PROTOCOL THS 9A-002 
REVISED 5/19/94 
PAGE 7 
tumors were confirmed by histologic analysis. The data of tumor measurements are 
summarized in Table II: only 25% of the Ad5CMV-p53-treated mice formed tumors, 
whereas in the vehicle or Ad5/RSV/GL2 control group, 70-80% of the treated mice 
formed tumors. The average tumor size of the Ad5CMV-p53 group was significantly 
smaller than those of the control groups. These results indicate that Ad5CMV-p53 
can prevent H226Br from forming tumors in the mouse model of orthotopic human 
lung cancer. 
Table II. Effect of Ad5CMV-p53 on tumorigenicity in 
H226Br In mouse model of orthotopic human lung cancer* 
Treatment 
No. mice w/Tumor 
Total Mice (%) 
Mean Volume 
(mm® ± SD) 1 
Vehicle 
7/10 (70) 
30 ± 8.4 
Ad5/RSV/GL2 
8/10 (80) 
25 ± 6.9 
Ad5CMV-p53 
2/8 (25) 
8 ± 3.3® 1 
• Mice were inoculated with 2x10® H226Br cells/mouse intratracheally. On the 3rd day 
post-inoculation, the mice were given either vehicle or viruses (5 xio’ each in 0.1 ml) 
intratracheally once a day for 2 days. Tumor formation was evaluated at the end of a 6-week 
period. 
** p <.0.05 by two-way analysis of variance when compared to the groups receiving vehicle 
(PBS) or virus control. 
Some human lung cancer cells such as H358a are resistant to both p53 and chemotherapy 
induced apoptosis. In an effort to confirm the feasibility of this combination therapy in 
human lung cancer, we examined whether Ad-p53 and CDDP given In a sequential 
combination could induce apoptosis in vivo. Following 3-day direct intratumoral Injection of 
Ad-p53, H358a tumors subcutaneously transplanted in nu/nu mice showed a modest 
slowing of growth; Ad-p53-lnjected tumors, however, regressed if CDDP was administered 
Intraperitoneally for 3 days, and the tumor size remained almost consistent for more than 12 
days by multiple cycle treatments with Ad-p53 and CDDP (Fig. 8/V, Appendix E). Histologic 
examination revealed a massive destruction of tumoral tissues in the area where Ad-p53 was 
injected in mice treated with CDDP. In situ staining of programmed cell death demonstrated 
many apoptotic cells around the dead space (Fig. 8B-E, Appendix E). In contrast, tumors 
treated with CDDP alone or Ad-p53 alone showed neither empty space nor apoptotic area. 
3.0 
SAFETY INFORMATION 
3.1 Continued absence of replication competent infectious virus was determined from sequential 
Infection experiments. No replicative virus was detectable by PCR analysis of DNA samples from 
HeLa cells treated with the frozen/thawed cell extracts from HeLa cells Initially Infected with 
Ad5CMV-p53 at 100 PFU/cell, Ad5CMV-p53 was confirmed as a replication-defective and helper- 
independent virus. Further confirmation of this was obtained by labeling viral supernatants with 
[’HjThymidlne. Absence of labeling in extracted DNA showed absence of replication competent 
adenovirus. These studies and the following safety studies will be performed by Microbiological 
Associates, Inc. 
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Recombinant DNA Research, Volume 19 
