectra. In addition, the most important mechanisms and methods to sense amino acids, protein, peptides, enzymes, carbohydrates, neurotransmitters, nucleic acids, vitamins, ions, metals, and electrolytes, blood gases, drugs (i.e., anti-inflammatory agents and antibiotics), toxins, alkaloids, antioxidants, cancer biomarkers, urinary metabolites (i.e., urea, uric acid, and creatinine), and pathogenic microorganisms were outlined and compared in terms of their selectivity and sensitivity. Altogether, the small dimensions and capability of these nanosensors for sensitive, label-free, real-time sensing of chemical, biological, and pharma-ceutical agents could be used in array-based screening and in-vitro or in-vivo diagnostics. Although fluorescent nanoprobes are widely applied in determining biological macromolecules, unfortunately, they present many challenges and limitations. Efforts must be made to minimize such limitations in utilizing such nanobiosensors with an emphasis on their commercial developments. We believe that the current review can foster the wider incorporation of nanomedicine and will be of particular interest to researchers working on fluorescence tech-nology, material chemistry, coordination polymers, and related research areas

Biomanufacturing Technologies for Regenerative Medicine

Regenerative Medicine has the potential to be a game-changer for patients who have damaged tissues or organs due to untreatable diseases, injuries, and congenital conditions. Lab-based innovations have shown great promise in restoring structure and function, but to deliver treatments to large numbers of patients in a clinical setting, new tools and technologies are needed. Regenerative Medicine is a new area of medical research that seeks to automate and scale-up the production and deployment of these groundbreaking solutions. The technologies discussed in this report are intentionally pre-competitive, meaning that the Federal Government may choose to play a role in additional growth via well-informed initiatives. Governmental support can come in the form of additional research & development (R&D) dollars that are magnified by private co-investment, or can be in the form of non-pecuniary actions such as modifications to the regulatory environment to better support this rapidly changing field. Ideally, a cooperative relationship between government and private industry will result in cross-industry, pre-competitive tools that decrease development cost and time while still respecting individual intellectual property ownership within a competitive environment. This report identifies promising biomanufacturing platforms that will provide a foundation for the automation and standardization of the processes associated with successful scale-up and scale-out. After evaluating a range of potential translational technology options according to their suitability for co-investment and cross-industry appeal, two platform technologies and two enabling tools were selected: Platform Technology #1: 3D Constructs, including Organoids, Scaffolds, and Printed Tissues; Platform Technology #2: Biomanufacturing Processes; Enabling tool #1: Scaled-up bioreactors for cell culture; Enabling tool #2: Improvements in cell harvesting, cell processing, and preservation technologies.National Science Foundation, Grant No. 1552534https://deepblue.lib.umich.edu/bitstream/2027.42/144783/1/RegenMedicine_Final.pdfDescription of RegenMedicine_Final.pdf : Repor

T Cell Calcium Flux And Clonal Proliferation Report On Antigen-Specific Myeloid Cell Encounters

Despite its clinical success, the mechanism underlying extracorporeal photopheresis (ECP) is not well understood, however, the plate-passage (PP) step appears integral to generating activated monocytes. In the first part of the project, we developed a functional assay to evaluate the efficacy of plate-passed myeloid cells (PPM) compared with freshly isolated, unstimulated monocytes (UM) and conventional dendritic cells derived from blood monocytes cultured with GM-CSF/IL4 (DC). Each of these three antigen presenting cell (APC) was co-cultured with purified, autologous CD8 cells, with or without CD4 cells. Cultures were carried out using melanoma antigen MART-1 long peptide (LP), a 25-amino acid peptide containing the binding sequences for the appropriate MHC class I and II, and for presentation to CD8+ and CD4+ cells. Results showed reliable expansions of freshly isolated naïve human T cells using the three types of APC, without any significant differences among the types, and the addition of CD4+ tended to enhance expansion of PPM and DC, but not UM. In the second part, we sought to develop a method for directly tracking early T cell responses during immunotherapy. Using calcium flux to indicate early T cell signaling, we focused mostly on the ovalbumin (OVA)-derived, SIINFEKL-specific transgenic mouse model (OT1). After a few protocol modifications, we were able to detect antigen-specific calcium flux (ASF) upon mixing naïve OT1 cells with SIINFEKL peptide-loaded DC compared with non-specific peptide counterparts. We could still detect ASF down to a peptide-loading concentration of ~10-3uM and at a frequency of ~0.1% OT1 cells among wild-type (WT), non-responding cells. We next identified the activation requirements of early effector and memory OT1 cells from the spleen, lymph nodes, and peripheral blood after adoptive transfer into WT recipients immunized with OVA. At 1 week, OT1 cells from all 3 tissues had become activated, effector cells (CD44hi and CD62 lo), and while detectable, ASF in all three tissues was reduced compared with naïve cells. At 6 weeks, only the peripheral blood OT1 cells had generated a memory response (CD127hi KLRG1lo), and ASF in all three tissues was further reduced. Herein, we have shown that ASF can be detected in naïve, and less so antigen-experienced and memory T cells in a single-antigen, transgenic system from which we hope to develop a multi-antigen tumor model

High-Throughput Screening for Drug Discovery

The book focuses on various aspects and properties of high-throughput screening (HTS), which is of great importance in the development of novel drugs to treat communicable and non-communicable diseases. Chapters in this volume discuss HTS methodologies, resources, and technologies and highlight the significance of HTS in personalized and precision medicine