The Oncolytic Adenovirus VCN-01 As Therapeutic Approach Against

Published OnlineFirst November 24, 2015; DOI: 10.1158/1078-0432.CCR-15-1899

Cancer Therapy: Preclinical Clinical Cancer Research The Oncolytic Adenovirus VCN-01 as Therapeutic Approach Against Pediatric Osteosarcoma Naiara Martínez-Velez 1,2,3, Enric Xipell1,2,3, Beatriz Vera1,2,3, Arlet Acanda de la Rocha1,2,3, Marta Zalacain1,4, Lucía Marrodan 1,4, Marisol Gonzalez-Huarriz1,2,3, Gemma Toledo5, Manel Cascallo6,Ramon Alemany7, Ana Patino~ 1,4, and Marta M. Alonso1,2,3

Abstract

Purpose: Osteosarcoma is the most common malignant bone were carried out to evaluate the cytotoxicity of VCN-01. Hexon tumor in children and adolescents. Despite aggressive chemo- assays were used to evaluate the replication of the virus. Western therapy, more than 30% of patients do not respond and develop blot analysis was performed to assess the expression levels of bone or lung metastasis. Oncolytic adenoviruses engineered to viral proteins and autophagic markers. The antitumor effect of specifically destroy cancer cells are a feasible option for osteosar- VCN-01 was evaluated in orthotopic and metastatic osteosarco- coma treatment. VCN-01 is a replication-competent adenovirus ma murine animal models. specifically engineered to replicate in tumors with a defective RB Results: This study found that VCN-01, a new generation pathway, presents an enhanced infectivity through a modified genetically modified oncolytic adenovirus, administered locally fiber and an improved distribution through the expression of a or systemically, had a potent antisarcoma effect in vitro and soluble hyaluronidase. The aim of this study is to elucidate in vivo in mouse models of intratibial and lung metastatic whether the use of VCN-01 would be an effective therapeutic osteosarcoma. Moreover, VCN-01 administration showed a strategy for pediatric osteosarcoma. safe toxicity profile. Experimental Design: We used osteosarcoma cell lines estab- Conclusions: These results uncover VCN-01 as a promising lished from patients with metastatic disease (531MII, 678R, strategy for osteosarcoma, setting the bases to propel a phase I/II 588M, and 595M) and a commercial cell line (143B). MTT assays trial for kids with this disease. Clin Cancer Res; 1–9. 2015 AACR.

Introduction Nowadays, the standard treatment for osteosarcoma is neoadjuvant chemotherapy followed by conservative surgery (in which, Osteosarcoma is the most common primary malignant bone as far as possible, function is preserved), followed by postoper- tumor (1). It appears principally in the metaphysis of the long ative chemotherapy. Despite aggressive chemotherapy protocols, bones of children and adolescents (2), often during the period 30% to 40% of patients do not respond and relapse, developing of rapid skeletal growth of the first two decades of life (3). bone or lung metastasis (8, 9). For these patients, survival remains Osteosarcoma is a highly invasive tumor that frequently metas- poor, with an overall five-year survival rate of about 20% (10). tasizes to the lungs. Pathogenesis involves a wide range of Thus, new treatments are needed. Oncolytic adenoviruses engi- different molecular pathways, but of fundamental relevance is neered to specifically destroy cancer cells are a feasible option. the inactivation of tumor suppressor genes, particularly p53 (4) VCN-01 is an oncolytic adenovirus that harbors a 24-base pair and the retinoblastoma susceptibility gene (RB1; refs. 5, 6); deletion in the E1A region, and the native E1A promoter has been further support for this model has recently come from whole- modified by the insertion of eight E2F-binding sites organized in genome sequencing (WGS; ref. 7). four palindromes and one Sp1-binding site. These modifications selectively restrict the replication of VCN-01 to cells with a defective pRB pathway (11, 12). In addition, the VCN-01 adeno- 1The Health Research Institute of Navarra (IDISNA), Pamplona, Spain. 2Program in Solid Tumors and Biomarkers, Foundation for the Applied virus displays improved infectivity and bioavailability as a result Medical Research, Pamplona, Spain. 3Department of Medical Oncol- of the inclusion of an RGDK motif in the heparin sulphate- ogy, University Hospital of Navarra, Pamplona, Spain. 4Department of – fi 5 glycosaminoglycans (HSG) binding domain KKTK of the ber Pediatrics, University Hospital of Navarra, Pamplona, Spain. Depart- fi ment of Pathology, MD Anderson Cancer Center, Madrid, Spain. 6VCN shaft (13); this modi cation improves antitumor potency com- Biosciences, Sant Cugat del Valles, Barcelona, Spain. 7Translational pared with the adenovirus modified with an RGD motif in the Research Laboratory, IDIBELL-Institut Catala d'Oncologia, L'Hospita- fiber HI loop (14). let de Llobregat, Barcelona, Spain. Athirdmodification engineered into the VCN-01 genome is Note: Supplementary data for this article are available at Clinical Cancer a novel element, an expression cassette with the human PH20 Research Online (http://clincancerres.aacrjournals.org/). gene (12). This cassette enables expression of a soluble hyal- Corresponding Author: Marta M. Alonso, University Hospital of Navarra, Uni- uronidase, active under physiologic conditions (pH 7), that can versity of Navarra, Av. Pio XII, 55, CIMA Building, Pamplona, Navarra 31008, degrade extracellular matrix hyaluronic acid. Hyaluronic acid is Spain. Phone: 349-4819-4700, ext. 2026; Fax: 349-4819-4717; E-mail: a negatively charged, high molecular weight polysaccharide [email protected] that forms part of and confers specific properties to the extra- doi: 10.1158/1078-0432.CCR-15-1899 cellular matrix (15). There is an association between hyaluronic 2015 American Association for Cancer Research. acid and malignancy, and this molecule is overproduced in

