The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model

BACKGROUND Endometriosis is still a highly underdiagnosed disease, and the current medical and surgical treatment of endometriosis is associated with a high recurrence rate. This study investigates the use of derivatives of the human antibody F8, specific to the alternatively spliced extra-domain A of fibronectin (Fn), for the imaging and treatment of endometriosis. METHODS Immunohistochemistry and immunofluorescence was used to evaluate antigen expression in endometriotic tissue of human endometriosis and of a syngeneic mouse model of the disease. The in vivo targeting performance of a fluorescent derivative of the F8 antibody was assessed by imaging mice with endometriosis using a near-infrared fluorescence imager, 24 h following i.v. injection of the antibody conjugate. Furthermore, the mouse model was used for therapy experiments using two recombinant F8-based immunocytokines [F8-interleukin-10 (IL10) and F8-IL2] or saline for the treatment groups. RESULTS A very strong vascular expression of splice isoforms of Fn and of tenascin-C was observed in human endometriotic lesions by immunohistochemistry and immunofluorescence techniques. After i.v. administration, a selective accumulation of the F8 antibody in endometriotic lesions could be observed in a syngeneic mouse model. These targeting data were used as a basis for therapy experiments with a pro-inflammatory (F8-IL2) and an anti-inflammatory (F8-IL10) cytokine fusion protein of the F8 antibody. The average lesion size in the F8-IL10 treatment group was clearly reduced compared with the saline control group and with the F8-IL2 group, for which no therapeutic effects were observed. CONCLUSIONS The F8 antibody targets endometriotic lesions in vivo in a mouse model of endometriosis and may be used for the non-invasive imaging of the disease and for the pharmacodelivery of anti-inflammatory cytokines, such as IL1

Preclinical characterization of DEKAVIL (F8-IL10), a novel clinical-stage immunocytokine which inhibits the progression of collagen-induced arthritis

Introduction In this article, we present a comparative immunohistochemical evaluation of four clinical-stage antibodies (L19, F16, G11 and F8) directed against splice isoforms of fibronectin and of tenascin-C for their ability to stain synovial tissue alterations in rheumatoid arthritis patients. Furthermore we have evaluated the therapeutic potential of the most promising antibody, F8, fused to the anti-inflammatory cytokine interleukin (IL) 10. Methods F8-IL10 was produced and purified to homogeneity in CHO cells and shown to comprise biological active antibody and cytokine moieties by binding assays on recombinant antigen and by MC/9 cell proliferation assays. We have also characterized the ability of F8-IL10 to inhibit arthritis progression in the collagen-induced arthritis mouse model. Results The human antibody F8, specific to the extra-domain A of fibronectin, exhibited the strongest and most homogenous staining pattern in synovial biopsies and was thus selected for the development of a fully human fusion protein with IL10 (F8-IL10, also named DEKAVIL). Following radioiodination, F8-IL10 was able to selectively target arthritic lesions and tumor neo-vascular structures in mice, as evidenced by autoradiographic analysis and quantitative biodistribution studies. The subcutaneous administration route led to equivalent targeting results when compared with intravenous administration and was thus selected for the clinical development of the product. F8-IL10 potently inhibited progression of established arthritis in the collagen-induced mouse model when tested alone and in combination with methotrexate. In preparation for clinical trials in patients with rheumatoid arthritis, F8-IL10 was studied in rodents and in cynomolgus monkeys, revealing an excellent safety profile at doses tenfold higher than the planned starting dose for clinical phase I trials. Conclusions Following the encouraging preclinical results presented in this paper, clinical trials with F8-IL10 will now elucidate the therapeutic potential of this product and whether the targeted delivery of IL10 potentiates the anti-arthritic action of the cytokine in rheumatoid arthritis patients.ISSN:1465-9905ISSN:1465-9913ISSN:1478-6362ISSN:1478-635

A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells

The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering researc

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trial

Complexity analysis of the fetal heart rate variability: early identification of severe intrauterine growth-restricted fetuses

The main goal of this work is to suggest new indices for a correct identification of the intrauterine growth-restricted (IUGR) fetuses on the basis of fetal heart rate (FHR) variability analysis performed in the antepartum period. To this purpose, we analyzed 59 FHR time series recorded in early periods of gestation through a Hewlett Packard 1351A cardiotocograph. Advanced analysis techniques were adopted including the computation of the Lempel Ziv complexity (LZC) index and the multiscale entropy (MSE), that is, the entropy estimation with a multiscale approach. A multiparametric classifier based on k-mean cluster analysis was also performed to separate pathological and normal fetuses. The results show that the proposed LZC and the MSE could be useful to identify the actual IUGRs and to separate them from the physiological fetuses, providing good values of sensitivity and accuracy (Se = 77.8%, Ac = 82.4%)

Global maps of soil temperature

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km² resolution for 0–5 and 5–15 cm soil depth. These maps were created by calculating the difference (i.e., offset) between in-situ soil temperature measurements, based on time series from over 1200 1-km² pixels (summarized from 8500 unique temperature sensors) across all the world’s major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (-0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in-situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Global maps of soil temperature.

