Specific Elimination of CD133 Tumor Cells with Targeted Oncolytic

Published OnlineFirst January 4, 2013; DOI: 10.1158/0008-5472.CAN-12-2221

Cancer Therapeutics, Targets, and Chemical Biology Research

Speciﬁc Elimination of CD133þ Tumor Cells with Targeted Oncolytic Measles Virus

Patricia Bach1, Tobias Abel1, Christopher Hoffmann3, Zoltan Gal4, Gundula Braun1, Iris Voelker1, Claudia R. Ball4, Ian C.D. Johnston5, Ulrich M. Lauer6, Christel Herold-Mende4, Michael D. Muhlebach€ 2, Hanno Glimm3, and Christian J. Buchholz1

Abstract Tumor-initiating cells (TIC) are critical yet evasive targets for the development of more effective antitumoral strategies. The cell surface marker CD133 is frequently used to identify TICs of various tumor entities, including hepatocellular cancer and glioblastoma. Here, we describe oncolytic measles viruses (MV) retargeted to CD133. The þ viruses, termed MV-141.7 and MV-AC133, infected and selectively lysed CD133 tumor cells. Both viruses exerted strong antitumoral effects on human hepatocellular carcinoma growing subcutaneously or multifocally in the peritoneal cavity of nonobese diabetic/severe combined immunodeﬁcient (NOD/SCID) mice. Notably, the CD133- targeted viruses were more effective in prolonging survival than the parental MV-NSe, which is currently assessed as oncolytic agent in clinical trials. Interestingly, target receptor overexpression or increased spreading kinetics through tumor cells were excluded as being causative for the enhanced oncolytic activity of CD133-targeted viruses. MV-141.7 was also effective in mouse models of orthotopic glioma tumor spheres and primary colon cancer. Our þ results indicate that CD133-targeted measles viruses selectively eliminate CD133 cells from tumor tissue, offering a key tool for research in tumor biology and cancer therapy. Cancer Res; 73(2); 865–74. 2013 AACR.

þ Introduction In glioma, small subpopulations of CD133 cells extracted from tumors were shown to be considerably more tumorigenic Tumor-initiating cells (TICs) form an attractive target for þ novel antitumoral strategies as they differentiate into cancer than the tumor bulk population. As few as a hundred CD133 fi cells forming the tumor mass and were shown to be resistant tumor cells were suf cient to induce malignant tumors in fi 5 toward conventional therapies, such as radio- and chemother- immunode cient mice, whereas 1 10 cells of the primary apy (for review see ref. 1). A putative marker for TICs is CD133, tumor material are required for tumor formation (4). More- also known as prominin-1. In the healthy organism, CD133 is over, poor prognosis correlates with CD133 expression in glioma biopsies (5). Yet, the relevance of CD133 as a sole mainly expressed on stem cells of the hematopoietic and the endothelial system with a yet unknown physiologic function marker for TICs in glioma is under debate, as CD133 cells (2). Two epitopes, CD133/1 and CD133/2, both located in the have been reported to exhibit TIC properties as well (6). It has been suggested that these cells constitute a lineage hierarchy extracellular domains of CD133, are often used for the detec- þ fi with primordial CD133 tumor stem cells (TSC), which can tion of CD133 cells using speci c monoclonal antibodies þ AC133 and AC141.7, respectively. The expression of CD133 give rise to CD133 progenitors (7). Other publications have underlies a complex regulation at different levels (3). shown that CD133 expression depends on oxygen, as a portion of glioma cells shows large phenotypic plasticity and can acquire stem-like features and AC133-positivity in response to a hypoxic microenvironment (8). In line with this, a subset of Authors' Affiliations: 1Medical Biotechnology and Gene Therapy and 2Oncolytic Measles Viruses and Vectored Vaccines, Paul-Ehrlich-Institut, endothelial cells lining the tumor vessels was recently shown to þ Langen; 3Department of Translational Oncology, National Center for Tumor be derived from CD133 TSCs (9, 10). Single multipotent 4 þ Diseases (NCT) and German Cancer Research Center (DKFZ); Division of CD133 tumor cells were identified in glioma, which differ- Neurosurgical Research, Department of Neurosurgery, University of Hei- delberg, Heidelberg; 5Miltenyi Biotec GmbH, Bergisch-Gladbach; and entiated into both, tumor cells and endothelial cells (10). 6Department of Internal Medicine I, Medical University Hospital, Tuebingen, Also for other tumors such as hepatocellular carcinoma Germany (HCC) and colon cancer, CD133 expression has been detected Note: Supplementary data for this article are available at Cancer Research on many human tumor cell lines (11, 12). Histologic analysis of Online (http://cancerres.aacrjournals.org/). HCC biopsy material revealed CD133 expression in HCC tissue P. Bach and T. Abel are joint first authors. only but not in noncancerous liver tissue (13). Moreover, þ fi Corresponding Author: Christian J. Buchholz, Paul-Ehrlich-Institut, Paul- CD133 HCC cells possess a greater colony forming ef cacy, Ehrlich-Str. 51-59, 63225 Langen, Germany. Phone: 49-6103-774011; Fax: a higher proliferative capacity and a greater ability to form 49-6103-771255; E-mail: [email protected] tumors in vivo (14). These examples for the relevance of CD133 doi: 10.1158/0008-5472.CAN-12-2221 in tumorigenesis and as potential TSC marker imply that there 2013 American Association for Cancer Research. is urgent need to develop TIC-directed therapeutic strategies.