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Cell viability assays Translational Relevance 531MII, 588M, 595M, and 678R cells were seeded at a density Osteosarcoma is the most common primary malignant of 1 103 cells per well in 96-well plates, and one day later, cells tumor of bone in children and adolescents. Despite significant were infected with VCN-01 at MOIs (multiplicity of infection) of improvements in treatment of the primary tumor, a significant 5, 10, 20, 40, and 100. Cell viability was assessed five days later proportion of osteosarcoma patients eventually develop lung using the MTT assay (Sigma-Aldrich) as previously described (23). metastases and succumb to their disease even after multistage Dose-response curves were analyzed using GraphPad Software, conventional chemotherapy and surgical excision. Thus, there which fits the dose–response curves to Chou–Talalay lines (24); is a need to develop new and safe approaches for the treatment IC50 is the median-effect dose (the dose causing 50% of cells to be of osteosarcoma. Oncolytic adenoviruses have already proven affected, i.e., in this case IC50 is the amount of virus that results in safe and effective in the clinic. VCN-01 is a replication-com- 50% cell death or, equivalently, 50% survival). In each experi- petent adenovirus specifically engineered to replicate in ment, cells were plated in triplicate and each experiment was tumors with a defective RB pathway, presents enhanced infec- performed three times. tivity through a modified fiber and improved distribution through the expression of a soluble hyaluronidase. Our results Viral replication assays show that VCN-01 showed a potent antisarcoma effect while Osteosarcoma cells were seeded at a density of 1 104 cells/ fi maintaining a safe toxicity pro le in relevant sarcoma animal well in 6-well plates and infected 20 hours later with 1 MOI of models. Our results offer a strong preclinical rationale for VCN-01. Three days later, cells were collected, and the final propelling a phase I/II study with VCN-01 for pediatric amount of virus was determined by means of a method based osteosarcoma. on anti-hexon staining in HEK293 cells (25).

