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Cancer Gene Therapy (2006) 13, 732–738 r

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ORIGINAL ARTICLE

Pharmacokinetics of oncolytic measles virotherapy: eventual equilibrium between virus and tumor in an ovarian cancer xenograft model K-W Peng1,2, EM Hadac1, BD Anderson1, R Myers1,2, M Harvey1,2, SM Greiner1,2, D Soeffker1,2, MJ Federspiel1,3 and SJ Russell1,2 1

Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN, USA; 2Toxicology Core, Mayo Clinic College of Medicine, Rochester, MN, USA and 3Viral Vector Production Facility, Mayo Clinic College of Medicine, Rochester, MN, USA

Because of their ability to replicate, the dose–response relationships of oncolytic viruses cannot easily be predicted. To better understand the pharmacokinetics of virotherapy in relation to viral dose and schedule, we administered MV-CEA intraperitoneally in an orthotopic mouse model of ovarian cancer. MV-CEA is an attenuated oncolytic measles virus engineered to express soluble human carcinoembryonic antigen (CEA), and the virus is currently undergoing phase I clinical testing in patients with ovarian cancer. Plasma CEA levels correlate with numbers of virus-infected tumor cells at a given time, and were used as a surrogate to monitor the profiles of viral gene expression over time. The antineoplastic activity of single- or multiple-dose MV-CEA was apparent over a wide range of virus doses (103–108 TCID50), with little reduction in observed antitumor efficacy, even at the lowest tested dose. However, analysis of CEA profiles of treated mice was highly informative, illustrating the variability in virus kinetics at different dose levels. The highest doses of virus were associated with higher initial levels of tumor cell killing, but the final outcome of MV-CEA therapy at all dose levels was a partial equilibrium between virus and tumor, resulting in significant slowing of tumor growth and enhanced survival of the mice. Cancer Gene Therapy (2006) 13, 732–738. doi:10.1038/sj.cgt.7700948; published online 10 March 2006 Keywords: measles virus; ovarian cancer; pharmacokinetics; soluble CEA

Introduction

Ovarian cancer is a devastating malignancy that frequently spreads within the peritoneal cavity (stage 3 disease), before it spreads via the bloodstream in stage 4 disease.1 In its advanced stages, when the disease has become refractory to chemotherapy, it often remains localized in the peritoneal cavity, and is therefore potentially amenable to regional therapy administered via the intraperitoneal (i.p.) route. Oncolytic viruses, which are selectively destructive to ovarian cancer cells, are emerging as a promising new experimental modality for the treatment of ovarian cancer.2,3 Several such viruses have been tested in mouse ovarian cancer models, and some have already entered clinical trials.4,5 The range of viruses showing promise as oncolytic therapies for ovarian cancer is quite broad and includes reovirus, Correspondence: Dr SJ Russell, Guggenheim 18, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA. E-mail: [email protected] Received 9 October 2005; revised 19 December 2005; accepted 8 January 2006; published online 10 March 2006

Newcastle Disease virus, echovirus Type I, Sindbis virus, mumps virus and engineered adenoviruses.6–13 We previously reported that a genetically modified measles virus expressing a soluble marker peptide (MV-CEA) is a potent and selective oncolytic agent, with promising activity in preclinical models of ovarian cancer.14 MV-CEA targets and causes extensive fusion in ovarian cancer cells through the CD46 receptor, which is expressed at high levels by many tumor types, including ovarian cancer.15–18 MV-CEA is derived from an attenuated strain of measles virus belonging to the Edmonston vaccine lineage (MV-Edm), and its inserted transgene codes for the soluble extracellular domain of human CEA, which serves as an inert soluble marker to facilitate noninvasive monitoring of virus spread in vivo.19 MV-CEA-infected cells release CEA into body fluids, and the circulating level of CEA, which can be easily measured, provides a convenient readout to determine if the number of virus-infected cells in the body is increasing or decreasing over time. Carcinoembryonic antigen is a well-established clinical marker that is used to monitor the growth of certain types of tumors, but CEA levels are rarely elevated in patients with ovarian cancer, making it a potentially suitable, nonimmunogenic marker for

