Self-assembled peptide nanostructures: production, structural characterization and in vivo application as cancer vaccine

Abstract

AIM OF THIS THESIS: In this thesis, we capitalize on the previous work done in our group with SA2 peptides and apply peptide self-assembly as a means to obtain peptide nanoparticles that can be used for vaccination. The main objectives of this thesis are: i) To better understand the dynamics of self-assembly of SA2 and characterize the selfassembling structures at the molecular level. ii) To utilize self-assembling peptides derived from SA2 to deliver peptide epitopes into antigen presenting cells for tumor vaccination. In addition to these two main objectives, we also attempted to optimize the recombinant production of SA2 peptides in E. coli. OUTLINE OF THIS THESIS Chapter 2 provides a general overview of self-assembling peptides. The first part of this chapter covers the different secondary structures that peptides can adopt which are important for the further assembly into highly-ordered nanostructures, such as micelles, vesicles, fibrils, and tubes. The second part gives an overview of the recent literature on studies in peptide self-assembly for application in drug delivery, vaccination, and tissue engineering. Chapter 3 focuses on the optimization of the recombinant production of SA2 peptides in E. coli as a cost-effective way to produce large quantities of these peptides for vaccination purposes. First, this chapter describes how the production yields of the SA2 peptide can be improved by using a SUMO fusion construct and a well-balanced autoinduction medium. Second, it describes the optimization of the purification steps by preventing premature self-assembly of the SA2 peptide within the E. coli host leading to increased yields during production and less loss of the peptide during purification. Chapter 4 presents an avenue to the high-resolution structural characterization of peptide-based nanovesicles and their assembly pathway based on the example of the nanocarrier formed by the SA2 peptide. By integrating a multitude of experimental techniques (i.e. solid-state NMR, AFM, SLS, DLS, FT-IR, CD) with large- and multi14 scale MD simulations, it is demonstrated that SA2 nanocarriers consist of interdigitated antiparallel β-sheets, which bear little resemblance to phospholipid liposomes. In Chapter 5 and 6, the potential use of the self-assembling peptide as a carrier for peptide-based vaccine in prophylactic and therapeutics models is investigated. Chapter 5 describes the capability of a designed vaccine based on peptide self-assembly to induce OVA-specific immune responses and to delay tumor growth were tested in mice bearing OVA-expressing B16 melanoma cells both prophylactic and therapeutic setting. To establish and explore the ability of peptide self-assembly as a platform for delivery of minimal soluble epitopes, Chapter 6 describes the in vivo effectiveness another designed self-assembling peptide bearing human papillomavirus antigens (HPV 16 E744-57) in a therapeutic setting. Chapter 7 is a brief summary of the aforementioned chapters, and future perspectives of self-assembling peptides for biomedical applications are proposed.