While chimeric antigen T (CAR) T cell therapy has shown remarkable success in leukemia, lymphoma, and multiple myeloma, its effectiveness in solid tumors including glioblastoma (GBM) remains limited. It is crucial to understand mechanisms that reduce the efficacy of CAR T cell therapies and develop strategies to prevent tumor resistance. In this study, we conjectured that alterations in tumor cell-intrinsic interferon (IFN) signaling pathways contribute to establishment of immunosuppressive tumor microenvironment in solid tumors, leading to resistance of solid tumor cells to CAR T cell-mediated killing. We established syngeneic IFN signaling-deficient tumor models for murine IL-13Ra2 targeted CAR T cell therapy and showed that these models modulate the tumor microenvironment (TME), leading to resistance to CAR T cell therapy. We identified variations in gene expression associated with IFN signaling components and cytokines between IFN signaling-deficient tumor cells and wild type (WT) tumor cells after CAR T cell treatment. Furthermore, single-cell RNA sequencing and mass cytometry analysis of the tumor immune cell infiltrates in IFN-signaling deficient tumors compared to WT controls identified the immune-mediated causal components for the resistance of Janus Kinase1 knockout (JAK1/KO) tumors to CAR T cell therapy. CAR T cell-treated IFN signaling-deficient tumors presented decreased T-cell transcripts, with decreased frequency of CD8-early active, CD8-naive like T cells. Conversely, there were more regulatory and follicular T cells, exhausted endogenous T cells , and exhausted CAR T cells in treated IFN signaling-deficient tumors compared to treated WT tumors. The analyses also showed the superior enrichment and crosstalk of genes that identified fibroblasts, neutrophils, and myeloid cells in IFN signaling-deficient tumors compared to those of WT tumors. Mass cytometry analysis on the immune cells infiltrates of JAK1/KO and WT tumors post CAR T cell treatment corroborated the results from gene expression analysis. The potential cause of immune suppressive crosstalk in IFN signaling-deficient tumor niches could be attributed to the varied enhancement of receptor-ligand interactions such as SPP1+ tumor-associated macrophages (TAMs) and CD44+ cancer-associated fibroblasts (CAFs), as well as SPP1+ TAMs and integrins present on other cell lineages. To overcome resistance to CAR T cell therapies, we employed two distinct actionable approaches: triggering the immune microenvironment and disrupting the extracellular matrix. Unconjugated interferon signaling gene-15 (ISG-15) enhanced CAR T cell efficacy in an INF-signaling deficient model, increasing the recruitment of endogenous T cells and reshaping the TME. Anti-SPP1 blocking antibody was used to prime the JAK1/KO tumors prior to the treatment with CAR T cell therapy potentially via enhancing the persistence and trafficking of CAR T cells in the TME.
\r\n\r\nWe next identified immune signatures of 32 GBM patients who had progressive disease after CAR T cell treatment compared to those who had relatively stable disease or showed improvement. We identified the presence of fibroblasts and SPP1+ APOE+ C1QA+ C1QC+ myeloid cells in GBM signatures that are associated with immune suppression and resistance to therapy. Patients with GBM who exhibited a relatively stable response to treatment and increased T cell recruitment had differential expression of interferon regulatory factors (IRFs) and ISGs compared to patients with less response to the treatment. Our findings uncover a correlation between tumor-intrinsic driver mutations, the composition of the TME, and the responsiveness of solid tumors to CAR T cell therapy, providing insights into potential approaches to address resistance in IFN non-responsive tumors.
