@phdthesis{Melzer2013,
  author    = {Melzer, Juliane},
  title     = {Die Funktion der p21-aktivierten Kinase Mbt in Neuroblasten w{\"a}hrend der Entwicklung des zentralen Nervensystems von Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85619},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {p21-aktivierte Kinasen regulieren zahlreiche zellul{\"a}re Prozesse, die w{\"a}hrend der Entwicklung, aber auch beispielsweise bei der Krebsentstehung, von zentraler Bedeutung sind. Mbt, das einzige Typ II PAK-Protein von Drosophila melanogaster, spielt eine Rolle bei der Gehirnentwicklung. Eine Nullmutation von mbt, mbtP1, bildet kleinere Gehirne mit stark verkleinerten Pilzk{\"o}rpern aus. In dieser Arbeit wurde die Funktion von Mbt in Neuroblasten untersucht. Mbt wurde als Teil des apikalen Proteinkomplexes in Neuroblasten des Zentralhirns nachgewiesen. Die apikale Lokalisation von Mbt ist Zellzyklus-abh{\"a}ngig und wird {\"u}ber Bindung an Cdc42 reguliert. Sie ist essentiell f{\"u}r die Funktion von Mbt in Neuroblasten. Trotz apikaler Mbt-Lokalisation in Neuroblasten zeigte die mbt Nullmutante keine Defekte des basalen Mechanismus der asymmetrischen Zellteilung. Mud zeigte geringf{\"u}gige Lokalisationsver{\"a}nderungen, die auf einen m{\"o}glichen Einfluss von Mbt hinweisen. Obwohl PAKs zentrale Regulatoren des Zytoskeletts sind, zeigte die mbtP1 Mutante keine offensichtlichen Ver{\"a}nderungen des Aktin- und Tubulin-Zytoskeletts. Armadillo, ein Aktin-assoziiertes Mbt-Substrat, zeigte ebenfalls keine Lokalisationsver{\"a}nderung in Neuroblasten. Mbt steuert jedoch die apikale Anreicherung von Cno, einem weiteren Aktin-assoziierten Protein, in Neuroblasten. Dar{\"u}ber hinaus beeinflusst Mbt die Zellgr{\"o}ße von Neuroblasten, sowie deren Proliferationspotenzial und {\"U}berleben. mbtP1 Neuroblasten sind kleiner als wildtypische Neuroblasten, haben ein geringeres Proliferationsverm{\"o}gen und eine geringere {\"U}berlebenswahrscheinlichkeit. Der Zelltod von Neuroblasten ist jedoch ein sekund{\"a}rer Effekt. Daher kann eine Blockierung von Apoptose den adulten Pilzk{\"o}rperph{\"a}notyp nicht retten. Signalwege, die Zellgr{\"o}ße und Proliferation regulieren, wurden auf eine Beteiligung von Mbt hin analysiert. mbtP1 induzierte leichte Effekte im Insulin-Signalweg und die Delokalisation eines nukleol{\"a}ren Proteins. Eine genetische Interaktion von mbtP1 mit Mutationen in Genen des klassischen MAPK-Signalweges identifzierte mbt als Positivregulator dieses Signalweges im Auge. Ein {\"a}hnlicher, schw{\"a}cherer Effekt wurde auch bzgl. der Proliferation und Gr{\"o}ße von Neuroblasten beobachtet. Eine 2D-Gelanalyse von Larvengehirnen identifizierte Bic und Hsp83 als m{\"o}gliche von Mbt regulierte Proteine. Diese Arbeit charakterisiert eine bisher unbekannte Funktion der p21-aktivierten Kinase Mbt in neuronalen Stammzellen und liefert damit Ansatzpunkte f{\"u}r eine detaillierte Aufkl{\"a}rung der Funktionsmechanismen von Typ II PAKs bei der Regulation von Zellproliferation und {\"U}berleben},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Hartel2013,
  author    = {Hartel, Andreas J. W.},
  title     = {Die laterale Diffusion des variablen Oberfl{\"a}chenglykoproteins in Trypanosomen und in artifiziellen Membranen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90997},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Die Diffusion von Membranproteinen spielt bei einer Vielzahl von zellbiologischen Prozessen eine zentrale Rolle. So hat die Beweglichkeit von Glykosyl-Phosphatidyl-Inositol-(GPI-) verankerten Proteinen zum Beispiel eine tragende Funktion bei der Alzheimer Krankheit, der Creutzfeldt-Jacob Krankheit und der Afrikanischen Schlafkrankheit. Der Erreger der Afrikanischen Schlafkrankheit, Trypanosoma brucei spec., pr{\"a}sentiert auf seiner Zelloberfl{\"a}che einen dichten Mantel aus identischen GPI-verankerten Proteinen. Diese sogenannten Variant Surface Glycoproteins (VSGs) stellen den zentralen Pathogenit{\"a}tsfaktor der Trypanosomen im Blutstrom des Wirtes dar und erm{\"o}glichen dem Parasiten die Antigene Variation. W{\"a}hrend der Antigenen Variation wird der VSGMantel durch einen immunologisch distinkten Mantel ersetzt. Hierf{\"u}r ist die Diffusion der VSG essentiell. In der vorliegenden Arbeit wird die Diffusion des VSG in lebenden Trypanosomen und in artifiziellen Membranen systematisch untersucht. Auf diese Weise werden