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• W2431416257 abstract "To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass. To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass. In 2012, breast and ovarian carcinoma accounted for 25.2 and 3.6% of worldwide 3.5 million cancer mortalities in women, respectively.1Ferlay J Soerjomataram I Ervik M Dikshit R Eser S Mathers C et al.GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11. GLOBOCAN, 2013http://globocan.iarc.frGoogle Scholar Especially aggressive forms of both tumor entities are characterized by some shared markers. Among these are the epithelial cell adhesion molecule (EpCAM) and the human epidermal growth factor receptor 2 (HER2/neu, or short HER2). Targeting of the tumor-associated antigen HER2 by monoclonal antibodies has been a showcase for the development of targeted cancer medicine, which has first been authorized for the treatment of metastatic breast cancer.2Goldenberg MM Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer.Clin Ther. 1999; 21: 309-318Abstract Full Text PDF PubMed Scopus (377) Google Scholar HER2, a receptor tyrosine kinase, is amplified in 15–30% of all breast3Eroles P Bosch A Pérez-Fidalgo JA Lluch A Molecular biology in breast cancer: intrinsic subtypes and signaling pathways.Cancer Treat Rev. 2012; 38: 698-707Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar,4Slamon DJ Clark GM Wong SG Levin WJ Ullrich A McGuire WL Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235: 177-182Crossref PubMed Scopus (10014) Google Scholar and 9–32% of ovarian cancer patients.5Meden H Kuhn W Overexpression of the oncogene c-erbB-2 (HER2/neu) in ovarian cancer: a new prognostic factor.Eur J Obstet Gynecol Reprod Biol. 1997; 71: 173-179Abstract Full Text PDF PubMed Scopus (109) Google Scholar This cellular surface glycoprotein is an active “driver” oncogene, which has no known ligand, and its expression is clinically associated with poor prognosis. HER2-targeted therapies radically changed the way HER2-overexpressing tumors are treated, but a significant number of patients with recurrent, HER2-positive breast cancer do not respond to antibody therapy with or without chemotherapy (only 25–50% response6Vu T Sliwkowski MX Claret FX Personalized drug combinations to overcome trastuzumab resistance in HER2-positive breast cancer.Biochim Biophys Acta. 2014; 1846: 353-365PubMed Google Scholar). For ovarian carcinoma, numbers are even worse.7Yap TA Carden CP Kaye SB Beyond chemotherapy: targeted therapies in ovarian cancer.Nat Rev Cancer. 2009; 9: 167-181Crossref PubMed Scopus (413) Google Scholar Moreover, even responsive tumors eventually become resistant. Reasons for the disappointing durability of response to HER2-targeted therapies in advanced breast and ovarian carcinoma are complex, but can be partially explained by acquired resistance or a priori heterogeneity in tumor cell populations.8Nahta R Yu D Hung MC Hortobagyi GN Esteva FJ Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer.Nat Clin Pract Oncol. 2006; 3: 269-280Crossref PubMed Scopus (724) Google Scholar High mutation rates and heterogeneity of tumor cells are a general hallmark of cancer. Consequently, cell populations of advanced tumors are inherently diverse, also with regard to expressed markers, and can acquire escape mutations. Moreover, the stem cell theory of cancer proposes that among all cancerous cells within a tumor, a few act as stem cells that reproduce themselves and sustain the tumor. In this view, it is especially these cancer stem cells (CSCs) that need to be targeted by a successful therapy in order to prevent the tumor from recurrence and becoming therapy resistant. Among the markers that identify potential CSCs9Jordan CT Guzman ML Noble M Cancer stem cells.N Engl J Med. 2006; 355: 1253-1261Crossref PubMed Scopus (1351) Google Scholar,10Meacham CE Morrison SJ Tumour heterogeneity and cancer cell plasticity.Nature. 2013; 501: 328-337Crossref PubMed Scopus (1618) Google Scholar is EpCAM.11Munz M Baeuerle PA Gires O The emerging role of EpCAM in cancer and stem cell signaling.Cancer Res. 2009; 69: 5627-5629Crossref PubMed Scopus (429) Google Scholar, 12Visvader JE Lindeman GJ Cancer stem cells in solid tumours: accumulating evidence and unresolved questions.Nat Rev Cancer. 