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Selective killing of defined cell types of interest can be colleagues with modifications (29). In short, the plasmid achieved by virotherapy, which is based on oncolytic viruses encoding the MV-AC133 genome was cotransfected with engineered to preferentially infect and destroy tumor cells. expression plasmids pCA-MV-N, pCA-MV-P, and pCA-MV-L Because of their promising safety profile (vector generation on into 293T cells using Lipofectamine 2000. Two days posttrans- the basis of viral vaccine strains) and their potential to over- fection, 293T cells were overlayed onto Vero-aHis cells to come resistance against irradiation or chemotherapy, oncoly- amplify rescued virus. tic viruses are currently investigated in numerous clinical trials Virus stocks were produced upon infection of Vero-aHis (15, 16). Measles virus (MV) vaccine strains were shown to be (23) cells at a multiplicity of infection (MOI) of 0.03. Two to 3 oncolytically active against various types of human cancer, days after infection, cells were harvested and lysed by 3 freeze- including HCC, glioma, breast cancer, ovarian cancer, lympho- thaw cycles. Virus stocks were titrated on Vero-aHis cells and ma, and mesothelioma (17). the tissue culture infective dose (TCID50) was determined. Measles virus entry into cells is dependent on attachment of the hemagglutinin envelope glycoprotein (H) to its cellular Cells receptors, CD46, CD150/SLAM, and nectin-4, followed by Vero and HT1080 cell lines were purchased from the Amer- fusion with the cell membrane via the envelope fusion (F) ican Type Culture Collection and HuH7 cells from JCRB glycoprotein. CD46 is expressed ubiquitously on nucleated (Japan), HT1080-CD133 and Vero-aHis cells have been cells including hematopoietic stem cells (HSC), whereas described (21, 23). Luciferase (pGL4.50, Promega) or red fluo- signalling lymphocyte activation molecule expression is con- rescent protein (RFP) gene (TurboFP635-C, Evrogen) expres- fined to immune cells (18). Nectin-4, which is selectively sing cell lines were generated by lentiviral transduction using expressed on lung epithelial cells, was very recently identified pSEW derived transfer vectors (21, 22) followed by bulk cell as a host exit receptor mediating horizontal transfer of measles sorting (MoFlow cell sorter, Beckmann Coulter) for homoge- virus (19, 20). We and others have shown that the envelope nous gene expression. All cell lines were cultivated in protein complex of measles virus can be engineered to restrict Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal calf infection to cell types defined by a cell surface marker of choice serum (FCS), 2 mmol/L glutamine and passaged for less than 4 (21–23). For this purpose, residues interacting with the measles months before another vial from the master cell bank was virus receptors are mutated and a single-chain antibody (scFv) taken into culture. All cell lines were regularly tested for specific for the target receptor of interest is genetically fused to mycoplasm and found to be negative. H. This way measles virus glycoproteins specific for a variety of Primary glioblastoma cells NCH644 were obtained from cell surface receptors, such as CD20, Her2/neu, CD105, gluta- patients undergoing surgical resection as approved by the mate receptor, and also CD133 have been generated and were Institutional Review Board of the Medical Faculty Heidelberg used to target lentiviral vectors (21, 22, 24) and oncolytic (Heidelberg, Germany). Tumor tissue was enzymatically dis- measles viruses (25) sociated and short-term cultures were established as floating Here, we present oncolytic measles viruses retargeted to the tumor spheres in DMEM/F-12 medium containing 20% bovine CD133 molecule. We show that these viruses are absolutely insulin transferrin serum-free supplement, basic fibroblast þ specific for CD133 cells and highly efficient in target cell growth factor (bFGF), and EGF at 20 ng/mL each (all Provitro). killing. We assess their oncolytic activity in 3 different cancer The TSC-03 tumor sphere culture was established from a types, that is, glioma, colon cancer, and hepatocellular cancer primary colorectal cancer lung metastasis, which was obtained by applying mouse tumor models. Our data suggest that at Heidelberg University Hospital (Heidelberg, Germany) in CD133-targeted measles virus can serve as a novel tool not accordance with the declaration of Helsinki and under an þ only to improve our understanding on the role of CD133 cells Institutional Review Board–approved protocol following writ- in oncology, but also providing an innovative strategy for ten informed consent. Sphere cultures were established and tumor therapy supplementing current treatment modalities. grown as previously described (30). Human HSCs were obtained from apheresis products after Materials and Methods mobilization with granulocyte colony-stimulating factor (G- þ Generation of CD133 measles viruses CSF). CD133 HSCs were purified and cultivated as described Cloning of the 141.7 scFv cDNA has been described (21). The (21). The scientific use of anonymous samples no longer AC133 scFv was PCR-assembled from the hybridoma line required for stem cell transplantation from deceased indivi- AC133 (Miltenyi-Biotec) as described for other scFv (21, 22). duals or from a remaining sample has been approved by the Both scFv reading frames were cloned into pCG-HmutXaCD20 University Hospital Ethics Committee (Frankfurt, Germany; (26) via SfiI/NotI resulting in pCG-HmutCD133scFv-(AC133) 163/03). and pCG-HmutCD133scFv-(141.7), respectively. The PacI/ SpeI–digested fragment of pCG-HmutCD133scFv-(141.7) was Immunoblotting inserted into the corresponding sites of pMV-eGFP(SpeI; Virus particles harvested from cell supernatant were dena- refs. 27, 28), whereas the PacI/SpeI fragment of pCG- tured for 5 minutes at 95Cin2 urea sample buffer (5% SDS, 8 HmutCD133scFv-(AC133) was transferred into p(þ)PolII- mol/L urea, 200 mmol/L Tris–HCl, 0.1 mmol/L EDTA, 0.03% MVNSe-GFP(N; ref. 29). bromphenol blue, 2.5% di-thiothreitol, pH 8.0), separated on a MV-141.7 was rescued using the hexa-histidine (His6) retar- 10% SDS-polyacrylamide gel and blotted onto a nitrocellulose geting system (23) and MV-AC133 as described by Martin and membrane (GE Healthcare). After blocking of membranes with

