TY - JOUR A1 - Rauert-Wunderlich, Hilka A1 - Siegmund, Daniela A1 - Maier, Eduard A1 - Giner, Tina A1 - Bargou, Ralf C. A1 - Wajant, Harald A1 - Stühmer, Thorsten T1 - The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NF kappa B Transcription Factors JF - PLoS ONE N2 - Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. KW - signal inhibition KW - necrotic cell death KW - cell viability testing KW - cell death KW - small interfering RNAs KW - HT29 cells KW - phosphorylation KW - multiple myeloma Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130140 VL - 8 IS - 3 ER - TY - JOUR A1 - El-Mesery, M. A1 - Trebing, J. A1 - Schafer, V. A1 - Weisenberger, D. A1 - Siegmund, D. A1 - Wajant, H. T1 - CD40-directed scFv-TRAIL fusion proteins induce CD40-restricted tumor cell death and activate dendritic cells JF - Cell Death & Disease N2 - Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction. KW - dendritic cells KW - apoptosis KW - CD40 KW - TRAIL Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128777 VL - 4 IS - e916 ER - TY - JOUR A1 - Rödel, Mark-Oliver A1 - Brede, Christian A1 - Hirschfeld, Mareike A1 - Schmitt, Thomas A1 - Favreau, Philippe A1 - Stöcklin, Reto A1 - Wunder, Cora A1 - Mebs, Dietrich T1 - Chemical Camouflage - A Frog's Strategy to Co-Exist with Aggressive Ants JF - PLOS ONE N2 - Whereas interspecific associations receive considerable attention in evolutionary, behavioural and ecological literature, the proximate bases for these associations are usually unknown. This in particular applies to associations between vertebrates with invertebrates. The West-African savanna frog Phrynomantis microps lives in the underground nest of ponerine ants (Paltothyreus tarsatus). The ants usually react highly aggressively when disturbed by fiercely stinging, but the frog is not attacked and lives unharmed among the ants. Herein we examined the proximate mechanisms for this unusual association. Experiments with termites and mealworms covered with the skin secretion of the frog revealed that specific chemical compounds seem to prevent the ants from stinging. By HPLC-fractionation of an aqueous solution of the frogs' skin secretion, two peptides of 1,029 and 1,143 Da were isolated and found to inhibit the aggressive behaviour of the ants. By de novo sequencing using tandem mass spectrometry, the amino acid sequence of both peptides consisting of a chain of 9 and 11 residues, respectively, was elucidated. Both peptides were synthesized and tested, and exhibited the same inhibitory properties as the original frog secretions. These novel peptides most likely act as an appeasement allomone and may serve as models for taming insect aggression. KW - amphibian skin secretions KW - antimicrobial peptides KW - paltothyreus tarsatus KW - dendrobates pumilio KW - anurans KW - microhylidae KW - hymenoptera KW - formicidae KW - mutualisms KW - alkaloids Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128181 SN - 1932-6203 VL - 8 IS - 12 ER - TY - JOUR A1 - Chopra, Martin A1 - Lang, Isabell A1 - Salzmann, Steffen A1 - Pachel, Christina A1 - Kraus, Sabrina A1 - Bäuerlein, Carina A. A1 - Brede, Christian A1 - Jordán Garrote, Ana-Laura A1 - Mattenheimer, Katharina A1 - Ritz, Miriam A1 - Schwinn, Stefanie A1 - Graf, Carolin A1 - Schäfer, Viktoria A1 - Frantz, Stefan A1 - Einsele, Hermann A1 - Wajant, Harald A1 - Beilhack, Andreas T1 - Tumor Necrosis Factor Induces Tumor Promoting and Anti-Tumoral Effects on Pancreatic Cancer via TNFR1 JF - PLoS ONE N2 - Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome. KW - Bioluminescence KW - cancer treatment KW - cell staining KW - cytokines KW - immune cells KW - metastasis KW - regulatory T cells KW - T cells Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97246 ER - TY - THES A1 - Banaszek, Agnes T1 - Dual Antigen-Restricted Complementation of a Two-Part Trispecific Antibody for Targeted Immunotherapy of Blood Cancer T1 - Von zwei Antigenen abhängige Komplementierung eines zweiteiligen trispezifischen Antikörpers zur gezielten Immuntherapie von Blutkrebs N2 - 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. N2 - Krebszellen entgehen der Immunüberwachung oftmals dadurch, dass sie zwei wichtige Komponenten der Immunabwehr, nämlich