Dario Neri

Ijuí Charity Hospital, Ijuhy, Rio Grande do Sul, Brazil

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Publications (306)1718.87 Total impact

  • Christian Hess, Dario Neri
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    ABSTRACT: We describe the expression and in vivo characterization of an antibody-cytokine fusion protein, based on murine Interleukin-13 (IL13) and the monoclonal antibody F8, specific to the alternatively spliced extra domain A of fibronectin, a marker of neo-angiogenesis. The IL13 moiety was fused at the C-terminal extremity of the F8 antibody in diabody format. The resulting F8-IL13 immunocytokine retained the full binding properties of the parental antibody and cytokine bioactivity. The fusion protein could be expressed in mammalian cells, purified to homogeneity and showed a preferential accumulation at the tumor site. When used as single agent at doses of 200 μg, F8-IL13 exhibited a strong inhibition of tumor growth rate in two models of cancer (F9 teratocarcinoma and Wehi-164), promoting an infiltration of various types of leukocytes into the neoplastic mass. This anticancer activity could be potentiated by combination with an immunocytokine based on the F8 antibody and murine IL12, leading to complete and long-lasting tumor eradications. Mice cured from Wehi-164 sarcomas acquired a durable protective antitumor immunity, and selective depletion of immune cells revealed that the antitumor activity was mainly mediated by cluster of differentiation 4-positive T cells. This study indicates that IL13 can be efficiently delivered to the tumor neo-vasculature and that it mediates a potent anticancer activity in the two models of cancer investigated in this study. The observed mechanism of action for F8-IL13 was surprising, since immunocytokines based on other payloads (e.g., IL2, IL4, IL12 and TNF) eradicate cancer by the combined contribution of natural killer cells and cluster of differentiation 8-positive T cells.
    Cancer immunology, immunotherapy : CII. 02/2015;
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    ABSTRACT: The antibody-based delivery of interleukin-2 (IL-2) to extracellular targets expressed in the easily accessible tumor-associated vasculature has shown potent anti-leukemic activity in xenograft and immunocompetent murine models of acute myeloid leukemia (AML), especially in combination with cytarabine. Here, we report our experiences in four patients with relapsed AML after allogeneic hematopoietic stem cell transplantation (allo-HSCT), who were treated with the immunocytokine F16-IL2, in combination with low-dose cytarabine (LDAC). One patient with disseminated extramedullary AML lesions achieved a complete metabolic response in PET/CT, which lasted three months. Two out of three patients with bone marrow (BM) relapse achieved a blast reduction with transient molecular negativity. One of the two enjoyed a short complete remission before AML relapse occurred two months after the first infusion of F16-IL2. In line with a site-directed delivery of the cytokine, F16-IL2 led to an extensive infiltration of immune effector cells in the BM. Grade 2 fevers were the only non-hematological side effects in two patients. Grade 3 cytokine-release syndrome developed in the other two patients, but was manageable in both cases with glucocorticoids. The concept of specifically targeting IL-2 to the leukemia-associated stroma deserves further evaluation in clinical trials, especially in patients who relapse after allo-HSCT. Copyright © 2015, American Association for Cancer Research.
    Cancer immunology research. 02/2015;
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    ABSTRACT: Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.
    Angewandte Chemie 02/2015;
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    ABSTRACT: The ability of antibodies to extravasate out of blood vessels is critical for therapeutic activity, because molecular targets for most diseases are located outside of the endothelial lining. By performing detailed biodistribution studies with a novel IL9-armed cancer-specific antibody, we identified a clear correlation between N-linked glycan structures and tumor-targeting efficiencies. Site-specific glycan analysis provided a detailed view of the glycan microheterogeneity present on the IL9 portion of the recombinant protein. Nonsialylated glycan structures have a negative impact on disease-homing activity, highlighting the importance of glycosylation control and characterization during process development.
    Proceedings of the National Academy of Sciences of the United States of America. 02/2015;
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    ABSTRACT: Antibody-cytokine fusion proteins (immunocytokines) represent a novel class of armed antibodies in oncology. In particular, IL2–and TNF–based immunocytokines targeting the EDB domain of fibronectin and the A1 domain of tenascin-C have demonstrated promising anti-tumor activity and are currently investigated in Phase I and Phase II clinical trials. To advance the development of immunocytokines for NSCLC, we here report on the therapeutic efficacy of F8-IL2, an immunocytokine directed against the alternatively-spliced EDA domain of fibronectin in a fully immunocompetent, orthotopic model of NSCLC, and the characterization of the target antigen expression in human NSCLC specimens.
    Lung Cancer. 01/2015;
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    ABSTRACT: In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5′ and 3′ ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanom
    Nature Chemistry 01/2015; 7(3):241. · 21.76 Impact Factor
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    ABSTRACT: A phase Ib/II trial was performed to evaluate safety, tolerability, recommended dose (RD) and efficacy of F16-IL2, a recombinant antibody-cytokine fusion protein, in combination with doxorubicin in patients with solid tumours (phase Ib) and metastatic breast cancer (phase II). Six patient cohorts with progressive solid tumours (n = 19) received escalating doses of F16-IL2 [5-25 Million International Units (MIU) of IL2 equivalent dose] in combination with escalating doses of doxorubicin (0-25mg/m(2)) on day 1, 8 and 15 every 4 weeks. Subsequently, patients with metastatic breast cancer (n = 10) received the drug combination at the RD. Clinical data and laboratory findings were analysed for safety, tolerability, and activity. F16-IL2 could be administered up to 25 MIU, in combination with the RD of doxorubicin (25mg/m(2)). No human anti-fusion protein antibodies (HAFA) response was detected. Pharmacokinetics of F16-IL2 was dose-dependent over the tested range, with half-lives of ca. 13 and ca. 8 hours for cohorts dosed at lower and higher levels, respectively. Toxicities were controllable and reversible, with no combination treatment-related death. After 8 weeks, 57% and 67% disease control rates were observed for Phase I and II, respectively (decreasing to 43% and 33% after 12 weeks), considering 14 and 9 patients evaluable for efficacy. One patient experienced a long lasting partial response (45 weeks), still on-going at exit of study. F16-IL2 can be safely and repeatedly administered at the RD of 25 Mio I.U. in combination with 25mg/m(2) doxorubicin; its safety and activity are currently being investigated in combination with other chemotherapeutics, in order to establish optimal therapy settings.
