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Chemovirotherapy of Malignant Melanoma with a Targeted and Armed Oncolytic Measles Virus

  • Johanna K. Kaufmann
    Affiliations
    Helmholtz University Group Oncolytic Adenoviruses, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany
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  • Sascha Bossow
    Affiliations
    Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • Christian Grossardt
    Affiliations
    Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • Author Footnotes
    7 Current address: II. Medical Clinic and Institute of Tumor Biology, University Comprehensive Cancer Center, University Hospital Hamburg-Eppendorf, Hamburg, Germany
    Stefanie Sawall
    Footnotes
    7 Current address: II. Medical Clinic and Institute of Tumor Biology, University Comprehensive Cancer Center, University Hospital Hamburg-Eppendorf, Hamburg, Germany
    Affiliations
    Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • Jörg Kupsch
    Affiliations
    RAFT Institute of Plastic Surgery, Mount Vernon Hospital, Northwood, UK
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  • Philippe Erbs
    Affiliations
    Transgene S.A., Strasbourg, France
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  • Jessica C. Hassel
    Affiliations
    Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany
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  • Christof von Kalle
    Affiliations
    Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • Alexander H. Enk
    Affiliations
    Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany
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  • Author Footnotes
    8 These authors contributed equally to this work.
    Dirk M. Nettelbeck
    Correspondence
    Helmholtz University Group Oncolytic Adenoviruses, F110, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, Heidelberg 69120, Germany
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Helmholtz University Group Oncolytic Adenoviruses, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany
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  • Author Footnotes
    8 These authors contributed equally to this work.
    Guy Ungerechts
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital, Heidelberg, Germany
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  • Author Footnotes
    7 Current address: II. Medical Clinic and Institute of Tumor Biology, University Comprehensive Cancer Center, University Hospital Hamburg-Eppendorf, Hamburg, Germany
    8 These authors contributed equally to this work.
      Effective treatment modalities for advanced melanoma are desperately needed. An innovative approach is virotherapy, in which viruses are engineered to infect cancer cells, resulting in tumor cell lysis and an amplification effect by viral replication and spread. Ideally, tumor selectivity of these oncolytic viruses is already determined during viral cell binding and entry, which has not been reported for melanoma. We engineered an oncolytic measles virus entering melanoma cells through the high molecular weight melanoma–associated antigen (HMWMAA) and proved highly specific infection and spread in melanoma cells. We further enhanced this oncolytic virus by inserting the FCU1 gene encoding the yeast-derived prodrug convertases cytosine deaminase and uracil phosphoribosyltransferase. Combination treatment with armed and retargeted MV-FCU1-αHMWMAA and the prodrug 5-fluorocytosine (5-FC) led to effective prodrug conversion to 5-fluorouracil, extensive cytotoxicity to melanoma cells, and excessive bystander killing of noninfected cells. Importantly, HMWMAA-retargeted MV showed antitumor activity in a human xenograft mouse model, which was further increased by the FCU1/5-FC prodrug activation system. Finally, we demonstrated susceptibility of melanoma skin metastasis biopsies to HMWMAA-retargeted MV. The highly selective, entry-targeted and armed oncolytic virus MV-FCU1-αHMWMAA may become a potent building block of future melanoma therapies.

      Abbreviations

      5-FC
      5-fluorocytosine
      5-FU
      5-fluorouracil
      ANOVA
      analysis of variance
      CD
      cytosine deaminase
      ciu
      cell infectious units
      CPE
      cytopathic effect
      EGFP
      enhanced green fluorescent protein
      FCU1
      yeast CD-UPRT fusion protein
      FCY1
      yeast CD
      H
      hemagglutinin
      HMWMAA
      high molecular weight melanoma–associated antigen
      MOI
      multiplicity of infection
      MV
      measles virus
      N
      nucleoprotein
      p.i.
      post infection
      scFv
      single-chain variable fragment
      UPRT
      uracil phosphoribosyltransferase

