Enhancing the Tumor Selectivity of a Picornavirus Virotherapy Promotes Tumor Regression and the Accumulation of Inﬁltrating Cd8þ T Cells Michael P

Published OnlineFirst January 28, 2016; DOI: 10.1158/1535-7163.MCT-15-0459

Models and Technologies Molecular Cancer Therapeutics Enhancing the Tumor Selectivity of a Picornavirus Virotherapy Promotes Tumor Regression and the Accumulation of Inﬁltrating CD8þ T Cells Michael P. Bell and Kevin D. Pavelko

Abstract

Picornaviruses have emerged as promising cancer therapies acteristics, despite their extensive sequence homology. These due to their ability to drive cytotoxic cellular immune responses hybrids exhibit a unique propensity to infect and replicate in and for promoting oncolysis. These properties include prefer- melanoma. We have identified GD7-KS1, a virus that is par- ential replication in tumor cells, the induction of strong innate ticularly effective at replicating and infecting B16 melanoma and adaptive immune responses, and the ease with which their in vitro and provides benefitasanoncolytictherapyin vivo genomes can be manipulated. We have developed Theiler's after intratumoral injection. In addition, this virus promotes þ murine encephalomyelitis virus (TMEV) as an immunotherapy the mobilization and accumulation of CD8 T cells within vector that promotes strong adaptive immune responses to treated tumors. Altogether, these findings demonstrate that tumor antigens embedded within its genome. To further picornavirus substrains can be used to rationally design virus explore its usefulness as cancer therapy, we investigated wheth- hybrids that promote antitumor responses and add to the er direct intratumoral delivery of TMEV could promote tumor known strategies identified by us and others to further enhance regression. We generated several picornavirus hybrids using the therapeutic potential of vectors used to treat cancer. substrains of TMEV that have unique immunopathologic char- Mol Cancer Ther; 15(3); 1–8. 2016 AACR.

Introduction pathology to human poliovirus in wild-type mice (10). Several TMEV isolates have been identified and are divided into two The use of viruses to directly target and kill tumor cells has subgroups, GDVII and TO, based on their virulence after intra- emerged as a promising treatment for a variety of cancers and has cranial infection (11). The GDVII subgroup viruses cause a severe been associated with some remarkable clinical successes (1, 2). acute infection that is often lethal, and the TO subgroup causes a This success has emerged from an increased understanding of less severe encephalitis that often persists in the central nervous virus attachment, entry, and replication in nontransformed and system (CNS). Although TMEV is recognized as a model of CNS transformed cells (3, 4). Consequently, much effort has been disease after direct intracranial infection, natural CNS infections placed on defining and modifying the molecular attributes asso- are rare and little is known about how this virus migrates to the ciated with virus attachment and replication and understanding brain and which organ systems are primarily involved in prop- how viruses can be manipulated to increase tumor-specific killing. agating the virus after natural infection. Although these two A drawback to their use is that receptor expression levels can limit substrains share extensive homology, the major differences their efficacy and broader use (5–8). Therefore, it is important to between these two viruses are within the virus capsid regions, not only identify new vectors but to identify strategies that best suggesting that cell entry and attachment may differ, a character- enhance their effectiveness as cancer therapy. Evidence further istic consistent with the differences in pathology induced using suggests that the immune system is critical for driving these these related virus strains (12–14). therapies (9), suggesting that engineering viruses to promote We have developed TMEV as an immunotherapy vector that strong immune responses may enhance their effectiveness as well. drives strong T-cell responses to tumor antigens embedded within Questions remain in regards to this role and whether the immune its genome (15, 16). Our vector was designed using the genetic response to oncolytic therapy focuses primarily on virus clearance backbone of the TO subgroup member Daniel's strain (DA), a or whether this therapy can support secondary responses to tumor strain that is most often readily cleared from intracranially antigens as well. infected mice after the development of an immunodominant Theiler murine encephalomyelitis virus (TMEV) is a naturally þ CD8 T-cell response (17). These studies identified a strategy for occurring pathogen that displays a similar neurotropism and driving cytotoxic T-cell responses that subsequently target tumors and inhibit their outgrowth. This strategy has been shown to effectively inhibit melanoma, breast cancer, and glioblastoma Department of Immunology, Mayo Clinic, Rochester, Minnesota. outgrowth when delivered systemically (15, 16, 18). Although our Corresponding Author: Kevin D. Pavelko, Mayo Clinic, 200 First Street SW, 3-23 studies focused on the immune potential of this vector, it is Guggenheim, Rochester, MN 55905. Phone: 507-284-4488; Fax: 507-266-0981; unclear whether direct infection of tumors with TMEV could E-mail: [email protected] provide further therapeutic benefit for the treatment of cancers. doi: 10.1158/1535-7163.MCT-15-0459 In this study, we examine the oncolytic potential of TMEV for 2016 American Association for Cancer Research. use as a virotherapy vector for breast cancer and melanoma. We

