Therapeutic Cancer Vaccination with Ex Vivo RNA-Transfected Dendritic Cells-An Update

Abstract

Over the last two decades, dendritic cell (DC) vaccination has been studied extensively as active immunotherapy in cancer treatment and has been proven safe in all clinical trials both with respect to short and long-term side effects. For antigen-loading of dendritic cells (DCs) one method is to introduce mRNA coding for the desired antigens. To target the whole antigenic repertoire of a tumor, even the total tumor mRNA of a macrodissected biopsy sample can be used. To date, reports have been published on a total of 781 patients suffering from different tumor entities and HIV-infection, who have been treated with DCs loaded with mRNA. The majority of those were melanoma patients, followed by HIV-infected patients, but leukemias, brain tumors, prostate cancer, renal cell carcinomas, pancreatic cancers and several others have also been treated. Next to antigen-loading, mRNA-electroporation allows a purposeful manipulation of the DCs' phenotype and function to enhance their immunogenicity. In this review, we intend to give a comprehensive summary of what has been published regarding clinical testing of ex vivo generated mRNA-transfected DCs, with respect to safety and risk/benefit evaluations, choice of tumor antigens and RNA-source, and the design of better DCs for vaccination by transfection of mRNA-encoded functional proteins.

Keywords: RNA; clinical trial; dendritic cells; electroporation; immunotherapy; therapeutic vaccination.

Figure 1

Schematic representation of an mRNA-transfected DC. Endocytosed and phagocytosed material is usually presented in MHC class II to CD4⁺ helper T cells and can be cross-presented in MHC class I only under certain circumstances (not depicted). Proteins from the cytoplasm are, in contrast, primarily presented in MHC class I. mRNA can be introduced into cells by passive pulsing, relying on intrinsic and yet unknown means of uptake. It can be complexed with lipid reagents that mediate entry into the cytoplasm, or it can be transfected by electroporation. By applying a short electric pulse, pores in the cell membrane temporarily open, allowing entry of the RNA molecules. The transferred mRNA is then translated in the cytoplasm, and the encoded antigens are hence presented in a MHC class I context. By encoding signaling and targeting sequences (green) fused to the antigenic protein, this can also be directed towards the endosomal pathway, resulting in efficient MHC class II presentation.

Figure 2

Concept of therapeutic vaccination with mRNA-transfected DCs. (1) Tumor material is isolated by surgery or biopsy. From this material, mRNA can be directly isolated. This RNA is usually amplified via a PCR-based method to gain sufficient amounts of mRNA. The tumor material can also be analyzed by sequencing, immunohistology, or other methods to identify antigens associated with this tumor, including somatic mutations. New bioinformatical methods can be used to identify the most promising T-cell epitopes for each individual patient. mRNA molecules that encode these antigens are transcribed in vitro from plasmid templates. (2) Cells (usually monocytes) that can be differentiated into DCs in vitro are isolated from the patient’s blood. Alternatively, DCs, which are present in the blood, can be used. These DCs are matured to become immunogenic. (3) DC are transfected with the RNA to express the encoded tumor antigens. The DCs’ own processing machinery degrades and presents the included T-cell epitopes in MHC. (4) The DCs are injected into the patient. Intradermal (id) and subcutaneous (sc) injection require migration via the lymphatic vessels towards the draining lymph node. Intravenous (iv) injection necessitates the transfer from the blood stream into lymphatic tissue. The direct injection into lymph nodes (i.n.) is an elegant approach, but is technically very difficult. (5) If the vaccine is successful, the DCs present the tumor-specific epitopes to T cells which are activated and attack the malignant tissue. Usually the DCs are injected repetitively to boost and maintain the responses. (The Motifolio Scientific Illustration Toolkit was used for the generation of this figure).