4. Forty-eight hours prior to harvest an aliquot of the cell supernatant from 
at least one flask will be sent for bacterial and fungal cultures and 
contamination by endotoxin. The final harvest of cells for delivery to the 
patient will proceed as follows: Cells in an appropriate number of triple 
flasks will be trypsinized and placed in 3-fold excess volume of culture 
medium at a concentration of 2-10 x 10^ cells/ml. Cells will then be 
transported on ice to the gamma-irradiator (Mark-1 irradiator: J.L. Shepard 
and Associates, San Fernando, Ca . ) , and 20,000 rads will be delivered over 
10.5 minutes. Cells will then be returned to the lab, washed three times in 
cold PBS, and counted. A gram stain will be performed. Depending on the 
cohort, cells will be resuspended in an appropriate volume of PBS for 
injection and aspirated into a capped syringe. Cells will be transported on 
ice to the clinic. The cells do not stick to the walls of the syringe. The 
cells will be administered subcutaneously as noted below immediately upon 
arrival to the clinic. 
IV. Eligibility/Exclusions 
Eligibility Criteria : 
1. Patients with metastatic melanoma. Patients who have stage IV 
disease that has been completely resected are eligible. The 
pathology must be reviewed and confirmed by the Surgical Pathology 
staff of the NCI. 
2. Patients must be typed for histocompatibility antigens (HLA-A, B, 
and C, HLA-DR) , and must be positive for HLA-A1 or HLA-A2 . 
3. Age > 18, male or female 
4. Performance .status 0 or 1 
5. No other treatment for their malignancy within the 4 weeks prior 
to entry to this study. 
6. Life expectancy of 3 months or more. 
Recombinant DNA Research, Volume 18 
[731] 