Pharmacokinetics of oncolytic measles virotherapy K-W Peng et al

tracking virus propagation in this disease.20,21 Based on its promising activity in preclinical models of ovarian cancer, and its favorable toxicity profile, MV-CEA is currently being tested in a phase I clinical trial in which it is administered by i.p. infusion to patients with advanced ovarian cancer. Oncolytic viruses are believed to destroy tumors by a process that involves multiple cycles of virus replication, release of progeny and infection of new tumor cells. The rate and extent of intratumoral virus propagation is subject to a multitude of influences such that in vivo dose– response relationships cannot be easily predicted. In our initial in vivo efficacy studies in mouse models of ovarian cancer, we used extremely high doses of MV-CEA that would be impractical for human therapy. In the light of these considerations, we performed comprehensive dose– response studies of MV-CEA therapy in the orthotopic SKOV3ip.1 human tumor xenograft model of ovarian cancer, with the aim of closely monitoring the pharmacokinetics of viral gene expression by using CEA as a surrogate marker. Results from the study revealed interesting dose-related viral pharmacokinetics data that were otherwise not evident from the survival curves of the treated mice.

Materials and methods

Cell culture Human epithelial ovarian cancer cells, SKOV3ip.1, were maintained in alpha-MEM (Irvine Scientific, CA) supplemented with 20% fetal bovine serum (Invitrogen). African green monkey Vero cells (ATCC CCL-81) used for production of MV-CEA were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 5% fetal bovine serum. Production of MV-CEA virus MV-CEA virus was propagated on Vero producer cells. Briefly, Vero cells were inoculated with MV-CEA at a multiplicity of infection (MOI) of 0.02 for 2 h at 371C. The cell monolayer was incubated at 371C until 80–90% of cells were recruited into syncytia. Cells were harvested by scraping into reduced serum medium Opti-MEM (Invitrogen), frozen and thawed twice and the supernatant containing virions was aliquoted and frozen at 761C until use. TCID50 (50% tissue culture infective dose) titrations were performed on Vero cells and virus titers were calculated according to the Spearman and Ka¨rber equation and expressed as number of plaqueforming units/ml viral stock. In vivo experiments All procedures involving animals were approved and performed according to the guidelines of the Mayo Clinic Institutional Animal Care and Use Committee. Female 5week-old NCR athymic mice (Taconic Laboratory, Germantown, NY) were maintained in the barrier facility of Mayo Clinic. Mice were injected i.p. with SKOV3ip.1 cells (3.5 106 cells/250 ml PBS) using a 25-gauge needle at

day 0. One week later when the tumors were well established, mice were given therapy i.p. using a 28-gauge needle. Mice in the treatment groups received one dose or six doses (three doses each week for 2 weeks) of 103, 104, 105 or 107 TCID50 MV-CEA diluted in saline to a final injection volume of 500 ml, whereas mice in the control group received 500 ml of non-diluted clarified Vero cell lysates. Mice were observed daily and were euthanized if they developed ascites, lost more than 15% of body weight or developed subcutaneous injection site tumors that were more than 10% of body weight. All surviving mice were euthanized at 3 months after tumor cell implantation, and necropsy performed. Blood samples were collected via the retroorbital plexus (see below). Residual tumors were harvested and weighed separately either as ‘peritoneal’ tumors (localized in greater omemtum and/or studded on the mesentery) or injection site subcutaneous tumors.

Plasma carcinoembryonic antigen levels Mice were bled from the retroorbital plexus while under general anesthesia with isoflurane (Abbott Laboratories, North Chicago, IL) using a non-heparinized capillary (Fisher Scientific, Pittsburgh, PA) weekly for 1 month, biweekly thereafter, and a terminal bleed was performed before euthanasia. Blood samples were collected into lithium heparin tubes, spun at 8000 g for 5 min to separate the plasma and CEA levels were analyzed by the Mayo Clinic Central Clinical Laboratory using the Bayer Centaur Immunoassay System. Statistical analysis Survival curves were compared using the Wilcoxon log rank test to determine if the treatment groups were statistically different from the control and if the singledose versus multiple-dose groups were different from each other. Correlation between plasma CEA and tumor weights was determined using the Spearman rank correlation test. Groups are considered statistically significant if Po0.05.

Results

Survival of MV-CEA-treated mice In our previously published study,14 mice bearing i.p. SKOV3ip.1 ovarian cancer xenografts were treated with 107 TCID50 MV-CEA, administered intraperitoneally three times a week up to a total of 16 doses. To determine whether a shorter duration of therapy could also be effective in this model, we compared the survivals of SKOV3ip.1 tumor-bearing mice treated with a single dose or six doses of 107 TCID50 MV-CEA (n ¼ 10 per treatment group). As shown in Figure 1a, median survivals of mice that were treated with one or six doses of MV-CEA were significantly prolonged compared to the control (Po0.001), and the outcome of treatment was not superior in the multidose group compared to the single-dose group (P ¼ 0.89).

