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Synthesis and Evaluation of Fc-Bifunctional Peptide Inhibitors for Different Disease States of Multiple Sclerosis
Mahadik, Rucha
Mahadik, Rucha
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Abstract
Multiple Sclerosis (MS) is a neurodegenerative autoimmune disease in which immune cells target and attack the myelin sheath, the fatty tissue surrounding and insulating neurons. Finding an effective treatment for MS has been particularly challenging because there are multiple disease states, each having varied onset mechanisms and requiring their own specific treatment. Despite their variation in disease onset, it is accepted that the start of the disease begins with the activation of autoreactive T cells against myelin sheath proteins, specifically proteolipid protein (PLP), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Most treatments for MS currently are limited to treating only certain forms of the disease and tend to be immunosuppressive, leaving patients vulnerable to pathogenic infections. Bifunctional peptide inhibitors (BPIs) have been developed to suppress MS in an antigen specific manner, allowing for disease remittance as well as leaving the general immune system uncompromised. BPIs have been proven to have efficacy in suppressing experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. Despite their efficacy, BPIs require high doses to suppress the disease and have been shown to sometimes induce hypersensitivity reactions such as anaphylaxis. To counter this, this work focuses on synthesis and evaluation of Fc-Bifunctional peptide inhibitors (BPIs), which use the Fc fragment of IgG1 to help optimize the BPI in terms solubility, stability, and efficacy.The PLP-Fc-BPI has been previously synthesized and evaluated for its therapeutic efficacy in suppressing PLP-stimulated EAE, the relapse-remitting model. Because of the promising results of the PLP-Fc-BPI study, Chapter 2 of this dissertation focuses on evaluating the benefits of adding the Fc fragment to the BPI, using MBP-Fc-BPI as our model. MBP-Fc-BPI was synthesized using sortase mediated ligations and then evaluated for its stability and pharmacokinetic profile. The stability of MBP-Fc-BPI was compared to its parent LABL-Fc-ST to see if the addition of an antigenic peptide affected the conformation and stability of the Fc region, The pharmacokinetic profile of MBP-Fc-BPI was assessed both in vitro and in vivo to see if the half-life of MBP-Fc-BPI was greater than that of the parent BPIs. Our studies found that adding the MBP peptide did not affect the conformation and stability of the Fc and the half-life of MBP-Fc-BPI was much greater than the half-life of the parent BPI. Last, the efficiacy of MBP-Fc-BPI in treating MBP-stimulated EAE was evaluated in vivo. The untreated mice experienced a mixed disease state, resulting in a mild, but progressive form of EAE. The efficacy of MBP-Fc-BPI was compared to the parent MBP-BPI and the starting protein, LABL-Fc-ST. Of all the treatment groups, MBP-Fc-BPI treated mice showed the lowest overall clinical scores during the course of the study and were the only ones to experience complete disease suppression by the end of the study. We show that MBP-Fc-BPI has higher stability, better half-life, and increased therapeutic efficacy compared to the original BPIs.The last Fc-BPI to be evaluated for its therapeutic efficacy was MOG-Fc-BPI, a conjugate of the third antigenic peptide, MOG. The third chapter focuses on the synthetic challenges of making MOG-Fc-BPI. MOG peptide is the most hydrophobic antigenic peptide, resulting in limited solubility and stability. This posed challenges in terms of finding a synthetic route that would allow for MOG to be conjugated to the Fc to produce a stable form of MOG-Fc-BPI. Terminal sequence modifications were required to increase the solubility of the MOG peptide and the final MOG-Fc-BPI, after ligation via sortase mediated ligations. Once MOG-Fc-BPI was synthesized, its therapeutic efficacy was evaluated in vivo in MOG-stimulated EAE mice. The untreated mice experienced a progressive form of the disease with increasing clinical scores and symptoms throughout the course of the study. In comparison, the MOG-Fc-BPI treated mice had very mild scores and experienced complete disease suppression, meaning they showed no symptoms of the disease by the end of the study.Fc-BPIs for the three main antigenic peptides of MS showed high therapeutic efficacy in suppressing their respective forms of MS. Each Fc-BPI could be potential treatments for their peptide’s respective disease state. However, it is often difficult to discern the different disease states of MS in patients, especially early in the disease course; this makes it difficult to dose the patients with the appropriate treatment. Ideally, Fc-BPIs with multiple antigens would create a treatment for any form of MS using a single molecule. At the same time, this approach would have the means to treat epitope spreading, an occurrence where T cells become activated against multiple antigens of MS. The fourth chapter of this dissertation focuses on the feasibility of making multi-antigen Fc-BPIs (MA-Fc-BPIs). This project focuses on determining a synthetic route to site-selectively add two antigenic peptides to the Fc region. This study uses the transglutaminase mediated reaction, click chemistry reaction, and thiol-malemide reaction to selectively add MOG to Gln295 of the Fc while MBP is added via the sortase mediated ligations to the C-terminal of LABL-Fc-ST, mentioned in chapter 2. We show that 2-antigen MA-Fc-BPIs can be synthesized and potentially used in the future for treatment of multiple disease states.In summary, this dissertation delves into the synthetic challenges of making Fc-BPIs while evaluating and assessing their potential as antigen-specific treatments for different disease states of MS. In the future, various MA-Fc-BPIs with two or three different antigenic peptides will be synthesized and evaluated in PLP-, MBP-, and MOG-stimulated EAE mice.
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2023-12-31
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University of Kansas
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Pharmaceutical sciences, antigen-specific treatment, experimental autoimmune encephalomyelitis, Fc-bifunctional peptide inhibitors, immune modulation, multiple sclerosis