", "doi": "10.7907/jf79-pv58", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14413", "collection": "thesis", "collection_id": "14413", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10302021-184745797", "primary_object_url": { "basename": "Magnus_Hoffmann_2021_Thesis_final version_No EBR.pdf", "content": "final", "filesize": 16843967, "license": "other", "mime_type": "application/pdf", "url": "/14413/4/Magnus_Hoffmann_2021_Thesis_final version_No EBR.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Nanoparticle Technologies to Cure and Prevent Infectious Diseases", "author": [ { "family_name": "Hoffmann", "given_name": "Magnus Adrian Gero", "orcid": "0000-0003-4923-9568", "clpid": "Hoffmann-Magnus-Adrian-Gero" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_bbe" } ], "abstract": "Despite almost 40 years of intensive research, there is still no curative treatment for HIV-1/AIDS. Anti-retroviral therapy (ART) prolongs the life expectancy of HIV-1-infected individuals but is associated with side effects, and multiple drugs need to be given in combination to prevent the development of viral resistance. In addition, treatment must continue for the lifetime of the individual due to the existence of a long-lived latent proviral reservoir. While a \"sterilizing\" cure remains difficult to achieve due to difficulties associated with identifying and clearing latently-infected cells, recent research has focused on designing a \"functional\" cure, i.e., a therapeutic strategy that enables long-term suppression of HIV-1 replication and remission of symptoms in the absence of ART. The work presented here describes a new therapeutic direction for the development of a functional cure against HIV-1. This approach is based on the hypothesis that HIV-1 is unable to escape from a nanoparticle (NP)-based decoy that presents clusters of the HIV-1 receptor CD4, because CD4-NPs mimic viral target cells more accurately than soluble CD4-based inhibitors and permit high-avidity interactions with trimeric HIV-1 Env proteins. We demonstrate that CD4-NPs are >10,000-fold more potent than soluble CD4 (sCD4) and prevent viral escape in vitro. AAV-mediated delivery of self-assembling CD4-NPs produced stable CD4-NP serum concentrations in mice that were almost 1,000-fold higher than concentrations required to neutralize HIV-1 in vitro, suggesting that these concentrations could be therapeutic. Viral challenge studies in non-human primates are underway to evaluate the potential of this therapeutic strategy.
\r\n\r\nAs an alternative approach to generate decoys against HIV-1, we generated engineered red blood cells (RBCs) that expressed viral receptors and potently inhibited HIV-1 infection of target cells in vitro. Because RBCs do not contain nuclei or functional organelles required for protein translation, infection of engineered RBCs represents a dead-end for a lentivirus such as HIV-1, which must integrate into the host cell genome as part of its lifecycle. We generated stable erythroid progenitor cell lines that continuously produced HIV-1 receptor-expressing RBCs that could be administered to HIV-1-infected individuals. As RBCs vastly outnumber CD4+ T-cells, HIV-1\u2019s main target cells, and have extended lifetimes, only a fraction of an individual\u2019s RBCs would need to be replaced with the engineered RBC viral traps in order to suppress HIV-1 infection in vivo.
\r\n\r\nMy work on CD4-NP therapeutics against HIV-1 also led to the invention and development of the EBR NP technology that is ideally suited for vaccine design applications. This technology can be used to modify any type of membrane protein to self-assemble into enveloped virus-like NPs without the need for additional proteins. EBR NP assembly is induced by inserting a short amino acid sequence into the cytoplasmic tail of the membrane protein, which was designed to recruit host proteins from the endosomal sorting complex required for transport (ESCRT) pathway. We applied this technology to design protein NP-based vaccines against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which elicited potent serum neutralizing antibody responses in mice. The EBR NP technology is also ideally suited for the development of hybrid vaccine approaches that allow genetic encoding of protein-based NPs, thereby combining attributes of mRNA and protein-based NP vaccines. Pilot studies demonstrated that mRNA and DNA vaccines encoding the self-assembling SARS-CoV-2 spike-EBR construct elicited ~10-fold higher neutralizing antibody responses than mRNA and DNA vaccines encoding the unmodified spike protein. This hybrid approach has the potential to substantially enhance the potency of mRNA vaccines and could become a leading vaccine platform technology. Future applications for the EBR NP technology are discussed, including the development of a universal coronavirus vaccine to prevent future pandemics, and engineering EBR NPs to mRNA vaccines or therapeutic cargoes for efficient and targeted delivery.
