Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 118-128). / A variety of cancers are marked by the over-expression and over-activity of the EGF receptor (EGFR), rendering this protein an attractive therapeutic target. Anti-EGFR therapeutics are a mainstay of clinical practice for the treatment of colorectal, lung and head and neck cancers but efficacy is limited and response rates low. Opportunities for improving EGFR antagonism include higher potency inhibition of ligand binding, inducing receptor downregulation, or creating synergistic therapeutic combinations. The Wittrup lab has previously made significant advances in EGFR antagonism by demonstrating the therapeutic potential of inducing receptor downregulation through multi-epitopic targeting. The lab has also pioneered the use of a novel protein scaffold, called Sso7d, for yeast surface display-based libraries and selections. In the first part of this work I show that a combination of traditional yeast display techniques with simple but novel in silico approaches can be applied to derive a panel of Sso7d binders against EGFR with diverse paratopes. I demonstrate the superior EGFR inhibition of antibody-Sso7d fusions in vitro, and discuss the lessons learned from applying these proteins in vivo. In the second part of this work I use a structure-guided yeast display approach to create a novel research tool, a minimally modified verstion of cetuximab called "mCetux", which essentially enables in vivo experiments of cetuximab. I apply this antibody tool in vitro and in vivo in a new and highly relevant model system for colorectal cancer and subsequently discuss future opportunities for its use. / Funded by NIH/NIGMS Biotechnology Training Grant / by Alison W. Tisdale. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 184-197). / Most malignant cancers are treated with chemotherapeutic agents that target and damage cellular DNA While genotoxic chemotherapies have proven to be highly effective agents for cancer therapy, it is well known that intrinsic and acquired cancer drug resistance is a problem that severely limits the successful elimination of a wide range of malignancies. This point is particularly important in the context of genotoxic chemotherapies, because the DNA damaging agents used in cancer treatment induce a diverse spectrum of toxic lesions that are recognized by a variety of DNA damage response (DDR) mechanisms. In this thesis I used CRISPR-Cas9 gene-editing and other molecular biology and biochemical techniques to examine the functional relevance of that Rev7, a multi-functional translesion synthesis (TLS) DNA damage tolerance protein in drug-resistant cancers. / In particular, I employed CRISPR-Cas9 gene-editing technologies to generate Rev7 knockout (KO) drug-resistant lung cancers cell to use as a tool to investigate the impact that Rev7 loss may have on chemotherapeutic efficacy. Excitingly, this work reveals that Rev7 loss sensitizes intrinsically drug-resistant lung tumors to cisplatin and drastically enhances the overall survival of syngeneic mice transplanted with drug-resistant lung tumors. Additionally, in this thesis I conducted immunoprecipitation and mass spectrometry to better elucidate the Rev7's functional relevance. Mass spectrometry findings in this thesis reveal that when Rev7 is immunoprecipitated under different cellular conditions (i.e. G2/M arrested or DNA damaged cells), Rev7 interacts with novel and diverse Rev7 protein interactors. Intriguingly, my mass spectrometry findings also reveal that Rev7 forms protein-protein interactions with many proteins that play a role in regulating double-strand break (DSB) repair. / Given these findings we conducted DSB repair studies investigating if Rev7 plays a role regulating DSB repair in drug-resistant lung cancer. Notably, these studies suggest that Rev7 loss results in a decrease in DSB repair capacity and increases in DSBs in drug-resistant lung cancer cells. Altogether, this thesis demonstrates that Rev7 has functional relevance in modulating chemotherapeutic response in drug-resistant lung cancer. Further, this thesis presents findings that strongly argue that the development of small molecule inhibitors targeting Rev7 may provide a new way to enhance chemotherapeutic efficacy in drug-resistant tumors in the clinic. / by Faye-Marie Vassel. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018 / Cataloged from PDF version of thesis. / Includes bibliographical references. / The humble red blood cell (RBC) is the most abundant cell in the human body. Every second, a normal adult generates some 2.5 million RBCs, which subsequently circulate through the blood vessels for a lifespan of 50 or 120 days in mouse and human, respectively. RBCs are also unique in that they are completely enucleated once fully mature. These two characteristics exist as distinct assets for cellular therapy applications utilizing RBCs as a platform, enabling long-lasting availability in vivo and the ability to genetically modify precursor cells without worry of the terminally differentiated progeny carrying any foreign genetic material. The first part of this thesis is devoted to the establishment of methodologies that allow for the covalent attachment of both natural and synthetic cargoes to the surface of red blood cells without compromising its biological properties. This system employs genetic engineering and sortase A, a bacterial transpeptidases. / We show that this strategy is able to efficiently engineer both mature