For cancer immunotherapy via tumor antigen vaccination in combination with an adjuvant, major challenges include the identification of a particular tumor antigen and efficient delivery of the antigen as well as adjuvant to antigen-presenting cells. In this study, we proposed an efficient exosome-based tumor antigens-adjuvant co-delivery system using genetically engineered tumor cell-derived exosomes containing endogenous tumor antigens and immunostimulatory CpG DNA. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a fusion streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) protein, yielding genetically engineered SAV-LA-expressing exosomes (SAV-exo). SAV-exo were combined with biotinylated CpG DNA to prepare CpG DNA-modified exosomes (CpG-SAV-exo). Fluorescent microscopic observation revealed the successful modification of exosomes with CpG DNA by SAV-biotin interaction. CpG-SAV-exo showed efficient and simultaneous delivery of exosomes with CpG DNA to murine dendritic DC2.4 cells in culture. Treatment with CpG-SAV-exo effectively activated DC2.4 cells and enhanced tumor antigen presentation capacity. Immunization with CpG-SAV-exo exhibited stronger in vivo antitumor effects in B16BL6 tumor-bearing mice than simple co-administration of exosomes and CpG DNA. Thus, genetically engineered CpG-SAV-exo is an effective exosome-based tumor antigens-adjuvant co-delivery system that will be useful for cancer immunotherapy.

中文翻译：

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基于外来体的肿瘤抗原-利用遗传工程肿瘤细胞衍生的外来体与免疫刺激性CpG DNA的佐剂共递送

对于通过与抗原佐剂组合的肿瘤抗原疫苗接种的癌症免疫疗法，主要挑战包括鉴定特定的肿瘤抗原以及将抗原以及佐剂有效递送至抗原呈递细胞。在这项研究中，我们提出了一个有效的基于外泌体的肿瘤抗原-佐剂共递送系统，该系统使用基因工程改造的肿瘤细胞衍生的含有内源性肿瘤抗原和免疫刺激性CpG DNA的外泌体。用编码融合链霉亲和素（SAV;高亲和力与生物素结合的蛋白质）-lacherherin（LA;外泌体向性蛋白）蛋白的质粒载体转染鼠黑色素瘤B16BL6细胞，产生基因工程表达SAV-LA的外泌体（ SAV-exo）。将SAV-exo与生物素化的CpG DNA结合以制备CpG DNA修饰的外泌体（CpG-SAV-exo）。荧光显微镜观察显示，通过SAV-生物素相互作用，成功地用CpG DNA修饰了外泌体。CpG-SAV-exo显示将带有CpG DNA的外来体有效并同时递送到培养的鼠类树突状DC2.4细胞中。用CpG-SAV-exo处理可有效激活DC2.4细胞并增强肿瘤抗原呈递能力。与携带外泌体和CpG DNA的简单共同给药相比，用CpG-SAV-exo免疫在B16BL6荷瘤小鼠中表现出更强的体内抗肿瘤作用。因此，基因工程CpG-SAV-exo是有效的基于外来体的肿瘤抗原-佐剂共递送系统，可用于癌症免疫治疗。CpG-SAV-exo显示将带有CpG DNA的外来体有效并同时递送到培养的鼠类树突状DC2.4细胞中。用CpG-SAV-exo处理可有效激活DC2.4细胞并增强肿瘤抗原呈递能力。与携带外泌体和CpG DNA的简单共同给药相比，用CpG-SAV-exo免疫在B16BL6荷瘤小鼠中表现出更强的体内抗肿瘤作用。因此，基因工程CpG-SAV-exo是有效的基于外来体的肿瘤抗原-佐剂共递送系统，可用于癌症免疫治疗。CpG-SAV-exo显示将带有CpG DNA的外来体有效并同时递送到培养的鼠类树突状DC2.4细胞中。用CpG-SAV-exo处理可有效激活DC2.4细胞并增强肿瘤抗原呈递能力。与携带外泌体和CpG DNA的简单共同给药相比，用CpG-SAV-exo免疫在B16BL6荷瘤小鼠中表现出更强的体内抗肿瘤作用。因此，基因工程CpG-SAV-exo是有效的基于外来体的肿瘤抗原-佐剂共递送系统，可用于癌症免疫治疗。与携带外泌体和CpG DNA的简单共同给药相比，用CpG-SAV-exo免疫在B16BL6荷瘤小鼠中表现出更强的体内抗肿瘤作用。因此，基因工程CpG-SAV-exo是有效的基于外来体的肿瘤抗原-佐剂共递送系统，可用于癌症免疫治疗。与携带外泌体和CpG DNA的简单共同给药相比，用CpG-SAV-exo免疫在B16BL6荷瘤小鼠中表现出更强的体内抗肿瘤作用。因此，基因工程CpG-SAV-exo是有效的基于外来体的肿瘤抗原-佐剂共递送系统，可用于癌症免疫治疗。