Anti-Tumor Efficacy of In Situ Vaccination Using Bacterial Outer Membrane Vesicles.

In situ vaccination (ISV) is a promising cancer immunotherapy strategy that consists of the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site, and therefore, to elicit an effective anti-tumor immune response, all that is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial outer membrane vesicles (OMVs) are potent adjuvants since they contain several microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work, we first show that the OMVs from E. coli BL21(DE3)Δ60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of three different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse model. Overall, our data support the use of the OMVs in ISV and indicate that ISV efficacy can benefit from the addition of properly selected tumor-specific neo-antigens.

Proteome-minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram-negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes-specific antibody titers and high frequencies of epitope-specific IFN-γ-producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV-based vaccines.

Bacterial outer membrane vesicles engineered with lipidated antigens as a platform for Staphylococcus aureus vaccine.

Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 µg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.

Vaccination With a FAT1-Derived B Cell Epitope Combined With Tumor-Specific B and T Cell Epitopes Elicits Additive Protection in Cancer Mouse Models.

Human FAT1 is overexpressed on the surface of most colorectal cancers (CRCs) and in particular a 25 amino acid sequence (D8) present in one of the 34 cadherin extracellular repeats carries the epitope recognized by mAb198.3, a monoclonal antibody which partially protects mice from the challenge with human CRC cell lines in xenograft mouse models. Here we present data in immune competent mice demonstrating the potential of the D8-FAT1 epitope as CRC cancer vaccine. We first demonstrated that the mouse homolog of D8-FAT1 (mD8-FAT1) is also expressed on the surface of CT26 and B16F10 murine cell lines. We then engineered bacterial outer membranes vesicles (OMVs) with mD8-FAT1 and we showed that immunization of BALB/c and C57bl6 mice with engineered OMVs elicited anti-mD8-FAT1 antibodies and partially protected mice from the challenge against CT26 and EGFRvIII-B16F10 cell lines, respectively. We also show that when combined with OMVs decorated with the EGFRvIII B cell epitope or with OMVs carrying five tumor-specific CD4+ T cells neoepitopes, mD8-FAT1 OMVs conferred robust protection against tumor challenge in C57bl6 and BALB/c mice, respectively. Considering that FAT1 is overexpressed in both KRAS+ and KRAS- CRCs, these data support the development of anti-CRC cancer vaccines in which the D8-FAT1 epitope is used in combination with other CRC-specific antigens, including mutation-derived neoepitopes.

Synergistic Protective Activity of Tumor-Specific Epitopes Engineered in Bacterial Outer Membrane Vesicles.

INTRODUCTION: Bacterial outer membrane vesicles (OMVs) are naturally produced by all Gram-negative bacteria and, thanks to their plasticity and unique adjuvanticity, are emerging as an attractive vaccine platform. To test the applicability of OMVs in cancer immunotherapy, we decorated them with either one or two protective epitopes present in the B16F10EGFRvIII cell line and tested the protective activity of OMV immunization in C57BL/6 mice challenged with B16F10EGFRvIII. MATERIALS AND METHODS: The 14 amino acid B cell epitope of human epidermal growth factor receptor variant III (EGFRvIII) and the mutation-derived CD4+ T cell neo-epitope of kif18b gene (B16-M30) were used to decorate OMVs either alone or in combination. C57BL/6 were immunized with the OMVs and then challenged with B16F10EGFRvIII cells. Immunogenicity and protective activity was followed by measuring anti-EGFRvIII antibodies, M30-specific T cells, tumor-infiltrating cell population, and tumor growth. RESULTS: Immunization with engineered EGFRvIII-OMVs induced a strong inhibition of tumor growth after B16F10EGFRvIII challenge. Furthermore, mice immunized with engineered OMVs carrying both EGFRvIII and M30 epitopes were completely protected from tumor challenge. Immunization was accompanied by induction of high anti-EGFRvIII antibody titers, M30-specific T cells, and infiltration of CD4+ and CD8+ T cells at the tumor site. CONCLUSION: OMVs can be decorated with tumor antigens and can elicit antigen-specific, protective antitumor responses in immunocompetent mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs makes the OMV platform particularly attractive for cancer immunotherapy.

A HER2-specific Modified Fc Fragment (Fcab) Induces Antitumor Effects Through Degradation of HER2 and Apoptosis.

FS102 is a HER2-specific Fcab (Fc fragment with antigen binding), which binds HER2 with high affinity and recognizes an epitope that does not overlap with those of trastuzumab or pertuzumab. In tumor cells that express high levels of HER2, FS102 caused profound HER2 internalization and degradation leading to tumor cell apoptosis. The antitumor effect of FS102 in patient-derived xenografts (PDXs) correlated strongly with the HER2 amplification status of the tumors. Superior activity of FS102 over trastuzumab or the combination of trastuzumab and pertuzumab was observed in vitro and in vivo when the gene copy number of HER2 was equal to or exceeded 10 per cell based on quantitative polymerase chain reaction (qPCR). Thus, FS102 induced complete and sustained tumor regression in a significant proportion of HER2-high PDX tumor models. We hypothesize that the unique structure and/or epitope of FS102 enables the Fcab to internalize and degrade cell surface HER2 more efficiently than standard of care antibodies. In turn, increased depletion of HER2 commits the cells to apoptosis as a result of oncogene shock. FS102 has the potential of a biomarker-driven therapeutic that derives superior antitumor effects from a unique mechanism-of-action in tumor cells which are oncogenically addicted to the HER2 pathway due to overexpression.