Background:Interleukin (IL)-33 is implicated in the initiation and progression of asthma. FRONTIER-3 (NCT04570657) was a phase 2a study to investigate tozorakimab, an anti-IL-33 monoclonal antibody, in adults with moderate-to-severe early-onset asthma receiving standard of care. Methods:Patients were randomised 1:1:1 to tozorakimab 600 mg, tozorakimab 300 mg or placebo, once every 4 weeks subcutaneously. The primary end-point was change from baseline to week 16 in pre-bronchodilator (pre-BD) forced expiratory volume in 1 s (FEV1) measured in clinic. Secondary and exploratory end-points included home pre-BD FEV1, home peak expiratory flow (PEF), rescue medication use, CompEx, pharmacokinetics, immunogenicity and safety. Results:The intent-to-treat (ITT) population included 235 patients with a median disease duration of >30 years; a majority had a baseline blood eosinophil count (BEC) <300 cells·μL-1. At week 16, tozorakimab did not show a statistically significant improvement in change from baseline in clinic pre-BD FEV1 versus placebo (least-squares mean difference (LSMD) (80% CI), 600 mg: 4 mL (-71-79), p=0.473; 300 mg: 36 mL (-38-111), p=0.267). Compared with the ITT population, a pre-specified subgroup of patients with ≥2 prior exacerbations showed greater improvements in clinic pre-BD FEV1 at week 16 with tozorakimab versus placebo (LSMD (80% CI), 600 mg: 212 mL (102-322), p=0.007; 300 mg: 77 mL (-34-187), p=0.186). Tozorakimab also showed greater improvements in home FEV1 and home PEF versus placebo at week 16 in patients with ≥2 prior exacerbations than in the ITT population. Tozorakimab was well tolerated. Conclusions:Although FRONTIER-3 did not meet the primary study end-point, tozorakimab treatment showed encouraging efficacy signals in patients with early-onset asthma and a history of exacerbations.

OBJECTIVE:Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE) and is partially driven by type I interferon signaling. Anifrolumab, an approved treatment for patients with SLE, has been investigated in a phase 2 trial in patients with LN receiving standard therapy (TULIP-LN, ClinicalTrials.gov identifier NCT02547922). We studied the impact of anifrolumab treatment on urinary biomarker expression in patients with LN through proteomic analysis of samples from TULIP-LN. METHODS:Urine samples were collected at weeks 0, 12, and 48 from patients treated with the anifrolumab basic regimen (n = 35), intensified regimen (n = 42), or placebo (n = 35), in addition to standard therapy, and analyzed for the presence of 197 proteins. The impact of anifrolumab relative to placebo on prespecified biomarkers linked to histologic activity was assessed, and a comparison of responders versus nonresponders was conducted on proteins detected in ≥75% of samples. RESULTS:Anifrolumab treatment significantly reduced urinary CD163 and monocyte chemoattractant protein 1 expression at week 12 versus placebo, including in patients classified as proteinuric nonresponders across all regimens. By week 48, biomarker levels declined in all groups, indicating that standard therapy alone can eventually suppress intrarenal inflammation but with slower kinetics. Proteomic analyses further revealed that anifrolumab was superior to placebo in reducing the proteomic inflammatory signature, regardless of responder status. CONCLUSION:Compared with placebo, anifrolumab treatment significantly reduced urinary biomarkers of renal histologic activity in patients with LN, which may accelerate the resolution of intrarenal inflammation, potentially preventing damage accrual.

