Assessment of p53 gene transfer and biological activities in a clinical study of adenovirus-p53 gene therapy for recurrent ovarian cancer Shu Fen Wen, 1 Vikas Mahavni, 2 Erlinda Quijano, 1 Jeremy Shinoda, 1 Michael Grace, 3 Mary Lynn Musco-Hobkinson, 3 Tong-Yuan Yang, 3 Yuetian Chen, 3 Ingo Runnenbaum, 4 JoAnn Horowitz, 3 Dan Maneval, 1 Beth Hutchins, 1 and Richard Buller 2 1 Canji, Inc., San Diego, California, USA; 2 The University of Iowa Hospital and Clinic, Iowa City, Iowa, USA; 3 Schering-Plough Research Institute, Kenilworth and Union, New Jersey, USA; and 4 Universita ¨ts-Frauenklinik, Freiburg, Germany. A cohort study was designed to evaluate the efficiency of gene transfer and whether biological activity from the expressed therapeutic gene resulted after administration of a recombinant adenovirus containing the human wild-type p53 (p53 wt ) gene (rAd- p53 SCH 58500). The cohort study was conducted in five trial subjects with recurrent ovarian cancer. Each trial subject received multiple cycles of rAd-p53 SCH 58500, each cycle comprised of doses of 7.5  10 13 particles on each of five consecutive days. Subjects were treated with rAd-p53 SCH 58500 alone during Cycle 1 and in combination with gemcitabine during the subsequent cycles. Both tumor biopsies and peritoneal aspirates were collected and evaluated for gene transfer and evidence of the biological activities of the expressed p53 wt gene. Using quantitative PCR and RT-PCR, and in situ PCR, gene transfer and expression were documented in tumor biopsies (four of five patients) collected from Cycle 1. Furthermore, upregulation of p21/WAF1, bax and mdm-2, and downregulation of survivin were observed in these same tumor biopsy samples, suggesting that intraperitoneal administration of rAd-p53 SCH 58500 leads to detectable p53 biological activity in target tumor tissue. In addition, gene transfer and its expression were observed in cells obtained from peritoneal aspirates. These fluids were mainly comprised of polymorphonuclear neutrophils, indicating that successful gene transfer can be achieved by multiple cycle intraperitoneal administration of recombinant adenovirus. Cancer Gene Therapy (2003) 10, 224–238. doi:10.1038/sj.cgt.7700562 Keywords: rAd-p53; ovarian cancer; gene therapy; QPCR; SCH 58500 r Ad-p53 SCH 58500 is a replication-deficient adenovirus encoding the p53 tumor-suppressor gene and has been studied in human subjects with melanoma, breast cancer, 1 small-cell lung cancers, 2,3 bladder cancers, 4 liver, 5 and ovarian cancers. 6,7 These studies established the safety and feasibility of regional injections of rAd-p53 SCH 58500 for cancer. Evidence of p53 gene transfer was also shown for most of the human subjects who received high doses of rAd- p53 SCH 58500. Extensive safety data and gene transfer information have also been reported by other investigators using recombinant adenoviral (rAd) gene transfer in various clinical trials. 8–12 While many of these initial protocols have demonstrated the feasibility of using recombinant adeno- virus to deliver genes to tumors in humans, further investigation is required to understand the potential for effective gene therapy with this delivery system. In this study we investigated: (1) whether the gene was delivered to the tumors of human subjects using a recombinant adenovirus vector, (2) whether the delivered gene demonstrated the anticipated transcriptional activity of human p53 wt , and (3) whether repeat administration of a recombinant adenovirus will result in significant gene transfer. To this end, we designed a clinical protocol to evaluate the efficiency of gene transfer after multiple cycles of rAd-p53 SCH 58500 and measured the expres- sion of genes known to be transcribed by p53. It has been reported in clinical studies that a single intraperitoneal administration of a recombinant adeno- virus vector carrying the gene encoding thymidine kinase 13 or anti-erbB-2 single-chain antibody 10 can result in detectable biological activity of the therapeutic gene. However, in these studies only low levels of gene transfer and expression were observed in tumors. The authors of these studies have also suggested that changing the dose regimen to include multiple administrations of the rAd might be a means to enhance the therapeutic benefit. In fact, favorable clinical responses have been reported in Received May 6, 2002. Address correspondence and reprint requests to: Dr Shu Fen Wen, Canji, Inc., 3525 John Hopkins Ct., San Diego, CA 92121, USA. E-mail: shufen.wen@canji.com Cancer Gene Therapy (2003) 10, 224–238 r 2003 Nature Publishing Group All rights reserved 0929-1903/03 $25.00 www.nature.com/cgt