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A Peptide-Based Platform for Displaying Antibodies to Engage T Cells Ying Zheng

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A Peptide-Based Platform for Displaying Antibodies to Engage T Cells Ying Zheng Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Fall 2010 A Peptide-Based Platform for Displaying Antibodies to Engage T Cells Ying Zheng Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Zheng, Y. (2010). A Peptide-Based Platform for Displaying Antibodies to Engage T Cells (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/1412 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. A PEPTIDE-BASED PLATFORM FOR DISPLAYING ANTIBODIES TO ENGAGE T CELLS A Dissertation Submitted to the Graduate School of Pharmaceutical Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmaceutics By Ying Zheng December 2010 Copyright by Ying Zheng 2010 A PEPTIDE-BASED PLATFORM FOR DISPLAYING ANTIBODIES TO ENGAGE T CELLS By Ying Zheng Approved October 14, 2010 _________________________________ ___________________________________ Wilson S. Meng, Ph.D. James K. Drennen, III, Ph.D. Associate Professor of Pharmaceutical Associate Professor of Pharmaceutics Sciences Associate Dean for Graduate Programs and (Committee Chair) Research (Committee Member) __________________________________ ___________________________________ Philip E. Auron, Ph.D Ellen Gawalt, Ph.D Professor and Chair of Biological Sciences Associate Professor of Chemistry and Biochemistry (Committee Member) (Committee Member) __________________________________ ___________________________________ Peter Wildfong, Ph.D J. Douglas Bricker, Ph.D Associate Professor of Pharmaceutics Professor of Pharmacology-Toxicology (Committee Member) Dean of the Mylan School of Pharmacy iii ABSTRACT A PEPTIDE-BASED PLATFORM FOR DISPLAYING ANTIBODIES TO ENGAGE T CELLS By Ying Zheng December 2010 Dissertation supervised by Wilson S. Meng, Ph.D. This study investigated a strategy by which antibodies were displayed on a gel-like substance to engage T cells. The substance, a peptidic composite, was characterized in vitro and explored as an injectable system in vivo. The composite consists of two amphiphilic peptides, AEAEAKAKAEAEAKAK (referred to as “EAK”) and AEAEAKAKAEAEAKAKHHHHHH (“EAKH6”). Spectroscopic analysis showed the two peptides integrated into a single structure. Prior to combination, conformational analysis revealed that EAKH6 adopts a mixed -helix/-strand conformation. In the presence of EAK, EAKH6 exists predominantly in a -strand conformation. Using nickel-bound horseradish peroxidase as a probe, the composite of EAK-EAKH6 was found to display His-tags. T-cell-specific antibodies were found stably displayed on the iv EAK-EAKH6 assembly using recombinant protein A/G and anti-hexahistidine antibody as an adaptor. When mounted with an anti-CD4 antibody, the system was shown to capture CD4 T cells in a mixed population of lymphocytes. Antibodies were concentrated in the subcutaneous space in mice when co-administered with EAK and EAKH6 along with protein A/G and anti-hexahistidine antibody as an aqueous (deionized water) solution. Taken together, these results indicate that the design can be used as a platform for engaging specific subsets of leukocytes for purpose of immune modulation. v ACKNOWLEDGEMENTS I would like to gratefully acknowledge my advisor, Dr. Wilson Meng, for his unlimited support, valuable advisory and continued guidance throughout my graduate study. His sincere dedication and tireless devotion to research has provided me with the courage to strive at difficult times and will continue to be indispensable for my professional development. I would also like to thank Dr. Philip E. Auron and Dr. Ellen Gawalt for their encouragement and tremendous technical guidance in confocal microscopy and FTIR spectroscopy. I also would like to extend my sincere appreciation to Dr. James K. Drennen III and Dr. Peter Wildfong for their contributions and commitment to this project. In addition, expertise and helpful discussions with Dr. Rita Mihaelescu, Dr. Hongmei Shen and Dr. Nick Giannoukakis have been extremely instrumental for circular dichroism and flow cytometry studies. I also want to extend my special thanks to our current and previous group members as well as undergraduate research assistants for all their help: Jeffery R. Kovacs, Liang Jia, Yi Wen, Megan Mitchell, Amanda George and Alison Steinbach. Their friendship, both professionally and personally, has been a wonderful source of support. I am grateful to Denise Butler-Bucchilli for her help in the animal studies. I am also greatly indebted to other faculty members: Dr. Carl A. Anderson, Dr. Laurence H. Block, Dr. Riccardo L. Boni for the high-quality training and education they have provided during my graduate studies in Pharmaceutical Sciences. vi Lastly, I would like to thank my family: my father and my brother, for their endless support and understanding in my graduate study. Even from afar, they have been my constant source of strength to achieve my academic and personal goals. vii TABLE OF CONTENTS ABSTRACT .................................................................................................................................. iv ACKNOWLEDGEMENTS ......................................................................................................... vi LIST OF FIGURES ....................................................................................................................... x ABBREVIATIONS .................................................................................................................... xiii CHAPTER 1: INTRODUCTION ................................................................................................ 1 CHAPTER 2: SIGNIFICANCE OF THE RESEARCH ............................................................ 6 CHAPTER 3: LITERATURE REIVEW .................................................................................. 13 T CELLS IN HEALTH AND DISEASES .......................................................................................... 13 BETA STRUCTURE-ΒASED SELF-ASSEMBLING PEPTIDE SCAFFOLDS ........................................ 21 CHAPTER 4: PHYSICAL CHARACTERIZATION OF EAK-EAKH6 COMPOSITE ..... 40 INTRODUCTION .......................................................................................................................... 40 MATERIALS AND METHODS ...................................................................................................... 43 RESULTS AND DISCUSSION ........................................................................................................ 48 SUMMARY AND CONCLUSIONS ................................................................................................. 65 CHAPTER 5: FUNCTIONALIZATION OF EAK-EAKH6 COMPOSITE WITH PROTEIN A/G AS THE ADAPTOR ........................................................................................ 66 INTRODUCTION .......................................................................................................................... 66 MATERIALS AND METHODS ...................................................................................................... 69 RESULTS AND DISCUSSION........................................................................................................ 72 SUMMARY AND CONCLUSIONS ................................................................................................. 83 viii CHAPTER 6: CAPTURE OF LEUKOCYTE SUBSETS IN FUNCTIONALIZED EAK- EAKH6 COMPOSITE ................................................................................................................ 84 INTRODUCTION .......................................................................................................................... 84 MATERIALS AND METHODS ...................................................................................................... 86 RESULTS AND DISCUSSION........................................................................................................ 90 SUMARRY AND CONCLUSIONS .................................................................................................. 97 CHAPTER 7: CHARACTERIZATION OF POL-(D, L-LACTIC-CO-GLYCOLIC ACID) PARTICLES AS CARRIERS OF NUCLEIC ACIDS INTO PRIMARY DENDRITIC CELLS AND T CELLS .............................................................................................................. 99 INTRODUCTION .......................................................................................................................... 99 MATERIALS AND METHODS .................................................................................................... 102 RESULTS AND DISCUSSION...................................................................................................... 112 SUMARRY AND CONCLUSIONS ................................................................................................ 131 CHAPTER 8: SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS FOR: A PEPTIDE-BASED PLATFORM FOR DISPLAYING ANTIBODIES TO ENGAGE T CELLS ........................................................................................................................................ 133 CHAPTER 9: MODULATION OF ALLOGRAFT IMMUNOGENICITY WITH IL-10 GENE PARTICLES .................................................................................................................
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