Feasibility of storing the genetically-altered tumor vaccines in liquid 
nitrogen until the day of vaccination. 
As outlined in the treatment plan, patient tumor specimens will be 
processed, expanded and transduced at Somatix which is located in Alameda, Ca. 
The genetically-altered tumor vaccine cells will then be frozen and sent back 
to Johns Hopkins Oncology Center where they will be stored in a liquid 
nitrogen freezer until the day that the patient is vaccinated. To determine 
the efficacy of the freshly thawed, genetically-altered tumor vaccine, mice 
were treated with freshly thawed B16 melanoma cells that had been genetically- 
altered to secrete GM-CSF, frozen, and stored in a liquid nitrogen freezer 
until the day of vaccination (Figure 6). On day 0, 10 mice per group were 
vaccinated with either 1 x 10 6 GM-CSF secreting vaccine cells grown in 
culture, 1 x 10 6 GM-CSF secreting vaccine cells freshly thawed from the liquid 
nitrogen freezer, or 1 x 10 6 untransduced B16 melanoma cells. All vaccines 
were irradiated with 5000 rads prior to the subcutaneous vaccination in the 
left flank. On day 14 following vaccination, mice were challenged with 1 x 
10 5 B16 wild-type tumor cells in the right flank. Mice were evaluated twice a 
week for the development of tumor. None of the mice treated with either the 
freshly thawed or in vitro grown vaccine cells developed tumors. 70% of the 
mice treated with the 1 x 10 6 irradiated wild-type vaccine cells developed 
tumors. These results were confirmed in a second experiment. This data shows 
that freshly thawed and irradiated genetically-altered tumor vaccine cells are 
at least as effective as the in vitro grown and irradiated genetically-altered 
tumor vaccine cells. 
TABLES 8, 9A, 9B, 10, 11 AND FIGURES 5 AND 6 FOLLOW ON PAGES 18-23. 
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