approximately 15.1-27.6 hours in males and from 9.9-15.5 hr. in females. The 
estimated systemic clearance (CLT) averaged 6.7 and 8.0 ml. hr. kg. for males 
and females, respectively. Estimated from the limited data, the apparent 
half-life following the SC dose was longer than that observed for the IV 
route, ranging from 26.6 hrs -48.3 hrs. in males and 9.7-19.7 hrs in females, 
presumed due to continuous absorption. Compared to the IV data for each 
animal, the estimated absolute bioavallabillty (F) of the SC dose ranged from 
14-29% in the 6 nonhuman primates studied (data on fllw. Genetics Institute 
and Hoffmann-La Roche; Minutes of Advisory Board Meeting for IL-12, New York; 
February 9, 1993). We would presume half life kinetics obtained with 
fibroblast delivery comparable to that observed with subcutaneous 
administration. 
10.2 Insertional Mutagenesis: 
The possibility of causing malignancy in cells secondary to the random 
insertion of the retroviral vectors in the genome exists, though the actual 
risk of this occurring is thought to be low. Nevertheless, transduced 
fibroblasts will be irradiated with 5,000 rads prior to vaccination. Tests of 
the viral supernatant as well as of the fibroblasts used for vaccination will 
be conducted to assure that no replication competent virus is present in 
either preparation. Since no other cells will be exposed to retroviral vector 
insertion, no other cells will be at risk for insertional mutagenesis. 
10.3 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 proteins necessary to package its RNA 
into an Intact infectious virus (14, 15). To assemble the retrovlms, a 
second-generation retrovirus packaging cell line (YCRIP) will be used that 
contains coding sequences that express the viral structural proteins in two 
separate plasmids. This packaging cell line does not produce replication 
competent retrovirus because of multiple modifications made to the gag, pol, 
and env coding sequences that prevent its replication. These modifications 
Include removal or signals required for RNA encapsidation, priming of reverse 
transcription, and integration (Y region) . Multiple assays will be performed 
on the packaging cell line, the retroviral vector supernatant as well as on 
the fibroblasts prior to vaccination to insure that no replication competent 
virus is present. These tests will Include S+/L- assays and 3T3 amplification. 
Any supernatants or fibroblasts 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 HGTAL and then subsequently in the 
IMDL/CT. 
Prior safety studies have shown that exposure of primates to large 
infusions of infectious murine amphotrophic virus produce no acute pathologic 
effects (16). In a study of 21 primates receiving retroviral-medlated, 
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 
(17). 
Patients in the proposed protocol will not be exposed to the vector 
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Recombinant DNA Research, Volume 19 
