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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 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 Multivalent antibody-scTRAIL fusion proteins for tumor therapy : impact of format and targeting(2017) Hutt, Meike; Kontermann, Roland (Prof. Dr.)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.Item Open Access Die Rolle von XIAP und Caspase-8 bei der Progression und Resistenz humaner Melanomen(2012) Hörnle, Manuela; Scheurich, Peter (Prof. Dr.)Das maligne Melanom zählt auch heute noch zu den gefährlichsten und tödlichsten Krebserkrankungen weltweit. Eukaryote Zellen stellen ein vielschichtiges multifaktorielles System dar, in welchem die Summe aus pro- und anti-apoptotischen Proteinen im Kontext der zellulären Gesamtsituation über das Schicksal der einzelnen Zelle zugunsten des gesamten Organismus entscheidet. Dieser Entscheidung liegt ein komplexes und mannigfaltiges System zugrunde, in dem eine Vielzahl von Variablen und Faktoren moduliert und reguliert werden. Das Verständnis dieser zugrundeliegenden molekularen Mechanismen stellt das Fundament zur Bekämpfung dieser tödlichen Krankheit dar. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der XIAP-Caspase-3-crosstalk das Schlüsselmoment der Entscheidung zwischen Leben und Sterben in humanen Melanomzellen darstellt. Initial TRAIL-resistente Melanomzellen können durch eine sublethale UVB-Bestrahlung für diese Rezeptor-vermittelte Apoptose sensitiviert werden. Dabei wird eine Beteiligung des intrinsischen mitochondrialen Apoptose¬wegs und den damit assoziierten pro-apoptotischen Faktoren nachgewiesen. Die Antagonisierung des anti-apoptotischen XIAP-Proteins führt zur vollständigen katalytischen Aktivierung der Effektor-Caspase-3 und resultiert final in einem positiven regulatorischen feedback-loop, in dem Caspase-3 durch proteolytische Spaltung die Degradierung des XIAP-Proteins initiiert. Zudem kann in der vorliegenden Arbeit eine weitere essentielle Funktion für Caspase-8 nachgewiesen werden. Diese vermeintlich ausschließlich pro-apoptotische Initiator-Caspase demonstriert eine fundamentale Bedeutung zur Aufrechterhaltung der zellulären Homöostase in humanen Melanomzellen. Neben ihrer etablierten Rolle bei der Initiierung der Rezeptor-vermittelten Apoptose zeigt sich diese Protease als essentiell für die Progression durch die S-Phase des Zellzyklus. Dabei kann von einer Beteiligung der Caspase-8-Prodomäne an der DNA-Reparatur sowohl während der regulären DNA-Replikation als auch nach DNA-Schaden ausgegangen werden. Die Abwesenheit von Caspase-8 führt zur Arretierung humaner Zellen in der S-Phase des Zellzyklus und resultiert in letzter Instanz in der Induktion des apoptotischen Zelltods über den intrinsischen mitochondrialen Signalweg.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 Serum albumin and its interaction with the neonatal Fc receptor (FcRn) : characterization of the albumin/FcRn-binding mechanism(2013) Färber-Schwarz, Aline; Kontermann, Roland (Prof. Dr.)In the past a lot of different biomolecular therapeutics were developed. A common problem using small molecules in therapy is their reduced serum concentration after short time period. The human body clears therapeutic molecules which have a size beneath the kidney clearance threshold in minutes to hours. The consequences are more frequent and high application doses. These facts demand the development of half-life prolonging strategies. plasma proteins, like IgG’s and albumin are recycled by the neonatal Fc receptor (FcRn) resulting in an extraordinary long circulating half-life which is used to prolong the serum circulation of small therapeutic proteins. Non-covalent binding or fusion to the Fc-part of IgG’s or albumin significantly improves the pharmacokinetic properties of small proteins. The aim of this study was to understand the interaction mechanism between albumin and the FcRn and to investigate the impact of point-mutations on the pharmacokinetic properties of albumin. A set of mouse serum albumin (MSA) mutants was generated and their pharmacokinetics were analyzed in vivo. Mutations of H464, E501, E505, D512, H510, S511, E531, H535, D565, T570 and E571 affect the in vivo half-life of albumin in mice. Even if these results are statistically not significant they still suggest the importance of the investigated amino acid residues for the albumin/FcRn binding. The highly conserved histidine residues H464, H510 and H535 play a major role in the albumin/FcRn interaction. In addition to the in vivo experiments a steered molecular dynamic (SMD) visualizes that the domain III of albumin removes as the last part of the protein from the receptor. Furthermore, the polar interactions beween MSA and the mouse FcRn were analyzed. Thus the amino acids S111 and L112 within the domain I and the amino acids E425, L466, T467 and P468 within the domain III of albumin were identified to be responsible for the FcRn interaction. Moreover, phage display was used to mature the affinity of MSA towards the mouse FcRn. A MSA library has been generated by site directed mutagenesis of five amino acids within the domain III of albumin. The selection of MSA variants with an increased affinity to the FcRn was not successful. Further approaches are required to mature the affinity of MSA towards the mouse FcRn.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.