Currently, we are further pursing this clinical protocol in patients diagnosed with renal 
cell cancer. Our initial impression is that additional methods may be required to generate 
therapeutic activated T cells for the treatment of melanoma. To address this problem, we 
have explored the use of gene-modified tumor cells to upregulate the sensitization of vaccine 
primed LN cells. These observations are summarized in the next section. 
3.3 Rationale for proposed clinical trial to utilize IL-4 gene modified tumor cells to 
prime LN in vivo. 
Conceptually, the modulation of tumor immunogenicity by genetic manipulation can be 
categorized into two categories: 1) the expression of novel immunogenic antigens on the 
cell surface which will stimulate the host immune response (ie. , allogeneic MHC antigens, 
B7, etc.), or 2) the elaboration of a gene product by the tumor cell which will facilitate the host 
immune response in a paracrine fashion. Supporting the latter, several recent studies have 
demonstrated that the transduction of murine tumor cells with genes encoding cytokines alter 
the tumorigenicity of the cells as well as upregulate the T cell response by the hosts 
inoculated with the tumors. Fearon et al. reported that the transduction of the murine tumor, 
CT26, with a gene coding for IL-2 resulted in the spontaneous regression of these tumors in 
the syngeneic host (20). After regression, these animals were found to have cytolytic T cells 
in their spleens reactive to parental tumor and developed systemic immunity documented by 
the rejection of a subsequent challenge of the parental tumor. Many other cytokines (ie, GM- 
CSF, IFNy, TNFa, IL-4) examined in different murine tumor models have demonstrated 
similar findings. Golumbek et al. found that mice inoculated with the Renca renal cell tumor 
genetically engineered to secrete IL-4 were rejected in a T cell-independent manner (21). 
More importantly, these animals developed T cell-dependent systemic immunity to the 
parental tumor. Tepper et al. examined five other distinct tumor types and confirmed that 
genetic modification of these tumors to secrete IL-4 would lead to decreased tumorigenicity; 
and that this phenomenon was directly correlated to the amount of IL-4 secretion (22). In the 
latter study, the local infiltration of eosinophils was found to be a dependent factor in 
regression of the transduced tumors. Despite the potent antitumor response observed locally 
with cytokine transfected tumors, these and other studies have failed to document the 
capability of inducing a sufficient host immune response to mediate the regression of 
established disseminated tumors. 
We have further examined the immunobiologic effects of IL-4 transduced tumors 
utilizing the poorly immunogenic B16BL6 murine melanoma. The B16BL6 tumor was cloned 
by limiting dilution technique and a clone, A9, was isolated. The A9 clone was transfected by 
electroporation with a murine IL-4 cDNA in the PLJ expression vector containing the 
neomycin-resistance gene (23). Transfectants were selected in G418 and clones were 
assayed for their ability to secrete IL-4. A clone, A94-3, was isolated and found to secrete 38 
u/ml (2 x 10 5 cells/ml/24 hr). The in vivo tumorigenicity of A94-3 was compared to a control 
tumor clone which was transfected with the expression vector only (A9PLJ1). Syngeneic B6 
mice (5/group) were inoculated with 10 6 tumor cells s.c. and tumor growth serially measured 
(see Figure, Appendix B). A94-3 tumor cells were found to have decreased tumorigenicity 
compared to A9PLJ1 tumors. Previous studies had documented that the tumorigenicity of 
A9PLJ1 was identical to unmodified parental A9 tumor cells (23). 
The ability to establish pulmonary metastases after i.v. inoculation of A94-3 was also 
impaired compared to parental A9 tumor. B6 mice were inoculated with 2 x 10 5 tumor cells 
(5/group) and lung metastases enumerated 2 weeks later. The mean number of lung 
metastases ± SEM for the A9 and A94-3 groups was 185 ± 15 and 51± 6, respectively. 
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