(G-276) Universal mRNA-loaded immunogenic exosomes for immunotherapy of glioblastoma

Introduction:: Immune checkpoint blockade (ICB) has shown great potential in the treatment of various cancers. However, ICB therapy is not effective against glioblastoma (GBM) because of the immunosuppressive tumor microenvironment and low tumor mutational load, which allow tumor cells to escape recognition by activated T cells. Here, we report a novel mRNA-loaded immunogenic exosome (imExo) that can restore tumor immunogenicity for GBM immunotherapy. Specifically, we combined a microfluidic electroporation approach with a nanosecond pulse electroporation system to generate large quantities of exosomes overexpressing CD64 on their surfaces that can selectively load mRNA for IFN-γ within the exosomes. We further added docking sites for both CD71 and programmed cell death-ligand 1 (PD-L1) antibodies to the surface of the exosome to facilitate GBM targeting and immunotherapeutic effects in vivo. We found that these “imExo” upregulated IFN-γ expression in GBM, which in turn increased the expression of major histocompatibility complex I (MHC-I) in the tumor microenvironment, further extending the anti-tumor effect. Our findings suggest that these universal imExo are promising candidates for GBM immunotherapy.


Materials and Methods:: In our project, we described a microfluidic electroporation approach combined with a nanosecond pulse electroporation system to generate large quantities of exosomes that overexpress CD64 on their surfaces and can actively load mRNA for IFN-γ within themselves. We further add docking sites to these exosomes for both anti-CD71 and anti-PD-L1 antibodies to facilitate exosome targeting to GBM tumors (Figure. 1). We explored this IFN-γ effect in two different GBM models, the moderately immunogenic GL261 model, which response slightly to anti-PD-L1 immunotherapy, and the non-immunogenic SB28 model, which does not respond to immunotherapy.

Results, Conclusions, and Discussions:: The results have shown that imExo was effective in these two GBM models. The administration of imExo drastically extended the survival time of the mice and greatly increased the number of CD8⁺ cells in the tumor microenvironment and MHC-I expression, suggesting that immunogenicity had been restored in these mice. We also noted that the number of pro-inflammatory M1-polarized macrophages increased after imExo treatment. That M0 and M2-polarized macrophages suppress tumor immunity by secreting anti-inflammatory cytokines, whereas M1-type macrophages can promote tumor immunogenicity. Details of the mechanisms underlying macrophage polarization during imExo treatment will be important to obtain in future studies. Our integrated microfluidic electroporation obtained great enhancement in the yield of exosomes. Our study engineered a universal drug delivery system (CD64-Exo) that was able to transport biomacromolecules to the brain across BBB, the presence of anti-CD71 and anti-PD-L1 achieved both GBM targeting and immunotherapeutic effects. In addition, the introduction of N pep/box B achieved the specific loading of targeted mRNA. Importantly, our CD64-Exo platform is a versatile platform that has broad application prospects.


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