Part:BBa_K1611004

DEC-205

This is a scFv antibody against DEC205. DEC205 is an endocytic receptor that is expressed by dendritic cells. Antigen endocytosed via DEC205 enters the MHC class I and MHC class II antigen presentation pathways and is subsequently presented to both CD4+ and CD8+ T cells. We used it to activate T-CD8 cells, with tumorous antigen that we binded to DEC205 scFv.

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 (1). 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 (2). In the same way, immunization with DNA vectors encoding antigens fused to a DEC-205 scFv elicits a strong specific CD8+ responses in vivo (3).

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 (4), 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 results

We cloned the full fusion protein OVA1-DEC205-HAtag inside our yeast with AGA1P. It was correctly displayed on yeast membrane (figure 1). HA-tag was fused at the end C-terminal of the protein to ensure complete translation upon detection. In both experiment, 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 1: 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 2). 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 3), suggesting the role of DEC205 in cross-presenting OVA1.

Figure 2: Flow cytometry of Dendritic cells CD11C+ with MHCII/CD86/80/MHCI labels

Figure 3: 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.

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 4). 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 4: 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.

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 5). 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 5: 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 6: Sequencing of DEC205 cloned in pSB1C3 vector. The black line shows the consensus sequence.

References

1. 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

2. 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

3. 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

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 533
Illegal XhoI site found at 414
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]