# Design of Outer Membrane Vesicles as Cancer Vaccines: A New Toolkit for Cancer Therapy
## 3 New OMV Applications: Tumor Vaccines
### Bacterial OMVs as Cancer Vaccines  
Over a century ago, [PERSON] developed the first bacteria-based cancer treatment by injecting killed bacteria directly into a tumor after having observed regression of the tumor subsequent to bacterial injection. This suggests that live-attenuated bacteria can potentially function as cancer antigen carriers through genetic modification [106]. In addition, the concept of exploiting bacteria as biological tumor vaccine vectors has existed for some time, and the emergence of the anti-tumor strategy of using live bacteria provided the theoretical basis for the development of OMV as a cancer vaccine by shedding from the bacterial outer membrane. Here, we summarize multiple live bacterial vectors used in preclinical research and clinical trials for anti-cancer therapy to better use these live bacterial vectors for the development of OMV-based cancer vaccines (Table 2). Use of bacteria as vectors for cancer vaccines has included intracellular _Salmonella_, _Listeria_, _Pseudomonas aeruginosa_ (_P. aeruginosa_), and _E. coli_, for which escape from the phagosome requires virulence factors like Listerolysin O and phospholipase C to degrade the phagosomal membrane and migrate to neighboring cells by a direct cell-to-cell transfer mechanism. In addition, _Listeria_ and _Salmonella_ vectors have been shown to activate innate immunity. This occurs through TLR binding, secretion of inflammatory cytokines and chemokines, upregulation of co-stimulatory molecules, stimulation of antigen specific CD4+ and CD8+ T cells, and suppression of regulatory T cells [107; 108]. Thus, these two attenuated bacteria are commonly used to deliver cancer antigens. Several other Gram-positive bacteria have also been used, including _Clostridium_ and _Bifidobacterium_, and these organisms can utilize MHC II molecules to deliver antigens [109]. Therefore, these Gram-positive bacteria can also purify their EVs for cancer vaccine development (Table 2) [110; 111]. Although many live bacteria have been used as cancer vaccines in preclinical studies, the biggest problem hindering the entry of such vaccines into clinical trials and clinical application is the balance between safety and immunogenicity. Due to its safety and high efficiency, OMVs provide new choices and ideas for the application of these bacterial vectors as cancer therapeutic vaccines. OMVs stimulate both humoral and cell-mediated immunity in a manner similar to bacteria; however, they are superior to bacteria in their safety profile and ease of production. Moreover, while intracellular bacteria could be used as live delivery vectors, they could also be utilized to purify OMVs genetically engineered for cancer therapy; several researchers have attempted this and achieved positive results. [PERSON] et al., established the AffiHER2 OMV vaccine by utilizing siRNA to target kinesin spindle protein and select human epidermal growth factor receptor 2 (HER2) as the receptor in tumor cells [105]. They derived OMVs displaying a HER2-specific affibody from a mutant _E. coli_ strain and injected them into mice. The results indicate that systemic injection of siRNA-packaged OMVs caused targeted gene silencing and induced highly significant tumor growth regression in an animal model. More importantly, the modified OMVs were well tolerated and showed no evidence of nonspecific side effects [105]. Moreover, [PERSON] et al., selected HPV16 E7 protein as a tumor antigen and utilized the Trx protein to present E7 both on the surface and in the lumen of OMVs [101]. In contrast to siRNA recombinant OMVs, these were taken up rapidly by DCs, significantly stimulating the expression of DC maturation markers CD80, CD86, CD83, and CD40. As a result, numbers of interferon-gamma (IFN-\(\gamma\))-expressing splenocytes and IFN-\(\gamma\)-expressing CD4+ and CD8+ cells increased, and growth of grafted TC-1 tumors in mice was significantly suppressed [101].