When the tolerated doses of selected and non-selected INF-modified TIL 
are established then the dose of IL-2 will be escalated to 720,000 lU IL-2 
(the exact IL-2 dose used in our previous studies of gene modified TIL protocol 
(86-C-183c) in the next series of at least 5 patients each. 
2. Growth and transduction of tumor infiltrating lymphocytes . The pro- 
cedures used here are the same as those used in our previous protocol involv- 
ing the infusion of TIL transduced with the Neo resistance gene (protocol 
86-C-183c) (25-27). 
At least two days prior to surgery, peripheral blood lymphocytes are 
collected by leukapheresis for four hours. These are Ficoll-Hypaque separated 
and the mononuclear cells collected from the interface, washed in saline, and 
placed in culture in roller bottles at 10^ cells/ml. Half are placed into AIMV 
(a serum free medium, Gibco Laboratories) with 6000 lU/ml IL-2 (Cetus), and half 
are placed into RPMI supplemented with 2% type-compatible human serum, penicillin 
(unless the patient is allergic), gentamicin, and 6000 lU/ml IL-2. After 3 to 4 
days cells are centrifuged and the supernatants are collected and filtered. 
These are referred to as LAK supernatants. 
Immediately upon tumor resection, the specimen(s) is transported to the 
laboratory in a sterile container and placed on a sterile dissection board in 
a laminar flow hood. A small representative portion is taken for pathologic 
analysis, and the rest is minced into pieces roughly 4 mm in diameter. These 
are placed into an enzyme solution of collagenase, DNAse type I, and 
hyaluronidase type V as previously described (26) for overnight digestion at 
room temperature. The resulting suspension is filtered through a wire mesh to 
remove any large debris, washed in saline, and placed on Ficoll-Hypaque gradients. 
The Interface containing viable lymphocytes and tumor cells is collected and 
washed in saline, and a portion is frozen for subsequent use as targets. 
Recombinant DNA Research, Volume 14 
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