(216,000 lU/kg) doses of IL-2 t.i.d. and lOug/kg of IL-4 before escalating to 
high dose IL-2 (720,000 lU/kg) in combination with IL-4 in 5 other patients. 
Selected patients (those with readily accessible and biopsiable lesions will have 
a portion of their cells transduced for trafficking purposes. No more than 10% 
of the cells administered will carry the marker gene. 5 patients will receive 
such transduced cells at low doses of IL-2 and 5 patients at high doses of IL-2. 
TIL will be grown from patients and will be transduced with the NeoR 
retroviral vector and expanded. Extensive safety and functional tests will be 
conducted on these TIL during this time. Patients will first receive a single 
infusion of up to 10'° Neo modified TIL along with the infusion of IL-2 at 
216,000 lU/kg every eight hours and IL-4 at lOug/kg for up to 5 days. This dose 
of IL-2 is approximately the equivalent of 30,000 Cetus units/kg which is one- 
third the dose of IL-2 used in our previous protocol using NeoR gene modified TIL 
(NIH86-C-183c) . If 5 patients do not reach Grade IV toxicity (see Appendix B) 
or if they reach Grade IV toxicity easily managed by concomitant medications (as 
in previous protocols) then patients will be escalated to receive up to 10'° TIL 
plus IL-2 in a similar fashion given at 720,000 lU/kg. At least five patients 
will be treated in this fashion. 
Detailed studies of patient toxicity as well as possible therapeutic 
effects will be noted. Detailed studies of the survival and distribution of 
these cells in patients will be conducted. Safety and monitoring studies (see 
Sec. 3.5 and 4.2) will be performed in a fashion virtually identical to those in 
our previously approved protocol for the use of TIL modified by the LNL6 
retroviral vector (86-C-183C; reference 21). 
3 . Growth and transduction of tumor infiltrating lymphocytes. 
The procedures used here are the same as those used in our previous 
protocol at the NIH involving the infusion of TIL transduced with the Neo 
resistance gene (protocol 86-C-183c) (29-31). These are detailed below. 
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 gas permeable bags or hollow fiber devices 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 seruim, 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 (30) 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. 
TIL cultures are initiated at 5 x 10^ viable cells/ml (tumor plus 
lymphocytes). For half the cells, the fresh medium is AIMV supplemented with 
penicillin, fungizone, and 6000 lU/ml IL-2 and 1000 U/ml IL-4 (Sterling); for the 
other half, the fresh medium is RPMI supplemented with 10% human serum, 
penicillin, gentamicin, fungizone, and 120 or 6000 lU/ml IL-2 with 1000 U/ml IL- 
4. The cultures are placed into 6 -well tissue culture dishes and incubated at 
37° in humidified incubators with 5% CO 2 . LAK supernatant at up to 20% will be 
used in some cultures . 
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Recombinant DNA Research, Volume 14 
