|dc.description.abstract||Recently, new discoveries in the field of cancer immunology have been able to increase the strength of immune responses against tumors. For example, monoclonal antibodies such as Ipilimumab and Nivolumab are able to drastically reduce the suppressive capabilities of cancer cells and regulatory T cells leading to complete or partial responses in a substantial portion of patients with non-small-cell lung cancer, melanoma and renal-cell cancer. Other immuno-therapeutic approaches are aimed at stimulating an immune response against specific molecular targets expressed by tumor cells. This could be achieved by the administration of modified immune cells derived from the patient or by inducing an immune response against tumor cells with cancer vaccines. In the case of cancer vaccines, the therapy involves the delivery of an antigen (in the form of protein or peptide) that is expressed by cancer cells to antigen presenting cells (APCs). To further modulate the activity of target APCs, the delivery system could be designed to enhance the function of APCs as an adjuvant therapy in order to stimulate a stronger and prolonged immune response.
Recent data suggest that the function and survival of APCs can be modulated by targeting genes involved in immune suppression/regulation with small interfering RNAs (siRNAs). siRNAs are a class of double stranded RNA molecules designed to interfere with the expression of specific genes with complementary nucleotide sequences. siRNAs are incorporated into the RNA interference specificity complex (RISC) leading to the cleavage and degradation of the target mRNA.
Since siRNAs are prone to ribonucleases and lysosomal degradation, they require a delivery system designed to rapidly target an appropriate cell population and to avoid degradation. This research work addresses whether implementation of a molecular release mechanism associated with an APC targeting vector would be advantageous to avoid the degradation of siRNAs and peptide antigens. A bacteria derived microparticle called MIS416 designed to target APCs was used as a delivery system to test this hypothesis.
MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes and comprises NOD-2 (nucleotide-binding oligomerization domain containing 2) and TLR-9 (toll-like receptor-9) ligands, both of which have well-described adjuvant activity. Given its inherent adjuvant properties, MIS416 microparticles could provide an ideal vehicle for co-delivery of cargo such as peptide antigens, as well as immunomodulatory siRNAs to APCs.
In this work, MIS416 microparticles were used as a vehicle for the delivery of the peptide antigen SIINFEKL and siRNAs to target Dendritic cells (DCs) in order to modulate the immune response against tumors. These conjugates were designed to facilitate the release of the attached molecular cargo by the inclusion of a glutathione sensitive cleavable bond (disulfide). The release strategy takes advantage of the different concentration of glutathione between the extracellular environment and the cytoplasm of target cells. This approach was hypothesized to facilitate the release of siRNAs and peptides from MIS416 after internalization of MIS416 conjugates in target cells to avoid lysosomal degradation. A conjugation strategy based on a streptavidin bridge was developed to link biotinylated peptide/siRNA to biotinylated MIS416. The conjugation strategy was validated by delivery of fluorophores and the model peptide antigen, SIINFEKL to DCs. MIS416/siRNA conjugates were also developed to investigate whether they could negatively regulate the expression of target proteins on DCs.
The results showed that conjugates containing a disulfide linker were able to more rapidily release SIINFEKL in the cytoplasm of DCs than conjugates not containing a disulfide. However, the inclusion of a cleavable bond in these conjugates did not improve the presentation of the antigen on MHC (major histocompatibility complex) molecules on DCs. Furthermore, DCs treated with MIS416/SIINFEKL conjugates were able to induce activation and expansion of specific CD8 T cells, in addition to a cytotoxic response against the peptide antigen SIINFEKL in treated mice. However, the cytotoxic response was greater in mice vaccinated with MIS416/SIINFEKL conjugates that did not possess a disulfide bond in the linker. Furthemore, following treatment of DCs with MIS416/siRNA conjugates target protein levels were significantly downregulated, leading to the conclusion that MIS416/siRNA conjugates should be investigated for in vivo use.
To conclude, the results suggest that a disulfide-based release strategy could be used for the delivery of siRNAs to DCs. However, the release mechanism does not improve the immune response generated by MIS416/SIINFEKL conjugates indicating that a more rapid release is not advantageous for peptide delivery. In a future extension of this research MIS416 microparticles could potentially be used in vivo for the co-delivery of peptide antigens and siRNAs, to modulate APC activity and to induce a specific immune response against molecular targets expressed by tumor cells.||