    Cell adhesion & migration 01/2015; · 3.40 Impact Factor
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    ABSTRACT: Purpose: Radiotherapy (RT) modifies the tumor microenvironment and causes the release of tumor antigens, which can enhance the effect of immunotherapy (IT). L19 targets the extra domain B (ED-B) of fibronectin, a marker for tumor neo-angiogenesis, and can be used as immunocytokine when coupled to interleukin-2 (IL2). We hypothesize that RT in combination with L19-IL2 provides an enhanced antitumor effect, which is dependent on ED-B expression. Experimental Design: Mice were injected with syngeneic C51 colon carcinoma, Lewis lung carcinoma (LLC), or 4T1 mammary carcinoma cells. Tumor growth delay, underlying immunological parameters and treatment toxicity were evaluated after single-dose local tumor irradiation and systemic administration of L19-IL2 or equimolar controls. Results: ED-B expression was high, intermediate and low for C51, LLC and 4T1, respectively. The combination therapy showed (i) a long-lasting synergistic effect for the C51 model with 75% of tumors being cured, (ii) an additive effect for the LLC model, and (iii) no effect for the 4T1 model. The combination treatment resulted in a significantly increased cytotoxic (CD8+) T-cell population for both C51 and LLC. Depletion of CD8+ T-cells abolished the benefit of the combination therapy. Conclusion: These data provide the first evidence for an increased therapeutic potential by combining RT with L19-IL2 in ED-B positive tumors. This new opportunity in cancer treatment will be investigated in a Phase I clinical study for patients with an oligometastatic solid tumor (NCT02086721). Copyright © 2014, American Association for Cancer Research.
    Clinical Cancer Research 12/2014; · 8.19 Impact Factor
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    ABSTRACT: Background The antibody-mediated delivery of cytokines (“immunocytokines”) to sites of pathological angiogenesis represents an attractive strategy for the development of innovative biopharmaceuticals, capable of modulating the activity of the immune system in cancer and in chronic inflammatory conditions. Objective Recombinant IL4 has previously been shown to be therapeutically active in patients with psoriasis. The antibody-mediated delivery of this cytokine to sites of chronic skin inflammatory conditions should lead to an improved potency and selectivity, compared to non-targeted IL4. Methods The therapeutic activity of F8-IL4, a fusion protein of the F8 antibody (specific to the alternatively-spliced EDA domain of fibronectin) with murine IL4, was investigated in three immunocompetent mouse models of skin inflammation: two induced by the TLR7/8 ligand imiquimod (in Balb/c and C57BL/6) and one mediated by the over-expression of VEGF-A. Results The EDA domain of fibronectin, a marker for angiogenesis, is expressed in the inflamed skin in all three models and F8-IL4 selectively localized to inflamed skin lesions following intravenous administration. The F8-IL4 fusion protein mediated a therapeutic benefit, which was superior to the one of a non-targeted version of IL4 and led to increased levels of key regulatory cytokines (including IL5, IL10, IL13, and IL27) in the inflamed skin, while IL2 levels were not affected in all treatment groups. A murine version of etanercept and a murine anti-IL17 antibody were used as positive control in the therapy experiments. Conclusion Skin inflammatory lesions can be selectively targeted using anti-EDA antibody–cytokine fusion proteins and the pharmacodelivery of IL4 confers a therapeutic benefit by shifting the cytokine balance.
    Journal of Dermatological Science 11/2014; · 3.34 Impact Factor
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    ABSTRACT: Advances in the understanding of tumor immunology and molecular biology of melanoma cells have favored a larger application of immunotherapy and targeted therapies in the clinic. Several selective mutant gene inhibitors and immunomodulating antibodies have been reported to improve overall survival or progression-free survival in metastatic melanoma patients. However, despite impressive initial responses, patients treated with selective inhibitors relapse quickly, and toxicities associated to the use of immunomodulating antibodies are not easily manageable. In this sense, the concept of using antibodies as delivery vehicles for the preferential in vivo localization of the drug at the site of disease with reduction of side effects has raised particular interest. Antibody-cytokine fusion proteins (termed immunocytokines) represent a new simple and effective way to deliver the immunomodulatory payload at the tumor site, with the aim of inducing both local and systemic antitumoral immune responses and limiting systemic toxicities. Several clinical trials have been conducted and are actually ongoing with different immunocytokines, in several tumor histotypes. In metastatic melanoma patients, different drug delivery modalities such as systemic, loco-regional and intratumoral are under investigation. In this review, the rationale for the use of L19-IL2 and L19-TNF, two clinical stage immunocytokines produced by the Philogen group, as well as opportunities for their future development will be discussed.
    Cancer Immunology and Immunotherapy 10/2014; · 3.94 Impact Factor
  • The Israel Medical Association journal: IMAJ 10/2014; 16(10):666. · 0.90 Impact Factor
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    ABSTRACT: Fibrin formation from fibrinogen is a rare process in the healthy organism, but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones), for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal aminoacids of fibrin with high-affinity (Kd=44 nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, SIP and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors, but did not exhibit any detectable staining towards normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.
