description of the vector containing supernatant is taken verbatim from that 
approved protocol. 
The TNF-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 (36,37). 
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 (11,12). 
The TNF-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 tumor cells after transduction . The vector includes the retroviral 
L TR promoting the TNF gene followed by the SV-40 early promoter and the gene 
ending f nr npnmyri n phosphotransferase. Testing will be the same as 
previously approved for the LNL6 supernatants used to introduce the NeoR gene 
into TIL (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 with titers greater than 5 x 10 4 colony forming units/ml will be 
used. 
2) Southern blots will be run on the producer line to detect the 
TNF gene. 
3) TNF production by the producer line will be measured and 
should be significantly above baseline control values. TNF will be assayed 
using standard biologic assays on the L929 sensitive cell line (15) or by 
ELISA assay (R&D Systems, Minneapolis, MN) . 
4) Sterility of the producer line and the supernatant will be 
assured by testing for aerobic and anaerobic bacteria, fungus and for 
mycoplasma. 
5) Viral testing will be performed including: 
a. MAP test 
Recombinant DNA Research, Volume 15 
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