# Design of Outer Membrane Vesicles as Cancer Vaccines: A New Toolkit for Cancer Therapy
## 3 New OMV Applications: Tumor Vaccines
### Advantages of OMVs as Cancer Vaccines  
Cancer vaccines commonly include three essential components: tumor special antigen or tumor associated antigen, potent adjuvants, and a delivery system. Loss of any of these components can lead to an ineffective immune response or even drive antigen loss and immune evasion [88]. The appropriate selection of tumor antigens has been reviewed [29]. Here, we restrict our discussion to OMVs as the suitable adjuvants and efficient delivery vectors of tumor antigens. Through the suitable design of an OMV, it can be more efficient in delivering tumor antigens or molecule drugs to fulfill antitumor treatment. OMVs can easily be decorated with foreign antigens or epitopes via different synthetic biology approaches, especially by utilizing protein antigen-coding RNA and siRNA interfering gene expression. Furthermore, OMVs are capable of carrying more than one cancer-specific epitope, corresponding to the diversity of tumor antigens (Figure 2). The formation of OMVs involves a sorting mechanism, where proteins are screened and selectively localized to the periplasm or outer membrane. Utilizing this mechanism, different tumor antigens can be delivered to the lumen or surface of vesicles by fusing their coding sequences to those proteins which act as leader peptides for delivery. Some tumor antigens can be located on the surface of OMVs to be better recognized by APCs and thus, induce immune response to kill tumor cells. However, in order to reach deeper tumor tissues to fulfill function, other tumor antigens and small molecule drugs need to be expressed inside the OMVs, which can be hydrophobically encapsulated by lipid bilayers to protect the targeted antigens from degradation of exogenous proteases and early recognition of the immune system [89; 90]. Nevertheless, several studies have demonstrated that various antigens delivered to the lumen of OMVs can elicit an effective antibody response and significant protection [19; 71]. Initially, the strategy of displaying heterologous antigens into OMVs was to transport the target antigens to the periplasm space by fusing to the N-terminal \(\beta\)-lactamase signal sequence, which was then encapsulated in lumen during the formation of OMVs [71]. Furthermore, [PERSON] et al., tested SlyB as a carrier to direct proteins to the interior of OMVs; cohesin domains were inserted between the Z-domain and INP and functionalized with a dockerin-tagged GFP for cancer cell imaging, indicating the potential role for SlyB and INP as leader peptides to carry antigens [91]. Another delivery system involves fusing antigens with specific proteins such as ClyA, AIDA-I, and OspA which act as leader sequences for antigen delivery [92; 93; 94]. Currently the most studied leader peptide directing heterologous antigens to the surface of OMVs is ClyA [95; 96; 97]; genetic fusion between recombinant polypeptides and the C-terminus of ClyA results in a functional display of recombinant protein on  
Figure 2: Schematic representation of mechanisms by which OMVs act as carriers to deliver tumor antigens in cancer therapy. Tumor antigens are targeted to the membrane by the leading peptide. OMVs are released from mutant bacterial cells and carry tumor antigens. OMVs travel via blood vessels to arrive at the target tissue. Cells mainly recognize OMVs by ligand-receptor interaction and internalize OMVs by membrane invagination or membrane fusion. the surface of _E. coli_ and their derived OMVs [96]. Moreover, several heterologous antigens have also been successfully exported to the surface of OMVs when fused to the \(\beta\)-barrel forming auto transporter AIDA or borrelial lipoprotein OspA [19; 94]. In addition to using protein fusion to display antigens, many studies demonstrated that a hemoglobin protease (Hbp) autotransporter platform, originally developed by [PERSON] et al., could also be used to display heterologous antigens on the surface of OMVs [98; 99; 100].