    Journal of Molecular Biology 10/2014; · 3.96 Impact Factor
  • Thomas List, Giulio Casi, Dario Neri
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    ABSTRACT: The combination of immunostimulatory agents with cytotoxic drugs is emerging as a promising approach for potentially curative tumor therapy, but advances in this field are hindered by the requirement of testing individual combination partners as single agents in dedicated clinical studies, often with suboptimal efficacy. Here we describe for the first time a novel multi-payload class of targeted drugs, the immunocytokine-drug conjugates (IDCs), which combine a tumor-homing antibody, a cytotoxic drug and a pro-inflammatory cytokine in the same molecular entity. In particular, the IL2 cytokine and the disulfide-linked maytansinoid DM1 microtubular inhibitor could be coupled to the F8 antibody, directed against the alternatively-spliced EDA domain of fibronectin, in a site-specific manner, yielding a chemically defined product with selective tumor homing performance and potent anticancer activity in vivo, as tested in two different immunocompetent mouse models.
    Molecular Cancer Therapeutics 09/2014; · 5.60 Impact Factor
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    ABSTRACT: Antibody-cytokine fusion proteins (immunocytokines) are innovative biopharmaceutical agents, which are being considered for the therapy of cancer and chronic inflammatory conditions. Immunomodulatory fusion proteins capable of selective localization at the sites of rheumatoid arthritis (RA) are of particular interest, as they may increase the therapeutic index of the cytokine payload. The F8 antibody recognizes the alternatively spliced extra domain A of fibronectin, a marker of angiogenesis, which is strongly overexpressed at sites of arthritis. In this study, we investigated the targeting and therapeutic activity of the immunocytokine F8-IL4 in the mouse model of collagen-induced arthritis. Different combination regimes were tested and evaluated by the analysis of serum and tissue cytokine levels. We show that F8-IL4 selectively localizes to neovascular structures at sites of rheumatoid arthritis in the mouse, leading to high local concentrations of IL4. When used in combination with dexamethasone, F8-IL4 was able to cure mice with established collagen-induced arthritis. Response to treatment was associated with an elevation of IL13 levels and decreased IL6 plasma concentrations. A fully human version of F8-IL4 is currently being developed for clinical investigations.
    Annals of the Rheumatic Diseases 08/2014; · 9.27 Impact Factor
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    ABSTRACT: DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.
    Bioconjugate Chemistry 07/2014; · 4.82 Impact Factor
  • Cancer Immunology and Immunotherapy 07/2014; · 3.94 Impact Factor
  • N. Krall, F. Pretto, D. Neri
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    ABSTRACT: There is a pressing need for the development of innovative chemical drug delivery strategies in oncology, since conventional chemotherapeutic agents typically do not localise to solid tumours in vivo. It is widely accepted that bivalent antibody formats accumulate in tumours more strongly than monovalent ones and that they should thus be preferred for antibody-based pharmacodelivery approaches. For small molecule-drug conjugates this is less clear. Here, we show that a bivalent ligand against the tumour marker carbonic anhydrase IX leads to an improved tumour-targeting performance compared with the corresponding monovalent counterpart in the SKRC52 model of constitutively CAIX-positive renal cell carcinoma. A bivalent disulfide-linked small drug conjugate with the potent cytotoxic maytansinoid DM1 as the payload can mediate complete eradication of the same tumours, which are resistant to standard-of-care therapeutics, in a proportion of treated mice. In the A375 melanoma model, which preferentially expresses CAIX at sites distant to blood vessels, no measurable tumour accumulation could be observed. Our results suggest that the use of bivalent small molecule ligand-drug conjugates against CAIX may represent an attractive chemical strategy for the treatment of constitutively CAIX-positive kidney cancer.
    Chemical Science 07/2014; · 8.60 Impact Factor
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    ABSTRACT: Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.
    PLoS ONE 06/2014; 9(6):e100000. · 3.53 Impact Factor
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    ABSTRACT: There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer.
    Experimental Cell Research 06/2014; · 3.37 Impact Factor
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    ABSTRACT: Antibody-cytokine fusion proteins (“immunocytokines”) represent a promising class of armed antibody products, which allow the selective delivery of potent pro-inflammatory payloads at the tumor site. The antibody-based selective delivery of interleukin-2 (IL2) is particularly attractive for the treatment of metastatic melanoma, an indication for which this cytokine received marketing approval from the US Food and drug administration. We used the K1735M2 immunocompetent syngeneic model of murine melanoma to study the therapeutic activity of F8–IL2, an immunocytokine based on the F8 antibody in diabody format, fused to human IL2. F8–IL2 was shown to selectively localize at the tumor site in vivo, following intravenous administration, and to mediate tumor growth retardation, which was potentiated by the combination with paclitaxel or dacarbazine. Combination treatment led to a substantially more effective tumor growth inhibition, compared to the cytotoxic drugs used as single agents, without additional toxicity. Analysis of the immune infiltrate revealed a significant accumulation of CD4+ T cells 24 h after the administration of the combination. The fusion proteins F8–IL2 and L19–IL2, specific to the alternatively spliced extra domain A and extra domain B of fibronectin respectively, were also studied in combination with tumor necrosis factor (TNF)-based immunocytokines. The combination treatment was superior to the action of the individual immunocytokines and was able to eradicate neoplastic lesions after a single intratumoral injection, a procedure that is being clinically used for the treatment of Stage IIIC melanoma. Collectively, these data reinforce the rationale for the use of IL2-based immunocytokines in combination with cytotoxic agents or TNF-based immunotherapy for the treatment of melanoma patients.
    Cancer Immunology and Immunotherapy 06/2014; · 3.94 Impact Factor

