9.1.1 Monoclonal antibodies to HLA-B7 and immunochemistry techniques will be 
used to detect the presence of the recombinant gene product in biopsied 
samples. Fluorescence staining of freshly dispersed cells will also be evaluated. 
9.1.2 Successful gene transfer will be assessed by the presence of DNA from the HLA- 
B7 gene as detected by PCR amplification of DNA from cells obtained by biopsy 
of the treated sites on various days after the injection of the study drug (see 
Schema). Genomic DNA will be isolated by standard methods and a portion of 
the HLA-B7 gene will be cimplified and sequenced. 
9.1.3 Circulating antibodies to HLA-B7 will be measured by immunoassay. The 
presence of antibody will be evaluated by FACS analysis of a matched pair of 
HLA-B7-positive and-negative cell lines. 
9.1.4 Autologous peripheral blood lymphocytes will be immortalized by EBV 
transformation eind subjected to in vitro gene transfer with the DNA/lijx>somal 
complex. These autologous cells expressing the HLA-B7 gene will be used to 
assess the specificity of patients' immune responses to the HLA-B7 antigen. 
Direct gene transfer and expression of the HLA-B7 gene is expected to sensitize 
the patient to HLA-B7 and lead to the generation of an immune response to this 
antigen. Limiting dilution analysis (LDA) wUl be done to evaluate alterations in 
the frequency in HLA-B7-spedfic helper and cytol)dic T cells in the peripheral 
blood following direct gene transfer. 
9.1.5 Peripheral blood lymphoc)des will be isolated and cryo-preserved prior to, and 
at 2 to 4-week intervals following, the initial treatment. At the completion of 
treatment, PBL samples from each time point will be simultaneously evaluated 
for responsiveness to HLA-B7 by culturing PBLs, under LDA conditions, with 
autologous EBV-transformed cells transfected with the FILA-B7 gene. Antigen 
sp>ecific elaboration of IL-2 or generation of CTLs to HLA-B7-positive target cells 
will be the indices evaluated in these studies. The presence of antibody will be 
evaluated by FACS analysis of a matched pair of HLA-B7-positive and -negative 
cell lines. 
9.1.6 In selected instances, lymphocytes will be isolated directly from the tumor, 
expanded in tissue culture, and analyzed for cytolytic activity. Tumor biopsies 
at 7 and 14 days after treatment wUl be analyzed by immunochemistry. An 
attempt wiU be made to isolate and expand draining lymph node T cells or TIL 
cells to test their cytolytic activity. 
9.1.7 When pxDSsible, autologous ceU lines wiU be derived from a pretreatment tumor 
specimen and used in a 5lCr-release assay with peripheral blood lymphocytes. 
Recombinant DNA Research, Volume 19 
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