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have generated a hybrid TMEV vector that integrates virulence Tumor experiments and virotherapy determinants from the GDVII subgroup and find that this mod- The implantation of B16, B16-OVA, and TUBO tumors into ification enhances its ability to target and inhibit melanoma the flank of B6 and Balb/c mice was performed as described outgrowth when used as an intratumoral virotherapy. In addition, previously (15, 16). Mice were monitored daily, and the tumor we find that direct tumoral injection of GD7-KS1 promotes the size was measured every second or third day after virus treatment. accumulation of an increased number of T cells, including a Balb/c mice bearing TUBO tumors were injected intratumorally þ substantial number of CD8 T cells that target an immunodo- with 5 105 pfu of wild-type TMEV-DA on day 9 after tumor minant virus antigen. These results reveal GDVII virus as a dual implantation. C57BL/6 mice bearing B16 or B16-OVA tumors þ threat able to both mobilize activated CD8 T cells and directly were injected with either a single injection of TMEV-DA (6 106 infect tumor cells. pfu) or with six consecutive injections of 2 105 pfu of GD7-KS1 or GD7-P1 starting on day 7 after implantation. Control animals were injected with PBS or media-absent virus. Tumor index Materials and Methods was calculated as the square root of the product of tumor width Tumor lines, cell lines, and animals and height. Mice bearing tumors with a diameter in excess of 17 The tumor lines EL4, TUBO, 4T1, B16, B16-F10, and B16-OVA mm were killed in accordance with Mayo IACUC requirements. were maintained in DMEM supplemented with 10% FCS (Gibco, Invitrogen). B16, B16-OVA, and B16-F10 cells lines were origi- Flow cytometric analysis of tumor-infiltrating lymphocytes nally obtained from Dr. Richard Vile (Mayo Clinic, Rochester, To analyze the tumor-infiltrating lymphocyte populations after MN) in 2005, and frozen stocks have been maintained in our administration of GD7-KS1 virus, tumor-bearing mice were intra- laboratory. EL4 and TUBO were obtained from Dr. Keith Knutson tumorally injected with virus on day 7 and day 8. On day 13, (Mayo Clinic, Jacksonville, FL) in 2007, and the 4T1 cell line was tumors were harvested, physically dissociated into a single-cell obtained from Dr. Haidong Dong (Mayo Clinic, Rochester, MN) suspension, and sieved through a 100 mm mesh filter. After red in 2014. The fibroblast lines baby hamster kidney (BHK) and blood cell lysis with ACK, cells were pelleted and resuspended in L929 (ATCC) were obtained in 2012 and were used to propogate FACS buffer for staining of appropriate markers. We stained with virus and to titer virus by plaque-forming assay and were main- mouse CD45-PerCP (BD Pharmingen; clone 30-F11) to discrim- tained in DMEM 10% FBS. B16-OVA cells were grown in media inate infiltrating lymphocytes from tumor cells. Antibodies to additionally supplemented with G418 (Life Technologies). No mouse CD8b (eBioscience; clone H35-17.2) and mouse CD4 authentication of the described cell lines was performed by the (eBioscience; clone GK1.5) were used to identify T-cell popula- authors. tions. Quantitation of absolute numbers of infiltrating CD8 and C57BL/6 and Balb/c mice were purchased from The Jackson CD4 T cells was performed using CountBright Absolute Counting Laboratory. All animals were housed in the Mayo Clinic Depart- Beads (Molecular Probes Inc.). The H-2Kb/OVA8 and H-2Db/ ment of Comparative Medicine (Rochester, MN) and cared for VP2121-130 tetramers were kindly provided by Dr. Aaron Johnson according to institutional and NIH guidelines for animals use and (Mayo Clinic, Rochester, MN) and have been described previously care. (17). Samples were run on a BD LSRII Flow Cytometer (BD Biosciences), and data were analyzed using FlowJo Software Recombinant viruses, in vitro killing, virus quantitation, and version 7.6.5 (Tree Star). growth kinetics Recombinant vectors and viruses were generated using techni- Statistical analysis ques previously described (15). The wild-type TMEV-DA used here Mean and SE values were calculated using Excel 2010. All was generated using the pciDAFL3 vector (15). To generate statistical analyses were performed using SigmaPlot for Win- GDVII chimeric viruses, we cloned a KpnI-StuI fragment corre- dows version 11.0. All parametric data were analyzed by t test sponding to nucleotides 932 to 3941 of GDVII (NC_001366) from or ANOVA with individual comparisons performed using the cDNA generated from virus supernatant (Dr. Charles Howe, Mayo Student–Newman–Keuls test. Survival analysis was analyzed Clinic, Rochester, MN). This fragment was subsequently cloned by Kaplan–Meier log rank survival analysis. Significance was into the vector pciDAFL3 to generate the vector encoding GD7- determined by P < 0.05. KS1. Subsequently, 30 and 50 flanking regions of the capsid-coding region (nucleotides 1296 to 3833) were replaced with the DA genome to produce the vectors GD7-P1S1, GD7-KP1, and GD7- Results P1. All viruses generated from these vectors were subsequently Direct intratumoral injection with DA does not significantly plaqued on L929 cells. Plaque sizes were determined using ImageJ inhibit breast cancer or melanoma outgrowth software (19) on scanned images of viral plaques. Virus growth As our previous studies demonstrated that a TMEV vector kinetics for the wild-type DA and GD7-KS1 virus were performed harboring tumor antigens could effectively inhibit both breast on BHK and B16 cells. Viruses were added to wells at an MOI of cancer and melanoma outgrowth, we chose to study these 0.01 and allowed to adhere for 1 hour at room temperature. models to discover whether TMEV could provide a therapeutic Supernatants were removed, and cells were washed twice with benefit through direct oncolysis of established tumors. To test media before incubation at 37C for designated times. Time zero this, we analyzed tumor growth in two implantable models of samples were those not placed at 37C. At each time point, plates cancer, the rat her2/neu–expressing breast cancer TUBO and the were freeze-thawed twice before sonication and pelleting of cell melanoma tumor B16. After 9 days of growth in vivo,breast debris. Supernatants were tested for plaque forming units (pfu). cancer tumors were treated by intratumoral injection with In vitro killing of cell lines and virus quantitation by semiquan- either the wild-type DA or with vehicle. We found that our titative RT-PCR was performed as described previously (15). strategy failed to inhibit the outgrowth of breast cancer at any of

