Cancer Therapy: Preclinical Single-Step Antigen Loading and Activation of Dendritic Cells by mRNA Electroporation for the Purpose of Therapeutic Vaccination in Melanoma Patients

Purpose: A critical factor determining the effectiveness of currently used dendritic cell (DC)–based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen–derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen–encoding mRNA. Experimental Design: The T-cell stimulatory capacity of TriMix DCs pulsed with the immunodominant MelanA-A2 peptide and that of TriMix DCs coelectroporated with MelanA mRNA were compared in vitro. TriMix DCs were also coelectroporated with mRNA encoding Mage-A3, Mage-C2, tyrosinase, or gp100. The capacity of these DCs to stimulate tumor-antigen–specific T cells in melanoma patients was investigated both in vitro before vaccination and after DC vaccination. Results: Like peptide-pulsed TriMix DCs, TriMix DCs coelectroporated with MelanA mRNA are very potent in inducing MelanA-specific CD8 T cells in vitro. These T cells have an activated phenotype, show cytolytic capacity, and produce inflammatory cytokines in response to specific stimulation. TriMix DCs coelectroporated with tyrosinase are able to stimulate tyrosinase-specific CD8 T cells in vitro from the blood of nonvaccinated melanoma patients. Furthermore, TriMix DCs coelectroporated with Mage-A3, Mage-C2, or tyrosinase are able to induce antigen-specific CD8 T cells through therapeutic DC vaccination. Conclusions: TriMix DCs coelectroporated with whole tumor-antigen mRNA stimulate antigen-specific T cells in vitro and induce antigen-specific T-cell responses in melanoma patients through vaccination. Therefore, they represent a promising new approach for antitumor immunotherapy. The past five decades have witnessed a steady increase in the incidence of malignant melanoma. Whereas early detection and appropriate surgery have improved outcomes, at least one third of patients with early-stage melanoma will develop metastases. The prognosis for patients with malignant metastatic melanoma remains poor. These patients have a median survival of approximately 6 to 8 months, and <5% will generally survive for 5 years or more (1). There is universal agreement that further research to address this problem is critically warranted. Many strategies to enhance specific or nonspecific immunity in melanoma patients have been explored in clinical studies (2). Although the field is relatively new and many clinical variables remain to be investigated, vaccination with tumor-associated antigen (TAA)–expressing dendritic cells (DC) might provide a therapeutic benefit (3). Roughly, the DC life cycle can be divided into two stages: the immature and the mature stage. Immature DCs reside in the periphery and are specialized Authors' Affiliations: Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel; Ludwig Institute for Cancer Research, Cellular Genetics Unit, Université Catholique de Louvain; and Department of Medical Oncology, UZ-Brussel, Brussels, Belgium Received 11/14/08; revised 2/19/09; accepted 2/20/09; published OnlineFirst 5/5/09. Grant support: Interuniversity Attraction Poles Program-Belgian State, Belgian Science Policy, Belgian Foundation against Cancer, Integrated Project and Network of Excellence sponsored by the European Union, and Fund for Scientific Research-Flanders (FWO-Vlaanderen). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: V. François is a doctoral fellow of the FNRS-Télévie. A. Bonehill is a postdoctoral fellow of the FWO-Vlaanderen. Requests for reprints: Aude Bonehill, Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. Phone: 32-2-477-4565; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2982 3366 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Cancer Research. on January 28, 2018. © 2009 American Association for clincancerres.aacrjournals.org Downloaded from in antigen recognition and uptake. On receiving a maturation signal such as a Toll-like receptor (TLR) ligand or other pathogen-associated molecular pattern, DCs undergo several morphologic, phenotypic, and functional changes and transform into efficient antigen-processing/presenting cells capable of stimulating both CD4 and CD8 T cells (4). Based on these characteristics, mature DCs seem to be the ideal cellular tools for use in cancer immunotherapy. Nonetheless, the process of maturation is highly complex and involves up-regulation of HLA and costimulatory molecules, changes in chemokine receptor repertoire, and enhanced secretion of inflammatory cytokines and chemokines. Different maturation stimuli lead to different “mature DC” that possess different effector functions (5). Thus, the activation state of DCs is a critical factor determining whether these cells will be potent inducers of an antitumor immune response through vaccination, and there is general belief that the effectiveness of currently used DC vaccination protocols could be improved by providing the DCs with a more potent activation signal. We have recently shown that the T-cell stimulatory capacity of peptide-pulsed DCs 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; ref. 6). Here, the combination of CD40L and caTLR4 electroporation would mimic CD40 ligation (7) and TLR4 signaling (8) of the DCs and generates phenotypically mature, cytokine/chemokine-secreting DCs, as has been shown for CD40 and TLR4 ligation through addition of soluble CD40L and lipopolysaccharide (9). On the other hand, the introduction of CD70 into the DCs would provide a costimulatory signal to CD27 naive T cells by inhibiting activated T-cell apoptosis and by supporting T-cell proliferation (10). Providing the DCs with a maturation signal through mRNA electroporation offers several advantages. There is no need to preincubate the DCs for up to 48 hours with soluble maturation signals like proinflammatory cytokines or TLR ligands to achieve DC activation, which can render the cells “exhausted” and inferior for vaccination purposes (11). As a result, TriMixelectroporated DCs, which can be injected into the patient within a few hours after electroporation, will mature and secrete most of their immunostimulatory cytokines and chemokines in situ. Furthermore, it has been postulated that maturation of DCs in situ resembles more closely the physiologic process involved in response to pathogen infection and may therefore lead to enhanced T-cell immunity (12). Here, we investigate whether TriMix DCs can be coelectroporated with TAA-encoding mRNA instead of being pulsed with antigenic peptides. This approach offers several additional advantages. First, the maturation and TAA loading of the DCs can be combined in one simple electroporation step. Obviating the peptide pulsing step in the vaccine production thus results in less manipulation of the cells and less cell loss and contamination risk. Second, by using full-length TAA-encoding mRNA, all possible antigenic epitopes of the TAA will be presented instead of some selected epitopes. Consequently, this strategy could induce a broader TAA-specific T-cell response, and it is not dependent on the knowledge of each patient's HLA haplotype or on the prior identification of TAA-derived epitopes (13). Third, the TAA-encoding plasmid can be genetically modified by adding an HLA class II targeting sequence. This not only routes the TAA to the HLA class II compartments for processing and presentation of HLA class II–restricted TAA-derived peptides but also enhances processing and presentation in the context of HLA class I molecules (14, 15). We show that TriMix DCs can stimulate specific T cells when coelectroporated with whole MelanA-encoding mRNA instead of being pulsed with MelanA-derived peptide. We also show that TriMix DCs are able, both in vitro and in vivo, to induce T cells specific for other TAA with a lower precursor frequency. Materials and Methods Genetic constructs. The pGEM-CD40L, pGEM-CD70, and pGEMcaTLR4 plasmids encoding CD40L, CD70, and caTLR4 (containing the intracellular and transmembrane fragments of TLR4, as described in ref. 8); the pGEM-NGFR plasmid encoding a truncated form of the nerve growth factor receptor (NGFR, containing the extracellular and transmembrane fragments); the pGEM-sig-MageA3-DCLamp plasmid encoding the full-length Mage-A3 antigen linked to the HLA class II targeting sequence of DC-Lamp (transmembrane/luminal region); and the pGEM-sig-MelanA-DCLamp plasmid encoding the full-length MelanA antigen, containing the optimized immunodominant MelanA-A2 epitope and linked to the DC-Lamp targeting signal, have previously been