Immunoblotting For immunoblotting assays, samples subjected to the same many cancers (16, 17). In some tumors, high levels of hya- treatment as described earlier and containing identical amounts luronic acid have been found to be related with low survival of protein, were subjected to SDS-PAGE. Membranes were incu- rates and also with development of chemoresistance (18, 19). bated with the following antibodies: E1A, (Santa Cruz Bio- Osteosarcoma is characterized by the production of malig- technology), fiber (NeoMarkers), and GRB2 (BD Transduction nant osteoid by tumor cells. It has been hypothesized that Laboratories). The membranes were developed according to the degradation of hyaluronic acid can destabilize such osteoids Amersham enhanced chemiluminescence protocol. thereby improving viral efficacy. Supporting this notion, several studies have shown that drugs capable of disturbing hyaluro- qRT-PCR analysis of gene expression nan-rich pericellular matrix cells result in less accumulation Total RNA was isolated and cDNA was synthesized using the of hyaluronan, and that, consequently, these drugs have a high-capacity cDNA Archive Kit (Applied Biosystems). Total RNA in vivo in vitro potent antisarcoma effect and (20). It remains was extracted from paraffin-embedded sections using RecoverAll to be demonstrated whether VCN-01 expressed-hyaluronidase Total Nucleic Acid Isolation (Ambion by Life Technologies). degrades the hyaluronic acid present in the pericellular matrix Reverse transcriptase reactions contained 1 mg of RNA, 1 RT of tumors, thereby improving viral spread and increasing the buffer, 4 mL of dNTPs (100 mmol/L), 1 random primers, 5 mLof oncolytic effect of VCN-01. MultiScribe Reverse Transcriptase (50 U/mL), and 0.005 mLof In this study, we analyzed the antisarcoma effect of VCN-01. RNase inhibitor (0.20 U/mL). The reactions were incubated in a Our data show that VCN-01 exerts a potent antitumor effect GeneAmp PCR System 2400 (Applied Biosystems) for 10 minutes in vitro in vivo and , in intratibial and lung metastatic models. at 25C and then 2 hours at 37C. Each cDNA sample was These results uncover VCN-01 as a promising strategy for osteo- analyzed in triplicate by fast quantitative-PCR analysis (Applied sarcoma, setting the bases to propel a phase I/II trial for pediatric Biosystems 7900 Sequence Detection System) using Fast SYBR osteosarcoma. Green PCR Master Mix (Applied Biosystems) and specific primers for fiber (forward, 50CAAACGCTGTTGGATTTATG-30; reverse, 50- Materials and Methods GTGTAAGAGGATGTGGCAAAT-30), PH20 (forward, 50-TACA- 0 0 Cell lines and culture conditions CACTCCTTGCTCCTGG-3 ; reverse, 5 -CTTAGTCTCACAGAGGC- 0 0 Primary osteosarcoma-derived cell lines 531MII, 588M, 595M, CAC-3 ), and the housekeeping gene GAPDH (forward, 5 -AGC- 0 0 and 678R were developed at the Clínica Universidad de Navarra CACATCGCTCAGACAC-3 ; reverse, 5 -GCCCAATACGACCAA- 0 (Pamplona, Spain) as previously described (15). In addition, we ATCC-3 ). For thermal cycling, fast real-time conditions were used the 143B cell line, obtained from the ATCC. All the cell lines 20 seconds at 95 C for one cycle, 1 second at 95 C, and 20 were maintained in minimum essential medium supplemented seconds at 60 C for 40 cycles. To determine relative levels of gene C with 10% FBS in a humidified atmosphere containing 5% CO2 expression, the comparative threshold cycle ( t) method was DDC at 37 C. All the cell lines were tested and authenticated at the used, and data were presented as 2 t. CIMA Genomic Core Facility (Pamplona, Spain) using short tandem repeats DNA profiling. Animal studies Ethical approval for animal studies was granted by the Animal Adenovirus construction and infection Ethical Committee of the University of Navarra (CEEA; Comite The construction of VCN-01 and method of viral infection Etico de Experimentacion Animal under the protocol number followed procedures described elsewhere (21, 22). CEEA/065–13 and CEEA/075–13).

