Surface display

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). CD4+ priming by tumor MHC II antigen expression in yeast cytosol Many papers show that CD4+ T cells are as important as CD8+ in the antitumor action. CD4+ T helper have a supporting role in the immune response because : • Depletion of CD4+ T cells with a monoclonal antibody just before tumor challenge resulted in the complete loss of tumor rejection, in contrast with mice with intact immune system (30), confirming the role of CD4+ in tumor suppression. • CD4+ T cells express both Th1 and Th2 cytokines and recruit other antitumor effector cells, in addition of priming CD8+ T cells through OX40. Through the local release of cytokines, CD4+ direct the effector response, recruiting and activating tumoricidal macrophages and eosinophils (31). Eosinophil peroxidase synergizes with macrophage reactive oxygen intermediates to kill tumor cells. • Adoptive transfer of activated CD4  T-cell clones specific for the murine leukemias have been demonstrated to confer systemic anti-tumor immunity upon transfer into tumor-bearing hosts (32). To benefit from the presence of activated CD4+, we created a yeast to prime CD4+ against the tumor antigen OVA2. To prime CD4+ against the tumor antigen, we transformed the yeast S. cerevisiae to express OVA2 in its cytosol. OVA2 is the peptide with the sequence SLKISQAVHAAHAEINEAGREVV, binding to MHC class II protein (33). OVA2 was cloned into yeast cytosol and conjugated to myc-c tag for detection purpose..