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

Abstract

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

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy

“…Although this technique allows substantial transfection of DCs, its efficiency is still relatively low in comparison to electroporation, where over 90% of the DCs can be transfected with mRNA. Indeed, with mRNA electroporation, TriMix does result in a significant up-regulation of maturation markers and cytokine expression, as previously reported by Bonehill et al [17]. In addition, based on the results obtained in the OT-I proliferation assay and the in vivo CTL assay, it would appear that TriO sonoporation works fine to enhance the quantity of antigen-specific CD8 + T lymphocytes (i.e.…”

“…TriMix is a mixture of 3 mRNAs, encoding CD40-ligand, a constitutively active form of TLR4 and CD70 (a co-stimulatory molecule required for effective CD8 + T cell priming) [16]. Co-delivery of these 3 nucleic acid sequences was already shown to modulate the DCs' functionality, resulting in APCs that display a more mature, T cell activating phenotype [17]. Previous studies have demonstrated the superiority of TriMix over other, more conventional, maturation stimuli after intranodal injection of TAA and TriMix mRNA in tumor-bearing mice [18].…”

Intratumoral Delivery of TriMix mRNA Results in T-cell Activation by Cross-Presenting Dendritic Cells

“…Delivery of tumor-associated antigen (TAA) and TriMix mRNA to DCs, ex vivo or in situ, reprograms them to mature antigen-presenting cells (18,19). These DCs and the T cells they activate are protected from regulatory T cells (Treg; ref.…”

Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells

“…[47][48][49] The vaccine concept may be further developed by novel strategies for enhancing DC-migration and immune stimulation. [50][51][52][53][54] We currently test the clinical use of DCs generated with a 3D protocol, based on a collaboration with Prof. Schendel and collegues. 55 This protocol includes a TLR seven out of eight agonist that enhance the IL-12 secretion of the DCs and their ability to generate Th1-like responses in vitro and in mouse models.…”