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Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg, GermanyDepartment of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital, Heidelberg, Germany
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.
analysis of variance
cell infectious units
enhanced green fluorescent protein
yeast CD-UPRT fusion protein
high molecular weight melanoma–associated antigen
multiplicity of infection
single-chain variable fragment
Malignant melanoma is the most common form of fatal skin cancer (
). 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 (
). 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.
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 (
). 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 (
)). 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 (
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.
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 (
)). 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, (
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.
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.
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.
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.
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.
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 (
), 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 (
). 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 (
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 (
). 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 (
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 (
). 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 (
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 (
) 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
Human melanoma cultures (A375M, Mel888, pMelL, and SK-MEL-28) and cell lines Vero, Vero-αHis, HT1080-CD20, and Raji were previously described (
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.
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 (
). 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).
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.
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 (
) 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.
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).
PE is an employee of Transgene S.A., owns stock options in this company, and is an inventor on patents and patent applications assigned to Transgene S.A. Transgene S.A. holds a patent on FCU1 with PE as an inventor. All other authors declare no conflict of interest.