der Einfluss der lateralen Proteindichte, der N-Glykosylierung und der Proteingr{\"o}ße auf die Diffusion der GPI-verankerten Proteine charakterisiert. Die Mobilit{\"a}t des VSG auf lebenden Trypanosomen ist an der Grenze zu einem Diffusionsschwellenwert, dieser wird allerdings nicht {\"u}berschritten. Die Mobilit{\"a}t des VSG in der N{\"a}he des Diffusionsschwellenwertes wird durch die N-Glykosylierung der VSG erm{\"o}glicht. Außerdem kann gezeigt werden, dass die Gr{\"o}ße der Proteine einen entscheidenden Einfluss auf den Diffusionskoeffizienten der GPI-verankerten Proteine aus{\"u}bt. Zusammengefasst zeigen die Ergebnisse der vorliegenden Arbeit deutlich, dass der VSG-Mantel der Trypanosomen ein, an seine Anforderungen, hoch-adaptiertes System darstellt. W{\"u}rde entweder die laterale Dichte, die N-Glykosylierung oder die Gr{\"o}ße der Proteine beeintr{\"a}chtigt werden, so w{\"a}re die Funktion der Antigenen Variation gest{\"o}rt und die Pathogenit{\"a}t des Parasiten gef{\"a}hrdet. Da die lokale Verteilung von GPI-verankerten Proteinen in biologischen Membranen ein wichtiges funktionelles Konzept darstellt, ist der Einfluss der untersuchten Faktoren nicht nur f{\"u}r den VSG-Mantel relevant, sondern kann auch f{\"u}r das generelle Verst{\"a}ndnis der Dynamik von Proteinen in zellul{\"a}ren Membranen dienen.},
  subject      = {Trypanosomen},
  language  = {de}
}
@phdthesis{Dietz2013,
  author    = {Dietz, Lena},
  title     = {Die Rolle von NFATc1 und NFATc2 bei der Immunpathogenese von Experimenteller Autoimmuner Enzephalomyelitis (EAE), dem Tiermodell der Multiplen Sklerose},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-94569},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Multiple Sklerose (MS) ist eine Autoimmunkrankheit, welche durch Infiltration autoreaktiver Immunzellen in das Zentrale Nervensystem (ZNS) gekennzeichnet ist. Hierbei gelten insbesondere Th1- und Th17-Zellen als wichtige Mediatoren der ZNS-Entz{\"u}ndungsreaktion. Beide T-Helfer-Zellarten k{\"o}nnen durch regulatorische T-Zellen (Tregs) in ihrer Funktion supprimiert werden. NFAT(Nuclear Factors of Activated T cells)-Transkriptionsfaktoren werden nach TCR-Antigen-Stimulation induziert und regeln - als pleiotrope Transkriptionsfaktoren - viele funktionelle Prozesse in T-Zellen. Um die Rolle dieser Faktoren bei der Immunpathogenese von MS zu analysieren, wurden unterschiedliche NFAT-defiziente Mausst{\"a}mme auf den Krankheitsverlauf des Tiermodells Experimentelle Autoimmune Enzephalomyelitis (EAE) hin untersucht. Es konnte gezeigt werden, dass sowohl der einzelne Verlust von NFATc1 und NFATc2 in CD4+ T-Zellen als auch das Fehlen einer spezifischen C-terminalen Proteinmodifikation von NFATc1, die SUMOylierung, sich abmildernd auswirkten. Der verminderte klinische Ausgang der EAE beruhte allerdings je nach knock-out auf unterschiedlichen Mechanismen. Im Fall des T-Zell-spezifischen Verlustes von NFATc1 (Nfatc1fl/fl x Cd4cre+ M{\"a}use), erwies sich die EAE aufgrund einer stark eingeschr{\"a}nkten Aktivierung und Effektorzellentwicklung von CD4+ T-Zellen als vermindert. Dies konnte durch eine reduzierte Produktion an pathogenen Effektorzytokinen, wie IFNγ, IL-17A, GM-CSF sowie IL-22 und weniger an IL-17A+ IFNγ+ Doppelproduzenten im ZNS gezeigt werden. Der Verlust von NFATc2 resultierte in einer starken Th2-Antwort im ZNS von Nfatc2-/- EAE-M{\"a}usen einhergehend mit protektiven IL-4- und IL-10-Produzenten. Interessanterweise konnten auch mehr nicht-pathogene Th17-Zellen nachgewiesen werden. Nfatc1/CΔSUMO CD4+ T-Zellen sezernierten sowohl nach in vitro als auch nach in vivo Stimulation erh{\"o}hte Mengen von IL-2. In vitro Kulturen von Th1- und Th17-Zellen wiesen neben dieser erh{\"o}hten IL-2-Sekretion eine verminderte Produktion von IFNγ und IL-17A auf. In {\"U}bereinstimmung mit diesen in vitro Befunden zeigte sich auch in der EAE ein reduziertes Krankheitsbild mit weniger Th1- und Th17-Zellen, daf{\"u}r aber eine IL-2-gef{\"o}rderte Erh{\"o}hung der Treg-Population. Anhand der Erkenntnis, dass NFAT-Faktoren die (Auto)-Immunreaktion entscheidend beeinflussen, k{\"o}nnte die Inhibition einzelner NFAT-Faktoren ein neues Ziel f{\"u}r eine MS-Therapie darstellen.},
  subject      = {Immunologie},
  language  = {de}
}
@article{SchubertSpahnKneitzetal.2013,
  author    = {Schubert, Maria and Spahn, Martin and Kneitz, Susanne and Scholz, Claus J{\"u}rgen and Joniau, Steven and Stroebel, Philipp and Riedmiller, Hubertus and Kneitz, Burkhard},
