in mediating the regression of established cancer in several murine tumor models 
(19) . These animal experiments led to clinical trials of the use of TIL in 
humans (22) . The results of the treatment of 50 patients with metastatic 
malignant melanoma are shown in Table 2. Thirty-eight percent of patients had 
objective, although often brief, regressions of their cancer. Currently all 
patients in the Surgery Branch, NCI have their TILs grown in combinations of IL-2 
and IL-4. Similar adoptive transfer studies using adherent LAK cells (23-24) 
cultured for up to two weeks and T- cells cultures following in vitro 
sensitization to tumor (25) have been performed safely here at the University of 
Pittsburgh. We have also proposed to administer TIL cells expanded in IL-2 and 
IL-4 to selected patients with melanoma while they are receiving this combination 
of cytokines in vivo . 
Extensive studies have been performed in the Surgery Branch, NCI to study 
the characteristics of human TIL and the mechanism of action of these cells in 
patients (26) . In an attempt to study the traffic of TIL following cell 
infusion, 19 infusions of TIL labelled with Indium-111 were given to 18 patients 
and the distribution of these cells assessed using gamma camera imaging and 
sequential biopsies (27). Clear tumor localization of TIL was seen on 13 of 18 
nuclear scans and sequential biopsy data confirmed the homing of TIL to tumor 
deposits. Unfortunately, information regarding persistence of cells was limited 
to the first several days following administration because of the short half-life 
of Indium- 111. Additional means to follow transferred cells in vivo were thus 
required. 
We have previously performed studies of the retroviral transduction of the 
gene coding for neomycin resistance into TIL (28) and the subsequent infusion of 
these TIL into autologous patients with advanced cancer (29) . The retroviral 
vector used in the current study is identical to that used in our prior studies. 
Reports of these prior studies are submitted with this protocol. 
Following extensive review by investigational review committees of the 
National Cancer Institute, National Heart, Lung and Blood Institute, the 
Recombinant DNA Advisory Committee (RAC) and the Food and Drug Administration we 
received permission to transfer TIL modified by insertion of the gene coding for 
neomycin resistance into cancer patients. The first patient was treated on May 
22, 1989, and we have subsequently treated a total of seven patients with these 
gene modified TIL. Extensive studies have been conducted on the first five 
patients and they are presented in Tables 3 and 4 and Figure 1 (29) . No 
toxicities of any kind could be attributed to the gene modification of the TIL. 
The expected toxicities associated wtih the concomitant administration of IL-2 
were seen. Patients received up to 1.45 x 10" gene transduced TIL populations. 
The percent of cells transduced in these populations varied between one and 11%. 
In all cases, integration and expression of the NeoR gene was demonstrated. As 
shown in Figure 1, gene modified TIL could be detected at tumor deposits as long 
as 64 days after gene transduction. 
All safety studies performed in these patients showed no evidence of 
exposure to replication competent virus. 3T3 amplification and S-1-/L- assays 
revealed no replication competent virus present in the TIL at the time they were 
infused. Pol 3 onerase chain reaction analysis for the viral envelope gene and 
reverse transcriptase assay of the gene-modified TIL culture were also negative. 
Western blot analyses of patient serum at various times after cell administration 
were all negative for evidence of exposure to virus as well. These studies 
demonstrated that gene modification of the TIL could be performed and that these 
TIL could be infused with no exposure of the patient to a replication competent 
virus. These studies have provided us sufficient experience to perform the 
proposed studies with such TIL at the University of Pittsburgh with safety. 
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Recombinant DNA Research, Volume 14 