Publication Stats

9k Citations
1,718.87 Total Impact Points

Institutions

  • 2014
    • Ijuí Charity Hospital
      Ijuhy, Rio Grande do Sul, Brazil
  • 1997–2014
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland
  • 2006–2013
    • VU University Medical Center
      • Department of Otolaryngology/Head and Neck Surgery
      Amsterdamo, North Holland, Netherlands
    • University of Liège
      • Metastasis Research Laboratory
      Liège, WAL, Belgium
  • 1989–2012
    • ETH Zurich
      • • Institute of Pharmaceutical Sciences
      • • Department of Chemistry and Applied Biosciences
      • • Institute of Molecular Biology and Biophysics
      Zürich, ZH, Switzerland
  • 2011
    • Universitätsklinikum Tübingen
      Tübingen, Baden-Württemberg, Germany
  • 1991–2010
    • Hochschule für Technik Zürich
      Zürich, Zurich, Switzerland
  • 2008
    • National Research Council
      • Institute of Biomedical Technologies ITB
      Roma, Latium, Italy
  • 2003
    • Università degli Studi di Genova
      • Dipartimento di Medicina sperimentale (DIMES)
      Genova, Liguria, Italy
  • 1998–2000
    • Università degli Studi di Siena
      • Department of Medicine, Surgery and Neuroscience
      Siena, Tuscany, Italy