@article{WeibelBasseLuesebrinkHessetal.2013, author = {Weibel, Stephanie and Basse-Luesebrink, Thomas Christian and Hess, Michael and Hofmann, Elisabeth and Seubert, Carolin and Langbein-Laugwitz, Johanna and Gentschev, Ivaylo and Sturm, Volker J{\"o}rg Friedrich and Ye, Yuxiang and Kampf, Thomas and Jakob, Peter Michael and Szalay, Aladar A.}, title = {Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI)}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0056317}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130311}, pages = {e56317}, year = {2013}, abstract = {Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.}, language = {en} } @article{WangChenMinevetal.2013, author = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Zimmermann, Martina and Aguilar, Richard J. and Zhang, Qian and Sturm, Julia B. and Fend, Falko and Yu, Yong A. and Cappello, Joseph and Lauer, Ulrich M. and Szalay, Aladar A.}, title = {Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {9}, doi = {10.1371/journal.pone.0071105}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130059}, pages = {e71105}, year = {2013}, abstract = {Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.}, language = {en} } @article{WangChenMinevetal.2012, author = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Szalay, Aladar A.}, title = {Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells}, series = {Journal of Translational Medicine}, volume = {10}, journal = {Journal of Translational Medicine}, number = {167}, doi = {10.1186/1479-5876-10-167}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130019}, year = {2012}, abstract = {Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.}, language = {en} } @article{TsonevaStritzkerBedenketal.2015, author = {Tsoneva, Desislava and Stritzker, Jochen and Bedenk, Kristina and Zhang, Qian and Cappello, Joseph and Fischer, Utz and Szalay, Aladar A.}, title = {Drug-encoded Biomarkers for Monitoring Biological Therapies}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {9}, doi = {10.1371/journal.pone.0137573}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125265}, pages = {e0137573}, year = {2015}, abstract = {Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.}, language = {en} } @article{TsonevaMinevFrentzenetal.2017, author = {Tsoneva, Desislava and Minev, Boris and Frentzen, Alexa and Zhang, Qian and Wege, Anja K. and Szalay, Aladar A.}, title = {Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis}, series = {Molecular Therapy Oncolytics}, volume = {5}, journal = {Molecular Therapy Oncolytics}, doi = {10.1016/j.omto.2017.03.001}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170786}, pages = {41-61}, year = {2017}, abstract = {Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb's in vitro binding ability and functionality in cell culture, beside the significant increase of CD56\(^{bright}\) NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded β-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our "patient-like" humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.}, language = {en} } @article{SzalayWeibelHofmannetal.2013, author = {Szalay, Aladar A and Weibel, Stephanie and Hofmann, Elisabeth and Basse-Luesebrink, Thomas Christian and Donat, Ulrike and Seubert, Carolin and Adelfinger, Marion and Gnamlin, Prisca and Kober, Christina and Frentzen, Alexa and Gentschev, Ivaylo and Jakob, Peter Michael}, title = {Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer}, series = {Journal of Translational Medicine}, journal = {Journal of Translational Medicine}, doi = {doi:10.1186/1479-5876-11-106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96016}, year = {2013}, abstract = {Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.}, subject = {Lungenkrebs}, language = {en} } @article{SchaeferWeibelDonatetal.2012, author = {Sch{\"a}fer, Simon and Weibel, Stephanie and Donat, Ulrike and Zhang, Quian and Aguilar, Richard J. and Chen, Nanhai G. and Szalay, Aladar A.}, title = {Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors}, series = {BMC Cancer}, volume = {12}, journal = {BMC Cancer}, number = {366}, doi = {10.1186/1471-2407-12-366}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140800}, year = {2012}, abstract = {Background Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.}, language = {en} } @article{SchaeferWeibelDonatetal.2012, author = {Sch{\"a}fer, Simon and Weibel, Stephanie and Donat, Ulrike and Zhang, Qian and Aguilar, Richard J. and Chen, Nanhai G. and Szalay, Aladar A.}, title = {Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78220}, year = {2012}, abstract = {Background: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.}, subject = {Biochemie}, language = {en} } @article{PatilGentschevAdelfingeretal.2012, author = {Patil, Sandeep S. and Gentschev, Ivaylo and Adelfinger, Marion and Donat, Ulrike and Hess, Michael and Weibel, Stephanie and Nolte, Ingo and Frentzen, Alexa and Szalay, Aladar A.}, title = {Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0047472}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130039}, pages = {e47472}, year = {2012}, abstract = {Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.}, language = {en} } @article{MinevLanderFelleretal.2019, author = {Minev, Boris R. and Lander, Elliot and Feller, John F. and Berman, Mark and Greenwood, Bernadette M. and Minev, Ivelina and Santidrian, Antonio F. and Nguyen, Duong and Draganov, Dobrin and Killinc, Mehmet O. and Vyalkova, Anna and Kesari, Santosh and McClay, Edward and Carabulea, Gabriel and Marincola, Francesco M. and Butterfield, Lisa H. and Szalay, Aladar A.}, title = {First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells}, series = {Journal of Translational Medicine}, volume = {17}, journal = {Journal of Translational Medicine}, doi = {10.1186/s12967-019-2011-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224105}, year = {2019}, abstract = {Background ACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML). Methods Twenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment. Results No serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days—an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients' blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition. Conclusions Treatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility. Trial registration Retrospectively registered (ISRCTN\#10201650) on October 22, 2018.}, language = {en} }