serum. Cultures will be split 1 to 3 when confluent monolayers develop. After 
the first passage, the fibroblasts will be transduced and replated as previously 
described . 
3.2 Transduction and Growth of Fibroblasts: 
The procedures used here are the same as those used in our previous protocols at 
the NIH and the PCI, involving the infusion of TIL transduced with the Neo 
resistance gene (protocol 86-C-183c and 90-C-183c) (12). These are detailed in 
the appendix (19.3). Cultured fibroblasts will be transduced during log phase 
of growth using a retroviral vector containing the gene for human IL-4. Twenty- 
four hours after transduction, they will be exposed to the selective pressure of 
0.3 mg/ml of G418. This concentration may be increased to as much as 1 mg/ml 
depending on the health of the culture. 
3.3 Preparation of the IL-4 Vector-Containing Supernatant: 
Supernatants from GTI will be prepared with retroviral vectors incorporating the 
IL-4 gene (appendix 19.4). They will be tested and shown to be free of 
mycoplasma and repl ication- competent virus using the NIH 3T3 and S+/L- assays. 
3.4 Preparation and Administration of Vaccine: 
When the total population of fibroblasts reaches about 5 X 10 7 cells, the medium 
will be changed and the amount of IL-4 appearing in the culture supernatant of 
representative flasks over 24 hours will be determined by ELISA. This will be 
used to estimate the amount of IL-4 that may be elaborated at each vaccine site 
per 10 7 cells per 24 hours. About 2 X 10 7 fibroblasts will then be harvested by 
brief trypsinization for vaccine preparation. At the same time, the frozen tumor 
cell suspension will be thawed and washed. The number of viable tumor cells will 
be counted by trypan blue exclusion. Suspensions with less than 50X viability 
will have viable cells separated with Ficoll -Hypaque centrifugation. 
In order to realize the stated objectives of this protocol, patients will first 
undergo primary vaccination at multiple sites using vaccines made of a fixed 
number of irradiated tumor cells but escalating numbers of transduced fibroblasts 
that will deliver varying doses of IL-4 to each vaccine site. This will define 
the local and systemic toxicity associated with IL-4-elaborating vaccines of 
different histologies. Five vaccines will be prepared for primary immunization 
by admixing 5 X 10° tumor cells with 3 log dilutions of fibroblasts with the most 
potent vaccine containing up to 10 7 fibroblasts elaborating no more than 10 5 U 
of IL-4 per 24 hours. In addition to a nontransduced control vaccine, for 
example, a patient would receive 4 vaccines that elaborated 10 5 , 10 4 , 10 3 , or 10 2 
U IL-4/ 24 hours. Each vaccine will be prepared in 0.1 ml of sterile saline. 
The vaccine will be irradiated with 5,000 rads prior to intradermal 
administration. At the time of vaccine administration, a small aliquot of 
fibroblasts will be set aside to test for lack of growth and for production of 
IL-4 following irradiation. 
Five sites for vaccination, oriented vertically and positioned 2 cm apart, will 
be placed over the left lumbar region of the patient's back between 2 points 
tattooed using India ink. This indelible tattoo will allow for the accurate 
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Recombinant DNA Research, Volume 16 
