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Magnus Nilsson,1 Magdalena Rhedin,2 Ramon Hendrickx,3 Susanne Berglund,1 Antonio Piras,2 Parmis Blomgran,2 Anders Cavallin,2 Mia Collins,2 Göran Dahl,4 Bilel Dekkak,5 Therese Ericsson,3 Niklas Hagberg,6 Ann Aurell Holmberg,3 Agnes Leffler,2 Anders J Lundqvist,3 Thomais Markou,5,7 James Pinkerton,5,7 Lars Rönnblom,6 Stacey Siu,8 Vanessa Taylor,8 Tiiu Wennberg,2 Dimitrios Zervas,5,7 Arian D J Laurence,9 Suman Mitra,2 Maria G Belvisi,5,7 Mark Birrell,5,7 Annika Borde2 1Medicinal Chemistry, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 2Bioscience, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 3DMPK, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 4Discovery Science, R&D, AstraZeneca, Gothenburg, Sweden; 5Respiratory Pharmacology Group, Division of Airway Disease, National Heart and Lung Institute, Imperial College London, London, UK; 6Rheumatology and Science for Life Laboratories, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; 7Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 8Rigel Pharmaceuticals, South San Francisco, CA, USA; 9Department of Haematology, University College London Hospitals NHS Foundation Trust, London, UKCorrespondence: Magnus Nilsson, Medicinal Chemistry, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, SE-431 83, Sweden, Tel +46722237222, Email Magnus.Nilsson@astrazeneca.comPurpose: Janus kinase 1 (JAK1) is implicated in multiple inflammatory pathways that are critical for the pathogenesis of asthma, including the interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin cytokine signaling pathways, which have previously been targeted to treat allergic asthma. Here, we describe the development of AZD0449 and AZD4604, two novel and highly selective JAK1 inhibitors with promising properties for inhalation.Methods: The effects of AZD0449 and AZD4604 in JAK1 signaling pathways were assessed by measuring phosphorylation of signal transducer and activator of transcription (STAT) proteins and chemokine release using immunoassays of whole blood from healthy human volunteers and rats. Pharmacokinetic studies performed on rats evaluated AZD0449 at a lung deposited dose of 52 μg/kg and AZD4604 at 30 μg/kg. The efficacy of AZD0449 and AZD4604 was assessed by evaluating lung inflammation (cell count and cytokine levels) and the late asthmatic response (average enhanced pause [Penh]).Results: Both compounds inhibited JAK1-dependent cytokine signaling pathways in a dose-dependent manner in human and rat leukocytes. After intratracheal administration in rats, both compounds exhibited low systemic exposures and medium-to-long terminal lung half-lives (AZD0449, 34 hours; AZD4604, 5 hours). Both compounds inhibited STAT3 and STAT5 phosphorylation in lung tissue from ovalbumin (OVA)-challenged rats. AZD0449 and AZD4604 also inhibited eosinophilia in the lung and reduced the late asthmatic response, measured as Penh in the OVA rat model.Conclusion: AZD0449 and AZD4604 show potential as inhibitors of signaling pathways involved in asthmatic immune responses, with target engagement demonstrated locally in the lung. These findings support the clinical development of AZD0449 and AZD4604 for the treatment of patients with asthma.Keywords: AZD0449, AZD4604, JAK, STAT

Intratumoral regulatory T (Treg) cell abundance associates with diminished antitumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high-affinity receptor subunit for interleukin (IL)-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their antitumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Treg cells, while preserving IL-2-STAT5 signaling on effector T cells and show synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both nonhuman primates and humanized mouse models, efficient Treg cell depletion with no overt immune-related toxicities. Our data support the clinical development of RG6292 and evaluation of new combination therapies incorporating non-IL-2-blocking anti-CD25 antibodies in clinical studies. Quezada and colleagues develop improved anti-CD25 antibodies that preserve IL-2 signaling and enhance single-agent antitumor immunity and immunotherapy through specific and efficient Treg cell depletion.

The EU Water Framework Directive foresees the ecological assessment of surface waters against identified pressures. Nutrient loading is the main pressure impairing the ecological quality of lake ecosystems, and aquatic macrophytes are considered good indicators of ecological response. In this study, we statistically assessed different aspects of aquatic plant (macrophyte) diversity in response to different trophic levels in Mediterranean lakes. We used 5690 relevés of aquatic vegetation, distributed over 305 transects, sampled in 18 freshwater lake ecosystems during 2013–2016. Our results show a significant decrease in taxonomic alpha diversity in lakes with a total phosphorus content above 100 μg/L. Syntaxonomic diversity followed the species richness pattern as well. Functional richness decreased along the trophic gradient, while functional dispersion was higher in lakes with high trophic levels. Taxonomic and functional beta partitioning presented changes in assembly processes leading to greater community homogeneity in lakes with higher trophic levels. In summary, we found no redundancy between taxonomic and functional diversity indices. These results provide novel insights into aquatic plant assembly processes of impacted freshwater lakes needed to forward conservation and restoration practices.

Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

The ecological water regime in lake water bodies refers to the water levels that enable the fulfillment of the ecosystem&rsquo, s multiple functions. Therefore, assessing the ecological water regime necessitates the consideration of hydrological, economic, social, and ecological factors. The present research is focused on the assessment of the ecological water level of four Mediterranean natural lake ecosystems, considering their morphological and biological features. Initially, suggestions on the ecological water regime of the studied lakes were made based on an analysis of the lakes&rsquo, morphometry. Further, the ecological and biological requirements of the present fish fauna and aquatic macrophytic vegetation were considered. For the latter, mapping was conducted by extensive sampling according to international standards, in order to assess macrophyte composition, abundance, and chorology, as well as species sensitivity to water level fluctuations. The above guided the proposals on the optimal water level regime that should be met by each lake regarding the macrophytic and fish communities&rsquo, sustainability, also taking into account the unique hydromorphological features of each lake. The differences in the outcoming results revealed that hydromorphological and biological approaches should be combined for assessing lakes&rsquo, ecological water regimes.

Introduction: Asthma is a chronic inflammatory disease of the airways with typical symptoms manifesting in coughing, wheezing and dyspnoea. Whilst current treatments can be effective, some patients are not properly controlled, and the medications are not ideal and are associated with unwanted side effects. Thus, new therapies are urgently required. Typically, the chronic inflammation endotype is associated with increased levels of type 2 cytokine such as IL-4, IL-5, IL-9 & IL-13. These pro-inflammatory signalling pathways critically depend on Janus Kinase (JAK) I or II signal transduction, therefore the inhibition of JAK pathways represents a promising therapeutic target in asthma. Aims and Objectives: To assess the potential benefit of JAK inhibition using two structurally distinct inhibitors in a in vivo model system. Methods: Two JAK inhibitors, AZD4604 & AZD0449, were intratracheally dosed into sensitised Brown Norway rats one hour prior to aerosolised allergen (OVA) challenge. In one set of animals we assessed target engagement (TE) in the lung tissue by measuring levels of pSTATs. In a parallel set, we measured the Late Asthmatic Response (LAR) and lung inflammation. Results: Administration of both AZD4604 and AZD0449 resulted in dose related reduction of pSTAT3 and pSTAT5, indicating TE in the lung. Furthermore, AZD4604 & AZD0449 administration suppressed LAR and airway inflammation to a similar level to that achieved with the ICS comparator, budesonide. Conclusions: These data indicate that JAK inhibition may be of benefit to patients suffering from allergic asthma.

Despite the advances in cancer immunotherapy, in particular in the field of checkpoint inhibitors (CPI), many patients fail to respond (primary resistance) or initially benefit but then progress upon treatment (secondary resistance). High regulatory T-cell (Treg) counts correlate with poor prognosis and reduced responsiveness to CPI therapy in humans, underscoring their potential as an immunotherapy target. Clinical attempts aiming to lower Treg counts, however, either failed to deliver convincing Treg reduction or lacked specificity for Treg over tumor antigen specific cytotoxic T cells (CTL). CD25 (the interleukin-2 receptor alpha (IL-2Ra) chain) is a recently revisited target for Treg depletion. For privileged access to IL-2, activated CTL up-regulate CD25 expression only transiently during clonal expansion. Further confirmation of an > 20 fold higher cell surface expression of CD25 on Tregs versus CTLs in human malignancies is provided. The novel compound RG6292 was developed as an ADCC and ADCP competent monoclonal antibody of human IgG1 isotype with afucosylated glycans in the Fc region. RG6292 binds with low monovalent affinity (KD 250 nM) to the extracellular domain of CD25 antigen. A high density of CD25 receptors promotes bivalent avidity of RG6292 increasing its binding strength to CD25 by at least 100 fold (KD 2-3 nM). RG6292 selectively favors the depletion of CD25 high Tregs over CD25 low activated CTLs, here shown in comparison to ipilimumab and mogamulizumab in human αCD3 activated PBMC, human tumor explants and immunopharmacodynamic studies in tumor bearing (BxPC-3), stem cell humanized mice and cynomolgus monkeys. IL-2 is an essential prerequisite for clonal expansion of CTLs, which is necessary to generate effective anti-tumor responses. Earlier immunosuppressant anti-CD25 antibodies (e.g. daclizumab and basiliximab) interfered with the formation of the high affinity IL-2R complex. Their evidenced lack of therapeutic activity in immunoncology tempered enthusiasm and highlights the pivotal role of IL-2. RG6292 is the first anti-human CD25 antibody developed to deplete Tregs selectively while fully preserving IL-2 signaling and CTL activity. Pre-clinically, a single administration of the RG6292 surrogate effectively promoted eradication of established tumors in several tumor mouse models and synergized with CPI in models of CPI resistance. RG6292 is expected to unleash the potential of selective Treg depletion while allowing for unrestricted access of IL 2 to CTLs and could therefore result in clinically superiority compared to other Treg depleting antibodies. RG6292 provides a novel therapeutic approach to alleviate a major mechanism of immune suppression in the tumor microenvironment. Clinical testing is currently ongoing to evaluate the safety and tolerability of RG6292 in patients with advanced solid tumors (NCT04158583). Citation Format: Maria Amann, Gabriel Schnetzler, Kolben Theresa, Isabelle Solomon, Christophe Boetsch, Estelle Marrer-Berger, Reto Flury, Claudio Murgia, Vaios Karanikas, Johannes Sam, Roger Sutmuller, Jan Eckmann, Hans Koll, Sara Belli, Frederic Arce Vargas, Dimitrios Zervas, Chen Qing, Mark A. Brown, Josephine Salimu, Anne Goubier, Sebastian Neumann, Karl S. Peggs, Sergio A. Quezada. The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4553.

