Periodic Reporting for period 1 - CNECT-VAX (C- and N-terminal Epitope Conjugate immune Cell Targeted Vaccines)
Reporting period: 2022-06-01 to 2023-11-30
Vaccination is arguably one of the greatest medical achievements, which has dramatically improved public health. Cancer vaccines hold great promises for the prevention and treatment of malignancies. Several gynaecological cancers can successfully be prevented with human papillomavirus (HPV) vaccines. However, vaccination against non-virally induced cancers remains a challenge. Advances in sequencing approaches led to the discovery of patient-specific neo-antigens derived from genetic mutations in tumor cells. These neo-antigens sparked research activities in the cancer vaccination field, since next to shared tumor-associated antigens these could be used to induce or boost cancer-specific immune responses. While initial research has focused on eliciting CD8+ killer T cell responses, it is becoming increasingly apparent that CD4+ helper T cells are crucial in generating optimal immune responses and should be considered in developing therapeutic strategies. Based on preclinical research it is well established that targeted delivery of antigenic vaccine cargo to dendritic cells (DCs) drastically improves vaccine efficacy, yet only very few of these strategies are available in the clinic. This project aims to investigate the potential for clinical translation of a nanobody-based targeted vaccine for specific co-delivery of CD8+ and CD4+ epitopes to DCs in vivo.
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.
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.
Based on conducted market research and the scientific outcomes during this project we were recommended by our business development team to terminate our patent filing and focus our efforts and resources on the newly gained insights to obtained new compositions of matter for future patent applications. Key needs to ensure further uptake and success include obtaining funding to conduct further research based on the findings of this project. Taken together, this project resulted in fundamental scientific insights, as well as market and IPR information, which are highly valuable for the advancement of DC targeted peptide epitope delivery towards a clinical vaccination approach.