3) Limitation of the usefulness of aminoglycoside antibiotics* The 
NeoR gene product, neomycin phosphotransferase (NPT), phosphorylates the 3’ 
hydroxyl group of the aminohexose I of neomycin and its analogues, thereby in- 
activating the antibiotic. While amikacin may be inactivated by this enzyme, 
gentamicin and tobramycin do not contain an hydroxyl at the 3’ position and are 
not inactivated (34,35). Therefore, introduction of the NeoR gene would not 
exclude the use of aminoglycosides or any other conventional antibiotic that 
may be needed in the clinical management of these patients. 
4) Production of increased TNF by TIL . The intravenous administra- 
tion of large amounts of recombinant TNF (greater than 8 ug/kg) to patients can 
lead to hypotension. One of the hypotheses upon which the protocol is based is 
that the localization of cells at the tumor site will lead to the local 
production of TNF that will lead to selective antitumor effects at the tumor 
site. It is possible, however, that released TNF can lead to hypotension and 
for this reason patients will be carefully monitored. Low numbers of cells 
will be administered in an escalating dose fashion separated at three weekly 
Intervals to monitor carefully for this potential side effect. 
The total amounts of TNF produced by the infused human TIL cells (assuming 
an average production of 500 pg TNF/10^ cells/24 hrs) will be 0.07 ug/kg/24 hrs 
at the first dose level of infusion (10^® cells) or less than 1/100 of that 
tolerated by humans following the Intravenous Infusion of recombinant TNF 
(8 ug/Kg/24 hrs). Further, nontransduced TIL secrete approximately 20 pg/lO^ 
cells/24 hours (see Table 4). Over 40 prior patients have tolerated from 2-6 x 
10^^ of these TIL (thus producing about 0.2 ug TNF/Kg/24 hours which is about 3 
times higher than the amount of TNF produced by the starting dose of transduced 
TIL). In addition we have demonstrated that lymphokine activated killer (LAK) 
cells, which do not selectively traffic to tumor deposits, secrete about 
570 pg TNF/106 cells (in four separate experiments LAK cells produced 974, 413, 
Recombinant DNA Research, Volume 14 
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