      Introduction

      Malignant melanoma is the most common form of fatal skin cancer (
      • Siegel R.
      • Naishadham D.
      • Jemal A.
      Cancer statistics, 2012.
      ) and incidences continue to rise (
      • MacKie R.M.
      • Hauschild A.
      • Eggermont A.M.
      Epidemiology of invasive cutaneous melanoma.
      ). Although the recent approval of the targeted therapeutics vemurafenib (
      • Heakal Y.
      • Kester M.
      • Savage S.
      Vemurafenib (PLX4032): an orally available inhibitor of mutated BRAF for the treatment of metastatic melanoma.
      ) and ipilimumab (
      • Cameron F.
      • Whiteside G.
      • Perry C.
      Ipilimumab: first global approval.
      ) is encouraging, for advanced malignant melanoma no curative therapy is currently available, creating an urgent need for effective combination therapies.
      An emerging approach to treat cancer is oncolytic virotherapy (
      • Russell S.J.
      • Peng K.W.
      • Bell J.C.
      Oncolytic virotherapy.
      ). Viruses of various families are being engineered to specifically infect and replicate in tumor cells, whereas healthy cells are spared (Figure 1a). In the course of infection, the therapeutic agent is locally amplified, spreads, and tumor cells are destroyed by viral cell lysis. Moreover, viral oncolysis has been reported to trigger systemic antitumor immune activation (
      • Boisgerault N.
      • Tangy F.
      • Gregoire M.
      New perspectives in cancer virotherapy: bringing the immune system into play.
      ). Ideally, oncolytic viruses are genetically modified to bind and enter tumor cells only. This has not been reported to date for malignant melanoma and is a major aim of our study.
      Figure thumbnail gr1
      Figure 1Concept of chemovirotherapy and recombinant measles virus (MV) genomes. (a) Schematic outline of chemovirotherapy. An entry-targeted and armed MV specifically infects tumor cells, leading to syncytia formation, viral replication and spread, and ultimately tumor cell death. In infected tumor areas, the prodrug convertase is expressed, allowing for activation of innocuous prodrug into active drug, and thus combined oncolysis and bystander killing of uninfected neighboring cells. Modified after
      • Kaufmann J.K.
      • Nettelbeck D.M.
      Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond.
      with permission of Elsevier. (b) Schematic representation of recombinant MV genomes. Vectors contain either an unmodified hemagglutinin (H) or a receptor-blinded H retargeted toward HMWMAA and CD20, respectively, and a transgene X. Transgenes are inserted behind the leader and encode enhanced green fluorescent protein (EGFP), yeast cytosine deaminase (yCD), or yCD in combination with uracil phosphoribosyltransferase (yCD-UPRT). Names of corresponding viruses are stated at the bottom right.
      A highly promising molecule for targeted melanoma therapy is the high molecular weight melanoma–associated antigen (HMWMAA, also called MCSP or CSPG-4), as >90% of melanomas are HMWMAA-positive with limited intratumoral heterogeneity and retained expression in advanced disease (
      • Campoli M.
      • Ferrone S.
      • Wang X.
      Functional and clinical relevance of chondroitin sulfate proteoglycan 4.
      ). In combination with restricted distribution in normal tissues, this has encouraged the development of diagnostic antibodies and HMWMAA-targeted immunotherapeutic agents (
      • Campoli M.
      • Ferrone S.
      • Wang X.
      Functional and clinical relevance of chondroitin sulfate proteoglycan 4.
      ).
      We and others have shown that derivatives of the measles virus (MV) vaccine strain, Edmonston B, have high oncolytic potency on various tumor entities (
      • Galanis E.
      Therapeutic potential of oncolytic measles virus: promises and challenges.
      ) which is currently investigated in clinical trials (
      • Russell S.J.
      • Peng K.W.
      • Bell J.C.
      Oncolytic virotherapy.
      ). Target cells are recognized by the MV attachment protein hemagglutinin (H) that can be engineered to bind to a specific tumor surface marker of interest by destroying the natural receptor tropism (
      • Nakamura T.
      • Peng K.W.
      • Vongpunsawad S.
      • et al.
      Antibody-targeted cell fusion.
      ;
      • Vongpunsawad S.
      • Oezgun N.
      • Braun W.
      • et al.
      Selectively receptor-blind measles viruses: Identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model.
      ) while adding a new target cell specificity by the fusion of single-chain antibodies (single-chain variable fragment, scFv’s, (
      • Nakamura T.
      • Peng K.W.
      • Harvey M.
      • et al.
      Rescue and propagation of fully retargeted oncolytic measles viruses.
      )).
      In addition to causing tumor cell lysis, oncolytic MVs have been utilized to deliver therapeutic transgenes (
      • Ungerechts G.
      • Springfeld C.
      • Frenzke M.E.
      • et al.
      An immunocompetent murine model for oncolysis with an armed and targeted measles virus.
      ;
      • Li H.
      • Peng K.W.
      • Dingli D.
      • et al.
      Oncolytic measles viruses encoding interferon beta and the thyroidal sodium iodide symporter gene for mesothelioma virotherapy.
      ). Prodrug-activating enzymes are of special interest, as they can locally convert innocuous prodrugs into chemotherapeutically active drugs in infected tumor cells (Figure 1a). This strategy aims at producing higher drug concentrations in the tumor than reached after systemic drug application in conventional chemotherapy. The combination of viral oncolysis and prodrug activation is coined chemovirotherapy and has proven to be efficient in various tumor models (
      • Ungerechts G.
      • Springfeld C.
      • Frenzke M.E.
      • et al.
      Lymphoma chemovirotherapy: CD20-targeted and convertase-armed measles virus can synergize with fludarabine.
      ;
      • Bossow S.
      • Grossardt C.
      • Temme A.
      • et al.
      Armed and targeted measles virus for chemovirotherapy of pancreatic cancer.
      ;
      • Zaoui K.
      • Bossow S.
      • Grossardt C.
      • et al.
      Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus.
      ). A popular prodrug activation system is based on cytosine deaminase (CD) converting 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU, (
      • Portsmouth D.
      • Hlavaty J.
      • Renner M.
      Suicide genes for cancer therapy.
      )). The FCU1 gene encodes a fusion protein of yeast CD (encoded by FCY1) and yeast uracil phosphoribosyltransferase (UPRT, encoded by FUR1). UPRT catalyzes the activation of 5-FU into 5-FU monophosphate, a function frequently diminished in tumor cells rendering them resistant to 5-FU treatment (
      • Erbs P.
      • Regulier E.
      • Kintz J.
      • et al.
      In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.
      ). Yeast CD, either by itself (yCD) or in combination with UPRT (yCD-UPRT), has been successfully used in context of various oncolytic viruses (
      • Nakamura H.
      • Mullen J.T.
      • Chandrasekhar S.
      • et al.
      Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil.
      ;
      • Fuerer C.
      • Iggo R.
      5-Fluorocytosine increases the toxicity of Wnt-targeting replicating adenoviruses that express cytosine deaminase as a late gene.
      ;
      • Conrad C.
      • Miller C.R.
      • Ji Y.
      • et al.
      Delta24-hyCD adenovirus suppresses glioma growth in vivo by combining oncolysis and chemosensitization.
      ;
      • Chalikonda S.
      • Kivlen M.H.
      • O’Malley M.E.
      • et al.
      Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene.
      ;
      • Foloppe J.
      • Kintz J.
      • Futin N.
      • et al.
      Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus.
      ;
      • Dias J.D.
      • Liikanen I.
      • Guse K.
      • et al.
      Targeted chemotherapy for head and neck cancer with a chimeric oncolytic adenovirus coding for bifunctional suicide protein FCU1.
      ;
      • Quirin C.
      • Rohmer S.
      • Fernandez-Ulibarri I.
      • et al.
      Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy.
      ), but not in oncolytic MVs.
      In this study we developed a targeted chemovirotherapy for malignant melanoma. We constructed an MV displaying an scFv directed against HMWMAA, and analyzed specificity and oncolytic efficacy in melanoma cell cultures, in a xenograft mouse model, and in biopsies of melanoma metastases. We investigated whether therapeutic efficacy in vitro and in vivo is significantly enhanced by arming the retargeted oncolytic MV with FCY1 or FCU1, and subsequent prodrug application.

