for infusion will be conducted to assure that no replication competent virus is 
present in either preparation. In addition, tests will be performed on the 
transduced TIL populations themselves to assure that they remain dependent on IL- 
2/IL-4 for their continued proliferation. TIL are dependent on IL-2 and IL-4 for 
their continued growth and will die in the absence of IL-2. IL-2 and IL-4 will 
be withdrawn from an aliquot of the culture prior to TIL infusion to assure that 
TIL retain their dependence on these cytokines. 
b) 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 (32 - 35). 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. Multiple assays will be performed on the 
packaging 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. Any supernatants or TIL 
with evidence of any replication competent virus will not be utilized. The 3T3 
amplification and S+/L- assays are thought to be capable of detecting a single 
replication competent viral particle per ml. These studies will initially be 
done at GTI . 
Prior safety studies have shown that exposure of primates to large 
infusions of infectious murine amphotrophic virus produce no acute pathologic 
effects (36) . In a study of 21 primates receiving retroviral mediated gene- 
modified autologous bone marrow cells no animal showed evidence of toxicity 
related to the gene transfer for as long as 4 years after Infusion (37, 
unpublished data) . 
More recently we have seen a cancer develop in a mouse that received large 
amounts of retroviral vector supernatant by the intraperitoneal route. We are 
currently conducting studies to see if this tumor is in an 3 ^way related to the 
retroviral transfer. It should be emphasized, however, that these mice were 
exposed to large amounts of retroviral vector and that the patients in the 
proposed protocol will not be exposed to the vector supernatant. TIL will be 
transduced with the retroviral vector supernatant and then the TIL will be washed 
extensively and then grown for several weeks in the absence of supernatant. The 
TIL will then be washed extensively again prior to reinfusion into the patient. 
c) 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 inactivating 
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 
(38,39). 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. 
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Recombinant DNA Research, Volume 14 
