TCR-transgenic T cells and YB-1-based oncolytic virotherapy improve survival in a preclinical Ewing sarcoma xenograft mouse model.

Background: Ewing sarcoma (EwS) is an aggressive and highly metastatic bone and soft tissue tumor in pediatric patients and young adults. Cure rates are low when patients present with metastatic or relapsed disease. Therefore, innovative therapy approaches are urgently needed. Cellular- and oncolytic virus-based immunotherapies are on the rise for solid cancers. Methods: Here, we assess the combination of EwS tumor-associated antigen CHM1319-specific TCR-transgenic CD8+ T cells and the YB-1-driven (i.e. E1A13S-deleted) oncolytic adenovirus XVir-N-31 in vitro and in a xenograft mouse model for antitumor activity and immunostimulatory properties. Results: In vitro both approaches specifically kill EwS cell lines in a synergistic manner over controls. This effect was confirmed in vivo, with increased survival using the combination therapy. Further in vitro analyses of immunogenic cell death and antigen presentation confirmed immunostimulatory properties of virus-infected EwS tumor cells. As dendritic cell maturation was also increased by XVir-N-31, we observed superior proliferation of CHM1319-specific TCR-transgenic CD8+ T cells only in virus-tested conditions, emphasizing the superior immune-activating potential of XVir-N-31. Conclusion: Our data prove synergistic antitumor effects in vitro and superior tumor control in a preclinical xenograft setting. Combination strategies of EwS-redirected T cells and YB-1-driven virotherapy are a highly promising immunotherapeutic approach for EwS and warrant further evaluation in a clinical setting.

YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells.

Oncolytic viruses (OVs) selectively replicate in tumor cells resulting in lysis, spreading of new infectious units and induction of antitumor immune responses through abrogating an immunosuppressive tumor microenvironment (TME). Due to their mode of action, OVs are ideal combination partners with targeted immunotherapies. One highly attractive combination is the inhibition of the 'don't-eat-me'-signal CD47, which is known to increase the phagocytic potential of tumor-associated macrophages. In this work, we analyzed the combination approach consisting of the YB-1-based oncolytic adenovirus XVir-N-31 (XVir) and the CD47 inhibitor (CD47i) B6.H12.2 concerning its phagocytic potential. We investigate phagocytosis of XVir-, adenovirus wildtype (AdWT)-, and non-infected established pediatric sarcoma cell lines by different monocytic cells. Phagocytes (immature dendritic cells and macrophages) were derived from THP-1 cells and healthy human donors. Phagocytosis of tumor cells was assessed via FACS analysis in the presence and absence of CD47i. Additional characterization of T cell-stimulatory surface receptors as well as chemo-/cytokine analyses were performed. Furthermore, tumor cells were infected and studied for the surface expression of the 'eat-me'-signal calreticulin (CALR) and the 'don't-eat-me'-signal CD47. We herein demonstrate that (1) XVir-infected tumor cells upregulate both CALR and CD47. XVir induces higher upregulation of CD47 than AdWT. (2) XVir-infection enhances phagocytosis in general and (3) the combination of XVir and CD47i compared to controls showed by far superior enhancement of phagocytosis, tumor cell killing and innate immune activation. In conclusion, the combination of CD47i and XVir causes a significant increase in phagocytosis exceeding the monotherapies considerably accompanied by upregulation of T cell-stimulatory receptor expression and inflammatory chemo/-cytokine secretion.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.

Intranasal Delivery of Oncolytic Adenovirus XVir-N-31 via Optimized Shuttle Cells Significantly Extends Survival of Glioblastoma-Bearing Mice.

A glioblastoma (GBM) is an aggressive and lethal primary brain tumor with restricted treatment options and a dismal prognosis. Oncolytic virotherapy (OVT) has developed as a promising approach for GBM treatment. However, reaching invasive GBM cells may be hindered by tumor-surrounding, non-neoplastic cells when the oncolytic virus (OV) is applied intratumorally. Using two xenograft GBM mouse models and immunofluorescence analyses, we investigated the intranasal delivery of the oncolytic adenovirus (OAV) XVir-N-31 via virus-loaded, optimized shuttle cells. Intranasal administration (INA) was selected due to its non-invasive nature and the potential to bypass the blood-brain barrier (BBB). Our findings demonstrate that the INA of XVir-N-31-loaded shuttle cells successfully delivered OAVs to the core tumor and invasive GBM cells, significantly prolonged the survival of the GBM-bearing mice, induced immunogenic cell death and finally reduced the tumor burden, all this highlighting the therapeutic potential of this innovative approach. Overall, this study provides compelling evidence for the effectiveness of the INA of XVir-N-31 via shuttle cells as a promising therapeutic strategy for GBM. The non-invasive nature of the INA of OV-loaded shuttle cells holds great promise for future clinical translation. However, further research is required to assess the efficacy of this approach to ultimately progress in human clinical trials.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

The Oncolytic Adenovirus XVir-N-31 Joins Forces with CDK4/6 Inhibition Augmenting Innate and Adaptive Antitumor Immunity in Ewing Sarcoma.