  title     = {Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer},
  series = {PLoS ONE},
  journal   = {PLoS ONE},
  doi       = {10.1371/journal.pone.0065064},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96825},
  year      = {2013},
  abstract  = {Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.},
  language  = {en}
}
@article{HerpinAdolfiNicoletal.2013,
  author    = {Herpin, Amaury and Adolfi, Mateus C. and Nicol, Barbara and Hinzmann, Maria and Schmidt, Cornelia and Klughammer, Johanna and Engel, Mareen and Tanaka, Minoru and Guiguen, Yann and Schartl, Manfred},
  title     = {Divergent Expression Regulation of Gonad Development Genes in Medaka Shows Incomplete Conservation of the Downstream Regulatory Network of Vertebrate Sex Determination},
  series = {Molecular Biology and Evolution},
  volume    = {30},
  journal   = {Molecular Biology and Evolution},
  number    = {10},
  doi       = {10.1093/molbev/mst130},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132262},
  pages     = {2328-2346},
  year      = {2013},
  abstract  = {Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.},
  language  = {en}
}
@phdthesis{Ott2013,
  author    = {Ott, Christine Kornelia},
  title     = {Diverse Aspects of the Sorting and Assembly Machinery in Human Mitochondria},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Mitochondria are organelles of endosymbiotic origin, which play many important roles in eukaryotic cells. Mitochondria are surrounded by two membranes and, considering that most of the mitochondrial proteins are produced in the cytosol, possess import machineries, which transport mitochondria-targeted proteins to their designated location. A special class of outer mitochondrial membrane (OMM) proteins, the β-barrel proteins, require the sorting and assembly machinery (SAM) for their OMM integration. Both mitochondrial β-barrel proteins and the central component of the SAM complex, Sam50, have homologs in gram-negative bacteria. In yeast mitochondria, bacterial β-barrel proteins can be imported and assembled into the OMM. Our group demonstrated that this, however, is not the case for human mitochondria, which import only neisserial β barrel proteins, but not those of Escherichia coli and Salmonella enterica. As a part of this study, I could demonstrate that β-barrel proteins such as Omp85 and PorB of different Neisseria species are targeted to human mitochondria. Interestingly, only proteins belonging to the neisserial Omp85 family were integrated into the OMM, whereas PorB was imported into mitochondria but not assembled. By exchanging parts of homologous neisserial Omp85 and E. coli BamA and, similarly, of neisserial PorB and E. coli OmpC, it could be demonstrated in this work that the mitochondrial import signal of bacterial β barrel proteins cannot be limited to one short linear sequence, but rather secondary structure and protein charge seem to play an important role, as well as specific residues in the last β-strand of Omp85. Omp85 possesses five conserved POTRA domains in its amino-terminal part. This work additionally demonstrated that in human mitochondria, at least two POTRA domains of Omp85 are necessary for membrane integration and functionality of Omp85. In the second part of this work, the influence of Sam50 on the mitochondrial cristae structure was investigated. This work contributed to a study performed by our group in which it was confirmed that Sam50 is present in a high molecular weight complex together with mitofilin, CHCHD3, CHCHD6, DnaJC11, metaxin 1 and metaxin 2. This connection between the inner and outer mitochondrial membrane was shown to be crucial for the maintenance of the mitochondrial cristae structure. In addition, a role of Sam50 in respiratory complex assembly, suggested by a SILAC experiment conducted in our group, could be confirmed by in vitro import studies. An influence of Sam50 not only on respiratory complexes but also on the recently described respiratory complex assembly factor TTC19 was demonstrated. It was shown that TTC19 not only plays a role in complex III assembly as published, but also influences the assembly of respiratory complex IV. Thus, in this part of the work a connection between the OMM protein Sam50 and maintenance of cristae structure, respiratory complex assembly and an assembly factor could be established.},
