Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

90 Nb: potential radionuclide for application in immuno-PET : development of appropriate production strategy and first in vivo evaluation of 90 Nb-labeled monoclonal antibody

Die Nuklearmedizin ist ein modernes und effektives Werkzeug zur Erkennung und Behandlung von onkologischen Erkrankungen. Molekulare Bildgebung, die auf dem Einsatz von Radiopharmaka basiert, beinhaltet die Einzel-Photonen-Emissions-Tomographie (SPECT) und Positronenemissions¬tomographie (PET) und ermöglicht die nicht-invasive Visualisierung von Tumoren auf nano-und picomolarer Ebene.rnDerzeit werden viele neue Tracer für die genauere Lokalisierung von kleinen Tumoren und Metastasen eingeführt und hinsichtlich ihrer Eignung untersucht. Die meisten von ihnen sind Protein-basierte Biomoleküle, die die Natur selbst als Antigene für die Tumorzellen produziert. Dabei spielen Antikörper und Antikörper-Fragmente eine wichtige Rolle in der Tumor-Diagnostik und Behandlung. Die PET-Bildgebung mit Antikörpern und Antikörperfragmenten bezeichnet man als immuno-PET. Ein wichtiger Aspekt hierbei ist, dass entsprechende Radiopharmaka benötigt werden, deren Halbwertszeit mit der Halbwertszeit der Biomoleküle korreliert ist.rnIn neueren Arbeiten wird 90Nb als potenzieller Kandidat für die Anwendung in der immuno-PET vorgeschlagen. Seine Halbwertszeit von 14,6 Stunden ist geeignet für die Anwendung mit Antikörperfragmenten und einige intakten Antikörpern. 90Nb hat eine relativ hohen Anteil an Positronenemission von 53% und eine optimale Energie für die β+-Emission von 0,35 MeV, die sowohl eine hohe Qualität der Bildgebung als auch eine niedrige Aktivitätsmenge des Radionuklids ermöglicht.rnErsten grundlegende Untersuchungen zeigten: i) dass 90Nb in ausreichender Menge und Reinheit durch Protonen-Bombardierung des natürlichen Zirkonium Targets produziert, ii) aus dem Targetmaterial in entsprechender radiochemischer Reinheit isoliert und iii) zur Markierung des monoklonalen Antikörpers (Rituximab) verwendet werden kann und iv) dieser 90Nb-markierte mAb eine hohe in vitro Stabilität besitzt. Desweiteren wurde eine alternative und schnelle Abtrennungsmethode entwickelt, die es erlaubt 90Nb, mit einer geeigneten radiochemischen und radionuklidischen Reinheit für eine anschließende Markierung von Biomolekülen in einer Stunde zu aufzureinigen. Schließlich wurden erstmals 90Nb-markierte Biomolekülen in vivo untersucht. Desweiteren wurden auch Experimente durchgeführt, um den optimalen bifunktionellen Chelatbildner (BFC) für 90Niob zu finden. Mehrere BFC wurden hinsichtlich Komplexbildung mit NbV untersucht. Desferrioxamin (Df) erwies sich als geeignetster Chelator für 90Nb. Der monoklonale Antikörper Bevacizumab (Avastin®) wurde mit 90Nb markiert und eine Biodistributionsstudie und eine PET-Untersuchung durchgeführt. Alle diese Ergebnisse zeigten, dass 90Nb ein vielversprechendes Radionuklid für die Immuno-PET ist, welches sogar für weitere kommerzielle Anwendungen in der klinischen Routine geeignet zu sein scheint.rn

Towards antibody-mediated targeting of dendritic cells for cancer immunotherapy with multivalent polymer-antigen conjugates

