Development of immunotherapy using antigen-loaded multifunctional small extracellular vesicles

概要

Small extracellular vesicles (sEVs) with a diameter of around 100 nm, are membrane vesicles released by a variety of cells and serve as intercellular transport carriers for proteins and nucleic acids. Because of their nature as endogenous delivery carriers, sEVs are expected to become an effective vaccine by delivering antigen proteins. For vaccine application of sEV, antigen loading, delivery to antigen- presenting cells (especially dendritic cells, DCs) and activation of the antigen-presenting cells are important factors. In this thesis, I investigated the effect of modifying endogenous antigen-containing sEVs with CD40L, a peptide ligand with DCs-directing and immunostimulatory properties. Moreover, to create more types of multifunctional sEVs, I explored the possibility of loading exogenous antigens onto the sEVs. Since the exogenous antigens can be loaded onto the outside or inside of the lipid bilayer of sEVs, I investigated the effects of antigen localization on the efficiency of antigen presentation by sEVs-engulfed immune cells. After optimizing the loading method of exogenous antigens, I attempted to construct multifunctional sEVs by modifying the adjuvant to the antigen- loaded sEVs and verified the possibility of using it to treat allergic rhinitis.

Chapter 1 Development of endogenous antigen containing multifunctional sEVs for effective induction of anti-tumor immune response
Tumor-derived small extracellular vesicles are considered for use in inducing tumor antigen-specific immune responses as they contain endogenous tumor antigens. The delivery of tumor antigens to the antigen presentation cells (especially DCs), and the activation of DCs are the main challenges of endogenous antigen containing sEV therapy. In this chapter, sEVs derived from B16BL6 cells were modified with CD40L, which can target CD40 expressed on the surface of DCs and can activate them via CD40L-CD40 interactions. It was found that CD40L-sEVs were efficiently taken up by DCs and activated them. Moreover, tumor antigens were efficiently presented to the T cells by DCs treated with CD40L-sEVs. These results indicate that CD40L-modified endogenous antigen containing multifunctional sEVs will be helpful for further development of sEV-based tumor vaccination.

Chapter 2 Evaluation of effects of exogenous antigen localization in antigen-loaded sEVs on efficiency of antigen presentation
To enable the development of new kinds of multifunctional sEVs, the incorporation of exogenous antigens into sEVs has become a critical component of multifunctional sEVs design. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of sEVs, might affect antigen presentation after sEVs are taken up by antigen-presenting cells. However, little is known about the effect of antigen localization on the efficiency of antigen presentation. In this chapter, lactadherin (LA) and group-specific antigen (Gag), sEV-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of sEVs, respectively, were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as sEVs producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) sEVs were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded sEVs were added to bone marrow-derived DCs. OVA-inner- loaded (Gag-OVA) sEVs showed enhanced class I antigen presentation capacity compared with OVA-outer-loaded (OVA-LA) sEVs. Using PKH-labeled sEVs, it was found that the localization of OVA had very little effect on the cellular uptake of sEVs. These results indicate that the loading of antigen proteins inside sEVs helps in efficient antigen presentation.

Chapter 3 Development of allergic rhinitis immunotherapy using exogenous allergen-loaded multifunctional sEVs
Allergic rhinitis is caused by a breakdown of the Th1/Th2 balance, in which the allergen-induced Th2 immune response predominates over the Th1 immune response, culminating in IgE-mediated anaphylaxis. In this chapter, I used sEVs as simultaneous delivery carriers for allergens (OVA) and CpG DNA, an adjuvant that can induce a Th1 immune response, for the treatment of allergic rhinitis. sEVs loaded with CpG DNA and OVA(CpG-OVA-sEVs) were successfully prepared. CpG DNA modification did not influence the uptake of sEVs by DCs and CpG-OVA-sEVs activated DCs. The CpG-OVA-sEVs were delivered to the nasopharynx-associated lymphoid tissue (NALT) of mice and were primarily taken up by the CD11c positive cells after intranasal administration. Intranasally administering CpG-OVA-sEVs significantly enhanced OVA-specific IgG antibody titers in mice models of allergic rhinitis, suggesting a transformed Th1/2 balance. Moreover, The CpG-OVA-sEVs administration alleviated allergic symptoms compared to the control group. Further, the amount of IgE secreted in mouse serum decreased. Thus, CpG DNA modified allergen-loaded multifunctional sEVs could be a useful therapeutic method for treating allergic rhinitis.

In conclusion, I successfully developed CD40L modified endogenous antigen-containing multifunctional sEVs, which can be efficiently delivered to DCs and can activate DCs to improve the antigen presentation efficacy of DCs. Moreover, when loading exogenous antigens onto sEVs to enable the possibility of new types of multifunctional sEVs, I found that the loading of antigen proteins inside sEVs resulted efficient antigen presentation. Finally, I modified allergen-loaded sEVs with CpG DNA, which showed a significant therapeutic effect in allergic rhinitis model mice. The findings in this thesis contribute to the development of antigen-loaded multifunctional sEVs-based immunotherapies.

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