To answer the question of whether the marked TIL were 
significantly altered by the process of inserting the gene, 
Dr. Blaese explained they had removed marked cells and analyzed 
them for cell surface phenotype changes. Using a fluorescence 
activated cell sorter, they found no significant alterations in 
phenotype over time in culture although some drifting and 
maturation of phenotype did occur which is normal in cultured 
cells over long periods of time. 
Dr. Blaese said another experiment was performed to discover 
whether absence of changes in cell surface phenotype may be 
reflective of other changes occurring, such as cytotoxicity. 
This was performed by looking at the ability to kill autologous 
tumor. TIL samples from four of the six patients studied were 
non-cytotoxic and did not acquire cytotoxic activity after gene 
insertion. Two of the patients TIL were cytotoxic against the 
autologous tumor but not against other targets both before and 
after vector insertion and selection. So, no changes in cell 
cytotoxic activity has been seen. Cytokine production was also 
looked at and found to be unaffected by vector insertion and 
selection, as was T cell receptor specificity and heterogeneity. 
Therefore, Dr. Blaese concluded that some modifications occur in 
some TIL populations; however, in the vast majority of cases the 
TIL are not significantly altered by the process of inserting the 
genes and selecting for expression of Neo*. 
Dr. Blaese described studies undertaken to detect the marked 
cells in animal models. He summarized data from one such model, 
the nude mouse. Normal mice were immunized with sperm whale 
myoglobin. The lymphoid tissues were then removed and expanded 
in culture. The SAX vector (an N2 based vector) was used to 
carry two genes: one coding for neomycin resistance, the other 
coding for human ADA, into these lymphoid cells. Then the 
tissues were reinfused into nude mice and the animals were 
analyzed at various times for presence of the inserted gene. 
Twenty-three days after receiving the cells containing these 
genes, blood in 5 animals was found to be positive for vector 
DNA. Furthermore in one animal, after 37 days, both blood and 
spleen were analyzed and the spleen was directly positive for 
vector DNA. After growing both spleen and white blood cells in 
culture, they were found to be G418 resistant and to express 
human ADA. The longest surviving animal transduced with the SAX 
vector is positive for DNA in the spleen 105 days after cell 
transfer . 
Dr. Blaese then presented the results of experiments in a murine 
system using SAX-transduced TILs recovered from lymphoid tissues. 
He also described an experiment in primates that was conducted 
using the N2 vector to transduce T lymphocytes in culture. 
Tetanus toxoid was used to stimulate cell proliferation after 
which the lymphocytes were reinfused intraperitoneally . Cells 
Recombinant DNA Research, Volume 13 
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