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1.  Structural and Functional Studies on the Interaction of Adenovirus Fiber Knobs and Desmoglein 2 
Journal of Virology  2013;87(21):11346-11362.
Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14, as well as a recently emerged strain of Ad14 (Ad14p1), use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interaction with CAR and CD46, structural details for Ad binding to DSG2 are still elusive. Using an approach based on Escherichia coli expression libraries of random Ad3 and Ad14p1 fiber knob mutants, we identified amino acid residues that, when mutated individually, ablated or reduced Ad knob binding to DSG2. These residues formed three clusters inside one groove at the extreme distal end of the fiber knob. The Ad3 fiber knob mutant library was also used to identify variants with increased affinity to DSG2. We found a number of mutations within or near the EF loop of the Ad3 knob that resulted in affinities to DSG2 that were several orders of magnitude higher than those to the wild-type Ad3 knob. Crystal structure analysis of one of the mutants showed that the introduced mutations make the EF loop more flexible, which might facilitate the interaction with DSG2. Our findings have practical relevance for cancer therapy. We have recently reported that an Ad3 fiber knob-containing recombinant protein (JO-1) is able to trigger opening of junctions between epithelial cancer cells which, in turn, greatly improved the intratumoral penetration and efficacy of therapeutic agents (I. Beyer, et al., Clin. Cancer Res. 18:3340–3351, 2012; I. Beyer, et al., Cancer Res. 71:7080–7090, 2011). Here, we show that affinity-enhanced versions of JO-1 are therapeutically more potent than the parental protein in a series of cancer models.
doi:10.1128/JVI.01825-13
PMCID: PMC3807342  PMID: 23946456
2.  Penton-Dodecahedral Particles Trigger Opening of Intercellular Junctions and Facilitate Viral Spread during Adenovirus Serotype 3 Infection of Epithelial Cells 
PLoS Pathogens  2013;9(10):e1003718.
Human adenovirus serotypes Ad3, Ad7, Ad11, and Ad14 use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. During Ad infection, the fiber and penton base capsid proteins are produced in vast excess and form hetero-oligomers, called pentons. It has been shown for Ad3 that pentons self-assemble into penton-dodecahedra (PtDd). Our previous studies with recombinant purified Ad3 PtDd (produced in insect cells) showed that PtDd bind to DSG2 and trigger intracellular signaling resulting in the transient opening of junctions between epithelial cells. So far, a definitive proof for a function of Ad3 PtDd in the viral life cycle is elusive. Based on the recently published 3D structure of recombinant Ad3 PtDd, we generated a penton base mutant Ad3 vector (mu-Ad3GFP). mu-Ad3GFP is identical to its wild-type counterpart (wt-Ad3GFP) in the efficiency of progeny virus production; however, it is disabled in the production of PtDd. For infection studies we used polarized epithelial cancer cells or cell spheroids. We showed that in wt-Ad3GFP infected cultures, PtDd were released from cells before viral cytolysis and triggered the restructuring of epithelial junctions. This in turn facilitated lateral viral spread of de novo produced virions. These events were nearly absent in mu-Ad3GFP infected cultures. Our in vitro findings were consolidated in mice carrying xenograft tumors derived from human epithelial cancer cells. Furthermore, we provide first evidence that PtDd are also formed by another DSG2-interacting Ad serotype, the newly emerged, highly pathogenic Ad14 strain (Ad14p1). The central finding of this study is that a subgroup of Ads has evolved to generate PtDd as a strategy to achieve penetration into and dissemination in epithelial tissues. Our findings are relevant for basic and applied virology, specifically for cancer virotherapy.
Author Summary
We have recently reported that a group of human Ads uses DSG2 as a receptor for infection. Among the DSG2-interacting Ads is serotype 3, which is widely distributed in the human population. During Ad3 infection, subviral particles (PtDd) formed by two capsid proteins are produced in vast excess and released early in infection. In this study, we demonstrate that PtDd trigger the opening of epithelial junctions and thus support the lateral spread of Ad3 progeny virus in epithelial tissues. Our study contributes to a better understanding of Ad3 infection and pathology. It also has implications for Ad-mediated gene transfer into epithelial tissues and tumors.
doi:10.1371/journal.ppat.1003718
PMCID: PMC3814681  PMID: 24204268
3.  Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells 
Gene therapy  2012;20(2):201-214.
Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site specific endonucleases. Two genomic sites within the MBS85 and CCR5 genes [AAVS1 and CCR5 zinc finger nuclease (CCR5-ZFN) site, respectively] have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells. Matrixchromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptionally inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and hematopoietic stem cells. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a zinc-finger nuclease was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.
doi:10.1038/gt.2012.25
PMCID: PMC3661409  PMID: 22436965
4.  Co-administration of epithelial junction opener JO-1 improves the efficacy and safety of chemotherapeutic drugs 
Purpose
Epithelial junctions between tumor cells inhibit the penetration of anti-cancer drugs into tumors. We previously reported on recombinant adenovirus serotype 3 derived protein (JO-1), which triggers transient opening of intercellular junctions in epithelial tumors through binding to desmoglein 2 (DSG2), and enhances the anti-tumor effects of several therapeutic monoclonal antibodies. The goal of this study was to evaluate whether JO-1 co-therapy can also improve the efficacy of chemotherapeutic drugs.
Experimental Design
The effect of intravenous application of JO-1 in combination with several chemotherapy drugs including paclitaxel/Taxol™, nanoparticle albumin bound paclitaxel/Abraxane™, liposomal doxorubicin/Doxil™ and irinotecan/Camptosar™, was tested in xenograft models for breast, colon, ovarian, gastric and lung cancer. Because JO-1 does not bind to mouse cells, for safety studies with JO-1, we also used human DSG2 (hDSG2) transgenic mice with tumors that overexpressed human DSG2.
Results
JO-1 increased the efficacy of chemotherapeutic drugs, and in several models overcame drug resistance. JO-1 treatment also allowed for the reduction of drug doses required to achieve anti-tumor effects. Importantly, JO-1 co-admininstration protected normal tissues, including bone marrow and intestinal epithelium, against toxic effects that are normally associated with chemotherapeutic agents. Using the hDSG2 transgenic mouse model, we demonstrated that JO-1 predominantly accumulates in tumors. Except for a mild, transient diarrhea, intravenous injection of JO-1 (2mg/kg) had no critical side effects on other tissues or hematological parameters in hDSG2-transgenic mice.
Conclusions
Our preliminary data suggest that JO-1 co-therapy has the potential to improve the therapeutic outcome of cancer chemotherapy.
doi:10.1158/1078-0432.CCR-11-3213
PMCID: PMC3547677  PMID: 22535153
5.  Strategies to Increase Drug Penetration in Solid Tumors 
Frontiers in Oncology  2013;3:193.
Despite significant improvement in modalities for treatment of cancer that led to a longer survival period, the death rate of patients with solid tumors has not changed during the last decades. Emerging studies have identified several physical barriers that limit the therapeutic efficacy of cancer therapeutic agents such as monoclonal antibodies, chemotherapeutic agents, anti-tumor immune cells, and gene therapeutics. Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. Furthermore, nests of malignant epithelial tumor cells are shielded by layers of extracellular matrix (ECM) proteins (e.g., collagen, elastin, fibronectin, laminin) whereby tumor vasculature rarely penetrates into the tumor nests. In this chapter, we will review potential strategies to modulate the ECM and epithelial junctions to enhance the intratumoral diffusion and/or to remove physical masking of target receptors on malignant cells. We will focus on peptides that bind to the junction protein desmoglein 2 and trigger intracellular signaling, resulting in the transient opening of intercellular junctions. Intravenous injection of these junction openers increased the efficacy and safety of therapies with monoclonal antibodies, chemotherapeutics, and T cells in mouse tumor models and was safe in non-human primates. Furthermore, we will summarize approaches to transiently degrade ECM proteins or downregulate their expression. Among these approaches is the intratumoral expression of relaxin or decorin after adenovirus- or stem cell-mediated gene transfer. We will provide examples that relaxin-based approaches increase the anti-tumor efficacy of oncolytic viruses, monoclonal antibodies, and T cells.