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Figure 1 Survival curves of mice bearing intraperitoneal (i.p.) SKOV3ip.1 ovarian tumors and treated 1 week after tumor cell implantation with one or six i.p. doses of (a) 107, (b) 105, (c) 104 or (d) 103 TCID50 MV-CEA virus. Mice in the control group (black line with asterisks) were given six doses of clarified Vero cell lysate.

On the basis of these encouraging efficacy data at a dose level of 107 TCID50 MV-CEA, we undertook a second study in which we greatly reduced the virus dose

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and compared the outcomes of single- or multidose therapy using 103, 104 or 105 TCID50 per dose (n ¼ 5 animals per treatment group). As shown in Figures 1b–d, significant prolongation of median survival was observed following single- and multidose therapy at all three dose levels (Po0.005). At 105 and 104 TCID50 dose levels, there was no significant difference in survival prolongation between single- and multi-dose groups. However, at the 103 dose level, the survival prolongation in animals treated with a single dose of MV-CEA was significantly less than the survival prolongation of animals treated with six doses (P ¼ 0.02). When SKOV3ip.1 cells are inoculated into the peritoneal cavity, they seed predominantly to the greater omentum and at later stages disseminate throughout the peritoneal cavity, at which point the animals develop ascites, which is one of the criteria for euthanasia. Sometimes, depending on the operator, animals also develop subcutaneous tumors at the injection site, presumably owing to leakage of a small number of tumor cells at this site at the time of initial administration. We therefore compared the cause of death (ascites or large injection site tumor) in all of the treatment groups in this experiment. As expected, ascites was the criterion for euthanasia in all of the control untreated animals. However, in all but two of the 30 mice that were treated with MV-CEA, the animals did not have ascites at the time of euthanasia and instead had large subcutaneous injection site tumors. The occurrence of these injection site tumors in MV-CEA-treated animals may be a significant confounding factor when comparing prolongation of survival between animals treated with different doses of the virus. Immunohistochemical analysis of injection site tumors indicated that they were not infected by MV-CEA (not shown), indicating that hematogenous spread of the virus from the initially infected tumors in the peritoneal cavity to more distant tumor sites is extremely inefficient in this model. Interestingly, the distribution of i.p. tumors in almost all of the MV-CEA-treated mice was confined to the peri-gastric region at the time of euthanasia. In contrast, control mice had tumors disseminated throughout the peritoneal cavity with associated ascites

Carcinoembryonic antigen expression profiles in MV-CEA-treated mice To evaluate the pharmacokinetics of the oncolytic MVCEA infections that led to the survival prolongations shown in Figure 1b–d, we monitored the plasma CEA levels at weekly intervals after initiation of therapy. Plasma CEA levels in MV-CEA-treated mice have been shown to provide a convenient surrogate indicator of the total number of virus-infected cells in the animal at a given time. Also, as these are nontransgenic mice, in which only the xenografted human SKOV3ip.1 cells are susceptible to MV-CEA, the plasma levels of CEA provide a quantitative marker for the number of virusinfected tumor cells. The CEA profiles for each of the tumor-bearing mice treated with single or multiple doses of 103, 104 or 105 TCID50 of MV-CEA are shown in


Figure 2. Also, the average concentrations of CEA on days 7 and 21 following therapy for each of the treatment groups are shown in Table 1. As expected, the plasma levels of CEA on day 7 after initiation of therapy are highest for the 105 TCID50 dose group, intermediate for the 104 TCID50 dose group and lowest for the 103 TCID50 dose group. However, the association between CEA level and virus dose is nonlinear with only threefold difference in CEA levels for a 10-fold difference in virus dose between the 103, 104 and 105 TCID50 groups. Between days 7 and 21, there is a significant change in the CEA levels, which increase substantially in the 103 TCID50 groups and decline in the 105 TCID50 groups. Thus, by day 21, plasma CEA levels in the low-dose group are 20- to 30-fold higher than the corresponding CEA levels in the higher dose group. Despite these substantial differences in CEA levels between the high- and low-dose groups at early and intermediate time points, it is apparent from the CEA profiles shown in Figure 2 that the longer term trend is for CEA levels to increase such that these initial differences are no longer apparent at

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Figure 2 Plasma carcinoembryonic antigen (CEA) (ng/ml) profiles of mice treated intraperitoneally with one or six doses of 103, 104 or 105 TCID50 MV-CEA virus. Arrows indicate start and end of virus therapy. The mice were bled 1 week after the initial virus injection and weekly thereafter. The limit of detection of the CEA assay is 2.5 ng/ml.