PURPOSE: Ewing sarcoma (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity. EXPERIMENTAL DESIGN: In vitro, viral toxicity, replication, and immunogenicity were studied in several EwS cell lines. In vivo tumor xenograft models with transient humanization were applied to evaluate tumor control, viral replication, immunogenicity, and dynamics of innate as well as human T cells after treatment with XVir-N-31 combined with CDK4/6 inhibition. Furthermore, immunologic features of dendritic cell maturation and T-cell-stimulating capacities were assessed. RESULTS: The combination approach significantly increased viral replication and oncolysis in vitro, induced HLA-I upregulation, and IFNγ-induced protein 10 expression and enhanced maturation of monocytic dendritic cells with superior capacities to stimulate tumor antigen-specific T cells. These findings were confirmed in vivo showing tumor infiltration by (i) monocytes with antigen-presenting capacities and M1 macrophage marker genes, (ii) TReg suppression in spite of adenovirus infection, (iii) superior engraftment, and (iv) tumor infiltration by human T cells. Consequently, survival was improved over controls with signs of an abscopal effect after combination treatment. CONCLUSIONS: The joint forces of the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition induce therapeutically relevant local and systemic antitumor effects. Innate as well as adaptive immunity against EwS is boosted in this preclinical setting, pointing toward high therapeutic potential in the clinic.

The Oncolytic Adenovirus XVir-N-31, in Combination with the Blockade of the PD-1/PD-L1 Axis, Conveys Abscopal Effects in a Humanized Glioblastoma Mouse Model.

Glioblastoma (GBM) is an obligatory lethal brain tumor with a median survival, even with the best standard of care therapy, of less than 20 months. In light of this fact, the evaluation of new GBM treatment approaches such as oncolytic virotherapy (OVT) is urgently needed. Based on our preliminary preclinical data, the YB-1 dependent oncolytic adenovirus (OAV) XVir-N-31 represents a promising therapeutic agent to treat, in particular, therapy resistant GBM. Preclinical studies have shown that XVir-N-31 prolonged the survival of GBM bearing mice. Now using an immunohumanized mouse model, we examined the immunostimulatory effects of XVir-N-31 in comparison to the wildtype adenovirus (Ad-WT). Additionally, we combined OVT with the inhibition of immune checkpoint proteins by using XVir-N-31 in combination with nivolumab, or by using a derivate of XVir-N-31 that expresses a PD-L1 neutralizing antibody. Although in vitro cell killing was higher for Ad-WT, XVir-N-31 induced a much stronger immunogenic cell death that was further elevated by blocking PD-1 or PD-L1. In vivo, an intratumoral injection of XVir-N-31 increased tumor infiltrating lymphocytes (TILs) and NK cells significantly more than Ad-WT not only in the virus-injected tumors, but also in the untreated tumors growing in the contralateral hemisphere. This suggests that for an effective treatment of GBM, immune activating properties by OAVs seem to be of greater importance than their oncolytic capacity. Furthermore, the addition of immune checkpoint inhibition (ICI) to OVT further induced lymphocyte infiltration. Consequently, a significant reduction in contralateral non-virus-injected tumors was only visible if OVT was combined with ICI. This strongly indicates that for an effective eradication of GBM cells that cannot be directly targeted by an intratumoral OV injection, additional ICI therapy is required.

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus.

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

Concepts in Oncolytic Adenovirus Therapy.