doi:10.3389/fonc.2013.00193
PMCID: PMC3724174  PMID: 23898462
epithelial junctions; tumor stroma; extracellular matrix; relaxin; junction opener; tumor-associated macrophages
6.  Overcoming physical barriers in cancer therapy 
Tissue Barriers  2013;1(1):e23647.
Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. These intercellular junctions represent a protective mechanism against attacks by the host’s immune system and pose as physical barriers that prevent intratumoral penetration and dissemination of cancer therapeutics. A key protein of epithelial junctions is desmoglein 2 (DSG2). DSG2 is consistently upregulated in all cancers analyzed. Recently, we demonstrated that a group of human adenoviruses (Ad serotypes 3, 7, 11 and 14) use DSG2 as a primary attachment receptor for the infection of cells. We subsequently created a small recombinant protein derived from Ad serotype 3, which binds to DSG2 and triggers transient opening of epithelial intercellular junctions. We named the protein “JO-1” (“junction opener -1”). JO-1 is a small protein that can easily be produced in E. coli. JO-1 binding to and clustering of DSG2 triggers an epithelial-to-mesenchymal-transition that results in transient opening of epithelial junctions. We have shown in over 25 xenograft tumor models that the intravenous injection of JO-1 increased the efficacy of monoclonal and chemotherapy, subsequently reducing the required treatment dose and concomitantly reducing the toxic side effect of these treatments. The application of JO-1 has not been associated with toxicities in safety studies performed in human DSG2-transgenic mice and monkeys.
doi:10.4161/tisb.23647
PMCID: PMC3875641  PMID: 24665377
adenovirus; desmoglein 2; EMT; chemotherapy; monoclonal antibodies
7.  A New Human DSG2-Transgenic Mouse Model for Studying the Tropism and Pathology of Human Adenoviruses 
Journal of Virology  2012;86(11):6286-6302.
We have recently reported that a group of human adenoviruses (HAdVs) uses desmoglein 2 (DSG2) as a receptor for infection. Among these are the widely distributed serotypes HAdV-B3 and HAdV-B7, as well as a newly emerged strain derived from HAdV-B14. These serotypes do not infect rodent cells and could not up until now be studied in small-animal models. We therefore generated transgenic mice containing the human DSG2 locus. These mice expressed human DSG2 (hDSG2) at a level and in a pattern similar to those found for humans and nonhuman primates. As an initial application of hDSG2-transgenic mice, we used a green fluorescent protein (GFP)-expressing HAdV-B3 vector (Ad3-GFP) and studied GFP transgene expression by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemistry subsequent to intranasal and intravenous virus application. After intranasal application, we found efficient transduction of bronchial and alveolar epithelial cells in hDSG2-transgenic mice. Intravenous Ad3-GFP injection into hDSG2-transgenic mice resulted in hDSG2-dependent transduction of epithelial cells in the intestinal and colon mucosa. Our findings give an explanation for clinical symptoms associated with infection by DSG2-interacting HAdVs and provide a rationale for using Ad3-derived vectors in gene therapy.
doi:10.1128/JVI.00205-12
PMCID: PMC3372198  PMID: 22457526
8.  Epithelial junction opener JO-1 improves monoclonal antibody therapy of cancer 
Cancer research  2011;71(22):7080-7090.
