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 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 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 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 Prognostication and prediction of cancer patient outcomes using AI-based classifiers(2023) Guttà, Cristiano; Morrison, Markus (Prof. Dr.)Item Open Access DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes(2020) Benz, David; Olayioye, Monilola (Prof. Dr.)Item Open Access Regulation of basal and activity-mediated AMPAR endocytosis by Protein Kinase D (PKD)(2021) Oueslati Morales, Carlos Omar; Hausser, Angelika (PD Dr.)AMPARs are one of the four different types of glutamate-gated ion channels in the mammalian brain, allowing influx of Na+ and efflux of K+ and thus depolarisation of the neuronal membrane. They are critical for correct brain functioning, as they represent the main mediators of excitatory synaptic transmission and as such are key for intercellular communication, brain development and, importantly, learning and memory. AMPARs are highly motile, undergoing constitutive and activity-mediated endocytosis, recycling and surface lateral diffusion among their different cellular pools. Notably, regulation of this trafficking is instrumental to mediate long-term potentiation (LTP) and long-term depression (LTD), cellular mechanisms of synaptic plasticity, which promote a long-lasting enhancement or decrease in synaptic strength, respectively, and are widely believed to be the main contributors to learning and memory. However, the mechanisms behind that regulation are not fully understood yet. The protein kinase D family of serine/threonine kinases consists of three isoforms in mammalian cells, and is activated downstream of DAG and PKC to participate in the regulation of processes such as vesicle fission from the trans-Golgi network and actin cytoskeleton remodelling. All three isoforms are expressed in neurons from an early embryonic stage, where they modulate tissue-specific processes such as the establishment and maintenance of neuronal polarity, neuroprotection against early oxidative stress and, importantly, synaptic plasticity through stabilisation of filamentous actin via cofilin inactivation during the expression of LTP and the phosphorylation of remaining surface NMDARs during the expression of LTD. Despite these observations, PKD has not been linked yet to AMPAR trafficking regulation. Therefore, in this thesis I aimed to elucidate whether PKD controls AMPAR trafficking in primary neuronal cells in basal and/or in activity-mediated conditions, and to shed light on the molecular mechanism that underlies this regulation. This work presents compelling evidence that PKD acts as a promoter of AMPAR endocytosis in primary hippocampal neurons in both basal and activity-mediated conditions. Short-term pharmacological inhibition of PKD via treatment with the small-molecule inhibitor CRT0066101 led to an increase in surface and synaptic AMPAR levels and slowed down AMPAR surface trafficking dynamics. Conversely, expression of a constitutively active PKD mutant promoted a decrease in AMPAR synaptic levels while increasing its localisation at early endosomes. Moreover, it was found that PKD activity is necessary for the decrease in surface AMPAR levels in response to both agonist- and NMDA treatments. Finally, I provide evidence that phosphorylation of the PKD substrate and Rab5 effector Rabaptin-5 at S407, the PKD phosphorylation site, is necessary for the correct regulation of basal AMPAR synaptic levels and for the endocytosis of AMPAR in response to NMDA treatment. Together, these findings identify PKD as a novel regulator of AMPAR endocytosis, presumably through the phosphorylation of Rabapatin-5 and subsequent Rab5 activation.Item Open Access Engineering of bispecific T cell receptors using mammalian display technologies(2022) Dilchert, Janine; Kontermann, Roland (Prof. Dr.)The main goal of immunotherapy in oncology is to use the power of the patient’s own immune system to fight cancer. Therefor many bispecific antibodies were developed mainly for redirecting T cells to cancer cells. A novel and promising class of biotherapeutics are increasingly recognized, namely bispecific T cell receptor (TCR)- based molecules capable of redirecting and activating T cells towards tumor-specific peptides presented by human leucocyte antigens (HLA). The usage of TCR-based molecules allows for targeting of novel tumor antigens including intracellular antigens and thus significantly widens the accessible target space in cancer immunotherapy. In contrast to antibodies, TCRs naturally exhibit a low binding affinity and stability and thus a complex maturation process is required for successful generation of TCR-based biotherapeutics. We developed a Chinese hamster ovary (CHO) cell display system for the maturation of TCR-based biomolecules, such as T cell engaging receptor (TCER®). Unlike previously used phage or yeast display systems, the mammalian system is capable of engineering TCRs in the final TCER® format making the step of reformatting of matured TCRs dispensable. The display approach is based on a recombinase-mediated cassette exchange for efficient and stable single copy integration of bispecific agents into a predefined genetic locus of the CHO cell. This work describes the setup of the CHO display, the membrane-bound expression of different TCR-based formats as well as its successful application for engineering of TCER® molecules using TCR variable domains from a model TCR recognizing preferentially expressed antigen in melanoma (PRAME). Affinity-improved TCER® molecules were isolated from a library encoding different complementarity determining region (CDR) variants in the final format. The selected TCER® candidates were evaluated in the CHO display system regarding their binding to the PRAME pHLA target as well as 11 peptides with high degree of sequence similarity to the PRAME peptide as part of specificity testing. TCER® variants expressed as soluble proteins showed strong reactivity against PRAME-positive tumor cells linked with a pronounced cytokine release from activated T cells. This study supports feasibility of the CHO-based maturation system for TCR affinity maturation in the final TCER® format and demonstrate data consistency between membrane-bound and soluble TCER® format.Item Open Access Predicting response to immunotherapy