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For the orthotopic intratibial model, 531MII osteosarcoma Statistical analysis cells (5 105) were engrafted by injection through the tibial Data were expressed as mean SD. For the in vitro experiments, plateau in the primary spongiosa of both tibias of female nude data were evaluated with the two-tailed Student t test. The same mice (Taconic Farms, Inc). Seven days after injection, animals test was used to compare data for treatment and control groups in were randomized to 3 groups (control with PBS and two groups of the in vivo experiments. The program GraphPad Prism 5 (Statis- VCN-01 treated with two different doses). PBS or VCN-01 [107 tical Software for Sciences) was used for the statistical analysis. plaque-forming unit (pfu)/animal and 108 pfu/animal] were administered into the tibia of the animals on days 7 and 14 after Results cell implantation. Tumor development was monitored every Characterization of the in vitro antiosteosarcoma effect of week until the end of the experiment (day 90 after cell implan- VCN-01 fi tation), and mice were then sacri ced. When a tumor became As mentioned in the introduction, VCN-01 is an improved visible, its size was measured weekly, and tumor volume was genetically modified adenovirus that presents in its genome calculated (26). several modifications that renders a more specific and potent For the lung-metastatic model, 531MII osteosarcoma cells virus; E2F-binding sites in the E1A promoter, a 24-bp deletion 6 (2 10 cells) were injected through the tail vein. Seven days in the E1A gene, modified fiber, and finally, VCN-01 has an later, animals were arbitrarily randomized to 3 groups (con- expression cassette inserted in its genome that allows the virus trol, PBS-treated, or VCN-01 with the same doses as above), to express PH20 (hyaluronidase; Fig. 1A). and the VCN-01 virus was intravenously administered in the First, we wanted to evaluate the in vitro antisarcoma effect tail vein. of VCN-01. To this end, we performed MTT analyses in 4 All animals were weighed every two weeks throughout the primary (531 MII, 588M, 595M, and 689R) and one estab- experiment to evaluate toxicity. Animals were weighed weekly lished (143B) osteosarcoma cell lines. MTT assays showed that during the period of VCN-01 administration. VCN-01 induced cell death in a dose-dependent manner in all cell lines. The IC50 of VCN-01 ranged between 1 and 30 MOIs. PET analyses 531MII was the most sensitive line and 588M, the most As part of the orthotopic study, at the end of the experimental resistant (Fig. 1B and Supplementary Fig. S1A). procedure, antitumoral effect was measured by PET with the Assessment of viral protein levels in cells previously infected radiotracer 18F-fluorodeoxyglucose (18F-FDG). Overnight prior with VCN-01 revealed robust expression of E1A, an early viral to PET, mice were fasted but allowed water. On the day of PET, protein, and fiber, a late protein. Levels of both proteins 18 followed a dose-dependent pattern (Fig. 1C and Supplemen- mice were anesthetized with 2% isoflurane in 100% O2 and F- FDG (17.7 MBq 2.6 in 80–100 mL) was injected into the tail tary Fig. S1B). These data suggest that there was effective viral vein. To avoid radiotracer uptake in the hindlimb muscle, transcription, and that the virus was replicating. 18F-FDG uptake was performed under continuous anesthesia for To confirm the existence of an effective viral cycle in osteosar- 50 minutes. PET imaging was performed in a dedicated small- coma cell lines, we used a replication assay based on anti-hexon 4 7 animal Philips Mosaic tomograph with 2 mm resolution, 11.9 cm staining. Viral counts ranged from 10 to 10 pfu/mL, indicating fi axial field of view (FOV), and 12.8 cm transaxial FOV. Anesthe- ef cient replication in all cell lines (Fig. 1D and Supplementary tized mice were placed horizontally on the PET scanner bed to Fig. S1C). In 531MII and 678R cell lines, the strength of cytotoxic perform a static acquisition (sinogram) of 15-minute duration. effect of VCN-01 correlated with viral titers. Images were reconstructed using the 3D Ramla algorithm (a true Finally, we assessed the expression levels of the PH20 pro- 3D reconstruction) with two iterations and a relaxation parameter duced by the virus and whether those levels were correlated of 0.024 into a 128 128 matrix with a 1 mm voxel size; with fiber expression. We performed mRNA expression analy- fi corrections were applied for dead time, decay and random, and sis, and we observed that PH20 and ber mRNA levels corre- 2 scattering corrections. For the assessment of 18F-FDG uptake, all lated with each other in both cell lines tested (R ¼ 0.99, P < studies were exported and analyzed using the PMOD software 0.0001) indicating that VCN-01, when transcribed, can express (PMOD Technologies Ltd.). Regions of interest(ROI) were drawn the PH20 cassette inserted in the viral genome (Fig. 1E and on coronal 1-mm thick small-animal PET images on consecutive Supplementary Fig. S1D). slices including entire hindlimbs. Finally, maximum standardized Altogether these data indicate that, in pediatric osteosarcoma– derived cell lines, VCN-01 replicated in vitro, expressed the PH20 uptake value (SUVmax) was calculated using the formula SUV ¼ [tissue activity concentration (Bq/cm3)/injected dose (Bq)] enzyme, and exerted a robust oncolytic effect. body weight (g). Evaluation of viral toxicity in vivo To evaluate the potential toxicity of local and systemic viral Immunohistochemical analysis administration, weight was controlled every week after local Paraffin-embedded sections of mouse leg, lung, and liver (intratumoral) or systemic (tail vein) viral administration. Mice were immunostained with specific antibodies for adenoviral did not lose weight with VCN-01 administration; route of admin- mouse–hexon (Chemicon International, Inc.), E1A (Santa Cruz istration (intratumoral in the intratibial model versus tail vein in Biotechnology), biotinylated hyaluronan binding protein the lung-metastatic model) did not affect weight loss and neither (AMS biotechnology), and vimentin clone V9 (IS30, Dako did viral concentration (Fig. 2A and B). Denmark A/S). Conventional procedures were followed in all Hematoxylin–eosin staining of liver sections did not reveal cases (27). For immunohistochemical staining, Vectastain ABC morphologic alterations associated with hepatotoxicity (Fig. 2B kits (Vector Laboratories Inc.) were used according to the and C). We observed no Councilman bodies, macrosteatosis, or manufacturer's instructions. necrotic areas. IHC did not detect expression of E1A and hexon

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A C E2F- LITR binding sites FiberRGDK SA K pA RITR Fiber (62 kDa) E1a-D24 MLP L1 L2 L3 L4 PH20

L5 143B

E1A (45 kDa) B

GRB2 (25 kDa)