The efficacy of monoclonal antibodies (mAbs) used to treat solid tumors is limited by intercellular junctions which tightly link epithelial tumor cells to each another. In this study, we define a small, recombinant adenovirus serotype 3-derived protein, termed junction opener 1 (JO-1), which binds to the epithelial junction protein desmoglein 2 (DSG2). In mouse xenograft models employing Her2/neu- and EGFR-positive human cancer cell lines, JO-1 mediated cleavage of DSG2 dimers and activated intracellular signaling pathways which reduced E-cadherin expression in tight junctions. Notably, JO-1-triggered changes allowed for increased intratumoral penetration of the anti-Her2/neu mAb trastuzumab (Herceptin) as well as improved access to its target receptor, Her2/neu, which is partly trapped in tight junctions. This effect translated directly into increased therapeutic efficacy of trastuzumab in mouse xenograft models using breast, gastric, and ovarian cancer cells that were Her2/neu-positive. Furthermore, combining JO-1 with the EGFR-targeting mAb cetuximab (Erbitux) greatly improved therapeutic outcomes in a metastatic model of EGFR-positive lung cancer. Taken together, our findings offer preclinical proof of concept to employ JO-1 in combination treatments which enhance the efficacy of trastuzumab treatment, by generating a transient degradation of tumor stroma proteins that can elicit eradication of tumors.
doi:10.1158/0008-5472.CAN-11-2009
PMCID: PMC3217128  PMID: 21990319
9.  Multimerization of Adenovirus Serotype 3 Fiber Knob Domains Is Required for Efficient Binding of Virus to Desmoglein 2 and Subsequent Opening of Epithelial Junctions▿ 
Journal of Virology  2011;85(13):6390-6402.
Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population (H. Wang et al., Nat. Med. 17:96–104, 2011). In this study, we have attempted to delineate structural details of the Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2-interacting domain(s) within Ad3 is formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered the transient opening of intercellular junctions in epithelial cells. The pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g., CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased the transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, the pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased the killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understanding of how Ads achieve high avidity to their receptors and the infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues.
doi:10.1128/JVI.00514-11
PMCID: PMC3112237  PMID: 21525338
10.  Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models 
Cancer gene therapy  2011;18(6):407-418.
Tumor-associated antigens (TAAs) include overexpressed self-antigens (e.g. Her2/neu) and tumor-virus antigens (e.g. HPV-16 E6/E7). Although in cancer patients, TAA-specific CD4+ and CD8+ cells are often present, they are not able to control tumor growth. In recent studies it became apparent that tumor-site located immune-evasion mechanisms contribute to this phenomenon and that regulatory T-cells play a major role. We tested in Her2/neu+ breast cancer and HPV-16 E6/E7+ cervical cancer mouse models, whether intratumoral expression of immunostimulatory proteins (ISPs), e.g. recombinant antibodies (αCTLA-4, αCD137, αCD3), cyto/chemokines (IL-15, LIGHT, mda-7), and costimulatory ligands (CD80), via adenovirus(Ad)-mediated gene transfer would overcome resistance. In both the breast and cervical cancer model, none of the Ad.ISP vectors displayed a significant therapeutic effect when compared with an Ad vector that lacked a transgene (Ad.zero). However, the combination of Ad.ISP vectors with systemic Treg depletion, using anti-CD25 mAb (breast cancer model) or low-dose cyclophosphamide (cervical cancer model) resulted in a significant delay of tumor growth in mice treated with Ad.αCTLA4. In the cervical cancer model we also demonstrated the induction of a systemic anti-tumor immune response that was able to delay the growth of distant tumors. Ad.αCTLA4 mediated tumor-destructive immune responses involved NKT- and CD8+ T-cells. In both models no auto-immune reactions were observed. The study shows that Ad.αCTLA4 in combination with systemic Treg depletion has potentials in the immunotherapy of cancer.
doi:10.1038/cgt.2011.8
PMCID: PMC3096725  PMID: 21394107
11.  Immuno-Therapy with Anti-CTLA4 Antibodies in Tolerized and Non-Tolerized Mouse Tumor Models 
PLoS ONE  2011;6(7):e22303.
Monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA4) are a novel form of cancer immunotherapy. While preclinical studies in mouse tumor models have shown anti-tumor efficacy of anti-CTLA4 injection or expression, anti-CTLA4 treatment in patients with advanced cancers had disappointing therapeutic benefit. These discrepancies have to be addressed in more adequate pre-clinical models. We employed two tumor models. The first model is based on C57Bl/6 mice and syngeneic TC-1 tumors expressing HPV16 E6/E7. In this model, the HPV antigens are neo-antigens, against which no central tolerance exists. The second model involves mice transgenic for the proto-oncogen neu and syngeneic mouse mammary carcinoma (MMC) cells. In this model tolerance to Neu involves both central and peripheral mechanisms. Anti-CTLA4 delivery as a protein or expression from gene-modified tumor cells were therapeutically efficacious in the non-tolerized TC-1 tumor model, but had no effect in the MMC-model. We also used the two tumor models to test an immuno-gene therapy approach for anti-CTLA4. Recently, we used an approach based on hematopoietic stem cells (HSC) to deliver the relaxin gene to tumors and showed that this approach facilitates pre-existing anti-tumor T-cells to control tumor growth in the MMC tumor model. However, unexpectedly, when used for anti-CTLA4 gene delivery in this study, the HSC-based approach was therapeutically detrimental in both the TC-1 and MMC models. Anti-CTLA4 expression in these models resulted in an increase in the number of intratumoral CD1d+ NKT cells and in the expression of TGF-β1. At the same time, levels of pro-inflammatory cytokines and chemokines, which potentially can support anti-tumor T-cell responses, were lower in tumors of mice that received anti-CTLA4-HSC therapy. The differences in outcomes between the tolerized and non-tolerized models also provide a potential explanation for the low efficacy of CTLA4 blockage approaches in cancer immunotherapy trials.
doi:10.1371/journal.pone.0022303
PMCID: PMC3136517  PMID: 21779410
12.  Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11, and 14 
Nature medicine  2010;17(1):96-104.
We have identified desmoglein 2 (DSG2) as the primary high-affinity receptor used by adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14. These serotypes represent important human pathogens causing respiratory tract infections. In epithelial cells, adenovirus binding to DSG2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This improves access to receptors, e.g. CD46 and Her2/neu, that are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDd), formed by viral penton and fiber in excess during viral replication, can trigger DSG2-mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDd. Our findings shed light on adenovirus biology and pathogenesis and have implications for cancer therapy.
doi:10.1038/nm.2270
PMCID: PMC3074512  PMID: 21151137
13.  Correction: Analysis of Epithelial and Mesenchymal Markers in Ovarian Cancer Reveals Phenotypic Heterogeneity and Plasticity 
PLoS ONE  2011;6(2):10.1371/annotation/8c637352-3614-406c-89dc-e78d10fa069c.
doi:10.1371/annotation/8c637352-3614-406c-89dc-e78d10fa069c
PMCID: PMC3039027
14.  Analysis of Epithelial and Mesenchymal Markers in Ovarian Cancer Reveals Phenotypic Heterogeneity and Plasticity 
PLoS ONE  2011;6(1):e16186.
In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherinlow/cytoplasmic E-cadherinhigh/CD133high, CD44high, Tie2low) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.
doi:10.1371/journal.pone.0016186
PMCID: PMC3021543  PMID: 21264259
15.  In situ adenovirus vaccination engages T effector cells against cancer 
Vaccine  2009;27(31):4225-4239.
The efficacy of cancer-immunotherapy is limited because of central and peripheral immune-tolerance towards tumor-antigens. We propose a novel approach based on the fact that the immune-system has not evolved tolerance towards adenoviruses (Ads) and that Ads have not evolved efficient mechanisms for immune-escape. The host-response to intratumoral Ad-vector-injection in mice that were immunologically tolerant to neu-positive syngeneic mammary-cancer (MMC) was investigated. Intratumoral injection with replication-deficient, transgene-devoid Ad induced immune-responses at two different anatomical sites: the tumor-draining lymph-nodes and the tumor-microenvironment. The lymph-nodes supported the generation of both neu- and Ad-specific T-effector-cells, while inside the tumor-microenvironment only Ad-specific T-cells expanded. Importantly, Ad-specific T-cells were anti-tumor-reactive despite the presence of active regulatory-T-cell-mediated immune-tolerance inside MMC-tumors and anti-tumor efficacy of Ad was increased by pre-immunization against Ad despite the production of Ad-neutralizing antibodies.
doi:10.1016/j.vaccine.2009.03.074
PMCID: PMC2727281  PMID: 19481312
16.  The epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses 
Cancer research  2009;69(12):5115-5125.
