conditions which allow only highly contemporary sequences of probe to remain bound to target DNA. 
The presence of specifically bound 32 P-labeled RNA probe is detected by autoradiography of the nylon 
membranes. The inherent specificity of nucleic acid hybridization is enhanced in this assay by the use 
of RNA probes. This approach allows an enzymatic digestion step to reduce nonspecific binding of 
probes, thus minimizing background signal. In addition, the performance of separate hybridizations 
with three discrete probe mixtures provides an in there control for nonspecific hybridization for each 
specimen. It is stated that approximately 70% of all HPV infected patients contain one or more of the 
HPV strains detectable by our assay. 
4.6.2 112 production of irradiated cells. 
Prior to injection the transfected, 112 producing SCLC cells will be irradiated with 12,000 rad. 
Table 10a, b, Appendix C shows that irradiated SCLC, similar to the murine LLC, continue to produce 
112 for up to 12 days. Thus, even though the cells are not replication competent, they still function to 
produce lymphokine and may induce a strong immune response. 
4.6.3 Clonogenic assay of irradiated cells: 
To ascertain the absence of surviving cells post irradiation (12,000 rad) of the 112 producing pBMG- 
Neo-112 transfected SCLC lines clonogenic assays were carried out in the absence or presence of feeder 
cells (Table 10a, b, see Appendix C). The cloning efficiency of two SCLC lines (non irradiated) 
measured in the presence of 10,000 irradiated feeder cells, plated in a volume of 0.2ml in microtiter 
plates, is 58 and 46%. In the absence of feeder cells, plating efficiency at 1 cell per well is 0 and 2%. 
Plating irradiated cells (12,000 rad) at 10 4 cells/well, no colony was detected after three weeks, 
which is ample time for colonies to form. A total of 15xl0 6 cells were plated in three separate 
experiments as described, and no colonies were detected. Allowing for the plating efficiency as 
determined above in the presence of irradiated feeder cells our data indicates that there are fewer than 
1 live cells in 6.8xl0 6 irradiated SCLC. It appears therefore that the injection of 5xl0 6 irradiated cells 
for patient treatment is safe and that tumor outgrowth is extremely unlikely. 
4.6.4 Microbacterial Studies 
Microbacterial studies include fungal cultures, aerobic and anaerobic cultures for bacteria and 
screening for mycoplasma. 
4.6.5 Endotoxin Assay 
Endotoxin assay by the Limulus amoebocyte lysate assay. 
4.7 GENE THERAPY 
f 
Not sooner than two months after the completion of chemotherapy, patients who continue to satisfy 
the eligibility criteria previously listed and who have cell lines with adequate numbers of cells stably 
transfected with interleukin genes will receive these transfected cells after all biosafety assessments 
required by the IRB, FDA and RAC- approved protocol have been completed. The gene-transfected 
cells will administered subcutaneously under the skin of the forearm. 
NOTE: All cells will be irradiated immediately before administration to the patient to prevent their 
subsequent replication. Our preliminary studies indicate that the dose of irradiation that prevents 
replication of cells while not impairing their ability to produce interleukin-2 is 6,000 cGy. 
Recombinant DNA Research, Volume 18 
[259] 