      Results

      Generation of HMWMAA-retargeted MV

      We constructed a recombinant MV based on the Edmonston B vaccine strain by modifying the attachment glycoprotein H to both ablate natural viral tropism toward CD46 and SLAM (
      • Vongpunsawad S.
      • Oezgun N.
      • Braun W.
      • et al.
      Selectively receptor-blind measles viruses: Identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model.
      ) and introducing a melanoma-directed tropism through C-terminal fusion of an scFv binding HMWMAA (RAFT3, (
      • Kang N.
      • Hamilton S.
      • Odili J.
      • et al.
      In vivo targeting of malignant melanoma by 125Iodine- and 99mTechnetium-labeled single-chain Fv fragments against high molecular weight melanoma-associated antigen.
      )). To monitor virus infection, an additional transcription unit encoding the enhanced green fluorescent protein (EGFP) was inserted into the genome upstream of the nucleoprotein (N) gene (MV-EGFP-αHMWMAA, Figure 1b). Control viruses contained an unmodified H (MV-EGFP) or H directed against the lymphoma-associated antigen CD20 (MV-EGFP-αCD20, (
      • Ungerechts G.
      • Springfeld C.
      • Frenzke M.E.
      • et al.
      Lymphoma chemovirotherapy: CD20-targeted and convertase-armed measles virus can synergize with fludarabine.
      )).

      Melanoma-specific infectivity of HMWMAA-retargeted MV

      Expression of the modified H by MV-EGFP-αHMWMAA-infected cells was verified by western blot analysis (Figure 2a). In MV producer cells, Vero-αHis, the expression levels of retargeted HαHMWMAA were comparable with those of the previously reported HαCD20 control.
      Figure thumbnail gr2
      Figure 2Specificity of oncolysis and replication of high molecular weight melanoma–associated antigen (HMWMAA)-retargeted measles virus (MV). (a) Detection of hemagglutinin (H) and nucleoproteins (N) by western blot analysis of Vero-αHis lysates infected with indicated viruses at a multiplicity of infection (MOI) of 1 for 24hours. (b) Melanoma cell cultures and control cells were infected with indicated viruses at MOI 0.5. Pictures of enhanced green fluorescent protein (EGFP) fluorescence and phase contrast were taken 120hours post infection (p.i.) and merged (top, bar = 200μm). The surface expression of HMWMAA and CD20 was determined by flow cytometry (bottom). Growth kinetics for (c) MV-EGFP-αHMWMAA and MV-EGFP in Mel888 cells (MOI 3), and for (d) MV-EGFP-αHMWMAA and MV-EGFP-αCD20 in Mel888 and HT1080-CD20 cells (MOI 3). Total viral particles were collected at indicated time points and titrated on Vero-αHis cells.
      To proof specific transduction of melanoma cells, we infected a panel of cell lines expressing either HMWMAA or CD20 with MV-EGFP-αHMWMAA and with the control viruses (Figure 2b). In cell culture, MV infection and spread manifest in a cytopathic effect (CPE) by formation of multinucleated syncytia due to the fusion of infected cells with neighboring cells. After infection of three melanoma cell lines and the low-passage melanoma cells pMelL with MV-EGFP-αHMWMAA, we detected CPE by typical, broad syncytia formation comparable to that after infection with MV-EGFP (Figure 2b, upper panel). In contrast, CPE for HMWMAA-negative cells was visible upon infection with MV-EGFP only (with the exception of Vero-αHis cells, see below). The CD20-targeted control virus could infect CD20-positive cells, but not HMWMAA-expressing cells. Thus, infection patterns matched the surface expression of the two target antigens, as determined by FACS analysis (Figure 2b, bottom panel). The MV producer cell line, Vero-αHis, was highly susceptible to all three viruses because of the presence of both CD46, the natural receptor for unmodified MV-EGFP, and a pseudoreceptor for the His6-tag in retargeted MV H variants. For this cell line, syncytia formation had already been followed by complete cell lysis at 120hours post infection (p.i.). Vero cells expressed HMWMAA at low levels and, consequently, were weakly susceptible to melanoma-retargeted MV.
      In conclusion, the infection pattern and CPE of MV-EGFP-αHMWMAA correlated with antigen expression, demonstrating its target cell specificity and success of our MV entry–targeting strategy for melanoma using the HMWMAA-specific scFv.