We studied the propensity of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to CAR or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, so far little explored adenovirus serotypes (Ad3, 7, 11, and 14) that use receptor(s) other than CAR and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed a number of interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (E/M cells). These E/M cells are the only xenograft-derived cells that can be cultured, and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology.
doi:10.1158/0008-5472.CAN-09-0645
PMCID: PMC2738419  PMID: 19491256
17.  Transduction of Liver Metastases After Intravenous Injection of Ad5/35 or Ad35 Vectors With and Without Factor X-Binding Protein Pretreatment 
Human Gene Therapy  2009;20(6):621-629.
Abstract
Inefficient tumor transduction with targeted adenoviral vectors is largely due to unspecific virus sequestration by blood components, including coagulation factor X, and Kupffer cell scavenging. In this study, we show that preinjection of snake venom factor X-binding protein (X-bp) reduces hepatocyte transduction and increases the circulation time in blood of an intravenously injected, fiber-chimeric Ad5/35 vector. X-bp pretreatment resulted in improved Ad5/35 transduction of liver metastases and increased the antitumor efficacy of an Ad5/35-based oncolytic adenovirus. Furthermore, we demonstrate that a vector based on adenoviral serotype 35, which is less sequestered by factor X, is efficient in tumor targeting. This gives a rationale for using Ad35-based vectors in virotherapy of cancer.
doi:10.1089/hum.2008.142
PMCID: PMC2828640  PMID: 19245296
18.  Receptor usage of a newly emergent adenovirus type 14 
Virology  2009;387(2):436-441.
Recently, cases of severe respiratory illness in military and civilian populations have been associated with a new genomic variant of adenovirus (Ad) serotype 14, designated Ad14a. Compared to the Ad14 reference strain (de Wit), this new virus had a deletion of two amino acid residues in the fiber protein knob. Here we tested whether this mutation changed receptor usage of Ad14a compared to Ad14-de Wit. Competition studies with radio-labeled viruses revealed that both Ad14-de Wit and Ad14a used the same receptor which is hitherto unknown. We also found that recombinant fiber knobs only partially blocked attachment of Ad14a, indicating that virus capsid proteins other than the fiber are involved in infection.
doi:10.1016/j.virol.2009.02.034
PMCID: PMC2674115  PMID: 19307010
adenovirus; species B; tropism
19.  Tightly regulated gene expression in human hematopoietic stem cells after transduction with helper-dependent Ad5/35 vectors 
Experimental hematology  2008;36(7):823-831.
Objective
Inducible and transient expression of transcription factors, growth factors, or mitogenic factors can be used to influence proliferation or differentiation of hematopoietic progenitor/stem cells (HSCs). Furthermore, transient expression of proteins with site-specific endonuclease activity, potentially, can support targeted integration of exogenous transgenes into specific sites in the genome, a task that is currently a focus in development of gene therapy vectors.
Methods
We constructed a set of helper-dependent adenovirus (Ad) vectors with serotype 35 fiber knob domains (HD-Ad5/35) which can efficiently transduce human CD34+ cells, particularly subsets with potential stem cell capacity. These vectors contain Tet-inducible expression systems that were shielded by insulators and transcription stop signals to minimize unspecific interference by transcriptional elements present in viral and stuffer DNA. We compared two vectors, containing a fusion between the Krüppel-associated box (KRAB) domain and the tetracycline repressor (HD-Ad5/35.Tet-1) or an autoregulated rtTA (HD-Ad5/35.Tet-2) for regulated transgene expression in Mo7e cells, a model for HSC, and primary human CD34+ cells.
