Browsing by Subject "antibody"

Now showing 1 - 5 of 5
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    Antibody Conjugation and Formulation
    (Oxford, 2019) Alves, Nathan J.; Emergency Medicine, School of Medicine
    In an era where ultra-high antibody concentrations, high viscosities, low volumes, auto-injectors, and long storage requirements are already complex problems with the current unconjugated monoclonal antibodies on the market the formulation demands for antibody-drug conjugates (ADCs) are significant. Antibodies have historically been administered at relatively low concentrations through intravenous (IV) infusion due to their large size and the inability to formulate for oral delivery. Due to the high demands associated with IV infusion and the development of novel antibody targets and unique antibody conjugates more accessible routes of administration such as intramuscular (IM), and subcutaneous (SC) are being explored. This review will summarize various site-specific and non-site-specific antibody conjugation techniques in the context of antibody-drug conjugates (ADCs) and the demands of formulation for high concentration clinical implementation.
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    Developing Novel Methods to Identify RNA-Associated Mechanisms for Inheritance
    (2020-11) Ettaki, Zacharia Nabil; Aoki, Scott T.; Georgiadis, Millie; Quilliam, Lawrence
    Animals depend on inheriting non-genetic information early in life to grow and develop naturally. This inherited, non-genetic information was previously thought to be limited to DNA modifications and DNA binding proteins. But recent studies have expanded our understanding of inheritance to include RNA and RNA binding proteins. We currently lack methods to identify and enrich for RNA binding proteins that might be involved in providing non-genetic information from mother to daughter cells. Others have developed a method using modified enzyme tags to pulse-label proteins with small molecule fluorescent ligands and follow these proteins as they are inherited by cells. Here I characterized and tested the application of a fluorescent small molecule targeting antibody to enrich for these labeled proteins. I first tested the ability of this antibody to bind to fluorescent ligand-labeled enzymes. I determined that the antibody can efficiently bind to at least one of the labeled enzymes. Second, I determined crystallization conditions for the ligand binding antibody fragment. This thesis sets the stage for structure determination and to test whether this antibody can work in vivo to enrich for RNA binding proteins involved in the delivery of non-genetic information to cells.
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    Glycosylphosphatidylinositol Anchor Deficiency Attenuates the Production of Infectious HIV-1 and Renders Virions Sensitive to Complement Attack
    (Mary Ann Liebert, 2016-11-01) Amet, Tohti; Lan, Jie; Shepherd, Nicole; Yang, Kai; Byrd, Daniel; Xing, Yanyan; Yu, Qigui; Microbiology and Immunology, School of Medicine
    Human immunodeficiency virus type 1 (HIV-1) escapes complement-mediated lysis (CML) by incorporating host regulators of complement activation (RCA) into its envelope. CD59, a key member of RCA, is incorporated into HIV-1 virions at levels that protect against CML. Since CD59 is a glycosylphosphatidylinositol-anchored protein (GPI-AP), we used GPI anchor–deficient Jurkat cells (Jurkat-7) that express intracellular CD59, but not surface CD59, to study the molecular mechanisms underlying CD59 incorporation into HIV-1 virions and the role of host proteins in virus replication. Compared to Jurkat cells, Jurkat-7 cells were less supportive to HIV-1 replication and more sensitive to CML. Jurkat-7 cells exhibited similar capacities of HIV-1 binding and entry to Jurkat cells, but were less supportive to viral RNA and DNA biosynthesis as infected Jurkat-7 cells produced reduced amounts of HIV-1 RNA and DNA. HIV-1 virions produced from Jurkat-7 cells were CD59 negative, suggesting that viral particles acquire CD59, and probably other host proteins, from the cell membrane rather than intracellular compartments. As a result, CD59-negative virions were sensitive to CML. Strikingly, these virions exhibited reduced activity of virus binding and were less infectious, implicating that GPI-APs may be also important in ensuring the integrity of HIV-1 particles. Transient expression of the PIG-A gene restored CD59 expression on the surface of Jurkat-7 cells. After HIV-1 infection, the restored CD59 was colocalized with viral envelope glycoprotein gp120/gp41 within lipid rafts, which is identical to that on infected Jurkat cells. Thus, HIV-1 virions acquire RCA from the cell surface, likely lipid rafts, to escape CML and ensure viral infectivity.
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    Immunoglobulin-Based Investigation of Spontaneous Resolution of Chlamydia trachomatis Infection
    (Oxford, 2017-06) Bakshi, Rakesh; Gupta, Kanupriya; Jordan, Stephen J.; Brown, Ladraka' T.; Press, Christen G.; Gorwitz, Rachel J.; Papp, John R.; Morrison, Sandra G.; Lee, Jeannette Y.; Morrison, Richard P.; Geisler, William M.; Medicine, School of Medicine
    Chlamydia trachomatis elementary body enzyme-linked immunosorbent assay (ELISA) was used to investigate serum anti-CT immunoglobulin G1 (IgG1; long-lived response) and immunoglobulin G3 (IgG3; short-lived response indicating more recent infection) from treatment (enrollment) and 6-month follow-up visits in 77 women previously classified as having spontaneous resolution of chlamydia. Of these women, 71.4% were IgG1+IgG3+, consistent with more recent chlamydia resolution. 15.6% were IgG3− at both visits, suggesting absence of recent chlamydia. Using elementary body ELISA, we demonstrated approximately 1 in 6 women classified as having spontaneous resolution of chlamydia might have been exposed to C. trachomatis but not infected. Further, we classified their possible infection stage.
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    A Novel Strategy for the Development of Vaccines for SARS-CoV-2 (COVID-19) and Other Viruses Using AI and Viral Shell Disorder
    (ACS, 2020-11-06) Goh, Gerard Kian-Meng; Dunker, A. Keith; Foster, James A.; Uversky, Vladimir N.; Biochemistry and Molecular Biology, School of Medicine
    A model that predicts levels of coronavirus (CoV) respiratory and fecal–oral transmission potentials based on the shell disorder has been built using neural network (artificial intelligence, AI) analysis of the percentage of disorder (PID) in the nucleocapsid, N, and membrane, M, proteins of the inner and outer viral shells, respectively. Using primarily the PID of N, SARS-CoV-2 is grouped as having intermediate levels of both respiratory and fecal–oral transmission potentials. Related studies, using similar methodologies, have found strong positive correlations between virulence and inner shell disorder among numerous viruses, including Nipah, Ebola, and Dengue viruses. There is some evidence that this is also true for SARS-CoV-2 and SARS-CoV, which have N PIDs of 48% and 50%, and case-fatality rates of 0.5–5% and 10.9%, respectively. The underlying relationship between virulence and respiratory potentials has to do with the viral loads of vital organs and body fluids, respectively. Viruses can spread by respiratory means only if the viral loads in saliva and mucus exceed certain minima. Similarly, a patient is likelier to die when the viral load overwhelms vital organs. Greater disorder in inner shell proteins has been known to play important roles in the rapid replication of viruses by enhancing the efficiency pertaining to protein–protein/DNA/RNA/lipid bindings. This paper suggests a novel strategy in attenuating viruses involving comparison of disorder patterns of inner shells (N) of related viruses to identify residues and regions that could be ideal for mutation. The M protein of SARS-CoV-2 has one of the lowest M PID values (6%) in its family, and therefore, this virus has one of the hardest outer shells, which makes it resistant to antimicrobial enzymes in body fluid. While this is likely responsible for its greater contagiousness, the risks of creating an attenuated virus with a more disordered M are discussed.