The development of prophylactic and therapeutic vaccine using cell derived extracellular vesicles

Abstract

The primary aim of this thesis is to extend the breadth of in vivo application of externally loaded cell-line derived and tumor derived exosomes as a prophylactic and therapeutic carrier against cancer treatment. Exosomes with a size between 30 to 150 nm are small extracellular vesicles secreted by all types of mammalian cells. They mediate a novel mode of intercellular communication through their bioactive cargos such as lipids, nucleic acids, metabolites, and proteins, which can be delivered to the target cells. Exosomes have successfully served as immunotherapeutic nanocarriers in cancer treatment using their natural delivery capabilities. Furthermore, they are attractive as a delivery system because of their stability in circulation, biocompatibility, and low toxicity. In the first part of this thesis, we used exosomes as a nanocarrier system to develop cancer vaccines in a therapeutic murine melanoma cancer treatment. We show that lyophilization of exosomes together with the CpG ODN, model antigen OVA and lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution is successfully accomplished. We analyzed the effect of the lyophilization on a cell line-derived exosomes and we characterized the exosomes by using bead-based technique via flow cytometry, qNano, Scaning electron microscopy, and western blotting. We showed that lyophilization does not harm exosomes’ vesicular integrity and fundamental biological features. Furthermore, we tested the biodistribution and activating capacity of encapsulated exosomes in mouse PEC, mesenteric lymph node, and spleen cells. We found out that loaded exosomes are mostly taken up by antigen-presenting cells. Also, we showed that loading CpG ODN into exosomes significantly improves APC activation markers in macrophages, B cells, and DCs and induced significantly higher IFNγ production from mouse mLN and splenocytes. Finally, we tested the therapeutic utility of the CpG ODN, OVA and αGC encapsulating exosome in the B16F10-OVA melanoma tumor-bearing mice. We found out that therapeutic vaccination with triple (CpG ODN, OVA, and αGC) ligand encapsulating exosomes suppressed the progression of established melanoma tumors in mice. Moreover, our triple ligand loaded exosomes triggered Th-1 biased anti-IgG OVA immunity and converted immune cells in tumor microenvironment to the tumor-suppressing phenotype. In the second part of this thesis, we used tumor-derived exosomes (TEXs) as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells, TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). We showed that engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic breast cancer model, we found out that administration of 4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regressed tumor growth in 4T1 tumor-bearing mice. As a result, this work implicated that an exosome based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. The last part of this thesis, we studied the therapeutic potential of cell line-derived exosomes loaded with superparamagnetic iron oxide nanoparticles(SPION) and immunostimulatory ligands. We showed that loading SPION enhanced the in vitro delivery of exosomes within immune cells. Also, we found out that spleen cells incubated with exosomes encapsulating with SPION and CpG ODN induced significantly higher levels of IL-12 and IFNγ. Finally, we tested our exosomal vaccine candidate in human hepatocellular carcinoma tumor model in athymic mice. We showed that TLR3 and TLR9 ligands encapsulated with SPION loaded exosomes induced pronounced innate immune activation and regressed tumors and improve survival rate of treated mice.