Results
HD-Ad5/35.Tet-1 conferred lower background expression than HD-Ad5/35.Tet-2, although levels of induced gene expression were higher for HD-Ad5/35.Tet-2. In CD34+ cells, while HD-Ad5/35.Tet-1 allowed for activated gene expression in all transduced cells, induced gene expression from HD-Ad5/35.Tet-2 was restricted to a small subset of CD34+ cells. Importantly, clonogeneic assays and repopulation studies in NOD/SCID mice showed that both HD-Ad5/35.Tet-1 and -2 vectors mediated induced gene expression in primitive hematopoietic cells. These studies also confirmed that transduction of CD34+ cells with HD-Ad5/35 vectors is not associated with cytotoxity, a problem observed with first-generation Ad5/35 vectors.
Conclusions
Both HD-Ad5/35 vector expression systems confer tightly regulated, transient transgene expression in human HSC.
doi:10.1016/j.exphem.2008.01.014
PMCID: PMC2517261  PMID: 18394775
gene therapy; adenovirus; human hematopoietic stem cells
20.  In Vitro and In Vivo Properties of Adenovirus Vectors with Increased Affinity to CD46▿  
Journal of Virology  2008;82(21):10567-10579.
Gene transfer vectors containing adenovirus (Ad) serotype 35 (Ad35) fibers have shown promise for cancer and stem cell gene therapy. In this study, we attempted to improve the in vitro and in vivo infection properties of these vectors by increasing their affinity to the Ad35 fiber receptor CD46. We constructed Ad vectors containing either the wild-type Ad35 fiber knob (Ad5/35) or Ad35 knob mutants with 4-fold- and 60-fold-higher affinity to CD46 (Ad5/35+ and Ad5/35++, respectively). In in vitro studies with cell lines, the higher affinities of Ad5/35+ and Ad5/35++ to CD46 did not translate into correspondingly higher transduction efficiencies, regardless of the CD46 receptor density present on cells. However, in vivo, in a mouse model with preestablished CD46high liver metastases, intravenous injection of Ad5/35++ resulted in more-efficient tumor cell transduction. We conclude that Ad5/35 vectors with increased affinity to CD46 have an advantage in competing with non-CD46-mediated sequestration of vector particles after intravenous injection.
doi:10.1128/JVI.01308-08
PMCID: PMC2573189  PMID: 18753195
21.  Biodistribution and Safety Profile of Recombinant Adeno-Associated Virus Serotype 6 Vectors following Intravenous Delivery ▿  
Journal of Virology  2008;82(15):7711-7715.
Recombinant adeno-associated virus vectors based on serotype 6 (rAAV6) efficiently transduce skeletal muscle after intravenous administration and have shown efficacy in the mdx model of muscular dystrophy. As a prelude to future clinical studies, we investigated the biodistribution and safety profile of rAAV6 in mice. Although it was present in all organs tested, rAAV6 was sequestered mainly in the liver and spleen. rAAV6 had a minimal effect on circulating blood cells and caused no apparent hepatotoxicity or coagulation activation. rAAV6 caused some neutrophil infiltration into the liver, with a transient elevation in cytokine and chemokine transcription/secretion. In summary, rAAV6 induces transient toxicity that subsides almost completely within 72 h and causes no significant side effects.
doi:10.1128/JVI.00542-08
PMCID: PMC2493321  PMID: 18480442
22.  Role of Cellular Heparan Sulfate Proteoglycans in Infection of Human Adenovirus Serotype 3 and 35 
PLoS Pathogens  2008;4(10):e1000189.
Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.