Inducible T cell co-stimulator (ICOS) is a co-stimulatory receptor expressed on tumor infiltrating regulatory and effector T cells. ICOS potential role in cancer control is supported by the correlation between ipilimumab’s efficacy and the presence of CD4+ICOShi T cells in PBMC, as well as loss of tumor control in ICOS−/− and ICOSL−/− mice treated with anti CTLA-4. 37A10 is an anti ICOS agonistic mAb reactive to mouse and human ICOS, with a human IgG1 Fc region to promote depletion of regulatory T cells through antibody-dependent cellular cytotoxicity. We hypothesized a pure agonistic, non-depleting anti ICOS would promote co-stimulatory signals on activated T cells while avoiding depletion of activated ICOS+ T cells. Hence, we aimed to compare the efficacy in tumor control of two isotypes of 37A10. Both mIgG1 (non-depleting) and mIgG2a (depleting) isotypes failed to promote survival, as a monotherapy or in combination with anti PD-1 or anti CTLA-4. This was consistent with both isotypes promoting the elimination of activated T cells in an Fc-independent manner. Ablation of activated effector T cells with anti ICOS replicated loss of activated effector T cells upon in vivo blockade of ICOSL reflecting their ligand blocking activity. Moreover, both isotypes impaired the activity of anti CTLA-4, associated to effector T cell depletion, raising concerns regarding the use of anti ICOS mAbs as combination substrates in tumor immunotherapy. The data suggest that agonistic antibodies that block the natural ligand binding site and have weak in vivo agonism could result in unwanted negative responses in cancer. Future work should aim to identify antibodies with robust agonistic activity in vivo while allowing signaling induced by the natural ligand.

The Water Framework Directive (WFD) requires Member States to develop appropriate assessment methods for the classification of the ecological status of their surface waters. Mediterranean region has lagged behind in this task, so we propose here the first developed method for Greek lakes, Hellenic Lake Macrophyte (HeLM) assessment method. This method is based on two metrics, a modified trophic index and maximum colonization depth Cmax that quantify the degree of changes in lake macrophytic vegetation, as a response to eutrophication and general degradation pressures. The method was developed on the basis of a data set sampled from 272 monitoring transects in 16 Greek lakes. Sites from three lakes were selected as potential reference sites by using a screening process. Ecological quality ratios were calculated for each metric and for each lake, and ecological status class boundaries were defined. For the evaluation of effectiveness of the method, the correlations between individual metrics and final HeLM values and common pressure indicators, such as total phosphorus, chlorophyll a and Secchi depth, were tested and found highly significant and relatively strong. In addition, the ability of HeLM values and its individual metrics to distinguish between different macrophytic communities’ structure was checked using aquatic plant life-forms and found satisfactory. The HeLM method gave a reliable assessment of the macrophytic vegetation’s condition in Greek lakes and may constitute a useful tool for the classification of ecological status of other Mediterranean lakes. Electronic supplementary material The online version of this article (10.1007/s10661-018-6708-1) contains supplementary material, which is available to authorized users.