125 143B 125 531MII Fiber (62 kDa) 100 100

75 75 531MII IC50 = 12.5 MOIs IC 50 = 1.0 MOIs 50 50 E1A (45 kDa)

25 25

Cell proliferation (%) Cell proliferation GRB2 (25 kDa) 0 0 0 20 40 60 80 100 01020 50 100 VCN-01 (MOIs) VCN-01 (MOIs) VCN-01 (MOIs): Mock 5 10 25 50

D E 143B 531MII 7 106 1.5 × 10 600 ) t 105 C –∆∆ 1.0 × 107 400 104

103 5.0 × 106 200 102 P < 0.0001 P < 0.0001 R2 = 0.99 R2 = 0.99 PH20 mRNA (2 101 0 0 Viral replication Viral replication (pfu/mL) 0 20,000 40,000 60,000 0 2,000 4,000 6,000 8,000 10,000 100 –∆∆C –∆∆C 143B 531MII Fiber mRNA (2 t) Fiber mRNA (2 t)

Figure 1. VCN-01 exerts a potent oncolytic effect in pediatric osteosarcoma cell lines, 531MII and in 143B. A, illustrative scheme of genetic modifications in VCN-01 genome. B, cell viability analyses of osteosarcoma cell lines after viral treatment. 143B and 531MII cell lines were infected with VCN-01 at the indicated MOIs. Cell viability was assessed using MTT assays 5 days after infection. Data are shown as the percentage (mean SD) of cells alive after infection with VCN-01. C, viral protein expression in osteosarcoma cell lines infected with VCN-01 assessed by Western blot analysis. D, quantification of VCN-01 replication in the indicated cell lines. Viral counts were determined three days after VCN-01 infection at 1MOI by culture infection antihexon staining–based method in HEK293 cells and expressed as pfu per millilitre. Data are shown as the mean SD of three independent experiments. E, correlation of viral PH20 and fiber mRNA expression measured by qRT-PCR in osteosarcoma cell lines previously infected with VCN-01. mRNA levels are presented as DD 2 Ct standardized with their constitutive gene and compared with an uninfected control.

(Supplementary Fig. S2A and S2B) in the liver, indicating absence tumors, whereas only 3 out of 10 tibias in the group treated with of virus (Fig. 3B). 107 pfu VCN-01 and none of the mice in the group treated with Thus, in mice, locally or systemically administrated VCN-01 108 pfu VCN-01 did (Fig. 3D and E). Tumor volumes of control was not significantly toxic and was well tolerated. mice ranged between 100 and 500 mm3; volumes for mice in the 107 pfu group were between 50 and 400 mm3. Antitumor effect of VCN-01 in an orthotopic osteosarcoma Pathologically, at the end of the experiment, tumors had the model characteristic morphology of osteosarcoma and a notable pro- Next, we analyzed the therapeutic effect of VCN-01 in an duction of malignant osteoid (Fig. 3F). In some control mice, orthotopic osteosarcoma model. For that purpose, we inoculated tumors had grown such that they had crossed the epiphysis of the 531MII cells in the tibial tuberosity of nude mice followed by tibiae, resulting in transarticular tumors. In the VCN-01 lower VCN-01 injection at days 7 and 21 administered at two different dose treated group, there was evidence of a certain tumoral burden dosages (107 pfu/animal or 108 pfu/animal). PBS was adminis- in some tibiae. Mice in the higher dose treated group showed no tered to the control group (Supplementary Fig. S3A). Tumor evidence of tumor. development was monitored every week until the end of the At the end of the experiment, approximately 70% of control experiment (day 90 after cell implantation), and mice were then mice presented lung metastases derived from the primary osteo- sacrificed. sarcoma tumor (Supplementary Fig. S4B). Of mice treated with By the end of the experiment, there were clearly visible differ- VCN-01, only one in the 107 pfu–treated group developed lung ences in tumors in the three groups of mice (Fig. 3A). PET imaging metastasis (Fig. 3E and F and Supplementary Figs S4B and S4C). also showed that tumor volumes were significantly bigger in the Hexon staining verified viral presence (Supplementary Fig. control group relative to those in the 108 pfu group (Fig. 3B and S4A). In hexon-positive areas, IHC indicated decreased hyaluro- C). All control mice (5 mice 10 tibias) developed visible tibial nic acid expression and a lower density of cancer cells. These

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A B Intratibial model Lung metastatic model 28 30 Control Control 28 7 26 7 10 pfu 10 pfu VCN-01 VCN-01 108 pfu 108 pfu 26 24 24 Weight (g) 22 Weight (g) 22

