Evaluation of the safety and biodistribution of M032, an attenuated herpes simplex virus type 1 expressing hIL-12, after intracerebral administration to aotus nonhuman primates.

Herpes simplex virus type 1 (HSV-1) mutants lacking the γ(1)34.5 neurovirulence loci are promising agents for treating malignant glioma. Arming oncolytic HSV-1 to express immunostimulatory genes may potentiate therapeutic efficacy. We have previously demonstrated improved preclinical efficacy, biodistribution, and safety of M002, a γ(1)34.5-deleted HSV-1 engineered to express murine IL-12. Herein, we describe the safety and biodistribution of M032, a γ(1)34.5-deleted HSV-1 virus that expresses human IL-12 after intracerebral administration to nonhuman primates, Aotus nancymae. Cohorts were administered vehicle, 10(6), or 10(8) pfu of M032 on day 1 and subjected to detailed clinical observations performed serially over a 92-day trial. Animals were sacrificed on days 3, 31, and 91 for detailed histopathologic assessments of all organs and to isolate and quantify virus in all organs. With the possible exception of one animal euthanized on day 16, neither adverse clinical signs nor sex- or dose-related differences were attributed to M032. Elevated white blood cell and neutrophil counts were observed in virus-injected groups on day 3, but no other significant changes were noted in clinical chemistry or coagulation parameters. Minimal to mild inflammation and fibrosis detected, primarily in meningeal tissues, in M032-injected animals on days 3 and 31 had mostly resolved by day 91. The highest viral DNA levels were detected at the injection site and motor cortex on day 3 but decreased in central nervous system tissues over time. These data demonstrate the requisite safety of intracerebral M032 administration for consideration as a therapeutic for treating malignant brain tumors.

Preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin-12.

Herpes simplex virus 1 (HSV-1) mutants that lack the γ(1)34.5 gene are unable to replicate in the central nervous system but maintain replication competence in dividing cell populations, such as those found in brain tumors. We have previously demonstrated that a γ(1)34.5-deleted HSV-1 expressing murine interleukin-12 (IL-12; M002) prolonged survival of immunocompetent mice in intracranial models of brain tumors. We hypothesized that M002 would be suitable for use in clinical trials for patients with malignant glioma. To test this hypothesis, we (i) compared the efficacy of M002 to three other HSV-1 mutants, R3659, R8306, and G207, in murine models of brain tumors, (ii) examined the safety and biodistribution of M002 in the HSV-1-sensitive primate Aotus nancymae following intracerebral inoculation, and (iii) determined whether murine IL-12 produced by M002 was capable of activating primate lymphocytes. Results are summarized as follows: (i) M002 demonstrated superior antitumor activity in two different murine brain tumor models compared to three other genetically engineered HSV-1 mutants; (ii) no significant clinical or magnetic resonance imaging evidence of toxicity was observed following direct inoculation of M002 into the right frontal lobes of A. nancymae; (iii) there was no histopathologic evidence of disease in A. nancymae 1 month or 5.5 years following direct inoculation; and (iv) murine IL-12 produced by M002 activates A. nancymae lymphocytes in vitro. We conclude that the safety and preclinical efficacy of M002 warrants the advancement of a Δγ(1)34.5 virus expressing IL-12 to phase I clinical trials for patients with recurrent malignant glioma.

Serial passage through human glioma xenografts selects for a Deltagamma134.5 herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice with experimental brain tumors.

Previous studies have described in vitro serial passage of a Deltagamma(1)34.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent Deltagamma(1)34.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early U(S)11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early U(S)11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.