      Replication efficiency and selectivity of HMWMAA-retargeted MV

      To assess a possible impact of retargeting on viral replication, we analyzed one-step growth kinetics of MV-EGFP-αHMWMAA and MV-EGFP control virus on Mel888 melanoma cells. Titration of progeny particles revealed a growth delay for the retargeted MV, whereas comparable maximal titers were eventually reached 72hours p.i. (Figure 2c). In addition, we compared the selective replication potential of MV-EGFP-αHMWMAA and the CD20-targeted control virus on both HMWMAA-positive Mel888 and antigen-negative HT1080-CD20 cells. MV-EGFP-αCD20 replicated efficiently in HT1080-CD20, but was strongly attenuated in Mel888 cells. Conversely, MV-EGFP-αHMWMAA produced high virus titers in Mel888, whereas replication was attenuated almost 1,000-fold on HMWMAA-negative HT1080-CD20, underlining its exceptional target cell specificity.

      Characterization and cytotoxicity of yCD-armed HMWMAA-retargeted MVs for chemovirotherapy

      To enhance cytotoxicity of the melanoma-specific oncolytic MV, the EGFP reporter gene was exchanged by yeast-derived genes encoding the prodrug-activating enzymes yCD (FCY1) or yCD-UPRT (FCU1, Figure 1b). The expression of yCD, yCD-UPRT, and EGFP by HMWMAA-retargeted viruses was verified by western blot analysis (Supplementary Figure S1a online). Analyzing growth kinetics of these armed viruses in comparison with MV-EGFP-αHMWMAA demonstrated all viruses to replicate equally (Supplementary Figure S1b online).
      Next we compared the cytotoxic impacts of the two armed variants in melanoma cells, A375M and Mel888. At 96hours p.i., virus-mediated cytotoxicity could clearly be detected with the more pronounced effects on Mel888 cells (Figure 3a). On addition of 5-FC 24hours p.i., cell killing was drastically enhanced after MV-FCU1-αHMWMAA infection of both cell lines. The most marked effect was observed for A375M cells. Here cell killing by combined oncolysis and prodrug activation was 81.0–99.6% dependent on the viral dose. Cell killing with the MV-FCU1-αHMWMAA/5-FC combination treatment was equally potent than that of the control treatment with 5-FU for both multiplicity of infection (MOI) 3 and 1 on both cell lines, alluding to effective prodrug activation. Cytotoxicity exerted by MV-FCY1-αHMWMAA was clearly less enhanced by the prodrug.
      Figure thumbnail gr3
      Figure 3Cytotoxicity of prodrug convertase-armed, high molecular weight melanoma–associated antigen (HMWMAA)-retargeted measles virus (MV). Melanoma cell lines A375M and Mel888 were infected with indicated viruses and multiplicities of infection (MOIs), or were mock-infected for 24hours and then treated with 5-fluorocytosine (5-FC), 5-fluorouracil (5-FU), or medium. Cell viability was determined 96hours post infection (p.i.). Means and SDs of quadruplicate infections are shown; viability of mock-treated cells was set to 100%. (a) 5-FC and 5-FU were used at 1,000μM. *P<0.001 (two-way analysis of variance (ANOVA) as compared with respective infected cells not treated with 5-FC). (b) Cells were infected at MOI 1. 5-FU was used at 1,000μM, 5-FC was used at doses from 10 to 1,000μM. *P<0.001 (one-way ANOVA as compared with mock-infected, 5-FC-treated cells); N.S., nonsignificant.
      Treatment efficacy of combined MV-FCU1-αHMWMAA/5-FC proved not only to be dependent on viral doses, but also on the 5-FC concentration as shown for A375M and Mel888 cells (Figure 3b).