", "doi": "10.7907/g0w1-rc77", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14565", "collection": "thesis", "collection_id": "14565", "cite_using_url": "https://resolver.caltech.edu/CaltechThesis:04282022-192922044", "type": "thesis", "title": "Targeting Fusion Proteins of HIV-1 and SARS-CoV-2", "author": [ { "family_name": "Jette", "given_name": "Claudia Angela", "orcid": "0000-0002-5085-8027", "clpid": "Jette-Claudia-Angela" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_chem" } ], "abstract": "Viruses are disease-causing pathogenic agents that require host cells to replicate. Fusion of host and viral membranes is critical for the lifecycle of enveloped viruses. Studying viral fusion proteins can allow us to better understand how they shape immune responses and inform the design of therapeutics such as drugs, monoclonal antibodies, and vaccines. This thesis discusses two approaches to targeting two fusion proteins: Env from HIV-1 and S from SARS-CoV-2. The first chapter of this thesis is an introduction to viruses with a specific focus on HIV-1 CD4 mimetic drugs and antibodies against SARS-CoV-2. It discusses the architecture of these viruses and fusion proteins and how small molecules, peptides, and antibodies can target these proteins successfully to treat and prevent disease. In addition, a brief overview is included of the techniques involved in structural biology and how it has informed the study of viruses. For the interested reader, chapter 2 contains a review article that serves as a more in-depth introduction for both viruses as well as how the use of structural biology has informed the study of viral surface proteins and neutralizing antibody responses to them. The subsequent chapters provide a body of work divided into two parts. The first part in chapter 3 involves a study on conformational changes induced in the HIV-1 Env protein by CD4-mimemtic drugs using single particle cryo-EM. The second part encompassing chapters 4 and 5 includes two studies on antibodies isolated from convalescent COVID-19 donors. The former involves classification of antibody responses to the SARS-CoV-2 S receptor-binding domain (RBD). The latter discusses an anti-RBD antibody class that binds to a conserved epitope on the RBD and shows cross-binding and cross-neutralization to other coronaviruses in the sarbecovirus subgenus.
", "doi": "10.7907/pxa2-dy41", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14073", "collection": "thesis", "collection_id": "14073", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02032021-180657616", "type": "thesis", "title": "Diverse Roles of RNA-protein Interactions: From Viral Antagonism to Mammalian Development", "author": [ { "family_name": "Banerjee", "given_name": "Abhik Kumar", "orcid": "0000-0002-9797-0104", "clpid": "Banerjee-Abhik-Kumar" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_bbe" } ], "abstract": "RNA is a widely utilized and integrated component of core cellular function because of its abilities to recognize and hybridize to nucleic acid templates, spatially localize to different compartments within the cell, bind combinatorially to effector molecules, and in some cases directly catalyze chemical reactions. In this thesis, I describe three cases, illustrating the biomolecule\u2019s unique importance in several different aspects of cellular homeostasis. Chapter 1 provides historical context for studying RNA-protein interactions within RNA biology and Virology. Chapter 2 details experiments in which we explored RNA as a central target of host cell takeover by SARS-CoV-2. In the process, we highlight the importance of RNA in many integral complexes within the cell, including components of the spliceosome, the eukaryotic ribosome, and signal recognition particle. Chapter 3 presents data from our consideration of RNA within the context of cis gene regulation. We specifically focus on a model RNA-binding protein, SMRT/HDAC1 Associated Repressor Protein (SHARP), and the paternally imprinted long non-coding RNA, Kcnq1ot1, as case studies. Chapter 4 describes our dissection of a transcriptional circuit involving SHARP and discusses implications of RNA-binding to developmentally sensitive circuits and processes. Finally, Chapter 5 poses new questions raised by these studies. Together these data emphasize the diverse and unique role RNA plays in cellular homeostasis and suggest additional roles in nuclear compartment stabilization and crosstalk.
