HUMAN GENE THERAPY 10:2757–2768 (November 20, 1999) Mary Ann Liebert, Inc. Disabled Infectious Single-Cycle Herpes Simplex Virus as an Oncolytic Vector for Immunotherapy of Colorectal Cancer STEPHEN TODRYK, 1 CORNELIA MCLEAN, 2 SELMAN ALI, 3 CLAIRE ENTWISTLE, 2 MICHAEL BOURSNELL, 2 ROBERT REES, 3 and RICHARD VILE 1,4 ABSTRACT New modalities of treatment for colorectal cancer are required to support and improve those currently avail- able. One such approach is immunotherapy by transfer of immunostimulatory genes to tumor cells. Here, we report the use of a herpes simplex virus (HSV) vector that is capable of a single round of infection (disabled infectious single-cycle [DISC]-HSV) as a gene transfer vehicle for colorectal cancer. This vector has potential advantages over other vectors for cancer immunotherapy in that it lyses infected tumor cells. Infection with DISC-HSV inhibited tumor cell growth both in vitro and in vivo. In addition, DISC-HSV-mediated cell killing occurs by both apoptotic and necrotic mechanisms. A range of colorectal tumor cell lines could be rapidly transduced with DISC-HSV/ lacZ (14–90% in 4 hr). Both tumor prevention and tumor therapy protocols showed clear antitumor effects with DISC-HSV/mGM-CSF. In the prophylactic approach, an infected/irra- diated whole cell vaccine protected up to 80% of mice from rechallenge. In addition, intratumoral injection of established tumors with DISC-HSV/GM-CSF caused rejection in 40% of mice and generated some pro- tection from subsequent rechallenge. In both cases, however, it is clear that a dominant therapeutic effect of the DISC-HSV vector derives from its oncolytic properties, irrespective of the transduced gene. As a prelude to taking these studies forward to human clinical trials, we demonstrate that tumor cells could be successfully grown from freshly obtained human colorectal cancer resections (within 1 week of surgery), were transduced with DISC-HSV/hGM-CSF, and secreted the cytokine. This study provides the preclinical basis for trials of immunotherapy of colorectal cancer using DISC-HSV. 2757 OVERVIEW SUMMARY The disabled infectious single-cycle herpes simplex virus (DISC-HSV) vector has been developed as a vehicle for transfer of immunotherapeutic genes to colorectal tumor cells. All human colorectal tumor cell lines examined could be rapidly transduced (within 4 hr) with DISC-HSV en- coding b -galactosidase (DISC-HSV/lacZ ). Infection inhib- ited tumor cell growth and caused apoptosis and necrosis. DISC-HSV-mediated transfer of GM-CSF to murine colo- rectal tumor cells, either ex vivo or directly in vivo by in- tratumoral injection, generated protection from rechallenge or regression of established tumors, respectively. Human colorectal tumor cells could be successfully isolated from surgical resections and transduced with DISC-HSV to pro- duce human GM-CSF. These preclinical studies suggest that the DISC vector should be of value for both ex vivo or in vivo delivery of immunotherapeutic genes to tumor cells for clinical trials. INTRODUCTION C OLORECTAL CANCER is the second most common cause of cancer-related death, with 17,900 deaths in the United Kingdom in 1995 (Imperial Cancer Research Fund/Office for National Statistics report). New modalities of treament of colo- rectal cancer would be of great benefit in this disease, where 1 Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial College of Science and Medicine, Hammersmith Hospital, Lon- don, W12 0NN, UK. 2 Cantab Pharmaceuticals Research, Ltd., 310 Cambridge Science Park, Cambridge, CB4 1LH, UK. 3 Department of Life Sciences, Nottingham Trent University, Nottingham, NG11 8NS, UK. 4 Molecular Medicine Program, Guggenheim 18, Mayo Clinic, Rochester, MN 55905.