  subject      = {Mitochondrien},
  language  = {en}
}
@article{WegenerKarsaiPollaketal.2013,
  author    = {Wegener, Christian and Karsai, Gergely and Poll{\´a}k, Edit and Wacker, Matthias and V{\"o}mel, Matthias and Selcho, Mareike and Berta, Gergely and Nachman, Ronald J. and Isaac, R. Elwyn and Moln{\´a}r, L{\´a}szl{\´o}},
  title     = {Diverse in- and output polarities and high complexity of local synaptic and non-synaptic signaling within a chemically defined class of peptidergic Drosophila neurons},
  series = {Frontiers in Neural Circuits},
  journal   = {Frontiers in Neural Circuits},
  doi       = {10.3389/fncir.2013.00127},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96914},
  year      = {2013},
  abstract  = {Peptidergic neurons are not easily integrated into current connectomics concepts, since their peptide messages can be distributed via non-synaptic paracrine signaling or volume transmission. Moreover, the polarity of peptidergic interneurons in terms of in- and out-put sites can be hard to predict and is very little explored. We describe in detail the morphology and the subcellular distribution of fluorescent vesicle/dendrite markers in CCAP neurons (NCCAP), a well defined set of peptidergic neurons in the Drosophila larva. NCCAP can be divided into five morphologically distinct subsets. In contrast to other subsets, serial homologous interneurons in the ventral ganglion show a mixed localization of in- and output markers along ventral neurites that defy a classification as dendritic or axonal compartments. Ultrastructurally, these neurites contain both pre- and postsynaptic sites preferably at varicosities. A significant portion of the synaptic events are due to reciprocal synapses. Peptides are mostly non-synaptically or parasynaptically released, and dense-core vesicles and synaptic vesicle pools are typically well separated. The responsiveness of the NCCAP to ecdysis-triggering hormone may be at least partly dependent on a tonic synaptic inhibition, and is independent of ecdysteroids. Our results reveal a remarkable variety and complexity of local synaptic circuitry within a chemically defined set of peptidergic neurons. Synaptic transmitter signaling as well as peptidergic paracrine signaling and volume transmission from varicosities can be main signaling modes of peptidergic interneurons depending on the subcellular region. The possibility of region-specific variable signaling modes should be taken into account in connectomic studies that aim to dissect the circuitry underlying insect behavior and physiology, in which peptidergic neurons act as important regulators.},
  language  = {en}
}
@article{KellerGrimmerSteffanDewenter2013,
  author    = {Keller, Alexander and Grimmer, Gudrun and Steffan-Dewenter, Ingolf},
  title     = {Diverse Microbiota Identified in Whole Intact Nest Chambers of the Red Mason Bee Osmia bicornis (Linnaeus 1758)},
  series = {PLoS One},
  journal   = {PLoS One},
  doi       = {10.1371/journal.pone.0078296},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-97305},
  year      = {2013},
  abstract  = {Microbial activity is known to have profound impact on bee ecology and physiology, both by beneficial and pathogenic effects. Most information about such associations is available for colony-building organisms, and especially the honey bee. There, active manipulations through worker bees result in a restricted diversity of microbes present within the colony environment. Microbial diversity in solitary bee nests remains unstudied, although their larvae face a very different situation compared with social bees by growing up in isolated compartments. Here, we assessed the microbiota present in nests and pre-adults of Osmia bicornis, the red mason bee, by culture-independent pyrosequencing. We found high bacterial diversity not comparable with honey bee colonies. We identified a variety of bacteria potentially with positive or negative interactions for bee larvae. However, most of the other diverse bacteria present in the nests seem to originate from environmental sources through incorporated nest building material and stored pollen. This diversity of microorganisms may cause severe larval mortality and require specific physiological or symbiotic adaptations against microbial threats. They may however also profit from such a diverse environment through gain of mutualistic partners. We conclude that further studies of microbiota interaction in solitary bees will improve the understanding of fitness components and populations dynamics.},
