nude mice showed temporary growth followed by complete regression. This is in 
contrast to the continued lethal growth of the MCA-207 and human tumor lines in 
normal and nude mice respectively when these cell lines were not transduced 
with the gene for TNF, In addition to our own data using TNF producing murine 
and human tumors, recent reports have demonstrated that the local injection of 
TNF directly into tumors can lead to tumor regression in cancer patients (20,21). 
These combined data support the hypothesis that the local production of 
TNF at the site of the tumor may well lead to tumor regression. 
3. Introduction of the gene coding for tumor necrosis factor into human 
tumor infiltrating lymphocytes. 
The retroviral vector construct selected for insertion of the TNF gene 
into human TIL contains the TNF gene (in the LN Moloney murine leukemia retro- 
viral vector backbone) (22). The expression of the TNF gene is promoted 
by the murine LTR and the expression of the neomycin resistance (NeoR) gene is 
promoted by the simian virus 40 (SV40) early promoter region. The two gene 
retroviral construct was introduced into the PA317 producer cell line (23) and 
the supernatant from this line was used to transduce the TIL from three 
patients with advanced metastatic melanoma. The transduction procedure was 
similar to that used in our previous protocol (24) to introduce the NeoR gene 
into TIL and is detailed later in this protocol. TIL from all three patients 
grew well after the transduction procedure. Studies of the transduced and 
non-transduced TIL from these three patients are shown in Table 4. Southern 
blots probed for the intact exogenous TNF gene were positive on DNA extracted 
from the transduced cells from all three patients. While all transduced and 
nontransduced TIL showed the 3.2 kilobase fragment characteristic of the 
endogenous TNF gene, only the transduced TIL from each of the patients exhibited 
the expected 1.0 kilobase (predicted 0.976 kb) fragment characteristic of the 
EcoRl digested DNA representing the exogenous TNF gene. 
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Recombinant DNA Research, Volume 14 
