Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

Oncolytic viral therapies have shown such success in preclinical testing as a novel cancer treatment modality that several phase I and II trials are already underway. Oncolytic vaccinia virus (VACV) strains have been of particular interest due to several advantages. VACV's large 192-kb genome enables a large amount of foreign DNA to be incorporated without reducing the replication efficiency of the virus, which has been shown not to be the case with some other viruses such as adenoviruses [1]. It has fast and efficient replication, and cytoplasmic replication of the virus lessens the chance of recombination or integration of viral DNA into cells. Perhaps most importantly, its safety profile after its use as a live vaccine in the World Health Organization's smallpox vaccination program makes it particularly attractive as an oncolytic agent and gene vector [2].

Currently, biopsy is the gold standard for monitoring the therapeutic effects of viral oncolysis [3‐5]. This may be feasible in preclinical or early clinical trials, however, a noninvasive method facilitating ongoing monitoring of therapy is needed for human studies. The tracking of viral delivery could give clinicians the ability to correlate efficacy and therapy and monitor potential viral toxicity. Furthermore, a more sensitive and specific diagnostic technique to detect tumor origin and, more importantly, presence of metastases may be possible [3].

Here, we report on the construction and testing of a VACV carrying the human sodium iodide symporter (hNIS) as a marker gene for non-invasive tracking of virus by imaging. This virus was derived from VACV GLV-1h68, which has already been shown to be a simultaneously diagnostic and therapeutic agent in several human tumor models including breast tumors [6], mesothelioma [7], canine breast tumors [8], pancreatic cancers [9], anaplastic thyroid cancers [10, 11], melanoma [12], and squamous cell carcinoma [13].

Oncolytic viral therapy is emerging as a novel cancer therapy. Preclinical and clinical studies have shown a number of oncolytic viruses to have a broad spectrum of anti-cancer activity and safety [18]. These are ongoing, and the first oncolytic viral therapy has now been approved in China as a treatment for head and neck cancers [19]. Clinical trials are underway to assess the effects of many other oncolytic viral therapies [20]. However, future clinical studies may benefit from the ability to noninvasively and serially identify sites of viral targeting and to measure the level of viral infection and spread in order to provide important information for correlation with safety, efficacy, and toxicity [3‐5]. Such real-time tracking would also provide useful information regarding timing of viral dose and administration for optimization of therapy, as well as distribution and replication of the oncolytic virus, and would alleviate the need for multiple and repeated tissue biopsies.

VACV is arguably the most successful biologic therapy agent, since versions of this virus were given to millions of humans during the smallpox eradication campaign [2]. More recently, engineered VACVs have also been successfully used as direct oncolytic agents, capable of preferentially infecting, replicating within, and killing a wide variety of cancer cell types [6‐11, 13, 21]. VACV displays many of the qualities thought necessary for an effective oncolytic antitumor agent. In particular, the large insertional cloning capacity allows for the inclusion of several functional and therapeutic transgenes. With the insertion of reporter genes not expressed in uninfected cells, viruses can be localized and the course of viral therapy monitored in patients.

One such promising virus strain is GLV-1h68[21]. This strain has shown efficacy in the treatment of a wide range of human cancers and is currently being tested in phase I human trials[20]. In this study we describe the generation of a novel recombinant VACV, GLV-1h153, derived from GLV-1h68, which has been engineered for specific targeted treatment of cancer and the additional capability of facilitating noninvasive imaging of tumors and metastases. To our knowledge, GLV-1h153 is the first oncolytic VACV expressing the hNIS protein.