", "doi": "10.7907/tfb9-n887", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14098", "collection": "thesis", "collection_id": "14098", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03032021-224823348", "primary_object_url": { "basename": "Caltech-Thesis-Mohamad-Final.pdf", "content": "final", "filesize": 7550092, "license": "other", "mime_type": "application/pdf", "url": "/14098/1/Caltech-Thesis-Mohamad-Final.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Thermal Bioswitches for Non-Invasive Control of Cellular Therapies", "author": [ { "family_name": "Abedi", "given_name": "Mohamad", "orcid": "0000-0001-9717-6288", "clpid": "Abedi-Mohamad" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Temperature is a unique input signal that could be used by engineered therapeutic cells to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, I present here a new class of thermal bioswitches that enables thermal control over bacterial and mammalian cells. For bacterial applications, we developed two new families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32\u201346 \u00b0C. We integrated these molecular bioswitches into thermal logic circuits and demonstrated their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications.
\r\n\r\nTo apply this technology in a relevant clinical scenario, we sought to engineer microbial immunotherapies that can be thermally controlled with focused ultrasound. This technology was enabled by rapid advances in synthetic biology that are driving the development of genetically modified microbes as therapeutic agents for a multitude of human diseases, including cancer. In particular, the reduced immune surveillance within the core of some solid tumors creates an ideal environment for microbes to engraft and release therapeutic payloads. However, these therapeutic payloads could be harmful if released in healthy tissues where microbes tend to also engraft in smaller numbers. As described in Chapter 2, my colleagues and I introduced a temperature-actuated state switch that enables tight spatiotemporal control over the activity of therapeutic microbes when combined with focused ultrasound hyperthermia. Through a combination of rational design and high throughput screening, we optimized the behavior of this switch to minimize leakage and maximize inducibility. When tested in a clinically relevant in vivo model, engineered microbes, successfully switched states, and induced a marked suppression of tumor growth upon focal activation. This bioswitch provides a critical tool to attain selective and sustained activity of therapeutic microbes in vivo.
\r\n\r\nEncouraged by the successful development of thermally actuated circuits in microbes, we aimed to establish equivalent technologies for thermal control of human T cells. Genetically engineered T cells are actively being developed to perform a variety of therapeutic functions with great clinical promise. However, no robust mechanisms exist to externally control the activity of T cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. In Chapter 4, my colleagues and I tested the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T cells in the well-tolerated temperature range of 37\u221242 \u00b0C, and we introduced genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrated the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. Overall, the technologies developed here provide critical tools to direct control therapeutic cells after they have been deployed deep inside the body.
", "doi": "10.7907/z7ac-2g66", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:11693", "collection": "thesis", "collection_id": "11693", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-181520170", "primary_object_url": { "basename": "PhD_Thesis_Ramesh_Pradeep_Final.pdf", "content": "final", "filesize": 4837595, "license": "other", "mime_type": "application/pdf", "url": "/11693/1/PhD_Thesis_Ramesh_Pradeep_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Imaging and Control of Engineered Cells using Magnetic Fields", "author": [ { "family_name": "Ramesh", "given_name": "Pradeep", "orcid": "0000-0001-6243-8145", "clpid": "Ramesh-Pradeep" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Making cells magnetic is a long-standing goal of synthetic biology, aiming to enable the separation of cells from complex biological samples and their non-invasive visualization in vivo using Magnetic Resonance Imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have largely been unsuccessful due to the stringent required chemical conditions. In this thesis, we introduce an alternative approach to making cells magnetic, focusing on biochemically maximizing cellular paramagnetism. Here, we show that a novel genetic construct combining the functions of ferroxidation and iron-chelation enables engineered bacteria to accumulate iron in 'ultraparamagnetic' macromolecular complexes, which subsequently allows for these cells to be trapped using strong magnetic field gradients and imaged using MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, an array of spectroscopic techniques, biochemical assays, and computational modeling to elucidate the unique mechanisms and implications of this 'ultraparamagnetic' concept.