2008; 8: 755-768Crossref PubMed Scopus (2837) Google Scholar, 13Gires O Klein CA Baeuerle PA On the abundance of EpCAM on cancer stem cells.Nat Rev Cancer. 2009; 9 (author reply 143): 143Crossref PubMed Scopus (109) Google Scholar It is often upregulated in epithelial tumors and is in general better accessible for therapeutics here, since in the course of transformation the polarity of EpCAM expression at tight junctions is lost and EpCAM molecules are homogenously distributed on the cancer cell surface.11Munz M Baeuerle PA Gires O The emerging role of EpCAM in cancer and stem cell signaling.Cancer Res. 2009; 69: 5627-5629Crossref PubMed Scopus (429) Google Scholar Interestingly, EpCAM is present at low levels in 48%, and overexpressed in approximately 35–42% of all breast tumor patients samples.14Gastl G Spizzo G Obrist P Dünser M Mikuz G Ep-CAM overexpression in breast cancer as a predictor of survival.Lancet. 2000; 356: 1981-1982Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 15Spizzo G Obrist P Ensinger C Theurl I Dünser M Ramoni A et al.Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer.Int J Cancer. 2002; 98: 883-888Crossref PubMed Scopus (89) Google Scholar, 16Spizzo G Went P Dirnhofer S Obrist P Simon R Spichtin H et al.High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer.Breast Cancer Res Treat. 2004; 86: 207-213Crossref PubMed Scopus (208) Google Scholar In ovarian cancer, EpCAM-expression is upregulated even in 69% of all patients.17Spizzo G Went P Dirnhofer S Obrist P Moch H Baeuerle PA et al.Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer.Gynecol Oncol. 2006; 103: 483-488Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar Coexpression of HER2 and EpCAM occurs in a significant number of breast cancer patients (13.2%) and further worsens prognosis.15Spizzo G Obrist P Ensinger C Theurl I Dünser M Ramoni A et al.Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer.Int J Cancer. 2002; 98: 883-888Crossref PubMed Scopus (89) Google Scholar Thus, the treatment of this particular subtype of carcinomas could be improved by combination of HER2- and EpCAM-targeted therapies. The potential for tumor escape might be reduced when therapy is combined in one setting or even a single drug, an approach that can be realized by an oncolytic virus (OV) therapy. OVs are novel cancer therapeutics and are intensively studied in preclinical and clinical studies. Most recently, an oncolytic herpesvirus named talimogene laherparepvec (Imlygic®) has received a positive recommendation for marketing authorization by the European Medicines Agency and the Federal Drug Agency for the treatment of melanoma.18Greig SL Talimogene Laherparepvec: first global approval.Drugs. 2016; 76: 147-154Crossref PubMed Scopus (85) Google Scholar Notably, being replication competent viruses, OVs have a completely different mode of action than conventional drugs.19Russell SJ Peng KW Bell JC Oncolytic virotherapy.Nat Biotechnol. 2012; 30: 658-670Crossref PubMed Scopus (996) Google Scholar,20Miest TS Cattaneo R New viruses for cancer therapy: meeting clinical needs.Nat Rev Microbiol. 2014; 12: 23-34Crossref PubMed Scopus (197) Google Scholar Due to the strongly lytic nature of its replication, measles virus (MV) appears as an ideal virus for use as OV with currently ongoing phase 1 trials for the treatment of six different tumor entities and a phase 2 study for multiple myeloma, which is recruiting patients.21US National Institutes of Health Keywords: Measles Virus & Tumor.http://www.clinicaltrials.govGoogle Scholar In general, MV is well tolerated and one patient with advanced multiple myeloma went into full remission after high-dose MV treatment, with flu-like symptoms during infusion reported as most striking side effects.22Russell SJ Federspiel MJ Peng KW Tong C Dingli D Morice WG et al.Remission of disseminated cancer after systemic oncolytic virotherapy.Mayo Clin Proc. 2014; 89: 926-933Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar MV is the prototypic Morbillivirus and belongs to the Paramyxoviridae family. It is the causative agent of the measles, but live attenuated vaccine strains have been developed, which are among the most efficient and safest vaccines known.23Moss WJ Griffin DE Global measles elimination.Nat Rev Microbiol. 