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 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 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 Development, characterisation, and testing of CNS deliverable TRAIL-receptor agonists for the treatment of Glioblastoma (GBM)(2022) Krishna Moorthy, Nivetha; Morrison, Markus (Prof. Dr.)Glioblastoma (GBM) is a grade IV glioma, which is the most malignant and aggressive form of glioma. It accounts for 80% of the primary malignant brain tumours with a median survival time of just ~14 months. Therefore, GBM is presented as a highly challenging tumour and continuous efforts are required to find innovative and more effective treatment options. Tumour Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)-based therapeutics potently induce apoptosis in cancer cells, including GBM cells, by binding and activating TRAIL receptors (TRAIL-R1 and R2). However, the blood-brain barrier (BBB) is a major obstacle for these biologics to enter the central nervous system (CNS). The BBB is mainly made up of tightly connected endothelial cells and therefore the penetration of large biologics into the CNS is generally controlled and prevented by the presence of the BBB, with approximately 0.1% of injected antibody doses reaching the brain parenchyma. Receptor-mediated transcytosis is a mode of transport capable of carrying large proteins and lipoproteins across the BBB. Therefore, in this research work, studies were performed to investigate if antibody-based fusion proteins that combine the apoptosis-inducing TRAIL with transcytosis-inducing angiopep-2 could be developed. It was observed that the addition of the ANG2 moiety does not interfere with the potent apoptosis induction of TRAIL and these hexavalent TRAIL-receptor agonists demonstrated robust cytotoxicity against GBM cells. TRAIL receptor quantification demonstrated that the BBB cells do indeed express TRAIL receptors although in significantly reduced amounts compared to cancer cells. In cytotoxicity studies, BBB cells remained highly resistant to this fusion protein in response to clinically relevant doses of TRAIL-receptor agonists. Binding studies indicated that ANG2 is active in these constructs, however, control peptides and TRAIL-blocking experiments demonstrated that TRAIL-ANG2 fusion construct binding to BBB cells is mainly TRAIL-mediated. TRAIL-agonists bind cells effectively at sub-nanomolar concentrations, whereas angiopep-2 binds its target Lrp1 with an affinity of 313 nM. As a result of the binding studies and the difference in affinity, it was hypothesized that low TRAIL receptor expression on BBB endothelial cells may interfere with efficient transport of TRAIL-ANG2 fusion proteins, which was indeed observed by transwell transport studies. However, ANG2-mediated transport can be restored by blocking the TRAIL moieties in the fusion proteins. Overall, this study showed that TRAIL-ANG2 fusion proteins are highly potent in inducing apoptosis in GBM cells, but it requires TRAIL-R masking or other innovative strategies to achieve efficient CNS-transport and utilize them for the treatment of GBM.Item Open Access Collective cell migration on collagen-I networks: the impact of matrix viscoelasticity(2022) Pajic-Lijakovic, Ivana; Milivojevic, Milan; Clark, Andrew G.Collective cell migration on extracellular matrix (ECM) networks is a key biological process involved in development, tissue homeostasis and diseases such as metastatic cancer. During invasion of epithelial cancers, cell clusters migrate through the surrounding stroma, which is comprised primarily of networks of collagen-I fibers. There is growing evidence that the rheological and topological properties of collagen networks can impact cell behavior and cell migration dynamics. During migration, cells exert mechanical forces on their substrate, resulting in an active remodeling of ECM networks that depends not only on the forces produced, but also on the molecular mechanisms that dictate network rheology. One aspect of collagen network rheology whose role is emerging as a crucial parameter in dictating cell behavior is network viscoelasticity. Dynamic reorganization of ECM networks can induce local changes in network organization and mechanics, which can further feed back on cell migration dynamics and cell-cell rearrangement. A number of studies, including many recent publications, have investigated the mechanisms underlying structural changes