04 Fakultät Energie-, Verfahrens- und Biotechnik

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    Targeting co-stimulatory receptors of the TNF superfamily for cancer immunotherapy
    (2022) Müller, Dafne
    The clinical approval of immune checkpoint inhibitors is an important advancement in the field of cancer immunotherapy. However, the percentage of beneficiaries is still limited and it is becoming clear that combination therapies are required to further enhance the treatment efficacy. The potential of strategies targeting the immunoregulatory network by “hitting the gas pedal” as opposed to “blocking the brakes” is being recognized and intensively investigated. Hence, next to immune checkpoint inhibitors, agonists of co-stimulatory receptors of the tumor necrosis factor superfamily (TNF-SF) are emerging as promising options to expand the immunomodulatory toolbox. In this review the development of different categories of recombinant antibody and ligand-based agonists of 4-1BB, OX40, and GITR is summarized and discussed in the context of the challenges presented by the structural and mechanistical features of the TNFR-SF. An overview of current formats, trends, and clinical studies is provided.
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    Indolyl-chalcone derivatives trigger apoptosis in cisplatin-resistant mesothelioma cells through aberrant tubulin polymerization and deregulation of microtubule-associated proteins
    (2023) Steinlein, Sophia; Essmann, Frank; Ghilardi, Amanda Franceschini; Horn, Heike; Schüler, Julia; Hausser, Angelika; Sun, Lijun; Ott, German; Kalla, Claudia
    Malignant pleural mesothelioma (MPM) is a neoplasm with dismal prognosis and notorious resistance to the standard therapeutics cisplatin and pemetrexed. Chalcone derivatives are efficacious anti-cancer agents with minimal toxicity and have, therefore, gained pharmaceutical interest. Here, we investigated the efficacy of CIT-026 and CIT-223, two indolyl-chalcones (CITs), to inhibit growth and viability of MPM cells and defined the mechanism by which the compounds induce cell death. The effects of CIT-026 and CIT-223 were analyzed in five MPM cell lines, using viability, immunofluorescence, real-time cell death monitoring, and tubulin polymerization assays, along with siRNA knockdown. Phospho-kinase arrays and immunoblotting were used to identify signaling molecules that contribute to cell death. CIT-026 and CIT-223 were toxic in all cell lines at sub-micromolar concentrations, in particular in MPM cells resistant to cisplatin and pemetrexed, while normal fibroblasts were only modestly affected. Both CITs targeted tubulin polymerization via (1) direct interaction with tubulin and (2) phosphorylation of microtubule regulators STMN1, CRMP2 and WNK1. Formation of aberrant tubulin fibers caused abnormal spindle morphology, mitotic arrest and apoptosis. CIT activity was not reduced in CRMP2-negative and STMN1-silenced MPM cells, indicating that direct tubulin targeting is sufficient for toxic effects of CITs. CIT-026 and CIT-223 are highly effective inducers of tumor cell apoptosis by disrupting microtubule assembly, with only modest effects on non-malignant cells. CITs are potent anti-tumor agents against MPM cells, in particular cells resistant to standard therapeutics, and thus warrant further evaluation as potential small-molecule therapeutics in MPM.
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    Mechanosensory feedback loops during chronic inflammation
    (2023) Saha, Sarbari; Müller, Dafne; Clark, Andrew G.
    Epithelial tissues are crucial to maintaining healthy organization and compartmentalization in various organs and act as a first line of defense against infection in barrier organs such as the skin, lungs and intestine. Disruption or injury to these barriers can lead to infiltration of resident or foreign microbes, initiating local inflammation. One often overlooked aspect of this response is local changes in tissue mechanics during inflammation. In this mini-review, we summarize known molecular mechanisms linking disruption of epithelial barrier function to mechanical changes in epithelial tissues. We consider direct mechanisms, such as changes in the secretion of extracellular matrix (ECM)-modulating enzymes by immune cells as well as indirect mechanisms including local activation of fibroblasts. We discuss how these mechanical changes can modulate local immune cell activity and inflammation and perturb epithelial homeostasis, further dysregulating epithelial barrier function. We propose that this two-way relationship between loss of barrier function and altered tissue mechanics can lead to a positive feedback loop that further perpetuates inflammation. We discuss this cycle in the context of several chronic inflammatory diseases, including inflammatory bowel disease (IBD), liver disease and cancer, and we present the modulation of tissue mechanics as a new framework for combating chronic inflammation.
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    Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol
    (2021) Hellwig, Christian T.; Delgado, M. Eugenia; Skoko, Josip; Dyck, Lydia; Hanna, Carol; Wentges, Alexa; Langlais, Claudia; Hagenlocher, Cathrin; Mack, Alexandra; Dinsdale, David; Cain, Kelvin; MacFarlane, Marion; Rehm, Markus
    Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.
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    Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids
    (2020) Stöhr, Daniela; Schmid, Jens O.; Beigl, Tobias B.; Mack, Alexandra; Maichl, Daniela S.; Cao, Kai; Budai, Beate; Fullstone, Gavin; Kontermann, Roland E.; Mürdter, Thomas E.; Tait, Stephen W. G.; Hagenlocher, Cathrin; Pollak, Nadine; Scheurich, Peter; Rehm, Markus
    The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.