Abstract Background Crohn’s Disease (CD) is a chronic condition that can impact any part of the gastrointestinal tract. The sustained inflammation is driven by the continuous influx of inflammatory leukocytes into the gut mucosa, which is regulated through chemokine gradients and adhesion molecules, including integrins. In the gut, the migration of inflammatory cells to specific segments is thought to be mediated by selected integrins and/or chemokine axes. The CCL25/CCR9 axis has been identified as a key pathway for immune cell migration into small bowel segments, such as the ileum. In this study, CITE-seq and spatial transcriptomic analysis was used to integrate proteomic and transcriptomic profiles to characterise CCR9+ immune cells in the ileum compared to the colon. Methods Biopsies were obtained via ileocolonoscopy from macroscopically inflamed and normal mucosa with appropriate consent from healthy volunteers and IBD patients visiting the Department of Gastroenterology at the Sahlgrenska Hospital. Biopsies were frozen in Cryostor CS10 or formalin fixed and paraffin embedded. Cells from frozen biopsies were sorted, CD45+ cells stained with CCR9 and Totalseq-C human universal antibody cocktail, followed by amplification, separation of antibody-derived DNA from mRNA derived cDNA, library construction and sequencing of both libraries. For spatial transcriptomics, tissue sections were deparaffinised, stained with H&E and decrosslinked before human whole transcriptome probe hybridisation, ligation, extension, library construction and sequencing. Cells were annotated using a transfer learning approach combining several reference single cell datasets including detailed CD4+ and CD8+ T cell datasets and utilising both antibody data and mRNA transcriptomic data. Results CCR9 positive cells appeared scattered throughout the lamina propria and expression at mRNA level was enriched in the ileum compared to the colon with about 10% of CD4 and 17% of CD8 T cells expressing CCR9. Interestingly at protein level, expression was more restricted with the highest expression on cells that exhibited proteomic and transcriptomic features associated with resident CD4 memory T cells (Trm). This population almost exclusively (>90%) consisted of CCR9 positive cells and their proteomic and transcriptomic profile consistent with the recently described effector and innate-like phenotype of Trm cells that is thought to contribute to Crohn’s disease pathology. Conclusion These data may point to CCR9 positive cells, and in particular Trm cells, in the ileum as important pathological cells in Crohn’s disease and provide further support for depleting CCR9 cells as a potential therapeutic target for the treatment of ileal Crohn’s disease.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by airway obstruction and inflammation. Non-typeable Haemophilus influenzae (NTHi) lung infections are common in COPD, promoting frequent exacerbations and accelerated lung function decline. The relationship with immune responses and NTHi are poorly understood. Herein, we comprehensively characterized the respiratory microbiome and mycobiome of patients while investigating microbial dynamics and host immune changes attributable to NTHi killing activity. Mild-to-moderate COPD patients encompassing frequent and infrequent exacerbators and healthy volunteers (HV) were enrolled. Microbial composition, proteomics and NTHi killing activity was analyzed using bronchoalveolar lavage fluid (BALF). In addition, antigen–antibody titers in sera to COPD pathogens were determined using a multiplex assay. Differential abundance analysis revealed an enrichment of Actinobacteria and Bacteroidetes in the BALF of COPD and HV subjects respectively. Significant differences in the IgA titer response were observed against NTHi antigens in COPD vs. HV. Notably, there was also significantly greater killing activity against NTHi in BALF from COPD vs. HV subjects (OR = 5.64; 95

OBJECTIVE:Patients with systemic lupus erythematosus (SLE) commonly develop lupus nephritis (LN), the most frequent severe organ manifestation of this systemic autoimmune disease. Using serum and urine samples from a phase 2 trial in LN, we investigated that how the LN metabolome is modulated in response to type I interferon receptor blockade with anifrolumab, an approved treatment for moderate to severe SLE. METHODS:Patients in TULIP-LN (NCT02547922) received standard therapy plus intravenous anifrolumab or placebo. Untargeted metabolomics analysis was performed on serum and urine samples from 128 and 119 patients, respectively. Metabolites impacted by anifrolumab and their associations with clinical, serological and kidney measures of LN disease activity were examined. An in vitro model was used to validate the impact of anifrolumab on metabolite-induced inflammation. RESULTS:In serum, indoxyl sulfate (IS) and cytosine were the metabolites most modulated by anifrolumab, while baseline levels correlated with measures of kidney damage. In urine, uracil and cytosine were the most modulated by anifrolumab and levels of these metabolites were associated with serological markers of disease activity. Anifrolumab reduced markers of vascular dysfunction and inflammation upregulated by IS in in vitro models. Baseline urine uracil levels predicted response to anifrolumab intensive regimen at Week 52. CONCLUSION:Our findings establish a connection between type I interferon signalling, pyrimidine metabolism and uremic toxins in patients with LN and support the evaluation of urine uracil as a potential biomarker of response to anifrolumab.

The role of eosinophils in COPD and their utility as biomarkers for cytokine targeting monoclonal therapies remains unclear. We investigated the distribution of eosinophils across different tissue compartments in COPD and analysed gene expression to understand the possible mechanistic drivers of eosinophilic inflammation in COPD. Blood and BAL from ex-smoking volunteers with mild/moderate COPD (n = 31) and healthy ex-smoking controls (n = 20), and bronchial biopsy tissue in a subcohort (n = 19 and n = 8, respectively) was analysed. Differentially-expressed genes (DEGs) were characterised using RNASeq. Proteomic analysis of BAL was conducted using mass-spectrometry. COPD subjects had more eosinophils in blood and lung tissue compared to controls, with increased eosinophil protein CLC/Galectin-10 in BAL. However, peripheral blood eosinophil counts related poorly to numbers in lung tissue (rho = -0.09192, p = 0.3541) or proportions in BAL (rho = 0.01762, p = 0.4632). Tissue IL-5Rα expression was higher in frequent exacerbators and related to tissue eosinophils, but not peripheral blood eosinophils. Higher blood eosinophils were associated with DEGs that differed with compartment. Higher tissue eosinophil levels were associated with IL-13-induced DEGs including POSTN in bronchial brushes and CCL26 in bronchial biopsies. Gene-set enrichment analysis on data from brushings revealed significant enrichment of IL-4/IL-13, but not IL-5, pathways associated with eosinophil presence. Eosinophilic lung inflammation is related to exacerbation frequency, but lung eosinophils are not predicted by blood eosinophil counts in COPD. Our data suggest IL-13-mediated pathways may be responsible for the presence of tissue eosinophils in COPD. Further work to establish more predictive biomarkers of lung eosinophil biology are required to unlock this axis to optimised treatment.

Rationale: Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin (TSLP). In patients with moderate to very severe chronic obstructive pulmonary disease (COPD) from the COURSE study (NCT04039113), tezepelumab numerically reduced the annualized rate of moderate or severe COPD exacerbations over 52 weeks versus placebo in the overall population (rate ratio: 0.83 [95% CI: 0.61-1.11; p=0.104]). Greater efficacy was observed in patients with a baseline blood eosinophil count (BEC) of ≥150 cells/µL (rate ratio: 0.63 [95% CI: 0.43, 0.93]) and in those with a BEC of ≥300 cells/µL (rate ratio: 0.54 [95% CI: 0.25-1.15]). This post hoc analysis evaluated the effect of tezepelumab on inflammatory biomarker levels and on COPD exacerbation rates by baseline inflammatory biomarker levels. Methods: COURSE was a phase 2a, multicenter, randomized, double-blind, placebo-controlled study. Patients (40-80 years old) with moderate to very severe COPD were randomized 1:1 to tezepelumab 420 mg or placebo subcutaneously every 4 weeks for up to 52 weeks. The least-squares (LS) mean percentage change from baseline to week 52 in BEC, serum eosinophil-derived neurotoxin (EDN) level (a marker of eosinophil activation/degranulation) and serum interleukin-5 level, and the annualized rate of moderate or severe COPD exacerbations over 52 weeks by baseline BEC (<150 cells/µL vs ≥150 cells/µL) and serum EDN level (low [<26.7 ng/mL] vs high [≥26.7 ng/mL] levels relative to median baseline serum EDN) were assessed. Results: Overall, 333 patients received tezepelumab (n=165) or placebo (n=168). A modest correlation was observed at baseline between BECs and serum EDN levels (r=0.51). Tezepelumab treatment reduced LS mean BECs, serum EDN levels and serum interleukin-5 levels over 52 weeks versus placebo; reductions were observed as early as week 12 (figure). Among those with baseline BEC and serum EDN level data available, tezepelumab reduced the annualized rate of moderate or severe COPD exacerbations over 52 weeks versus placebo by 35% (95% CI: 3-57) in patients with a BEC of ≥150 cells/µL, by 40% (95% CI: 8-61) in patients with high EDN levels and by 47% (95% CI: 14-68) in patients with a BEC of ≥150 cells/µL and high EDN levels (figure). Conclusions: In patients with moderate to very severe COPD, tezepelumab reduced type 2 inflammatory biomarker levels over 52 weeks. A numerical improvement in tezepelumab efficacy was observed in patients with high baseline EDN levels, overall and in patients with a BEC of ≥150 cells/µL.