Universität Stuttgart
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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 Molecular dynamics simulations of the substrate- and product specificity and mechanism of DNA- and protein lysine methyltransferases(2024) Schnee, Philipp; Jeltsch, Albert (Prof. Dr.)Protein Lysine Methyltransferases (PKMTs) regulate the epigenetic code of cells and their alteration via somatic mutations are often associated with cancer. The aim of this project is to rationalize the product and substrate specificity of this enzyme family by a combination of biochemical experiments and molecular dynamics simulations. Based on this, a detailed view of the underlying mechanism behind the disease associated mutations shall be gained, which may provide new possibilities for personalized cancer therapies.Item Open Access Deciphering metabolic pathways in high-seeding-density fed-batch processes for monoclonal antibody production : a computational modeling perspective(2024) Bokelmann, Carolin; Ehsani, Alireza; Schaub, Jochen; Stiefel, FabianDue to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism.Item Open Access Determination of muscle shape deformations of the tibialis anterior during dynamic contractions using 3D ultrasound(2024) Sahrmann, Annika S.; Vosse, Lukas; Siebert, Tobias; Handsfield, Geoffrey G.; Röhrle, OliverPurpose: In this paper, we introduce a novel method for determining 3D deformations of the human tibialis anterior (TA) muscle during dynamic movements using 3D ultrasound. Materials and Methods: An existing automated 3D ultrasound system is used for data acquisition, which consists of three moveable axes, along which the probe can move. While the subjects perform continuous plantar- and dorsiflexion movements in two different controlled velocities, the ultrasound probe sweeps cyclically from the ankle to the knee along the anterior shin. The ankle joint angle can be determined using reflective motion capture markers. Since we considered the movement direction of the foot, i.e., active or passive TA, four conditions occur: slow active, slow passive, fast active, fast passive. By employing an algorithm which defines ankle joint angle intervals, i.e., intervals of range of motion (ROM), 3D images of the volumes during movement can be reconstructed. Results: We found constant muscle volumes between different muscle lengths, i.e., ROM intervals. The results show an increase in mean cross-sectional area (CSA) for TA muscle shortening. Furthermore, a shift in maximum CSA towards the proximal side of the muscle could be observed for muscle shortening. We found significantly different maximum CSA values between the fast active and all other conditions, which might be caused by higher muscle activation due to the faster velocity. Conclusion: In summary, we present a method for determining muscle volume deformation during dynamic contraction using ultrasound, which will enable future empirical studies and 3D computational models of skeletal muscles.Item Open Access Native mechano-regulative matrix properties stabilize alternans dynamics and reduce spiral wave stabilization in cardiac tissue(2024) Erhardt, Julia; Ludwig, Sebastian; Brock, Judith; Hörning, MarcelThe stability of wave conduction in the heart is strongly related to the proper interplay between the electrophysiological activation and mechanical contraction of myocytes and extracellular matrix (ECM) properties. In this study, we statistically compare bioengineered cardiac tissues cultured on soft hydrogels ( E≃12kPa) and rigid glass substrates by focusing on the critical threshold of alternans, network-physiological tissue properties, and the formation of stable spiral waves that manifest after wave breakups. For the classification of wave dynamics, we use an improved signal oversampling technique and introduce simple probability maps to identify and visualize spatially concordant and discordant alternans as V- and X-shaped probability distributions. We found that cardiac tissues cultured on ECM-mimicking soft hydrogels show a lower variability of the calcium transient durations among cells in the tissue. This lowers the likelihood of forming stable spiral waves because of the larger dynamical range that tissues can be stably entrained with to form alternans and larger spatial spiral tip movement that increases the chance of self-termination on the tissue boundary. Conclusively, we show that a dysfunction in the excitation-contraction coupling dynamics facilitates life-threatening arrhythmic states such as spiral waves and, thus, highlights the importance of the network-physiological interplay between contractile myocytes and the ECM.Item Open Access Quantification of metabolic interactions between microorganisms(2024) Ulmer, Andreas; Takors, Ralf (Prof. Dr.-Ing.)Item Open Access Life cycle assessment for early-stage process optimization of microbial biosurfactant production using kinetic models : a case study on mannosylerythritol lipids (MEL)(2024) Bippus, Lars; Briem, Ann-Kathrin; Beck, Alexander; Zibek, Susanne; Albrecht, StefanIntroduction: This study assesses the environmental impacts of mannosylerythritol lipids (MELs) production for process optimization using life cycle assessment (LCA). MELs are glycolipid-type microbial biosurfactants with many possible applications based on their surface-active properties. They are generally produced by fungi from the family of Ustilaginaceae via fermentation in aerated bioreactors. The aim of our work is to accompany the development of biotechnological products at an early stage to enable environmentally sustainable process optimization. Methods: This