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    Membrane trafficking in breast cancer progression : protein kinase D comes into play
    (2023) Gutiérrez-Galindo, Elena; Yilmaz, Zeynep Hazal; Hausser, Angelika
    Protein kinase D (PKD) is a serine/threonine kinase family that controls important cellular functions, most notably playing a key role in the secretory pathway at the trans-Golgi network. Aberrant expression of PKD isoforms has been found mainly in breast cancer, where it promotes various cellular processes such as growth, invasion, survival and stem cell maintenance. In this review, we discuss the isoform-specific functions of PKD in breast cancer progression, with a particular focus on how the PKD controlled cellular processes might be linked to deregulated membrane trafficking and secretion. We further highlight the challenges of a therapeutic approach targeting PKD to prevent breast cancer progression.
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    Role of peripheral immune cells for development and recovery of chronic pain
    (2021) Bethea, John R.; Fischer, Roman
    Chronic neuropathic pain (CNP) is caused by a lesion or disease of the somatosensory nervous system. It affects ~8% of the general population and negatively impacts a person's level of functioning and quality of life. Its resistance to available pain therapies makes CNP a major unmet medical need. Immune cells have been shown to play a role for development, maintenance and recovery of CNP and therefore are attractive targets for novel pain therapies. In particular, in neuropathic mice and humans, microglia are activated in the dorsal horn and peripheral immune cells infiltrate the nervous system to promote chronic neuroinflammation and contribute to the initiation and progression of CNP. Importantly, immunity not only controls pain development and maintenance, but is also essential for pain resolution. In particular, regulatory T cells, a subpopulation of T lymphocytes with immune regulatory function, and macrophages were shown to be important contributors to pain recovery. In this review we summarize the interactions of the peripheral immune system with the nervous system and outline their contribution to the development and recovery of pain.
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    Influence of antigen density and immunosuppressive factors on tumor-targeted costimulation with antibody-fusion proteins and bispecific antibody-mediated T cell response
    (2020) Sapski, Sabrina; Beha, Nadine; Kontermann, Roland E.; Müller, Dafne
    Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-β, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.
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    Applying a GAN-based classifier to improve transcriptome-based prognostication in breast cancer
    (2023) Guttà, Cristiano; Morhard, Christoph; Rehm, Markus
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    IL-15-based trifunctional antibody-fusion proteins with costimulatory TNF-superfamily ligands for cancer immunotherapy
    (2018) Beha, Nadine; Kontermann, Roland (Prof. Dr.)
    IL-15 shows great potential to support an antitumor immune response and emerges as a promising agent in cancer immunotherapy. However, the systemic application of IL-15 is associated with toxicity and, as a monotherapy the efficacy of IL-15 is still limited. This study focusses on the development of novel trifunctional fusion proteins enforcing the activity of IL-15 with costimulatory ligands of the TNF superfamily and targeting the therapeutic activity to the tumor site by an antibody moiety. The homotrimeric trifunctional fusion proteins of the first generation was comprised of an antibody moiety (scFv), IL-15 fused to the extended sushi domain of the IL-15Rα chain (RD), and the extracellular domain (ECD) of 4-1BBL. Non-covalent trimerization of the ECD of 4-1BBL led to a homotrimeric fusion protein with three antibody moieties and three RD_IL-15 units. Based on the first generation trifunctional fusion protein, a novel second generation trifunctional fusion protein incorporating the ligand of the TNF superfamily in the single-chain format, i.e. genetic fusion of three extracellular domains by linkers on the same polypeptide chain, was generated, resulting in a monomeric trifunctional fusion protein with only one functional unit of each component. Similar T cell stimulation in a non-targeted setting, even improved capacity to enhance T cell stimulation when target bound and a clear antitumor effect in a mouse model in vivo was observed for the novel trifunctional fusion protein in the single-chain format. Furthermore, OX40L and GITRL were successfully incorporated into the novel trifunctional fusion protein in the single-chain format demonstrating stable protein configuration. Advantageous costimulatory properties in comparison to the combination of the respective bifunctional fusion proteins were observed for all trifunctional fusion proteins. Strongest synergistic effects were shown for RD_IL-15_scFvFAP_scGITRL in terms of enhancing the cytotoxic potential of CD8+ T cells and enhanced proliferation of CD4+ T cells. Finally, in a syngeneic lung tumor mouse model evaluating the antitumor potential of RD_IL-15_scFvFAP_scGITRL revealed a strong, targeting-dependent antitumor response. Additionally, the effect of an EGFR-directed trifunctional fusion protein on Trastuzumab-mediated ADCC was evaluated. Strong enhancement of the ADCC was achieved by the trifunctional fusion protein RD_IL-15_scFvEGFR_sc4-1BBL and the bifunctional fusion protein RD_IL-15_scFvEGFR. Thus, the trifunctional fusion protein format incorporating the ligand of the TNF superfamily in the single-chain format appears as a promising platform with versatile opportunities for further development.