Single-step antigen loading and TLR activation of dendritic cells by mRNA electroporation for vaccination in stage III and IV melanoma patients

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. We 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-1β, IL-6, TNFα) 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. We 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 IFNγ and TNFα 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.

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.

This study is an open label prospective exploratory intervention study.

Stage III and IV melanoma patients will be vaccinated three times biweekly with
mature DC injected directly into the lymph node loaded with mRNA encoding
tumor-associated antigens gp100 and tyrosinase and pulsed with KLH as an immune
control antigen. In part I we will perform a dose-finding study in 5 stage IV
patients with DC electroporated with CD40L, CD70 and caTLR4 (Trimix DC) and 5
stage IV patients with DC matured in the presence of the vaccines BCG, Typhim
and Act-HIB (TLR-DC). If no toxicity is observed, we will continue with part II
of the study. In part II we aim to include 24 evaluable patients. In this part
we aim to include in both arm A (Trimix DC) and arm B (TLR-DC), 7 stage IV
patients and 5 stage III patients within 2 months after radical regional
lymphnode dissection.

Based on the experience with our cytokine/PGE2-matured DC and TLR-DC, and the
studies performed by Dr. Kris Thielemans and Dr. Bart Neyns (VU Brussels,
Belgium) exploiting Trimix DC (thusfar 29 patients treated without toxicity: 4
times 12.5 million Trimix DC injected intradermally per vaccination timepoint),
we expect that the DC will be well tolerated. Common and expected side effects
of DC vaccination are usually mild and include flu-like symptoms and local
reaction at injection site, both CTC grade 1.
Patient material that will be requested during the study.