Idar-Oberstein, Germany
Idar-Oberstein, Germany

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Boehrer S.,University Paris XIII | Schroeder P.,EUFETS GmbH | Mueller T.,Fresenius Biotech GmbH | Atz J.,Fresenius Biotech GmbH
Anti-Cancer Drugs | Year: 2011

Monoclonal antibodies such as rituximab and alemtuzumab show considerable therapeutic efficacy in chronic lymphocytic leukaemia (CLL). Aiming to further improve antineoplastic efficacy, the trifunctional bispecific antibody FBTA05 was developed. FBTA05 is thought to function by simultaneously binding B cells and T cells by its variable regions and by recruiting FcγR-positive accessory immune cells by its intact Fc region. As it was previously shown that this antibody shows considerable cytotoxicity towards a spectrum of B-cell lymphoma cell lines, we here tested its potential efficacy ex vivo against malignant B-CLL cells. Therefore, we assessed the capacity of increasing concentrations of FBTA05 to bind to neoplastic cells, to induce cytotoxicity (comparing it with rituximab and alemtuzumab) and cytokine release. We evaluated the results with respect to the extent of CD20 expression, the effector:target cell ratio as well as with the patients' overall effector cell status. Thus, we show that, although FBTA05-elicited cytotoxicity was comparable with that induced by alemtuzumab, it considerably exceeded the antineoplastic effects of rituximab. Noteworthy, FBTA05 shows effective elimination of malignant B cells even if CD20 surface expression is low. Importantly, a high grade of cytotoxicity was associated with the induction of T-cell proliferation and the concomittant release of interferon-γ and interleukin-6, thus overcoming the detrimental effects of an unfavourable effector:target cell ratio. In conclusion, we here present novel evidence for the therapeutic efficacy of the trifunctional, bispecific antibody FBTA05 in CLL and provide evidence for the importance of immune-mediated mechanisms conveying the cytotoxic effects against malignant B lymphocytes. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.


Thinon E.,Imperial College London | Thinon E.,Rockefeller University | Serwa R.A.,Imperial College London | Broncel M.,Imperial College London | And 10 more authors.
Nature Communications | Year: 2014

Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells. © 2014 Macmillan Publishers Limited. All rights reserved.


Stein S.,Institute for Biomedical Research | Ott M.G.,University Medical School | Schultze-Strasser S.,Institute for Biomedical Research | Jauch A.,University of Heidelberg | And 27 more authors.
Nature Medicine | Year: 2010

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia. © 2010 Nature America, Inc. All rights reserved.


Braun C.J.,Ludwig Maximilians University of Munich | Boztug K.,Hannover Medical School | Boztug K.,Austrian Academy of Sciences | Paruzynski A.,German Cancer Research Center | And 27 more authors.
Science Translational Medicine | Year: 2014

Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.


Loew R.,EUFETS GmbH | Loew R.,University of Heidelberg | Heinz N.,EUFETS GmbH | Heinz N.,Hannover Medical School | And 4 more authors.
BMC Biotechnology | Year: 2010

Background: The performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state.Results: In this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems.Conclusions: Until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system. © 2010 Loew et al; licensee BioMed Central Ltd.


Briesemeister D.,Institute of Immunology | Sommermeyer D.,Max Delbrück Center for Molecular Medicine | Loddenkemper C.,Institute of Pathology | Loew R.,Eufets GmbH | And 5 more authors.
International Journal of Cancer | Year: 2011

It has been shown that injecting a suspension of IFN-Iγ-secreting tumor cells results in their rejection. This effect has been attributed to IFN-Iγ preventing tumor stroma formation but not to a direct effect on the cancer cells. However, it is not known, which influence IFN-Iγ has on tumors with an established stroma. To address this question, the plasmacytoma cell line J558L was transduced with a vector allowing doxycycline-inducible IFN-Iγ gene expression. After the injection of the tumor cells into mice, IFN-Iγ was induced at different time points. Tumors did not grow when inducing IFN-Iγ immediately after tumor cell inoculation, while approximately half of the tumors were rejected when IFN-Iγ was induced in early established tumors within 2 weeks. Induction of IFN-Iγ 2-3 weeks after tumor cell inoculation was less efficient (0-17% rejection). IFN-Iγ induction in established tumors led to a reduction of CD146+ endothelial cells and massive necrosis. Together, we show that vascularized tumors can be rejected by local IFN-Iγ expression, but that rejection of established tumors was less efficient over time. This suggests that transplanted tumors became less susceptible to local IFN-Iγ treatment the better they are established. Copyright © 2010 UICC.


