Single-step Antigen Loading and TLR Activation of Dendritic Cells by mRNA Electroporation for Vaccination in Stage III and IV 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
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.
Allocation: Randomized, Endpoint Classification: Safety/Efficacy Study, Intervention Model: Parallel Assignment, Masking: Open Label, Primary Purpose: Treatment
The primary objective of the study isto investigate immunological responses upon vaccination
The immunological response induced with TLR-DC and Trimix DC loaded with mRNA encoding melanoma-associated tumor antigens (gp100 and tyrosinase) will be evaluated by using 1. tetramer screening of skin-test biopsy derived cell-cultures and peripheral blood, 2. cytokine-bead assay to measure specific cytokine production of skin-test biopsy derived cell-cultures upon differential stimulation and 3. KLH-specific antibody and proliferative responses
C.J.A. Punt, prof.dr.
Netherlands: The Central Committee on Research Involving Human Subjects (CCMO)