in metastatic melanoma by a personalized mathematical model(2020) Tsur, Neta; Morrison, Markus (Prof. Dr.)At present, immune checkpoint inhibitors, such as pembrolizumab, are widely used for the treatment of advanced non resectable melanoma, as they induce more durable responses than other available treatments. However, the overall response rate does not exceed 50%, and prediction of the individual benefit of patients from these therapeutics remains an unmet clinical need. Mathematical models that predict the response of patients with advanced melanoma to immune checkpoint inhibitors can contribute to better informed clinical decisions. The aim of this work was to develop a new personalization algorithm for predicting time to disease progression under pembrolizumab treatment, based on personal mathematical models of patients with advanced melanoma. First, a simple mathematical model was developed for describing the interactions of an advanced melanoma tumor with both the patient’s immune system and the immunotherapeutic drug, pembrolizumab. The local and global dynamics of the treatment-free model was analyzed analytically and numerically. The complete model was then implemented in conjunction with clinical pre-treatment data, in an algorithm which enables prediction of the personal response to the drug. To develop the algorithm, clinical data of 54 patients with advanced melanoma under pembrolizumab were collected retrospectively. In the following, clinical parameters before checkpoint inhibition and in the early course of pembrolizumab treatment were correlated to the mathematical model parameters. Using the algorithm together with the longitudinal tumor burden of each patient, the personal mathematical models were identified and used in simulations to predict the patient’s time to progression. The prediction capacity of the algorithm was validated by the Leave-One-Out cross validation methodology. The results show that zero, one, or two steady states of the treatment-free mathematical model exist in the phase plane, depending on the parameter values of individual patients. Without treatment, the simulated tumors grew uncontrollably. At increased efficacy of the immune system, i.e. due to immunotherapy, two steady states were found, one leading to uncontrollable tumor growth, whereas the other resulted in tumor size stabilization. The model analysis indicates that a sufficient increase in the activation of CD8+ T cells results in stable disease, whereas a significant reduction in T-cell exhaustion, which indirectly promotes CD8+ T cell activity, entails a temporal reduction in the tumor mass, but fails to control disease progression in the long run. Importantly, the initial tumor burden influences the in-treatment dynamics: small tumors responded better to treatment than larger tumors. Among the analyzed clinical parameters, the baseline tumor load, the Breslow tumor thickness, and the status of nodular melanoma were significantly correlated with the activation and exhaustion rates of CD8+ T cells. Using the measurements of these correlates to personalize the mathematical model, the time to progression of individual patients was predicted (Cohen’s κ = 0.489). Comparison of the predicted and clinical time to progression in patients progressing during the follow-up period showed moderate accuracy (R2=0.505). The treatment-free model analysis suggests that disease progression and response to immune checkpoint inhibitors depend on the ratio between activation and exhaustion rates of CD8+ T cells as well as the tumor growth rate. This analysis provides a foundation for the use of computational methods to personalize immunotherapy. Furthermore, the results suggest that a relatively simple mathematical mechanistic model, implemented in a computational algorithm, can be personalized by baseline clinical data before immunotherapy onset. The algorithm, currently yielding moderately accurate predictions of individual patients’ response to pembrolizumab, can be improved by using a larger number of patient files. Validation of the algorithm by an independent clinical dataset will enable its use as a tool for treatment personalization.Item Open Access Evaluating necroptosis competency in malignant melanoma(2020) Podder, Biswajit; Morrison, Markus (Prof. Dr.)Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies, as well as immunotherapies, frequently remain inefficient. Therefore, alternative means to treat melanoma, particularly through the induction of programmed cell death modalities such as apoptosis or necroptosis, still need to be explored. Apoptotic and necroptotic cell death depends on cysteine aspartate-specific proteases (caspases) and receptor-interacting serine/threonine kinase (RIPK) 1/3, respectively. RIPK1/3 and caspases can directly interact with the TNFR signalling complex, thereby inducing programmed cell death. Thus, these cell death regulations need to be explored in melanoma cancer. In the course of this thesis, a panel of melanoma cell lines was studied for cell death susceptibility in response to the activation of TNF family receptors. Apoptosis and necroptosis responsiveness, as well as cell death sensitization approaches, were tested, followed by mechanistic signal transduction studies making use of pharmacological and genetic interventions. In addition, phosphoprotein and secretome analyses were performed by xMAP (Luminex) assays. This study reports that melanoma cell lines responded heterogeneously to either single treatment of death ligands (DLs) or in combination with an IAP antagonist. Furthermore, it was observed that melanoma cell lines expressing notable amounts of RIPK1, RIPK3, and MLKL, the key players in necroptosis signal transduction, failed to execute necroptotic cell death. Interestingly, the activity of transforming growth factor β-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5z)-7-Oxozeaenol, deletion of MAP3K7, or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNF-α or TRAIL-based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling, as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Collectively, these results demonstrate that TAK1 suppresses susceptibility to RIPK1-dependent cell death. These findings were in line with high expression of TAK1 indicating an increased risk for disease progression in melanoma, as shown by subsequent collaborative work.
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