Author Summary
In this study, we attempted to identify binding receptors that are used by the two human adenovirus (Ad) serotypes 3 and 35. Ad3 uses yet-unknown receptors and is one of the most common Ads causing epidemic conjunctivitis, and respiratory and gastrointestinal diseases. Ad35 uses the complement receptor CD46 as an attachment receptor and mainly causes infections of the kidney and urinary tract. We utilized novel high-throughput techniques in combination with the recombinant viral proteins (fiber knobs), which mediate the initial interaction of Ads with host cells. We found that both serotypes interacted with cellular heparan sulfate proteoglycans (HSPGs). In subsequent assays, we show that HSPGs were not major receptors, but acted as low-affinity co-receptors for both Ad3 and Ad35. Ad3 and Ad35 used different viral proteins in order to interact with HSPGs. Both serotypes, however, used the same regions within HSPGs that show high levels of sulfation and are important for binding of extracellular located physiologic ligands. In summary, we show that Ad3 and Ad35 evolved to “highjack” yet another class of cellular surface molecules that are essential for the function of the target host cells and are ubiquitously expressed. This provides new insights into the emerging picture of the infection mechanism of Ad3 and Ad35.
doi:10.1371/journal.ppat.1000189
PMCID: PMC2568953  PMID: 18974862
23.  Identification of CD46 Binding Sites within the Adenovirus Serotype 35 Fiber Knob▿  
Journal of Virology  2007;81(23):12785-12792.
Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a KD (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.
doi:10.1128/JVI.01732-07
PMCID: PMC2169084  PMID: 17898059
24.  Evaluation of adenovirus vectors containing serotype 35 fibers for vaccination. 
In contrast to commonly used serotype 5 based adenovirus (Ad) vectors, Ads containing fibers derived from B-group serotype 35 (Ad5/35) efficiently transduce human DCs ex vivo and appear to target antigen-presenting cells after intravenous injection into baboons. Based on this, Ad5/35 vectors could be valuable tools for immunotherapy and vaccination. On the other hand, a number of studies indicate that signaling through the B-group Ad receptor, CD46, can cause tolerance or immuno-suppression. Since mice do not express CD46 in a human-like pattern, we studied the in vivo properties of Ad5/35 in transgenic mice that express CD46 in a pattern and at a level similar to humans. Hypersensitivity assays and analyses of frequencies of regulatory T-cells and T-cell responses did not indicate that Ad5/35 injection exerts detrimental effects on the host's immune system. An Ad5/35 vector expressing a model antigen was able to trigger a strong T-cell response against the test antigen after intramuscular injection. Overall, compared to Ad5 vectors, Ad5/35 vectors had a better safety profile, reflected by lower serum levels of pro-inflammatory cytokines.
doi:10.1016/j.ymthe.2005.12.008
PMCID: PMC1424671  PMID: 16461009
25.  Effect of adenovirus mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on anti-tumor immune responses 
Cancer research  2006;66(2):960-969.
Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen presenting cells. In this study we tested whether adenovirus (Ad)-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an anti-tumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing subcutaneous tumors derived from syngeneic TC-1 cells, a cell line that expresses HPV16 E6 and E7 proteins. We found that secreted gp96 can induce protective and therapeutic anti-tumor immune responses. Our data also indicate that the anti tumor effect sgp96 expression appears to be limited by induction of suppressive regulatory T cells (Tregs). TC-1 tumor transplantation increased the number of splenic and tumor infiltrating Tregs. Importantly, treatment of mice with low-dose cyclophosphamide decreased the number Tregs and enhanced the immunostimulatory effect of sgp96 expression. We also tested whether an oncolytic vector (Ad.IR-E1A/TRAIL), that is able to induce tumor cell apoptosis and, potentially, release cryptic tumor epitopes in immunogenic form, can stimulate anti-tumor immune responses. While tumor cells infected ex vivo with Ad.IR-E1A/TRAIL had no anti-tumor effect when used as a vaccine alone, the additional treatment with low-dose cyclophosphamide resulted in elimination of pre-established tumors. This study gives a rationale for testing approaches that suppress Tregs in combination with oncolytic or immunostimulatory vectors.
doi:10.1158/0008-5472.CAN-05-2388
PMCID: PMC1360184  PMID: 16424031
adenovirus; oncolytic; heat shock protein; gp96; regulatory T-cells; cyclophosphamide

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