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portion of 2C. Subsequently, vectors were generated that con- tained the P1 region and 30 flanking region, 50 flanking region, and P1 region and a P1-only region (Fig. 2A). These vectors were transfected into BHK cells to generate the viruses GD7-KS1, GD7-P1S1, GD7-KP1, and GD7-P1 (Fig. 2B). As both capsid and noncapsid regions have been implicated in promoting virulence in the TMEV substrains (20, 21), we hypoth- esized that changes in these regions would modulate cell tropism, replication efficiency, or the lytic potential of this virus. We used plaque morphology on L929 cells as an indicator of infection, viral replication, and lytic potential. To determine how each of the GDVII fragments contributes to changes in plaque morphology, we measured plaque diameters after 3 days of in vitro infection. When compared with DA, all GD7 chimeric viruses demonstrated increased plaque size, demonstrating that the components of GD7 capsid and noncapsid sequences contribute to these changes. Furthermore, the GD7-KS1 virus had the largest plaque size, GD7-P1S1 virus had the second largest plaques, and the GD7-P1 and GD7-KP1 had smaller plaques than both of the other GD7 chimeras (Fig. 2B). These viruses provide a variety of plaque phenotypes for the design and implementation of vectors with modified cellular tropism and potential for enhanced virulence and immunogenicity when used as immunotherapy or as virotherapy.

