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A number of widely distributed adenoviruses use the epithelial junction protein DSG2 as a receptor for infection and lateral spread. Interaction with DSG2 not only allows the virus to enter cells but also to open epithelial junctions which form a physical barrier to virus spread. Our study elucidates the mechanism beyond virus triggered junction opening with a focus on adenovirus serotype 3. Ad3 binds to DSG2 with its fiber knob domain and triggers intracellular signaling that culminates in the cleavage of the extracellular domain of DSG2 thereby disrupting DSG2 homodimers between epithelial cells. We confirmed this pathway with a second DSG2-interacting serotype, Ad14 and its recently emerged stain Ad14P1. These new insights in basic adenovirus biology can be employed to develop novel drugs to treat adenovirus infection as well as be used as tools for gene delivery into epithelial tissues or epithelial tumors.
Journal of Virology 08/2015; 89(21). DOI:10.1128/JVI.01425-15 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A central treatment resistance mechanism in solid tumors is the maintenance of epithelial junctions between malignant cells that prevent drug penetration into the tumor. We have developed a small recombinant protein (JO-1) that triggers the transient opening of intercellular junctions and thus increases the efficacy of monoclonal antibodies and chemotherapeutic drugs without causing toxicity in mouse tumor models. Here, we provide data toward the clinical translation of an affinity-enhanced version of JO-1, which we call JO-4, in combination with PEGylated liposomal doxorubicin (PLD)/Doxil for ovarian cancer therapy. We have presented X-ray crystallography data suggesting a structural basis for the higher affinity of JO-4 to DSG2. We also confirmed JO-4 efficacy in a xenograft model with primary ovarian cancer cells showing that JO-4 can salvage Doxil therapy when given at a dose that was threefold lower than the therapeutic dose. Furthermore, we tested the safety of intravenous JO-4 alone and in combination with Doxil in Macaca fascicularis, an adequate animal model for predicting toxicity in humans. Our studies did not show critical JO-4-related toxicity or an increase of Doxil-related side effects. Our efficacy and safety data will help to support an Investigational new drug-filing for a JO-4/Doxil combination treatment.
[Show abstract][Hide abstract] ABSTRACT: Adenoviruses are common pathogens. The localization of their receptors coxsackievirus and adenovirus receptor, and desmoglein-2 in cell-cell junction complexes between polarized epithelial cells represents a major challenge for adenovirus infection from the apical surface. Structural proteins including hexon, penton base and fiber are excessively produced in serotype 5 adenovirus (Ad5)-infected cells. We have characterized the composition of structural protein complexes released from Ad5 infected cells and their capacity in remodeling cell-cell junction complexes. Using T84 cells as a model for polarized epithelium, we have studied the effect of Ad5 structural protein complexes in remodeling cell-cell junctions in polarized epithelium. The initial Ad5 infection in T84 cell culture was inefficient. However, progressive distortion of cell-cell junction in association with fiber release was evident during progression of Ad5 infection. Incubation of T84 cell cultures with virion-free supernatant from Ad5 infected culture resulted in distortion of cell-cell junctions and decreased infectivity of Ad5-GFP vector. We used gel filtration chromatography to fractionate fiber containing virion-free supernatant from Ad5 infected culture supernatant. Fiber containing fractions were further characterized for their capacity to inhibit the infection of Ad5-GFP vector, their composition in adenovirus structural proteins using western blot and LC-MS/MS and their capacity in remolding cell-cell junctions. Fiber molecules in complexes containing penton base and hexon, or mainly hexon were identified. Only the fiber complexes with relatively high content of penton base, but not the fiber-hexon complexes with low penton base, were able to penetrate into T84 cells and cause distortion of cell-cell junctions. Our findings suggest that these two types of fiber complexes may play different roles in adenoviral infection.
PLoS ONE 02/2015; 10(2):e0117976. DOI:10.1371/journal.pone.0117976 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oncolytic gene therapy using viral vectors may provide an attractive therapeutic option for malignant gliomas. These viral vectors are designed in a way to selectively target tumor cells and spare healthy cells. To determine the translational impact, it is imperative to assess the factors that interfere with the anti-glioma effects of the oncolytic adenoviral vectors. In the current study, we evaluated the efficacy of survivin-driven oncolytic adenoviruses pseudotyping with adenoviral fiber knob belonging to the adenoviral serotype 3, 11 and 35 in their ability to kill glioblastoma (GBM) cells selectively without affecting normal cells. Our results indicate that all recombinant vectors used in the study can effectively target GBM in vitro with high specificity, especially the 3 knob-modified vector. Using intracranial U87 and U251 GBM xenograft models we have also demonstrated that treatment with Conditionally Replicative Adenovirus (CRAd-S-5/3) vectors can effectively regress tumor. However, in several patient-derived GBM cell lines, cells exhibited resistance to the CRAd infection as evident from the diminishing effects of autophagy. To improve therapeutic response, tumor cells were pretreated with tamoxifen. Our preliminary data suggest that tamoxifen sensitizes glioblastoma cells towards oncolytic treatment with CRAd-S-5/3, which may prove useful for GBM in future experimental therapy
[Show abstract][Hide abstract] ABSTRACT: Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35) vectors for zinc-finger nuclease (ZFN)- or transcription activator-like effector nuclease (TALEN)-mediated genome editing in human CD34+ hematopoietic stem cells (HSCs) from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA)-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35.ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells). The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2) within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.