      Bystander killing by MV-FCU1-αHMWMAA in combination with 5-FC

      An important feature of the yCD/5-FC prodrug activation system is based on the diffusibility of 5-FU, enabling a toxic bystander effect on neighboring and more distant tumor cells that have not been infected initially. We tested this phenomenon in vitro by infection of cells (converting cells) and subsequent prodrug addition. Supernatants were collected upon complete lysis of the cell layer and viral particles were heat-inactivated. Subsequently, serial dilutions were transferred onto newly seeded A375M cells (bystander cells). After treatment of converting cells, 5-FU in the supernatant then exerts cytotoxicity on bystander cells. We first tested bystander activity of the superior yCD-UPRT-armed virus with Vero-αHis cells as converting cell line and, thus, independent of entry through HMWMAA. Supernatants from MV-FCU1-αHMWMAA/5-FC-treated cells elicited marked, dose-dependent cell killing similar to equal dilutions of 5-FU (Figure 4). In contrast, neither mock-infected nor MV-EGFP-αHMWMAA-treated cells produced cytotoxic supernatants when supplied with 5-FC. Furthermore, supernatants from cells infected with yCD-UPRT-armed virus, but not treated with prodrug, did not transfer cytotoxicity. These controls show that prodrug conversion was virus-dependent, and that inactivation of viruses in the transferred supernatants was successful. In addition, we confirmed the bystander activity of MV-FCU1-αHMWMAA in a more relevant setting by using A375M and Mel888 melanoma cell lines as converting cells. Again, cytotoxicity similar to the 5-FU-treated positive control was reached, demonstrating virtually complete prodrug conversion in melanoma cells after chemovirotherapy.
      Figure thumbnail gr4
      Figure 4Bystander cytotoxicity of MV-FCU1-αHMWMAA/5-fluorocytosine (5-FC). Vero-αHis, A375M, or Mel888 cells were infected with indicated viruses at multiplicity of infection (MOI) 0.1 and treated with 1,000μM 5-FC or medium at 36hours post infection (p.i; Vero-αHis), 72hours p.i. (A375M), or 60hours p.i. (Mel888), respectively. Supernatants were collected 12hours (Vero-αHis, Mel888) or 24hours (A375M) later on complete lysis of the cell layer, and were heat-inactivated. Serial dilutions were transferred onto fresh A375M in quadruplicates and viability was determined 72hours later. Means and SDs are shown; viability of mock-infected cells was set to 100%. Equivalent dilutions of 5-fluorouracil (5-FU) served as controls. *P<0.001 (two-way analysis of variance as compared with respective cells treated with supernatant not supplied with 5-FC); N.S., nonsignificant.

      Oncolysis and chemovirotherapy with HMWMAA-retargeted MVs in vivo

      To assess the efficacy of a chemovirotherapeutic regimen in vivo, we subcutaneously engrafted human A375M tumors in NOD/SCID mice. When reaching an average volume of 50mm3, tumors were injected on 5 consecutive days with 1.44 × 105 cell infectious units MV-FCU1-αHMWMAA, MV-EGFP-αHMWMAA, or carrier fluid. Starting 3 days after the last virus injection, 200mgkg−1 5-FC or saline were administered intraperitoneally twice daily on 5 consecutive days. Measurement of tumor volumes revealed a highly significant growth retardation of virus-treated xenografts when compared with control groups. Demonstrating effective oncolysis in vivo, this translated into a significant extension of median survival from 43 to 54 days (Figure 5a). Average tumor volumes and survival did not differ between armed and reporter virus-treated groups. However, no additional prodrug activation effect was observed.
      Figure thumbnail gr5
      Figure 5Oncolysis and chemovirotherapy with high molecular weight melanoma–associated antigen (HMWMAA)-retargeted measles viruses (MVs) in vivo. NOD/SCID mice harboring subcutaneous A375M xenografts were treated with five daily intratumoral virus injections or mock injections, followed by intraperitoneal injections of 5-fluorocytosine (5-FC) or saline twice daily on 5 consecutive days as indicated. Tumor volumes (left, middle) and survival (right) were monitored. Left: ***P<0.001 (two-way analysis of variance (ANOVA), mock vs. every virus). Middle: ***P<0.001 (unpaired t-test). (a) Virus injections started when mean tumor volume was 50mm3 with daily doses of 1.44 × 105 cell infectious units (ciu). Right: P<0.0001 (log-rank test, survival distributions of mock vs. each virus-treated group). (b) Virus injections started when mean tumor volume was 40mm3, with daily doses of 3.05 × 105 ciu. Left: §§§P<0.001 (two-way ANOVA, MV-FCU1-αHMWMAA vs., MV-FCU1-αHMWMAA+5-FC). Middle: **P=0.0025, §P=0.0106 (unpaired t-test). Right: P=0.0024 (log-rank test, mock vs. MV-FCU1-αHMWMAA), P=0.0037 (log-rank test, MV-FCU1-αHMWMAA vs. MV-FCU1-αHMWMAA+5-FC).
      We then modified the regimen by starting treatment at an average tumor volume of 40mm3, doubling the viral dose, and administering 5-FC earlier, i.e., on days 1 to 5 after the last virus application. With this regimen, we could reproduce the highly significant oncolysis-only effect of MV-FCU1-αHMWMAA (Figure 5b). The expression of yCD-UPRT and viral N mRNAs in xenografts was confirmed (Supplementary Figure S2 online). Most notably, we observed a clear and highly significant additional prodrug activation effect on both tumor growth and survival. Thus, our schedule-adjusted chemovirotherapy represents a beneficial treatment regimen in this model of melanoma.

