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 $-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 {3-2 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 malignant melanoma. 
1.2 Background 
1.2.1 Direct Gene Transfer and Modulation of the Immune System 
The utilization of catheter-based gene delivery in vivo provided a model system for the 
introduction of recombinant gene-specific sites in vivo. Early studies focused on the 
demonstration that specific reporter genes could be expressed in vivo (13,14). 
Subsequent studies were designed to determine whether specific biologic responses 
could be induced at sites of recombinant gene transfer. To address this question, a 
highly immunogenic molecule, a foreign major histocompatibility complex (MHC), was 
used to elicit an immune response in the iliofemoral artery using a porcine model. The 
human HLA-B7 gene was introduced using direct gene transfer with a retroviral vector 
or DNA lipid complex (12). With either delivery system, expression of the recombinant 
HLA-B7 gene product could be demonstrated at specific sites within the vessel wall. 
More importantly, the expression of this foreign histocompatibility antigen induced an 
immunologic response at the sites of genetic modification. This response included a 
granulomatous mononuclear cell infiltrate beginning 10 days after introduction of the 
recombinant gene. This response resolved by 75 days after gene transfer; however, a 
specific cytolytic T cell response against the HLA-B7 molecule was persistent. This 
study demonstrated that a specific immunologic response could be induced by the 
introduction of a foreign recombinant gene at a specific site in vivo. Moreover, this 
provided one of the first indications that direct gene transfer of specific recombinant 
genes could elicit an immune response to the product of that gene in vivo (12). 
Recombinant DNA Research, Volume 18 
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