Expression of FMS-like Tyrosine Kinase 3 Ligand by Oncolytic Herpes Simplex Virus Type I Prolongs Survival in Mice Bearing Established Syngeneic Intracranial Malignant Glioma

Recently, there has been increased interest in immunotherapy approaches for treatment of malignant glioma. Better understanding of the cellular entities that drive or suppress antitumor immunity, enhanced ex vivo cellular engineering techniques, and recognition of a growing number of glioma-associated antigens have lead to successful preclinical models of vaccination, and early-phase clinical trials have demonstrated safety, systemic biological effect, and suggestions of disease stabilization and extended survival. Currently, phase II multicenter dendritic cell vaccination1 and epidermal growth factor variant III (EGFRvIII) peptide vaccination2 protocols are being conducted for patients with newly diagnosed glioblastoma. Although the immune system is able to develop antibody and T-lymphocte responses against growing glioblastomas, tolerance wins out over antitumor immunity, and the tumor effectively shields itself from immune effectors. Therefore, the key to clinical efficacy is the successful breaking of tolerance. In some fashion, tumor-associated antigens require unveiling so that they can be presented to effector lymphocytes which can be activated and positioned to infiltrate and target the tumor. Given the lack of draining lymphatics in the central nervous system and the lack of potent antigen presenting cells in the immunosuppressed brain tumor microenvironment, driving an effective anti-glioma response presents particular challenges. Treatment of malignant tumors with oncolytic herpes simplex virus 1 (oHSV) vectors is promising because of the opportunity to target cancerous cells while sparing neighboring normal tissues. Cancer clinical trials examining direct intratumoral or intravascular injection of oHSV in patients with solid tumors inside and outside of the brain have been completed without evidence of treatment-associated toxicity and with some objective clinical and radiographic responses3–6. The dynamic interplay between oHSV with the immune system is a critical factor in understanding how to optimize the vigor and the durability of the antitumor effect7. As expected, antiviral immunity develops or re-emerges after infection and can limit the viral replicative cycle and abrogate the direct cytocidal impact of the therapy8. In fact, pre-infection suppression of innate immunity with cyclophosphamide or inhibitors of complement is associated with enhanced oHSV replication and tumor killing in rodent models. Our group and others have demonstrated that oHSV infection of flank tumors initiates an inflammatory cascade that results in the development of systemic and specific adaptive antitumor immunity9. In an effort to take advantage of this anticancer vaccine effect, investigators have armed oHSV with genes for immunostimulatory cytokines such as GM-CSF6 and IL-1210, which have variably yielded improved tumor control in several models. Dendritic cells (DCs) are professional antigen-presenting cells that have the capacity to migrate to sites of inflammation, to ingest and process antigenic material, and, then, to traffic to draining lymph nodes where cross-presentation of tumor antigens to lymphocyte receptors occurs. DCs may represent the link between the initial innate immune response to viral infection and subsequent adaptive antiviral or, antitumor immunity. This is underscored by the fact that combining oHSV infection of flank tumors with intratumoral injection of ex vivo generated immature DC’s generates a powerful antitumor immune response that is nearly 100% curative 11. oHSV infection appears to break tolerance to tumors by exposing tumor-associated antigens and by elaboration of inflammatory danger signals, but the subsequent enhancement of antigen presentation by DCs appears to be requisite 12, 13. We, therefore, set out to engineer an oHSV that expresses soluble Flt3L, a cytokine and growth factor associated with the development of hematopoietic precursors into both plasmacytoid (pDCs) and conventional (cDCs) dendritic cells, as well as their mobilization out of bone marrow 14. We hypothesized that infection of an intracranial glioma by an oHSV armed with Flt3-L transgene would exert an antitumor immune effect by creating an inflammatory environment in situ, while systemically mobilizing DC precursors.