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Abstract Introduction Spesolimab, a selective, humanised monoclonal antibody targeting the interleukin-36 receptor, is approved for the treatment of generalised pustular psoriasis (GPP). As a result of the limited patient numbers in GPP trials of spesolimab, analysing safety events across dermatological and non-dermatological diseases helps to further characterise the known safety profile of spesolimab. Here, we analyse predefined safety events from nine randomised, placebo-controlled spesolimab trials across dermatological (including GPP) and gastrointestinal conditions. Methods Predefined safety events were based on the known safety profile of spesolimab across all diseases investigated to date and potential risks of biological therapeutics, and included serious/severe/opportunistic infections, hypersensitivity, malignancies and peripheral neuropathy. Results Including placebo-controlled trials and open-label periods/trials, 589 patients received ≥ 1 dose of spesolimab (772.2 patient-years; mean exposure 1.31 patient-years). Overall, 452 patients had long-term exposure (≥ 6 months) to spesolimab, with 31 patients up to ≥ 3 years. In placebo-controlled periods, 445 patients had exposure to spesolimab (162.0 patient-years; mean exposure 0.36 patient-years). Severe/serious/opportunistic infections occurred in 0–3.2% of spesolimab-treated patients and 0–14.3% of placebo-treated patients. Malignancies occurred infrequently across trials (0–6.7% in spesolimab, 0–2.3% in placebo). Peripheral neuropathy events also occurred infrequently, with single events reported in the placebo arm of EFFISAYIL® 2, and the spesolimab and placebo arms of palmoplantar pustulosis Study 2. Potential hypersensitivity events occurred in all trials, except for Crohn’s disease, and were largely balanced between spesolimab (7.7–33.3%) and placebo (4.3–44.4%). Conclusions Across placebo-controlled periods of spesolimab trials in dermatological and non-dermatological conditions, severe/serious/opportunistic infections, malignancies and peripheral neuropathy events were low, with no evidence for an increased risk with spesolimab versus placebo. Potential hypersensitivity events were similar between spesolimab and placebo. These results support the favourable safety profile of spesolimab observed in EFFISAYIL® 2, the largest GPP trial conducted to date. Graphical Abstract

Introduction Generalized pustular psoriasis (GPP) is a rare, potentially life-threatening disease characterised by recurrent flares of widespread neutrophilic aseptic skin pustular eruption. Despite the availability of approved biologics for GPP in Japan, Taiwan and Thailand, associated evidence is largely based on uncontrolled studies in which acute flares were not directly assessed. Therefore, there is a high unmet need to investigate new rapid-acting effective treatments that resolve symptoms associated with acute GPP flares. A prior Phase I proof-of-concept study showed rapid improvements in skin and pustule clearance with a single intravenous dose of spesolimab, a novel anti-interleukin-36 receptor antibody, in patients presenting with an acute GPP flare. Here, we present the design and rationale of Effisayil 1, a global, Phase II, placebo-controlled study to evaluate the efficacy, safety and tolerability of spesolimab in patients presenting with an acute GPP flare.Methods and analysis At least 51 patients with an acute GPP flare will be randomised 2:1 to receive a single 900 mg intravenous dose of spesolimab or placebo and followed for up to 28 weeks. The primary endpoint is a Generalized Pustular Psoriasis Physician Global Assessment (GPPGA) pustulation subscore of 0 (pustule clearance) at Week 1. The key secondary endpoint is a GPPGA score of 0 or 1 (clear or almost clear) at Week 1. Safety will be assessed over the study duration by the occurrence of treatment-emergent adverse events. Blood and skin biopsies will be collected to assess biomarkers. Superiority of spesolimab over placebo in the proportion of patients achieving the primary and key secondary endpoints will be evaluated.Ethics and dissemination The study complies with the ethical principles of the Declaration of Helsinki, the International Council for Harmonisation’s Good Clinical Practice and local regulations. Ethics committee approvals have been obtained for each centre from all participating countries and are listed in online supplementary file 1. Primary results will be published in a peer-reviewed journal.Trial registration details ClinicalTrials.gov identifier: NCT03782792; Pre-results.

Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.

Experimental studies on hepatitis B virus (HBV) replication are commonly done with human hepatoma cells to reflect the natural species and tissue tropism of the virus. However, HBV can also replicate, upon transfection of virus coding plasmids, in cells of other species. In such cross-species transfection experiments with chicken LMH hepatoma cells, we previously observed the formation of HBV genomes with aberrant electrophoretic mobility, in addition to the those DNA species commonly seen in human HepG2 hepatoma cells. Here, we report that these aberrant DNA forms are mainly due to excessive splicing of HBV pregenomic RNA and the abundant synthesis of spliced DNA products, equivalent to those also made in human cells, yet at much lower level. Mutation of the common splice acceptor site abolished splicing and in turn enhanced production of DNA from full-length pgRNA in transfected LMH cells. The absence of splicing made other DNA molecules visible, that were shortened due to the lack of sequences in the core protein coding region. Furthermore, there was nearly full-length DNA in the cytoplasm of LMH cells that was not protected in viral capsids. Remarkably, we have previously observed similar shortened genomes and non-protected viral DNA in human HepG2 cells, yet exclusively in the nucleus where uncoating and final release of viral genomes occurs. Hence, two effects reflecting capsid disassembly in the nucleus in human HepG2 cells are seen in the cytoplasm of chicken LMH cells.

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.