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5% skim milk powder in TBS supplemented with 0.05% Triton Image 4.1 analysis software (Caliper Life Sciences). For imag- X-100 for 30 minutes at room temperature, they were incu- ing, mice were intraperitoneally injected with 250 mL luciferin bated with rabbit antimeasles virus nucleoprotein N antibody (15 mg/mL) in PBS and anesthetized with 2.5% isoflurane. (Abcam) diluted 1:5,000 or anti-MV-Hcyt antiserum 1:5,000 Images were acquired ventral, within 10- to 20 minutes after (Eurogentec), which were both detected by a peroxidase- luciferin injection. For quantification of luciferase activity, conjugated goat anti-rabbit immunoglobulin (DAKO) diluted total light emission per mouse was determined after back- 1:2,000 as secondary antibody. ground subtraction. A total of 1 105 TSC-03 sphere culture cells were mixed in a Virus spreading assay 1:1 ratio with Matrigel (BD Biosciences) 24 hours postinfection HuH7 cells (5 104) were cultivated in triplicates in 24-well and transplanted under the kidney capsule of NOD.Cg- plates and infected at an MOI of 0.005. Twenty-four, 40, 48, and Prkdcscid Il2rgtm1Wjl/SzJ (Il2rg / ) mice (The Jackson Labora- 60 hours after infection, cells were fixed with 0.25% glutaral- tory). Mice were sacrificed and tumors were compared 58 days dehyde in PBS. Twenty-five adjacent tiles (total area: 8.35 mm2; after transplantation. For retransplantation experiments 1 resolution: 1.34 pcx/mm) were acquired at 10 magnification 105 cells were transplanted under the kidney capsule of using a motorized Axio-Observer-Z1 microscope. Infected area secondary mice. was determined by computational analysis using the Cell A total of 1 105 NCH644 tumor sphere cells were infected Profiler software 2.0 (31). For quantification of GFP-expression, at an MOI of 0.5, dissociated 16 hours later and stereotactically acquired images were converted to binary files, using threshold implanted into the right hemispheres of NOD/SCID mice as þ levels as determined on uninfected control cells. The GFP described (32). area was normalized to the total area for quantification. Statistical analysis Flow cytometry Evaluation of curves describing tumor volumes was con- Cells were trypsinized and staining was conducted using ducted comparing treatment groups versus control groups by anti-human CD133/1-PE antibody (clone AC133, MiltenyiBio- means of the area under the curve (AUC) for each single mouse. tec), anti-human CD133/2-PE (clone 293C3, MiltenyiBiotec), Survival data were depicted with Kaplan–Meier curves. Com- anti-CD46-PE antibody (AbD Serotec), or anti-CD46-FITC parisons between different virus types and the control group (Biolegend). Appropriate isotype controls were used according were conducted by a log-rank test, adjusted for multiple to the manufacturer's instructions. Cells were subjected to comparisons according to Bonferroni or Dunnet method. P fl ow-cytometry analysis using a FACS LSRII cytometer (Becton 0.05 was regarded significant. Dickinson) and the BD FACS DIVA Software (Becton Dick- inson). Only cells with staining intensities above the maximal þ level of isotype controls were defined as CD133 cells. Receptor Results densities of CD133 and CD46 on HuH7 cells were determined Cell specificity of CD133-retargeted oncolytic measles using QantiBRITE-PE beads (Becton Dickinson) according to viruses the manufacturer's instructions. To generate CD133-specific measles viruses, scFv-reading frames cloned from the hybridoma cell lines AC141.7 or AC133 Cell viability assay were fused to that of the measles virus H protein mutant The MTT (Cell Proliferation Kit I, Roche) assay was used to deficient in receptor recognition. Virus genomes encoding an measure cell viability in quadruplicate as recommended by the additional GFP-reading frame for easy detection of virus manufacturer. Viability was calculated as ratio of mean values infection were then assembled and the corresponding viruses measured for infected and uninfected cells. MV-AC133 and MV-141.7 rescued (23). Western blot analysis of virus stocks confirmed that the anti-CD133 scFv displaying H Animal experiments proteins were properly expressed and incorporated into virus All animal experiments were carried out according to the particles (Fig. 1A). Next, we determined the spreading kinetics þ German animal protection law. Animals in tumor experiments of the viruses in CD133 HuH7 hepatoma cells upon infection were regularly checked for health condition and sacrificed if at a low MOI. There was no significant difference between the they had lost 20% of body weight or showed severe signs of spreading kinetics of targeted MV-CD133 viruses and the illness. parental MV-NSe, showing that the retargeted entry via CD133 For the subcutaneous tumor model, HuH7 cells (5 106/ had neither impaired nor enhanced virus spreading and mea- 100mL) were injected into the right flank of 6-week-old non- sles virus-induced syncytia formation (Fig. 1B). þ obese diabetic/severe combined immunodeficient (NOD/ Selectivity of the MV-CD133 viruses for CD133 cells was þ SCID) mice (Harlan). Tumor diameters were measured and determined on HuH7 (CD133 ), Vero (CD133 ), and HT1080 the volume calculated (0.5 length length width). (CD133 ) cells. While MV-NSe infected all cell types, MV-141.7 For the multifocal tumor model, 6- to 8-week-old athymic and MV-AC133 infected HuH7 cells only (Fig. 1C; Supplemen- nude mice (HSD: Athymic Nude-FoxN1NU, Harlan) were intra- tary Fig. S1A). To easily discriminate between the infection of þ peritoneally injected with 5 106 HuH7-luc cells. Tumor CD133 and CD133 cells and to exclude any influence by cell formation was analyzed by in vivo imaging using the IVIS type specific differences, we generated HT1080-CD133 cells Spectrum optical imaging system equipped with the Living stably expressing CD133 and HT1080-RFP cells, which are