Oncolytic adenovirus therapy is gaining importance as a novel treatment option for the management of various cancers. Different concepts of modification within the adenovirus vector have been identified that define the mode of action against and the interaction with the tumour. Adenoviral vectors allow for genetic manipulations that restrict tumour specificity and also the expression of specific transgenes in order to support the anti-tumour effect. Additionally, replication of the virus and reinfection of neighbouring tumour cells amplify the therapeutic effect. Another important aspect in oncolytic adenovirus therapy is the virus induced cell death which is a process that activates the immune system against the tumour. This review describes which elements in adenovirus vectors have been identified for modification not only to utilize oncolytic adenovirus vectors into conditionally replicating adenoviruses (CRAds) that allow replication specifically in tumour cells but also to confer specific characteristics to these viruses. These advances in development resulted in clinical trials that are summarized based on the conceptual design.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

Combination of Talazoparib and Palbociclib as a Potent Treatment Strategy in Bladder Cancer.

The use of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors represents a potent strategy for cancer therapy. Due to the complex molecular network that regulates cell cycle progression, cancer cells often acquire resistance mechanisms against these inhibitors. Previously, our group identified molecular factors conferring resistance to CDK4/6 inhibition in bladder cancer (BLCA) that also included components within the DNA repair pathway. In this study, we validated whether a combinatory treatment approach of the CDK4/6 inhibitor Palbociclib with Poly-(ADP-Ribose) Polymerase (PARP) inhibitors improves therapy response in BLCA. First, a comparison of PARP inhibitors Talazoparib and Olaparib showed superior efficacy of Talazoparib in vitro and displayed high antitumor activity in xenografts in the chicken chorioallantoic membrane (CAM) model. Moreover, the combination of Talazoparib and the CDK4/6 inhibitor Palbociclib synergistically reduced tumor growth in Retinoblastoma protein (RB)-positive BLCA in vitro and in a CAM model, an effect that relies on Palbociclib-induced cell cycle arrest in G0/G1-phase complemented by a G2 arrest induced by Talazoparib. Interestingly, Talazoparib-induced apoptosis was reduced by Palbociclib. The combination of Palbociclib and Talazoparib effectively enhances BLCA therapy, and RB is a molecular biomarker of response to this treatment regimen.

STAT3/5 Inhibitors Suppress Proliferation in Bladder Cancer and Enhance Oncolytic Adenovirus Therapy.

The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.

Functional genomics identifies predictive markers and clinically actionable resistance mechanisms to CDK4/6 inhibition in bladder cancer.

BACKGROUND: CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer. METHODS: A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois membrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal. RESULTS: In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in in vivo xenografts. CONCLUSIONS: Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.

The Oncolytic Adenovirus XVir-N-31 as a Novel Therapy in Muscle-Invasive Bladder Cancer.

Muscle-invasive bladder cancer represents approximately 25% of diagnosed bladder cancer cases and carries a significant risk of death. Oncolytic viruses are novel antitumor agents with the ability to selectively replicate and lyse tumor cells while sparing healthy tissue. We explored the efficiency of the oncolytic YB-1-selective adenovirus XVir-N-31 in vitro and in an orthotopic mouse model for bladder cancer by intramural injection under ultrasound guidance. We demonstrated that XVir-N-31 replicated in bladder cancer cells and induced a stronger immunogenic cell death than wild-type adenovirus by facilitating enhanced release of HMGB1 and exosomal Hsp70. The intratumoral delivery of XVir-N-31 by ultrasound guidance delayed tumor growth in an immunodeficient model, demonstrating the feasibility of this approach to deliver oncolytic viruses directly into the tumor.

Virotherapy Research in Germany: From Engineering to Translation.

Virotherapy is a unique modality for the treatment of cancer with oncolytic viruses (OVs) that selectively infect and lyse tumor cells, spread within tumors, and activate anti-tumor immunity. Various viruses are being developed as OVs preclinically and clinically, several of them engineered to encode therapeutic proteins for tumor-targeted gene therapy. Scientists and clinicians in German academia have made significant contributions to OV research and development, which are highlighted in this review paper. Innovative strategies for "shielding," entry or postentry targeting, and "arming" of OVs have been established, focusing on adenovirus, measles virus, parvovirus, and vaccinia virus platforms. Thereby, new-generation virotherapeutics have been derived. Moreover, immunotherapeutic properties of OVs and combination therapies with pharmacotherapy, radiotherapy, and especially immunotherapy have been investigated and optimized. German investigators are increasingly assessing their OV innovations in investigator-initiated and sponsored clinical trials. As a prototype, parvovirus has been tested as an OV from preclinical proof-of-concept up to first-in-human clinical studies. The approval of the first OV in the Western world, T-VEC (Imlygic), has further spurred the involvement of investigators in Germany in international multicenter studies. With the encouraging developments in funding, commercialization, and regulatory procedures, more German engineering will be translated into OV clinical trials in the near future.

