effective anti-tumor immunity, 10^ irradiated tumor cells mixed with 3T3-IL-12 
or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge 
was subsequently examined (Fig. 2). With a tumor challenge of less than 1 x 
10^ non-irradiated BL-6 cells, significant delay of establishment of tumor 
was noted with a relatively small amount of IL-12 secretion (1.2 Hoffmann-La 
Roche units / 5 x 10^ cells / 48 hours). 
These results suggest that local delivery of IL-12 inhibits tumor growth in 
a dose dependent manner and leads to the development of an anti-tumor immune 
response . 
1.4 Development of TFG-IL-12 Retroviral Vector (Murine and Human) 
Our previous system uses allogeneic 3T3 cells as a means to continuously 
deliver IL-12 at the site of tumor. However, these cells probably induce an 
allogeneic immune response thus effectively eliminating long term IL-12 
production. This ma)ces direct interpretation of our results somewhat 
complicated. Development of efficient vectors for transfection into 
appropriate autologous cells will enable us to as)c questions raised by our 
studies and also test more clinically relevant systems. Thus, we have 
constructed a retroviral vector which allows us to express genes of both 
subunits of mIL-12 with a selectable marker. The requirement for simultaneous 
expression of p35 and p40 for production of biologically active IL-12 (Wolf et 
al, 1991), presented unique problems in the creation of these vectors. The 
existing strategies using regulated splicing or heterogeneous promoters raised 
substantial concern about discordant production of the two chains from the 
transfected cells. To overcome this problem, we developed vectors utilizing an 
internal ribosome entry site (IRES) sequence. The IRES sequence used was 
obtained from the 5' nontranslated region of equine encephalomyocarditis virus 
(EMCV) . Other picorna-viruses have similar sequences which allow cap- 
independent translation initiation from the internal ribosome entry site. A 
single polycistronic transcript from two separate genes can thus be 
transcribed following a single promoter and a second gene can be translated in 
a cap-independent manner. A modified MFC based retroviral vector (DFG-mIL-12, 
Fig. 3a) was constructed using an IREiS fragment of EMCV, obtained from the 
pCITE plasmid (Novagen, Madison, WI) . The resultant construct is shown in 
Fig. 3a. DFG-human IL-12 has also been constructed in a similar manner to 
prepare for this clinical application (Zitovogel et al. 1994). To obtain a 
retrovirus which carries both the mIL-12 genes and the neomycin 
phosphotranspherase gene as a selectable marker, the IRES-Neo (pMClneo Poly A, 
Strategene, La Jolla, CA) cassette was cloned into the site adjacent to the 
p35 cDHA in DFG-mIL-12 to create a vector capable of coordinately expressing 
three independent genes, termed TFG-mIL-12-Neo, Fig. 3b) . Retroviral 
supernatant was generated using DFG-roIL-12 and the CRIP packaging cell line 
(Danoa et al, 1988) or using TFG-mIL-12-Neo and the BOSC23 packaging cell line 
(Pear et al, 1993). We now have over six months of experience with a stable 
producer cell line ('FCRIP-TFG-IL-12-Neo) . 
1.5 Production of IL-12 Following Retriviral Transduction 
Elxpression of these retroviral vectors was measured using NIH3T3 cells. 
These cells were infected with the supernatant of the producer cell line, and 
their supernatant was measured for expression of the transfected genes (Table 
1 ) . 
Table 1 Expression of the transfected NIH-3T3 cells 
Vector (s) Transfection msthod Selection IL-1 2 .secretion* 
BL-pSV-p35 L p40 
Ca-Ph. 
+ (Colony #8) 
2.1 
DFG-mIL-12 
Infection 
- 
5 
TFG-mIL-12 
Infection 
^ (bulk) 
12Q. 
( 650 ) 
Recombinant DNA Research, Volume 19 
