Part:BBa_K1611002
OVA1
OVA1 is an antigen expressed in tumor melanocytes cell line B16-OVA1. In the immune system, it is presented by the MHC-I to activate T-CD8 cells. It activated CD8+ in vivo against OVA1 in C57BL/6 mice, as tetramer CD8+ assays demonstrated.
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 (1). 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 (1).
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 (2) 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 (3). 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 (4).
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
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 3). 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 4), suggesting the role of DEC205 in cross-presenting OVA1.
Figure 3: Flow cytometry of Dendritic cells CD11C+ with MHCII/CD86/80/MHCI labels
Figure 4: 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 5). 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 5: 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.
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 6). 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 6: 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.
Part sequencing
Figure 7: Sequencing of OVA1 cloned in pSB1C3 vector. The black line shows the consensus sequence. LEQLESIINFEKLTEWTSA correspond to OVA1
References
1. Howland SW, Wittrup KD, Antigen release kinetics in the phagosome are critical to cross-presentation efficiency. J Immunol 2008;180:1576–1583
2. 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.
3. 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.
4. 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.
Sequence and Features
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