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km2 resolution for 0-5 and 5-15 cm soil depth. These maps were created by calculating the difference (i.e. offset) between in situ soil temperature measurements, based on time series from over 1200 1-km2 pixels (summarized from 8519 unique temperature sensors) across all the world's major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (-0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications

Verdingkinder in der Schweiz. Qualitative Untersuchung der Behandlung von Verdingkindern in der Schule

„Du kannst nichts, du bist nichts und du wirst nichts" (U. Mäder, Gewalt und Machtmissbrauch, 2008, S. 219) Aussagen wie diese haben viele Verdingkinder immer wieder zu hören bekom-men. Bis tief ins 20. Jahrhundert wurden schulpflichtige Knaben und Mädchen als Verdingkinder in Pflegefamilien untergebracht und dienten dort vornehmlich als billige Arbeitskräfte (M. Leuenberger, Einleitung, 2008, S. 14f). Die vorliegende Arbeit verschafft dem Leser/der Leserin in einem ersten Teil einen Überblick über das Verdingkinderwesen in der Schweiz. Er zeigt die soziale und wirtschaftliche Lage der Schweiz während des 19. und 20. Jahrhunderts auf und beleuchtet die gesetzlichen und rechtlichen Grundlagen, sowie deren Entwicklung und Veränderung im Laufe der Zeit. Im zweiten Teil der Arbeit wird der Schwerpunkt auf die einzelnen Schicksale der Verdingkinder gelegt. In diesem Zusammenhang folgen Beschreibungen von Rol-lenverständnissen und Haltungen der Schule und deren Lehrpersonen. Die Mas-terarbeit verwendet dazu Datenmaterial aus der Nationalfondstudie „Verdingkin-der, Schwabengänger, Spazzacamini und andere Formen von Fremdplatzierung und Kinderarbeit in der Schweiz im 19. und 20. Jahrhundert“. Dabei geht es um die Erfahrungen, die ehemalige Verdingkinder mit der Fremdplatzierung gemacht haben. Die Lehrpersonen haben sehr unterschiedlich reagiert im Umgang mit den Verdingkindern. Zum einen boten sie Unterstützungen und ermöglichten positive Schulerlebnisse, zum anderen gab es Lehrpersonen, die ihre Machtposition ge-genüber den Verdingkindern missbrauchten

Verdingkinder in der Schweiz. Qualitative Untersuchung der Behandlung von Verdingkindern in der Schule

„Du kannst nichts, du bist nichts und du wirst nichts" (U. Mäder, Gewalt und Machtmissbrauch, 2008, S. 219) Aussagen wie diese haben viele Verdingkinder immer wieder zu hören bekom-men. Bis tief ins 20. Jahrhundert wurden schulpflichtige Knaben und Mädchen als Verdingkinder in Pflegefamilien untergebracht und dienten dort vornehmlich als billige Arbeitskräfte (M. Leuenberger, Einleitung, 2008, S. 14f). Die vorliegende Arbeit verschafft dem Leser/der Leserin in einem ersten Teil einen Überblick über das Verdingkinderwesen in der Schweiz. Er zeigt die soziale und wirtschaftliche Lage der Schweiz während des 19. und 20. Jahrhunderts auf und beleuchtet die gesetzlichen und rechtlichen Grundlagen, sowie deren Entwicklung und Veränderung im Laufe der Zeit. Im zweiten Teil der Arbeit wird der Schwerpunkt auf die einzelnen Schicksale der Verdingkinder gelegt. In diesem Zusammenhang folgen Beschreibungen von Rol-lenverständnissen und Haltungen der Schule und deren Lehrpersonen. Die Mas-terarbeit verwendet dazu Datenmaterial aus der Nationalfondstudie „Verdingkin-der, Schwabengänger, Spazzacamini und andere Formen von Fremdplatzierung und Kinderarbeit in der Schweiz im 19. und 20. Jahrhundert“. Dabei geht es um die Erfahrungen, die ehemalige Verdingkinder mit der Fremdplatzierung gemacht haben. Die Lehrpersonen haben sehr unterschiedlich reagiert im Umgang mit den Verdingkindern. Zum einen boten sie Unterstützungen und ermöglichten positive Schulerlebnisse, zum anderen gab es Lehrpersonen, die ihre Machtposition ge-genüber den Verdingkindern missbrauchten