title: Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma.
abstract: BACKGROUND
  Dendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines.
  
  
  METHODS
  We prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma.
  
  
  RESULTS
  Three preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 +/- 12 percent) in comparison to non-electroporated cells (82 +/- 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections.
  
  
  CONCLUSIONS
  Standardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.
authors.affiliation: Division of Hematology, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA. markovic.svetomir@mayo.edu
authors.forename: Svetomir N
authors.initials: SN
authors.lastname: Markovic
authors.forename: Allan B
authors.initials: AB
authors.lastname: Dietz
authors.forename: Carl W
authors.initials: CW
authors.lastname: Greiner
authors.forename: Mary L
authors.initials: ML
authors.lastname: Maas
authors.forename: Greg W
authors.initials: GW
authors.lastname: Butler
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Padley
authors.forename: Peggy A
authors.initials: PA
authors.lastname: Bulur
authors.forename: Jacob B
authors.initials: JB
authors.lastname: Allred
authors.forename: Edward T
authors.initials: ET
authors.lastname: Creagan
authors.forename: James N
authors.initials: JN
authors.lastname: Ingle
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Gastineau
authors.forename: Stanimir
authors.initials: S
authors.lastname: Vuk-Pavlovic