mouse and human RBCs in a site-specific and covalent manner. The next portion of this work describes how these established methodologies can be mixed and matched according to the diverse needs of engineered RBC applications. We provide a proof of concept that utilizes engineered RBCs to prolong prophylactic protection against deadly toxins. By expressing chimeric proteins of single domain antibodies (VHHs) against botulinum neurotoxin A (BoNT/A) with RBC-specific proteins, we demonstrated that mouse RBCs expressing anti-BoNT/A VHHs can provide resistance up to 10,000 times the lethal dose (LD₅₀) of BoNT/A. We validate this finding by repeating our results in a human RBC culture system that we have improved to achieve 90% enucleation, illustrating the broad translatability of our strategy for therapeutic applications. / Finally, drawing upon knowledge that the body clears 2.5 millions RBCs every second to maintain homeostasis, we use sortase to attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells (RBCs). Such modified RBCs masquerade with these autoantigens as their own, and hijack the non-inflammatory nature of the RBC clearance pathway to promote tolerance to their carried payload. We show that this blunts the immune contribution of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of RBCs expressing self-antigen epitopes alleviates and even prevents signs of disease in an experimental system for autoimmune encephalomyelitis, and also maintains normoglycemia in a mouse model of type 1 diabetes, highlighting the potential of engineered RBCs for treating autoimmune diseases. / Taken together, the results of applying our engineered RBCs in areas of both acute infectious and toxic agents, as well as for longer-term chronic and autoimmune diseases, hint at the tremendous potential of this system, and we have only begun to scratch the surface. / by Novalia Pishesha. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. "June 2019." / Includes bibliographical references. / The ability to map the molecular organization of cells and tissues with nanoscale precision would open the door to understanding their biological functions as well as the mechanisms that lead to pathologies. Though recent technological advances have expanded the repertoire of biological tools, this crucial ability remains an unmet need. Expansion Microscopy (ExM) enables the 3D, nanoscale imaging of biological structures by physically magnifying cells and tissues. Specimens, embedded in a swellable hydrogel, undergo uniform expansion as covalently anchored labels and tags are isotropically separated. ExM thereby allows for the inexpensive nanoscale imaging of biological samples on conventional light microscopes. In this thesis, I describe the development of a method called Expansion FISH (ExFISH) that uses ExM to enable the nanoscale imaging of RNA throughout cells and tissues. A novel chemical approach covalently retains endogenous RNA molecules in the ExM hydrogel. After expansion, RNA molecules can be interrogated with in situ hybridization. ExFISH opens the door for the investigation of the nanoscale organization of RNA molecules in various contexts. Applied to the brain, ExFISH allows for the precise localization of RNA in nanoscale neuronal compartments such as dendrites and spines. Furthermore, the optical homogeneity of expanded samples enables the imaging of RNA in thick tissue-sections. ExFISH also supports multiplexed imaging of RNA as well as signal amplification techniques. Finally, this thesis describes strategies for the multiplexed characterization of biological specimens. Taken together, these approaches will find applications in developing an integrative understanding of cellular and tissue biology. / by Asmamaw T. Wassie. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017 / Cataloged from PDF version of thesis. "February 2017." / Includes bibliographical references (pages 157-170). / The local cytokine milieu shapes the nature and function of immune cells and by extension the overall course of tissue-specific immune responses. In the context of cancer and autoimmunity, two opposing immune responses, the local immune environment can lead to dysfunctional T cell states. Understanding the mechanisms that distinguish these two states is key to identifying unique pathways that could be induced in autoimmunity and relieved without causing autoimmunity in cancer. We sought to characterize unique and shared T cell states in the context of multiple sclerosis (MS) and glioblastoma (GBM) using a combination of immunophenotyping and functional approaches, including ultra low-input transcriptional profiling. First we use a series of unbiased approaches to identify functional differences between auto-reactive T cells derived from MS or healthy donors. / We found that MS-derived CCR6⁺ T cells produce pathogenic inflammatory cytokines IFN-[upsilon], IL-17, and GM-CSF, while healthy controls produce IL-10 in response to myelin antigen. We also identified a transcriptional signature that characterizes these cells from MS donors and highlights the role of CTLA-4 signaling in autoreactive T cells derived from healthy donors. Next, we sought to better understand the role of another co-inhibitory receptor, PD-1, specifically in the context of Treg and CD4⁺ T effector function in GBM, a central nervous system cancer. We identify unique signatures of dysfunction in both the CD4⁺ T cell and Treg compartment correlated with PD-1 expression. Finally, adverse development of autoimmunity in the context of treatment with blocking CTLA-4 antibodies suggests that studies directly comparing T cells in the context of autoimmunity and cancer can yield valuable insight into mechanisms of T cell dysfunction. / Through a transcriptional comparison of isolated T cell subsets from the spinal fluid of MS patients, tumors of GBM patients, and matched blood we identify common biological mechanisms of CNS T cells and identify unique signatures of CNS exhaustion. Taken together, our data suggests that the CNS may be enriched for pathogenic cells in the context of both MS and GBM. / by Brittany A. Goods. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references. / The human microbiome is essential for health and has been implicated in many diseases. DNA sequencing has enabled the detailed characterization of these human-associated microbial communities, leading to a rapid expansion in studies investigating the human microbiome. In this thesis, I describe multiple projects which overcome various data analysis challenges to extract useful clinical insights from microbiome data. In the first project, I present an analysis of lung, stomach, and oropharyngeal microbiomes. I leverage data collected from multiple sites per patient to identify aspiration-associated changes in the relationships between these communities, discovering new properties of the aerodigestive microbiome and suggesting new approaches for treatment. In the second project, I perform a meta-analysis of case-control gut microbiome datasets with standard data processing and analysis methods. / I find consistent patterns characterizing disease-associated microbiome changes and a set of shared associations which could inform clinical treatment and therapeutic development approaches for different microbiome-mediated diseases. Enabled by this work, in the third project I contribute to the development of a method to correct for batch effects in case-control microbiome studies. In the fourth project, I describe a framework for rational donor selection in fecal microbiota transplant clinical trials in which knowledge derived from clinical and basic science research is used to inform which donor is selected for fecal transplants, increasing the likelihood of successful trials. Finally, I present preliminary results analyzing the microbiome and metabolome of residential sewage as a novel platform for community-level public health surveillance. / Together, these projects demonstrate a variety of approaches to mine the human microbiome for clinically-relevant insights and suggests multiple avenues forward for translating findings from microbiome data analyses into clinical and public health impact. / by Claire Marie Noëlle Duvallet. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 151-167). / Medulloblastoma (MB) is a chiefly pediatric cancer of the cerebellum that has been studied extensively using genomic, epigenomic, and transcriptomic data. It comprises at least four molecularly distinct subgroups: WNT, SHH, Group 3, and Group 4. Despite the detailed characterization of MB, many disease-driving events remain to be elucidated and therapeutic targets to be nominated. In this thesis, we describe three studies that contribute to a better understanding of this devastating disease: First, we describe a study that aims to fully describe the genomic landscape in the largest medulloblastoma cohort to date, using 491 sequenced MB tumors and 1,256 epigenetically analyzed cases. This work describes subgroup-specific driver alterations including previously unappreciated actionable targets; and, based on epigenetic data, identifies further heterogeneity within Group 3 and Group 4 tumors. Second, we focus on the proteomes and phospho-proteomes of 45 medulloblastoma samples. / We identified distinct pathways associated with two subsets of SHH tumors that showed robustly distinct proteomes, but similar transcriptomes, and found post-translational modifications of MYC that are associated with poor outcomes in Group 3 tumors. We also found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. This study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies. Third, we characterize the metabolomic space of MB on largely the same 45 tumors as used in the proteome-focused study. Here, we present preliminary insights from derived from integrative network and other analyses. We find that MB consensus subgroups are preserved in metabolic space, and that certain classes of metabolites are elevated in MYC-activated MB. / We also show that, similar to other cancers, a previously described gain-of-function mutation in IDH1 may cause elevated 2-hydroxyglutarate levels in MB. The work described in this thesis significantly enhances previous knowledge of medulloblastoma and its subgroups, and provides insights that may aid in the development of medulloblastoma therapies in the near future. / by Tobias Ehrenberger. