Using our mouse dendritic cell targeting nanobody we validated our preclinical data. Optimal nanobody conjugation site and linkage type was determined for MHCI and MHCII epitope (MHCIp and MHCIIp) delivery. C-terminal epitopes linked via a dipeptide protease cleavage motif outperform N-terminal and C-terminal epitope without such motif. Suboptimal in vitro activation of CD8+ T cell by N-terminally fused MHCIp is rescued by nanobody-based codelivery of MHCIIp via site-specific conjugation. Both CD4+ and CD8+ T cell activation are enhanced via codelivery compared to separate delivery on independent nanobodies. We hypothesized that at lower doses, not all DCs could prime both CD8+ and CD4+ T cells when antigens were delivered on separate nanobodies. This suggested a role for direct interactions between CD8+, CD4+ T cells, and DCs in eliciting an increased CD8+ T cell response. Microscopy analysis revealed that codelivery of MHCIp and MHCIIp conjugated to our nanobody to the same DCs increased recruitment of both CD8+ and CD4+ T cells during early T cell priming compared to delivery using separate nanobody conjugates.
In an adoptive transfer model of T cell memory response nanobody mediated codelivery of MHCIp and MHCIIp to DCs increased CD8+ T cell activation in vivo. In line with our in vitro findings, the cytotoxic activity of CD8+ T cells was also more robust when both epitopes were codelivered by the same nanobody. This suggested a role for direct interactions between CD8+ and CD4+T cells onto the same DCs to provide optimal CD4+ help signals. We next examined the effect of codelivery on primary T cell response. In a prime-boost strategy mice were vaccinated twice 21 days apart and challenged with target cells seven days after the second injection. At the dose investigated, all vaccines led to a similar proportion of lysis, suggesting no advantage to vaccinating animals with a CD4+ epitope. This could mean two things: this prime-boost strategy does not require CD4+ T cells in a primary setup. Or nanobody mediated MHCIIp delivery did not induce primary CD4+ T cell activation / expansion. This is currently still under investigation, also in the context of an analogous strategy in which a ‘common’ MHCII epitope was used.
In parallel, an engineering pipeline to generate nanobody - peptide epitope conjugates targeting human DCs has been established. We identified poor solubility of in particular MHCIp to be a crucial bottleneck in the process and we have identified several potential solutions which will be further examined in follow-up work.
Overall, this project delivered crucial fundamental insights both from an immunological, as well as an engineering perspective, which are highly valuable for the advancement of DC targeted peptide epitope delivery towards a clinical vaccination approach.