Adenovirus Particle Quantification in Cell Lysates Using Light Scattering.

Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand. Light scattering is an established process to measure virus particles' size, though due to cell impurities, adequate quantification of adenovirus particles in cell lysates by light scattering has been impossible until today. This report describes a new method using light scattering to measure virus concentration in nonpurified cell lysates. Here we report application of light scattering, a routine method to measure virus particle size, to virus quantification in enzymatically conditioned crude lysates. Samples are incubated with phospholipase A2 and benzonase and filtered through a 0.22 µm filter cartridge prior to quantification by light scattering. Our results show that this treatment provides a precise method for fast and easy determination of total adenovirus particle numbers in cell lysates and is useful to monitor virus recovery throughout all downstream processing.

YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells.

BACKGROUND: The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo. METHODS: YB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1. RESULTS: YB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment. CONCLUSION: The results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts.

Effects of nanoparticle coatings on the activity of oncolytic adenovirus-magnetic nanoparticle complexes.

Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized virus. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.

Boosting oncolytic adenovirus potency with magnetic nanoparticles and magnetic force.

Oncolytic adenoviruses rank among the most promising innovative agents in cancer therapy. We examined the potential of boosting the efficacy of the oncolytic adenovirus dl520 by associating it with magnetic nanoparticles and magnetic-field-guided infection in multidrug-resistant (MDR) cancer cells in vitro and upon intratumoral injection in vivo. The virus was complexed by self-assembly with core-shell nanoparticles having a magnetite core of about 10 nm and stabilized by a shell containing 68 mass % lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 32 mass % 25 kDa branched polyethylenimine. Optimized virus binding, sufficiently stable in 50% fetal calf serum, was found at nanoparticle-to-virus ratios of 5 fg of Fe per physical virus particle (VP) and above. As estimated from magnetophoretic mobility measurements, 3,600 to 4,500 magnetite nanocrystallites were associated per virus particle. Ultrastructural analysis by electron and atomic force microscopy showed structurally intact viruses surrounded by magnetic particles that occasionally bridged several virus particles. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to nonmagnetic virus when infections were carried out under the influence of a magnetic field. Increased virus internalization resulted in a 10-fold enhancement of the oncolytic potency in terms of the dose required for killing 50% of the target cells (IC(50) value) and an enhancement of 4 orders of magnitude in virus progeny formation at equal input virus doses compared to nonmagnetic viruses. Furthermore, the full oncolytic effect developed within two days postinfection compared with six days in a nonmagnetic virus as a reference. Plotting target cell viability versus internalized virus particles for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with magnetic nanoparticles. Hence, we conclude that the mechanism of boosting the oncolytic effect by magnetic force is mainly due to the improved internalization of magnetic virus complexes resulting in potentiated virus progeny formation. Upon intratumoral injection and application of a gradient magnetic field in a murine xenograft model, magnetic virus complexes exhibited a stronger oncolytic effect than adenovirus alone. We propose that this approach would be useful during in vivo administration to tumor-feeding blood vessels to boost the efficacy of the primary infection cycle within the tumor. For systemic application, further modification of magnetic adenovirus complexes for shielding and retargeting of the whole magnetic virus complex entity is needed.

Ribozyme to TGF-beta1 mRNA abrogates immunosuppressive effects of human colorectal adenocarcinoma HRT-18 cells in vitro and in vivo.

Transforming growth factor-beta1 (TGF-beta1) is overexpressed in a variety of malignant epithelial tumors and was suggested to be a marker of colorectal cancer. Moreover, there is growing evidence that TGF-beta1 contributes to tumor progression by regulating tumor cell proliferation and differentiation, inducing a favorable tumor microenvironment, promoting migration and invasion, and suppressing macrophage cytotoxicity. Therefore, we stably transfected an anti-TGF-beta1 hammerhead ribozyme into the human colorectal adenocarcinoma cell line HRT-18. Expression of this ribozyme resulted in significant inhibition of TGF-beta1 expression on mRNA and protein level. This was associated with an enhanced tumor cell differentiation and a reduced tumor growth in vivo. The capability of tumor cells to suppress ROI production of co-cultivated human macrophages was abrogated in transfectants. Taken together, inhibition of TGF-beta1 in colorectal carcinoma cells might be an interesting therapeutic tool leading to reduced tumor cell growth and increased macrophage cytotoxicity. Thus, a gene-therapeutic approach using anti-TGF-beta1 ribozyme in combination with established anti-tumor agents is of great promise.