\r\n\r\nIn addition to making cells magnetic, remote control of cellular localization in deep tissue is another long-standing goal of synthetic biology. Such an ability to non-invasively direct cells to sites of interest will not only improve therapeutic outcomes by minimizing off-target activity, but more broadly enable new research on complex cellular communities, such as the gut microbiome, in living animals. Given their deep penetrance through tissues, magnetic fields are ideally suited for facilitating non-invasive targeting of cells; however, the rapid decay of magnetic flux density from its source currently limits the depths to which magnetic targeting can be employed to within 1-2 mm from the surface. Here, we demonstrate a new approach wherein the retention of orally-administered and synthetically magnetized cell-like-particles is selectively enhanced within the murine intestinal tract to depths of up to 13 mm from the surface. Our cellular localization assisted by magnetic particles (CLAMP) strategy can potentially be generalized to any cell (bacterial, mammalian) or drug-containing nanoparticle of interest, and can be combined with existing non-invasive imaging modalities thereby facilitating remote environmental sensing at sites of interest.
\r\n \r\nFinally, while magnetic fields in MRI scanners are widely used today to safely and non-invasively image anatomical structures in living animals, much of the image contrast in MRI is the result of microscale magnetic-field variations in tissues. However, the connection between these microscopic patterns and the appearance of macroscopic MR images has not been the subject of direct experimental studies due to a lack of methods to map microscopic fields in biological samples under ambient conditions. Here, we optically probed magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy (NV) diamond magnetometry and combined these measurements with simulations of nuclear-spin precession to predict the corresponding MRI contrast. Additionally, we demonstrate the broad utility of this technology for imaging an in vitro model of cellular iron uptake, as well as imaging histological samples from a mouse model of hepatic iron overload. Taken together, our approach bridges a fundamental intellectual gap between a macroscopic MRI voxel and its microscopic constituents.
\r\n", "doi": "10.7907/KY00-7Y74", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:9864", "collection": "thesis", "collection_id": "9864", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082016-141626444", "primary_object_url": { "basename": "Galimidi Thesis.pdf", "content": "final", "filesize": 70011604, "license": "other", "mime_type": "application/pdf", "url": "/9864/1/Galimidi Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Combating HIV with Novel Antibody Architectures ", "author": [ { "family_name": "Galimidi", "given_name": "Rachel P.", "clpid": "Galimidi-Rachel-P" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Zack", "given_name": "Jerome", "clpid": "Zack-J-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "More than 30 years has passed since the discovery of Human Immunodeficiency Virus (HIV) yet it remains one of the most important current threats to global public health. HIV is a T-lymphotrophic retrovirus that is the causative agent of Acquired Immune Deficiency Syndrome, and despite decades of research, there remains no cure. Vaccines are most effective when they are able to induce broadly neutralizing antibodies at concentrations capable of blocking viral infection. Notwithstanding all of the effort, a successful vaccine that is capable of inducing complete protection from the immune system has yet to be found. In this thesis, the first chapter provides a history of the discovery of HIV, the origins of the virus, description of the HIV genome, focusing primarily on the envelope glycoprotein, a trimeric spike on the surface of the HIV virion necessary for viral fusion and the sole epitope for broadly neutralizing antibodies. Lastly, the first chapter reviews an overview of the antiviral immune response specifically the role of humoral immune branch and broadly neutralizing antibodies, as well as their limitations in protection against HIV. Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. In addition to structural features of HIV\u2019s envelope spike that facilitate antibody evasion, we proposed that the low-density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The work described in subsequent chapters attempts to overcome HIV\u2019s evasion strategy of low spike density through the design of novel antibody architectures.
\r\n\t\r\nWe postulated that anti-HIV-1 spike antibodies primarily bind monovalently because HIV\u2019s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike trimer structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and neutralization potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a \u201cmolecular ruler\u201d to measure intra-epitope distances on virion-bound spikes and to construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude of increased potency (>100-fold average increases across a virus panel) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection. These results shed light on dynamic spike conformations and are relevant to therapeutic interventions.