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 128-137). / Effective cancer treatment of the future requires incorporating diverse and innovative aspects of immunity to fight against cancer, accounting for pharmacokinetic and temporal barriers of therapeutics, and engineering approaches to understand and improve upon current immunotherapies. This thesis addresses these challenges in three projects utilizing the Wittrup Lab's quantitative, engineering approach to protein-based cancer immunotherapy. In the first project, I attempted to harness the potency of allergic reactions against cancer by designing IgE class antibodies against two mouse tumor antigens and comparing them with traditional IgG antibodies. These IgE antibodies elicited modest or no tumor control, and limited efficacy could be due to fast pharmacokinetic clearance, absence of human-like allergic effector cells in mice, or tumor-suppressive effects from mast cells responding to IgE. / The second project described in this thesis focused on synchronizing combination immunotherapies with the temporal progression of the anti-cancer immune response. In this work, anti-tumor antibodies were combined with the cytokines interleukin 2 (IL2) and interferon alpha (IFNa). The order of administration of these therapies decoupled strong efficacy from dose-limiting toxicity in two tumor models. Given before IFN[alpha], IL2 activated natural killer cells and heightened their responsiveness to subsequent IFN[alpha], which was ultimately toxic and unnecessary for therapeutic efficacy. This project's proof of concept that efficacy and toxicity could be unlinked in immunotherapy began to establish a framework to use for rational combination therapy treatment schedule design, with the goal of treating with each agent when that piece of the immune system is active. / Finally, the third project used the Wittrup Lab's system of yeast surface display to engineer novel antibodies against the checkpoint blockade target cytotoxic T lymphocyte associated protein 4 (CTLA-4) as tools to improve understanding of the anti-CTLA-4 mechanism of action against cancer. Although the first wave of antibodies made had favorable characteristics against CTLA-4 as a soluble target, they bound a CTLA-4 epitope too close to the cell surface and so could not be used for therapeutic studies. Next generation sequencing on the yeast libraries identified alternative CTLA-4 binding antibody sequences, and these will be tested in future mechanistic and therapeutic studies. / by Adrienne Marie Rothschilds. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 153-173). / The field of synthetic biology aims to control cellular behavior using programmable gene circuits. Generally these gene circuits sense molecular biomarkers, process these inputs and execute a desired calculated response. This is especially relevant for gene and cell therapies where integrating multiple disease-related inputs and/or sophisticated control could lead to safer and more effective approaches. While mammalian synthetic biology has made great progress, few gene circuit-based therapies have entered the clinic. Regulatory issues aside, this lag may be due to several technical impediments. First, the computing part of circuits is often accomplished via transcriptional regulation, which presents challenges as we move toward the clinic. Second, the field relies on a limited set of sensors; the detection of other types of disease biomarkers will help robustly identify cell state. / Finally, the design cycle currently used to develop gene circuits is laborious and slow, which is not suitable for clinical development, especially applications in personalized medicine. In this thesis I describe how I address these three limitations. I develop a new posttranscriptional regulation platform based on RNA cleavage that I term "PERSIST" (Programmable Endonucleolytic RNA Scission-Induced Stability Tuning). CRISPR-specific endonucleases are adapted as RNA-level regulators for the platform and we demonstrate several genetic devices including cascades, feedback, logic functions and a bistable switch. I explore sensor designs for relevant biomolecules including mRNAs, miRNAs and proteins via the PERSIST and other platforms. Finally, I present a "poly-transfection" method, associated advanced data analysis pipelines, and computational models that make circuit engineering faster and more predictive. / Taken together, the expanded RNA toolkit that the PERSIST platform offers as well as advancements in sensing and circuit design will enable the more straightforward creation of robust gene circuits for gene and cell therapies. / by Breanna Elizabeth DiAndreth. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 190-200). / The transcription factor Myc is a basic helix-loop-helix leucine zipper (bHLHLZ) protein with crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Myc transcriptional activity is dependent on dimerization with its obligate partner Max, another bHLHLZ transcription factor. Max also forms homodimers as well as heterodimers with other proteins including the Mxd family of proteins, creating a dynamic network of protein-protein interactions to regulate transcriptional programs. Despite the significance of this network, the arsenal of chemical probes to interrogate these proteins in cancer biology remains limited. Here, we utilized small molecule microarrays and luciferase-based reporter assays to identify compounds that bind Max and modulate Myc transcriptional activity. We discovered the small molecule KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in mouse models of Myc-driven cancers. In a cancer cell line model treated with KI-MS2-008, the equilibrium of protein-protein interactions shifts toward a transcriptionally repressed state over time by recruiting Mxd4 and other repressive machinery to Max. This study establishes that perturbing Max dimerization with small molecules is a tractable approach to targeting Myc activity in cancer. / by Andrew Chen. / Ph. D. / Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering