Dancing with dendritic cells: Targeting human skin dendritic cells for anti-tumor immunity

In the studies described in this thesis we explored the potential of human skin DC to induce anti-tumor immunity. By the design of various vaccines we explored how the multiple human skin DC subsets responded for the induction of robust adaptive anti-tumor immunity. For intradermally injected particulate-vaccines, we need to ensure that the tumor-antigen containing particle will reach the targeted DC subset to facilitate the induction of cytotoxic CD8+ T-cells with tumor cell killing capacity. For this DC need to shuttle exogenously derived antigens into the endogenous presentation pathway, better known as cross-presentation. The expression of DC specific CLR gives us the opportunity to both specifically target skin DC whilst inducing cross-presentatio in combinaiton with TLR activation. In chapter 2 we show Langerin to be superior in the activation of specific CD8+ T-cells. In order to explore whether targeting dermal DC is beneficial in facilitating cross-presentation, we used DC-SIGN as target candidate. In chapter 3 we elucidated the exact intracellular routingof DC-SIGN and its cargo. Furthermore, we show that simultaneous triggering of DC-SIGN using an antibody conjugated to the melanoma specific gp100 SLP and TLR4 using LPS, efficiently enhances cross-presentation. This dual stimulation ensured SLP degradation by the proteasome and antigen processing for MHC I loading. In chapter 4, we used the overlapping glycan binding profiles for the Lewis Y type antigens of Langerin and DC-SIGN to target both receptors. Using Lewis Y (LeY ) as targeting glycan we aimed to generate a single glycovaccine targeting multiple human skin DC subsets simultaneously. As carrier system we used PAMAM-dendrimers, to covalently link the gp100 SLP and create two differentially sized vaccines. We show glycodendrimers of approximately 50nm efficiently target both Langerin and DC-SIGN,thereby reaching multiple human skin DC subsets when injected in situ. Furthermore,this enhanced activation of tumor specific CD4+ and CD8+ T-cells compared to nontargeting dendrimers. In order to investigate whether we could further optimize our cancer vaccine, we investigated whether we could improve DC maturation for cytokine skewing and co-stimulation and antigen processing, by combining various PRR agonists. TLR and NOD-like receptors (NLR) that are expressed by different human skin DC and can synergize for DC maturation and antigen handling. In chapter 5 we used the PAMAMdendrimer, to develop a multivalent antigenic vaccine containing both the gp100 SLP and NOD2 agonist MDP. A synthetic NOD2-agonist, was covalently linked to the multivalent antigenic dendrimer. We show that the combination of the NLR agonist-antigen complex with the soluble TLR4 agonist MPLA enhances cytokine secretion within the skin micromilieu. Furthermore, it enhances cross-presentation by human skin DC for CD8+ T-cell activation. Intradermal vaccine delivery is usually achieved through injection, though efforts are made to design systems that simplify intradermal vaccine delivery. In chapter 6 we made use of an ablative fractional laser to verify whether it might benefit vaccination with our anti-tumor vaccine particles. We show that in our human skin explant model intradermal injection was more efficient for vaccine delivery to and uptake by skin DC, resulting in higher level CD8+ T-cell activation. This thesis aimed to develop a human skin DC targeting cancer vaccine, exploiting the expression by DC of the CLR Langerin and DC-SIGN. By the design of a multivalent glyco-vaccine incorporating melanoma specific gp100 epitopes and the targeting moiety LeY we could efficiently target multiple human skin DC for enhanced (cross)- presentation using a single vaccine formulation. This dual targeting, multivalent vaccine can be used for inclusion of a multitude of epitopes and PRR agonists. Thereby we developed a flexible intradermal vaccine platform which has merit for clinical studies aiming to cure different types of cancer