The advantages of using exogenous genes as "markers* for cells are several: 
1. Genes inserted into the chromosomes of the host are stable markers which can 
be readily detected by sensitive methods (the PGR technique will detect 1 cell in 
a population of 100,000). 
2. The inserted, integrated gene will mark that cell and its progeny for as 
long as the cells survive. The label does not leach away or become sequestered 
in a phagocyte or reutilized after the original marked cell has died. Cell 
proliferation does not dilute the label so that long term population studies can 
be performed. 
3. The use of a modified retrovirus as the gene transfer vector is a very 
simple technical procedure which does not expose the marked cell to toxic 
compounds or radioactivity which might alter the function of the marked cell. 
4. Of the gene transfer technologies available, retroviral -mediated gene 
transfer is the most efficient by far, averaging a success rate of 10-25% for 
lymphocytes in our preliminary experiments. 
5. The inserted marker gene can be chosen to provide a selective advantage to 
the marked cell (ie., resistance of a drug) so that the labeled cell population 
can be treated with the drug in vitro . killing the non- gene -containing cells, 
thereby insuring that all the cells infused into the patient will carry the 
marker gene. This same property can be used later so that the marked cells car. 
be recovered from the body and selectively expanded in culture to evaluate their 
functional properties. Specifically, TIL can be transduced with the N2 vector 
(see below. Section III.B.) carrying the neomycin resistance (NeoR) gene (that 
produces a neomycin phosphotransferase) at high efficiency. The TIL can then be 
grown in the presence of the neomycin analogue G418 for several days to insure 
that 100% of the cells carry an active NeoR gene. In theory, any marked TIL or 
its offspring could be selected from a blood or biopsy sample by incubating the 
specimen in T cell-IL-2 medium together with G418. These recovered marked cells 
could then be expanded and studied. 
6) Although the primary value of the present Addition is to gain information 
about the distribution and survival of TIL in the patients studied in order to 
optimize TIL immunotherapy, information gained could be important for determining 
if retroviral -mediated gene transfer is a technique of sufficient efficacy and 
safety to be used for the insertion of therapeutic genes (e.g., IL-2, TNF, etc.) 
into TIL. Studies using mouse TIL models are now underway. These clinical 
applications of this advanced technology are not covered by this Addition and 
would require a new protocol. 
Since we are proposing to use a retrovirus to transfer the marker gene into 
the TIL, what potential hazards does this procedure pose to the patient? Is it 
safe to use a recombinant murine retroviral vector in man? We believe that, 
under the conditions defined in this Addition, it is safe. The murine retrovirus 
used as our gene transfer vector has been modified to remove all intact viral 
coding sequences so that it no longer carries any viral genes. The marker gene 
to be transferred to the TIL is the only coding genetic information contained in 
the virus. Since this vector has no remaining viral genes, it is incapable of 
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Recombinant DNA Research, Volume 14 