to collagen networks in response to mechanical force as well as the role of collagen rheology and topology in regulating cell behavior. In this mini-review, we explore the cause-consequence relationship between collagen network viscoelasticity and cell rearrangements at various spatiotemporal scales. We focus on structural alterations of collagen-I networks during collective cell migration and discuss the main rheological parameters, and in particular the role of viscoelasticity, which can contribute to local matrix stiffening during cell movement and can elicit changes in cell dynamics.Item Open Access Dual- and triple-targeting of the HER-family members using combinations of mono- and bispecific antibodies(2023) Rau, Alexander; Olayioye, Monilola A. (Prof. Dr.)Epidermal growth factor receptor (EGFR)-targeted cancer treatments with antibodies like Cetuximab are successfully used in the clinic for about 20 years. However, intrinsic, as well as newly developed resistance mechanisms to EGFR-targeted therapies, are the main reason for their failure. Activation of human epidermal growth factor receptor 3 (HER3)-signaling upon EGFR-targeted therapies is frequently observed and has motivated the development of combination therapies that simultaneously block EGFR and HER3. In this study, bispecific and bivalent, or tetravalent, respectively, single-chain diabody (scDb) and scDb-Fc molecules were developed comprising the antigen-binding sites of a humanized version of Cetuximab (hu225) as well as a recently developed anti-HER3 antibody (3-43). In total, eight molecules (two scDb and six scDb-Fc) with varying linkers were engineered. The scDb hu225x3 43 Fc showed the most favorable properties regarding production yield, purity, homogeneity and linker setup. Binding of the scDb-Fc to recombinant receptors, as well as to HER-family receptor expressing cell lines revealed retained binding properties, compared to parental antibodies. Furthermore, the scDb hu225x3 43 Fc showed strong and long-lasting inhibition of downstream signaling by EGF, HRG or combination of both ligands. Proliferation studies on head and neck squamous cell carcinoma (HNSCC), triple negative breast cancer (TNBC), and colorectal cancer (CRC) cell lines revealed either similar, or stronger inhibition, compared to parental antibodies as single or combination treatment, which translated into to long-lasting growth suppression in a s.c. xenograft tumor model. Treatment with the bispecific antibody inhibited in vitro HRG-stimulated oncosphere formation of two TNBC cell lines. In an orthotopic MDA-MB-468 tumor model, superior antitumor effects were observed compared to those obtained by the parental antibodies alone or in combination. Furthermore, this was associated with a reduced number of cells with stem-like properties demonstrating that the bispecific antibody not only efficiently blocks TNBC proliferation but also the survival and expansion of the cancer stem cell population. The high degree of plasticity and compensatory signaling within the HER-family not only leads to compensatory crosstalk by HER3 but also HER2 giving the rational to combine the EGFR- and HER3-targeting scDb-Fc with a HER2-targeting antibody like Trastuzumab. The triple-targeting approach with the scDb-Fc and Trastuzumab was superior in inhibition of HRG-stimulated proliferation of the CRC cell line LIM1215 compared to the combination of IgG hu225, Trastuzumab and IgG 3 43. This was also observed in primary and secondary CRC oncosphere formation assays. Finally, in CRC patient derived organoids (PDOs) grown in HRG-supplemented medium the triple-targeting of EGFR, HER2 and HER3 provided broader efficacy than dual- or mono-targeting of receptors of the HER family. In contrast to Afatinib (anti-EGFR, -HER2, -HER4), the triple-targeted antibody approach showed efficient inhibition in all tested PDOs. Thus, the bispecific scDb-Fc alone or in combination with Trastuzumab represents a superior strategy to deal with primary and acquired resistances compared to targeting a single receptor with different antibodies or any combination of antibodies targeting two receptors of the HER-family.Item Open Access 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, ClaudiaMalignant 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.Item Open Access Prognostication and prediction of cancer patient outcomes using AI-based classifiers(2023) Guttà, Cristiano; Morrison, Markus (Prof. Dr.)