  language  = {en}
}
@article{DeekenGohlkeScholzetal.2013,
  author    = {Deeken, Rosalia and Gohlke, Jochen and Scholz, Claus-Juergen and Kneitz, Susanne and Weber, Dana and Fuchs, Joerg and Hedrich, Rainer},
  title     = {DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors},
  series = {PLoS Genetics},
  journal   = {PLoS Genetics},
  doi       = {10.1371/journal.pgen.1003267},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96318},
  year      = {2013},
  abstract  = {Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.},
  language  = {en}
}
@phdthesis{Banaszek2013,
  author    = {Banaszek, Agnes},
  title     = {Dual Antigen-Restricted Complementation of a Two-Part Trispecific Antibody for Targeted Immunotherapy of Blood Cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90174},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Cancer cells frequently escape from immune surveillance by down-regulating two important components of the immune defence: antigen-presenting MHC and costimulatory molecules. Therefore several novel anti-tumour compounds that aim to assist the immune system in recognising and fighting cancer are currently under development. Recombinant bispecific antibodies represent one group of such novel therapeutics. They target two different antigens and recruit cytotoxic effector cells to tumour cells. For cancer immunotherapy, bispecific T cell-engaging antibodies are already well characterised. These antibodies target a tumour-associated antigen and CD3ε, the constant molecule of the T cell receptor complex. On the one hand, this study presents the development of a bispecific antibody targeting CD3ε and the rhabdomyosarcoma-associated fetal acetylcholine receptor. On the other hand, it describes a novel two-part trispecific antibody format for the treatment of leukaemia and other haematological malignancies in the context of haematopoietic stem cell transplantation (HSCT). For HSCT, an HLA-identical donor is preferred, but very rarely available. In an HLA-mismatched setting, the HLA disparity could be exploited for targeted cancer treatment. In the present study, a two-part trispecific HLA-A2 × CD45 × CD3 antibody was developed for potential cases in which the patient is HLA-A2-positive, but the donor is not. This holds true for about half the cases in Germany, since HLA-A2 is the most common HLA molecule found here. Combinatorial targeting of HLA-A2 and the leucocyte-common antigen CD45 allows for highly specific dual-antigen restricted tumour targeting. More precisely, two single-chain antibody constructs were developed: i) a single-chain variable fragment (scFv) specific for HLA-A2, and ii) a scFv against CD45, both linked to the VL and the VH domain of a CD3ε-specific antibody, respectively. It turned out that, after the concomitant binding of these constructs to the same HLA-A2- and CD45-expressing cell, the unpaired variable domains of a CD3ε-specific antibody assembled to a functional scFv. In a therapeutic situation, this assembly should exclusively occur on the recipient's blood cancer cells, leading to T cell-mediated cancer cell destruction. In this way, a relapse of disease might be prevented, and standard therapy (radiation and chemotherapy) might be omitted. For both approaches, the antibody constructs were periplasmically expressed in E. coli, purified via His tag, and biochemically characterised. Their binding to the respective targets was proven by flow cytometry. The stimulatory properties of the antibodies were assayed by measuring IL-2 release after incubation with T cells and antigen-expressing target cells. Both the bispecific antibody against rhabdomyosarcoma and the assembled trispecific antibody against blood cancer mediated T-cell activation in a concentration-dependent manner at nanomolar concentrations. For the trispecific antibody, this effect indeed proved to be dual antigen-restricted, as it could be blocked by prior incubation of either HLA-A2- or CD45-specific scFv and did not occur on single-positive (CD45+) or double-negative (HLA-A2- CD45-) target cells. Furthermore, antibodies from both approaches recruited T cells for tumour cell destruction in vitro.},
  subject      = {Immuntherapie},
  language  = {en}
}