Novel HER3 and IGF-1R Peptide Mimics and Synthetic Cancer Vaccines DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Megan Miller Graduate Program in Microbiology The Ohio State University 2014 Dissertation Committee: Dr. Pravin Kaumaya, Advisor Dr. Larry Schlesinger Dr. Abhay Satoskar Dr. Nicanor Moldovan Copyright by Megan Miller 2014 Abstract Overexpression and constitutive activation of protein tyrosine kinases, including HER1 and HER2, are found in many human cancers and are critical factors in the development and malignancy of tumors. The downstream signaling networks of HER1 and HER2 have been extensively targeted by cancer therapeutics, and agents such as therapeutic monoclonal antibodies and small tyrosine kinase inhibitors (TKI) have been developed to block ligand binding, receptor dimerization, and intracellular tyrosine kinase activity. Drugs approved by the FDA include TKIs such as gefitinib and erlotinib and therapeutic monoclonal antibodies such as cetuximab, pertuzumab and trastuzumab. HER3 (ErbB3) and IGF-1R are receptor tyrosine kinases that have only recently been recognized as important for the development and progression of cancer. These receptors are frequently upregulated in cancer and also may provide routes for resistance to agents that target HER1 or HER2. Several recent studies have shown that HER3 and IGF-1R may be attractive targets against many types of cancer, including breast, ovarian, pancreatic, prostate, colon, head and neck, etc. Although there are no FDA approved therapies that target HER3 or IGF-1R, several monoclonal antibodies have been developed and are currently being evaluated in clinical trials. Our laboratory has proposed using peptides as candidate vaccines or therapeutic agents that target the HER family and IGF-1R. In particular, this study evaluates novel HER3 and IGF-1R peptide mimics expected to elicit an enduring immune response with protein reactive high affinity peptide antibodies. Our ii laboratory has identified several peptides of the HER3 and IGF-1R extracellular domain as potential B cell epitopes for active immunotherapy against HER3 and IGF-1R driven cancers. The peptide vaccines were immunogenic in outbred rabbits, and the vaccine antibodies and peptidomimetics induced anti-tumor responses, such as: inhibition of cancer cell proliferation, inhibition of receptor phosphorylation, induction of apoptosis and antibody dependent cellular cytotoxicity (ADCC). In addition, treatment with the peptidomimetics or vaccine antibodies significantly inhibited growth of xenografts originating from both pancreatic and breast cancers. In conclusion, we provide novel candidates for HER3 and IGF-1R targeted cancer therapy. iii Dedication This document is dedicated to my husband. iv Acknowledgments I wish to thank The Ohio State University Microbiology Department for their willingness to support me as a Graduate Research/Teaching Associate. I would also like to thank my advisor, Dr. Pravin Kaumaya for the opportunity to work in his lab, as well as for his support, encouragement and constructive criticism. I thank all of the members of the Kaumaya lab, especially Dr. Kevin Chu Foy and Jay Overholser for their assistance, stimulating conversations and advice over the past 5+ years. I am also grateful for my committee members, Dr. Abhay Satoskar, Dr. Larry Schlesinger and Dr. Nicanor Moldovan for their time and guidance. Finally, I would like to thank God, my family and friends. I thank my parents, grandmother, mother-in-law and father-in-law for their love, advice, inspiration and encouragement to pursue my interests. I thank my brother, sister, sister-in-law, brother- in-laws and friends for being there for me and helping me maintain my sanity throughout this process. Lastly, I would like to thank my beloved husband for always believing in me and pushing me to be better in all areas of my life. Thank you, Luke, for being my rock! v Vita June 2003 ......................................................Westland High School, Galloway, OH June 2007 .......................................................B.S. Life Sciences, Otterbein University 2008 to present ..............................................Graduate Teaching and Research Associate, Department of Microbiology, The Ohio State University Publications Foy KC, Liu Z, Phillips G, Miller MJ, Kaumaya PT. Combination Treatment with HER-2 and VEGF Peptide Mimics Induces Potent Anti-tumor and Anti-angiogenic Responses in Vitro and in Vivo. J Biol Chem 2011; 286, 13626-13637. Foy KC, Miller MJ, Moldovan N, Carson III WE, Kaumaya PTP. Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo. OncoImmunology 2012; 1:1048–60 Foy KC, Wygle RM, Miller MJ, Overholser JP, Bekaii-Saab T, Kaumaya PT. Peptide vaccines and peptidomimetics of EGFR (HER-1) ligand binding domain inhibit cancer vi cell growth in vitro and in vivo. J Immunol. 2013; 191(1):217-27. Miller MJ, Foy KC, Kaumaya PT. Cancer immunotherapy: present status, future perspective, and a new paradigm of peptide immunotherapeutics. Discov Med 2013; 15(82):166-76. Fields of Study Major Field: Microbiology vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Abbreviations ................................................................................................................... xiv Chapter 1 : Introduction to Peptide Immunotherapy and HER3/IGF-1R Signaling Pathways in Cancer ............................................................................................................. 1 Targeted therapeutics in oncology .................................................................................. 1 Protein Tyrosine Kinases ................................................................................................ 2 Monoclonal antibodies and tyrosine kinase inhibitors .................................................... 3 Peptide therapeutics in oncology..................................................................................... 6 Human epidermal growth factor receptors and cancer.................................................... 9 HER3 and Cancer .......................................................................................................... 11 HER3-targeted therapeutics in cancer ........................................................................... 13 The type I insulin-like growth factor receptor (IGF-1R) .............................................. 15 viii Therapeutically targeting IGF-1R ................................................................................. 18 Drug resistance associated with HER-targeted therapeutics and receptor crosstalk ..... 19 HER3 and IGF-1R peptide mimics and vaccine antibodies .......................................... 20 Central Hypothesis and Overview of Chapters Two-Five ............................................ 21 Chapter 2 : Novel HER3 peptide mimics exhibit antitumor activity against HER3 positive cancers in vitro and in vivo ............................................................................................... 23 Introduction ................................................................................................................... 23 Materials and Methods .................................................................................................. 26 Results: .......................................................................................................................... 30 Discussion ..................................................................................................................... 35 Chapter 3 : Immunogenicity of HER3 peptide mimics and evaluation of cancer cell treatment with anti-HER3 peptide mimic antibodies in vitro and in vivo. ....................... 49 Introduction: .................................................................................................................. 49 Materials and Methods: ................................................................................................. 51 Results: .......................................................................................................................... 56 Discussion ..................................................................................................................... 59 Chapter 4 : IGF-1R peptide mimics, immunogenicity and anti-tumor activity ................ 70 Introduction: .................................................................................................................. 70 Materials and Methods: ................................................................................................. 72 ix Results: .........................................................................................................................