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Background: Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, used in combination with 5-fluorocytosine (5-FC) kills tumor by local production of 5-fluorouracil (5-FU), inducing local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. Toca 511 and Toca FC (oral extended-release 5-FC) are under investigation in patients with recurrent high-grade glioma. Lomustine is a treatment option for patients with high-grade glioma., Methods: We investigated the effects of lomustine combined with Toca 511 + 5-FC in syngeneic orthotopic glioma models. Safety and survival were evaluated in immune-competent rat F98 and mouse Tu-2449 models comparing Toca 511 + 5-FC to lomustine + 5-FC or the combination of Toca 511 + 5-FC + lomustine. After intracranial implantation of tumor, Toca 511 was delivered transcranially followed by cycles of intraperitoneal 5-FC with or without lomustine at the first or fourth cycle., Results: Coadministration of 5-FC with lomustine was well tolerated. In F98, combination Toca 511 + 5-FC and lomustine increased median survival, but "cures" were not achieved. In Tu-2449, combination Toca 511 + 5-FC and lomustine increased median survival and resulted in high numbers of cure. Rejection of tumor rechallenge occurred after treatment with Toca 511 + 5-FC or combined with lomustine, but not with lomustine + 5-FC. Mixed lymphocyte-tumor cell reactions using splenocytes from cured animals showed robust killing of target cells in an effector:target ratio-dependent manner with Toca 511 + 5-FC and Toca 511 + 5-FC + lomustine day 10., Conclusion: The combination of Toca 511 + 5-FC and lomustine shows promising efficacy with no additive toxicity in murine glioma models. Immunotherapeutic responses resulting in long-term survival were preserved despite lomustine-related myelosuppression., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.

We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).