2006; 4: 900-908Crossref PubMed Scopus (189) Google Scholar MV vaccine strains use CD46 as entry receptor24Dörig RE Marcil A Chopra A Richardson CD The human CD46 molecule is a receptor for measles virus (Edmonston strain).Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (878) Google Scholar,25Naniche D Varior-Krishnan G Cervoni F Wild TF Rossi B Rabourdin-Combe C et al.Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus.J Virol. 1993; 67: 6025-6032Crossref PubMed Google Scholar in addition to the receptors used by pathogenic wildtype MV strains: SLAM on activated lymphocytes,26Tatsuo H Ono N Tanaka K Yanagi Y SLAM (CDw150) is a cellular receptor for measles virus.Nature. 2000; 406: 893-897Crossref PubMed Scopus (863) Google Scholar or nectin-4 on epithelial cells lining the upper airways.27Noyce RS Bondre DG Ha MN Lin LT Sisson G Tsao MS et al.Tumor cell marker PVRL4 (nectin 4) is an epithelial cell receptor for measles virus.PLoS Pathog. 2011; 7: e1002240Crossref PubMed Scopus (377) Google Scholar,28Mühlebach MD Mateo M Sinn PL Prüfer S Uhlig KM Leonard VH et al.Adherens junction protein nectin-4 is the epithelial receptor for measles virus.Nature. 2011; 480: 530-533Crossref PubMed Scopus (430) Google Scholar CD46 is expressed on all human nucleated cells, but regularly found upregulated in certain tumors.29Fishelson Z Donin N Zell S Schultz S Kirschfink M Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors.Mol Immunol. 2003; 40: 109-123Crossref PubMed Scopus (329) Google Scholar Accordingly, the tumor tropism of unmodified oncolytic MV derived from the Edmonston B vaccine strain has been correlated to CD46 upregulation.30Anderson BD Nakamura T Russell SJ Peng KW High CD46 receptor density determines preferential killing of tumor cells by oncolytic measles virus.Cancer Res. 2004; 64: 4919-4926Crossref PubMed Scopus (254) Google Scholar However, this tropism is only relative, since the CD46 is ubiquitously expressed on human cells. On the virus surface, the viral glycoprotein hemagglutinin (H) is responsible for receptor attachment followed by triggering cell-entry.31Navaratnarajah CK Oezguen N Rupp L Kay L Leonard VH Braun W et al.The heads of the measles virus attachment protein move to transmit the fusion-triggering signal.Nat Struct Mol Biol. 2011; 18: 128-134Crossref PubMed Scopus (88) Google Scholar MVs’ receptor usage can be altered by changing the binding specificity of H. This can be achieved by introducing four specific point mutations to ablate recognition of the native receptors32Vongpunsawad S Oezgun N Braun W Cattaneo R Selectively receptor-blind measles viruses: Identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model.J Virol. 2004; 78: 302-313Crossref PubMed Scopus (169) Google Scholar and the genetic fusion of binding domains specific for the desired target receptor to the C-terminus of H. Usually, these have been single-chain antibody fragments (scFv’s),33Nakamura T Peng KW Harvey M Greiner S Lorimer IA James CD et al.Rescue and propagation of fully retargeted oncolytic measles viruses.Nat Biotechnol. 2005; 23: 209-214Crossref PubMed Scopus (224) Google Scholar natural receptor ligands34Schneider U Bullough F Vongpunsawad S Russell SJ Cattaneo R Recombinant measles viruses efficiently entering cells through targeted receptors.J Virol. 2000; 74: 9928-9936Crossref PubMed Scopus (103) Google Scholar or peptides.35Hallak LK Merchan JR Storgard CM Loftus JC Russell SJ Targeted measles virus vector displaying echistatin infects endothelial cells via alpha(v)beta3 and leads to tumor regression.Cancer Res. 2005; 65: 5292-5300Crossref PubMed Scopus (56) Google Scholar Thereby, MV tropism can be redirected to virtually any surface-exposed structure of choice.33Nakamura T Peng KW Harvey M Greiner S Lorimer IA James CD et al.Rescue and propagation of fully retargeted oncolytic measles viruses.Nat Biotechnol. 2005; 23: 209-214Crossref PubMed Scopus (224) Google Scholar As an alternative binding domain, we recently developed a strategy36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar using designed ankyrin repeat proteins (DARPins)37Plückthun A Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy.Annu Rev Pharmacol Toxicol. 