[Show abstract][Hide abstract] ABSTRACT: The protein CD46 protects cells from complement attack by regulating cleavage of C3b and C3d. CD46 also regulates the adaptive immune response by controlling T cell activation and differentiation. Co-engagement of the T cell receptor and CD46 notably drives T cell differentiation by switching production of IFNγ to secretion of anti-inflammatory IL-10. This regulatory pathway is altered in several chronic inflammatory diseases highlighting its key role for immune homeostasis. The manipulation of the CD46 pathway may therefore provide a powerful means to regulate immune responses. Herein, we investigated the effect of recombinant proteins derived from the fiber knob of the adenovirus serotype 35 (Ad35) that uses CD46 as its entry receptor, on human T cell activation. We compared the effects of Ad35K++, engineered to exhibit enhanced affinity to CD46, and of Ad35K-, mutated in the binding site for CD46. Ad35K++ profoundly affects T cell activation by decreasing the levels of CD46 at the surface of primary T cells, and impairing T cell co-activation, shown by decreased CD25 expression, reduced proliferation and lower secretion of IL-10 and IFNγ. In contrast, Ad35K- acts a potent coactivator of T cells, enhancing T cell proliferation and cytokine production. These data show that recombinant Ad35 proteins are potent modulators of human T cell activation, and support their further development as potential drugs targeting T cell responses.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The complement system is composed of soluble factors in plasma that enhance or "complement" immune-mediated killing through innate and adaptive mechanisms. Activation of complement causes recruitment of immune cells; opsonization of coated cells; and direct killing of affected cells through a membrane attack complex (MAC). Tumor cells up-regulate complement inhibitory factors - one of several strategies to evade the immune system. In many cases as the tumor progresses, dramatic increases in complement inhibitory factors are found on these cells. This review focuses on the classic complement pathway and the role of major complement inhibitory factors in cancer immune evasion as well as on how current protein engineering efforts are being employed to increase complement fixing or to reverse complement resistance leading to better therapeutic outcomes in oncology. Strategies discussed include engineering of antibodies to enhance complement fixation, antibodies that neutralize complement inhibitory proteins as well as engineered constructs that specifically target inhibition of the complement system.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Most current cancer therapies focus on killing malignant cells, but these cells are often genetically unstable and can become resistant to chemotherapy. Tumor-associated macrophages (TAMs) facilitate disease progression by promoting angiogenesis and tumor cell growth, as well as by suppressing the adaptive immune response. TAMs are therefore potential targets for adjuvant anticancer therapies. However, resident macrophages are critical to host defense, and preferential ablation of TAMs remains challenging. Macrophage activation is broadly categorized as classically activated, or M1, and alternatively activated, or M2, and TAMs in the tumor microenvironment have been shown to adopt the anti-inflammatory, M2-like phenotype. To date, there are no methods for specific molecular targeting of TAMs. In this work, we report the discovery of a unique peptide sequence, M2pep, identified using a subtractive phage biopanning strategy against whole cells. The peptide preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes. Confocal imaging demonstrates the accumulation of M2pep in TAMs in vivo after tail vein injection. Finally, tail vein injection of an M2pep fusion peptide with a proapoptotic peptide delays mortality and selectively reduces the M2-like TAM population. This work therefore describes a molecularly targeted construct for murine TAMs and provides proof of concept of this approach as an anticancer treatment. In addition, M2pep is a useful tool for murine M2 macrophage identification and for modulating M2 macrophages in other murine models of disease involving M2 cells.
Proceedings of the National Academy of Sciences 09/2013; 110(40). DOI:10.1073/pnas.1312197110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, Ad14, and a recently emerged new 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 E.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 to the EF loop of the Ad3 knob that resulted in several orders of magnitude higher affinities to DSG2 compared with 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. Here we show that affinity-enhanced versions of JO-1 are therapeutically more potent than the parental protein in a series of cancer models.
Journal of Virology 08/2013; 87(21). DOI:10.1128/JVI.01825-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer stem cells (OCSCs) are in a transitional phase between epithelial and mesenchymal cell stages. Consequently, OCSCs possess a high degree of plasticity that complicates their identification and characterization. However, we recently demonstrated that the combined assessment of key antigens associated with cancer stem cells and the epithelial-mesenchymal transition can distinguish the phenotype of OCSCs from more differentiated cells. In this chapter we describe in detail an appropriate sample preparation for the analysis of epithelial, mesenchymal, and cancer stem cell markers by flow cytometry and immunohistochemistry in ovarian cancer. Furthermore, we provide methods for the establishment of primary ovarian cancer cultures from solid tumors.