      Infection of biopsies of human melanoma metastases with HMWMAA-retargeted MV

      Importantly, we validated infection and spread of the entry-targeted oncolytic MV in highly relevant human primary material. Biopsies of melanoma skin metastases were sectioned into living tissue slices, infected with MV-EGFP-αHMWMAA, and reporter gene expression was detected by fluorescence microscopy. We could show that melanoma metastasis tissue was readily infectable with retargeted MV (Figure 6a). In parallel, we established a monolayer cell culture originating from the same metastasis. These freshly purified melanoma cells strongly expressed HMWMAA (Figure 6b, bottom panel). Infection with MV-EGFP-αHMWMAA led to extended syncytia formation (Figure 6b, upper panel), resulting in complete eradication of the cell monolayer within 96hours p.i.. Infection was confirmed in living tissue slices from a melanoma skin metastasis biopsy of a second patient (not shown). Taken together, susceptibility of primary melanoma material supports the suitability of the HMWMAA-retargeted oncolytic MV for treatment of advanced melanoma.
      Figure thumbnail gr6
      Figure 6Infection of explanted human melanoma skin metastasis by high molecular weight melanoma–associated antigen (HMWMAA)-retargeted measles virus (MV). An excised human melanoma skin metastasis, cultivated as living tissue slices (a) and freshly purified melanoma cells (b), were infected with 106 cell infectious unit MV-EGFP-αHMWMAA per slice or at multiplicity of infection (MOI) 0.5, respectively. Pictures were taken 72hours post infection (p.i.). HMWMAA expression on primary cells was detected by flow cytometry (b, bottom panel). Bar = 200μm.

      Discussion

      In this study, we have established an entry-targeted oncolytic virus for treatment of malignant melanoma. HMWMAA-retargeted MV showed highly specific infection of and spread in melanoma cell lines and in biopsies of melanoma metastases. Cytotoxicity in vitro and therapeutic activity in a melanoma xenograft model were further increased by arming with FCU1.
      Our study demonstrates MV susceptibility in a panel of established and low-passage melanoma cell cultures. In line with the results of a recent study with untargeted MV-EGFP (
      • Donnelly O.G.
      • Errington-Mais F.
      • Steele L.
      • et al.
      Measles virus causes immunogenic cell death in human melanoma.
      ), we observed various degrees of cytotoxicity. Thus, combination of MV-mediated oncolysis with other therapeutic regimens is essential. Indeed, MV oncolysis is highly suitable for combination regimens, as the mode of cell killing differs from established therapies. Here we report that chemovirotherapy is an effective approach to achieve complete cell killing of melanoma cell lines that have different sensitivities to MV-mediated oncolysis alone.
      In addition to molecular chemotherapy, antitumor immune activation has high potential to increase the therapeutic impact of oncolytic viruses. In fact, a recent study has demonstrated that untargeted MV induces immunogenic cell death in human melanoma cells triggering activation of innate and adaptive antitumor responses (
      • Donnelly O.G.
      • Errington-Mais F.
      • Steele L.
      • et al.
      Measles virus causes immunogenic cell death in human melanoma.
      ). This supports the relevance of intratumoral injection of our HMWMAA-targeted MV, followed by systemic antitumor immunity for clinical translation. In this line, our observation of infection and spread of HMWMAA-targeted MV in biopsies of melanoma skin metastases provides a rationale for injecting skin metastases of melanoma patients, followed by monitoring of oncolysis and immune responses.
      HMWMAA is an appealing antigen for targeted therapies, as it is expressed not only on cells making up the tumor bulk, but also on cells maintaining melanoma growth (
      • Campoli M.
      • Ferrone S.
      • Wang X.
      Functional and clinical relevance of chondroitin sulfate proteoglycan 4.
      ). In addition, HMWMAA is expressed on activated pericytes in various tumor tissues (
      • Schlingemann R.O.
      • Rietveld F.J.
      • de Waal R.M.
      • et al.
      Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds.
      ). This may in fact be beneficial as implied by HMWMAA-directed vaccination studies in mice in which tumor neovascularization was impaired, whereas side effects in other tissues were not observed (
      • Maciag P.C.
      • Seavey M.M.
      • Pan Z.K.
      • et al.
      Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature.
      ).
      To enhance the therapeutic potential of the melanoma-specific MV, we inserted the FCU1 gene (yeast CD-UPRT fusion protein) into the virus genome. This resulted in efficient conversion of 5-FC into chemotherapeutic 5-FU and its active metabolites, bystander activity, and complementation of MV oncolysis in vitro. Our results for melanoma reveal that yCD-UPRT activity is superior to that of yCD alone and to bacterial CD-UPRT (not shown). This is accordant with a systematic evaluation of yeast and bacterial 5-FC-mediated prodrug activation systems identifying yCD-UPRT as the most effective enzyme combination (
      • Johnson A.J.
      • Ardiani A.
      • Sanchez-Bonilla M.
      • et al.
      Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications.
      ).
      5-FU is typically not included in melanoma chemotherapeutic regimens. However, melanoma cell lines are highly susceptible to 5-FU treatment in vitro (
      • Quirin C.
      • Rohmer S.
      • Fernandez-Ulibarri I.
      • et al.
      Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy.
      ). In fact, the aim of our chemovirotherapeutic approach is to achieve locally effective 5-FU concentrations while minimizing 5-FU-related systemic side effects. It is noteworthy that the FCU1-encoded UPRT activity converts 5-FU into its active metabolites, further increasing the sensitivity and overcoming resistance of melanoma cells to 5-FU (
      • Quirin C.
      • Rohmer S.
      • Fernandez-Ulibarri I.
      • et al.
      Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy.
      ).
      The in vitro results of the present study are in accordance with our recent report on effectivity of yCD-UPRT/5-FC prodrug activation in melanoma in combination with untargeted oncolytic adenoviruses (
      • Quirin C.
      • Rohmer S.
      • Fernandez-Ulibarri I.
      • et al.
      Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy.
      ). Importantly, we now show that prodrug activation is similarly functional in melanoma in vivo, augmenting the antitumor activity of the HMWMAA-retargeted MV. The combined chemovirotherapy only proved to be beneficial when virus treatment began at 40mm3 mean tumor volume, and when 5-FC application was started 1 day after the last virus administration. This emphasizes the importance of the exact application regimen, and as shown for other chemovirotherapeutic settings there is room for improvement with respect to timing and time span of prodrug administration (
      • Yamada S.
      • Kuroda T.
      • Fuchs B.C.
      • et al.
      Oncolytic herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation.
      ), as well as number and timing of multiple treatment cycles (
      • Ungerechts G.
      • Frenzke M.E.
      • Yaiw K.C.
      • et al.
      Mantle cell lymphoma salvage regimen: synergy between a reprogrammed oncolytic virus and two chemotherapeutics.
      ).
      In summary, MV-FCU1-αHMWMAA is a highly selective and potent oncolytic MV for chemovirotherapy of malignant melanoma showing efficient cell killing in vitro and therapeutic activity in vivo. The unique combination of oncolysis involving syncytia formation, bystander killing by prodrug activation, and induction of immunogenic cell death (
      • Donnelly O.G.
      • Errington-Mais F.
      • Steele L.
      • et al.
      Measles virus causes immunogenic cell death in human melanoma.
      ) may give an advantage over other virotherapeutic approaches for this disease. Corroborated by our infection studies of living primary human melanoma metastasis slices, further investigations shall support the translation of HMWMAA-retargeted MV toward clinical trials of virotherapy and chemovirotherapy for malignant melanoma.