The GD7-KS1 virus preferentially kills and replicates in melanoma tumor lines To determine whether the large plaque virus GD7-KS1 or the Figure 1. small plaque virus TMEV-DA preferentially replicates in specific The use of TMEV-DA as oncolytic therapy fails to inhibit outgrowth of breast tumor lines, we tested infectivity in melanoma, breast cancer, and cancer and melanoma. A, left, average tumor index of TUBO breast tumors in lymphoma lines. These viruses killed all melanoma lines tested by Balb/c mice beginning on the day of intratumoral injection of TMEV-DA (day 9); right, individual tumor growth over time. No increase in survival due 48 hours after infection; however, only the DA line killed breast to virus therapy was observed by Kaplan–Meier analysis. Median survival, cancer and neither killed the thymic lymphoma EL4 (Fig. 3A). TMEV-DA, 19 days; vehicle-ctrl., 34 days. B, left, average tumor index Consistent with these results, both viruses replicate their virus of B16 tumors treated with intratumoral TMEV-DA (day 7). No significance at genome in melanoma; DA replicated in breast cancer and neither any time point tested (Student t test); right, growth of individual tumors in replicated significantly in EL4 (Fig. 3B). To determine whether fi TMEV-DA and vehicle control groups. No signi cant survival advantage by there was preferential replication of either virus in the B16 Kaplan–Meier analysis was observed. Median survival, TMEV-DA, 22 days; vehicle-ctrl., 17 days. melanoma line, we performed one-step growth kinetics of the two viruses in the cell line BHK, which is used to propagate both viruses, and in the melanoma line B16. Both viruses propagated to the time points measured when compared with the vehicle similar levels after infection in BHK cells; in contrast, the GD7-KS1 control group (Fig. 1A). We then tested this strategy on estab- virus propagated to higher titers by 12 hours postinfection lished melanoma tumors using the tumor line B16 and found a when compared with DA. In addition, at the 24-hour time point, modest but insignificant delay in tumor outgrowth and survival the GD7-KS1 virus had replicated 1.5 logs more virus than in the time points measured (Fig. 1B). DA, and the overall yield of virus was 370 pfu/cell compared with 9 pfu/cell with DA (Fig. 3C). Engineering DA-GDVII chimeric vectors and the generation of recombinant viruses with unique plaque Oncolytic viruses containing the GDVII capsid region inhibit phenotypes B16 outgrowth after direct intratumoral injection Having determined that wild-type DA does not provide a As the GD7-KS1 virus preferentially replicates in melanoma, we significant therapeutic benefit in models of breast cancer or tested whether this virus could delay tumor outgrowth when used melanoma when used as oncolytic therapy, we chose to design as an oncolytic therapy. We found that the GD7-KS1 virus inhibits and engineer a series of virus vectors that encoded fragments of the tumor outgrowth with a significant delay detected 6 days after GDVII genome, a substrain previously characterized for its initial intratumoral delivery of the virus compared with vehicle- enhanced neurovirulence (10). The initial vector generated con- only controls (Fig. 4A). We also found that GD7-KS1 treatment sists of the DA backbone vector with a subgenomic fragment of the provides a survival advantage when compared with DA, with an GDVII virus cloned into KpnI and StuI restriction sites present increase in overall survival of 5 days. To determine the role of the in both virus genomes (Fig. 2A). This fragment consists of the capsid region in promoting this increase, we tested oncolytic entire capsid-coding region of GDVII (P1) along with a portion of potential of the GD7-P1 virus for delaying tumor outgrowth. 0 0 the 5 UTR, the leader protein-coding region as well as a 3 Similar to the result obtained with GD7-KS1, we found that GD7- fragment that encodes the noncapsid proteins 2A, 2B, and a P1 inhibits outgrowth of melanoma and increases survival by 7

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Figure 2. Generation of chimeric viruses composed of genomic elements from the TMEV substrains DA and GDVII. A, cloning strategy and map of DA/GDVII chimeric vectors. A 3 kb KpnI and StuI restriction fragment was used to generate an initial DA/GDVII vector. Site-directed mutagenesis and directional cloning were used to replace capsid ﬂanking regions with wild-type TMEV-DA sequences. GD7-P1 contains the complete capsid structure of GDVII, and all noncapsid-encoding elements are derived from TMEV-DA. B, virus plaques generated after the transfection of virus-encoding vectors into BHK cells. Plaque measurements from each virus (right). , signiﬁcant by ANOVA; P < 0.05.

