04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Development of novel bispecific antibodies for cancer therapy targeting the receptor tyrosine kinases HER4 and EGFR(2024) Kühl, Lennart; Kontermann, Roland E. (Prof. Dr.)In this study, novel mono- and bispecific antibodies targeting the ErbB receptor family members EGFR and HER4 were investigated. Dual targeting of EGFR and HER4 by a bispecific, tetravalent antibody comprising a novel, antagonistic HER4-targeting antibody showed inhibition of proliferation and migration for a HB-EGF-stimulated ovarian cancer cell line. No inhibitory effects in a breast cancer cell line expressing EGFR and HER4 indicated that successful dual targeting does not solely rely on target expression. The complexity of HER4 with its isoforms and their different signaling properties makes HER4 a challenging cancer target that needs further in-depth research. To overcome resistances based on escape mutations located in the epitopes of clinically approved antibodies, novel antagonistic EGFR-targeting antibodies binding to a different epitope were developed. This epitope was mapped to domain III of EGFR and binding to clinically relevant EGFR ectodomain mutations resulted in inhibition of EGFR signaling in stable cell lines used as test systems. Favorable activities in comparison to clinically approved antibodies regarding inhibition of EGFR signaling and proliferation were observed for cancer cell lines expressing the EGFR wildtype. Bispecific T-cell engagers can lead to a T-cell mediated target cell killing independent of intracellular downstream signaling in the cancer cell. One challenge for the applicability of T-cell engagers in solid tumors is to keep the balance between T-cell mediated tumor cell killing and severe side-effects caused by a systemic activation of the immune system. Studies on eleven different eIg-based formats for EGFR-binding T-cell engagers showed that valency, geometry, and size influenced their activity profile. Furthermore, one bivalent and one trivalent, bispecific format were investigated for two novel EGFR-targeting moieties. As these molecules bind to clinically relevant escape mutations located in the ectodomain of EGFR, they are expected to show activity in patients with an acquired resistance to approved EGFR-targeting antibodies. These molecules led to a robust T-cell mediated cytotoxicity of cancer cells expressing EGFR. Additionally, benefits regarding an EGFR-level dependent cytotoxicity were observed for reduced binding to EGFR. An initial in vivo study using surrogate molecules in a syngeneic mouse model showed reduction of tumor growth and prolonged survival for treatment with a trivalent, bispecific T-cell engager comprising a novel EGFR-binding moiety. Taken together, beneficial effects of the novel molecules may contribute to improved therapies for patients with both pre-existing and acquired resistances to EGFR-targeting antibodies.Item Open Access Transcriptional CDK inhibitors CYC065 and THZ1 induce apoptosis in glioma stem cells derived from recurrent GBM(2021) Juric, Viktorija; Düssmann, Heiko; Lamfers, Martine L. M.; Prehn, Jochen H. M.; Rehm, Markus; Murphy, Brona M.Glioma stem cells (GSCs) are tumour initiating cells which contribute to treatment resistance, temozolomide (TMZ) chemotherapy and radiotherapy, in glioblastoma (GBM), the most aggressive adult brain tumour. A major contributor to the uncontrolled tumour cell proliferation in GBM is the hyper activation of cyclin-dependent kinases (CDKs). Due to resistance to standard of care, GBMs relapse in almost all patients. Targeting GSCs using transcriptional CDK inhibitors, CYC065 and THZ1 is a potential novel treatment to prevent relapse of the tumour. TCGA-GBM data analysis has shown that the GSC markers, CD133 and CD44 were significantly upregulated in GBM patient tumours compared to non-tumour tissue. CD133 and CD44 stem cell markers were also expressed in gliomaspheres derived from recurrent GBM tumours. Light Sheet Florescence Microscopy (LSFM) further revealed heterogeneous expression of these GSC markers in gliomaspheres. Gliomaspheres from recurrent tumours were highly sensitive to transcriptional CDK inhibitors, CYC065 and THZ1 and underwent apoptosis while being resistant to TMZ. Apoptotic cell death in GSC subpopulations and non-stem tumour cells resulted in sphere disruption. Collectively, our study highlights the potential of these novel CKIs to induce cell death in GSCs from recurrent tumours, warranting further clinical investigation.Item Open Access Overcoming glioblastoma intractability : pre-clinical characterisation of TRAIL sensitisation by marizomib and novel treatment perspectives(2022) Boccellato, Chiara; Morrison, Markus (Prof. Dr.)Glioblastoma (GBM) is the most aggressive cancer of the central nervous system (CNS). Surgical resection, adjuvant temozolomide-based chemotherapy and radiation are the primary treatments, yet the outcome of GBM patients remains poor with a median life expectancy of 15 to 17 months. Therefore, novel and effective treatment options are required, as are reliable pre-clinical