*: Measured as (ng / 10^ cells / 48 hours) 
These results demonstrate the ability of our vectors to efficiently 
transfect and Induce production of large quantities of biologically active 
IL-12 from transfected fibroblasts. 
We used the TFG-mIL-12*Neo vector to genetically engineer fibroblasts which 
were injected directly into the subcutaneous tumor. Our results clearly showed 
that IL-12 not only has a profound effect on suppressing tumor growth, but 
also in promoting protective immunity to a subsequent tumor challenge. Thus, 
if local injection of fibroblasts expressing IL-12 can induce a well 
established tumor to regress and promote antl-tvimor immunity at the same time, 
this strategy might be applicable in the clinical setting. 
A preliminary experiment was done using Infected 3T3 cells expresslng- 
high levels of IL-12 (3T3-TFG-IL-12: 120 ng / 10^ cells / 48 hours). 3 x 10^ 
MCA207 (murine methycholanthrene sarcoma) cells were inoculated intradermally 
into the left flank of C57BL/6 mice on day 0. Seven days after the 
Inoculation, saline, 1 x 10° 3T3-Neo, or 1 x 10° 3T3-TFG-IL-12 were injected 
peri- tumorally into the well established tumor (mean size of tumors at the 
time of IL-12 treatment; 36 mm^), and the tumor area was measured in a blinded 
fashion (Fig. 4). In the 3T3-Neo treatment group, 7/8 tumors grew 
progressively after injection. One of these animals exhibited rejection of 
their tumors following injection of the allogeneic 3T3 line, however it 
recurred. 8 of 9 tumors treated with 3T3-TFG-IL-12 transfected fibroblasts 
disappeared within 2 weeks after treatment. Three of these animals experienced 
recurrence of their tumors around day 25, but one of them dissappeared again. 
In summary, 6 of 9 animals with well established tumor were apparently cured 
(>37 days). These mice were challenged with 5 x 10^ MCA- 207 tumor cells in 
the other flank, and all of them rejected the subsequent challenge. A profound 
anti- tumor effect of local IL-12 delivery was observed in this experiment. 
This approach is appealing since large number of live tumor cells existing in 
the tumor nodule can be used as antigen in contrast to the existing gene 
therapy vaccination protocols previously approved. 
To date, in vivo gene therapy models which demonstrate such anti- tumor 
effects have utilized only a few different strategies. These include in vivo 
gene transfer of the herpes simplex thymidine kinase (HSV-TK) gene using 
direct injection of the retroviral producer cell line (Culver et al, 1992) and 
one using a liposome-DNA conjugate (Nable et al, 1992). Although clinical 
application of our approach still uses a somewhat complicated procedure with 
propagation and transfection of autologous fibroblasts in culture, there are 
significant theoretical advantages compared with some of these other 
approaches. Our procedure 1) is independent of transfection efficiency which 
might be very different in vivo 2) can avoid delivery of infectious viral 
particles to the host, and 3) does not include potential immunogens other than 
the nominal tumor antigen. These hypothetical advantages provide an additional 
rationale for proceeding with this IL-12 gene therapy protocol. 
2.0 OBJECTIVES 
2.1 Define the local and systemic toxicity associated with peritumoral 
injection of fibroblasts genetically engineered to secrete IL-12 
2.2 Examine the local and systemic immunmodulatory effects of peritumoral 
Injection of fibroblasts genetically engineered to secrete IL-12 into human 
tiimors. 
2.3 Evaluate the clinical efficacy of this procedure. 
Recombinant DNA Research, Volume 19 
[651] 