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3 Figure 1. Basic characterization of CD133-targeted viruses. A, Western blot analysis of virus particles (5 10 TCID50). Signals derived from H proteins and N protein are indicated. B, spreading kinetics of MV-NSe, MV-141.7, and MV-AC133 through HuH7 hepatoma cells. There were no statistically significant differences between the curves (P ¼ 1.00; N ¼ 3). C, specificity of infection. Vero, HT1080 and HuH7 cells were infected with the indicated viruses at an MOI of 1, respectively. At 72 hours, after infection cells were analyzed by fluorescence microscopy. Scale bar, 200 mm. D, cell specificity in mixed cultures. CD133þ HT1080 cells (HT1080-CD133) and CD133 HT1080-red cells were cocultivated and infected with MV-141.7, MV-AC133, and MV-NSe at an MOI of 0.5, respectively. In the uninfected control culture (top left) HT1080-red cells appear red (labeled with arrow head CD133), whereas HT1080-CD133 are invisible (labeled with arrow head CD133þ). While MV-141.7 (bottom left) and MV-AC133 (bottom right) infection resulted in green cells only, MV-NSe infected both cell types, resulting in green and yellow color (top right).

CD133 and express a RFP (Supplementary Fig. S2). Infection were subcutaneously implanted into NOD/SCID mice. We then with GFP-encoding measles virus was thus expected to result followed tumor growth upon intratumoral injection of viruses þ in green fluorescence when CD133 cells, and yellow fluores- MV-NSe, MV-AC133, and MV-141.7, respectively. Compared cence (resulting from the overlay of red and green) when with mock-treated animals, tumor growth was significantly CD133 cells became infected. Both cell lines were mixed in reduced in all virus-treated animals. However, there were equal amounts and infected with MV-141.7, MV-AC133, or MV- substantial differences between the respective groups. NSe, respectively. While MV-NSe infection resulted in green Remarkably, both groups treated with CD133-targeted measles and yellow fluorescence, only green fluorescence was detected viruses showed a slower tumor growth and a considerably upon infection with MV-141.7 or MV-AC133 (Fig. 1D). More- prolonged survival than the MV-NSe–treated group (Fig. 2B over, MV-141.7- and MV-AC133-infected cells formed large and C). Among the CD133-targeted viruses, the oncolytic green colored syncytia, completely sparing out all red-labeled activity and overall survival were most pronounced in MV- CD133 cells (Fig. 1D). Thus, MV-141.7 and MV-AC133 are also 141.7–treated animals (P ¼ 0.0002). Further studies therefore þ highly selective for CD133 cells even when these are in close focused on MV-141.7. contact to CD133 cells. Oncolytic activity of MV-141.7 in primary tumor spheres Enhanced oncolytic activity of CD133-specific measles To test the oncolytic activity of MV-141.7 on primary tumor viruses cells, we cultivated tumor cells derived from colon cancer and We next assessed the oncolytic activity of MV-141.7 by glioblastoma, 2 cancer types that have been described to þ infection of HuH7 hepatoma cells at different MOIs. Compared contain CD133 TICs (3, 7, 33, 34). Both formed spheres under with mock-treated HuH7 cells, infected cells were substantially serum-free conditions (Figs. 3 and 4) and contained about 90% þ reduced in their viability. Interestingly, MV-141.7-infected cells of CD133 cells (Supplementary Figs. S3 and S5). were significantly less viable than MV-NSe infected cells (Fig. Both, MV-NSe and MV-141.7, efficiently infected the primary 2A). To determine the in vivo oncolytic activity, HuH7 cells colon tumor sphere culture and induced syncytia formation