20 20

0204060 80 0204060 Time (days) Time (days) C D Control 108 pfu VCN-01 Control 108 pfu VCN-01 H&E H&E IHC E1A IHC E1A

Figure 2. VCN-01 local or systemic administration showed a safe toxicity profile in osteosarcoma models in vivo. A and B, body weight plotting of animals treated locally (A) or systemically (B) with VCN-01 at the indicated pfu. Mice from the different groups were weighed every week during treatment. Data are shown as the median SD within each group at each time point. C, histologic analysis of mice livers bearing an orthotopic osteosarcoma and treated locally with VCN-01 at 108 pfu. H&E (top, magnification 400) and E1A IHC (bottom, magnification 400). D, Histologic analysis of mice livers bearing a lung metastatic osteosarcoma and treated systemically with VCN-01 at 108 pfu. Representative micrographs of H&E and E1A IHC of mice livers from indicated groups of the metastatic osteosarcoma model. Images (400 magnification) showed no viral presence in mice livers, and no signs of hepatotoxicity were found. observations, beyond an indirect demonstration of the oncolytic the tail vein of athymic nude mice and, seven days later, mice effect of VCN-01, suggest that VCN-01 was able to express PH20, were treated with four intravenous injections at doses of 107 and that this PH20 was functional at digesting hyaluronic acid pfu/animal of VCN-01 or 108 pfu/animal of VCN-01. Mice were (Supplementary Fig. S4A). sacrificed at day 60 (Supplementary Fig. S3B). The objective of RNA extracted from tibial sections was used to assess the this experiment was the assessment of tumor burden in mice expression of fiber and PH20. Expression of fiber and hyal- lungs and the evaluation of the capacity of VCN-01 to reach the uronidase mRNA was higher in mice treated with VCN-01– tumors and to replicate in tumor cells after systemic adminis- 108pfu group relative to those in the VCN-0 107pfu–treated tration. This virus has a modification in the fiber that improves group (Fig. 3G). As in the in vitro experiments, there was a its half-life in blood, which might improve the antitumor effect significant correlation between the levels of expression of compared with the other tested virus. hyaluronidase PH20 and fiber mRNA. The pathologic analysis of lung metastases showed the char- In conjunction, these results show that VCN-01 replicated acteristic appearance produced by 531MII. This phenotype was in vivo inside tumors, where it produced functional hyaluro- verified by V9-positive staining (Fig. 4A and C). Lung tumor nidase and had a potent antiosteosarcoma effect. burden was significantly reduced in both VCN-01–treated groups compared with the control group (PBS-treated mice): Therapeutic effect of VCN-01 in a metastatic osteosarcoma by the end of the experiment (day 60), a mean of 2.06% of lung model surface area in untreated mice presented tumor, whereas this As the lungs are preferred metastasis organs for osteosarco- percentage in the 108 pfu and 107 pfu groups was 0.4 and ma, we evaluated the antitumor effect of VCN-01 in a lung 0.14%, respectively (Fig. 4B). In addition, mice treated with metastatic osteosarcoma model using the cell line 531MII. This VCN-01 had less tumor nodules (Fig. 4C). cell line recapitulates the pathology found in osteosarcoma Viral fiber and PH20 expression in the lungs was analyzed lung metastasis in patients. We administrated 2 106 cells in by qRT-PCR, which demonstrated that the virus was able to

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A B VCN-01 Control 107 pfu 108 pfu

) 300 Control 3 7 10 pfu 8 VCN-01 10 pfu 200

100

Tumor volume (mm 0 0 15 30 45 60 75 Days D C

2.5 P = 0.0057 2.0

1.5

(FDG-PET) 1.0 ** max 0.5

SUV F 0.0 Control 107 pfu 108 pfu VCN-01 E P = 0.006 ) 3 400

300 G ) Fiber ) t t 200 25 PH20 ) 30 PH20/Fiber t

C 400 C C –∆∆ 20 –∆∆

100 –∆∆ 300 - 15 20 0 ** Tumor volume (mm 200 Control 107 pfu 108 pfu 10 10 VCN-01 5 100 P < 0.001 R2 = 0.899 PH20 mRNA (2 0 PH20 mRNA (2 Fiber mRNA (2 0 0 Control 107 pfu 108 pfu Control 107 pfu 108 pfu 0 100 200 300 –∆∆ C VCN-01 VCN-01 Fiber mRNA (2 t)

