Maraba Virus as a Potent Oncolytic Vaccine Vector

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© The American Society of Gene & Cell Therapy

Maraba Virus as a Potent Oncolytic Vaccine Vector Jonathan G Pol1, Liang Zhang1, Byram W Bridle1,2, Kyle B Stephenson1,3, Julien Rességuier1, Stephen Hanson1, Lan Chen1, Natasha Kazdhan1, Jonathan L Bramson1, David F Stojdl4, Yonghong Wan1 and Brian D Lichty1 McMaster Immunology Research Center, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; Ontario Veterinary College, University of Guelph, Toronto, Ontario, Canada; 3Centre for Innovative Cancer Research, Ottawa Hospital Research ­Institute, Ottawa, Ontario, Canada; 4Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada 1 2

The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity. Received 11 April 2013; accepted 10 October 2013; advance online publication 10 December 2013. doi:10.1038/mt.2013.249

INTRODUCTION

Oncolytic viruses (OVs) specifically infect, replicate in and kill malignant cells, leaving normal tissues unaffected. Several OVs have reached advanced stages of clinical evaluation for the treatment of various neoplasms.1 Data from clinical trials and preclinical models have demonstrated that these viral agents alone or in combination with standard cancer therapies hold great promise for improved therapeutic efficacy.2–4 We have previously reported that a naturally attenuated vesicular stomatitis virus (VSVΔM51), a prototypical rhabdovirus, is a compelling oncolytic agent due to its safety profile and the lack of pre-existing neutralizing antibodies in human populations, a practical problem associated with several other OV platforms.5–8 To expand our current array of safe and potent OVs from the Rhabdoviridae, we have recently identified several new vesiculoviruses that display strong oncolytic activities.9,10 Specifically, the Maraba virus showed the broadest oncotropism in vitro and specific genetic modifications were shown to dramatically improved its tumor selectivity and reduced its virulence in normal cells.

In vivo, this attenuated strain, called MG1, demonstrated potent antitumor activity in xenograft and syngeneic tumor models in mice, with superior therapeutic efficacy than VSVΔM51.9 Data accumulated over the past several years has revealed that antitumor efficacy of OVs not only depends on their direct oncolysis but may also depend on their ability to stimulate antitumor immunity.11 This immune-mediated tumor control seems to play a critical role in the overall efficacy of OV therapy. Indeed, tumor-specific adaptive immune cells can patrol the tissues and destroy tumor cells that have been missed by the OV. Moreover, their memory compartment can prevent tumor recurrence. Various strategies have been developed to improve OV-induced antitumor immunity.12 Some groups have genetically engineered OV expressing immunostimulatory cytokines. An herpes simplex and a vaccinia virus expressing granulocyte-macrophage colonystimulating factor have respectively reached phase III and IIB of the clinical evaluation for cancer therapy while a VSV expressing interferon (IFN)-β has just entered phase I.1 Another strategy, defined as oncolytic vaccine, consists of expressing a tumor antigen from the OV. Previously, we and others have demonstrated that VSV could also be used as a cancer vaccine vector.13,14 When applied in an heterologous prime-boost setting to treat a murine melanoma model, our VSV oncolytic vaccine not only induced an increased tumor-specific immunity but also a concomitant reduction in antiviral adaptive immunity. As a result, the therapeutic efficacy was dramatically improved with an increase of both median and long-term survivals.13 Given that both VSV and Maraba are classified as vesiculoviruses, we hypothesized that Maraba MG1 could share the vaccine properties of VSV. In the present study, we demonstrated that oncolytic Maraba MG1 can be used as an oncolytic vaccine platform. While unable to prime detectable responses against a melanoma-associated antigen, Maraba MG1 vaccine displayed a potent ability to boost pre-existing tumor-specific CD4+ and CD8+ T-cell immunity. When applied to the treatment of syngeneic murine melanoma tumor models, Maraba MG1-mediated recall immunization resulted in a dramatic extension of the median survival with complete remission in more than 20% of the animals treated. Together with our previous study involving VSV,13 this work confirmed that vesiculoviruses can be potent vaccine vectors for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive cell immunity.

Correspondence: Brian D Lichty, McMaster Immunology Research Centre, Department of Pathology and Molecular Medicine, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada. E-mail: [email protected]

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Maraba Virus as a Potent Oncolytic Vaccine Vector

RESULTS Characterizing the oncolytic activity of Maraba MG1 in vitro and its oncotropism in vivo in the murine B16-F10 melanoma model