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Moreover, reisolated tumors from both groups contained þ about 70% CD133 cells (Supplementary Fig. S4). Of note, also the glioma tumor spheres became readily infected upon incubation with MV-141.7 and MV-NSe, respectively. Especially tumor spheres infected with MV-141.7 showed strong syncytia formation (Fig. 4), which resulted in þ loss of CD133 cells (Supplementary Fig. S5). To follow tumor cell killing in vivo, we injected infected tumor spheres into the brains of NOD/SCID mice. Mice that had received control- treated tumor cells had to be sacriﬁced before day 35 due to cachexia, gasping, and anergy, whereas animals that had obtained MV-141.7 or MV-NSe–treated spheres survived up to 70 to 90 days after implantation (Fig. 4B). Interestingly, the survival period of MV-NSe–injected mice was heterogeneous with half of the mice showing reduced survival times compared with MV-141.7–treated mice. Overall, no signiﬁcant difference was evident between both groups. However, also in this primary orthotopic tumor model for glioma, MV-141.7 was at least as effective as MV-NSe in prolonging survival times (Fig. 4B).

MV-141.7 does not infect hematopoietic stem cells Besides TSCs, CD133 is a marker for early progenitors of the hematopoietic system (35, 36). It is therefore theoretically possible that MV-141.7 infects and lyses HSCs. Fluores- cence-activated cell sorting (FACS) analysis confirmed that HSCs expressed similar high cell surface levels of CD133 and CD46 as HuH7 cells (Fig. 5A). HSCs were incubated with þ different doses of MV-141.7 or MV-NSe. While no GFP cells were detected at low dose of MV-141.7 (MOI ¼ 0.1), single þ GFP cells were detected at MOI ¼ 1 (0.08%) and MOI ¼ 10 (0.17%; mean values of 2 samples). Notably, the percentages of þ GFP cells were slightly higher for MV-NSe (0.14% at MOI ¼ 1 and 0.3% at MOI ¼ 10; mean values of 2 samples; Fig. 5B). Figure 2. Oncolytic activity on HuH7 hepatoma cells. A, oncolytic activity þ in vitro. HuH7 cells were infected with MV-141.7 or MV-NSe at the Importantly, the fraction of GFP cells did not increase over indicated MOI, respectively. Ninety-six hours postinfection, cell viability time. Also when cocultivated with infected HuH7 cells, we þ was quantified by MTT assay. , P < 0.0001 (N ¼ 4; SD) as determined could not detect GFP HSCs (Fig. 5B). The data confirm that by 2-sided unpaired t tests. B, tumor growth curves monitored in mice the tropism of MV-141.7 is restricted to tumor cells and that its carrying subcutaneously applied HuH7 tumors after intratumoral virus fi administration. Once tumors had reached 5 mm in diameter, mice safety pro le does not differ from that of measles virus vaccine 6 received intratumoral injections of 1 10 TCID50 on 4 consecutive days, strains currently in clinical use as oncolytic viruses. respectively. Control animals (mock) were injected with an equal volume of DMEM. , P < 0001; N ¼ 7. C, survival curves. , P < 0.05; Oncolytic activity of MV-141.7 in a multifocal tumor , P < 0.0001; N ¼ 7, SEM. model To further assess the therapeutic potential of MV-141.7, we (Fig. 3A). Long-term cultivation of the infected cells revealed established a multifocal cancer model in which labeled HuH7 that MV-141.7 induced rapid loss of CD133 (Supplementary hepatoma cells form many tumor foci scattered through the Fig. S3; compare day 5 data for MV-NSe and MV-141.7). By day peritoneal cavity and allow quantitative monitoring of tumor 25, all living cells had been eliminated from the cultures by the growth over time. For this purpose, HuH7 hepatoma cells were viruses (data not shown). Next, infected tumor spheres were stably transduced with the firefly luciferase (luc) gene gener- implanted under the renal capsule of NSG mice. MV-141.7 ating HuH7-luc cells. Athymic nude mice injected intraperi- infection resulted in significantly smaller tumors than the toneally with these cells developed multiple tumor foci cov- control, whereas the difference between MV-NSe and mock- ering most parts of the peritoneal cavity, which became infected cells was not significant (Fig. 3B and C). However, detectable by in vivo imaging 7 days after injection (Supple- when the same number of resected cells from noninfected and mentary Fig. S6). From then on, luciferase activity slowly but MV-141.7-infected tumors, respectively, was transplanted in steadily increased. This was reflected by steady tumor growth secondary mice, no significant difference in the tumor volumes resulting in an increasing tumor burden that finally caused was observed (Fig. 3D), indicating that self-renewing TICs had significant body weight loss between days 80 and 200 after not been specifically depleted by MV-141.7 in this model. tumor cell implantation so that the animals had to be