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Figure 3 Plasma carcinoembryonic antigen (CEA) (ng/ml) levels at necropsy versus weight (g) of residual intraperitoneal tumors in MV-CEA-treated mice from two separate experiments. Mice received a range of MV-CEA doses ranging from (a) 105 to 6 107 TCID50 or (b) 103 to 6 105 TCID50. The association between plasma CEA levels and tumor burden was analyzed using Spearman rank correlation analysis and the respective P and r values are shown.

equilibrium state in which a fixed proportion of the tumor cells are infected by MV-CEA at a given time.19 To test this hypothesis, we weighed both i.p. tumors and subcutaneous injection site tumors of MV-CEA-treated animals at the time of euthanasia, and plotted these tumor weights against the plasma level of CEA determined at the same time (Figure 3). Spearman rank correlation analysis of the data revealed a significant association between plasma level of CEA and i.p. tumor burden. In contrast, there was no correlation between plasma CEA concentration and weight of subcutaneous injection site tumor (data not shown), confirming our earlier conclusion that the virus does not spread from the peritoneal cavity to infect tumor deposits at distant sites.

Discussion

MV-CEA is an oncolytic measles virus expressing the soluble extracellular domain of human CEA that was designed to facilitate the noninvasive analysis of virus kinetics in living animals and in human subjects.19 Based on promising preclinical efficacy studies in an ovarian cancer model,14 the virus is currently undergoing phase I clinical testing in patients with advanced ovarian cancer.

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Each patient will receive i.p. infusions of MV-CEA, administered at monthly intervals up to a total of six doses. The starting dose of MV-CEA for this human study is 103 TCID50, which is considerably lower than the dose of virus that was originally used to demonstrate efficacy in the murine ovarian cancer model.14 The dose–response studies reported in this manuscript show that MV-CEA is effective in the SKOV3ip.1 rodent xenograft model of ovarian cancer down to a dose as low as 103 TCID50. The magnitude of the survival prolongations (approximately doubled) did not differ significantly between groups of mice receiving single- or multiple-dose MV-CEA over a very wide range of doses from 104 to 107 TCID50. However, at the dose level of 103 TCID50, there was a significant difference in survival prolongation between mice receiving a single dose and those receiving six doses of MV-CEA in favor of the multi-dose group. Unfortunately, most of the MV-CEA-treated animals in these experiments had to be euthanized because of the development of large subcutaneous injection site tumors on their abdominal wall. These tumors were not readily apparent in the control animals, which had to be euthanized at a much earlier time point owing to rapidly progressive, disseminated i.p. disease leading to the development of ascites. It is therefore possible that the MV-CEA-treated animals might have survived considerably longer if it were not for the development of these injection site tumors. Analysis of injection site tumors at the time of euthanasia did not reveal the presence of MVCEA-infected foci, indicating that the virus does not disseminate from the peritoneal cavity to distant sites, at least in this ovarian cancer model. It is therefore quite possible that the magnitude of the therapeutic effect of MV-CEA has been significantly underestimated in those groups of animals that received doses higher than 103 TCID50. Thus, the dose–response relationship may be more complex than is apparent from the survival curve shown in Figure 1. In a previously published study using MV-CEA to treat SKOV3ip.1 orthotopic xenografts, in contrast to the current study, we did not see subcutaneous injection site tumors.14 We believe that this difference is operator dependent, probably a consequence of subtle differences in the i.p. injection technique between the two studies. Our working hypothesis to explain the observation that injection site tumors were not infected by MV-CEA is that the blood vessels supplying these tumors were relatively impermeable to blood-borne measles virus. Analysis of the plasma CEA concentration profiles in MV-CEA-treated animals was of considerable interest, not only to shed further light on the relationship between virus dose and tumor response, but also to gain insights into the correlations between virus kinetics and tumor response. Carcinoembryonic antigen profiles in mice receiving single or multiple doses of 105 TCID50 of MV-CEA showed an early peak at day 7, followed by a rapid decline in CEA levels during the next 2 weeks and a subsequent more gradual rise thereafter. An identical pattern was observed at a higher dose of 107 TCID50 (data not shown), but at the lower doses of 104 and 103 TCID50, the early peak and subsequent decline of CEA levels were