The oncolytic property of Maraba virus has recently been characterized in five melanoma-derived cell lines from the NCI-60 panel.9 However, the B16-F10 metastatic melanoma cell line, used as the tumor model in the present study, was not included. Before evaluating the therapeutic efficacy of Maraba MG1 in tumorbearing mice, we evaluated its ability to lyse B16-F10 cells in vitro. For this purpose, a monolayer culture of B16-F10 melanoma cells was infected with MG1-GFP at a multiplicity of infection of 0.01 and cell viability and viral replication were measured. By visualizing green fluorescent protein (GFP) expression, Maraba infection was noticeable at 12 hours and spread through the whole culture within 24 hours (Figure 1a). A burst of virus production was achieved within 20 hours (Figure 1b), along with clearance of the B16-F10 cell population (Figure 1c and visible field on Figure 1a). These data demonstrated that Maraba MG1 was able to infect, replicate in and kill B16-F10 melanoma cells. We subsequently evaluated the ability of Maraba virus to selectively infect B16-F10 melanoma cells in vivo. Immunocompetent mice bearing B16-F10 lung metastases, as well as tumor-free controls, were inoculated intravenously (i.v.) with MG1-GFP. Infectious particles were quantified in the lungs and in the secondary lymphoid organs (spleen and inguinal lymph nodes) at 24 hours and 48 hours after virus injection (Figure 2a). Maraba MG1 was detected at high titers in the spleen 24 hours after inoculation

a

Maraba MG1-hDCT alone is insufficient to improve the therapeutic outcome and to induce antitumor immunity Although Maraba MG1 could lyse B16-F10 cells in vitro and replicates at the tumor site in vivo, i.v. administration of MG1-GFP at its highest tolerable dose (109 pfu—Brun et al.)9 did not result

12 hours

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(5 log plaque-forming units (pfu)/g tissue) but cleared from the organ by 48 hours (0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. Ad, adenoviral vector; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.

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Figure 6  Maraba MG1 vaccine accelerates secondary CD8+ T-cell responses. C57Bl/6 mice received 2 × 108 pfu Ad-hDCT by intramuscular injection (DCT prime). At 12, 9 or 4 days after Ad-hDCT prime, MG1-hDCT was administered by intravenous injection at a dose of 109 pfu. DCT-specific T-cell responses were measured 5 days after Maraba injection in the blood. Immune response measured at the same timepoint in Ad-hDCT prime only animals is indicated (“No boost”). (a) Percentage of CD8+ T cells secreting IFN-γ after ex vivo exposure to the MHC-I restricted DCT immunodominant epitope SVY. (b) Percentage of CD4+ T cells secreting IFN-γ after ex vivo exposure to the MHC-II restricted DCT immunodominant epitope KFF. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values (n = 10 mice per group). P value considered nonsignificant (NS) when >0.05, *P < 0.05 and ***P < 0.001. Ad, adenoviral vector; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.

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Ad-hDCT prime-MG1-hDCT boost vaccination generated a very strong DCT-specific CD8+ T-cell response (mean % IFN-γ+ CD8+ T cells = 27.54 ± 2.17, Figure 7b) that was 14 times higher than in

model. Five days following i.v. injection of B16-F10 cells, animals received a sequential administration of Ad-hDCT and MG1-hDCT at a 9-day interval (Figure 7a). We confirmed that a Days

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Immune analysis Ad-empty Ad-hDCT Ad-hDCT Ad-hDCT

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Figure 7  Maraba MG1-hDCT administered in a heterologous prime-boost setting allowed to generate potent antitumor immunity and to extend survival of melanoma lung tumor-bearing animals. (a) C57Bl/6 mice were challenged intravenously with 2.5 × 105 B16-F10 cells in order to establish syngeneic lung melanoma metastases. Five days later, mice received 2 × 108 pfu Ad-hDCT by intramuscular injection. Control mice received empty Ad vector. Nine days after Ad injection, animals were administered intravenously with 109 pfu MG1-hDCT or its GFP control. Immune responses against the tumor antigen DCT were measured 5 days after Maraba injection in the blood and mouse survival was monitored daily. Percentage of CD8+ or CD4+ T cells reacting to SVY (b) or KFF (c) peptide exposure, respectively. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values. Pooled data from several experiments: n = 9 for Ad-empty group, n = 23 for Ad-hDCT group, n = 29 for Ad-hDCT + MG1-hDCT group, n = 11 for Ad-hDCT + MG1-GFP group. (d) Kaplan–Meier curves illustrating survival of treated melanoma lung-tumor bearing mice. Pooled data from several experiments: n = 16 for Ad-empty group, n = 23 for Ad-hDCT group, n = 30 for Ad-hDCT + MG1-hDCT group, n = 11 for Ad-hDCT + MG1-GFP group. (e) T-cell populations were selectively depleted to evaluate their respective therapeutic contribution. Kaplan–Meier curves illustrating survival of treated melanoma lung-tumor bearing mice (n = 5 per group). (-CD8) indicates CD8+ T-cells depletion while (-CD4) indicates CD4+ T-cells depletion. (f) Lungs extracted at day 19 from untreated and Ad-hDCT + MG1-hDCT treated mice. (g) Autoimmunity (vitiligo) induced in long-term survivors following Ad-hDCT + MG1-hDCT treatment. Pictures taken 280 days (mice on the left) or 220 days (mice on the right) after tumor challenge. P value considered nonsignificant (NS) when >0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. Ad, adenoviral vector; GFP, green fluorescent protein; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.

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Maraba Virus as a Potent Oncolytic Vaccine Vector

nonboosted mice (1.95% ± 0.29 in Ad-hDCT group and 1.91% ± 0.59 in Ad-hDCT + MG1-GFP group, Figure 7b). Similarly, DCT-specific CD4+ T-cell responses were measured in MG1hDCT boosted animals while rarely detected in primed only mice (mean % IFN-γ+ CD4+ T cells = 0.25% ± 0.06 in Ad-hDCT + MG1-hDCT group versus
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