Enhanced inhibition of syngeneic murine tumors by combinatorial therapy with genetically engineered HSV-1 expressing CCL2 and IL-12 Jacqueline N Parker, 1 Sreelatha Meleth, 2 Kenneth B Hughes, 3 G Yancey Gillespie, 4,5 Richard J Whitley, 1,3,6 and James M Markert 5 1 Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama, USA; 2 Department of Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama, USA; 3 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; 4 Brain Tumor Research Laboratories, University of Alabama at Birmingham, Birmingham, Alabama, USA; 5 Division of Neurosurgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA; and 6 Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA. Herpes simplex viruses type 1 (HSV-1) that lack the g 1 34.5 gene are unable to replicate in the central nervous system (CNS), but maintain replication competence in actively dividing tumors. To determine if antitumor therapy by M002, a g 1 34.5  HSV that expresses interleukin-12 (IL-12), could be augmented by combinatorial therapy with another g 1 34.5-deleted HSV-1 engineered to express the chemokine CCL2, Neuro-2a tumors were established subcutaneously in the syngeneic A/J mouse strain. Tumors received multiple injections intratumorally either of saline, the parent, non-cytokine-expressing virus R3659, M002, M010 (g 1 34.5  HSV expressing CCL2), or a combination of M002 and M010. Efficacies were evaluated by monitoring inhibition of tumor growth over time. Results demonstrated the following: (1) inhibition of tumor growth was most pronounced in tumors treated with a combination of M002 and M010; (2) enhanced tumor growth inhibition for the combinatorial treatment group was statistically significant compared to either M002 or M010 alone; and (3) the variability between slopes of the tumor growth rates within an individual treatment group appeared to be virus-dependent, and was reproducible between experiments. Our results demonstrate that combinatorial cytokine/chemokine g 1 34.5  HSV therapies can provide superior antitumor effects in experimental tumors as a model for malignancies arising in the brain. Cancer Gene Therapy (2005) 12, 359–368. doi:10.1038/sj.cgt.7700784 Published online 28 January 2005 Keywords: herpes simplex virus 1; IL-12; CCL2; Neuro-2a; immunohistochemistry E radication of malignancies arising in the brain has proventobeaformidabletask.Asanexample,high- grade malignant gliomas, the most common primary brain tumor, are almost always fatal despite aggressive surgical resection, radiotherapy and chemotherapy. Moreover, the overall 5-year survival rate for glioblas- toma (GBM), the most malignant glioma, is less than 5.5% and the median survival is approximately 1 year. 1 Because of poor survival of patients with primary intracranial malignancies, novel therapeutic approaches, most notably viral and gene therapy, have been investi- gated (for reviews, see Markert et al 2,3 and Andreansky et al 4). We and others have reported on the efficacy of using neuroattenuated replication-competent herpes simplexviruses(HSV)forthetreatmentofprimarybrain tumors. These viruses typically contain one or more mutations within the viral genome, including thymidine kinase (tk), 5 ribonucleotide reductase (U L 39), 6,7 UTPase 8 or g 1 34.5. 9,10 The g 1 34.5 gene of HSV is present in two copies and located within the inverted repeats of the unique long segment. Mutations within this gene have been shown previously to cause reduction in viral replication and associated neurovirulence of HSV. 11 Thus, g 1 34.5-deleted HSV are able to selectively replicate and destroy glioma cells in vivo, without damaging surrounding brain tissue. Moderate increases in long- termsurvivalforengineeredHSV-treated versus untreated animals have been reported in both syngeneic and xenogeneic murine tumor models of GBM. 5,9,10,12–16 In addition, Phase I studies in humans with malignant glioma have demonstrated that two g 1 34.5-deleted HSV, G207 and 1716, are safe for intracranial inoculation, 17–19 albeit at different quantities of inoculated virus. G207, whichcontainsmutationswithinbothcopiesofthe g 1 34.5 Received March 10, 2004. Address correspondence and reprint requests to: Dr James M Markert, MD, Division of Neurosurgery, Department of Surgery, University of Alabama at Birmingham, FOT 1050, 510 20th Street South, Birmingham, AL 35294-3410, USA. E-mail: jmarkert@uabmc.edu Cancer Gene Therapy (2005) 12, 359–368 r 2005 Nature Publishing Group All rights reserved 0929-1903/05 $30.00 www.nature.com/cgt