2015; 55: 489-511Crossref PubMed Scopus (366) Google Scholar to target tumor markers HER2 (ref. 38Zahnd C Wyler E Schwenk JM Steiner D Lawrence MC McKern NM et al.A designed ankyrin repeat protein evolved to picomolar affinity to Her2.J Mol Biol. 2007; 369: 1015-1028Crossref PubMed Scopus (199) Google Scholar), EpCAM,39Stefan N Martin-Killias P Wyss-Stoeckle S Honegger A Zangemeister-Wittke U Plückthun A DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency.J Mol Biol. 2011; 413: 826-843Crossref PubMed Scopus (101) Google Scholar or EGFR.40Steiner D Forrer P Plückthun A Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display.J Mol Biol. 2008; 382: 1211-1227Crossref PubMed Scopus (212) Google Scholar The unique structural properties of DARPins enabled us to generate a bispecific MV using HER2 and/or EpCAM, as entry receptors.36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar This virus was generated to address resistance development to mono-targeted therapies, but its qualities have only been partially characterized.36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Here, we aim to demonstrate the advantage of bispecific compared to monospecific MVs and to optimize bispecific targeting by variation of the linker connecting both DARPins. All bispecific binding cassettes were demonstrated to bind to cells expressing HER2/neu or EpCAM, albeit to different extent. Linkers composed of nine proline residues or constituting two α-helical turns were identified to convey best fusion helper function in vitro. Viruses generated with these linkers as well as the prototype virus MV-Ec4-G3 (ref. 36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) specifically infected cells via EpCAM or HER2. The viruses were found to be cytotoxic for two human carcinoma cell lines, while only one of these replicated the virus efficiently. Their oncolytic potential was shown in a disseminated ovarian cancer xenograft model in mice. Furthermore, the superior efficacy of bispecific over monospecific viruses was demonstrated in a cell culture model mimicking tumor heterogeneity, thus providing evidence for the versatility and efficacy of such viruses for the therapy of advanced tumors. With the final aim to further enhance the oncolytic efficacy of MV by targeting two receptors of choice simultaneously, new bispecific binding cassettes were constructed by exchanging the linker peptide connecting both DARPin domains (Figure 1a). The prototype virus encoded a glycine-serine linker ((G4S)2) between DARPins Ec4 (binding EpCAM with a KD of 1.7 nmol/l) and G3 (binding HER2 with a KD of 0.09 nmol/l), genetically fused in tandem to the carboxy-terminus of the mutated H protein.36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar The same orientation of DARPins was used for testing different linkers, since for initial experiments using H-DARPin-linker-DARPin proteins with the prototypic ((G4S)2-linker, the order of the DARPins did not matter with respect to fusion helper-activity (data not shown). Three different types of linkers were used: highly flexible glycine-serine linkers, more rigid poly-proline linkers, and α-helical peptide linkers, which however can unfold. The linkers were varied in length covering between 5 (G4S) and 20 amino acids (aa) ((G4S)4), 9 aa (two full helical turns; αHelix2) and 17 aa (4 turns; αHelix4), or 9 and 18 aa of consecutive prolines. Furthermore, simple glycine-serine hinges (hi) were added to both ends of the rigid linkers to increase flexibility (Figure 1a). The respective genes encoding the different H-DARPin-linker-DARPin constructs were first cloned into pCG-1-derived expression plasmids. After transfection of these plasmids into 293T cells, all recombinant H-proteins were detected by immunoblot analysis (Figure 1b). All proteins revealed the expected shift in molecular weight, compared to unmodified H, of approximately 30 kDa, consistent with the size of two connected DARPin units. No proteins with lower molecular weight became evident suggesting that there was no loss of the distal DARPin due to linker proteolysis. Furthermore, all proteins were successfully transported to the cell surface of