Advances in exosome-mediated immunotherapy and diagnostics

Abstract: Exosomes are small vesicles with immune-stimulatory capacity, which can activate T cell responses in a B cell dependent manner, and therefore may serve as immune therapeutic tools. Peptide-loaded dendritic cell (DC)-derived exosomes are proven safe in clinical trials, although with limited ability to induce cytotoxic T lymphocyte (CTL) responses or prolong patient survival. Therefore, we aimed to investigate the role of exosomal MHC/peptide complexes in immune activation and explore how to enhance exosome induced immunotherapies by applying additional stimuli to the exosomes. Bone marrow-derived dendritic cell (BMDC) exosomes loaded with ovalbumin (OVA) and α-galactosylceramide (αGC) were used for this purpose. Exosomes lacking major histocompatibility complex (MHC) class I or those that were MHC mismatched were thoroughly studied in vivo for their ability to stimulate effector T cells and humoral responses. In addition, we applied a novel strategy, lyophilization, for exosomal loading of antigen and adjuvants. Here, OVA, CpG-ODN and αGC were added to RAW 264.7-derived exosomes and assessed for their immune-stimulatory capacity. We demonstrated that exosomal MHC/peptide complexes were redundant for T cell stimulation in vivo in the presence of whole OVA, as MHCI-/- and allogeneic exosomes could successfully induce CD8+ T cell responses and inhibit tumor progression (study I). Importantly, allogeneic exosomes served as an adjuvant by the upregulation of T follicular helper (Tfh) cells and increased antigen-specific antibody production (study II). We also discovered that lyophilization was feasible for loading exosomes without markedly altering exosome characteristics. Notably, additional use of the TLR9 ligand CpG-ODN improved their immune-stimulatory properties and achieved tumor regression (study III). Selective loading and accumulation of certain tissue-specific proteins and RNA into exosomes provides a platform for potential biomarker analysis, the advantages of which include the accessibility of vesicles in body fluids (“liquid biopsies”), and the ability to trace cellular origin. However, limited material often restricts exosome proteomic analyses. Therefore, we aimed at applying the highly sensitive proximity extension assay (PEA) on cell line- and body fluid-derived exosomes to investigate the potential of using PEA for exosome protein evaluation. We confirmed that PEA can be applied on exosomes to trace their cellular source and to identify accumulated vesicle proteins. Also, the protein content of the body fluid-derived exosomes from breast milk and seminal fluid displayed diverse protein profiles (study IV), suggesting the cell/tissue traceability of exosomes by PEA and motivating their future use as biomarkers. In conclusion, this thesis provides increased understanding of the mechanisms underlying exosome-based immunotherapies and suggests the use of impersonalized exosomes and allogenicity as a possible means of enhancing their immune-stimulatory effects in a clinical setting. In addition, this thesis offers insight into novel technologies for improved exosomal loading and the use of PEA for exosome proteomic research.