Dario Neri

Eawag: Das Wasserforschungs-Institut des ETH-Bereichs, Duebendorf, Zurich, Switzerland

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Publications (317)1799.2 Total impact

  • Jörg Scheuermann, Dario Neri
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    ABSTRACT: In contrast to single-pharmacophore DNA-encoded libraries, where only one chemical moiety is linked to DNA, dual-pharmacophore DNA-encoded chemical libraries feature the display of two independent small-molecules in close proximity. This, in principle, allows to explore adjacent epitopes on a pharmaceutical target of choice and hence the discovery of simultaneously binding pairs of fragments, by virtue of the chelate effect. Copyright © 2015. Published by Elsevier Ltd.
    Current Opinion in Chemical Biology 06/2015; 26. DOI:10.1016/j.cbpa.2015.02.021 · 7.65 Impact Factor
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    ABSTRACT: The extracellular matrix protein ED-B fibronectin (ED-B) is upregulated in inflammatory atherosclerotic lesions. However, functional in vivo imaging of ED-B-containing plaques has not been explored. This study evaluated whether [99mTc]-conjugated AP39 ([99mTc]-AP39), a single-chain antibody specific to ED-B, can be used for in vivo detection of atherosclerotic plaques in Western diet (WD)-fed, apolipoprotein E-deficient (apoE-/-) mice as compared to wildtype (WT) control mice. Using SPECT, 12-month-old WD-fed apoE-/- and WT mice were studied 4 hours after injecting [99mTc]-AP39 (148 MBq). Subsequently, mice were sacrificed, thoracic aortas measured in a g-counter, and plaques analyzed using histology, immuno-histochemistry, autoradiography, and morphometry. In vivo [99mTc]-AP39-SPECT imaging of apoE-/- mice demonstrated a significant signal activity in the plaque-ridden thoracic aorta (52.236±40.646 cpm/cm3) that co-localized with the aortic arch and the supra-aortic arteries in MRI scans. Low signal activity (9.468±4.976 cpm/cm3) was observed in WT mice. In apoE-/- mice, the strongest signals were detected in the aortic root, aortic arch and along the abdominal aorta. Autoradiography analysis of aortas from apoE-/- mice confirmed the in vivo observation by demonstrating signal localization in atherosclerotic plaques. The size of autoradiography-positive plaque areas correlated significantly with the size of ED-B-positive (r=0.645, P=0.044) or macrophage-infiltrated (r=0.84, P<0.002) plaques. A significant correlation was found between the sizes of ED-B-positive and macrophage-infiltrated plaque areas (r=0.93, P<0.01). [99mTc]-AP39-SPECT in vivo imaging detects inflammatory plaque lesions in WD-fed apoE-/- mice.
    The quarterly journal of nuclear medicine and molecular imaging: official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of.. 06/2015; 59(2):228-37. · 1.72 Impact Factor
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    ABSTRACT: There is increasing interest in the site-directed pharmacodelivery of therapeutic payloads to the tumor site using antibodies as transport vehicles. Here, we investigated the efficacy of L19-IL2, an antibody-cytokine fusion protein that specifically delivers IL-2 to the tumor site by homing to the extra-domain B of fibronectin (EDB-Fn) expressed on tumor-associated blood vessels, against mantle cell lymphoma (MCL) in mice. L19-IL2 was shown to selectively localize at lymphoma lesions in vivo and to mediate significant lymphoma growth retardation, which was potentiated by co-administration of the anti-CD20 antibody rituximab. When co-injected with rituximab, L19-IL2 induced complete remissions of localized MCL xenografts in 6/8 mice (75%), whereas the combination of rituximab and equivalent doses of non-targeted IL-2 only slightly delayed tumor growth. In disseminated MCL, combination therapy with L19-IL2 and rituximab exhibited a significant survival benefit over treatment with IL-2 and rituximab and completely eradicated the disease in 2/7 cases (28.6%). Mechanistically, histological analyses of post-therapeutic lymphoma tissues revealed a strong intratumoral accumulation of macrophages and natural killer cells after a single dose of the immunocytokine, whereas L19-IL2 had no significant impact on microvessel density or on tissue penetration of co-injected rituximab. Collectively, these results provide the scientific rationale for the clinical evaluation of L19-IL2 in combination with anti-CD20 immunotherapy in patients with MCL.
  • Jörg Scheuermann, Dario Neri
    Current opinion in chemical biology 04/2015; DOI:10.1016/j.cbpa.2015.03.014 · 7.65 Impact Factor
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    ABSTRACT: Endometriosis is caused by the displacement of endometrium outside the uterus contributing heavily to infertility and debilitating pelvic pain. Ectopic adhesion and growth are believed to occur under the influence of a favorable hormonal environment and immunological factors. The objective of this study is to analyze the effect of a targeted therapy with an antibody-based pharmacodelivery of interleukin 4 (F8-IL4) in a mouse model of experimentally induced endometriosis. Endometriosis-like lesions were induced in Balb/c mice. The animals were treated intravenously with F8-IL4 or with untargeted IL4 (KSF-IL4). Twelve days after disease induction, the lesions were isolated. A significant reduction in the number of total lesions/mouse and in the total volume of lesions/mouse was observed in mice treated with F8-IL4 compared to controls (P = .029 and P = .006, respectively), while no difference was found between KSF-IL4-treated mice and their controls. Gene expression was evaluated by quantitative real-time polymerase chain reaction. Expression of genes involved in cell adhesion, extracellular matrix invasion, and neovascularization was significantly downregulated in F8-IL4-treated mice compared to their controls (integrin β1: P = .02; metalloproteinase [MMP] 3: P = .02; MMP9: P = .04; vascular endothelial growth factor: P = .04). Gene expression of inflammatory cytokines (tumor necrosis factor α, IL1β, IL1α, and IL6) did not vary in the ectopic lesions isolated from F8-IL4-treated mice compared to their controls. Immunohistochemistry demonstrated a significantly reduced expression of E-cadherin and β-catenin in the lesions of mice treated with F8-IL4. Our results show that the antibody-mediated targeted delivery of IL4 inhibits the development of endometriosis in a syngeneic mouse model by likely impairing adhesion, invasion, and vascularization of the ectopic endometrium. © The Author(s) 2015.
    Reproductive sciences (Thousand Oaks, Calif.) 04/2015; DOI:10.1177/1933719115578930 · 2.18 Impact Factor
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    ABSTRACT: Parallel affinity screening of a DNA-encoded chemical library against rat, bovine and human serum albumin allowed the identification of small-molecule ligands with distinctive binding specificities to the individual proteins.
    Chemical Communications 03/2015; DOI:10.1039/c5cc01230a · 6.72 Impact Factor
  • Giulio Casi, Dario Neri
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    ABSTRACT: Conventional cancer chemotherapy is limited by the fact that small organic cytotoxic agents typically do not preferentially localize at the tumor site, causing unwanted toxicities to normal organs and limiting dose escalation to therapeutically active regimens. In principle, antibodies and other ligands could be used for the selective pharmacodelivery of cytotoxic agents to the tumor environment. While traditionally internalizing ligands have been used for such targeting applications, increasing experimental evidence suggests that the ligand-based delivery of anti-cancer drugs to the extracellular space in the tumor, followed by suitable release strategies, may mediate a potent anti-cancer activity. In this review, we outline the main requirements for the development of non-internalizing targeted cytotoxics.
    Molecular Pharmaceutics 03/2015; DOI:10.1021/mp500798y · 4.79 Impact Factor
  • 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.
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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.
    02/2015; DOI:10.1158/2326-6066.CIR-14-0179
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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; 127(13). DOI:10.1002/ange.201410736
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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.
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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.
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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. DOI:10.1038/nchem.2158 · 23.30 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; DOI:10.4161/19336918.2014.983785 · 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; DOI:10.1158/1078-0432.CCR-14-2676 · 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; 76(2). DOI:10.1016/j.jdermsci.2014.07.012 · 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; 64(1). DOI:10.1007/s00262-014-1621-0 · 3.94 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; 426(21). DOI:10.1016/j.jmb.2014.07.023 · 3.96 Impact Factor
  • The Israel Medical Association journal: IMAJ 10/2014; 16(10):666. · 0.90 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; 13(11). DOI:10.1158/1535-7163.MCT-14-0599 · 6.11 Impact Factor