\r\n", "doi": "10.7907/Z9QC01FR", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8878", "collection": "thesis", "collection_id": "8878", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212015-194407438", "primary_object_url": { "basename": "Wu_Yunji_2015_thesis.pdf", "content": "final", "filesize": 64667179, "license": "other", "mime_type": "application/pdf", "url": "/8878/1/Wu_Yunji_2015_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein", "author": [ { "family_name": "Wu", "given_name": "Yunji", "clpid": "Wu-Yunji" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design. ", "doi": "10.7907/Z98050K6", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8857", "collection": "thesis", "collection_id": "8857", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112015-230250866", "primary_object_url": { "basename": "AWebster_PhDThesis_2015.pdf", "content": "final", "filesize": 127970725, "license": "other", "mime_type": "application/pdf", "url": "/8857/1/AWebster_PhDThesis_2015.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells", "author": [ { "family_name": "Webster", "given_name": "Alexandre", "orcid": "0000-0002-1416-5872", "clpid": "Webster-Alexandre" } ], "thesis_advisor": [ { "family_name": "Aravin", "given_name": "Alexei A.", "orcid": "0000-0002-6956-8257", "clpid": "Aravin-A-A" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Aravin", "given_name": "Alexei A.", "orcid": "0000-0002-6956-8257", "clpid": "Aravin-A-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of \u2018selfish\u2019 genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins\u2014Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)\u2014which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.
\r\n\r\nThe work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially \u2018cross-talk\u2019 with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.
", "doi": "10.7907/Z9WQ01RS", "publication_date": "2015-04-28", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8501", "collection": "thesis", "collection_id": "8501", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062014-175608943", "primary_object_url": { "basename": "SamyHamdoucheThesis2014.pdf", "content": "final", "filesize": 9265737, "license": "other", "mime_type": "application/pdf", "url": "/8501/1/SamyHamdoucheThesis2014.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineered Antibody and Monobody Domains with T Cell Receptor-Like Selectivity for Tumor Associated Peptide-MHC Antigens", "author": [ { "family_name": "Hamdouche", "given_name": "Samy", "clpid": "Hamdouche-Samy" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Miller", "given_name": "Thomas F.", "orcid": "0000-0002-1882-5380", "clpid": "Miller-T-F" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Monoclonal antibody (mAb)-based therapeutics have established themselves as meaningful components of the treatment paradigm for a variety of tumors. However, since the approval of rituximab in 1997 as the first mAb-based therapy for cancer, there has been a paucity of novel, validated cancer targets for therapeutic intervention by mAbs. In effect, numerous challenges lie in the discovery of suitable extracellular or transmembrane antigens that permit the differentiation of tumor from healthy tissue. The adaptive immune system, though, mediates recognition of foreign antigens derived from the intracellular proteome by T cell receptor (TCR) binding to peptide-loaded major histocompatibility complex (pMHC) molecules. Because cancer is associated with large-scale alterations in the genome, there are a vast number of novel epitopes presented to the adaptive immune system. Although natural TCRs have exquisite functionality in distinguishing these foreign epitopes, and several tumor-reactive TCRs have, in fact, been characterized, the molecules themselves are poorly developable as therapeutic candidates. Thus, in order to enable TCR-like binding of a broader class of protein agents, this study explores the transfer of TCR binding domains to other mAb-based scaffolds, including the fibronectin-derived Fn3 and the IgG-derived 4D5 scaffolds. By using a combination of rational design and directed evolution to guide binding domain transfer, evidence for TCR-like binding was demonstrated for several engineered molecules. In addition to conferring binding functionality, the grafted TCR domains had a deleterious effect on the biophysical properties of these inherently robust protein scaffolds. Thus, this work provides novel insight into the objective of developing mAb-based agents with TCR-like binding specificity for pMHC antigens, informing future efforts to target the abundance of intracellular tumor epitopes.", "doi": "10.7907/Z9WH2N02", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8504", "collection": "thesis", "collection_id": "8504", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072014-140700155", "type": "thesis", "title": "Biophysics of V(D)J Recombination and Genome Packaging: In Singulo Studies on RAG, HMGB1, and TFAM", "author": [ { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-Geoffrey-A" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.