Difference between revisions of "Team:Evry/Project/SurfaceDisplay"

Surface display of tumor antigen for CD8+ cross-priming

We chose to express our antigen on the membranes of S. cerevisiae because surface displayed antigen is cross-presented much more efficiently than yeast cytosol antigen (22). This is due to a particular kinetics inside the early phagosome, allowing the external antigen to escape from the phagosome. Cross-presentation can be further enhanced by inserting linkers susceptible to Cathepsin S cleavage between the antigen and Aga2p, supporting the evidence that early antigen release is important for cross-presentation (22).

Enhancing cross-priming with the antibody anti-DEC205

Our surface display antigen for ovalbumin was fused to DEC205 scFv. DEC205 is a lectin receptor expressed by some DCs subsets, including mouse spleen DC (23). It was shown that antibody targeting DEC-205, fused to tumor antigen, can induce T cell stimulation if administered with an additional stimulus triggering DC maturation, like anti-CD40 agonistic antibody (24). In the same way, immunization with DNA vectors encoding antigens fused to a DEC-205 scFv elicits a strong specific CD8+ responses in vivo (25).

The scFv of DEC205 was fused to our ovalbumin tumor antigen and surface displayed in order to get the yeast internalized in a DC endosome through DEC205 receptor, favoring CD8+ cross-presentation. We used the scFv instead of the whole antibody for the possibility to perform repeated immunisations without inducing deleterious host responses against the Fc part of the immunoglobulin chains.

Advantages of Yeast expressing DEC205 over DEC205 protein vaccines

Pure protein vaccines with DEC205 are far less immunogenic than vaccine with micro-organisms mimicking pathogens and request an additional adjuvant. Moreover, the protein needs a prior step of antigen purification (26), leading us to develop this yeast surface display of DEC205 scFv fused to the antigen. The advantages of our system include better concentration of the yeast due to less diffusion than the protein DEC205 alone, the codelivery of both antigen and adjuvant, the possibility to target multiple DCs compartments at the same time (MHCI and MHC II) and the absence of purification step.

Surface display design

Several surface display systems exist for the yeast S. cerevisiae. In the context of cancer immunotherapy, whole yeast cells has been coated with several layers of cancer-testis antigen NY-ESO-1 with a chemical conjugation (27) and this system was able to cross-prime naive CD8+ T cells in vitro. Antigen was also linked chemically to the surface of a capsular yeast shell instead of the whole yeast (28). The advantage of chemical conjugation is the ability to reach a high antigen loading. However, this technique is limited to soluble antigens and most antigens are not soluble, leading us to reject this solution in order to broaden our system to any tumor antigen. In addition, chemical conjugation requires a purified antigen, increasing therapeutic application costs.

We selected the surface display system based on the mating adhesion receptor Aga2p and Aga1p. This system is widely used for antibody affinity studies and was used to anchor the antibody ScFv DEC205 fused to the ovalbumin tumor antigen to the yeast surface. Aga1p was expressed separately and aga2p fused in C-terminal to our displayed protein.

To establish a proof of concept, our system was tested in vivo on C57BL/6 mice injected with the melanoma cell line B16-OVA expressing the ovalbumin antigen. We also tested the system in vitro on hybridoma B3Z T-cells specific for SIINFEKL. The tumor antigen cloned in our vector was OVA1 corresponding to the sequence QLESIINFEKLTEW, class I (Kb)-restricted peptide epitope of ovalbumin (OVA) plus 3 amino acids around the epitope to allow better digestion by the proteasome. It is presented by the class I MHC molecule H-2Kb (29).

Figure 1: Yeast surface display expressing troll antigen to carry out immunotherapy via MHC-I

(1) Surface Display of tumor antigen OVA1 fused to DEC205 scFv

(2) Yeast internalization in cross-presenting endosomes specific for DEC205

(3) CD8+ T cell cross-priming with tumor antigen OVA1

(4) Cancer cell lysis with antigen OVA1 targetin

Cloning results

We transformed the yeast to express OVA1 or OVA1-DEC205 on surface. We used AGA1P co-expression and AGA2P C-terminal fusion to the protein in order to get membrane presentation.

Figure 2: Plasmids with the constructions : A) AGA1P (B) AGA2P-OVA1-DEC205 (C) AGA2P-OVA1 (D) AGA2P-DEC205

Surface display results

Before displaying our tumor antigen fused to the scFv DEC205, we first cloned the fusion protein AGA2P-GFP in order to observe surface display of GFP with our without the coexpression AGA1P. We obtained a GFP signal located around the membrane only in presence of AGA1P coexpression.

Figure 3: GFP fused to AGA2P observed in fluorescence microscopy. A) without AGA1P (B) with AGA1P (C) with AGA1P with Z-scale on one single yeast.

Next, 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.

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