Publication Stats

10k Citations
1,799.20 Total Impact Points

Institutions

  • 1997–2014
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland
    • ETH Zurich
      • • Institute of Pharmaceutical Sciences
      • • Department of Chemistry and Applied Biosciences
      • • Institute of Molecular Biology and Biophysics
      Zürich, Zurich, Switzerland
  • 2013
    • VU University Medical Center
      • Department of Otolaryngology/Head and Neck Surgery
      Amsterdamo, North Holland, Netherlands
  • 2008
    • National Research Council
      • Institute of Biomedical Technologies ITB
      Roma, Latium, Italy
  • 2006
    • University of Liège
      • Metastasis Research Laboratory
      Liège, WAL, Belgium
  • 1999–2006
    • University of Zurich
      Zürich, Zurich, Switzerland
  • 2004
    • HELIOS Klinikum Erfurt
      Erfurt, Thuringia, Germany
  • 2003
    • Università degli Studi di Genova
      • Dipartimento di Medicina sperimentale (DIMES)
      Genova, Liguria, Italy
  • 1991–2003
    • Hochschule für Technik Zürich
      Zürich, Zurich, Switzerland
  • 2002
    • Universitätsklinikum Jena
      Jena, Thuringia, Germany
  • 1997–2002
    • Università degli Studi di Siena
      • Department of Medicine, Surgery and Neuroscience
      Siena, Tuscany, Italy
  • 1996
    • Medical Research Council (UK)
      Londinium, England, United Kingdom