Revision as of 22:13, 18 September 2015

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).

e

(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

References

22. Howland SW, Wittrup KD, Antigen release kinetics in the phagosome are critical to cross-presentation efficiency. J Immunol 2008;180:1576–1583

23. Anjuere F, Martin P, Ferrero I, Fraga ML, del Hoyo GM, Wright N, Ardavin C, Definition of dendritic cell subpopulations present in the spleen, Peyer’s patches, lymph nodes, and skin of the mouse, 1999, Blood 93, 590–598

24. Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii SI, Soares H, Brimnes MK, Moltedo B, Moran TM, Steinman RM, In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. 2004, J. Exp. Med. 199, 815–824

25. Demangel C, J Zhou, A BH Choo, G Shoebridge, GM. Halliday, WJ Britton, Single chain antibody fragments for the selective targeting of antigens to dendritic cells, 2005 May, Mol Immunol. 42(8):979-85

26. Petrovsky N, Aguilar JC, Vaccine adjuvants: current state and future trends Immunol Cell Biol. 2004;82:488–496

24. Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii SI, Soares H, Brimnes MK, Moltedo B, Moran TM, Steinman RM, In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. 2004, J. Exp. Med. 199, 815–824.

25.Demangel C, J Zhou, A BH Choo, G Shoebridge, GM. Halliday, WJ Britton, Single chain antibody fragments for the selective targeting of antigens to dendritic cells, 2005 May, Mol Immunol. 42(8):979-85.

26. Petrovsky N, Aguilar JC, Vaccine adjuvants: current state and future trends Immunol Cell Biol. 2004;82:488–496.

27. Howland SW, T Tsuji, S Gnjatic, G Ritter, LJ. Old and K D Wittrup, Inducing Efficient Cross-priming Using Antigen-coated Yeast Particles, J Immunother. 2008 September ; 31(7): 607. doi:10.1097/CJI.0b013e318181c87f.

28. Pan Y, Li X, Kang T, Meng H, Chen Z, Yang L, Wu Y, Wei Y, Gou M, Efficient delivery of antigen to DCs using yeast-derived microparticles, 2015, Sci. Rep. 5, 10687; doi: 10.1038/srep10687.

29. Rötzschke O, Falk K, Stevanović S, Jung G, Walden P, Rammensee HG, Exact prediction of a natural T cell epitope, 1991, Eur J Immunol.21(11):2891-4.

30. Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, Jackson V, Hamada H, Pardoll D, Mulligan RC. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony–stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA. 1993;90:3539–3543.

31. Hun K, R Hayashi, A Lafond-Walker, C Lowenstein, D Pardoll, H Levitsky, The central role of CD4+ T cells in the antitumor immune response, 1998, J. Exp. Med. 188, pp. 2357–2368.

32. Bookman MA, Swerdlow R, Matis LA: Adoptive chemoimmunotherapy of murine leukemia with helper T lymphocyte clones. J Immunol 1987, 139:3166-3170.

33. Johnsen G & Elsayed S, Antigenic and allergenic determinants of ovalbumin-111. MHC Ia-binding peptide (OA 323-339) interacts with human and rabbit specific antibodies, 1990, Mol Immunol. 27821-7.