studies were generated. In fact, even murine cell lines lacking vector can be induced to produce 
xenotropic RCRV (38) which can replicate in human cells. 
Another safety concern with murine PCLs used for retroviral vector production is that endogenous 
defective proviruses in the murine genome are expressed (e.g., VL30 sequences), recognized by 
the retroviral structural gene products of murine PCLs, and delivered and expressed in target cells 
with an efficiency at least comparable to that of the desired vector (39). The delivery of these 
undesirable retroviral-like sequences and the possibilities for generating replication-competent 
retrovirus suggest that murine cell lines may be less than ideal for the production of murine 
retroviral vectors for human therapeutics. 
Due to the safety risks associated with the generation of RCRV by homologous recombination with 
endogenous sequences, we have developed MLV-based retroviral PCLs from a canine cell line, 
D17. The MLV structural genes ("gag/pol" and "env") are also genetically unlinked, to decrease 
the chance of retrovirus generated by homologous recombinations between the therapeutic pro- 
vector DNA and the DNA encoding the MoMLV structural genes present in the PCL. These 
changes should help alleviate many of the safety concerns described above. 
2.3 CLINICAL APPROACH 
We propose to utilize retroviral vectors to deliver and express the y-IFN gene to human tumors in 
order to improve ASI for human malignancies. It is hoped that this approach will improve 
antigenic presentation and subsequent tumor-specific immune activation in cancer patients by 
creating an optimal microenvironment around the tumor cells for the activation of cell mediated 
immune responses. Short-term tumor cell cultures will be established from human tumor biopsy 
material. The resultant cell cultures will be transduced with the y-IFN retroviral vector. After 
G418 selection, lethal irradiation, and quality control testing, the lymphokine-expressing 
autologous tumor cells will be injected back into the patient. The resultant increase in specific 
immunity against the highly immunogenic gene-modified tumor cells may result in the clearance of 
the endogenous unmodified metastatic tumors (see Fig 1 for a diagrammatic summary of the 
approach). 
Although the lymphokine gene delivery approach could theoretically be applied to almost any 
tumor, certain tumor types offer greater chance for success than others. Some of the criteria used 
to evaluate tumor types include: general availability (tumors that are commonly accessible), tumor 
cell culturability in vitro, under expression of Class I MHC, known involvement of immune 
response (especially CTL), and known clinical response to immune therapies. By these criteria, 
the most logical choice is melanoma. Melanomas are relatively easy to establish as cell lines in 
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