added, Bam HI digested, and cloned into the plasmid pMFG (28). The plasmid pMFG was 
Bam HI digested, the 470 base pair fragment containing the IL-4 cDNA was isolated, and 
cloned into the retrovirus backbone gagBA. The orientation of the IL-4 sequence, and the 
integrity of the plasmid provirus DNA structure were confirmed by restriction endonuclease 
digestion. A schematic of the plasmid map is shown in Appendix D, followed by the 
restriction map of the provirus and the anticipated complete sequence of the provirus. The 
plasmid provirus vector is currently being completely sequenced to confirm that no 
inadvertent mutations have occurred during plasmid DNA subcloning. 
4.3.b. Generation of recombinant retroviruses encoding for human IL-4 
Virus producing cell lines were isolated for the vector using the amphotropic 
packaging cell line H'crip. kindly provided by Dr. James Wilson (29). The ^crip cell line was 
constructed by transfecting the gag-pol and env functions of the retrovirus on separate 
plasmid constructs into NIH3T3 cells. The 3' LTR's of the constructs were replaced by 
heterologous polyadenylation signals. These modifications were performed to minimize the 
chance that recombination could result in the production of replication competent virus. The 
plasmids used to make the packaging cell line were described in the original paper. 
The retrovirus vector GBAH4 was cotransfected into ^Fcrip with pSV2Neo, and stably 
transfected clones were selected in 1 mg/mL G418. The producer cell lines are maintained 
in Dulbecco's modified Eagle's media supplemented with 10% calf serum and 
penicillin/streptomycin. After screening 22 virus producing clones, the best clone (GBAH4- 
18) was further characterized. Virus supernatant was used to transduce exponentially 
growing NIH3T3 cells and human melanoma cells. IL-4 secretion after transduction with 
GBAH4-18 ranged from 100-2000 pg/ml (Appendix E). Southern blot analysis was 
performed with cells transduced with the GBAH4-18 virus (Appendix F). This revealed 
approximately 1 copy of unrearranged virus in the NIH3T3 cells. Optimization of the 
transduction protocol with melanoma cell lines is continuing to attempt to improve this 
transduction efficiency. Virus supernatant has undergone a helper assay with 15 days of 3T3 
amplification. This showed no evidence of helper virus. 
The virus producer cell line is currently undergoing further safety testing as required by 
the FDA of clinical grade retrovirus stocks. This includes testing for replication competent 
virus, for the presence of bacterial, viral and fungal pathogens. The retrovirus supernatant 
will be produced by a commercial supplier and will not be used to transduce melanoma cells 
for human trials until approval is received from the FDA. 
5.0 ELIGIBILITY CRITERIA 
5.1 Eligible patients will include those with the following (see Appendix G): 
a. Histologically proven metastatic melanoma. 
b. Performance status ECOG 0-1 (see Appendix H). 
c. No symptomatic or acutely life-threatening tumor which is judged likely to 
require intervention with alternative modalities for 3 months. 
d. Non-tumor involved superficial inguinal or axillary lymph nodes which 
are surgically accessible. 
e. Absolute WBC >3000/mm 3 . 
f. BUN < 25 mg, creatinine < 1 .8 mg% 
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Recombinant DNA Research, Volume 18 