antigenpräsentierende MHC- und kostimulatorische Moleküle, herunter regeln. Zurzeit befindet sich daher eine Reihe neuartiger Anti-Krebs-Substanzen in der Entwicklung, die darauf abzielen, das Immunsystem beim Erkennen und Bekämpfen von Krebs zu unterstützen. Rekombinante bispezifische Antikörper stellen eine Gruppe solch neuartiger Therapeutika dar. Sie erkennen zwei unterschiedliche Antigene und rekrutieren gezielt zytotoxische Effektorzellen zu Tumorzellen. Zur Krebsimmuntherapie sind BiTE-Antikörper (bispecific T cell engager) bereits gut untersucht. Diese Antikörper sind gegen ein tumorassoziiertes Antigen sowie gegen CD3ε, das konstante Molekül des T Zell-Rezeptor-Komplexes, gerichtet. Diese Arbeit beschreibt zum einen die Entwicklung eines bispezifischen Antikörpers, der CD3ε und den mit Rhabdomyosarkom assoziierten fetalen Acetylcholinrezeptor erkennt. Zum anderen präsentiert sie ein neues, zweiteiliges trispezifisches Antikörperformat, das zur Behandlung von Leukämie und anderen bösartigen Erkrankungen des blutbildenden Systems im Zusammenhang mit hämatopoetischer Stammzelltransplantation (HSZT) genutzt werden könnte. Für eine HSZT wird ein HLA-identischer Spender bevorzugt. Dieser steht jedoch nur sehr selten zur Verfügung. In Fällen mit nur einer Unstimmigkeit in den HLA-Merkmalen zwischen Patient und Spender könnte diese HLA-Unstimmigkeit nun zur gezielten Krebsbehandlung ausgenutzt werden. In dieser Arbeit wurde ein trispezifisches HLA-A2 × CD45 × CD3 Antikörperkonstrukt speziell für solche Fälle entwickelt, in denen der Patient HLA-A2-positiv ist, der Spender jedoch nicht. Dies trifft in Deutschland auf ungefähr die Hälfte aller Fälle zu, da HLA-A2 hier als häufigstes HLA-Molekül vorkommt. Mit der Kombination aus HLA-A2 und dem Pan-Leukozytenmarker CD45 (leucocyte-common antigen) als Ziel, wird eine hochspezifische, von zwei Antigenen abhängige, zielgerichtete Tumoransteuerung (tumour targeting) möglich. Genauer gesagt wurden zwei Einzelketten-Antikörperkonstrukte entwickelt: i) ein HLA A2-spezifisches single-chain variable fragment (scFv) und ii) ein CD45-spezifisches scFv, jeweils verbunden mit der VL- bzw. der VH-Domäne eines CD3ε-spezifischen Antikörpers. Es stellte sich heraus, dass nach gleichzeitiger Bindung der beiden Konstrukte an dieselbe HLA-A2- und CD45-exprimierende Zelle sich die beiden einzelnen, ungepaarten variablen Domänen eines CD3ε-spezifischen Antikörpers zu einem funktionellen scFv zusammenfügen. Dieses Zusammenfügen sollte in einer therapeutischen Situation ausschließlich auf den Blutkrebszellen des Empfängers geschehen, was zur T-Zell-vermittelten Zerstörung der Krebszellen führen würde. Auf diese Weise könnte ein Rückfall der Erkrankung vermieden und eventuell sogar auf die Standardtherapie (Bestrahlung und Chemotherapie) verzichtet werden. Für die beiden beschriebenen Ansätze wurden die Antikörperkonstrukte periplasmatisch in E. coli exprimiert, über einen His-Tag aufgereinigt und biochemisch charakterisiert. Ihre Bindung an die jeweiligen Zielantigene wurde mittels Durchflusszytometrie nachgewiesen. Die stimulatorischen Eigenschaften der Antikörper wurden durch eine Messung der IL-2-Freisetzung nach Inkubation zusammen mit T-Zellen und antigenexprimierenden Zielzellen untersucht. Sowohl der gegen Rhabdomyosarkom gerichtete BiTE-Antikörper, als auch der zusammengefügte trispezifische Antikörper gegen Blutkrebs vermittelten konzentrationsabhängig eine T Zellaktivierung bei nanomolaren Konzentrationen. Für den trispezifischen Antikörper erwies sich dieser Effekt tatsächlich als abhängig von zwei Antigenen, da er durch eine vorausgehende Inkubation mit entweder einem HLA-A2- oder einem CD45-spezifischen scFv-Fragment geblockt werden konnte und nicht auf Zellen auftrat, die nur ein Antigen (CD45+) oder keins von beiden (HLA-A2- CD45-) tragen. Darüber hinaus rekrutierten die Antikörper beider Ansätze T-Zellen zur Zerstörung von Tumorzellen in vitro. KW - Immuntherapie KW - Antikörper KW - Cytotoxischer Antikörper KW - Leukämie KW - Rhabdomyosarkom KW - bispecific antibodies KW - antibody engineering KW - cancer immunotherapy KW - rekombinante Antikörper KW - bispezifische antikörper Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-90174 ER -