Der Fokus dieser Arbeit lag auf der definierten Synthese multifunktioneller Polymer-Konjugate zur Anwendung in der Krebs-Immunotherapie. Durch gezielte Variation der Kon-jugationsbedingungen wurde Zusammensetzung, Größe und Aggregationsverhalten in Zell-medium sowie in humanem Serum untersucht. Nach definierter physikalisch-chemischer Charakterisierung wurde dann die induzierte Antigen-Präsentation zur Aktivierung der T-Zellproliferation analysiert.rnDafür wurden zwei verschiedene polymere Carrier-Systeme gewählt, lineares Poly-L-lysin und eine Polylysinbürste (PLL-Bürste). Es wird vermutet, dass die PLL-Bürste aufgrund der anisotropen Form eine bessere Verteilung im Körper und eine verlängerte Zirkulationsdauer zeigen wird. Die zu konjugierenden biologisch aktiven Komponenten waren der antiDEC205-Antikörper (aDEC205) für die gezielte Adressierung CD8-positiver dendritischer Zellen (DC), ein Ovalbumin (OVA)-spezifisches Antigen mit der Kernsequenz SIINFEKL für die Spezifität der Immunantwort gegen Krebszellen, die dieses Antigen tragen, und ein immunaktivieren-der TLR9-Ligand, CpG1826. Die Effizienz dieses Konjugates dendritische Zellen zu aktivieren, welche wiederum eine Immunantwort gegen OVA-exprimierende Krebszellen induzieren, wurde durch die Konjugation aller Komponenten am identischen Trägermolekül deutlich höher erwartet.rnLineares Poly-L-lysin diente als Modellsystem um die Konjugationschemie zu etablieren und dann auf die zylindrische Polylysinbürste zu übertragen. Anhand dieser polymeren Träger wurde das Verhalten der verschiedenen Topologien des Knäuels und der Bürste im Hinblick auf den Einfluss struktureller Unterschiede sowohl auf Konjugationsreaktionen als auch auf das in situ und in vitro Verhalten untersucht.rnFluoreszenzmarkiertes Antigen und der CpG Aktivator konnten jeweils aufgrund einer Thiol-Modifizierung an die Thiol-reaktive Maleimidgruppe des heterobifunktionellen Linkers Sulfo-SMCC an PLL-AlexaFluor48 konjugiert werden. Anschließend wurde aDEC205-AlexaFluor647 an PLL gekoppelt, entweder durch Schiff Base-Reaktion des oxidierten Antikörpers mit PLL und anschließender Reduzierung oder durch Click-Reaktion des PEG-Azids modifizierten An-tikörpers mit Dicyclobenzylcyclooctin (DIBO)-funktionalisiertem PLL. Die Konjugation der biologisch aktiven Komponenten wurde mit Durchflusszytometrie (FACS) und konfokaler Laser Scanning Mikroskopie (CLSM) untersucht und die Zusammensetzung des Konjugatesrnmittels UV/Vis-Spektroskopie bestimmt. Die PLL-Bürste alleine zeigte eine hohe Zytotoxizität bei HeLa und JAWS II Zelllinien, wohingegen lineares PLL und PLL-Konjugate sowie die PLL Bürsten-Konjugate keine ausgeprägte Zytotoxizität aufwiesen. Die Polymer-Konjugate wie-sen keine Aggregation in Zellmedium oder humanem Serum auf, was mittels winkelabhängi-ger dynamischer Lichtstreuung bestimmt wurde. CLSM Aufnahmen zeigten Kolokalisation der an die einzelnen Komponenten gebundenen Fluoreszenzfarbstoffe in dendritischen Zel-len, was die erfolgreiche Konjugation und Internalisierung der Konjugate in die Zellen bele-gen konnte. FACS Messungen ergaben eine geringfügig erhöhte Aufnahme des adressierten PLL-Antigen-Antikörper-Konjugates verglichen mit dem PLL-Antigen-Konjugat. Experimente mit dem „Specific Hybridization Internalization Sensor“ (SHIP) zeigten jedoch nur Aufnahme der PLL-Konjugate in CD8+ unreife DC, nicht in reife DC, die nicht mehr unspezifisch, sondern nur noch über Rezeptoren internalisieren. Dies bewies die unspezifische Aufnahme des Kon-jugates, da Antikörper-Konjugation keine Rezeptor-vermittelte Endozytose in reife DC indu-zieren konnte. T-Zell-Proliferationsassays ergaben eine Aktivierung von CD8+ T-Zellen indu-ziert durch Antigen-tragende Konjugate, wohingegen Konjugate ohne Antigen als Negativ-kontrollen dienten und keine T-Zell-Proliferation erzielten. Es konnte jedoch kein Unter-schied zwischen adressierten und nicht adressierten Konjugaten aufgrund der unspezifischen Aufnahme durch das Polymer beobachtet werden. Lösliches SIINFEKL alleine bewirkte schon bei geringeren Konzentrationen eine T-Zell-Proliferation.rnEs war somit möglich, drei biologischen Komponenten an einen polymeren Träger zu konju-gieren und diese Konjugate im Hinblick auf Zusammensetzung, Größe, Internalisierung in dendritische Zellen und Aktivierung der T-Zell-Proliferation zu untersuchen. Außerdem wur-de die Konjugationschemie erfolgreich von dem Modellsystem des linearen PLL auf die PLL-Bürste übertragen. Die Polymer-Konjugate werde unspezifisch in DC aufgenommen und in-duzieren T-Zellproliferation, die mit Antigen-Präsentationsassays nachgewiesen wird. Es konnte jedoch durch Konjugation des Antikörpers keine Rezeptor-vermittelte Aufnahme in CD8+ DC erzielt werden.rnDiese Studien stellen einen erfolgsversprechenden ersten Schritt zur Entwicklung neuer Na-nomaterialien für die Anwendung in Krebs-Immuntherapie dar.

