DESIGN AND EVALUATION OF NOVEL CONTRAST AGENTS FOR MR MOLECULAR IMAGING OF CANCER by ZHENG HAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Zheng-Rong Lu Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of: Zheng Han Candidate for the Doctor of Philosophy degree Zheng-Rong Lu Nicole F. Steinmetz (chair of the committee) William P. Schiemann Julian Kim March 17, 2017 We also certify that written approval has been obtained for any proprietary material contained therein ii Dedication This dissertation is dedicated to my wife, Yuechen Wu and my parents Ziqing Du and Wenhan Han, who are offering me love and support. iii TABLE OF CONTENTS Abstract .................................................................................................................. 1 Chapter 1 Diagnosis of Prostate and Breast cancer .......................................... 3 1.1 ABSTRACT...................................................................................................... 3 1.2 PROSTATE CANCER......................................................................................... 3 1.2.1 Prostate cancer epidemiology and risk factors ....................................... 3 1.2.2 Current tools for prostate cancer diagnosis ............................................ 6 1.2.3 Prostate cancer biomarkers .................................................................. 12 1.3 BREAST CANCER .......................................................................................... 16 1.3.1 Breast cancer epidemiology and risk factors ....................................... 16 1.3.2 Current tools for breast cancer diagnosis ............................................. 18 1.4 TARGETING FIBRONECTIN FOR CANCER IMAGING DIAGNOSIS ..................... 21 1.4.1 Fibronectin as a biomarker for cancer ................................................. 22 1.4.2 Targeting Fibronectin for Cancer Imaging .......................................... 35 1.5 PHAGE DISPLAY SELECTION OF LIGANDS FOR CANCER BIOMARKERS ......... 51 1.5.1 Principle of phage display .................................................................... 51 1.5.2 Filamentous phage systems .................................................................. 53 1.5.3 T7 phage library ................................................................................... 55 1.5.4 T4 bacteriophage system ...................................................................... 56 1.5.5 How to choose phage display system: our rationale ............................ 57 1.6 DISCUSSION AND CONCLUSIONS .................................................................. 59 Chapter 2 Phage Display Selection of Peptides Specific to EDB-FN ............. 78 iv 2.1 ABSTRACT.................................................................................................... 78 2.2 INTRODUCTION ............................................................................................ 79 2.3 MATERIALS AND METHODS ......................................................................... 81 2.4 RESULTS ...................................................................................................... 90 2.5 DISCUSSIONS AND CONCLUSIONS ............................................................... 103 Chapter 3 Design and development of a MRI molecular imaging agent that targets EDB-FN for the differential diagnosis of prostate cancer ................ 110 3.1 DESIGN AND SYNTHETIC ROUTE ................................................................. 110 3.2 MATERIALS AND METHODS ....................................................................... 111 3.3 RESULTS .................................................................................................... 120 3.4 DISCUSSION ............................................................................................... 137 Chapter 4 MRI differential diagnosis of breast cancer using a high-relaxivity Gadolinium metallofullerene targeted to extradomain-B fibronectin ......... 145 4.1 ABSTRACT.................................................................................................. 145 4.2 INTRODUCTION .......................................................................................... 146 4.3 MATERIAL AND METHODS .......................................................................... 148 4.4 RESULTS AND DISCUSSIONS ....................................................................... 155 Chapter 5 MR molecular imaging of triple-negative breast cancer and micrometastases ................................................................................................ 169 5.1 ABSTRACT.................................................................................................. 169 5.2 INTRODUCTION .......................................................................................... 170 5.3 MATERIALS AND METHODS ........................................................................ 173 v 5.4 RESULTS .................................................................................................... 178 5.5 DISCUSSIONS .............................................................................................. 188 Chapter 6 Future directions and concluding remarks .................................. 198 6.1 SUMMARY OF CURRENT WORK ................................................................... 198 6.2 NUCLEAR IMAGING BASED ON EDB-TARGETING PROBES ........................... 199 6.2.1 Non-metallic elements ....................................................................... 199 6.2.2 Metallic elements ............................................................................... 201 6.3 DELIVERY THERAPEUTIC AGENTS BASED EDB-TARGETING STRATEGY ...... 203 6.3.1 Radiotherapy ...................................................................................... 203 6.3.2 Delivery of anti-tumor agents ............................................................ 204 vi LIST OF FIGURES Chapter 1 Figure 1. Fibronectin structure and its binding sites to cell and other ECM molecules .............................................................................................................. 24 Figure 2. Upregulation of FN is crucial for cancer invasion and metastasis. ....... 27 Figure 3. Tumor uptake of 131I-L19SIP in a patient with metastatic prostate cancer. ................................................................................................................... 43 Figure 4. MRI contrast agents based on FN-targeting peptides............................ 47 Figure 5. MRI Detection of breast cancer micrometastases using FibFN targeting contrast agent, CREKA-Tris(Gd-DOTA)3. ........................................................... 49 Figure 6. Use of EDB-FN specific peptide, APTEDB, for development of imaging probes. ................................................................................................................... 50 Figure 7. Morphology of phages that have been engineered as phage display systems. ................................................................................................................. 54 Chapter 2 Figure 1. Construction of the EDB-expressing plasmid and SDS-PAGE assay of lysates from EDB-expressing E. coli strain BL21. ............................................... 91 Figure 2. Schematic illustration of the (A) synthesis of ZD2-Cy5 and (B) MALDI-TOF mass spectrum of ZD2-Cy5. .......................................................... 94 Figure 3. Characterization of the interaction between cyclic ZD2 and the EDB fragment using SPR .............................................................................................. 94 Figure 4. ZD2-Cy5 binding in PC3 cells. ............................................................. 96 vii Figure 5. In vivo binding of ZD2-Cy5 in mice bearing PC3-GFP prostate cancer flank xenografts. ................................................................................................... 99 Figure 6. Histological analysis of tissue sections of PC3 tumors from mice injected with ZD2-Cy5 or CERAK-Cy5............................................................. 100 Figure 7. ZD2-Cy5 binding in human prostate sections. .................................... 102 Chapter 3 Figure 1. Synthesis and characterization of ZD2-Gd(HP-DO3A). ..................... 122 Figure 2. In vitro complexation stability and transmetallation analysis of ZD2- Gd(HP-DO3A). ................................................................................................... 123 Figure 3. Upregulated EDB-FN expression is a promising biomarker for high-risk prostate cancer .................................................................................................... 126 Figure 4. The EDB-FN targeting contrast agent, ZD2-Gd(HP-DO3A), is capable of differentiating between PC3 and LNCaP tumors in T1-weighted MRI. ......... 129 Figure 5. Whole body signal changes in T1-weighted MRI with the contrast agents in tumor bearing mice. ........................................................................................ 131 Figure 6. Contrast enhanced MRI with CREKA-Gd(HP-DO3A) in the PC3 and LNCaP tumors. ................................................................................................... 132 Figure 7. T1 maps and accumulation of ZD2-Gd(HP-DO3A) validate its