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Figure 3. MV-141.7 inhibits tumor formation of primary colon cancer cells. Primary colon tumor sphere cultures were infected with MV- NSe or MV-141.7 at an MOI of 1, respectively. A, tumor spheres are shown in phase contrast (left) and fluorescent microscopy pictures (right). Scale bar, 100 mm. B and C, infected or uninfected tumor spheres (control) were transplanted under the kidney capsule of 4 mice per group 24 hours after infection. Mice were sacrificed 8 weeks after transplantation. Tumors were reisolated and used for photographs (B) and mass quantification (C). Statistics: , P < 0.01. D, equivalent numbers of recultivated tumor cells from noninfected and MV-141.7- infected mice were transplanted into secondary mice. Tumor volumes were determined 8 weeks later. Error bars indicate results of 2 transplanted mice per group; n.s., difference not significant.

sacrificed. Gross pathology of the animals revealed perfect Discussion colocalization of bioluminescent signals and tumor foci (Sup- CD133 is currently among the most intensely investigated plementary Fig. S6). molecules in molecular medicine being an important surface The oncolytic activity of MV-141.7 was then compared with marker not only for HSCs but also for TICs in a variety of that of MV-NSe upon intraperitoneal injection of virus into tumor entities. Here, we describe oncolytic viruses retargeted mice harboring multiple tumor foci in the peritoneal cavity. At the time point of virus injection, mice from all groups showed an almost identical luciferase activity. This changed dramat- ically after virus injection. While the signal steadily increased in the control group, it slowly decreased in the MV-NSe–treated group until day 50 (Fig. 6A and B). From then on, signals in this group rapidly increased and became as high as in the control group by day 95 (Fig. 6B; Supplementary Fig. S6). Accordingly, overall survival of control and MV-NSe–treated mice did not differ significantly (Fig. 6C). In sharp contrast, the luciferase signals in MV-141.7–treated mice declined rapidly immediate- ly after virus injection (Fig. 6B). Later on, only in 1 animal signals increased reaching a similar level as those of the MV- NSe–treated group (Supplementary Fig. S7). All other MV- 141.7–treated animals remained healthy over the whole observation period, which correlated nicely with a low or even undetectable luciferase activity (Fig. 6B; Supplementary Fig. S7). These long-term survivors were sacrificed after 247 days of observation. One of the animals showed a few distinct signals of low intensity, whereas no luminescence was emitted from the other 2 animals (Supplementary Fig. S7). Gross pathology was negative for all 3 animals (Supplementary Fig. S8A). Microscopic examination of histologic sections of the lumi- Figure 4. MV-141.7 inhibits tumor formation of primary glioma cells. A, nescent tissue showed presence of a single tumor site located microscopic pictures of glioma tumor spheres infected with MV-NSe or MV-141.7 at an MOI of 0.5, respectively. Bright field (left) and in the intraperitoneal adipose tissue of 1 animal (Supplemen- fluorescence (right) to indicate infected cells. Scale bar, 100 mm. B, in vivo tary Fig. S8B). These data showed that MV-141.7 treatment had tumorigenicity of MV-141.7 and MV-NSe–infected tumor spheres. fully cleared 2 animals from tumor cells in the peritoneal cavity. Survival over time was analyzed. , P < 0.05; N ¼ 5.

