Evaluation of thawed vs in vitro grown GM-CSF 
tumor vaccines 
Figure 6. Feasibility of vaccinating with a freshly thawed genetically- 
altered tumor vaccine. Mice were treated with freshly thawed B16 
melanoma cells genetically-altered to secrete GM-CSF and frozen down and 
stored in a liquid nitrogen freezer until the day of vaccination. On 
day 0, 10 mice per group were vaccinated with either 1 x 10^ GM-CSF 
secreting vaccine cells grown in culture, 1 x 10 6 GM-CSF secreting 
vaccine cells freshly thawed from the liquid nitrogen freezer and washed 
twice with Hank's balanced salt solution, or 1 x 10^ untransduced B16 
melanoma cells. All vaccines were irradiated with 5000 rads prior to 
subcutaneous vaccination in the left flank. On day 14 following 
vaccination, mice were challenged with 1 x 10^ B16 wild type melanoma 
cells in the right flank. Mice were evaluated twice a week for the 
development of tumor. These results were confirmed in a second 
experiment . 
Other Preclinical Data 
The murine tumor studies summarized above are detailed in the manuscript 
by Dranoff et. al., PNAS in press (Appendix 14. A. 3) and in appendix 14. A. 4. 
In addition, data relating to the serum pharmacokinetics of GM-CSF and 
toxicity studies involving mice injected with GM-CSF transduced kidney tumor 
cells can be found in Appendices 14. A. 1 and 14. A. 2. A protocol for 
preparation of human RCC tumor vaccines was developed on the basis of the 
above studies and is described in detail in Appendix 14. C. 
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Recombinant DNA Research, Volume 17 