Figure 3. VCN-01 antisarcoma effect in the orthotopic osteosarcoma model with the 531MII cell line. Tumors were developed by orthotopic injection of 500,000 531MII cells in the tibial tuberosity of female nude mice and were sacrificed 90 days later. A, analyses of tumor burden development. Tumor volume in the mice tibias was measured at different days until the end of experiment. B, PET images of tumor burden. Representative images of PET analyses of 7 8 18 control mice or treated with VCN-01 (10 or 10 pfu).C,quantification of tumor burden by PET with the radiotracer F-FDG. SUVmax was calculated using the formula SUV ¼ [tissue activity concentration (Bq/cm3)/injected dose (Bq)] body weight. Represented are the mean SD, SUV values of the tumors of all animals in the same group (Wilcoxon test). D, representative macroscopic images of mice tibias from all treated groups. E, tumor volume evaluation. Quantification of tumor volume was calculated by the formula: volume (mm3) ¼ [(long diameter perpendicular diameter 2)/2]. Represented are the mean SD of each tumor in the same group (Student t test). F, H&E staining of tibias from mice of indicated therapy groups. Representative photomicrographs of a control and two animals that received the different dose protocols of VCN-01 (magnification 100). G, quantification of fiber and DD PH20 mRNA expression of cDNA from orthotopic osteosarcoma tumors by qRT-PCR and its correlation graph. mRNA levels are presented as 2 Ct standardized with their constitutive gene and compared with an uninfected control. Data are given as the mean SD.

target and to replicate in tumor cells after systemic adminis- virus, administered locally or systemically, had a potent antisar- tration (Fig. 4D). The overall mRNA expression of viral coma effect in vitro and in vivo in mouse models of intratibial and fiber and PH20 in the lungs of mice treated with VCN-01– lung metastatic osteosarcoma. 107 pfu/animal was higher than that in mice treated with VCN-01 was engineered by inclusion of eight E2F response VCN-01 108 pfu/animal. This result can be explained by elements inside its genome and the deletion of 24 bp in the hypothesizing that at 108 pfu/animal, VCN-01 was left with E1A gene, to replicate preferentially in cancer cells with an few tumoral cells in which to replicate. aberrant RB pathway. We observed VCN-01 to have its stron- In summary, VCN-01 displayed a significant and potent gest antitumor effect in the 531MII cell line, which harbors antisarcoma effect in the 531MII lung metastatic osteosarcoma an Rb mutation. Because Rb mutations are associated with model. malignance and poor prognosis in osteosarcoma patients (32),thatVCN-01workedwellinthis scenario is of particular Discussion relevance. Several studies have found a relationship between levels of Previously, others and ourselves have shown that oncolytic hyaluronic acid and tumoral resistance to chemotherapy, with adenoviruses present a potential therapeutic approach for treat- associated poor prognosis for patients (17, 18). Therefore, in ment of pediatric osteosarcoma (28–31). This study found that the treatment of osteosarcoma, VCN-01 can have two thera- VCN-01, a new generation genetically modified oncolytic adeno- peutic effects: an intrinsic oncolytic effect on tumor cells and

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P A = 0.029 B VCN-01 P = 0.012 P = 0.021 7 8 3 Control 10 pfu 10 pfu

1 * ** Tumor area/total area 0 Control 107 pfu 108 pfu VCN-01 C D P = 0.001 80 P = 0.015

60 * 40 **

Number tumor nodules 0 Control 107 pfu 108 pfu P = 0.003 P = 0.003 P = 0.003 VCN-01 E 2.5 25 ** ) ) t t C C 2.0 20 –∆∆ –∆∆

1.5 15

1.0 10

0.5 5 ** Fiber mRNA (2 PH20 mRNA (2 0.0 0 7 8 Control 10 pfu 10 pfu Control 107 pfu 108 pfu VCN-01 VCN-01