Development of a highly potent bispecific antibody format targeting the novel tumor-specific antigen CLDN18.2

Due to multiple immune evasion mechanisms of cancer cells, novel therapy approaches are required to overcome the limitations of existing immunotherapies. Bispecific antibodies are potent anti-cancer drugs, which redirect effector T cells for specific tumor cell lysis, thus enabling the patient’s immune system to fight cancer cells. The antibody format used in this proof of concept study–bispecific ideal monoclonal antibodies termed BiMAB–is a tailor-made recombinant protein, which consists of two fused scFv antibodies recognizing different antigens. Both are arranged in tandem on a single peptide chain and the individual variable binding domains are separated by special non-immunogenic linkers. The format is comprised of a scFv targeting CLDN18.2–a gastric cancer tumor associated antigen (TAA) –while the second specificity binds the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) on T cells. For the first time, we compared in our IMAB362-based BiMAB setting, four different anti-CD3-scFvs, respectively derived from the mAbs TR66, CLB-T3, as well as the humanized and the murine variant of UCHT1. In addition, we investigated the impact of an N- versus a C-terminal location of the IMAB362-derived scFv and the anti-CD3-scFvs. Thus, nine CLDN18.2 specific BiMAB proteins were generated, of which all showed a remarkably high cytotoxicity towards CLDN18.2-positive tumor cells. Because of its promising effectiveness, 1BiMAB emerged as the BiMAB prototype. The selectivity of 1BiMAB for its TAA and CD3ε, with affinities in the nanomolar range, has been confirmed by in vitro assays. Its dual binding depends on the design of an N-terminally positioned IMAB362 scFv and the consecutive C-terminally positioned TR66 scFv. 1BiMAB provoked a concentration and target cell dependent T cell activation, proliferation, and upregulation of the cytolytic protein Granzyme B, as well as the consequent elimination of target cells. Our results demonstrate that 1BiMAB is able to activate T cells independent of elements that are usually involved in the T cell recognition program, like antigen presentation, MHC restriction, and co-stimulatory effector molecules. In the first in vivo studies using a subcutaneous xenogeneic tumor mouse model in immune incompetent NSG mice, we could prove a significant therapeutic effect of 1BiMAB with partial or complete tumor elimination. The initial in vitro RIBOMAB experiments correspondingly showed encouraging results. The electroporation of 1BiMAB IVT-RNA into target or effector cells was feasible, while the functionality of translated 1BiMAB was proven by induced T cell activation and target cell lysis. Accordingly, we could show that the in vitro RIBOMAB approach was applicable for all nine BiMABs, which proves the RIBOMAB concept. Thus, the CLDN18.2-BiMAB strategy offers great potential for the treatment of cancer. In the future, administered either as protein or as IVT-RNA, the BiMAB format will contribute towards finding solutions to raise and sustain tumor-specific cellular responses elicited by engaged and activated endogenous T cells. This will potentially enable us to overcome immune evasion mechanisms of tumor cells, consequently supporting current solid gastric cancer therapies.

Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

Improvement of non-paraneoplastic voltage-gated potassium channel antibody-associated limbic encephalitis without immunosuppressive therapy

We describe a 61-year-old patient with clinical evidence of limbic encephalitis who improved with anticonvulsant treatment only, that is, without the use of immunosuppressive agents. Three years following occurrence of anosmia, increasing memory deficits, and emotional disturbances, he presented with new-onset temporal lobe epilepsy, with antibodies binding to neuronal voltage-gated potassium channels and bitemporal hypometabolism on FDG-PET scan; the MRI scan was normal. This is most likely a case of spontaneous remission, illustrating that immunosuppressive therapy might be suspended in milder courses of limbic encephalitis. It remains open whether treatment with anticonvulsant drugs played an additional beneficiary role through the direct suppression of seizures or, additionally, through indirect immunomodulatory side effects.