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Oncology 07/2013; 3:193. DOI:10.3389/fonc.2013.00193
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We have developed a technology that depletes the complement regulatory protein (CRP) CD46 from the cell surface, and thereby sensitizes tumor cells to complement-dependent cytotoxicity triggered by therapeutic monoclonal antibodies (mAbs). This technology is based on a small recombinant protein, Ad35K++, which induces the internalization and subsequent degradation of CD46. In preliminary studies, we had demonstrated the utility of the combination of Ad35K++ and several commercially available mAbs such as rituximab, alemtuzumab, and trastuzumab in enhancing cell killing in vitro as well as in vivo in murine xenograft and syngeneic tumor models. We have completed scaled manufacturing of Ad35K++ protein in Escherichia coli for studies in nonhuman primates (NHPs). In macaques, we first defined a dose of the CD20-targeting mAb rituximab that did not deplete CD20-positive peripheral blood cells. Using this dose of rituximab, we then demonstrated that pretreatment with Ad35K++ reconstituted near complete elimination of B cells. Further studies demonstrated that the treatment was well tolerated and safe. These findings in a relevant large animal model provide the rationale for moving this therapy forward into clinical trials in patients with CD20-positive B-cell malignancies.Molecular Therapy (2012); doi:10.1038/mt.2012.212.
[Show abstract][Hide abstract] ABSTRACT: Twenty-five patients with chemotherapy refractory cancer were treated with a fully serotype 3-based oncolytic adenovirus Ad3-hTERT-E1A. In mice, Ad3 induced higher amounts of cytokines but less liver damage than Ad5 or Ad5/3. In humans, the only grade 3 adverse reactions were self-limiting cytopenias and generally the safety profile resembled Ad5-based oncolytic viruses. Patients that had been previously treated with Ad5 viruses presented longer lasting lymphocytopenia but no median increase in Ad3-specific T-cells in blood, suggesting immunological activity against antigens other than Ad3 hexon. Frequent alterations in antitumor T-cells in blood were seen regardless of previous virus exposure. Neutralizing antibodies against Ad3 increased in all patients, whereas Ad5 neutralizing antibodies remained stable. Treatment with Ad3-hTERT-E1A resulted in re-emergence of Ad5 viruses from previous treatments into blood and vice versa. Signs of possible efficacy were seen in 11/15 (73%) patients evaluable for tumor markers, four of which were treated only intravenously. Particularly promising results were seen in breast cancer patients and especially those receiving concomitant trastuzumab. Taken together, Ad3-hTERT-E1A seems safe for further clinical testing or development of armed versions. It offers an immunologically attractive alternative, with possible pharmacodynamic differences and a different receptor compared to Ad5.
[Show abstract][Hide abstract] ABSTRACT: Recombinant adenovirus vectors (Ads) are used as delivery vehicles either to express an antigen-encoding gene or to transfer an antigen protein in the context of the Ad capsid. Recent preclinical studies indicate that the combination of Ad with other vaccine platforms in heterologous prime-boost regimens has the potential to induce protective immunity, and the use of adjuvants or inhibition of regulatory T cells might further increase the efficacy of these approaches. A major problem with the currently used Ads is pre-existing anti-Ad immunity in humans and the induction of strong anti-Ad immune responses that can interfere with vaccination. Recent efforts in the understanding of the interaction of Ad with the host and the development of new Ad vectors based on rare serotypes can potentially address these problems.In this chapter, we will present principal approaches and considerations for the use of Ad as a vaccine, and discuss recent applications of Ads for prevention and treatment of infectious diseases and cancer.
[Show abstract][Hide abstract] ABSTRACT: Epithelial junctions between tumor cells inhibit the penetration of anticancer 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 antitumor effects of several therapeutic monoclonal antibodies. The goal of this study was to evaluate whether JO-1 cotherapy can also improve the efficacy of chemotherapeutic drugs.
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 hDSG2.
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 antitumor effects. Importantly, JO-1 coadmininstration 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 showed that JO-1 predominantly accumulates in tumors. Except for a mild, transient diarrhea, intravenous injection of JO-1 (2 mg/kg) had no critical side effects on other tissues or hematologic parameters in hDSG2-transgenic mice.
Our preliminary data suggest that JO-1 cotherapy has the potential to improve the therapeutic outcome of cancer chemotherapy.
Clinical Cancer Research 04/2012; 18(12):3340-51. DOI:10.1158/1078-0432.CCR-11-3213 · 8.72 Impact Factor