Team:Evry/Project/Encapsulation

The pathological interactions between cancer cells and host immune cells in the tumour microenvironment create an immunosuppressive network that promotes tumour growth, protects the tumour from immune attack and attenuates immunotherapeutic efficacy (3,8).

The differentiation and maturation of myeloid DCs is profoundly suppressed by factors present in the tumour microenvironment. Indeed, cancer cells can massively produce molecules like VEGF (9),(10) or IL-12, causes an aberrant cytokine pattern in the tumour environment. That imbalance in the tumour microenvironment blocks DC differentiation and maturation. Thus, it induces either suppressive Treg cells (11,12) or T-cell unresponsiveness (13).

In this context, we’re aiming to overcome the immunosuppressive environment created by cancer cells. Our strategy is based on Interferon gamma (IFNγ), a cytokine that stimulates immune response and creates a favorable environment to enable tumor regression when coupled to an appropriate therapy. To deliver IFNγ into the tumor, we engineered yeasts capable of secreting it.

Figure 1 : Yeast design to secrete IFN gamma through alginate beads.

IFNγ is also known by the name canonical Th1 cytokine, and belongs to the cytokine family. This protein has a crucial role in the immune system adaptive responses. It contributes to the macrophages activation (1), controls the differentiation of naïve T-CD4 and T-CD8 cells, enhances lymphocyte recruitment and finally prolongs the activation of TCD4 and TCD8 in tissues (7). Indeed, IFNγ can stimulate the production of MHCI and MHCII on dendritic cells, resulting on the activation of T-Cells (4). Moreover, IFNγ is an immunomodulator. Indeed, it can up regulates pro-inflammatory or anti-inflammatory cytokines (6).

Nowadays Interferon gamma is used in therapies for its immunomodulator properties. In the case of cancer it is used in combination with chemotherapies or radiotherapies. Multiple cancer type were studied and attested the interferon positive effect in tumor regression (5). Moreover, IFNγ is approved by the FDA to treat Granulomatous chronic, an auto immune disease (2). Imukin® is an example of medicine sold nowadays.

We choose IFNγ to perform an Interferon gamma recombinant in yeast because it has glycosylated site and Disulfide Bridge. Consequently, all sequences used were codon optimized for yeast. We designed IFN-gamma fused with MAT alpha, which is the peptide leader used as a secretion signal. In addition, the S. cerevisiae’s ADH1 constitutive promoter was chosen to ensure a continuous secretion of Interferon gamma.

Upon injection, yeasts are targeted by the immune system. To protect them and extend their activities, they were encapsulated in alginate beads. A mix with alginate and Calcium chloride composed the polymer. Bead size is a central point; the idea is to have a balance between yeast protection and IFNγ liberation in the environment. We realized the proof of concept with two millimeters diameters beads and we will continue with a smaller size (25µm), which are adapted to therapeutic conditions. The polymer is very stable in time. The first sign of degradation appeared at 12 weeks and can stay six weeks.

As a proof of concept, we first encapsulated successfully yeasts producing GFP. GFP was placed under the galactose inducible promoter GAL1. Induction by addition of galactose in the media confirmed that yeasts were living inside the beads. (figure 3)

Figure 2 : Encapsulated yeasts producing GFP observed with Transilluminator

Figure 3 : Encapsulated yeasts producing GFP with or without galactose observed with UV light.

Figure 4 : Plasmid of ADHI-Matalpha-Interferon gamma

Yeasts were cloned by golden gate assembly to secrete IFN gamma. The secretion signal Matalpha was fused to Interferon gamma and placed under the strong constitutive promoter ADHI after removal of GAL1 by site-directed mutagenesis. Control digestion confirmed the correct removal and assembly inside plasmid pYGG1:

Figure 5 : Control Digestion with HindIII confirmed insert assembly

Finally, we encapsulated the yeasts in beads and checked the supernatant IFN gamma by ELISA after 48h induction in galactose media. We did not measure detectable IFN gamma secretion and we need further characterization to finalize the project.

References

1. Boehm, U., Klamp, T., Groot, M., and Howard, J. C. (1997). Cellular responses to interferon-gamma. Annu. Rev. Immunol.15, 749 –795. 

2. R. Alan B. Ezekowitz, M.B., Ch.B., D.Phil., Mary C. Dinauer, M.D., Ph.D., Howard S. Jaffe, M.D., Stuart H. Orkin, M.D., and Peter E. Newburger, M.D. (1998) Partial Correction of the Phagocyte Defect in Patients with X-Linked Chronic Granulomatous Disease by Subcutaneous Interferon Gamma, N Engl J Med 1988; 319:146-151 

3. Gabriel A Rabinovich, Dmitry Gabrilovich, Eduardo M Sotomayor, (2007) Immunosuppresive strategies that are mediated by tumor cells, Annu Rev Immunol. 2007; 25: 267–296.

4. Zhou F (2009) Molecular mechanisms of IFN-gamma to up-regulate MHC class I antigen processing and presentation Int Rev Immunol. 2009;28(3-4):239-60. doi: 10.1080/08830180902978120.

5. Ehtesham, M., Samoto, K., Kabos, P., Acosta, F. L., Gutierrez, M. A., Black, K. L., & Yu, J. S. (2002). Treatment of intracranial glioma with in situ interferon-gamma and tumor necrosis factor-alpha gene transfer. Cancer Gene Therapy, 9(11), 925–934. doi:10.1038/sj.cgt.7700516 

6. Mühl, H., & Pfeilschifter, J. (2003). Anti-inflammatory properties of pro-inflammatory interferon-γ. International Immunopharmacology, 3(9), 1247–1255. doi:10.1016/S1567-5769(03)00131-0 

7. Schroder, K., Hertzog, P. J., Ravasi, T., & Hume, D. A. (2004). Interferon: an overview of signals , mechanisms and functions, (February). doi:10.1189/jlb.0603252.Journal 

8. Weiping Zou (April 2005) Immunosuppressive networks in the tumour environment and their therapeutic relevance Nature Reviews Cancer 5, 263-274 | doi:10.1038/nrc1586 

9. Kryczek, I. et al. CXCL12 and vascular endothelial growth factor synergistically induce neoangiogenesis in human ovarian cancers. Cancer Res. 65, 465–472 (2005). 

10. Carmeliet, P. & Jain, R. K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000). 

11. Jonuleit, H., Schmitt, E., Schuler, G., Knop, J. & Enk, A. H. Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192, 1213–1222 (2000).

12. Dhodapkar, M. V., Steinman, R. M., Krasovsky, J., Munz, C. & Bhardwaj, N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J. Exp. Med. 193, 233–238 (2001).

13.Hawiger, D. et al. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194, 769–779 (2001).