Anti-interleukin-5 antibody treatment (mepolizumab) in active eosinophilic oesophagitis: a randomised, placebo-controlled, double-blind trial

Eosinophilic oesophagitis (EoO) is a clinicopathological condition defined by proton pump inhibitor-refractory oesophageal symptoms combined with oesophageal eosinophilia. The pharmacodynamic effect of mepolizumab (a humanised anti-interleukin-5 monoclonal antibody) in EoO was evaluated.

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.

Sperm analysis of patients after successful treatment of childhood acute lymphoblastic leukemia with chemotherapy

Survivors of childhood acute lymphoblastic leukemia (ALL) treated with radiotherapy are at risk for impaired fertility. Whether chemotherapy alone is also long-term gonadotoxic is unclear. We assessed gonadal function in 11 male ALL-survivors treated with the same chemotherapy regimen and compared sperm analysis to healthy men. While sex hormone levels were normal in all subjects, 5/11 survivors showed pathological sperm concentration and 4/11 a decreased total sperm count compared to WHO criteria. Compared to healthy controls, all quantitative parameters in semen analysis of survivors were decreased. This suggests that treatment with chemotherapeutic agents alone, even in moderate doses, might have a gonadotoxic effect.

Mannan-binding lectin deficiency results in unusual antibody production and excessive experimental colitis in response to mannose-expressing mild gut pathogens

In Crohn's disease (CD) the deficiency of mannan-binding lectin (MBL) is associated with an increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of the disease. However, the role of MBL in intestinal inflammation is currently unclear. A study was undertaken to analyse local MBL expression in human intestine and the consequences of MBL deficiency in experimental colitis and yeast infection.

Effects of TNF antagonists on sperm characteristics in patients with spondyloarthritis

To study the influence of tumour necrosis factor (TNF) antagonists on spermatogenesis in a cohort of patients with spondyloarthritis (SpA).

A double role of sperm in scorpions: the mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm

Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel-like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel-like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3-5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry.

Sperm of colourful males are better protected against oxidative stress

Sperm cells are highly vulnerable to free radicals, and sperm quality and male fertility are critically affected by oxidative stress. Recently, sexual ornaments, particularly carotenoid-based colourful traits, have been proposed to depend on a male's capacity to resist oxidative stress, and thus to signal sperm quality. We conducted an experimental test of this hypothesis on great tits Parus major, in which adults are sexually dichromatic in carotenoid-based breast plumage. We report the first evidence that ornaments and sperm quality may be linked through oxidative stress. When experimentally subjected to oxidative stress resulting from increased workload, less colourful males suffered a greater reduction in sperm motility and swimming ability, and increased levels of sperm lipid peroxidation compared to more colourful males. Moreover, the level of sperm lipid peroxidation was negatively correlated with sperm quality. Finally, carotenoid supplementation increased sperm quality of less colourful males, suggesting that pale males are deficient in carotenoid antioxidants.

Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus

Sperm competition exerts strong selection on males to produce spermatozoa with an optimal morphology that maximizes their fertilization success. Long sperm were first suggested to be favored because they should swim faster. However, studies that investigated the relationship between sperm length and sperm competitive ability or sperm swimming velocity yielded contradictory results. More recently, ratios of the different sections of a spermatozoon (the head, midpiece, and flagellum) were suggested to be more crucial in determining swimming velocity. Additionally, sperm ability to remain and survive in the female storage organs may also influence fertilization success, so that optimal sperm morphology may rather maximize sperm longevity than velocity. In this study, we investigated how sperm morphology is related to sperm velocity and sperm longevity in the house sparrow Passer domesticus. Sperm velocity was found to be correlated with head/flagellum ratio. Sperm with small heads relative to their flagellum showed higher swimming velocity. Additionally, shorter sperm were found to live longer. Finally, we found sperm morphological traits to vary substantially within males and the head/flagellum ratio to be unrelated to total sperm length. We discuss the hypothesis that the substantial within-male variation in sperm morphology reflects a male strategy to produce a diversity of sperm from long, fast-swimming to short, long-living sperm to maximize their fertilization success in a context of sperm competition.