IV. SUMMARY OF STUDY DESIGN 
A. Pre-paration of N2 Vector Contalnlne Supernatant 
Retroviral vector supernatant is produced by harvesting the cell culture 
medium from the fibroblast packaging line. There are currently no guidelines for 
the preparation of retroviral supemate intended for human use. Over the past 
year we have been working with the FDA to develop these guidelines. Retroviral 
supemate would not be used in patients until its production and characterization 
are approved by the FDA. 
B . Preparation of TIL Containing a Selectable Marker Gene 
1 . Incubation protocol 
TIL harvested from the patient would be cultured using the standard 
procedure now in use. Once the TIL are growing well (7-14 days after initiation 
of culture) , an aliquot of the cells would be removed for retroviral gene 
transfer. This aliquot of TIL would be cultured in the N2 viral supernatant for 
a 2 hour incubation along with protamine sulfate at 5 ng/ml. The cells would 
then be centrifuged and resuspended in fresh AIM-V medium. G418 would be added 
at 0.3 mg/ml the following day. Selection would continue until essentially all 
of the cells were expressing the NeoR gene as measured by ^ [H] -thymidine 
incorporation or methylcellulose colony assay. After safety analysis (see below) . 
the expanded G418 -resistant TIL population would be added back immediately prior 
to infusion to the mass TIL population being prepared separately or would be 
administered to the patient as a separate infusion after the mass TIL population 
is given. At least 50% of all TIL administered to patients would be conventional 
non- transduced TIL cultured in the usual fashion. 
2 . Analysis of the marked TIL prior to infusion 
a) Demonstration of the vector DNA. Southern blot analysis would 
be performed to verify that the selected cells contain the vector. 
b) Determination of NeoR gene expression. The continued growth of 
TIL in G418 would docxament the expression of the NeoR gene. To confirm gene 
expression, TIL would be evaluated using ^ [H] -thymidine incorporation or 
methylcellulose colony assay as well as by enzymatic assay for NPT. 
c) Evaluation for possible helper virus contamination. 
Using the sarcoma positive/leukemia negative (S+/L-) assay, it would be verified 
that TIL infected with the N2 vector were not producing replication competent 
murine retrovirus. In addition, as another test to insure the lack of infectious 
virus , TIL would be cultured with NIH 3T3 cells in an attempt to rescue 
retroviral sequences. DNA from infected TIL would also be probed for helper virus 
sequences. Finally, the medium in which the TIL are growing would be assayed for 
the presence of reverse transcriptase. 
d) Evaluation of IL-2 dependence. In order to evaluate the 
possibility of autonomous growth of the TIL caused by insertional mutagenesis, we 
would determine if the cells continue to be IL-2 dependent. If cells continue to 
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Recombinant DNA Research, Volume 14 
