3.3 SETTING 
Most patients will be treated in an inpatient oncology ward although some 
may require transfer to the ICU to receive some of their therapy, especially if 
it requires pressors. 
3.4 PREPARATION OF THE Neo-R VECTOR CONTAINING SUPERNATANT. 
The NeoR vector was constructed by modifying the Moloney murine leukemia 
vector by techniques similar to those previously described. Retroviral vector 
supernatant is produced by harvesting the cell culture medium from the PA317 
packaging line developed by Dr. A. Dusty Miller (33, 35). This line has been 
extensively characterized and was used by us in our previous studies of the 
infusion of TIL modified by the LNL6 vector. These studies and preparation of 
supernatant will all be carried out by Dr. Moen and colleagues at GTI (see 
letter) . The Neo vector preparations from PA317 will be extensively tested to 
assure that no detectable replication competent virus is present. Tests for 
replication competent virus will be conducted on both the vector supernatant and 
on the TIL after transduction. Testing will be the same as previously approved 
for the LNL6 supernatants used to introduce the NeoR gene into TIL (NCI-5B 
protocol 86-C-183c). The following tests will be run on the producer line and/or 
the viral supernatant: 
1. The viral titer will be determined on 3T3 cells. Viral preparations 
wijth titers greater than 5 x 10^ colony forming units/ml will be used. 
2. Southern blots will be run on the producer line to detect the NeoR 
gene . 
3. Sterility of the producer line and the supernatant will be assured by 
testing for aerobic and anaerobic bacteria, fungus and for mycoplasma. 
4. Viral testing will be performed including: 
a. Map test 
b. LCM virus 
c. Thymic agent 
d. S+/L- assay for ecotropic virus 
e. S+/L- for xenotropic virus 
f. S+/L- for ampho tropic virus 
g. 3T3 amplification. 
The retroviral supernatant will not be used to transduce TIL infused into 
patients until approval is received from the Food and Drug Administration. 
3.5 TESTS ON THE TRANSDUCED TIL POPULATION . 
Following transduction and growth of the TIL populations the following 
tests will be performed on the TIL prior to infusion into patients. 
1. Cell viability will be greater than 70% as tested by trypan blue dye 
exclusion. 
2. As in all prior TIL protocols, cytologic analysis will be performed 
on over 200 ceTls prior to infusion to assure that tumor cells are 
absent . 
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Recombinant DNA Research, Volume 14 