is done by identifying hotspots and potentials for improvement based on a reliable quantification of the environmental impacts. The production processes of MELs are evaluated in a cradle-to-gate approach using the Environmental Footprint (EF) 3.1 impact assessment method. The LCA model is based on upscaled experimental data for the fermentation and purification, assuming the production at a 10 m³ scale. In the case analyzed, MELs are produced from rapeseed oil and glucose, and purified by separation, solvent extraction, and chromatography. Results: The results of the LCA show that the provision of substrates is a major source of environmental impacts and accounts for 20% of the impacts on Climate Change and more than 70% in the categories Acidification and Eutrophication. Moreover, 33% of the impacts on Climate Change is caused by the energy requirements for aeration of the bioreactor, while purification accounts for 42% of the impacts respectively. For the purification, solvents are identified as the main contributors in most impact categories. Discussion: The results illustrate the potentials for process optimization to reduce the environmental impacts of substrate requirements, enhanced bioreactor aeration, and efficient solvent use in downstream processing. By a scenario analysis, considering both experimental adaptations and prospective variations of the process, the laboratory development can be supported with further findings and hence efficiently optimized towards environmental sustainability. Moreover, the presentation of kinetic LCA results over the fermentation duration shows a novel way of calculating and visualizing results that corresponds to the way of thinking of process engineers using established environmental indicators and a detailed system analysis. Altogether, this LCA study supports and demonstrates the potential for further improvements towards more environmentally friendly produced surfactants.Item Open Access Algorithms for the global mapping of RNA-RNA interactomes(2024) Schäfer, Richard A.; Voß, Björn (Prof. Dr.)RNA-RNA intra- and intermolecular interactions are fundamental for numerous biological processes. While there are reasonable approaches to map RNA secondary structures genome-wide, understanding how different RNAs interact to carry out their regulatory functions requires mapping of intermolecular base pairs. RNA-RNA interaction prediction algorithms alone are not capable to consider all biological factors, thus, they suffer from low accuracy. Recently, different strategies to detect RNA-RNA duplexes in living cells, so called direct duplex detection (DDD) methods, have been developed. Common to all is the psoralen-mediated in vivo RNA crosslinking followed by RNA Proximity Ligation to join the two interacting RNA strands. Sequencing of the RNA via classical RNA-Seq and subsequent specialised bioinformatic analyses, which results in the prediction of intra- and intermolecular RNA-RNA interactions. Existing approaches adapt standard RNA-seq analysis pipelines but often neglect inherent features of RNA-RNA interactions that are useful for filtering and statistical assessment. In this work, RNAnue is presented, a general pipeline for the inference of RNA-RNA interactions from DDD experiments that takes into account hybridisation potential and statistical significance to improve prediction accuracy. RNAnue was applied to data from different DDD studies, and the results were compared to those of the original methods. This showed that RNAnue performs better in terms of prediction quantity and quality.Item Open Access Cu-doped calcium phosphate supraparticles for bone tissue regeneration(2024) Höppel, Anika; Bahr, Olivia; Ebert, Regina; Wittmer, Annette; Seidenstuecker, Michael; Carolina Lanzino, M.; Gbureck, Uwe; Dembski, SofiaCalcium phosphate (CaP) minerals have shown great promise as bone replacement materials due to their similarity to the mineral phase of natural bone. In addition to biocompatibility and osseointegration, the prevention of infection is crucial, especially due to the high concern of antibiotic resistance. In this context, a controlled drug release as well as biodegradation are important features which depend on the porosity of CaP. An increase in porosity can be achieved by using nanoparticles (NPs), which can be processed to supraparticles, combining the properties of nano- and micromaterials. In this study, Cu-doped CaP supraparticles were prepared to improve the bone substitute properties while providing antibacterial effects. In this context, a modified sol-gel process was used for the synthesis of CaP NPs, where a Ca/P molar ratio of 1.10 resulted in the formation of crystalline β-tricalcium phosphate (β-TCP) after calcination at 1000 °C. In the next step, CaP NPs with Cu 2+ (0.5-15.0 wt%) were processed into supraparticles by a spray drying method. Cu release experiments of the different Cu-doped CaP supraparticles demonstrated a long-term sustained release over 14 days. The antibacterial properties of the supraparticles were determined against Gram-positive ( Bacillus subtilis and Staphylococcus aureus ) and Gram-negative ( Escherichia coli ) bacteria, where complete antibacterial inhibition was achieved using a Cu concentration of 5.0 wt%. In addition, cell viability assays of the different CaP supraparticles with human telomerase-immortalized mesenchymal stromal cells (hMSC-TERT) exhibited high biocompatibility with particle concentrations of 0.01 mg mL -1 over 72 hours.Item Open Access Hydrogele aus Polyethylenglykol und methacryloylierter Gelatine für biosensorische Oberflächen(2024) Grübel, Jana; Tovar, Günter E. M. (Prof. Dr.)