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A principle concern when targeting CD133 with oncolytic viruses is spreading to extratumoral cells, such as HSC or endothelial progenitors. In this context, nontargeted MV-NSe, being already in clinical testing, does not differ from CD133 - targeted measles virus-based viruses. We therefore analyzed infection of cultivated HSC for both, MV-141.7 and MV-NSe, but could not detect any infected cells. At high virus dose a few reporter-positive cells were detected for both virus types but these did not give rise to productive infection. This is in line with the safety data obtained from ongoing clinical trials with oncolytic measles viruses, where for most patients spreading to nontumor tissue has not been reported (38–40). Our data confirm that receptor attachment is not the crucial step for vaccine-derived measles viruses to infect HSC. It is rather likely that the innate immune response of these cells, similarly as that of other nontumor cells, controls infection by measles virus vaccine strains, which carry mutations in the V protein, a viral inhibitor of the b-interferon response (28, 41). Accordingly, a low level of HSC infection with clinical measles virus isolates encoding a fully active V protein counteracting innate immunity has been previously reported (42, 43). CD133-retargeted measles viruses can therefore be expected to be at least as safe as untargeted oncolytic measles viruses currently investigated in clinical trials, whereas spreading through tumor cells that are often deficient in innate immunity proceeds with wild-type efficiency (37). An unexpected outcome of this study was the substantially enhanced oncolytic activity of CD133-specific viruses as compared with the unmodified MV-NSe, which is closely related to an oncolytic measles virus currently in clinical trials. This effect was especially pronounced on the HCC-derived HuH7 cell line but also evident on primary colon cancer tumor spheres in which a high number of cells expressed CD133. We determined CD46 as the relevant entry receptor for MV-NSe on HuH-7 cells, as nectin-4, the recently identified alternative measles virus receptor on epithelial cells (19), could not be detected by FACS analysis using a mouse anti-human nectin-4 (PVRL4) antibody (own unpublished data and ref. 20). Quantification of the receptor densities on HuH7 cells revealed a higher density of CD46 compared with that of CD133 (Supplementary Fig. S1B). Reduced expression of the MV-NSe receptor or increased þ Figure 5. MV-141.7 does not infect HSCs. A, FACS analysis of CD34 CD133 expression was therefore excluded as potential reasons human HSCs and HuH7 cells for CD133 and CD46 expression. Cells were for the enhanced oncolytic activity of MV-141.7. Moreover, stained with the CD133-specific antibody CD133/2-PE (top) or the CD46- specific phycoerythrin (PE)-labeled antibody (bottom). B, fluorescent spreading kinetics through tumor cells did not differ between microscopic pictures of HSC or mixed HuH7/HSC cultures infected with MV-141.7 and MV-NSe and were therefore excluded as caus- MV-NSe (top) or MV-141.7 (bottom) at the indicated MOI, respectively. ative as well. Pictures were taken 72 hours after infection. Scale bar, 100 mm. Alternative explanations must thus be based on the interaction of the particle-exposed scFv with CD133. We have to CD133. Our data show that targeting to the CD133 previously shown that a lentiviral vector pseudotyped with epitopes AC133 or AC141.7 is equally well possible with the measles virus glycoproteins displaying a CD20-specific recombinant measles viruses. While these epitopes have not scFv forms multiple contacts with CD20, thereby triggering been mapped exactly, antibody binding to these epitopes is mitotic activation in resting lymphocytes (44, 45). It is well glycosylation-dependent, and cell differentiation results in conceivable that also MV-141.7 establishes multiple scFv- loss of epitope recognition (37). Both viruses showed very CD133 contacts on the tumor cell surface. Such contacts can þ similar spreading kinetics through CD133 tumor cells but also be formed by noninfectious particles that do usually build did not infect CD133 cells. Their oncolytic activity was the vast majority of particles in virus stocks. This could shown on 3 different tumor entities including primary tumor substantially contribute to the oncolytic activity of MV-141.7 material. provided that this interaction triggers apoptosis in tumor cells.

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Figure 6. Oncolytic activity of MV- 141.7 in a multifocal HuH7 tumor model. Mice carrying intraperitoneally applied HuH7-luc tumor cells were treated with 2 6 10 TCID50 of MV-141.7 (N ¼ 4) or MV-NSe (N ¼ 4), respectively, or injected with OptiMEM (untreated; N ¼ 5). A, images of 2 representative mice from each group taken before (day 5) or after injection (days 19 and 47) of MV- 141.7 (top), MV-NSe (middle), and DMEM (bottom). B, luciferase bioluminescence intensities of tumors infected with MV-141.7 (red), MV-NSe (gray), or control (black) over time. Mice were imaged on the first day after injection (day 1) and subsequently every 3 days. Error bars represent mean 95% confidence intervals. Arrows indicate time points of virus injection. , P < 0.05, N ¼ 4; n.s., not significant, N ¼ 5. C, survival curves of MV-141.7- (red), MV- NSe- (gray), or control (black)- treated mice. One mouse died accidentally during in vivo imaging.