      Materials and methods

      Cell culture

      Human melanoma cultures (A375M, Mel888, pMelL, and SK-MEL-28) and cell lines Vero, Vero-αHis, HT1080-CD20, and Raji were previously described (
      • Nettelbeck D.M.
      • Rivera A.A.
      • Kupsch J.
      • et al.
      Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA.
      ;
      • Ungerechts G.
      • Springfeld C.
      • Frenzke M.E.
      • et al.
      Lymphoma chemovirotherapy: CD20-targeted and convertase-armed measles virus can synergize with fludarabine.
      ).

      Generation of recombinant MVs

      Genomic MV cDNA plasmids are based on the Edmonston B vaccine lineage strain with an additional transcription unit between leader and N gene. Here the transgenes EGFP, FCY1, and FCU1 (
      • Erbs P.
      • Regulier E.
      • Kintz J.
      • et al.
      In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.
      ) were inserted. The scFv fragment against HMWMAA (RAFT3, (
      • Kang N.
      • Hamilton S.
      • Odili J.
      • et al.
      In vivo targeting of malignant melanoma by 125Iodine- and 99mTechnetium-labeled single-chain Fv fragments against high molecular weight melanoma-associated antigen.
      )) was C-terminally fused to a receptor-blinded H protein and incorporated into the viral genomes. For details, see Supplementary Materials and Methods section.
      Recombinant MV particles were generated from cDNA constructs and amplified on Vero-αHis cells, as described previously (
      • Nakamura T.
      • Peng K.W.
      • Harvey M.
      • et al.
      Rescue and propagation of fully retargeted oncolytic measles viruses.
      ;
      • Martin A.
      • Staeheli P.
      • Schneider U.
      RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication.
      ). To prepare virus stocks, Vero-αHis cells were infected at MOI 0.03 and incubated at 32°C for 58hours. Viral particles were collected and titers were determined, as previously described (
      • Bossow S.
      • Grossardt C.
      • Temme A.
      • et al.
      Armed and targeted measles virus for chemovirotherapy of pancreatic cancer.
      ).

      Infection

      All infection experiments were conducted with viral stocks from the fourth passage. Cell lines were infected with the respective MV at indicated MOIs in Opti-MEM (Life Technologies, Darmstadt, Germany) for 2hours at 37°C, followed by exchange to standard medium. Infected cells were photographed using the Cell Observer fluorescence microscope and Axiovision 4.7 software (both from Zeiss, Jena, Germany), scraped into their medium for analysis of growth kinetics, or subjected to cell viability assay.

