about 30 ug of TNF for 24 hours. We and others have previously shown that 70 
kg patients can tolerate approximately 600 ug TNF iv every 24 hours (8- 
10/ug/kg). Thus for a 70 kg human, the amount of TNF being produced by these 
gene modified cells would be about l/50th the amount of TNF already shown to 
be well tolerated by patients. It should be emphasized, however, that it is 
not expected that these tumor cells will grow based on experimental animal 
models. Further, if patients do develop signs of toxicity due to TNF exposure 
then it will be possible to excise the local nodules in the anterior thigh. 
If tumor spreads from the local site, however, it may not be possible to 
remove all erf the TNF producing tumor. 
5. Risk from murine retrovirus . Exposure of the cancer patient to 
retrovirus could theoretically pose a risk of insertional mutagenesis. It 
should be emphasized, however, that careful tests will be conducted to assure 
that the patient is not exposed to replication competent virus. The 
retrovirus derived from the Moloney murine leukemia virus has been modified so 
that it no longer contains any intact viral genes and thus cannot produce the 
envelope proteins necessary to package its RNA into an intact infectious virus 
(36,37,41,43,44). To assemble the retrovirus, a retrovirus packaging cell 
line was used that contained a second defective retrovirus which expresses the 
viral structural proteins. This packaging cell line does not produce 
replication competent retrovirus because of multiple modifications made to the 
second retrovirus that prevent its replication, including removal of signals 
required for RNA encapsidation, reverse transcription, and integration (37). 
Multiple assays will be performed on the final producer cell line, the 
retroviral vector supernatant as well as on the TIL prior to infusion to 
insure that no replication competent virus is present. These tests will 
include S+L- assays including 3T3 amplification, PCR assays for the envelope 
gene, and assays for reverse transcriptase (42,42). Any supernatants or TIL 
with evidence of any replication competent virus will not be utilized. The 
3T3 amplif ication and S+L- assays are thought to be capable of detecting a 
single replication competent viral particle per ml (41). 
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Recombinant DNA Research, Volume 15 
