inoculation. Such therapeutic effects were also reported in similar models by 
Brunda, et al. (1993). In their studies, therapeutic Intervention with 
systemic administration of IL-12 can be initiated as late as day 28 after 
inlectlon of tumor cells (M5076 reticulum cell sarcoma) resulting in 
inhibition of tximor growth, reduction in the number of metastases and an 
Increase in survival time. Although IL-12 has demonstrated potent anti -tumor 
effects when injected systemically , Induction of long term immunity is less 
frequent and variable from experiment to experiment (Brunda et al, 1993. 
Nastala et al, 1994). Interestingly, Brunda et al observed that the best 
results with systemic IL-12 administration (complete regression of the tumor 
and induction of protective immunity against tumor re-challenge) was observed 
following perl- tumoral injections of IL-12 in a subcutaneous tumor model using 
Renca cells (Bininda et al, 1993). Since IL-12 is secreted only by 
"professional" antigen presenting cells (i.e. macrophages and B cells), it is 
possible that local administration of IL-12 at the site of tumor might 
approximate the role of IL-12 in eliciting an endogenous immune response. 
More recent evidence suggest that IL-12 plays a critical co-stimulatory role 
with B7 on APC in inducing proliferation and IFN-g production from clones 
(G Trinchieri, personal communication). Thus, IL-12 may be the ideal cytokine 
for inducing tumor specific immunity when administered at the tumor site. 
Local, secretion of the prototypic T-cell growth factors, IL-2 and IL-4 (Fearon 
et al, 1990. Gansbacher et al , 1990. Tapper et al, 1989), has been previously 
achieved through the introduction of genes directly into tumor cells. This 
strategy, however, has many obstacles precluding successful clinical 
application: tumor cells may be difficult to culture and transfect, and 
selection for transfected cells may require prolonged culture and alter 
expression of nominal tumor antigens. To avoid these potential disadvantages, 
we have developed an alternative approach to obtain paracrine cytokine 
secretion using fibroblasts genetically modified to secrete IL-12. The 
advantages of this strategy are that fibroblasts 1) can produce 
physiologically relevant levels of cytokine after transfection, 2) are readily 
available to culture, transfect and select, and 3) are less likely to migrate 
to other sites compared to tumor or lymphoid cells. 
1.3 IL-12 Paracrine Delivery 
To examine the effects of the rationale of IL-12 paracrine therapy 
described above, we have established animal models using fibroblasts 
genetically engineered to secrete murine IL-12 and examined its effects on 
tumor growth (Tahara et al, 1994). NIH3T3 cells were transfected with 
expression plasmids carrying both murine IL-12 genes, p35 and p40, using the 
calcium phosphate method (Sambrook et al, 1989) to express 100 - 240 
Hoffmann-La Roche units/10° cells/48 hours of IL-12. IL-12 levels were 
measured in a 4 d. human PHA blast assay and confirmed to be IL-12 by a PHA 
blast assay performed on the protein "captured" in microtiter wells by IL-12 
specific antibody. The effects of paracrine secretion of IL-12 on tumor 
establishment and vaccination models were examined using the poorly 
immunogenic murine melanoma cell line BL-6 in C57BL/6 (B6) mice. To determine 
the effects of IL-12 on tumor formation, non- irradiated BL-6 cells were 
subcutaneously Inoculated into B6 mice admixed with NIH3T3 cells transfected 
with both subunits of mIL-12(3T3-IL-12) or with cells transfected with only 
the neomycin phosphotransferase gene (3T3-Neo). Compared to mice injected with 
BL-6 alone, the day of emergence of detectable tumors was significantly 
delayed in mice injected with BL6 admixed with 3T3- IL-12, but not in mice with 
BL6 admixed with 3T3-Neo (Fig. la). Effectiveness in this system was related 
to the amount of JL-12 expressed by the 3T3-IL-12 (Fig. lb). 
To determine whether locally secreted IL-12 at the tumor site induces 
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