less apparent. Although the levels of CEA observed on day 7, after high-dose MV-CEA, were typically higher than those observed on day 14, we cannot conclude that the peak CEA level coincided with day 7 and it may have been higher at a slightly earlier or later time point. However, it is clear that the magnitude of the day 7 plasma CEA concentration is related to the dose of virus administered. Interestingly and for unknown reasons, this relationship is not linear, and the day 7 CEA level increases only threefold for a 10-fold increase in virus dose. Possible explanations for the smaller than expected increases in peak CEA levels with increasing virus dose include the limited accessibility of SKOV3ip.1 tumor cells to viruses in the peritoneal cavity, or a more robust innate immune response at higher virus doses with release of soluble antiviral cytokines such as interferon a and b that restrict viral gene expression.22,23 By day 21, following MV-CEA therapy, the CEA levels had changed dramatically from their day 7 levels, and both the magnitude and direction of these changes were completely different, depending on the dose of virus administered. Thus, in animals receiving a dose of 103 TCID50, there was a steady increase in CEA levels between days 7 and 21, whereas in animals receiving 105 TCID50, there was a steady decline in CEA levels over the same time periods. In animals receiving 104 TCID50 of MV-CEA, the CEA levels did not change significantly between days 7 and 21. The end result of these changes was that animals receiving the higher dose of virus had very few MV-CEA-infected tumor cells in their peritoneal cavity by day 21, whereas animals receiving 103 TCID50 had approximately 20- to 30-fold higher i.p. burden of virus-infected tumor cells on the same day. Our working hypothesis for these differences is that efficient destruction of SKOV3ip.1 tumor cells proceeds rapidly in the animals receiving 105 TCID50 such that they have very few remaining tumor cells (and hence few remaining virus-infected tumor cells) in the peritoneal cavity on day 21. Conversely, the lower virus dose of 103 TCID50 is insufficient to mediate substantial reduction of the tumor cell burden at early time points such that the virus continues to propagate in the plentiful tumor substrate, resulting in a much higher number of virus-infected tumor cells by day 21 post-initiation of therapy. In keeping with this interpretation, CEA levels in animals receiving very high-dose (107 TCID50) MV-CEA were even lower at day 21 than those observed in the 105 TCID50 group (data not shown). Irrespective of whether a low or higher dose of virus is administered, the CEA profiles reveal a fairly uniform, longer term pattern in which the CEA levels slowly and steadily increase over time. Our interpretation of this observation is that, at later stages after virus administration, the tumor has become relatively resistant to the virus such that an equilibrium state is established in which tumor growth is slowed but not inhibited, and virus growth simply keeps pace with the reduced rate of growth of the tumor. This type of equilibrium relationship has been predicted based on mathematical models of oncolytic virus kinetics,24,25 a prediction that is now potentially

validated by our experimental observations. Overall, our observations suggest that i.p. SKOV3ip.1 tumor xenografts are initially quite sensitive to killing by MV-CEA, but that they later become more resistant to the virus, allowing the establishment of a tumor/virus equilibrium. Various mechanisms can be envisaged for this acquired resistance to MV-CEA killing, and this question is the subject of ongoing investigations in our laboratory. In summary, we have conducted a detailed study of the dose–response relationships of MV-CEA, an engineered oncolytic measles virus in a murine xenograft model of ovarian cancer, and we have shown that the virus is effective even at a very low dose of 103 TCID50. The kinetics of intratumoral virus spread are quite distinct at different dose levels, and point to an initial phase of high tumor susceptibility to virus-mediated killing, which is later followed by a stage in which intratumoral virus replication is greatly slowed. At higher virus doses, there is an initial peak in CEA expression, which subsequently declines, corresponding to death of the initially infected tumor cells, and then steadily increases over time. The later progressive increase in CEA is attributed to the establishment of an equilibrium state between virus and tumor wherein the growth of the tumor is slowed rather than halted or reversed by the persistent virus infection. At lower virus doses, the initial peak in CEA expression is not seen, but the equilibrium state between virus and tumor is rapidly established and tumor growth is effectively retarded. Acknowledgements

We thank Maureen Craft for excellent secretarial support and the Molecular Medicine Program Viral Vector Production Laboratory (Guy Griesmann, Kirsten Langfield, Julie Sauer, Sharon Stephan, Henry Walker and Troy Wegman) for supply of the MV-CEA virus. This work was supported by the Olivier S and Jennie O Donaldson Charitable Trust, George M Eisenberg Foundation for Charities and NIH Grants CA100634, CA15083 and HL66958.

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