systemic immunity. Subsequent studies have shown that irradiated cells, while 
unable to replicate, are metabol ical ly active, and express inserted genes for 
over one week in vitro. 
Based on the above findings, experiments were than performed to compare 
the relative potency of irradiated tumor cells expressing the ten molecules 
previously examined using live, transduced tumor cells. As shown in Figure 
IB, GM-CSF was the most potent molecule tested, IL-4 and IL-6 possessed some 
activity, and notably, IL-2, IFN-gamma, and TNF-alpha, showed no activity. 
These results provided the major rationale for selecting GM-CSF, rather than 
other molecules, for our clinical studies. 
Figure 1A: Irradiated B16 Melanoma Cells Expressing GM-CSF Confer Potent 
Systemic Antitumor Effects. Mice were vaccinated with 5 x 10 6 irradiated 
(3,500 rads) GM-CSF transduced or non-transduced B16 cells. o animals 
displaying growth of tumor challenge. • animals protected from tumor 
challenge . 
Figure IB: Vaccinations with irradiated B16 melanoma cells expressing 
cytokines and adhesion molecules. Mice were vaccinated with 5 x 10 5 cells 
irradiated with 5000 rads. Animals were challenged 7 days later with 1 x 10® 
live, wild-type B16 tumor cells. 
Immunologic analysis of the response generated by GM-CSF transduced 
tumor cells indicated that systemic immunity was dependent on both CD4+ and 
CD8+ T cells and was long lived, since mice challenged several months after 
vaccination continued to be protected (Appendix 14. A. 4). 
Experiments were performed to determine the importance of immunizing 
cell dose on systemic protection. As shown in figure 2, immune protection 
against a challenge with live wild type tumor was highly sensitive to the 
total innoculum of vaccinating cells; increasing doses of GM-CSF transduced 
tumor cells resulted in enhanced systemic antitumor immunity. These results 
suggest that higher immunizing cell doses will have greater therapeutic 
efficacy in human cancers. 
[294] 
Recombinant DNA Research, Volume 17 