experimental models that are suitable for exploratory studies on novel drugs and drug combinations. In this work, patient-derived cell line models (PDCL), generated from fresh primary or recurrent glioblastoma tumours, have been examined to assess prevalence of responsiveness to a highly stable hexavalent format of TRAIL receptor agonist (IZI1551) and to the blood brain barrier (BBB)-permeant proteasome inhibitor marizomib (MRZ). Serum-free medium and limited cultivation times of both 2D and 3D cancer cell cultures were adopted to maintain the characteristics of primary tumour cells. The degree of BBB permeability of marizomib was evaluated in the human hCMEC/D3 cell line model, which was also employed to test the efficacy of the IZI1551+MRZ combination in pre-clinical settings. It was found that IZI1551 and marizomib acted synergistically to induce apoptosis in the majority of low-passage PDCLs, both under 2D and 3D cultivation conditions. Altering the relative timing of drug exposure, specifically marizomib pre-treatment, led to even enhanced responses and allowed to lower drug concentrations without losing treatment efficacy. Importantly, the amount of marizomib that can cross the simple BBB model was sufficient to confer sensitisation to IZI1551. In cases of treatment resistance against IZI1551 and marizomib, lowering the mitochondrial apoptosis threshold with BH3 mimetics appeared sufficient to restore apoptosis sensitivity. Taken together, these results demonstrated that marizomib is a potent sensitiser of apoptosis induced by a 2nd generation TRAIL receptor agonist in glioblastoma. The optimized synergism between marizomib and IZI1551 in time-shifted treatment schedules, together with the ability of marizomib to cross the BBB, suggests this combination as a promising strategy to be tested in clinical settings. In the second part of this work, an alternative cell death pathway, namely ferroptosis, has been investigated as a strategy to bypass the obstacle of the apoptosis refractory state of highly resistant cancers such as glioblastoma. Ferroptosis is a recently identified form of iron-dependent regulated cell death that presents distinct features compared to apoptosis and that is characterised by the accumulation of toxic lipid peroxides. Here it was shown that the U-87 MG, a bona-fide glioblastoma cell line that was reported to be TRAIL resistant, displays a dose-dependent cell death response to the ferroptosis inducer RSL3. Surprisingly, it was found that BH3 mimetics antagonised this cytotoxicity. The unexpected consequences of combining these agents highlight the need to better understand the interactions between these drugs in order to advance their use as cancer therapeutics. Overall, this thesis presents diverse treatment options against glioblastoma that exploit either drugs classically inducing apoptosis or the alternative cell death modality of ferroptosis. Considering the limited availability of approved treatments, studies aiming at expanding the choice of glioblastoma therapeutics, such as those conducted in this work, are of particular importance and pave the way for their implementation at a clinical and pre-clinical level.Item Open Access Population and single-cell based quantitative analysis of protein kinase D-mediated regulation of the cell cycle(2014) Räth, Sebastian; Pfizenmaier, Klaus (Prof. Dr.)The cell cycle consists of G1, G2, S, and M phase and is a tightly regulated process with various checkpoints to control order and length of the separate phases. A multitude of signal molecules and pathways are involved in this process. In cancer, cell cycle control is often changed and understanding of these changes may result in new therapeutic targets in the treatment of patients. Additionally, cell cycle control is of special interest in stem cells as important decisions of cell fate – to proliferate or to differentiate - are part of cell cycle control. The success of adult stem cell therapeutic applications is thus dependent on in-depth understanding of this regulation. The Fluorescent ubiquitination-based cell cycle indicator (Fucci) is a sophisticated technology, which can easily determine G1 and/or S/G2/M phases of the cell cycle. The technology analyzes living cells in a spatio-temporal manner using fusion proteins consisting of two distinct cell cycle proteins fused to two fluorophores - a dual color scheme of orange and green. The aim of this thesis was to characterize the influence of Protein kinase D (PKD) using this technology in cells with adult stem cell characteristics and an established human cancer cell line. At first, a characterization of primary human mesenchymal stromal cells (MSC) derived from umbilical cord (UC) and bone marrow (BM) was performed. Furthermore, murine bone marrow stromal cells (mBMSCs) were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and host derived hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy. Consequently, multipotent mBMSC lines were established from transgenic Fucci mice. Single cell analysis of cell cycle progression was performed in these Fucci-mBMSCs and Fucci