However, such properties have not yet been reported for the characterizing HuH7 cells that survived MV-NSe infection for monoclonal antibodies AC133 or AC141.7. Moreover, HSCs CD133 expression and infectability by MV-141.7. were unimpaired in growth and differentiation into all hemato- The enhanced oncolytic activity of MV-141.7 was also evi- poietic lineages when transduced with lentiviral vectors pseu- dent on primary colon cancer cells in which we observed a dotyped with the same 141.7-specific measles virus glycopro- significantly reduced growth of MV-141.7 versus MV-NSe on teins as used here on oncolytic measles viruses (21). It is the initially infected tumor cells, although both viruses elim- therefore rather unlikely that CD133-scFv contact enhanced inated all spheres in long-term cultures. We did, however, not the oncolytic activity of MV-141.7. detect any effect of MV-141.7 on secondary transplants. While þ In an alternative scenario, MV-141.7 infects a cell population formally it cannot be excluded that in vivo not all CD133 cells that is protected from infection by MV-NSe but highly relevant have been eliminated by the virus, it is more likely that similarly for tumor growth. Friedman and colleagues compared the as in glioblastoma (7), CD133 TSCs were among the colon þ oncolytic activity of herpes virus on glioblastoma-derived cancer cells of this patient, which gave rise to CD133 cells. þ CD133 and CD133 cells (46). They observed a substantially This is in line with previous publications showing that colon þ reduced sensitivity of CD133 glioma cells toward virus treat- cancer–derived CD133 cells can possess tumor-initiating ment as compared with CD133 cells, which correlated with a properties (30, 47). A final conclusion on this issue will require reduced CD111 (nectin-1) expression, a cell entry receptor for testing of many more patient samples, which is now possible HSV. Transferred to oncolytic measles virus, this suggests with the approach described here. þ downmodulation of CD46 expression in some CD133 TICs. MV-141.7 and MV-AC133 are to our knowledge the first Future work will have to test both hypotheses by, for example, oncolytic viruses rationally designed to target putative TICs or

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Targeting CD133þ Tumor Cells

CSC. Their primary application will be in basic research Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Bach, T. Abel, C. Hoffmann, Z. Gal, G. Braun, C.R. addressing the relevance of CD133 as marker for TICs. They Ball, H. Glimm will allow a systematic investigation on how tumors react to Analysis and interpretation of data (e.g., statistical analysis, biostatistics, þ the selective elimination of CD133 cells. Importantly, this will computational analysis): P. Bach, T. Abel, C. Hoffmann, I. Voelker, C.R. Ball, C. Herold-Mende, M.D. Muhlebach,€ H. Glimm, C.J. Buchholz be possible not only in colon cancer but basically in any cancer Writing, review, and/or revision of the manuscript: P. Bach, T. Abel, C. entity for which CD133 has been suggested as marker of TICs Hoffmann, Z. Gal, I. Voelker, I.C.D. Johnston, U.M. Lauer, C. Herold-Mende, M.D. Muhlebach,€ H. Glimm, C.J. Buchholz and/or to be of prognostic value. Moreover, the same approach Administrative, technical, or material support (i.e., reporting or orga- can be used to determine the relevance of other putative TIC nizing data, constructing databases): I. Voelker markers, such as CD44, EpCAM, or CD13 (48–50) by generating Study supervision: C. Herold-Mende, C.J. Buchholz oncolytic measles viruses retargeted to these markers. Beyond that, MV-141.7 also warrants further investigation as Acknowledgments a highly target specific virotherapeutic agent for cancer ther- The authors thank Ulrike Kohl€ (University Hospital, Frankfurt) for providing apheresis material, Kay-Martin Hanschmann (Paul-Ehrlich-Institut) for help apy. The antitumoral effect we observed in the multifocal with statistics, and Martin Selbert (Paul-Ehrlich-Institut) for help with cell tumor model of HuH7 with MV-141.7 was remarkable. MV- sorting by flow cytometry. 141.7 did not only prevent further tumor spreading through the peritoneum but even cleared 2 animals from tumor cells. Grant Support fi Future work will have to de ne the optimal therapeutic setting This work was supported by grants 01GU0504 (to C.J. Buchholz) and for clinical studies on MV-141.7. 01GU0503 (U.M. Lauer) of the Federal ministry for Education and Research of Germany (BMBF), by the LOEWE Center for Cell and Gene Therapy Frankfurt € fl funded by Hessisches Ministerium fur Wissenschaft und Kunst [III L 4-518/ Disclosure of Potential Con icts of Interest 17.004 (2010)] to C.J. Buchholz, the Deutsche Forschungsgemeinschaft to H. I.C.D. Johnston is an employee of Miltenyi Biotec GmbH. No potential Glimm (KFO 227) and C.J. Buchholz (BU 1301/3-1), and by contract research fl con icts of interest were disclosed by the other authors. "Adulte Stammzellen" of the Baden-Wurttemberg€ Stiftung (H. Glimm). The costs of publication of this article were defrayed in part by the payment of Authors' Contributions page charges. This article must therefore be hereby marked advertisement in Conception and design: P. Bach, T. Abel, C. Herold-Mende, M.D. Muhlebach,€ accordance with 18 U.S.C. Section 1734 solely to indicate this fact. H. Glimm, C.J. Buchholz Development of methodology: P. Bach, T. Abel, C. Hoffmann, Z. Gal, I.C.D. Received June 6, 2012; revised November 5, 2012; accepted November 7, 2012; Johnston, C. Herold-Mende, M.D. Muhlebach€ published OnlineFirst January 4, 2013.

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874 Cancer Res; 73(2) January 15, 2013 Cancer Research

Specific Elimination of CD133+ Tumor Cells with Targeted Oncolytic Measles Virus

Patricia Bach, Tobias Abel, Christopher Hoffmann, et al.

Cancer Res 2013;73:865-874. Published OnlineFirst January 4, 2013.

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