transfected 293T cells, as demonstrated by flow cytometry detecting the C-terminal surface-exposed His6-tag (Supplementary Figure S1). To analyze functionality, i.e., fusion helper function, the H protein variants were transiently coexpressed with the MV fusion protein (F) in CHO-HER2-K6 or CHO-EpCAM #6 cells with defined receptor density.36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar After binding its receptor, H initiates fusion of neighboring receptor-positive cells by triggering F to fuse the cell membranes, resulting in syncytia, i.e., multi-nucleated giant cells. Compared to the monospecific H-Ec4 or H-G3, the best fusion activity of the evaluated bispecific formats, indicated by formation of syncytia, was observed with medium sized linkers and regardless of their flexibility: (G4S)2 (10 aa), αHelix2 (9 aa), or Pro9 (9 aa). After binding to HER2, these proteins revealed almost completely conserved fusion helper function of between 80% (αHelix2 & Pro9) and 90% ((G4S)2) as compared to monospecific H-G3 (Figure 1c). After binding to EpCAM, fusion helper function of bispecific constructs ranged from 40% ((G4S)2) to 60% (αHelix2 & Pro9) compared to H-Ec4. It should be noted that the anti-EpCAM DARPin is placed between the H-protein and the anti-HER2 DARPin, and may thus be more sterically constrained than the latter, shown in lower percentages compared to Ec4 itself. Longer linker peptides or additional hinge regions impaired function, as well as shorter linker peptides such as Gly-Ser (X/B; introduced XmaI/BstBI restriction sites) or a Gly4-Ser sequence. Hence, the linker variants αHelix2 and Pro9 were chosen as alternatives to the prototype (G4S)2-linker for the generation of bispecific MVs. For additional characterization, soluble versions of the bispecific binding domains were generated, which were fused to the N-terminus of human IgG1-Fc-portion. The Fc-part induces homo-dimerization and thus potentially results in bivalent binding of the DARPin-Fc complexes. Recombinant DARPin-Fc proteins were expressed in 293T cells, purified (Supplementary Figure S2), and tested for binding to cell lines differing in HER2/neu or EpCAM surface expression (Table 1) by flow cytometry (Figure 1d, Supplementary Figure S3). Most interestingly, Fc-dimerized (G4S)2- and αHelix2-linked proteins bound either HER2- or EpCAM-positive target cells with comparable efficacy also to monospecific DARPin-Fc-proteins. These comparably constructed proteins demonstrated unimpaired binding capacity of the DARPin units when fused to the Fc-portion. The Pro9-connected soluble bispecific cassette bound both HER2 and EpCAM-expressing cells significantly less efficiently indicating a negative impact of the Pro9 linker in the binding efficiencies of both DARPins, as discussed below.Table 1Surface density of targeted receptors as determined previouslyData taken from ref. 36Friedrich K Hanauer JR Prüfer S Münch RC Völker I Filippis C et al.DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.Mol Ther. 2013; 21: 849-859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar.Cell lineHER2/cellaNumber of respective HER2/neu, EpCAM, or CD46 molecules per cell.EpCAM/cellaNumber of respective HER2/neu, EpCAM, or CD46 molecules per cell.CD46/cellaNumber of respective HER2/neu, EpCAM, or CD46 molecules per cell.SK-OV-35.72 × 1054.59 × 1039.10 × 104Caco-21.81 × 1041.01 × 1051.77 × 105LN-3084.76 × 103−bBackground level as determined by isotype control.6.30 × 104MCF-71.24 × 1049.57 × 1047.82 × 104a Number of respective HER2/neu, EpCAM, or CD46 molecules per cell.b Background level as determined by isotype control. Open table in a new tab After extension of the carboxy-terminal His6-tag by one extra histidine to coincide with the rule-of-six,41Calain P Roux L The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.J Virol. 