3 . 6. c. 
Starting Vaccine Tumor Cell Number and Treatment Cycles 
The starting Dose Level 4xl0 6 RCC vaccine cells is chosen for its 
apparent safety as it represents l/200th the maximum reported dose of tumor 
vaccine cells without propagation in short-term culture administered in 
previous reported trials. 56-59 A direct comparison between this trial and 
previous trials is limited: no study has prepared RCC vaccine cells in 
primary culture like this one. Despite local, reversible, cutaneous reactions 
of erythema and ulceration of 1-2 weeks duration following vaccination, life 
threatening toxicities were not described in earlier human tumor vaccine 
studies. 56-59 The ulcerations were thought due to adjuvants such as BCG used 
with uncultured tumor cells. The choice of 3 treatments is arbituary to 
evaluate cumulative toxicity. In our preclinical models multiple doses have 
not shown increased efficacy over a single dose. 
3.6.d. Highest Dose Level Choice 
Dose Level 4, at lxlO 9 vaccine cells is chosen as an upper limit 
because experience has shown that this limit can be reached within 4 tissue 
culture passages on occasions when one starts with large viable tumors. In 
order to preserve maximum antigenicity of tumor cells in primary culture, 
candidate vaccine cells will only be expanded to a maximum of 4 passages prior 
to use. Immunogenicity has been shown to decrease with long-term culture of 
cancer cell lines although precise data for humans on this point are not 
available . 
3.6.e. Choice of GM-CSF Dose 
The strategy of GM-CSF dosing to be followed in this study is set 
to enhance the evaluation of the paracrine effects of the cytokine on the 
immune response as opposed to the systemic effects of GM-CSF on hematopoiesis. 
Our preclinical studies suggest that a range of GM-CSF secretion of 24 to 
48 ng/10 6 cells/24 hours will achieve this result and insure maximal specific 
antitumor immunity against later challenge by the inoculation of wild-type 
tumor cells. 
In this study we are defining the amount of immunost imulat ing cytokine 
secreted by a fixed number of autologous tumor antigen expressed in ng/vaccine 
cells/per 24 hours. This form of dosing is different from the actual total 
number of retrovirally inserted genes (integrants) per vaccine tumor cell. In 
other words, the total numbers of GM-CSF genes inserted per vaccine cell could 
be different between vaccine preparations, but the absolute amount of 
secretion of the GM-CSF gene product into the microenvironment per hour is 
measurable, and can be standardized to the desired secretion rate of 24-48 
ng/10 6 cells/24 hr called for in this study. Final cell injection formulation 
is described in Appendix 14. C. 
The effect of fixing the range of secretion of GM-CSF from 24-48 ng/106 
cells/24 hours on the daily dose of the cytokine received by the patients may 
be seen in Table 14. The total daily dose received by patients will increase 
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