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  • 1
    Keywords: PHASE-I ; THERAPY ; antibody ; B-CELL LYMPHOMA ; MELANOMA-CELLS ; SOLID TUMORS ; cancer therapeutics ; immunotoxin ; REPLICATION-DEFICIENT ; RIBONUCLEASES
    Abstract: Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONCEGFR ) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONCEGFR expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONCEGFR induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONCEGFR -encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse tumor xenograft model. The latter demonstrates that ONCEGFR is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONCEGFR -encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof. (c) 2014 Wiley Periodicals, Inc.
    Type of Publication: Journal article published
    PubMed ID: 25303768
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  • 2
    Keywords: IN-VIVO ; lentiviral vectors ; INTRACELLULAR TRAFFICKING ; RECEPTOR INTERACTION ; ANTITUMOR EFFICACY ; MEASLES-VIRUS ; NEUTRALIZING ANTIBODIES TARGET ; FIBER KNOB DOMAIN ; TUMOR TRANSDUCTION ; SEROTYPE-5 VECTOR
    Abstract: Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies.
    Type of Publication: Journal article published
    PubMed ID: 22633438
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  • 3
    Keywords: EXPRESSION ; IN-VIVO ; THERAPY ; RNA ; VECTORS ; RECOMBINANT ADENOASSOCIATED VIRUS ; VIROTHERAPY ; PROGRESS ; cancer therapeutics ; SELF-CLEAVAGE
    Abstract: Therapeutic gene transfer by replication-defective viral vectors or, for cancer treatment, by replication-competent oncolytic viruses shows high promise for treatment of major diseases. To ensure safety, timing or dosing in patients, external control of therapeutic gene expression is desirable or even required. In this study, we explored the potential of artificial aptazymes, ligand-dependent self-cleaving ribozymes, as an innovative tool for regulation of therapeutic gene expression. Importantly, aptazymes act on RNA intrinsically, independent of regulatory protein-nucleic acid interactions and stoichiometry, are non-immunogenic and of small size. These are key advantages compared with the widely used inducible promoters, which were also reported to lose regulation at high copy numbers, e.g. after replication of oncolytic viruses. We characterized aptazymes in therapeutic gene transfer utilizing adenovectors (AdVs), adeno-associated vectors (AAVs) and oncolytic adenoviruses (OAds), which are all in advanced clinical testing. Our results show similar aptazyme-mediated regulation of gene expression by plasmids, AdVs, AAVs and OAds. Insertion into the 5'-, 3'- or both untranslated regions of several transgenes resulted in ligand-responsive gene expression. Notably, aptazyme regulation was retained during OAd replication and spread. In conclusion, our study demonstrates the fidelity of aptazymes in viral vectors and oncolytic viruses and highlights the potency of riboswitches for medical applications.
    Type of Publication: Journal article published
    PubMed ID: 22885302
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  • 4
    Keywords: IN-VIVO ; THERAPY ; GENE-EXPRESSION ; INFECTION ; DELETION ; IDENTIFICATION ; VECTORS ; POSTTRANSCRIPTIONAL REGULATION ; TRANSGENE EXPRESSION ; VIRUS-ASSOCIATED RNA
    Abstract: RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.
    Type of Publication: Journal article published
    PubMed ID: 23455436
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  • 5
    Keywords: EXPRESSION ; COMBINATION ; antibody ; MELANOMA ; SAFETY ; CANCER-IMMUNOTHERAPY ; RESCUE ; IPILIMUMAB ; CHEMOVIROTHERAPY
    Abstract: We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (〉 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application.Molecular Therapy (2014); doi:10.1038/mt.2014.160.
    Type of Publication: Journal article published
    PubMed ID: 25156126
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  • 6
    Keywords: RECEPTOR ; CELLS ; SURVIVAL ; OVARIAN-CANCER ; T-LYMPHOCYTES ; CUTANEOUS MELANOMA ; GENE-THERAPY ; COLONY-STIMULATING FACTOR ; ANTITUMOR ; CHEMOTHERAPY-REFRACTORY CANCER
    Abstract: Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.
    Type of Publication: Journal article published
    PubMed ID: 25821063
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  • 7
    Keywords: EXPRESSION ; IN-VIVO ; THERAPY ; GENE ; GENE-EXPRESSION ; GENES ; GENOME ; PROTEIN ; DNA ; INFECTION ; cell cycle ; CELL-CYCLE ; IDENTIFICATION ; MICROARRAY DATA ; VECTOR ; CANCER-CELLS ; ORIGIN ; COMPETENT ADENOVIRUS ; E1A
    Abstract: Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication
    Type of Publication: Journal article published
    PubMed ID: 22140489
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  • 8
    Keywords: APOPTOSIS ; EXPRESSION ; INFECTION ; mechanisms ; VECTORS ; CANCER-CELLS ; REPLICATION ; GENE-THERAPY ; ADENOASSOCIATED VIRUS ; AUTONOMOUS PARVOVIRUSES
    Abstract: In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.
    Type of Publication: Journal article published
    PubMed ID: 22787235
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  • 9
    Keywords: GROWTH ; FACTOR-I RECEPTOR ; UP-REGULATION ; TUMOR PROGRESSION ; p53 ; HUMAN-MELANOMA CELLS ; p73 ; CLINICAL-IMPLICATIONS ; EPITHELIAL-MESENCHYMAL TRANSITION ; CANCER-CELL INVASION
    Abstract: Dissemination of cancer cells from primary tumors is the key event in metastasis, but specific determinants are widely unknown. Here, we show that DNp73, an inhibitor of the p53 tumor suppressor family, drives migration and invasion of nonmetastatic melanoma cells. Knockdown of endogenous DNp73 reduces this behavior in highly metastatic cell lines. Tumor xenografts expressing DNp73 show a higher ability to invade and metastasize, while growth remains unaffected. DNp73 facilitates an EMT-like phenotype with loss of E-cadherin and Slug upregulation. We provide mechanistic insight toward regulation of LIMA1/EPLIN by p73/DNp73 and demonstrate a direct link between the DNp73-EPLIN axis and IGF1R-AKT/STAT3 activation. These findings establish initiation of the invasion-metastasis cascade via EPLIN-dependent IGF1R regulation as major activity of DNp73.
    Type of Publication: Journal article published
    PubMed ID: 24135282
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  • 10
    Keywords: IN-VIVO ; INHIBITION ; THERAPY ; transcription ; ATTENUATION ; ADENOVIRUS ; INTERFERENCE ; MEASLES-VIRUS ; REGULATORY SYSTEMS ; RIBOZYMES
    Abstract: Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule-triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses.
    Type of Publication: Journal article published
    PubMed ID: 24449891
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