Construction Of Exosome-Based Immunomodulators And Their Application In Tumor Immunotherapy

Tumor immunotherapy refers to a treatment that relies on the patient’s own immune function to eliminate the tumor.Classic tumor immunotherapy methods include immune checkpoint blockade therapy and chimeric antigen receptor T cell（CAR-T）therapy.With the widespread applications of immunotherapy in the clinic,the limitations and the toxic side effects of immunotherapy have gradually attracted the attention of people.Exosomes,as a class of endogenous extracellular vesicles,have significant advantages such as low immunogenicity and good biocompatibility,and play an important role in mediating intercellular communication and regulating immunity.Based on this,in this thesis,different exosome-based strategies were developed for enhancing tumor immunotherapy and reducing immunotherapy-related toxic side effects,which will provide new ideas for exosome-based tumor treatment.（1）In response to the problem that the extreme immunosuppressive microenvironment of tumor tissue limits the effect of immunotherapy,we have firstly revealed that dendritic cell（DC）-derived exosomes（Dexo）could modulate the anti-tumor immune response by polarizing macrophages.The experimental results showed that after Dexo treatment,the markers CD80,i NOS,TNF-α,IL-6 and NO of M1 macrophages were upregulated or secreted increasingly,while the markers CD206,Arg-1 and IL-10 of M2 macrophages were downregulated or secreted decreasingly,confirming that Dexo could effectively polarize macrophages to M1 phenotype and suppress tumor growth.Moreover,eukaryotic transcriptome sequencing results showed that Dexo promoted macrophage polarization primarily by inhibiting the biosynthesis of unsaturated fatty acids.Notably,unlike the results of Dexo-based studies,intratumor injection of DC could promote tumor growth,mainly due to the immunosuppressive factors of the tumor microenvironment which induced DC to transform into inhibitory DC（i DC）,while i DC-derived exosomes could polarize macrophages to the M2 phenotype.（2）Aiming at the problems of CAR-T therapy,such as the low treatment efficiency of solid tumors,the time-consuming production of CAR-T cells and the difficulty of storage and transportation,we have constructed a CAR-T-like treatment system based on antigen-feeding dendritic cell-derived exosomes.The results showed that the surface of antigen-stimulated DC-derived exosomes（Exo-OVA）contained the main histocompatibility complex（MHC）-antigen peptide complex and the co-stimulating factor CD86 of in situ activated T cells.In addition,in order to further increase the recognition and killing of tumor cells by activated T cells,the anti-CD3 and anti-EGFR were further engineered on the surface of Exo-OVA to finally form Exo-OVA-a CD3/a EGFR,and the killing effect of T cells on cancer cells was enhanced by means of anti-CD3 targeting of activated T cells and anti-EGFR targeting of tumor cells.In vitro experimental results indicated that the inherent immunostimulatory molecules on the surface of Exo-OVA-a CD3/a EGFR could exert the basic functions of the intracellular and extracellular domains of CAR,effectively promoting the proliferation and differentiation of T cell,and further enhance the killing effect of T cell on cancer cell by facilitating the binding of activated T cells to cancer cells.Furthermore,in vivo experimental results confirmed that Exo-OVA-aCD3/aEGFR had excellent anti-tumor activity and could effectively inhibite tumor recurrence and metastasis.In addition,the combination of Exo-OVA-a CD3/a EGFR and anti-PD-L1 could further enhance the anti-tumor immunotherapy efficiency of T cells by reducing immune escape of tumor cells.（3）In order to solve the problem of severe cardiotoxicity induced by immune checkpoint inhibitors,we proposed a strategy to construct an immunomodulator based on tumor circulating exosomes by biological orthogonal reactions for the prevention of myocardial injury induced by immune checkpoint inhibitors.The inhibitory effect of tumor cells-derived exosomes（TuExo）on T cell activity in the local area of myocardium,as well as the alleviation effect of myocardial toxicity induced by immune checkpoint inhibitors was investigated.As the in vitro experiments results,the N₃ groups on the surface of metabolically labeled B16-F10 cell-derived exosomes was confirmed,with the binding and loading ability towards target molecules PCM through biological orthogonal reactions.Thus,the immunomodulator TuExo-PCM composed of TuExo and myocardial-targeted peptide PCM was constructed with myocardial targeting function.Moreover,in vivo experiments results showed that TuExo-PCM was enriched to myocardial tissue,which significantly reduced myocardial inflammation by increasing the PD-L1 level on the surface of cardiomyocytes,inhibiting T cell activity,and promoting macrophage polarization to M2 phenotype.In addition,the targeted delivery of TuExo in the circulatory system was conducive to retaining T cell activity in the circulatory system and enhancing the infiltration of T cells into the tumor tissue.