days (Fig. 4B), thus demonstrating the increased effectiveness of protein ovalbumin, we tested for the presence of tumor-specific þ this therapy over wild-type DA is likely dependent on incorpo- CD8 T cells by using H-2Kb/SIINFEKL tetramers. We found that ration of the GDVII capsid region. the percent and absolute number of H-2Kb/SIINFEKL–specific cells was not different between the groups (Fig. 4B); however, the GD7-KS1 virus promotes the accumulation of activated cell surface expression level of CD8 on the CD45 population was þ CD8 T cells specific for virus and tumor antigen decreased in the GD7-KS1–treated group (Fig. 4D), a marker for As the TMEV virus GD7-KS1 promotes tumor regression after activation (22), demonstrating that GD7-KS1 modulates the direct intratumoral delivery and directly kills B16, we asked tumor environment in a manner that promotes tumor regression whether infection of the tumor promotes the mobilization and and tumor-specific T-cell activation. accumulation of T cells within tumors that had responded to GD7-KS1 therapy (Fig. 5A). Using dissociated tumor tissue, we stained for CD8 and CD4 to determine the percentage and Discussion number of T cells that had infiltrated into the B16 tumor. We The goal of this work was to determine the efficacy of an found that this therapy increased the percentage and absolute engineered picornavirus vector as an oncolytic therapy. In the þ number of CD8 T cells within the tumor but did not alter the absence of an appreciable affect using TMEV-DA, we sought to þ overall number of CD4 T cells (Fig. 5B). To further characterize identify a strategy for enhancing the potential of the picornavirus þ this population, we tested for the presence of CD8 T cells that as an oncolytic. Using two substrains of TMEV, we generated a recognize the immunodominant virus antigen VP2. We found chimeric virus that contains genomic material from both the DA þ that over 30% of the infiltrating CD8 T cells were specific for the strain and the GDVII strain. The chimeric virus GD7-KS1 showed b immunodominant H-2D antigen VP2121-130 (Fig. 5C), demon- an increased ability to kill melanoma in vitro and in vivo.In þ strating that therapy drives a strong influx of CD8 T cells, addition, we find that the introduction of capsid-coding regions including a large percentage that are specific for the immunodo- primarily accounts for this increase in efficacy, implicating minant VP2 peptide. enhanced virus infectivity and replication in B16 melanoma as Although the GD7-KS1 treatment did show an enhancement the driver of enhanced therapy. Although direct virus killing of the þ in the accumulation of CD8 T cells within the tumor, we wanted tumor may account for this therapeutic effect, we found that the to test this therapy using the immunogenic melanoma model virus increases the accumulation of virus-specific CD8þ T cells þ B16-OVA to determine whether virotherapy modulates tumor- and promotes activation of tumor-specific CD8 T cells. These specific immunity in tumors responding to virotherapy (Fig. 6A). findings demonstrate that TMEV can be rationally designed to The average tumor index for the control tumors at harvest target an established tumor and that oncolytic therapy using this was 9.3 þ 1.4 and was 7.7 þ 0.4 for tumors treated with GD7- vector modulates the immune cell infiltrate associated with tumor KS1 (P < 0.05 by Student t test). We analyzed the percentage and regression. þ þ total number of CD8 and CD4 T cells that had infiltrated these Although precise receptors for TMEV substrains have not been tumors. We found that there was an increase in the percentage of identified, infectivity of the low pathogenic strains like DA is þ þ CD8 T cells but not CD4 T cells on day 6 after treatment, and the associated with sialic acid moieties on N-linked oligosaccharides þ þ ratio of CD8 T cells to CD4 T cells was increased with GD7-KS1 (23), whereas GDVII attachment is thought to use heparan sulfate treatment (Fig. 6B). As this tumor expresses the immunogenic as a coreceptor (24). Tumor-specific restriction of picornaviruses

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may also decrease replication capacity, promote less efficient infectivity or tumor killing, and may disrupt the genomic stability of the vector (15). We found that the introduction of the GDVII capsid region enhances melanoma-specific killing of GD7-KS1, suggesting that it replicates more efficiently in B16 melanoma and is more cytolytic in vivo than DA, consistent with what we find in vitro. Similar to previous findings (21), we found that the virus assembly machinery from the DA strain can package and assemble GDVII capsids, and these virus hybrids have increased replication capacity and assembly upon infection. The association with sialylation or surface polysaccharides and TMEV attachment to cells may provide an opportunity for specific targeting of nonhuman pathogens to human cancers (28–30). Variations in cell surface glycosylation are observed in various cell types, providing unique functions to cells and potentially making them more or less susceptible to virus infection (31, 32). Cancer metastasis and progression are often associated with altered regulation of posttranslational modifications, including glycosylation (33, 34). This provides an opportunity for designing therapeutics to target these patterns, including viruses that