Heinz N.,Hannover Medical School | Schambach A.,Hannover Medical School | Galla M.,Hannover Medical School | Maetzig T.,Hannover Medical School | And 3 more authors.
Human Gene Therapy | Year: 2011

The regulated expression of therapeutic genes may become crucial in gene therapy when their constitutive expression interferes with cell fate in vivo. The efficient regulation of transgene expression requires tightly controlled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its application requires the introduction of two components into the target cell genome: the tet-responsive transactivator and the regulated expression cassette. In order to facilitate the usage of the tet-system for approaches in gene therapy, both components have to be transferred by a single vector, thus eliminating the preselection of transactivator positive cells. Published "all-in-one" vectors for regulated transgene expression display a relatively low signal-to-noise ratio, resulting in regulatory windows of around 500-fold even in selected clones. In this study, we show that a modified vector architecture combined with the introduction of new tet-responsive promoters, Ptet, improved the dynamic range of such all-in-one vectors to levels up to 14,000-fold for viral and 25,000-fold for nonviral transfer vectors in nonclonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improved regulation was the result of a strong reduction of background expression in the off-state, even if cells were transduced at high multiplicity of infection, while induction remained at high levels. In addition, the results indicated that successful regulation of gene expression in different target cells depended on vector architecture as well as the choice of the Ptet-promoter. © 2011, Mary Ann Liebert, Inc.


Maetzig T.,Hannover Medical School | Galla M.,Hannover Medical School | Brugman M.H.,Hannover Medical School | Loew R.,EUFETS AG | And 3 more authors.
Gene Therapy | Year: 2010

Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O 6-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression. © 2010 Macmillan Publishers Limited All rights reserved.


Heinz N.,EUFETS GmbH | Heinz N.,Hannover Medical School | Hennig K.,EUFETS GmbH | Loew R.,EUFETS GmbH
BMC Biotechnology | Year: 2013

Background: Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an " All-In-One" vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral " All-In-One" vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter.Results: Determination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose-response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence.Conclusion: Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations. © 2013 Niels et al.; licensee BioMed Central Ltd.


Dettmar K.,Fresenius Biotech GmbH | Seitz-Merwald I.,Fresenius Biotech GmbH | Lindemann C.,EUFETS GmbH | Schroeder P.,EUFETS GmbH | And 2 more authors.
Clinical and Translational Oncology | Year: 2012

Introduction In patients, a transient decrease in peripheral blood lymphocyte counts was observed following intraperitoneal administration of the trifunctional monoclonal antibody catumaxomab (anti-human EpCAM x anti-human CD3). The aim of this study was to clarify the observed effect in a preclinical mouse model and to analyse the related mechanism of action in vitro. Materials and methods A related antibody, BiLu (antihuman EpCAM x anti-mouse CD3), was administered to mice and blood leukocytes were analysed. In vitro studies measured activation and cytokine secretion from human peripheral blood mononuclear cells (PBMC). For the analysis of T cell adhesion, PBMC were preincubated with catumaxomab and then co-cultured with human endothelial cells (HUVEC); T cell adhesion was assessed in the presence or absence of endothelial cell preactivation by TNFα. Adherent T cells were determined by flow cytometry. Results Treatment of mice with BiLu resulted in a dosedependent transient decrease in CD3+ T cells (both CD4+ and CD8+) that returned to the normal range within 48 h. Catumaxomab physiologically activated T cells in vitro (increased CD69 expression) and induced cytokine release (TNFα, IFNγ). TNFα increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently enhanced adhesion of T cells to endothelial cells. Adhesion was further increased when endothelial cells were preactivated with TNFα. Conclusions Catumaxomab increases adhesion of T cells to endothelial cells due to antibody-mediated activation of T cells and production of T cell cytokines that up-regulate endothelial cell adhesion molecules. These results provide a mechanistic rationale for the transient, reversible decrease in lymphocyte counts observed following catumaxomab administration in patients, which is likely to be due to redistribution of lymphocytes.

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