      Western blot

      Vero-αHis cells (5 × 104 per 24 well) were infected at MOI 1. Cells were collected 24hours p.i., lysed, denatured at 96°C for 10minutes, and subjected to SDS–PAGE electrophoresis and immunoblotting, as described before (
      • Quirin C.
      • Rohmer S.
      • Fernandez-Ulibarri I.
      • et al.
      Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy.
      ). Membranes were probed with polyclonal rabbit-anti-H and rabbit-anti-N antibodies (kind gifts of R. Cattaneo, Mayo Clinic, Rochester, MN). Proteins were detected with respective horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technologies, Beverly, MA) and visualized using Pierce ECL reagents (Thermo Fisher Scientific, Bonn, Germany).

      FACS analysis

      Surface expression was determined using monoclonal antibodies against HMWMAA (clone 9.2.27; Millipore, Schwalbach/Ts, Germany) and CD20 (clone 2H7; BD Biosciences, Heidelberg, Germany) at 5μgml−1 in phosphate-buffered saline, 10% fetal calf serum, 0.01% NaN3. Isotype controls (IgG2a clone MOPC-173 and IgG2b clone MPC-11; BioLegend, Uithoorn, The Netherlands) and unconjugated antibodies were detected with polyclonal PE-coupled goat-anti-mouse secondary antibody (BD Biosciences; 0.5μgml−1). Samples were analyzed using FACSort (BD Biosciences) and FCS Express Version 3 (De Novo Software, Los Angeles, CA).

      One-step growth curves

      Cells (105 per 12 well) were infected at MOI 3 in duplicates. Cells were scraped into their medium at indicated time points and subjected to one freeze-thaw cycle. Total viral particles were titrated as mentioned above.

      Cytotoxicity and bystander assays

      For cytotoxicity and bystander assays, 104 cells per 96 well or 4 × 105 cells per 6 well were infected. 5-FC, 5-FU (both from Sigma, Steinheim, Germany), or medium was administered at indicated concentrations and time points. For measurement of bystander activity, converting cell supernatants were heat-inactivated at 60°C for 30minutes. Serial dilutions were transferred onto freshly seeded cells (104 per 96 well). Viability of cells was measured by the cell proliferation kit III (XTT; PromoKine, Heidelberg, Germany), according to the manufacturer’s protocol. Sample and reference absorbances were measured using Labsystems Multiskan MS and Ascent Software 2.6 (both from Thermo Fisher Scientific) at wavelengths of 477 and 620nm.

      Xenograft model

      All animal experimental procedures were approved by the responsible Animal Protection Officer at the German Cancer Research Center and by the regional authorities, according to the German Animal Protection Law. A375M cells (107) were implanted subcutaneously into right flanks of 6- to 8-week-old female NOD/SCID mice (Charles River, Sulzfeld, Germany). On an average volume of 40 or 50mm3, tumors were injected with 1.44 × 105 or 3.05 × 105 viral particles per dose or Opti-MEM on 5 consecutive days (n=10). Mice were then intraperitoneally injected with 200mgkg−1 5-FC or saline twice daily on 5 consecutive days at the indicated time points. Tumor volumes ((largest diameter) × (smallest diameter)2 × 0.5) and weight were monitored every third day. Animals were killed when tumor volumes exceeded 1,500mm3 or on weight loss of more than 20%.

      Human biopsy material

      Human melanoma skin metastasis biopsies were obtained according to the Declaration of Helsinki Principles and to the local Ethics Committee’s guidelines, with informed consent from the Department of Dermatology, University Hospital Heidelberg, Germany. Tissues were sliced and cultivated as previously described (
      • Zimmermann M.
      • Weiland T.
      • Bitzer M.
      • et al.
      Preclinical testing of virotherapeutics for primary and secondary tumors of the liver.
      ), and infected with 106 viral particles per slice. For establishment of primary cell cultures, blocks of melanoma tissue were cultivated in primary melanoma medium (
      • Nettelbeck D.M.
      • Rivera A.A.
      • Kupsch J.
      • et al.
      Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA.
      ) and regularly washed to eliminate all debris and cells in suspension until an adherent cell layer had formed. Fibroblast growth was suppressed by 100μgml−1 gentamycin medium supplement. Experiments were conducted after 10 passages.

      ACKNOWLEDGMENTS

      We thank Wilfried Roth (Molecular Tumor Pathology, German Cancer Research Center, Heidelberg, Germany) for support with tissue slicing. We acknowledge Sarah Engelhardt, Christine Engeland, and Jessica Albert for technical assistance. We are grateful to Roberto Cattaneo (Mayo Clinic, Rochester, MN) for measles-specific antibodies, as well as discussion and support of our work. We thank Isaiah J. Fiedler (MD Anderson Cancer Center, Houston, TX) and Jeffrey Schlom (NIH, Bethesda, MD) for providing melanoma cell lines. This work was supported by the Helmholtz International Graduate School for Cancer Research (JKK), the Helmholtz University Young Investigator Group Grant VH NG 212 (DMN), and the German Cancer Aid, Max Eder grant 108307 (GU).

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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