Figure 3. GD7-KS1 demonstrates enhanced cytotoxicity towards and increased replication in melanoma. A, melanoma lines (B16, B16-OVA, and B16-F10), breast cancer lines (TUBO and 4T1), and the thymic lymphoma line (EL4) were exposed to TMEV-DA or GD7-KS1 for 24 and 48 hours. At the given time points, MTT assays were performed to assess percent killing. B, relative fold change in VP2-specific virus transcript levels assessed in melanoma, breast cancer, and lymphoma at time 0 and 24 hours. Values are relative to uninfected control. C, growth kinetics of TMEV-DA and GD7-KS1 in the virus propagating cell line BHK and in B16 melanoma assessed at given time points Figure 4. GDVII capsid–containing viruses delay tumor outgrowth and promote and expressed as log10 virus pfu. Cells were infected at an MOI of 0.01. pfu/cell and were calculated on the basis of plating 106 cells and the total virus yield increased survival when used as oncolytic therapy. A, tumor-bearing mice from 2 mL of media (, significant by t test; P < 0.05). Data points and were treated with GD7-KS1 for 6 days, beginning on day 8. Tumor index was n ¼ SD for A–C, triplicate independent measurements. calculated until overwhelming tumor burden was reached ( 5/group). Right, individual tumor growth curves through completion of the study. The median survival was 18 days for vehicle control and 23 days for GD7-KS1– has consequently focused on modulating replication once the treated animals ( , significant by log rank; P, 0.021). B, tumor-bearing mice were treated with the GDVII capsid–only virus GD7-P1 for 6 days beginning on virus has entered the target cell. Several strategies, including the day 7, and tumors were monitored as above (n ¼ 5/group). Right, tumor incorporation of miRNA targets or tumor antigens and the mod- growth curves for individual animals. The median survival was 18 days for ification of noncapsid regions, have been used to restrict virus vehicle control and 25 days for the GD7-P1–treated group. , significant by log replication (25–27). Although these strategies are effective, they rank; P, 0.003.

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Figure 5. Intratumoral delivery of GD7-KS1 promotes the accumulation of CD8þ T cells. A, tumor index of B16-bearing hosts treated with intratumoral vehicle or GD7-KS1. Tumors were measured until tumor harvest. B, percentage (top) þ þ and absolute number of CD8 and CD4 T cells recovered from B16 host Figure 6. tumors treated with vehicle or GD7-KS1 in A. C, tumor-infiltrating þ þ GD7-KS1 promotes the activation of tumor-specificCD8 T cells. A, CD45 gated cells were assessed for CD8 and VP2-specific tetramer tumor index of B16-OVA tumors implanted into B6 hosts treated with reactivity. , significant by t test; P < 0.05 (A–C). control vehicle alone or GD7-KS1 virus. Tumors were measured until harvest. B, tumors from A were dissociated and analyzed by FACS. þ þ Total CD45 cells were gated and analyzed for the percentage of CD8 preferentially target human cancers through their dysregulated þ glycosylation. Although a specific receptor has not been identified and CD4 cells within dissociated tumors. Ratio of CD8 cells to CD4 cells comparing control with GD7-KS1 treatment. C, percent of CD8þ for TMEV, much is known about the virus capsid structure and the T cells specific for the tetramer H-2Kb-SIINFEKL. Cells within plot are fi þ speci c amino acids within these structures that contribute to from CD45 gate. D, mean fluorescence intensity of CD8 staining from virus attachment and cell entry (23, 35). Several virus mutations tumor-derived T cells observed in vehicle and GD7-KS1–treated groups. within the outer capsid regions have been identified and these are ,significant by t test; P < 0.05.

