Lymphocytes and eosinophils have also been observed (4-6). IL-4 may play a 
critical role in the homing of these cells to sites of tumor by inducing the 
expression of the vascular cell adhesion molecule VCAM by the tumor microvascular 
endothelium (7). Lymphocyte adhesion to these cells is thought to be mediated 
by interaction through cell surface VLA-4 (7). The effects of IL-4 on macrophages 
include the upregulation of class II molecule expression (8) and the induction 
of antigen-presenting capacity (9). T-cell activity is also modulated by IL-4. 
Specifically, IL-4 increases IL-2 production, increases IL-2 receptor expression, 
and enhances proliferation of mitogen-activated T cells (10). When IL-4 is 
combined with IL-2, LAK activity is suppressed while specific antitumor lytic 
activity is enhanced (11). These findings are concordant with the demonstration 
by Golumbek et al (6) that antitumor effects are abrogated by depleting mice of 
CD8+ cells prior to vaccination. Based on these observations, we have targeted 
tumor vaccines, designed to elaborate IL-4 at the site of vaccination, as a 
rational approach to immunotherapy. 
In light of the limited ability to consistently and quickly generate tumor cell 
lines for use in these studies, we have identified cultured fibroblasts a cells 
that can be quickly expanded to large numbers, easily transduced, and used as 
vehicles to deliver high, constant levels of IL-4 to the vaccine site. We have 
routinely been able to get 10 6 fibroblasts from small (1cm x 1cm) biopsies of 
skin after 2 to 3 weeks of culture and to expand them 2 to 4 fold each week 
thereafter. We plan to take 4cm diameter biopsies that are expected to yield 
about 10 to 20 fold more cells for these vaccine studies. We have been able to 
induce fibroblasts growing in log phase to incorporate the gene for IL-4 and 
produce bioactivity of. <0.1 to 10 2 units/10 6 cells/24 hours without selection, 
and as much as 10 4 U with selection. After irradiation with 5000 rads, these 
cells slowly involute and disappear after 1-2 weeks of additional culture. 
Irradiated, autologous fibroblasts transduced with the gene encoding IL-4 will 
be admixed with irradiated single-cell tumor suspensions and administered 
intradermally to patients with disseminated cancer. 
We already have extensive experience with the preparation of single cell 
suspensions of tumor for laboratory studies as well as for vaccine trials at the 
Pittsburgh Cancer Institute. This study is seen as an extension of our previous 
trials. We also have extensive experience with retroviral transduction of tumor 
infiltrating lymphocytes (TIL) using the gene that encodes resistance to the 
neomycin analog, G418. While at the National Cancer Institute, we treated ten 
patients with gene-marked TIL (12). No toxicities of any kind could be 
attributed to these cells. The expected toxicities associated with the 
concomitant administration of IL-2 were seen. Patients received up to 1.45 x 
10 11 gene- transduced TIL. The percent of cells transduced among the adoptively 
transferred TIL populations varied between 1 and 11 per cent. In each case, 
integration and expression of the NeoR gene was demonstrated. Gene -modified TIL 
could be detected at tumor deposits as long as 64 days after infusion. We have 
now treated two patients with disseminated cancer at the University of Pittsburgh 
with IL-2, IL-4, and TIL that have been tranduced with the NeoR gene. Toxicities 
of treatment were those usually associated with the systemic administration of 
IL-2 and IL-4 and are primarily those associated with a vascular leak. 
[500] 
Recombinant DNA Research, Volume 16 