Modern water resource management requires biomonitoring of the structure and functioning of freshwater ecosystems, which may be better illuminated by functional trait distribution patterns and responses across human-induced pressure gradients. In this study, we applied the RLQ, the fourth-corner and their novel combination methods, in order to assess the relationship between the distribution of 30 aquatic macrophyte functional traits and 14 indicators of anthropogenic pressures across 16 freshwater lakes. Our findings showed that there is a statistically significant relationship between the distribution of specific functional traits and anthropogenic pressures. Eutrophication was the dominant pressure and the shift from a submerged-macrophyte dominated vegetation community to an emergent one was the most important functional response. Aerial reproduction and dispersal traits were found to replace water-related ones under higher nutrient concentrations. Trophic and light preferences of macrophytic species increased, while their leaf morphology was found to change from tubular/capillary leaf types with low leaf area values to entire leaf types with greater leaf area. These results provide hints on the changes in ecosystem functioning occurring as a response to human-induced drivers. Therefore, this assessment approach could provide important support to the tasks of biomonitoring, conservation and management planning in freshwater ecosystems.

The accumulation of regulatory T-cells (Tregs) in the tumor hampers effector antitumor activity and correlates with a poor prognosis in several human cancers. Increasing the effector T-cell (Teff) to regulatory T-cell (Treg) ratio is known to result in improved control of established tumors. Studies demonstrating high levels of CD25 expression on Tregs and only low levels on Teff in human tumors have underscored its relevance as a target for Treg depletion. This supported the development of anti-CD25 depleting monoclonal antibodies (mAbs) as a promising monotherapy as well as combination partner in cancer immunotherapies. To date, anti-CD25 antibodies for clinical use have been designed to block IL-2 binding and signaling through the IL-2 receptor complex and also depletes CD25 positive cells. Since IL-2 is a critical cytokine involved in T-cell activation and proliferation, we hypothesized that a depleting antibody targeting CD25 but unable to block IL-2 signaling would promote a more potent anti-tumor response by preferentially depleting Tregs while still allowing IL-2 to stimulate effector T-cells expressing low levels of CD25 on their surface. We therefore compared the functional activity of anti-mouse CD25 depleting mAbs with and without IL-2 blocking activity. After having confirmed their differential impact on IL-2 signaling in vitro and in vivo, we evaluated the therapeutic activity of these mAbs in various syngeneic tumor models. While both the IL-2 blocking and non-IL-2 blocking mAbs showed equivalent Treg-depleting activity, the antibody sparing IL-2 signaling (αCD25NIB) promoted stronger antitumor effect than the IL-2 blocking mAb, with complete tumor regression observed in 70-100% of the mice after a single administration of the antibody. We demonstrated that the αCD25NIB would be an ideal partner for combination against several tumor types including “immune cold” tumors and αPD-L1 resistant tumors. Our data demonstrate that targeting CD25 with ADCC enabled antibodies preserving IL-2 signaling is a novel and powerful strategy for rejection of established tumors via depletion of Treg cells and enhanced, cell intrinsic, IL-2-driven effector T-cell activation. Citation Format: Anne Goubier, Isabelle Solomon, Frederick Arce Vargas, Dimitrios Zervas, Chen Qing, Josephine Salimu, Mark Brown, Pascal Merchiers, Karl S. Peggs, Sergio A. Quezada. Non-IL-2 blocking anti-CD25-targeting antibodies: depletion of regulatory T-cells driving optimal effector response for rejection of established tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A192.

Natural killer (NK) cells are cytotoxic lymphocytes able to kill tumor cells and virus-infected cells. Human-resting NK cells can be activated by co-culture with NK-resistant CTV-1a cells. These tumor-activated cells (TaNKs) are cytotoxic to a range of NK-resistant tumor cells in vitro. This potential, however, has not been explored in multiple myeloma (MM). In this study, we demonstrate that TaNK cells from 21 MM patients lyse a variety of myeloma targets, including primary isolates of autologous and allogeneic CD138+ myeloma cells whilst sparing CD138-ve bone marrow cells. Myeloma patients’ TaNK-induced lysis of the U266 cell line was significantly higher compared to normal controls (median-specific lysis 79.1% vs. 69.5%) (P = 0.003). In addition, TaNKs induced substantial lysis of autologous and allogeneic CD138+ myeloma cells (median-specific lysis 52.5% and 37.4%, respectively). The percentage of specific lysis did not correlate with important disease characteristics (ISS, age, and high-risk molecular abnormalities) or with the disease status and antimyeloma treatment, including novel agents and dexamethasone. In conclusion, tumor-primed NK cells are able to induce substantial lysis of myeloma targets including autologous and allogeneic CD138+ myeloma plasma cells and could be an additional therapeutic approach in MM, particularly in the era of novel agents. Am. J. Hematol., 2011. (c) 2011 Wiley-Liss, Inc.