FDA approval of this vector is pending 
15.3 Testing of retroviral supernatants 
The following tests will be performed on LNL6 and GIN cells and supernatants 
by GTI:a. titre, b. sterility, c. Map test, d. Leu virus, e. thymic agent, 
f. S+/L-assay for ecotropic virus, g. S+/L- for xenotropic virus, h. S+/L- for 
amphotropic virus, i. 3T3 amplification. 
15.4 Transduction of Bulk, CD8(+) and CD4(+) TILs and PBL 
Frozen LNL6 and GIN supernatants will be provided by GTI and stored at -70°C. 
On the day of TIL transduction, aliquots will be thawed and passed through 
Coming 0.22 micron filters. TIL will be resuspended at a concentration of 10 6 
cells/ml in a transduction mixture consisting of 50% AIM V + lOOOu/ml IL-2 
and 50% viral supernatant (an infectivity ratio of 2-3:1) supplemented with 
protamine sulfate at a final concentration of 5 ^g/ml. One half of TIL and all 
PBL willl be transduced. After incubation at 37°C for 4 hr, TIL will be washed 
3 x in AIMV and introduced into tissue culture bags for large scale expansion of 
cells for therapy. 
CD8(+) and CD4(+) TIL and PBL are prepared as described in Appendix II. 
These cells are cultured for 1-2 weeks in T 150 flasks. After satisfactory cell 
growth is confirmed, TIL are then transferred to tissue culture bags for large scale 
expansion. Retroviral transduction will be performed during the transition. One 
half of the cells destined for large scale expansion and clinical use will be 
transduced The other half will be expanded in parallel bags. In this way, if 
transduced TIL/PBL are found to be unsuitable for clinical use (e.g. sterility 
problems, etc.), patients can still be treated with a satisfactory number of 
untransduced cells. 
15.5 Tests on transduced TIL and PBL populations 
a. Viability - greater than 70% by trypan blue exclusion 
b. Cytology - prior to infusion a cytologic analysis will be performed on 
Cytospin preps to assure that no tumor cells are present 
c. Sterility - TIL cultures will be cultured for aerobic and anaerobic bacteria, 
fungus and mycoplasma 
d. S+/L-assay - negative including 3T3 amplification 
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Recombinant DNA Research, Volume 15 
