PMID- 16911798
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20070726
LR  - 20201226
IS  - 1479-5876 (Electronic)
IS  - 1479-5876 (Linking)
VI  - 4
DP  - 2006 Aug 15
TI  - 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.
PG  - 35
AB  - 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. 
      CONCLUSION: Standardized preparation of viable clinical-grade DCs transfected 
      with tumor-derived and in vitro amplified mRNA is feasible and their 
      administration is safe.
FAU - Markovic, Svetomir N
AU  - Markovic SN
AD  - Division of Hematology, Department of Internal Medicine, Mayo Clinic College of 
      Medicine, Rochester, Minnesota, USA. markovic.svetomir@mayo.edu
FAU - Dietz, Allan B
AU  - Dietz AB
FAU - Greiner, Carl W
AU  - Greiner CW
FAU - Maas, Mary L
AU  - Maas ML
FAU - Butler, Greg W
AU  - Butler GW
FAU - Padley, Douglas J
AU  - Padley DJ
FAU - Bulur, Peggy A
AU  - Bulur PA
FAU - Allred, Jacob B
AU  - Allred JB
FAU - Creagan, Edward T
AU  - Creagan ET
FAU - Ingle, James N
AU  - Ingle JN
FAU - Gastineau, Dennis A
AU  - Gastineau DA
FAU - Vuk-Pavlovic, Stanimir
AU  - Vuk-Pavlovic S
LA  - eng
GR  - N01 CA015083/CA/NCI NIH HHS/United States
GR  - P30 CA015083/CA/NCI NIH HHS/United States
PT  - Journal Article
DEP - 20060815
PL  - England
TA  - J Transl Med
JT  - Journal of translational medicine
JID - 101190741
PMC - PMC1570143
EDAT- 2006/08/17 09:00
MHDA- 2006/08/17 09:01
PMCR- 2006/08/15
CRDT- 2006/08/17 09:00
PHST- 2006/05/11 00:00 [received]
PHST- 2006/08/15 00:00 [accepted]
PHST- 2006/08/17 09:00 [pubmed]
PHST- 2006/08/17 09:01 [medline]
PHST- 2006/08/17 09:00 [entrez]
PHST- 2006/08/15 00:00 [pmc-release]
AID - 1479-5876-4-35 [pii]
AID - 10.1186/1479-5876-4-35 [doi]
PST - epublish
SO  - J Transl Med. 2006 Aug 15;4:35. doi: 10.1186/1479-5876-4-35.