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, 1993). 
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: 
• an improved cationic lipid formulation, DMRIE/DOPE; 
• 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, et al. 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 without toxicity. Because of these 
properties, it now becomes possible to introduce 100-1000 times more DNA which could 
allow the study of an expanded dose response gene expression in vivo. 
The vector improvements are divided into two categories for this proposal. In the first case 
expression of the HLA-B7 vector has been improved by the addition of a consensus 
translation initiation sequence and removal of an intron. In addition, the inclusion of the 6- 
2 microglobulin gene, with which class I MHC genes normally associate, allows synthesis 
of the complete histocompatibility molecule, which is composed of these two chains. 
Ordinarily, these two gene products are co-transported to the cell surface. This is 
important because some human melanoma cells do not express endogenous p-2 
microglobulin, thus limiting their ability to stably express class I on the cell surface. It has 
been found that the inclusion of the P-2 microglobulin gene on the same plasmid allows for 
the expression in these otherwise resistant cells and improve expression in other cells, thus 
overcoming a potential mechanism of resistance. These modifications have been 
incorporated in the study drug to be used in this submission. The study drug is identical to 
the study drug fully characterized in Dr. Nabel's RAC submission of June 7, 1993, which 
was unanimously allowed. This study will investigate the administration of the study drug 
in metastatic renal cell cancer. 
1.2 Background 
1.2.1 Direct Gene Transfer and Modulation ofthe Immune System 
Recombinant DNA Research, Volume 19 
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