foaming. Routinely one gram of tumor specimen will be digested in a minimum volume of 10 
ml enzyme mixture. The resulting cell suspension will be centrifuged (500 g, 15 min.), filtered 
through a double layer of no. 100 nylon mesh and resuspended in 100 ml RPMI 1640 
medium containing 10% heat-inactivated human AB serum. If there are significant residual 
tumor pieces remaining in the trypsinizing flask they will be digested by repeating the above 
protocol. An aliquot of harvested tumor cells will be sent for cytologic analysis and for 
bacteriological testing. The remaining tumor cells will be cryopreserved in 90% human AB 
serum plus 10% dimethylsulfoxide at -178°C in liquid nitrogen for patient skin testing and in 
vitro assays. Each freezing vial will contain 2 to 4 x 10 7 tumor cells. The procedures for 
tumor procurement and preparation will be carried out by sterile techniques. 
In addition to cryopreservation, a tumor cell line will be initiated by suspending 
approximately 60 ml of complete media (CM) in 150 cm 2 flasks with 1 -5 x 1 0 6 freshly 
dissociated tumor cells/flask. CM is composed of RPMI 1640 with 10% human AB serum, 2 
mM glutamine and 50 pg/ml gentamicin. Flasks are incubated at 37°C in 5° CO 2 . It is 
anticipated that only 3-4 gm of tumor will be required from each patient for therapy. 
6.2 Transduction of melanoma cells with the IL-4 encoding retrovirus vector. 
All procedures for tumor transduction will be performed in the Human Applications 
Laboratory of the CRC. This is a BL-2 facility dedicated for human gene therapy applications. 
FDA certified, frozen virus supernatant will be thawed in preparation for melanoma cell 
transduction. The thawed virus will be filtered through a 0.22 u filter, supplemented with 
protamine (5 ug/mL) and placed on cultured melanoma cells. The virus will be exposed to 
the cultured melanoma cells for a 16 hour period. The virus will be removed, and the cells 
will be placed in CM for an additional 24 hour period. The conditioned media will be 
harvested and assayed for IL-4 production. If IL-4 production is of high enough quantity to 
prepare a tumor cell vaccine (ie, greater than 50 pg/mL/10 6 cells/24 hours), the cells will be 
washed, harvested, and used for vaccine preparation as described below. 
6.3 Vaccination with IL-4 modified tumor cells 
Gene-modified tumor cells will be harvested and irradiated with 5,000 cGy. The cell 
suspension will be adjusted so that a volume of 0.5 ml HBSS contains 10 7 -10 8 irradiated 
tumor cells. The tumor cells will be placed on ice until injection. If 10 8 cells are not available, 
fewer may be given but not less than 10 7 will be injected per site. Patients will be immunized 
i.d. with 0.5 ml of the cell suspension in one or two separate sites drained by an accessible 
nodal basin (ie. inguinal or axillary region). An india ink tatoo will be placed to mark each 
site of the injection. Lymph nodes draining the sites of vaccine will be selected so that the 
chosen LN group will not have clinically apparent metastases. Before immunization, patients 
will have been skin tested with a standard panel of microbial recall antigens (Merieux multi- 
test) as well as irradiated (10,000 cGy) irradiated autologous tumor cells in graded doses. 
These skin tests are performed to determine the immunocompetence of the patient as well as 
obtain pre-treatment evaluation of cutaneous reactivity to autologous tumor antigens. 
6.4 Preparation of single cells from Ivmph nodes 
Patients will undergo excision of hypertrophied LN draining the site 7 to 10 days after 
the vaccination. This can be performed under local anesthesia as an outpatient procedure. 
Lymph nodes will be transported to the laboratory in sterile containers containing HBSS. 
Single cell suspensions will be prepared mechanically by mincing with scissors to 
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