transgenic human HeLa cells. Specifically, the influence of protein kinase D (PKD) and the RAF/MEK/ERK pathway on progression through S/G2/M phase was investigated in detail. Inhibition of PKD but not of MEK resulted in a delay in progression through S/G2/M phase in HeLa cells and mBMSCs. Furthermore, MAPK pathway activation was quantitatively assessed during the synchronous progression of HeLa cells through S/G2/M and successfully used to develop a quantitative mathematical model describing this pathway. Taken together this study demonstrates the benefit of quantitative and single cell analysis in cells with stem cell characteristics and an established cell line to enlighten the role of PKD in cell cycle control and, on top of that, support the notion that PKD is a potential new target for cancer therapy.Item Open Access Combinations of costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy(2013) Hornig, Nora; Kontermann, Roland (Prof. Dr.)Combinatorial strategies are of emerging interest in cancer immunotherapy. Costimulation by individual members of the Ig- or TNF-superfamily have already revealed promising antitumor potential, thus prompting the exploration of their synergistic abilities in combinatorial approaches. Here, in order to avoid systemic side effects, costimulation was restricted to the tumor site by pursuing a targeted strategy with antibody-ligand fusion proteins composed of tumor antigen-directed antibodies and the extracellular domain of the costimulatory ligands B7 or 4-1BBL, respectively. Costimulatory activity was assessed in an experimental model system where tumor cells coexpressed the antigens fibroblast activation protein (FAP) and endoglin (EDG) and initial MHC-independent T cell activation and tumor-targeting was mediated by a bispecific antibody (scDbFAPxCD3). Combined costimulation with B7- and 4-1BBL-fusion proteins (B7-DbFAP, scFvEDG-4-1BBL) was shown to be superior to the individual effects in terms of cytokine release (IL-2/IFN-γ), proliferation and activation marker expression (CD25), leading to a T cell population with enhanced levels of an activation-experienced memory phenotype and with a higher capability for target cell killing. Furthermore, the model system was adapted for a time-shift costimulation setting. Here, enhanced T cell proliferation and granzyme B expression as well as reduced levels of PD-1 expression demonstrated the benefit of B7.1- and 4-1BBL-costimulation-assisted restimulation. Consequently, the antitumor activity of this combinatorial setting was confirmed in vivo in a lung metastasis mouse model. Finally, the combinatorial spectrum was expanded by the generation and subsequent incorporation of antibody-fusion proteins comprising the extracellular domains of the TNF-superfamily ligands Ox40L, LIGHT or GITRL. Here, advantages of combined costimulation with either B7.1- or 4-1BBL- fusion proteins were shown in terms of T cell proliferation and IFN-γ release. In summary, combinatorial approaches with tumor-directed costimulatory ligands in form of antibody-ligand fusion proteins were shown to be feasible, revealing a great potential for the modulation and enhancement of a T cell response. Thus, they appear to be a promising strategy in cancer immunotherapy that should be considered for further investigation.Item Open Access Targeting co-stimulatory receptors of the TNF superfamily for cancer immunotherapy(2022) Müller, DafneThe 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.Item Open Access Identification of models of heterogeneous cell populations from population snapshot data(2011) Hasenauer, Jan; Waldherr, Steffen; Doszczak, Malgorzata; Radde, Nicole; Scheurich, Peter; Allgöwer, FrankBackground: Most of the modeling performed in the area of systems biology aims at achieving a quantitative description of the intracellular pathways within a "typical cell". However, in many biologically important situations even clonal cell populations can show a heterogeneous response. These situations require study of cell-to-cell variability and the development of models for heterogeneous cell populations. Results: In this paper we consider cell populations in which the dynamics of every single cell is captured by a parameter dependent differential equation. Differences among cells are modeled by differences in parameters which are subject to a probability density. A novel Bayesian approach is presented to infer this probability density from population snapshot data, such as flow cytometric analysis, which do not provide single cell time series data. The presented approach can deal with sparse and noisy measurement data. Furthermore, it is appealing from an application point of view as in contrast to other methods the uncertainty of the resulting parameter distribution can directly be assessed. Conclusions: The proposed method is evaluated using artificial experimental data from a model of the tumor necrosis factor signaling network. We demonstrate that the methods are computationally efficient and yield good estimation result even for sparse data sets.Item Open Access Multivalent antibody-scTRAIL