To avoid the use of infectious virus as a DNA transfer vehicle, we have used the Lipofectin 
technique, which also was successful in the murine LLC model. To date we have transfected 4 SCLC 
lines previously established by Dr. Savaraj. Since the doubling time of these cells is usually around 30h, 
a minimum period of two to three weeks is required to establish neomycin resistant lines. The same 
period again is required to expand the cells to sufficient numbers to be used for therapy. Monitoring 
of the growth rate and morphology of transfected cells showed no change in comparison to untransfected 
cells during the time span monitored (2 months). The transfected cell lines tested for biological activity 
produce the expected product. Moreover, the levels produced are quite high (> 1000U 112 in 24h by 
10 6 cells) and will certainly be sufficient to induce a vigorous immune response upon immunization of 
patients. 
In the Lipofectin technique, 5xl0 6 SCLC are washed twice with serum free medium (Opti-MEM, 
Gibco) and plated in 6cm tissue culture petri dishes (Falcon 3002) in 3ml OPTI-MEM. The Lipofectin 
DNA complex is generated by 5 min incubation at room temperature of 10 fxg plasmid DNA with 50^g 
Lipofectin in a volume of lOOjd H 2 0 and then slowly added with swirling to the cell culture. The cells 
are incubated without further additions in the C02 incubator overnight and then an equal volume of 
culture medium (IMDMEM) containing 20% FCS is added. After 48h 1 mg/ml G418 geneticin is 
added. The culture medium is changed and replaced with fresh G418 every two days until new colonies 
start growing. Then the medium is changed twice per week. The cells are expanded by continuous 
culture in standard medium [10% FCS, Iscove’s modified Dulbecco’s minimum essential medium 
(IMDMEM)], certified free of contaminants. For mycoplasma detection prior to patient treatment, 
samples are sent to the American Type Culture Collection (ATCC), for culture and staining. 112 
production in murine and human cells is summarized in Tables 8 and 9, Appendix C. Typically, both 
murine LLC and human SCLC produce a total of 1,000 to 2,000 U 112 in 24h when cells are 1- 
5xl0 6 /ml. This is sufficient to result in tumor rejection in mice. 
4.6 Other Safety Considerations 
Prior to transfection the established cell lines will be tested for the content of human papilloma virus 
(HPV) sequences. Cells that are positive for HPV will be excluded from further studies. After selection 
for neomycin resistance in G418 the cells will be expanded and tested for the production of the 
transfected lymphokine by standard biological assays. 
Transfected cells for use in patients will be screened for contamination as indicated below. 
4.6.1 Papilloma Virus Tests 
Papilloma virus tests will be performed to detect human papilloma virus sequences which could 
possibly interact with the bovine papilloma virus component of the transfecting vector pBMG Neo 
possibly resulting in a promoting effect on human papilloma infections. The ViraType HPV DNA 
Typing Kit is based on the specific binding of complementary 32 P-labeled RNA probes to target DNA 
from infected cells. Viral DNA released from disrupted cells is denatured and bound to a solid support 
by filtration through nylon membranes having a high affinity for nucleic acids. Three replicate 
membranes are prepared by filtration of an aliquot of the sample onto each of three membranes. HPV 
target DNA bound to the three replicate membranes is then hybridized to three distinct probe groups 
specific for HPV types 6/11, 16/18 or 31/33/35. The ViraType HPV DNA Typing Kit employs 32 P- 
labeled RNA probes prepared by in vitro transcription of recombinant plasmids containing substantially 
the entire DNA sequences of HPV types 6, 11, 16, 18, 31, 33, and 35. Following hybridization, the 
nylon membranes are treated with ribonuclease to remove unhybridized probe and washed under 
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Recombinant DNA Research, Volume 18 
