Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Summer 2014 Modulation of Immunity via Antibodies Displayed on in situ Forming Peptide Co-assemblies Yi Wen Follow this and additional works at:https://dsc.duq.edu/etd Recommended Citation Wen, Y. (2014). Modulation of Immunity via Antibodies Displayed on in situ Forming Peptide Co-assemblies (Doctoral dissertation, Duquesne University). Retrieved fromhttps://dsc.duq.edu/etd/1354 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]. MODULATION OF IMMUNITY VIA ANTIBODIES DISPLAYED ON IN SITU FORMING PEPTIDE CO-ASSEMBLIES 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 By Yi Wen August 2014 Copyright by Yi Wen 2014 MODULATION OF IMMUNITY VIA ANTIBODIES DISPLAYED ON IN SITU FORMING PEPTIDE CO-ASSEMBLIES By Yi Wen Approved June 2, 2014 ________________________________ ________________________________ Wilson S. Meng, Ph.D. Ellen S. Gawalt, Ph.D. Associate Professor of Pharmaceutical Associate Professor of Chemistry and Sciences Biochemistry Duquesne University, Pittsburgh, PA Duquesne University, Pittsburgh, PA (Committee Chair) (Committee Member) ________________________________ ________________________________ Ira S. Buckner, Ph.D. Jelena M. Janjic, Ph.D. Associate Professor of Pharmaceutics Assistant Professor of Pharmaceutics Duquesne University, Pittsburgh, PA Duquesne University, Pittsburgh, PA (Committee Member) (Committee Member) ________________________________ ________________________________ Lauren A. O’Donnell, Ph.D. David A. Johnson, Ph.D. Assistant Professor of Pharmacology Associate Professor of Pharmacology Duquesne University, Pittsburgh, PA Duquesne University, Pittsburgh, PA (Committee Member) iii ABSTRACT MODULATION OF IMMUNITY VIA ANTIBODIES DISPLAYED ON IN SITU FORMING PEPTIDE CO-ASSEMBLIES By Yi Wen August 2014 Dissertation supervised by Wilson S. Meng, Ph.D. Many diseases are associated with disruption of immune homeostasis. Monoclonal antibodies (mAb) targeting soluble antigens or immune cell surface molecules have been developed to modulate immunity. However, systemic administration can lead to severe immune related side effects. This dissertation investigated a versatile platform to display antibodies locally in diseased tissues to minimize unwanted systemic exposure. This system is a stimuli-responsive and injectable formulation utilizing in situ forming peptide co-assemblies and two linker proteins. The self-assembling peptides EAK16-II and EAKIIH6 co-assemble into fibrous structures with accessible His-tags when exposed to salts. Anti-His-tag antibodies (αH6-IgG) bind to these His-tags and provide fragment crystallizable region (Fc) for protein A/G (pAG). A therapeutic antibody can be displayed through non-covalent Fc-pAG interactions. Proof-of-concept studies demonstrate that antibodies were displayed via His-tags through the linker proteins, iv namely His-tag/αH6-IgG/pAG interactions. Displayed antibodies remained biologically active and were able to bind to cognate antigens. Upon administration to normal subcutaneous space, tumors, or transplant sites, a fluorescent dye labeled IgG was retained effectively at the site of injection. Antibodies targeting a cellular surface antigen were loaded onto the peptide co-assembly. Displayed antibodies were able to interact with cellular surface antigens and impede trafficking of target cells, resulting in potential therapeutic response in a murine skin transplantation model. Characterization of the peptide co-assembly indicates the His-tagged peptide (EAKIIH6) co-assembled with the parent peptide (EAK16-II) in a concentration dependent manner. Comparing the co- assembly efficiency of EAKIIH6 to other control peptides implies that EAKIIH6 might adopt a β-strand conformation and align with existing EAK16-II β-sheets during co- assembly. It was also found that the accessibility of His-tags could be tuned by changing the ratio of peptides. To conclude, these results demonstrate the peptide co-assembly based system could be used as a versatile antibody display system to modulate immunity. It may have the potential to improve therapeutical outcomes of mAbs as a local antibody delivery system, and it could also be utilized as a material strategy to immobilize target cells. v DEDICATION This dissertation is dedicated to my loving and supportive wife, Tiantian Gong, our sweet little boy, Luke Boteng Wen, and to my loving and encouraging parents Jianqing Wen and Huifang Li. vi ACKNOWLEDGEMENT I would like to gratefully and sincerely thank my advisor, Dr. Wilson Meng, for his guidance, understanding, and support through my PhD study. His mentorship and friendship not only lead me to develop essential scientific and professional skills but also to mature mentally as an individual. I would like to express my deepest gratitude to my dissertation committee members, Dr. Ellen Gawalt, Dr. Ira S. Buckner, Dr. Jelena M. Janjic, and Dr. Lauren A. O’Donnell for their valuable input and insightful comments as well as access to instruments. I also want to extend my special thanks to Dr. Rita Mihaelescu, Dr. Rehana K. Leak, and Mrs. Denise Butler-Buccilli for their help on circular dichroism, ex vivo imaging, and animal studies. I would like to thank the pharmaceutics faculty Dr. James K. Drennen III, Dr. Carl A. Anderson, Dr. Peter Wildfong, and Dr. Laurence H. Block for the high quality training and education I received at Duquesne. I am grateful to our current and former group members for their help and friendship: Ying Zheng, Wen Liu, Shana L. Roundebush, Jeffery R. Kovacs, Amanda George, and Harrold R. Kolonich. I also want to thank Kristen M. Kruszewski and Gavin A. Buckholtz for their technique help on SEM and AFM. In addition, the friendship and encouragement from all the graduate students has been a wonderful source of support. Finally and most importantly, I would like to thank my wife, my son, and my parents, for their endless love and support, without which, I cannot be where I am today. vii TABLE OF CONTENTS ABSTRACT∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ iv DEDICATION∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ vi ACKNOWLEDGEMENT∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ vii TABLE OF CONTENTS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ viii LIST OF TABLES∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ xiii LIST OF FIGURES∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ xiv LIST OF ABBREVIATIONS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙xvii CHAPTER 1 Introduction ··········································································· 1 Statement of the problem ········································································· 1 Significance ························································································· 7 Antibody display system ········································································ 12 Project overview ················································································· 19 CHAPTER 2 Design, Mechanisms, and Applications of Ionic Complementary β-sheet Self-Assembling Peptides ········································································· 22 Abstract ··························································································· 22 Introduction ······················································································· 22 Self-assembly of ionic complementary peptides ············································ 24 Rational design of self-assembling peptides ·············································· 24 viii Effect of peptide composition on self-assembly ········································· 26 Effect of salts on self-assembly ····························································· 30 Effect of peptide concentration on self-assembly ········································ 31 Effect of pH on self-assembly ······························································ 31 Mechanism and hierarchy of self-assembly ··············································· 32 Applications of ionic complementary self-assembling peptides ·························· 34 Tissue engineering and regeneration ······················································· 35 Drug delivery ·················································································· 39 Hemostasis ····················································································· 45 Pathogenesis of β-amyloids ································································· 45 Summary ·························································································· 46 CHAPTER 3 Retaining Antibodies in Tumors with a Self-assembling Injectable System ········································································································· 48 Abstract ··························································································· 48 Introduction ······················································································· 49 Materials and Methods ·········································································· 52 Materials ······················································································· 52 Sample preparations ·········································································· 53 Optical and scanning electronic microscopy ·············································· 53 Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) ·············· 54 Antigen binding activity of immobilized antibodies····································· 54 Mice, tumor inoculation, and imaging ····················································· 55 ix