Single-step Antigen Loading and TLR Activation of Dendritic Cells in Melanoma Patients

1. Rationale Immunotherapy applying ex vivo generated and tumor-antigen-loaded dendritic cells (DC) has now successfully been introduced in the clinic. A limited, but consistent, number of objective immunological and clinical responses have been observed. Thusfar it remains unclear why some patients respond and others not, but there is a general consensus that the current protocols applied to generate DC may not result in the induction of optimal Th1 responses. The investigators and others have demonstrated that DC maturation is one of the crucial factors, not only for effective DC migration but also to induce effective anti-tumor immune responses in cancer patients. Currently, the "golden standard" used to mature DC consists of a cocktail of pro-inflammatory cytokines (IL-1b, IL-6, TNFa) and prostaglandin E2 (PGE2). Recent mouse data demonstrated, however, that maturation of DC by solely pro-inflammatory cytokines yielded DC that supported T cell clonal expansion, but failed to efficiently direct effector T cell differentiation. Interestingly, DC matured in the presence of Toll like receptor (TLR) ligands were able to induce full T cell effector function and unleashed more potent immune responses. The investigators recently identified vaccines against infectious diseases that contain TLR ligands and are capable of inducing DC maturation. This knowledge provides a new application for these clinical applicable agents: clinical grade DC stimulators. A clinical grade DC maturation protocol is developed in which TLR ligands (preventive vaccines) and PGE2 are combined which resulted in the generation of mature DC that secrete high levels of the key cytokine IL-12. Moreover, these TLR-ligand matured DC (TLR-DC) induced T cells secreting at least 20-fold higher levels of the effector cytokines IFNa and TNFa as compared to DC matured in the absence of TLR ligands. In the group of Kris Thielemans and it was shown that the T-cell stimulatory capacity of peptide-pulsed DC can be greatly enhanced by providing them with three different molecular adjuvants through electroporation with mRNA encoding a so-called TriMix of CD40 ligand (CD40L), CD70, and a constitutively active form of TLR4 (caTLR4). The combination of CD40L and caTLR4 electroporation would mimic CD40 ligation and TLR4 signaling of the DC and generates phenotypically mature, cytokine/chemokine-secreting DC, as has been shown for CD40 and TLR4 ligation through addition of soluble CD40L and lipopolysaccharide. On the other hand, the introduction of CD70 into the DC would provide a costimulatory signal to CD27+ naive T cells by inhibiting activated T cell apoptosis and by supporting T cell proliferation. In conclusion, these in vitro data demonstrate that both TLR-DC and Trimix DC are promising candidates to improve immunological and clinical responses in cancer immunotherapy. 2. Objectives This is an exploratory study, consisting of two parts. In part I dose escalation is performed and the primary objective is the safety of different doses of TLR-DC and Trimix DC. In part II Trimix DC vaccination will be compared with TLR-DC vaccination and the primary objective of this part is the immunological response, with toxicity and clinical efficacy being secondary objectives. These studies will provide important data on the safety and immunological effects of TLR-DC and Trimix DC. 3. Study design Part I of this study is an open label dose escalation study. Part II of this study is an open label randomized phase II study. 4. Study population Our study population consists of melanoma patients, with proven expression of melanoma associated tumor antigens gp100 and tyrosinase. Melanoma patients with regional lymph node metastasis in whom a radical lymph node dissection is performed within 2 months of inclusion in this study (further referred to as stage III) and melanoma patients with measurable distant metastases (further referred to as stage IV) will be included. 5. Main study endpoints The primary objectives of the study are to investigate the toxicity of TLR-DC and Trimix DC by dose escalation of DC numbers in part I, and to investigate immunological responses upon DC vaccination in part II of the study. Immunological responses are: 1. The activation of immune cells in vivo. 2. The immunological response induced with TLR-DC and Trimix DC loaded with mRNA encoding melanoma-associated tumor antigens (gp100 and tyrosinase). Safety and clinical efficacy are secondary objectives.