Preclinical full-scale evaluation of dendritic cells transfected with autologous tumor-mRNA for melanoma vaccination

Abstract

Most cancer vaccines to date have made use of common tumor antigens or allogenic cancer cell lines. The majority of tumor antigens may, however, be unique patient-specific antigens. Dendritic cells (DCs) are the most potent antigen-presenting cells known. The present report is a full-scale preclinical evaluation of autologous DCs transfected with autologous tumor-mRNA (tDCs) for vaccination in malignant melanoma. By using autologous tumor-mRNA, we intend to make the DCs present a broad spectrum of tumor-associated antigens relevant to each individual patient. Previously, we have described effective methods for mRNAtransfection into DCs by square-wave electroporation and for generating large numbers of DCs. Here, we demonstrate the ability of tDCs, made under full-scale vaccine conditions, to generate in vitro T-cell responses specific for antigens encoded by the transfected tumor-mRNA. T-cell proliferation assays demonstrated tDC-specific responses for all six patients tested. Responses were further studied by IFNg ELISPOT and Bioplex cytokine assays (two patients) and by experiments on isolated CD4 þ and CD8 þ T cells, including HLA-blockage (one patient). Moreover, we describe the results of extensive tumor-RNA analysis using Agilent Bioanalyser, a method that we have implemented in the clinical protocol. Based on this preclinical evaluation, a vaccine trial has been started.

Immunotherapy with allotumour mRNA-transfected dendritic cells in androgen-resistant prostate cancer patients

“…The nature of the immune response was not studied in detail in this clinical study, but in a preclinical evaluation, we analysed a broad range of cytokines using the Bioplex system. These results demonstrated that a broad spectrum of cytokines was produced in the in vitrogenerated T-cell response (LJ Mu et al, unpublished;Kyte et al, 2005) All of the patients who mounted a specific T-cell response also developed a component of MLR reactive cells recognising DCn. This also held true for most of the patients who failed to raise a specific T-cell component.…”

“…Selected mRNA preparations were also evaluated on an Agilent Bioanalyser instrument using RNA 6000 Nano Reagent & Supplies (Agilent Technologies, Palo Alto, CA, USA). The Bioanalyser quantifies RNA by calculating size and concentration of each separated band (Kyte et al, 2005). Transfection efficiency was monitored in separate experiments with enhanced green fluorescent protein mRNA instead of tumour mRNA as described (Mu et al, 2003).…”

Phase I/II trial of melanoma therapy with dendritic cells transfected with autologous tumor-mRNA

“…Extraction of autologous tumor-RNA was performed as a two-step procedure, as previously described. 25 First, total-RNA was extracted by use of Trizol (Invitrogen Life Technologies, Carlsbad, CA). Then mRNA was purified by use of polyT-coated magnetic beads (Genoprep mRNA Beads, Qiagen, Hilden, Germany).…”

“…For 16 patients, the RNA samples were also evaluated on an Agilent Bioanalyser instrument (Agilent Technologies, Palo Alto, CA), as previously described. 25 The RNA concentration, rRNA percentage and mRNA content were estimated, according to the manufacturer's protocol.…”

“…24 Recently, we reported a full-scale preclinical evaluation of the melanoma DC-vaccine. 25 In the preclinical evaluation, we established the methods applied in the present trial for full-scale vaccine produc-tion and for monitoring of immune responses. Further, we demonstrated that tumor-mRNA transfected DCs (tDCs) were capable of inducing in vitro T-cell responses specific for antigens encoded by the transfected mRNA.…”