Engineered discoidin domain from factor VIII binds αvβ3 integrin with antibody-like affinity Thesis by Gene Kym In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2014 (Defended April 23rd, 2014) ii © 2014 Gene Kym All Rights Reserved iii ACKNOWLEDGEMENTS I would like to thank my family for encouraging my studies and keeping me focused on long-term goals. I am thankful for the teachers who educated me, the mentors who trained me, my colleagues in student organizations who showed me the power of teamwork, and my friends for teaching me about the world outside the of academia. I am grateful to Steve, the Mayo Lab, and the individuals at Caltech who made my doctoral years a time of exploration, learning, and fun. iv ABSTRACT Alternative scaffolds are non-antibody proteins that can be engineered to bind new targets. They have found useful niches in the therapeutic space due to their smaller size and the ease with which they can be engineered to be bispecific. We sought a new scaffold that could be used for therapeutic ends and chose the C2 discoidin domain of factor VIII, which is well studied and of human origin. Using yeast surface display, we engineered the C2 domain to bind to αvβ3 integrin with a 16 nM affinity while retaining its thermal stability and monomeric nature. We obtained a crystal structure of the engineered domain at 2.1 Å resolution. We have christened this discoidin domain alternative scaffold the “discobody.” v TABLE OF CONTENTS Acknowledgements ........................................................................................................ iii Abstract ........................................................................................................................... iv Table of contents ............................................................................................................. v Figures and tables ........................................................................................................... vi Abbreviations ............................................................................................................... viii Chapter I: Introduction to alternative scaffolds .............................................................. 1 Chapter II: Discobody engineering and characterization ............................................. 25 Appendix A: Detailed discobody data .......................................................................... 61 Bibliography .................................................................................................................. 82 vi FIGURES AND TABLES Figure 1-1. Antibody domain schematic .............................................................. 19 Figure 1-2. Early antibody alternatives ................................................................ 21 Figure 1-3. Alternative scaffold structures ........................................................... 23 Figure 2-1. Topology diagrams of discobody and traditional scaffolds .............. 40 Figure 2-2. Flow cytometry plots of discobody loop grafts ................................. 41 Figure 2-3. FACS plots of sequential library sorts and Eng-Db sequence .......... 42 Figure 2-4. Binding characterization of Eng-Db .................................................. 43 Figure 2-5. Soluble Eng-Db competition binding ................................................ 43 Figure 2-6. Crystal structure of Eng-Db ............................................................... 44 Figure 2S-1. Potential linker orientations of bispecific Dbs .................................. 45 Figure 2S-2. On rate of Eng-Db using multiple antigen concentrations ................ 46 Figure 2S-3. Off rate of Eng-Db using single antigen concentration ..................... 48 Figure 2S-4. On rate of Eng-Db using single antigen concentration ..................... 50 Figure 2S-5. Equilibrium binding constant of Eng-Db .......................................... 52 Figure 2S-6. Melting temperature of Eng-Db ......................................................... 54 Figure 2S-7. Gel filtration elution profiles of Eng-Db ........................................... 54 Figure 2S-8. Displayed Eng-Db competition with Dbs ......................................... 55 Figure 2S-9. Displayed fibronectin competition with Dbs ..................................... 56 Figure 2S-10. Eng-Db structure compared to RGD peptide .................................... 57 Table 2-1. Eng-Db characterization summary .................................................... 58 Table 2S-1. Crystal structure data collection and refinement statistics ................ 59 Figure A-1. Discoidin family structural information ............................................ 61 Figure A-2. Factor VIII and the C2 domain .......................................................... 62 Figure A-3. Hydrophobic feet and the 4-Ala mutation of the C2 domain ............ 62 Figure A-4. αvβ3 integrin and the RGD motif ...................................................... 63 Figure A-5. Yeast surface display schematics ....................................................... 63 Figure A-6. Yeast display expression of 4-Ala by flow cytometry ..................... 64 Figure A-7. Soluble expression of 4-Ala ............................................................... 64 Figure A-8. AgRP binding control by flow cytometry ......................................... 65 Figure A-9. AgRP equilibrium titration by flow cytometry ................................. 66 vii Figure A-10. AgRP equilibrium titration curve fit .................................................. 66 Figure A-11. Measurements for loop grafting ......................................................... 67 Figure A-12. Close look at loop graft measurements .............................................. 67 Figure A-13. SGRGDNDLV loop graft equilibrium titration ................................ 68 Figure A-14. SGRGDNDLV loop graft curve fit ................................................... 68 Figure A-15. AVTGRGDSPASS loop graft equilibrium titration ......................... 69 Figure A-16. AVTGRGDSPASS loop graft curve fit ............................................. 69 Figure A-17. Normalized binding curves for control and loop grafts .................... 70 Figure A-18. Library construction schematics ........................................................ 71 Figure A-19. FACS library population lift per sorting round ................................. 71 Figure A-20. Sequences from sort round I-III ......................................................... 72 Figure A-21. Converged sequences from sort round V .......................................... 72 Figure A-22. RPRGDIE equilibrium titration by flow cytometry .......................... 73 Figure A-23. RPRGDIE curve fit ............................................................................ 73 Figure A-24. ACRGDTC equilibrium titration by flow cytometry ........................ 74 Figure A-25. ACRGDTC curve fit .......................................................................... 74 Figure A-26. Db constructs for bacterial expression ............................................... 75 Figure A-27. Db bacterial expression gel ................................................................ 75 Figure A-28. TEV cleavage of Eng-Db ................................................................... 76 Figure A-29. Db constructs for yeast secretion expression ..................................... 76 Figure A-30. Db yeast secretion expression gel filtration and gel .......................... 77 Figure A-31. Db yeast display levels compared to traditional scaffolds ............... 77 Figure A-32. Db yeast display levels quantified ..................................................... 78 Figure A-33. Db soluble protein thermal melts ....................................................... 78 Figure A-34. Crystals collected for X-ray diffraction ............................................. 79 Figure A-35. Diffraction during pre-data collection ............................................... 79 Table A-1. Sequences of Db constructs ............................................................... 80 viii ABBREVIATIONS Da Daltons Db Discobody ECM Extracellular matrix E. coli Escherichia coli FACS Fluorescence-activated cell sorting fVIII Factor VIII IgG Immunoglobulin G Kd dissociation constant kDa kilodalton koff dissociation rate kon association rate LB lysogeny broth, also known as Luria-Bertani medium mAb Monoclonal antibody µM micromolar mM millimolar nM nanomolar PCR polymerase chain reaction PS phosphatidylserine S. cerevisea Saccharomyces cerevisea 1 Chapter 1 INTRODUCTION TO ALTERNATIVE SCAFFOLDS Monoclonal antibodies (mAbs) can be engineered to bind specific molecular targets with high affinity, and were heralded as being “magic bullets” to target cancer. Despite their increasing commercial success, mAbs have had limited therapeutic value for many diseases [1-4]. A richer understanding of how mAbs engage with and utilize the human immune system is beginning to change how mAbs are engineered [5]. Additionally, antibody engineering innovations [6] have lead to novel mAb formats, including bispecifics [7-9], small domains [10-12], and fusions to cytotoxic payloads [13] or nanoparticles [14]. Small domain alternative scaffolds such as fibronectin or DARPins are driving many of these later generation technologies [11,12,15]. While alternative