fusion proteins for tumor therapy : impact of format and targeting(2017) Hutt, Meike; Kontermann, Roland (Prof. Dr.)Item Open Access Pharmacokinetic engineering of OX40-blocking anticalin proteins using monomeric plasma half-life extension domains(2021) Siegemund, Martin; Oak, Prajakta; Hansbauer, Eva-Maria; Allersdorfer, Andrea; Utschick, Karoline; Winter, Alexandra; Grasmüller, Christina; Galler, Gunther; Mayer, Jan-Peter; Weiche, Benjamin; Prassler, Josef; Kontermann, Roland E.; Rothe, ChristineAnticalin® proteins have been proven as versatile clinical stage biotherapeutics. Due to their small size (∼20 kDa), they harbor a short intrinsic plasma half-life which can be extended, e.g., by fusion with IgG or Fc. However, for antagonism of co-immunostimulatory Tumor Necrosis Factor Receptor Superfamily (TNFRSF) members in therapy of autoimmune and inflammatory diseases, a monovalent, pharmacokinetically optimized Anticalin protein format that avoids receptor clustering and therefore potential activation is favored. We investigated the suitability of an affinity-improved streptococcal Albumin-Binding Domain (ABD) and the engineered Fab-selective Immunoglobulin-Binding Domain (IgBD) SpGC3Fab for plasma Half-Life Extension (HLE) of an OX40-specific Anticalin and bispecific Duocalin proteins, neutralizing OX40 and a second co-immunostimulatory TNFRSF member. The higher affinity of ABD fusion proteins to human serum albumin (HSA) and Mouse Serum Albumin (MSA), with a 4 to 5-order of magnitude lower KD compared with the binding affinity of IgBD fusions to human/mouse IgG, translated into longer terminal plasma half-lives (t1/2). Hence, the anti-OX40 Anticalin-ABD protein reached t1/2 values of ∼40 h in wild-type mice and 110 h in hSA/hFcRn double humanized mice, in contrast to ∼7 h observed for anti-OX40 Anticalin-IgBD in wild-type mice. The pharmacokinetics of an anti-OX40 Anticalin-Fc fusion protein was the longest in both models (t1/2 of 130 h and 146 h, respectively). Protein formats composed of two ABDs or IgBDs instead of one single HLE domain clearly showed longer presence in the circulation. Importantly, Anticalin-ABD and -IgBD fusions showed OX40 receptor binding and functional competition with OX40L-induced cellular reactivity in the presence of albumin or IgG, respectively. Our results suggest that fusion to ABD or IgBD can be a versatile platform to tune the plasma half-life of Anticalin proteins in response to therapeutic needs.Item Open Access Regulation of endocytic membrane trafficking by the GTPase-activating protein Deleted in Liver Cancer 3 (DLC3)(2015) Braun, Anja Catharina; Olayioye, Monilola (Prof. Dr.)Small GTPases of the Rho family are key regulators of the actin and microtubule cytoskeleton, whereby many cellular functions including cell migration, adhesion and polarity, as well as cell cycle progression are controlled. Increasing evidence suggests that Rho proteins are also critically involved in the regulation of membrane trafficking pathways within exocytosis and endocytosis. Although the molecular mechanisms are not well understood, Rho GTPases apparently have to govern and finely tune cytoskeletal remodeling, in order to support the formation, fusion and motility of transport carriers. However, the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure the balanced GTPase activation in space and time is largely elusive. The ‘Deleted in Liver Cancer’ (DLC1/2/3) proteins are a structurally conserved subfamily of RhoGAP proteins that act as negative regulators of Rho GTPases. In addition to the catalytically active GAP domain, all DLC proteins contain a sterile alpha motif (SAM) and steroidogenic acute regulatory protein-related lipid transfer (START) domain. Expression of the best studied member, DLC1, is frequently lost in various types of human cancers and a tumor suppressive function associated with its RhoGAP activity has been established in vivo. Although DLC3 was also observed to be downregulated in several cancer cell lines and primary tumors, the cellular functions of DLC3 are still poorly characterized. So far, GAP activity for RhoA has only been demonstrated in vitro and, associated with its localization at cell-cell contacts, a Rho-regulatory role in adherens junction stability was described. Thus, the aims of this thesis were to further investigate the subcellular localization of DLC3 and to shed light on the role of DLC3 in the regulation of Rho-mediated cellular processes, in particular endocytic membrane trafficking. This study provides convincing evidence that DLC3 is a functional, Rho-specific GAP protein in living cells and that its loss enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment, which is restored by the simultaneous downregulation of RhoA and RhoB. As a consequence, DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation and causes prolonged receptor signaling. Furthermore, it was found that DLC3-depleted cells show reduced surface N-cadherin levels, leading to decreased cell aggregation. Together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport via the control of local Rho activity.