The reporter gene chosen for insertion into GLV-1h153 was based on the already successful PET and SPECT imaging characteristics of the human sodium iodide symporter (hNIS) and carrier-free radioiodine reporter imaging system. hNIS is an intrinsic plasma membrane protein that mediates the active transport and concentration of iodide in thyroid gland cells and some extra-thyroidal tissues [16, 17]. Although endogenous NIS is physiologically and functionally expressed in several normal tissues, so far only 2 human cancers - some thyroid cancers, and around 80% of breast cancers including ductal carcinomas - have been shown to express endogenous NIS functionally, making them amenable to radiotherapy [22]. It is one of several human reporter genes that are currently being used in preclinical studies and has even been used in clinical studies imaging prostate cancer [20, 23]. hNIS gene transfer via viral vector may allow infected tumor cells to concentrate several commercially available, relatively inexpensive radionuclide probes, such as ¹²³I, ¹²⁴I, ¹²⁵I, and ^99mTcO₄, all of which have long been approved for human use by the U.S. Food and Drug Administration, allowing noninvasive imaging of tumors expressing NIS [22]. In contrast to a study published by McCart et al.[24] using an oncolytic VACV expressing the human somatostatin receptor hSSTR2, hNIS is a transporter-based reporter gene system. Whereas receptors usually have a 1:1 binding relationship with a radiolabeled ligand, transporters provide signal amplification through transport-mediated concentrative intracellular accumulation of substrate. hNIS use has also been shown to be comparable to the commonly used HSV1-tk reporter gene [25] and correlated with ^99mTcO₄[26]. This can be very useful for viral distribution with scintigraphy or PET scanning during and after viral therapy, and may allow for correlation with efficacy and toxicity during clinical trials and treatment thus offering potential clinical translation of this dual therapy.

In order to take advantage of the therapeutic and imaging potential of hNIS, several groups have attempted exogenous NIS gene transfer in several human cancers including head and neck squamous cell cancers, non-small cell lung, thyroid, liver, colorectal, and prostate cancers, as well as glioma and multiple myeloma [22]. Studies have shown that hNIS gene delivery to both thyroidal and non-thyroidal, non-organifying tumor cells is capable of inducing accumulation of therapeutically effective radioiodine doses. For example, a single therapeutic ¹³¹I dose of 3 mCi was shown to elicit a dramatic therapeutic response in NIS-transfected prostate cell xenografts, with an average volume reduction of more than 90% [27]. Transfection of an hNIS-defective follicular thyroid carcinoma cell line with the hNIS gene was able to reestablish iodide accumulation activity both in cell culture and in animal models [28]. Furthermore, transfection of pancreatic cancer cells with a replication-deficient adenoviral vector expressing hNIS lead to a more than 15-fold increase in iodide uptake visualized with ¹²³I scintigraphy, and an over 75% reduction in volume in vivo after treatment with 3mci of ¹³¹I [29].

We have previously reported on the use of a novel recombinant VACV, GLV-1h99, a derivative of GLV-1h68, which was constructed to carry the human norepinephrine transporter gene (hNET) under the VACV synthetic early promoter placed at the F14.5L locus for deep tissue imaging [30, 31]. The parental virus GLV-1h68, a recombinant VACV (LIVP strain), was constructed by inserting 3 expression cassettes (Renilla luciferase-Aequorea green fluorescent protein (RUC-GFP) fusion, β-galactosidase, and β-glucuronidase) into the F14.5L, J2R, and A56R loci of the viral genome, respectively [6]. The hNET protein was expressed at high levels on the membranes of cells infected with GLV-1h99, and expression of the hNET protein did not negatively affect virus replication in cell culture or in vivo virotherapeutic efficacy. GLV-1h99-mediated expression of the hNET protein in infected cells resulted in specific uptake of the radiotracer [¹³¹I]-meta-iodobenzylguanidine ([¹³¹I]-MIBG). In mice, GLV-1h99-infected tumors, including pancreatic and mesothelioma, were readily imaged by [¹²⁴I]-MIBG PET. However, one of the disadvantages of using hNET is that it requires the carrier MIBG for radioiodine uptake.

GLV-1h153 was effective at infecting and replicating within pancreatic cancer PANC-1 cells as efficiently as its parental virus GLV-1h68. This indicated that insertion of the hNIS protein did not negatively affect virus replication in cell culture. With the hNET-expressing GLV-1h99 virus, on the other hand, there was slight improvement in viral replication and oncolytic effect, which may have been due to the exchange of expression cassettes under the control of promoters with different strengths.

