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Characterization of Conformational B-Cell PEPTIDE-BASED B-CELL EPITOPE VACCINES TARGETING HER-2/NEU DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Joan T. Garrett, B.S./B.A. ***** The Ohio State University 2007 Dissertation Committee: Approved by Professor Pravin Kaumaya, Advisor ___________________________ Advisor Professor Dehua Pei, Advisor Professor Ross Dalbey ___________________________ Advisor Professor Thomas Magliery Graduate Program in Chemistry ABSTRACT HER-2/neu (ErbB2), a member of the epidermal growth factor family of receptors (EGFR) is overexpressed in a significant fraction of breast cancers. It is an attractive target for receptor-directed antitumor therapy using monoclonal antibodies. Trastuzumab and pertuzumab are growth-inhibitory humanized antibodies targeting the oncogenic protein HER-2/neu. Although passive immunotherapy with trastuzumab is approved for treatment of breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive, and has a limited duration of action, necessitating repeated administrations of the monoclonal antibody. Active immunotherapy with conformational B-cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide antibodies. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the antigen binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide bonding pattern. In order to minimally dissect the interacting binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion ‘promiscuous’ T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformationally restricted peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell ii proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/n mice with antibodies against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting antibodies which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their antibody- dependent cell-mediated cytotoxicity (ADCC). Moreover, immunization with the 597- 626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. Based on the three-dimensional structure of the HER-2: pertuzumab Fab fragment complex, we have designed three conformational peptide constructs to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo anti- tumor efficacy. All the constructs elicited high affinity anti-peptide antibodies and all the anti-peptide antibodies showed ADCC to varying degrees with the 266-296 constructs being equally effective as compared to trastuzumab. The 266-296 peptide vaccine statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in a transgenic mouse tumor model (Balb-neuT). Finally, we report on a phase I clinical trial using the first generation peptide vaccines MVF 316-339 and MVF 628-647 with nor-MDP as adjuvant. The goals of the trial were to determine the safety and toxicity of the vaccine as well as the maximum tolerable dose. The vaccine was well-tolerated and the maximum tolerable dose was iii identified as the highest dose level, 1.5 mg of each peptide. Additionally, patients produced antibodies of the IgG isotype against the vaccine, and patients receiving the highest dose level had a statistically significant increase in the IgG antibody response compared to patients receiving the lowest dose level. iv Dedicated to my family particularly my husband, Steve Garrett and my parents, Bob and Diane Steele v ACKNOWLEDGMENTS I wish to thank my advisor, Dr. Pravin Kaumaya, for his support and encouragement. His enthusiasm and interest have been essential for the success of this project. I am also grateful to my committee members, Drs. Ross Dalbey, Thomas Magliery, Dehua Pei, as well as Ming-Daw Tsai and Robert Coleman for their time and guidance. My sincere acknowledgements to Dr. Stephanie Allen, who worked with me on several of the studies described in Chapter 3. I thank Dr. Sharad Rawale for his assistance with the synthesis of many of the peptides used in this project. I acknowledge Drs. John Morris, Guido Forni, and Todd Reilly for providing cell lines and transgenic mice breeding pairs as well as critical reading of manuscripts. I am grateful to those who assisted in the clinical trial studies including Jacquie Lieblein, Danielle Carbin, Abby Short, and Stephen Vincent. Additionally, I thank the clinical trial coordinators I have worked with over the years including