Smaller size packs a stronger punch - Recent advances in small antibody fragments targeting tumour-associated carbohydrate antigens

Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness in vivo. To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies

Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4‐/‐ mice

The role of CC chemokine receptor 4 (CCR4) during the development and maintenance of Th2type allergic airway disease is controversial. In this study, we examined the role of CCR4 in the chronic allergic airway response to live Aspergillus fumigatus spores, or conidia, in A. fumigatussensitized mice. After the conidia challenge, mice lacking CCR4 (CCR4‐/‐ mice) exhibited significantly increased numbers of airway neutrophils and macrophages, and conidia were more rapidly eliminated from these mice compared with control CCR4 wild‐type (CCR4+/+) mice. Significant airway hyperresponsiveness to intravenous methacholine was observed at day 3 in CCR4‐/‐ mice, whereas at days 7 and 30, airway hyperresponsiveness was attenuated in these mice compared with control mice. A major reduction in peribronchial and airway eosinophilia was observed in CCR4‐/‐ mice at all times after conidia challenge in contrast to CCR4+/+ mice. Further, whole lung levels of interleukin (IL) 4 and IL‐5 were significantly increased in CCR4‐/‐ mice at day 3, whereas these Th2 cytokines and IL‐13 were significantly decreased at day 30 in CCR4‐/‐ mice compared with their wild‐type counterparts. Peribronchial fibrosis and goblet cell hyperplasia were similar in both groups of mice throughout the course of this model. In summary, CCR4 modulates both innate and acquired immune responses associated with chronic fungal asthma.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154441/1/fsb2fasebj16100193-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154441/2/fsb2fasebj16100193.pd

Cytokine-dependent and membrane-associated interactions between microbe- stimulated macrophages and natural killer cells

Titelblatt Inhaltsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung LiteraturDie Aktivierung von Makrophagen (MF) durch Interferon (IFN-) g ist ein bedeutender Abschnitt der zellulären Immunreaktion auf Erreger. Die Mechanismen der in vitro Induktion der IFN-g Produktion durch Mikroorganismen sind Thema der vorliegenden Arbeit. In Abwesenheit von T-Lymphozyten wird die Bildung von IFN-g durch zytokinvermittelte Interaktionen zwischen MF und Natürlichen Killer (NK) Zellen initiiert. Allerdings wurde beobachtet, daß a) die IFN-g Produktion abgeschwächt ist, wenn ein Membrankontakt zwischen MF und NK Zellen fehlt und b) in manchen Erregermodellen eine enge Nachbarschaft dieser Zellen für eine IFN-g Produktion sogar unabdingbar ist. Daher wurde die Hypothese aufgestellt, daß, neben den zytokinvermittelten, auch membranständige Mechanismen an der Induktion von IFN-g beteiligt sind. Es wurde untersucht, ob weitere lösliche MF-Faktoren an der Induktion der IFN-g Produktion beteiligt sind. Während der Koinkubation mit entweder Pneumocystis carinii oder Listeria monocytogenes wurden MF in einem Zweikammernsystem durch eine für Makromoleküle durchlässige Membran von NK Zellen getrennt. Eine Inkubation mit P. carinii induzierte keine Produktion von IFN-g. Nach einer Inkubation mit L. monocytogenes waren geringere IFN-g Konzentrationen zu beobachten, als in einer Kokultur mit der Möglichkeit eines direkten Membrankontaktes zwischen MF und NK Zellen. Die Beteiligung von löslichen, bislang unbekannten Mediatoren an der durch P. carinii-Organismen ausgelösten IFN-g Induktion konnte demnach ausgeschlossen werden. Die Rolle von zellständigen Antigenen bei der Induktion der IFN-g Produktion wurde durch Blockadeversuche mit monoklonalen Antikörpern untersucht. Eine Koinkubation von MF, NK Zellen und L. monocytogenes oder P. carinii in Gegenwart von Antikörpern gegen das F4/80 Molekül führte sowohl auf Ebene der mRNA als auch auf Proteinebene zu einer verringerten Bildung von IFN-g, IL-12 und TNF-a. Diese Daten wurden dahingehend interpretiert, daß der Antikörper das Antigen blockiert und somit eine Signalübertragung von MF zu NK Zellen verhindert. Die Stärke der F4/80 Expression hat keinen Einfluß auf die Produktion von IFN-g. Milzzellen von Mäusen hingegen, die aufgrund einer Mutation das F4/80 Antigen nicht exprimieren, konnten nach Inkubation mit L. monocytogenes weitaus weniger IFN-g bilden, als Tiere des Wildtyps. Die These eines membranabhängigen Signalweges zwischen MF und NK Zellen zur Induktion von IFN-g wurde dadurch erhärtet, daß gegen NK Zellen gerichtete monoklonale Antikörper generiert werden konnten, welche eine erregerinduzierte Produktion von IFN-g inhibierten. Die Daten dieser Arbeit weisen auf eine interzellulär wirksame Funktion des F4/80-Antigens hin und stellen damit die erste Funktionsbeschreibung dieses Moleküls dar. Weiterhin wird durch diese Arbeit zum ersten Mal eine Beteiligung membrangebundener Antigene an der mikrobiell induzierten IFN-g Produktion gezeigt. Es wird vermutet, daß ein membranabhängiger Signalweg zwischen MF und NK Zellen je nach Typ des Erregers entweder eine essenzielle (P. carinii) oder eine zumindest kostimulatorische Rolle (L. monocytogenes) besitzt.Activation of macrophages (MF) by gamma-interferon (IFN-g) is an important step in cellular immune reactions against microbial pathogens. In the absence of T lymphocytes, production of IFN-g is initiated by cytokine-dependent interactions between MF and natural killer (NK) cells, triggered by the pathogen. We found that a) Listeria monocytogenes-triggered release of IFN-g was significantly reduced when cell contact between MF and NK cells was inhibited, and b) Cell contact between MF and NK cells was even prerequisite for IFN-g release when triggered by Pneumocystis carinii. This lead to the hypothesis that, in addition to cytokine-release, membrane associated mechanisms play a role in inducing NK-dependent IFN-g. First, we examined whether other soluble MF-derived factors contribute to the induction of IFN-g. Macrophages were incubated with L. monocytogenes or P. carinii organisms in the same well as purified NK cells but separated by a semipermeable membrane. While P. carinii organisms did not trigger any IFN-g release, L. monocytogenes triggered significantly lower amounts of IFN-g than either pathogen when cell contact was uninhibited. These and other results made it unlikely, that as yet unknown soluble MF products played a significant role in this system. Next, we investigated whether known MF surface antigens were involved by inhibition experiments with monoclonal antibodies (mAb). Co-incubation of MF, NK cells and L. monocytogenes or P. carinii with mAb against the F4/80 antigen inhibited the production of IFN-g, interleukin (IL)-12 and tumor necrosis- factor (TNF)-a, both on mRNA and on protein levels. These data were interpreted in that the mAb blocked F4/80,thereby preventing a necessary or accessory signaling between microbe-triggered MF and NK cells. The intensity of F4/80 expression on MF did not seem to affect IFN-g release in vitro. In contrast, spleen cells of gene-deleted F4/80 knock-out mice produced much less IFN-g in the presence of L. monocytogenes than spleen cells of wild-type mice. The hypothesis of a membrane-dependent signaling pathway between MF an NK cells was affirmed by the development of NK-specific mAb which also inhibited pathogen-triggered, T cell-independent IFN-g release. Our data suggest an intracellular regulatory role for F4/80. Although widely used as a cell marker, this is the first report on a function for this murine MF-specific antigen. Taken together, our work suggests a membrane-associated signaling pathway between MF and NK cells. Its relative importance in initiating natural cellular immune response mechanisms depends on the nature of primary microbial stimulus

Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses.National Institutes of Health - 1R21AI139387-01Center for Companion Animal Health - 2016-21-F, 2017-11-F, 2018-53-F, and 2019-12-FDeutsche Forschungsgemeinschaft (DFG) - 251133687/GRK 2046German One-Health InitiativeEuropean Union’s Horizon 2020 Research and Innovation programDepto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against disinfectants and irradiation has been attributed to the oocyst and sporocyst walls by acting as physical and permeability barriers. However, the genetic basis for their resistance against stressors like changes in temperature, salinity, or humidity, is unknown. We show that a cluster of four genes encoding Toxoplasma Late Embryogenesis Abundant (TgLEA)-related proteins are important for this resistance to environmental stresses. TgLEAs have features of intrinsically disordered proteins, explaining some of their properties. Recombinant TgLEA proteins show cryoprotective effects on the parasite's lactate dehydrogenase, an abundant enzyme in oocysts, and expression in E. coli of two TgLEAs has a beneficial effect on growth after cold stress. Moreover, oocysts from a strain lacking all four TgLEA genes were more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts, highlighting the importance of the four TgLEAs for oocyst resilience

Generation of glycan-specific nanobodies

The development of antibodies that target specific glycan structures on cancer cells or human pathogens poses a significant challenge due to the immense complexity of naturally occurring glycans. Automated glycan assembly enables the production of structurally homogeneous glycans in amounts that are difficult to derive from natural sources. Nanobodies (Nbs) are the smallest antigen-binding domains of heavy-chain-only antibodies (hcAbs) found in camelids. To date, the development of glycan-specific Nbs using synthetic glycans has not been reported. Here, we use defined synthetic glycans for alpaca immunization to elicit glycan-specific hcAbs, and describe the identification, isolation, and production of a Nb specific for the tumor-associated carbohydrate antigen Globo-H. The Nb binds the terminal fucose of Globo-H and recognizes synthetic Globo-H in solution and native Globo-H on breast cancer cells with high specificity. These results demonstrate the potential of our approach for generating glycan-targeting Nbs to be used in biomedical and biotechnological applications