1993; 67: 4822-4830Crossref PubMed Google Scholar the targeted H-ORFs were cloned into the genome of attenuated Edmonston MV strain NSe (MVNSe) encoding eGFP as reporter protein (Figure 2a). All respective viruses were successfully rescued. These recombinant MVs were amplified on Vero-αHis cells and subsequently analyzed by immunoblot for H protein expression in infected cells (Figure 2b). Both MV-Ec4-Pro9-G3 and MV-Ec4-αHelix2-G3 showed expression of the recombinant H proteins with an approximately 30 kDa increase in molecular weight, as expected (Figure 2b). In multi-step growth curves on Vero-αHis cells, similar maximal cell-associated titers around 1 × 105 TCID50/ml were reached 48 hours after infection by all MVs (Figure 2c). As expected, the replication kinetics of the bispecific constructs closely reflected those seen for monospecific MV-Ec4: Virus titers did not drop after reaching their maximum, but stayed at a plateau until 6 days post infection (Figure 2c). Titers of all recombinant MV in the supernatant of infected Vero-αHis revealed a similar pattern, but were reduced by half an order of magnitude and delayed by 24 hours (Figure 2d). The receptor tropism of retargeted bispecific MVs was assessed by infecting a panel of transgenic CHO cell lines expressing either one of the native MV vaccine strain receptors (CD46, hSLAM, or nectin 4), or the targeted receptors (HER2 or EpCAM). As expected, neither CHO-K1 cells (not expressing any putative receptor) nor the CHO lines expressing one of the native MV receptors were infected by any retargeted virus, demonstrating successful detargeting from natural MV receptors. While monospecific MVs infected either CHO-HER2 or CHO-EpCAM cells according to their respective specificity, bispecific MVs were able to infect both (Figure 3). Thus, target specificity was retained irrespective of the peptide linkers connecting both targeting domains of the bispecific MVs. Next, the oncolytic properties of the bispecific MVs were characterized using the human carcinoma cell lines SK-OV-3 (ovarian) and Caco-2 (colorectal), expressing different levels of HER2 or EpCAM (Table 1, Figure 4). First, cytotoxicity of the bispecific MVs was assessed by monitoring cell viability in MTT-assays in comparison to nontargeted or monospecific MV. Both tumor cell lines revealed a rapid decline in viability within the first 72 hours after infection with any of the viruses, except Caco-2 infected with MV-G3. This virus exhibited only moderate cytotoxicity in this setting (71.7% viability 72 hours after infection, which declined to 28.7% 168 hours after infection). However, MV-G3 was more cytotoxic for SK-OV-3 cells than MV-Ec4 (27.7 versus 44.7% viability 72 hours after infection), which is likely explained by the 100-fold higher density of HER2 versus EpCAM in these cells. Interestingly, all bispecific viruses were at least as cytotoxic as MV-G3 on either cell line. In comparison to the nontargeted parental virus MVNSe, whose efficiency can be explained by the robust overexpression of its receptor CD46 on both cell lines (Table 1), MV-Ec4-Pro9-G3 even reached comparable cytotoxicity (15.3% versus 9.5% residual viability of infected SK-OV-3 72 after infection) (Figure 4a). In Caco-2 cells, residual viability dropped to almost undetectable levels 168 hours after infection with any bispecific virus (Figure 4b). Thus, bispecific MVs demonstrated high oncolytic efficacy in vitro irrespective of the targeted receptor. Surprisingly, analysis of viral replication on both target cell lines revealed a different picture. On SK-OV-3 cells, cell-associated virus titers peaked 48 hours (MVNSe) or 72 hours after infection (all targeted MVs). MVNSe and monospecific MV-Ec4 reached maximal cell-associated titers of 8.2 ± 2.7 × 103 TCID50/ml and 0.7 ± 1.0 × 104 TCID50/ml, respectively. Ninety-six hours after infection, titers of both viruses stabilized at around 1 × 103 TCID50/ml. Peak titers of MV-G3 were reduced (2.7 ± 2.1 × 103 TCID50/ml) and dropped below the limit of detection (1 × 102 TCID50/ml) 96 hours after infection. Titers of the bispecific viruses were only slightly above the detection limit on SKOV-3 cells (Figure 4c), but within the range of the monos" @default.
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• W2431416257 title "Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2 /neu or EpCAM as target receptors" @default.
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• W2431416257 doi "https://doi.org/10.1038/mto.2016.3" @default.
• W2431416257 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4824561" @default.
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