\r\n", "doi": "10.7907/Z9W9573H", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:6447", "collection": "thesis", "collection_id": "6447", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252011-233927917", "primary_object_url": { "basename": "Tadmor-thesis_complete.pdf", "content": "final", "filesize": 19775295, "license": "other", "mime_type": "application/pdf", "url": "/6447/55/Tadmor-thesis_complete.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Phage-Host Interaction in Nature", "author": [ { "family_name": "Tadmor", "given_name": "Arbel David", "clpid": "Tadmor-Arbel-David" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Though viruses may be the most abundant biological entities on the planet, very little is known about phage-host interaction in the wild due to the absence of proper experimental tools. In the present work we report of a method to pair environmental phages with their bacterial hosts at the single-cell level without having to culture either host or virus. The method utilizes microfluidic digital PCR in conjunction with a metagenome data mining tool that was developed to find a viral marker gene in an unknown environment. We implemented this technique on the microbial community residing in the hindgut of termites. Consequently, we discovered genus-wide infection patterns displaying remarkable intra-genus selectivity, with viral alleles displaying limited lateral gene transfer and/or host switching despite host proximity. To try and explain phage-host interactions from a theoretical perspective, we formulated a simple biophysical model describing the interaction of bacteria and viruses in aqueous environments. We predict that the radius r of a bacterium is the most critical parameter determining its fixed point concentration, which scales as r-4. Given the hypothesis that there is no selection pressure on bacterial radii, our model predicts that the size spectrum of marine bacteria follows a power law with slope -1, close to the observed average spectrum. Moreover, given the total concentration of bacteria in the ocean, our model enables us to estimate the total number of bacterial \u201cspecies\u201d per volume of water providing a lower and upper bound on the total number of species in the oceans. To elucidate the concept of a \u201cspecies\u201d, we consider a bacterial-viral co-speciation model, which is consistent with the observed narrow host range of phages. Our model hints that the bacterial-viral \u201carms race\u201d may be a critical component in the process of co-speciation. We suggest further experiments to test both models. Finally, we consider a recent high resolution measurement of the force as a function of time generated by stress fibers within a single fibroblast cell and suggest a stochastic model that is capable of accounting for the observed data.", "doi": "10.7907/68Q5-D532", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:2453", "collection": "thesis", "collection_id": "2453", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06042009-131723", "primary_object_url": { "basename": "11Complete.pdf", "content": "final", "filesize": 115499712, "license": "other", "mime_type": "application/pdf", "url": "/2453/12/11Complete.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigations in the Design and Characterization of HIV-1 Neutralizing Molecules", "author": [ { "family_name": "Klein", "given_name": "Joshua Simon", "clpid": "Klein-Joshua-Simon" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Human Immunodeficiency Virus (HIV) is a T-lymphotrophic retrovirus that is the causative agent of Acquired Immunodeficiency Syndrome and is estimated to currently infect approximately 40 million people worldwide. Life-extending therapies are credited for the precipitous drop in HIV-related mortality in developed countries, but their high costs prevent widespread distribution in developing countries. To date, all attempts to produce a vaccine capable of preventing or controlling an HIV infection have failed, but a comprehensive explanation for these failures has yet to emerge from the available data. In this thesis the first chapter provides an overview of the pandemic, the antigenic properties of gp120 and gp41, which are the two glycoproteins that comprise the outer envelope spike of the virus, and the broadly neutralizing antibodies that have been isolated against them. The second and third chapters discuss biophysical characterizations of these monoclonal antibodies and newly designed molecules derived from them. Based on a comparison of these data with pre-existing research, a novel hypothesis called the \"island effect\" was developed and is presented as a possible explanation for the consistent failure of the human immune system to respond to infection or vaccination with an effective humoral response. The final chapter summarizes ongoing investigations in the capacities of broadly neutralizing monoclonal antibodies to recruit antibody-dependent cellular cytotoxicity, a mechanism by which antibodies can trigger the lysis of HIV-infected cells by the innate immune system. ", "doi": "10.7907/QPEC-8Y25", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" } ]