Figure 4. VCN-01 administered systemically reduced osteosarcoma lung metastasis in vivo. Lung metastases were induced by endovenous injection of 2 106 531-MII cells in the tail of female nude mice. Animals were randomized as positive control (no treatment), VCN-01 group 107 pfu (107 pfu/week 4weeks) and VCN-01 group 108 pfu (108 pfu/week 4 weeks). At day 60 animals were sacrificed. A, Quantification of lung tumor burden (531-MII) in mice at the end of the experiment. Bar representation of tumor area relative to total lung area. The values represent mean percentage tumor area of animals from each group, as obtained from a specifically designed program that measured both tumor area and total lung area from which was calculated the percentage of tumor area. B, Representative H&E photomicrographs at 200 of lung lesions from mice of the three groups described. C, Quantification of number of lung tumor nodules. Bar representation of tumor number represent different quantifications of different observers. D, 200 vimentin (V9antigen) immunochemistry images showing metastatic implants in lung of mice from untreated and VCN-01 treated groups. E, Quantification of DD fiber and PH20 mRNA expression of cDNA from osteosarcoma tumors by qRT-PCR and its correlation graph. mRNA levels are presented as 2 Ct standardized with their constitutive gene and compared to an uninfected control. the effect of hyaluronidase on the extracellular matrix (33). In sequence into the fiber protein produced by VCN-01 consid- respect of the latter effect, inhibitors of hyaluronic acid reten- erably improved its biodistribution after systemic administration have been shown to be able to modify the pericellular tion and enhanced the therapeutic effect of VCN-01 in vivo in matrix in an osteosarcoma, restraining tumor formation in vivo two immunocompetent models (11, 38). Currently, there are and also reducing the occurrence of metastasis (34). In addi- two clinical trials underway to evaluate the therapeutic effect of tion, several works have found that hyaluronidase disturbed systemically administered VCN-01; one of these concerns VCN- cell–cell interaction within the tumor mass, thereby enhancing 01 alone and the other VCN-01 in combination with gentami- the diffusion of antitumor agents (33, 35). Following this line cin (NTC02045602 and NTC02045589). of thinking, further studies are needed to assess the promising In summary, VCN-01 exerted a potent anti-osteosarcoma possibility that the complementary effects of VCN-01 and effect in vitro and in vivo and was safe in terms of toxicity. Our chemotherapy used in conjunction will result in enhanced results consolidate the case for pursuing VCN-01 as a realistic diffusion of both virus and drugs and an improved antisarcoma therapeutic option for kids with osteosarcoma. efficacy. An important limitation of therapy with systemically admin- Disclosure of Potential Conflicts of Interest istered adenovirus is clearance of virus by the immune system M. Cascallo has ownership interests (including patents) in and reports (36). Virtually, the entire human population has IgG antibodies receiving commercial research support from VCN Biosciences. R. Alemany is to adenovirus serotype 5, and 55% of these antibodies effec- a consultant/advisory board member for VCN Biosciences. No potential tively neutralize adenoviruses (37). The insertion of an RGDK conflicts of interest were disclosed by the other authors.

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Martínez-Velez et al.

Authors' Contributions Grant Support Conception and design: N. Martínez-Velez, E. Xipell, A. Patino,~ M.M. Alonso This work was supported by the European Union (Marie Curie IRG270459; Development of methodology: N. Martínez-Velez, E. Xipell, M. Zalacain, to M.M. Alonso), by the Instituto de Salud Carlos III y los Fondos Feder L. Marrodan, G. Toledo, M. Cascallo, A. Patino,~ M.M. Alonso Europeos (PI13/125; to M.M. Alonso), the Spanish Ministry of Science and Acquisition of data (provided animals, acquired and managed patients, Innovation (Ramon y Cajal contract RYC-2009–05571; to M.M. Alonso), The provided facilities, etc.): N. Martínez-Velez, E. Xipell, B. Vera, A.A. de la Rocha, L'OREAL-UNESCO Foundation (to M.M. Alonso), The Department of Health of M. Zalacain, L. Marrodan, M. Gonzalez-Huarriz, G. Toledo the Government of Navarra (to M.M. Alonso), The Basque Foundation for Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Health Research (BIOEF, BIO13/CI/005), and Fundacion Caja Navarra (Con- computational analysis): N. Martínez-Velez, E. Xipell, B. Vera, M. Gonzalez- vocatoria de Ayudas 2011 to A. Patino).~ E. Xipell is supported by a fellowship Huarriz, G. Toledo, A. Patino,~ M.M. Alonso from the Credit Andorra Foundation. A.A. de la Rocha is supported by a Writing, review, and/or revision of the manuscript: N. Martínez-Velez, fellowship from Friends of the University of Navarra (ADA). R. Alemany is E. Xipell, M. Gonzalez-Huarriz, M. Cascallo, R. Alemany, A. Patino,~ M.M. Alonso supported by BIO2011–30299-C02–01 from the Spanish Ministry of Education Administrative, technical, or material support (i.e., reporting or organizing and Science. data, constructing databases): N. Martínez-Velez, E. Xipell, M. Cascallo, The costs of publication of this article were defrayed in part by the payment R. Alemany, M.M. Alonso of page charges. This article must therefore be hereby marked advertisement Study supervision: M. Cascallo, A. Patino,~ M.M. Alonso in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments Received August 7, 2015; revised October 19, 2015; accepted November 12, The authors thank David Burdon for editorial assistance. 2015; published OnlineFirst November 24, 2015.

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The Oncolytic Adenovirus VCN-01 as Therapeutic Approach Against Pediatric Osteosarcoma

Naiara Martínez-Vélez, Enric Xipell, Beatriz Vera, et al.

Clin Cancer Res Published OnlineFirst November 24, 2015.

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