Protocol ID93-008 
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malignant melanoma who were treated with rlL-2 expanded neo^^ transduced and non 
transduced TIL. The proportion of neo^^ transduced cells ranged from 1 - 1 1% and the 
number of transduced cells that were injected raged from 0.6 to 1.8x10® (37). The neo 
marked TlLs were detected in cells from metastatic tumors even at 64 days after IV injection 
of the TIL. 
Certain safety concerns remain regarding the clinical application of retroviral vectors in 
general, a larger clinical experience with these vectors and the introduction of stricter quality 
assurance measures utilized in the production of these vectors is providing reassurance 
about their safety (41). A recent report states that more than 1 10 patients (from different 
studies) have received cells infected with safety-modified retrovirus vectors without any 
adverse reports of clinical toxicity (42). 
2.8.3 Experimental data demonstrating our ability to conduct in vivo studies with neo ^ 
marked TlLs. 
We have established in our laboratory all the methods needed to accomplish the objectives of 
this protocol (see 3.2(d) p. 7-9 and Appendix 1). These methods have been repeated many 
times and have been found to be reproduceable and employable as a routine. Results from 
our experiments are provided in Appendix 1 and are also summarized briefly below. Purified 
ovarian CD8+ TlL-derived T-cell lines were infected with GlNa which has the same backbone 
as the retroviral vectors LNL6 and N2. Infection of CD3+CD8+ TIL with GlNa were 
accomplished with 3 infections at a virus concentration of 2-4 MOl per cell. Oligonucleotide 
primer sequences utilized for the detection of the 424 b.p. PCR product of the neo'^ gene were 
provided by Ed Otto (G.T.I., Gaithersburg. MD). The neo^ gene segment was detected in the 
GlNa transduced cells using a sensitive PCR assay based on procedures established by 
Morgan et al (43). The 424 b.p. gene product was detected by ethidium bromide (EtBr) 
staining and by Southern blotting followed by detection with labeled neo*^ probe. Some 
additional refinements to the PCR method have been incorporated following suggestions by 
Ed Otto (G.T.I.). Assays performed in our laboratory are capable now of detecting a single 
copy of neo^. i.e.. the number of neo^^ gene copies expected from an analysis of a 2 ^g DNA 
sample obtained by diluting neo^ transduced cells in nontransduced cells at a ratio of 
1:100,000 cells (43). The following modifications have led to a significant improvement in our 
retrovirus vector infection protocol: (a) infections with GlNa were performed at an MOl of 3, 
(b) three infections 24 hrs apart were performed after a vector incubation period of 6 hrs. (c) 
The culture medium was replaced with complete and conditioned AIM V medium between 
infections. In studies previously reported by Rosenberg and associates (37), infections with 
LNL6 were performed at an MOl of 2 and only two infections were utilized. Under the 
experimentaJ conditions that we have used, the numbers of CD8+ T cells transduced with 
neo^ were at least 10% in 4 of 5 CD8+ TIL-derived T-cell lines and 5% in one line (Appendix 
1). Moreover, no substantial differences were found in the lymphocyte phenotypes or in the 
rIL-2 dependency of these T-cell lines following infection with GlNa in comparison to those 
that were not infected. In vitro cytotoxicity was also not diminished after infection. Because 
of the improved transduction efficiency, the number of neo^^ marked T cells injected IP will be 
approximately 5x10® cells or higher. 
2.8.4 Summary of marker protocol 
Marking of the CD8‘*’ TIL-derived T cells by infection with GlNa will be performed after 
positive selection has been accomplished and the purified CD8+ T cells are observed to be 
multiplying In a T-flask. At this point consenting patients will be registered. A separate 
informed consent (attached to this protocol) will be obtained. The bulk expanded 
transduced and nontransduced T cells will be injected IP with low dose rIL-2. Following 
treatment with the adoptively transferred TIL, and IP rIL-2, biopsies will be obtained at 
laparoscopy performed at 1 month after injection of the TIL, from previously documented 
tumor sites and from normal tissues (peritoneum, omentum (if present) and lymph node 
tissue). Although animal and human studies show that injected TIL can be recovered beyond 
2 months we believe that selection of an earlier time point will maximize detection with a 
Recombinant DNA Research, Volume 19 
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