"eligible patients" in this mock-up, 20 (91%) could have received the GM-CSF- 
secreting tumor vaccine based on our ability to expand and transduce the cells 
in culture. 
In these studies, 7 consecutive renal cell tumors were initially 
expanded and transduced with the MFG vector carrying the Lacz marker gene. 
The last in vitro passage obtained and the percentage of cells expressing 
beta-galactosidase activity is shown in table 8. The percentage of Lacz 
expression among all of the optimally transduced tumor cells ranged from 40 to 
100 %. 
Feasibility of vaccine production at Somatix Therpy Corporation . 
We have found that it is also feasible to sterilely transport on wet ice 
overnight to Somatix in Alameda, California, in a thermally secure container, 
the excised tumors that have first been mechanically dissociated into 0.5 to 1 
cm fragments. These fragments can be subsequently digested into single cell 
suspension, and expanded and transduced with the MFG vector carrying the human 
GM-CSF gene. The results of enzymatic digestion and in vitro expansion are 
shown in Table 9A. Eight consecutive renal cell tumors were evaluated. A 
range of 6 x 10 6 to 1 x 10 9 total viable cells with a mean of 2.2 x 10 8 cells 
were obtained from the digested tumors. The total number of passages of 
expanded cells ranged from P2 to P5. 
Table 9B shows the results of transduction of 5 renal cell tumors with 
the MFG vector carrying the human GM-CSF gene. All transductions were 
performed at Somatix using retroviral supernatants that were produced using 
the cell cube system described in Appendix 14. B. This system routinely 
produces retroviral supernatants with titers at least 10 fold higher than what 
had been obtained when supernatants were collected from cells grown in 
traditional tissue culture flasks. Furthermore, these supernatants can be 
frozen for at least one month at -70°C with minimal loss of virus titer. 
Therefore, large quantities of retroviral supernatants can be titered and 
stored, providing greater consistency between transductions. All 5 of the 
renal tumors transduced with different batches of GM-CSF retroviral 
supernatant were shown to express between 24 and 57.6 ng/10 6 tumor cells/24 
hours, as determined by ELISA. This is between 10 and 50 fold greater levels 
of GM-CSF than endogenously produced and measured in untransduced patient 
derived controls. Based on our murine data, this level of expression is 
within the range that is expected to be therapeutic in patients. The 
procedures used for processing the human tumor cells, establishing primary 
cultures, and transducing the cells with the retroviral vector can be found in 
Appendix 14. C. 
Determining tumor cell purity of primary renal cell cultures . 
Determining the relative purity of the cancer cell population that is 
expanded in the primary cultures is a difficult problem because renal tumor- 
specific markers for quantitation are not yet developed. Several criteria can 
be used to evaluate these cultures for overwhelming presence of cancer cells. 
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