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often associated with virus attachment (36, 37). Our results here capsid sequences promotes enhanced targeting of melanoma demonstrate that the GDVII capsid proteins can be interchanged using this approach and that direct delivery of a hybrid virus with DA and provide a more specific targeting of melanoma. enhances its potential as an oncolytic therapy. The use of viruses for the treatment of cancer has led to the The use of viral oncolytics has shown promise in clinical development of vectors that not only target tumors through settings; however, the precise contributions of direct virus infec- direct lysis but have also been used as tools to directly stim- tion, infection of host tissues, and immune responses to the virus ulate immunity against tumors. Although direct tumor killing or to the tumor are not fully understood. We sought to determine by viruses is often the goal, the mechanisms for protection whether the direct infection of tumors with the picornavirus from tumor outgrowth are often linked to the immune system TMEV could inhibit tumor outgrowth. We found that TMEV (38, 39). We find that intratumoral delivery of GD7-KS1 substrains provide useful tools for engineering tumor-specific þ promotes the accumulation of CD8 T cells specificforthe vectors that can effectively target and inhibit melanoma. This virus antigen VP2 at the tumor site. This immunodominant virus provides a unique set of properties that will allow us to response is critical for virus clearance (40), providing a basis further investigate its use as on oncolytic therapy, and studies for CD8-mediated killing of virus-infected melanoma cells. In using TMEV will provide further mechanistic insight to the role of addition to the virus-specific T cells that accumulate after infection and immunity and how they contribute to tumor þ infection, we found that the tumor-specificCD8 Tcellsare regression. present prior to the treatment; however, their activation status þ is diminished compared with CD8 T cells acquired after Disclosure of Potential Conflicts of Interest intratumoral virus. This suggests that the development of No potential conflicts of interest were disclosed. immunity to virus and the accumulation of virus-specificT fl cells provide a proin ammatory environment and the poten- Disclaimer tial to overcome the immunosuppressive tumor microenviron- The funders had no role in study design, data collection and analysis, ment. Studies to explore the role of viruses in overcoming decision to publish, or preparation of the manuscript. factors that include T-cell exhaustion, myeloid-derived sup- pressor cells, and T-regulatory cells will be important for Authors' Contributions understanding the underlying immunologic mechanisms that Conception and design: M.P. Bell, K.D. Pavelko drive oncolytic immunotherapy. Development of methodology: M.P. Bell, K.D. Pavelko Unlike other viruses, the replication, translation, and assem- Acquisition of data (provided animals, acquired and managed patients, bly of picornaviruses occur exclusively in the cytoplasm of provided facilities, etc.): K.D. Pavelko infected cells (41). In addition, their genomes are translated Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.D. Pavelko as one polyprotein that is cleaved by specific proteases embed- Writing, review, and/or revision of the manuscript: K.D. Pavelko ded within this long protein. This makes the rational design of Administrative, technical, or material support (i.e., reporting or organizing picornaviruses challenging, as the virus replication and assem- data, constructing databases): M.P. Bell, K.D. Pavelko bly machinery is most often encoded within the carboxyl- Study supervision: K.D. Pavelko terminus and must specifically recognize cleavage, assembly, and replication signals that may be unique to a particular virus. Acknowledgments Nevertheless, several picornavirus hybrids have been described. The authors thank Kathy S. Allen for her technical expertise involving One focus of this work has been on the interchange of 50 UTR virological assays and Dr. Larry R. Pease for careful review of the data in this manuscript. sequences between several picornavirus strains that aid in attenuating their replication or restricting cell-specificreplica- Grant Support tion (42–45). Although these hybrids have demonstrated cell This work was financially supported by a grant from the NIH type and target specificity, the ability to attach and enter specific (5R01CA104996-08; to K.D. Pavelko). celltypesisunalteredusingthis approach. Alternatively, our The costs of publication of this article were defrayed in part by the payment of advertisement findings here demonstrate that virus tropism can be altered page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. through the introduction of related virus substrain sequences, similar to results obtained with coxsackie B3 capsid variants Received June 4, 2015; revised November 5, 2015; accepted November 29, (46). In addition, we found that the manipulation of TMEV 2015; published OnlineFirst January 28, 2016.

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OF8 Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics

Enhancing the Tumor Selectivity of a Picornavirus Virotherapy Promotes Tumor Regression and the Accumulation of Infiltrating CD8+ T Cells

Michael P. Bell and Kevin D. Pavelko

Mol Cancer Ther Published OnlineFirst January 28, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-15-0459

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