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Development of a Lectin-Fc Fusion Protein with Antiviral and Anti-Cancer Activity

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Development of a Lectin-Fc Fusion Protein with Antiviral and Anti-Cancer Activity University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2019 Development of a lectin-Fc fusion protein with antiviral and anti-cancer activity. Matthew iW lliam Dent University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Amino Acids, Peptides, and Proteins Commons, Biotechnology Commons, Macromolecular Substances Commons, Molecular Biology Commons, Other Immunology and Infectious Disease Commons, Other Plant Sciences Commons, Pharmaceutics and Drug Design Commons, and the Pharmacology Commons Recommended Citation Dent, Matthew William, "Development of a lectin-Fc fusion protein with antiviral and anti-cancer activity." (2019). Electronic Theses and Dissertations. Paper 3237. https://doi.org/10.18297/etd/3237 This Master's Thesis is brought to you for free and open access by ThinkIR: The nivU ersity of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The nivU ersity of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. DEVELOPMENT OF A LECTIN-FC FUSION PROTEIN WITH ANTIVIRAL AND ANTI-CANCER ACTIVITY By Matthew William Dent B.S., Arizona State University, 2014 M.Sc., University of East Anglia, 2015 A Thesis Submitted to the Faculty of the University of Louisville School of Medicine in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacology and Toxicology Department of Pharmacology and Toxicology University of Louisville School of Medicine Louisville, Kentucky May 2019 Copyright 2019 by Matthew William Dent All rights reserved DEVELOPMENT OF A LECTIN-FC FUSION PROTEIN WITH ANTIVIRAL AND ANTI-CANCER ACTIVITY By Matthew William Dent B.S., Arizona State University, 2014 M.Sc., University of East Anglia, 2015 A Thesis Approved on February 20, 2019 By the following Thesis Committee __________________________________________________________ Dr. Nobuyuki Matoba __________________________________________________________ Dr. Kenneth Palmer __________________________________________________________ Dr. Sucheta Telang __________________________________________________________ Dr. Jun Yan __________________________________________________________ Dr. Donald Miller ii DEDICATION This thesis is dedicated to: My mother and father, Dr. Jason C. Dent and Ms. Kimberly A. Dent, NP, for the years of support, inspiration, and opportunity that they have given me. My wife, Dr. Milena Mazalovska, who has been by my side through it all and to whom I owe my sanity. And lastly my mentor, Dr. Nobuyuki Matoba, and all my colleagues in the Biopharmaceutical Research Unit and the Department of Pharmacology and Toxicology for all their help and support. iii ABSTRACT DEVELOPMENT OF A LECTIN-FC FUSION PROTEIN WITH ANTIVIRAL AND ANTI-CANCER ACTIVITY Matthew William Dent February 20, 2019 This thesis describes the development of a novel lectin-Fc fusion protein and its antiviral and anti-cancer activity. The molecule, Avaren-Fc (AvFc), is a fusion of a variant of the actinomycete lectin actinohivin (Avaren) and the Fc region of human IgG1, and is selective for the terminal α1,2-mannose residues found at the ends of high-mannose-type glycans that can be found on the surface of certain heavily glycosylated viruses and cancer cells. Here, AvFc was found to be able to neutralize simian immunodeficiency virus as well as Hepatitis C virus with nanomolar IC50 values. Furthermore, AvFc recognizes a number of cell surface receptors and is capable of protecting mice against lung and flank B16F10 challenges primarily through Fc- mediated mechanisms. This work builds upon the framework developed by our group supporting the preclinical development of AvFc as a novel antiviral and anti-cancer protein. iv TABLE OF CONTENTS DEDICATION .................................................................................................................................... iii ABSTRACT ........................................................................................................................................ iv LIST OF FIGURES ............................................................................................................................. vii LIST OF TABLES .............................................................................................................................. viii CHAPTER I: INTRODUCTION ............................................................................................................. 1 Glycosylation of Proteins ............................................................................................................. 1 Description of N-glycans .......................................................................................................... 1 Aberrant Glycosylation in Cancer and Viruses ......................................................................... 3 Lectins .......................................................................................................................................... 4 Antiviral Lectins ........................................................................................................................ 5 Lectins as Cancer Therapy ........................................................................................................ 7 Transient Expression and Manufacturing of Biopharmaceuticals in Plants ................................ 8 Therapy of HIV ........................................................................................................................... 11 Therapy of Hepatitis C ............................................................................................................... 12 Avaren-Fc ................................................................................................................................... 13 CHAPTER II: EXPERIMENTAL PROCEDURES .................................................................................... 18 Plant Growth .............................................................................................................................. 18 Expression and Purification of AvFc and Variants ..................................................................... 18 HIV gp120 ELISA ......................................................................................................................... 20 Culture of Primary Simian Immunodeficiency Virus (SIV) Strains .............................................. 21 SIV Titration for Neutralization Assays ...................................................................................... 22 SIV Neutralization Assay ............................................................................................................ 23 Flow Cytometry of SIV-Infected Mesenteric Lymph Node Cells ................................................ 24 Pharmacokinetic Analysis of AvFc in Rhesus Macaques ............................................................ 24 Production of HCV...................................................................................................................... 25 TCID50 Assay for HCVcc ............................................................................................................. 26 HCVcc Neutralization Assay ....................................................................................................... 26 Flow Cytometry Analysis of AvFc Binding to B16F10 Cells ........................................................ 27 ADCC Reporter Assay ................................................................................................................. 27 v B16F10 Melanoma Lung Metastasis Model .............................................................................. 28 B16F10 Flank Tumor Model ....................................................................................................... 28 Proteomic Analysis of AvFc Binding Partners on Cancer Cells ................................................... 29 MTS Assay .................................................................................................................................. 30 Scratch Cell Migration Assay ...................................................................................................... 30 Pharmacokinetics of AvFc in C57bl/6 Mice ................................................................................ 31 A549-GFP Lung Cancer Model ................................................................................................... 31 Experimental Animals ................................................................................................................ 31 CHAPTER III: RESULTS..................................................................................................................... 33 Anti-SIV Activity of AvFc ............................................................................................................. 33 Anti-HCV Activity of AvFc ........................................................................................................... 37 Anti-Cancer Activity of AvFc ....................................................................................................... 38 CHAPTER IV: DISCUSSION .............................................................................................................
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