In vitro and in vivo proof of concept

After creating software prediction of tumor antigen and a functional surface displaying system, we intend to deliver the predicted antigen to the immune system. As a proof of concept, we tested in vitro on dendritic cells and macrophages extracted from mice spleen the yeast displaying system.

Then, we performed an in vivo assay on melanoma bearing mice. The melanoma chosen was the very aggressive cell line B16-OVA. Our yeast chassis was injected after the large tumoral development of melanoma in order to assess immune induction in a true clinical context with patients presenting advance cancer. Now it's time to turn to the results.

We cloned the full fusion protein OVA1-DEC205-HAtag inside our yeast with AGA1P. It was correctly displayed on yeast membrane (figure 4). HA-tag was fused at the end C-terminal of the protein to ensure complete translation upon detection. In both experiment (figure 2), the yeast was transformed to express AGA2P-OVA1-DEC205-HAtag and labelled with antibody anti-HA conjugated to the fluorochrome emitting at 650 nm.

Figure 4: Immunofluorescence microscopy with Ab anti-HA (650 nm). A. Yeast expressing AGA2P-OVA1-DEC205-HAtag B. Yeast expressing AGA2P-OVA1-DEC205-HAtag and membrane protein AGA1P

Incubation of yeast resulted in DC up-regulates MHC class I, MHC class II, CD80 and CD86 molecules, indicating efficient maturation of this cells. OVA1-DEC205 induces strong DC presentation of immune markers (figure 5). Immune markers show the induction of the DC with increasing CD80/CD86 and MHCI/MHCII for all transformed yeast in comparison with the wild type yeast. The most potent DC immune markers up regulation was obtained for OVA1-DEC205 surface displaying yeasts (figure 6), suggesting the role of DEC205 in cross-presenting OVA1.

Figure 5: Flow cytometry of Dendritic cells CD11C+ with MHCII/CD86/80/MHCI labels

Figure 6: In vitro phenotyping of murine dendritic cells extracted from spleen with wild type yeast (WT), yeast displaying OVA1 (OVA1), DEC205 or OVA1-DEC205. Khi tests were calculated between WT and each construction (black asteriks) or pair-colored asterisks for paired conditions.

Macrophages markers CD80/CD86 were not induced in comparison with DC.

DCs are considered to be the best candidate for T-cells activation against cancer because they bear MHC products 10-100 times higher than others APCs and because they secrete T cells co-stimulatory molecules (5). To support this evidence, we studied in vitro priming of macrophages that we isolated from mouse spleen (figure 7). Flow cytometry showed that MHCI was more strongly induced in all recombinant yeasts in comparison with wild type with a level matching DCs MHC I in contradiction with previous results (5). However, co-stimulatory CD80/CD86 molecules were not produced by macrophages. The well-known « two signal model » indicates that DCs must bear MHC I tumor antigen and costimulatory CD80/CD86 molecules to stimulate T-cells proliferation. This absence of CD80/CD86 impairs CD8+ activation, confirming our DC targeting strategy.

Figure 7: In vitro phenotyping of murine macrophages extracted from spleen with wild type yeast (WT), yeast displaying OVA1 (OVA1), DEC205 or OVA1-DEC205. Khi tests were calculated between pair-colored asterisks for paired conditions.

High number of yeasts is detrimental to DC and fixed yeasts improve priming.

The higher yeast concentration was, the lower immune markers were upregulated in DCs (figure 8). This suggest a toxicity caused by the yeasts in high concentration. To assess this hypothesis, S. cerevisiae were fixed in 0.5 % paraformaldehyde and loaded on to DCs for 24h. Immunomarkers were significantly upregulated for yeasts fixed in comparison with yeasts not fixed. Our result suggest that fixation does not abrogate MHC I presentation. Fixation increases safety of the vector and facilitates the vaccine supply chain while maintaining the efficiency of the yeasts and therefore will be applied for the next in vivo assays. We performed a CFU assay for fixed yeasts (data not shown) and results came negative, showing that all yeasts were effectively killed by fixation.

Figure 8: In vitro phenotyping of murine DCs extracted from spleen with OVA1-DEC205 yeasts. Khi tests were calculated between each condition for MHC-I+. MOI10 corresponds to 10 yeasts for 1 DC, MOI1 to 1 yeast for 1 DC, MOI10 FIXED and MOI1 FIXED correspond to the same MOI with fixed yeasts in 0,5 % PFA.

Yeast detrimental effect could be observed even at MOI1 with fixed yeasts. A viability assay on DC confirmed that many DCs were dead after 24h of co-incubation with yeasts (red dots on figure below).

Figure 9: Flow cytometry data of DCs coincubated at MOI1 with fixed yeasts during 24h.

Red dots correspond to dead DC and purple dots to living DCs.

The ability of recombinant yeasts to elicit anti-tumor immune response in vivo was examined. In vivo assay on melanoma mice confirmed T-cell induction against the tumor antigen OVA1 (figure 10). Mice were transfected with the melanoma cell line B16-OVA at day 0, and yeasts were injected at day 10 inside a large tumor reaching 7 mm. In this experiment, a tetramer assay for blood CD8+ OVA1 was performed after mice sacrifice at day+18. Vaccination with yeast OVA1-DEC205/OVA2 resulted in a significant CD8+ OVA1 induction compared with PBS control and wild type yeast. This is coherent with in vitro DC immunophenotyping. Because of the large and heterogeneous tumor size, tumor regression cannot be measured accurately at this stage of late injection.

Figure 10: Tetramer assay for CD8+ OVA1 specific T cells extracted from blood on melanoma mice C57BLC/6. Mice were sacrifice 18 days after tumor challenge with B16-OVA melanoma cell line. Mice received 3 injections of 2.10^7 yeasts.

Conclusion

We have designed a new chassis that was tested in vivo to induce a strong immune response against tumor antigen. In combination with our software predicting the best tumor antigen from the patient sequencing data. This chassis establishes a new paradigm. Instead of adapting the patient to the therapies available, we can adapt the therapy to the patient. Our chassis is scalable because it targets immune cells in vivo, making a personalized medicine a reality. In addition of tumor antigen delivery system, we have developed a recombinant encapsulated yeast to break the tolerogenic tumor environment. Finally, a yeast bio-sensor could detect and target hypoxic resistant cancer cells and prevent further metastasis.

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