Microarray analysis revealed an almost 2000-fold change increase in hNIS mRNA and an almost 5000-fold change increase by 24 hours after PANC-1 infection with GLV-1h153 at a multiplicity of infection of 5.0. Western blot studies showed hNIS protein expression as a band between 75 and 100 KiloDalton in PANC-1 cells infected with GLV-1h153, with higher concentrations of the protein at higher MOIs. This band also appears in normal human thyroid lysates at a slightly lower molecular weight, which is likely explained by differences in glycosylation within cells [16]. The hNIS protein was successfully transported and inserted into the cell membrane, as demonstrated by fluorescence microscopy. In vitro, GLV-1h153-mediated expression of the hNIS protein in infected PANC-1 cells resulted in specific uptake of the radiotracer ¹³¹I, indicating that the hNIS protein was functional, with the uptake reaching a >70-fold increase compared with uninfected control at an MOI of 1.0.

GLV-1h153 was also effective at infecting, replicating within, and killing PANC-1 cells and eradicating tumor xenografts as efficiently as parental virus GLV-1h68. This indicated that insertion of the hNIS protein did not negatively affect virus replication in vivo which was already demonstrated in vitro, or the cytolytic activity in cell culture and in vivo virotherapeutic efficacy. Similar effects were seen between the IT and IV groups treated with GLV-1h153 or GLV-1h68, indicating the inherent affinity of both genetically modified vaccinia viruses to tumors. Furthermore, administration of GLV-1h153 did not have any significant effects on mean net body weights of the animals 34 days after treatment, with the IT groups even gaining weight as compared to untreated control.

Finally, in mice, three PANC-1 tumors infected with GLV-1h153 were readily detectable by PET, with no enhancement above background of either GLV-1h68- or PBS-treated tumors. Mice were treated intratumorally with GLV-1h153, non-hNIS expressing parent virus GLV-1h68, and PBS, and imaged 48 hours after with carrier free ¹²⁴I. The quantitative ¹²⁴I -PET showed that imaging of GLV-1h153 infection of PANC-1 tumors is feasible after direct tumor injection.

As with any translational therapy, concerns over immune responses remain. Since hNIS is a human derived gene, it is unlikely to be immunogenic. However, application of GLV-1h153-mediated hNIS transfer raises concerns over the possibility of autoimmunity in patients. Several papers have already shown that hNIS is not a major candidate for autoimmune disease in patients with patients with Graves' disease and Hashimoto's thyroiditis [32, 33]. Moreover, a clinical trial assessing adenoviral-mediated hNIS transfer in humans did not report any serious adverse effects due to autoimmunity in patients treated for prostate cancer [23]. Further studies and caution will be needed to assess the potential of autoimmunity with hNIS transfer in humans. Studies are also now underway to determine the viral biodistribution, as well as the dose and timing related aspects of in vivo imaging using this novel virus.

We have therefore shown that a novel new oncolytic VACV engineered to carry the human sodium iodide symporter gene (hNIS) can successfully replicate within and kill pancreatic cancer cells as efficiently as its parental virus GLV-1h68. GLV-1h153 expresses the hNIS reporter gene, which enhances radiouptake in vitro and is readily imaged with the clinically approved radiopharmaceutical ¹²⁴I via PET. The ability of GLV-1h153 to infect and enhance cellular uptake of iodine in cells of pancreatic cancer origin, a uniformly fatal disease resistant to conventional therapy, justifies further studies as well as the initiation of clinical trials. Further, this imaging system could be directly translated to human studies, as clinical trials of oncolytic viral therapy would benefit from this noninvasive monitoring modality.

Nanhai G. Chen, Qian Zhang, Yong A. Yu, and Aladar A. Szalay are affiliated with Genelux Corporation. No competing financial interests exist for Dana Haddad, Chun-Hao Chen, Pat Zanzonico, Lorena Gonzalez, Susanne Carpenter, Joshua Carson, Joyce Au, Arjun Mittra, Mithat Gonen, and Yuman Fong.