gene directly into tumors in vivo has induced partial tumor regressions, as well as 
specific cytotoxic T cell responses to other antigens. 
In a preliminary trial in humans with malignant melanoma Nabel treated 5 patients 
with malignant melanoma. Three patients received 3 treatments, totaling 0.87 pg of 
DNA intratumorally, and 3 patients (2 additional patients plus one of the original 3 
patients) received cumulative dose of 2.58 pg of DNA via three treatments. No toxicity 
resulted from this form of treatment and there was no formation of anti DNA antibody 
or autoantibody. There was no plasmid DNA detectable in the blood by PCR following 
gene transfer (tested on days 3-7 post transfection at ~2 pg/ml sensitivity). 
Evidence of gene transfer was found on biopsy of the injected tumor. The biopsy 
samples were analyzed for plasmid DNA, mRNA coding for HLA-B7 and the 
expression of HLA-B7 protein. In 4 of the 5 patients, plasmid DNA and HLA-B7 
mRNA were detected within the treated nodules by PCR. HLA-B7 expression was 
confirmed in all treated nodules by immunohistochemical staining with a monoclonal 
antibody to the gene product. Two patients, where cell lines were established from the 
tumor, showed an immune response by lysing autologous tumor cells. One of the 5 
patients had a partial remission which involved cutaneous and visceral disease. 
(Nabel, et al., PNAS, in press). 
These data suggest that tumor cells modified with the HLA-B7 gene not only 
stimulate CTLs and potentially other immune system cells to recognize tumors 
expressing HLA-B7, but they may also provide a stimulus to immune cells to eliminate 
tumor cells at other sites which express tumor associated antigens in association with 
the patient's own HLA antigens. 
Several improvements that may increase the convenience, safety and efficacy of the 
procedure have been introduced since the original Nabel studies including: 
o an improved cationic lipid formulation, DMRIE/ DOPE*; 
o DNA plasmid construction to optimize expression 
The efficacy of transfection was improved for the following reasons. Briefly, a new 
formulation of cationic lipids has been described recently by Dr. Phillip Feigner (Vical) 
in which a different cationic lipid, l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl 
ammonium bromide (DMRIE), is utilized with dioleoyl phosphatidylethanolamine 
(DOPE). This has two properties which make it more suitable for these studies. First, it 
shows up to 10-fold improved transfection efficiency in vitro compared to the 
formulation previously used by Nabel. More importantly, this formulation does not 
aggregate at high concentrations. This characteristic thus allows higher absolute 
concentrations of DNA and lipid complex to be introduced into experimental animals 
DMRIE: l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium DOPE: dioleoyl 
phosphatidylethanolamine bromide. 
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Recombinant DNA Research, Volume 18 