Tammy Lamb and Yahaira Kane. I am grateful to all members of the Kaumaya lab past and present, especially Daniele Vicari, Marcus Lynch, and Naveen Dakappagari for advice, problem-solving, and a helping hand. Finally, I would like to thank my family and friends. I thank my parents for their unconditional love and support. I am grateful to my sister, Marie, for her support given through many hours of phone conversations. I thank my husband, Steve, for all the love vi and encouragement he has given me. He has helped me to maintain my sanity throughout the completion of this thesis as well as in all areas of life. vii VITA March 13, 1980..............................................Born – Cincinnati, Ohio 2002…………………………………………B.A./B.S. Chemistry/Computer Science, Ohio University 2002 – present………………………………Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. Allen, S.D., Garrett, J.T.*, Rawale, S., Jones, A.L., Phillips, G., Forni, G., Morris, J.C., Oshima, R.G., and Kaumaya, P.T. Peptide Vaccines of the HER-2/neu Dimerization Loop are Effective in Inhibiting Mammary Tumor Growth in vivo. J Immunol, 179: 472-482, 2007. (* joint first author) 2. Garrett, J.T., Rawale, S., Allen, S.D., Phillips, G., Forni, G., Morris, J.C., and Kaumaya, P.T. Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu. J Immunol, 178: 7120-7131, 2007. 3. Steele, J.T., Rawale, S., Kaumaya, P.T.P. Cancer Immunotherapy with Rationally Designed Synthetic Peptides. In: Kastin, A. (ed.), Handbook of Biologically Active Peptides. Elsevier. 511-518, 2006. 4. Dakappagari, N.K., Lute, K.D., Rawale, S., Steele, J.T., Allen, S.D., Phillips, G., Reilly, R.T., and Kaumaya, P.T. Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities. J Biol Chem, 280: 54-63, 2005. FIELDS OF STUDY Major Field: Chemistry viii TABLE OF CONTENTS Page Abstract………………………………………...………………………………………….ii Dedication……………………………………………………………………………...….v Acknowledgments…………………………………………………………………..……vi Vita……………………………………………………………………………………...viii List of Tables…………………………………………………………………………….xi List of Figures……………………………………………………………………………xii Abbreviations……………………………………………………………………………xv Chapters: 1. Introduction…………………………………………………………………………….1 1.1 Breast Cancer Epidemiology and AvailableTreatments…………………...….1 1.2 HER-2…………..……………………………………………………………..2 1.3 Cancer Immunotherapy………………………………………………………15 1.4 Hypothesis and Overview of Chapters 2-4.………………………………….30 1.5 Tables and Figures……….…………………………………………………..32 2. Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu………………….………..………40 2.1 Introduction…………………………………………………………………..40 ix 2.2 Materials and Methods……………………………………………………….44 2.3 Results………………………………………………………………………..52 2.4 Discussion………………..…………………………………………………..61 2.5 Tables and Figures……….…………………………………………………..67 3. Peptide Vaccine Strategies Targeting the HER-2 Dimerization Loop…………….…86 3.1 Introduction…………………………………………………………………..86 3.2 Materials and Methods……………………………………………………….89 3.3 Results………………………………………………………………………..94 3.4 Discussion………………..…………………………………………………100 3.5 Tables and Figures……….…………………………………………………107 4. Effect of Dose on Humoral Immune Response in Patients Vaccinated with Multi- Epitope Peptide-Based Vaccines Targeting HER-2…………………….…...…119 4.1 Introduction…………………………………………………………………119 4.2 Patients and Methods………..………………………………...……………121 4.3 Results………………………………………………………………………127 4.4 Discussion………………..…………………………………………………132 4.5 Tables and Figures……….…………………………………………………137 5. Summary and Future Perspectives…………………………………...……………...148 Bibliography…………………………………………………………………………....152 x LIST OF TABLES Table Page 2.1 Amino Acid Sequence of HER-2 Trastuzumab-Binding Epitopes…..………………67 3.1 Amino Acid Sequence of HER-2 Pertuzumab-Binding Epitopes…..….…………..107 4.1 Dose Escalating Trial Design for Multi-Epitope HER-2 Peptide Vaccine………....137 4.2 Patient Characteristics………………………………………………………………138 4.3 Partial Response and Stable Disease Patients……………………………...……….139 4.4 Maximum Mean IgG Response to HER-2 Peptide Vaccines per Dose Level…...…140 xi LIST OF FIGURES Figure Page 1.1 Proposed mechanisms of action of trastuzumab. …..……………………..…………32 1.2 Comparison of soluble EGFR monomer, soluble EGF-EGFR monomer complex, soluble 2:2 EGF-EGFR complex, and soluble
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