Protocol HNS 94-001 
October 4, 1994 
Page 8 
Table II. Effect of Ad5CMV-p53 on Tumorigenicity in a Microscopic Residual Disease 
Model of HNSCC. 8 
Cell line 
Treatment 
No. mice w/ 
Total Mice (%) 
Vehicle (PBS) 
dl31 2 
Ad5CMV-p53 
Tu138 (homozygous mutation p53) 
8/8 
8/8 
0/8 
Tu177 (homozygous mutation p53) 
8/8 
8/8 
0/8 
686 LN (homozygous wild-type p53) 
5/8 
5/8 
0/8 
886 (homozygous wild-type p53) 
6/6 
6/6 
2/6 
a Mice were inoculated with 2.5 x 10 6 /flap subcutaneously. On the 2nd day post-inoculation, 
the mice were given either vehicle or viruses (1 xl 0 8 P.F.U. each in 0.1 ml) in the same flap 
site in a single intervention strategy. Tumor formation was evaluated at the end of a 8-week 
period. 
In a treatment strategy to determine the role of repeat treatment of AdCMVp53 in established 
tumors, subcutaneously established HNSCC tumor cell lines were peritumorally infiltrated with 
either vehicle (PBS) or viruses (1 xl 0 9 P.F.U. each in 0.1 ml) three times weekly for two 
consecutive weeks. Tumor burdens exceeding 1 cm in greatest dimension were utilized in 
these studies. A greater than 50% reduction in Tu138 tumor mass was seen in 5/5 
animals treated in this study with significant reduction in size compared to control-treated sites 
(p< 0.04). (Figure 10, Appendix D ) No evidence of systemic toxicity was clinically or 
histologically noted in whole organ necropsy studies. In additional repeat treatment 
studies using smaller established tumors that had reached 75mm 3 , were similarly 
peritumorally treated with identical controls as described above. In both Tu177 and Tu138, 
following 7 peritumoral AdCMVp53 treatments (3 times weekly), 8 of 9 animals had complete 
regression of disease whereas control animals showed continued progression of tumors as 
well as no evidence of systemic toxicity in every animal (10 of 10). 
5.0 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 [ 3 H]Thymidine. 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. 
3.2 Sterility will be assured by testing for aerobic and anaerobic bacteria, fungus, and mycoplasma. Other 
tests to be performed by Microbiological Associates, Rockville, MD include: 
Recombinant DNA Research, Volume 20 
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