Immunoassays are commonly used to assess airway inflammation in sputum samples from chronic obstructive pulmonary disease (COPD) patients. However, assay performance and validation in this complex matrix is inconsistently reported. The aim of this study was to assess the suitability of various immunoassays for use with sputum samples, followed by use of validated immunoassays to evaluate biomarker levels in COPD patients. Assays were assessed for recombinant reference standard suitability, optimal sample dilution, standard recovery in the biological matrix and reproducibility. Validated assays were used to assess sputum supernatants in Cohort A (n = 30 COPD, n = 10 smokers, n = 10 healthy) and Cohort B (n = 81 COPD, n = 15 smokers, n = 26 healthy). Paired baseline and exacerbation samples from 14 COPD patients were assessed in cohort A, and associations with sputum cell counts and bacterial colonisation investigated in cohort B. 25/32 assays passed validation; the primary reason for validation failure was recombinant reference standard suitability and sample dilution effects. Interleukin (IL-)6 and IL-8 were significantly increased in COPD patients compared to healthy subjects and smokers for both cohorts. Tumour necrosis factor (TNF)α and IL-1β were higher in COPD compared to smokers using one immunoassay but not another, partly explained by different absolute recovery rates. IL-1β, IL-2, IL-4, IL-8, IL-17A, Granulocyte colony stimulating factor (G-CSF), Interferon (IFN-)γ, Interferon gamma induced protein (IP-)10, Macrophage inflammatory protein (MIP)-1α, MIP-1β and TNF-α levels correlated with sputum neutrophil percentage in COPD patients. IL-1β, IL-4, IL-8, G-CSF and IFN-γ levels were associated with Haemophilus influenzae colonisation in COPD patients. Current smokers had lower levels of IL-1β, IL-4, IL-8, G-CSF, IFN-γ, IP-10, Monocyte chemoattractant protein (MCP)-1, MIP-1α, MIP-1β and TNF-α. Validated immunoassays applied to sputum supernatants demonstrated differences between COPD patients and controls, the effects of current smoking and associations between Haemophilus influenzae colonisation and higher levels of selected cytokines. Immunoassay validation enabled inflammatory mediators associated with different COPD characteristics to be determined.

Background: Chronic obstructive pulmonary disease (COPD) inflammatory endotypes are associated with different airway microbiomes. We used quantitative polymerase chain reaction (qPCR) analysis of sputum samples to establish the bacterial load upper limit in healthy controls; these values determined the bacterial colonisation prevalence in a longitudinal COPD cohort. Bacteriology combined with sputum inflammatory cells counts were used to investigate COPD endotypes. Methods: Sixty COPD patients and 15 healthy non-smoking controls were recruited. Sputum was analysed by qPCR (for Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Psuedomonas aeruginosa) and sputum differential cell counts at baseline and 6 months. Results: At baseline and 6 months, 23.1% and 25.6% of COPD patients were colonised with H. influenzae, while colonisation with other bacterial species was less common, e.g., S. pneumoniae—1.9% and 5.1%, respectively. H. influenzae + ve patients had higher neutrophil counts at baseline (90.1% vs. 67.3%, p < 0.01), with similar results at 6 months. COPD patients with sputum eosinophil counts ≥3% at ≥1 visit rarely showed bacterial colonisation. Conclusions: The prevalence of H. influenzae colonisation was approximately 25%, with low colonisation for other bacterial species. H. influenzae colonisation was associated with sputum neutrophilia, while eosinophilic inflammation and H. influenzae colonisation rarely coexisted.

Verekitug, a novel, high‐affinity, fully human monoclonal antibody targeting thymic stromal lymphopoietin receptor (TSLPR), is in development as a potential treatment for severe asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and chronic obstructive pulmonary disease (COPD). This phase 1b, double‐blind, randomized, placebo‐controlled, multiple ascending‐dose trial assessed the safety, tolerability, immunogenicity, pharmacokinetics, and pharmacodynamics of verekitug administered subcutaneously in patients with mild–moderate asthma. Thirty‐two participants were randomized in 4 placebo‐controlled dosing cohorts (3 × 100‐mg or 200‐mg doses, once every 4 weeks; 2 × 300‐mg doses, once every 12 weeks; single 25‐mg dose) and observed for 32 weeks. The primary endpoint was safety; secondary endpoints were pharmacokinetics and immunogenicity. Exploratory endpoints included TSLPR occupancy and biomarker effects. Treatment‐emergent adverse events were mild or moderate. Complete TSLPR occupancy was observed at the first timepoint (2 weeks post dose) and maintained for 24 weeks (doses ≥100 mg). Rapid mean reductions in fractional exhaled nitric oxide, eosinophils, and interleukin‐5 (up to −54%, −65%, and −64%, respectively) were sustained up to 24 weeks (doses ≥100 mg). The mean verekitug half‐life was ~20 days. Low‐titer antidrug antibody response was observed in some participants, without clinically meaningful impact on pharmacokinetics, pharmacodynamics, or safety. Verekitug was generally well tolerated, with rapid, substantial, and sustained effects on asthma biomarkers. These findings support further development of verekitug for treating severe asthma, CRSwNP, and COPD.

Introduction: Sputum biomarker measurements are used to measure airway inflammation in COPD patients. We have previously validated a Luminex assay able to quantify 15 analytes in COPD sputum supernatant. This assay demonstrated sputum protein expression profiles associated with neutrophilia, airway bacterial colonisation and current smoking in COPD. Methods: We report repeat sputum supernatant analysis at 6 months from a sub-group of COPD patients who participated in the original study. 48 COPD patients provided a repeat sputum sample at 6 months. 15 panel analytes were detectable. Results: Repeated sputum cytokine measurements showed a significant positive correlation between baseline and 6 months for all analytes except IL-1RA with intraclass correlation coefficient (ICC) indicating good to excellent repeatability. IL-1β, IL-2, IL-8, IL-17A, G-CSF, MIP-1α, MIP-1β and TNF-α were significantly correlated with sputum neutrophil percentage at 6 months. IL-1β, IL-4, IL-8 and G-CSF were significantly increased in Haemophilus influenzae colonised patients and IL-1β, IL-4, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β and TNF-α were significantly higher in ex-smoking COPD patients. Discussion: A multiplex immunoassay used at repeated visits in COPD patients showed a high degree of reproducibility for the majority of analytes. There were reproducible inflammatory signatures in sputum associated with clinical characteristics in COPD patients.

Lipopolysaccharide (LPS) inhalation causes neutrophilic airway inflammation. We used LPS produced to Good Manufacturing Practice (GMP) standards to characterise the dose response. A total of 15 healthy non-smoking subjects inhaled 5-, 15- and 50-µg LPS. Whole blood cell counts and serum C-reactive protein (CRP) were measured at baseline and up to 24 h post challenge. Sputum was induced at baseline and 6 h post challenge for cell counts and quantification of myeloperoxidase (MPO), interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor α (TNF-α) in supernatants. LPS inhalation was well tolerated. Blood neutrophil counts increased at 6 h post LPS with all doses. Serum CRP significantly increased with 15- and 50-µg LPS. All LPS doses significantly increased sputum neutrophil percentage (P < 0.001). IL-1β, IL-6 and TNF-α were significantly increased in sputum supernatant following challenge with 50-µg LPS, there was no change in MPO or IL-8. The 50-µg LPS was well tolerated and produced a robust inflammatory response. This study supports the use of 50-µg GMP-grade LPS as a suitable challenge agent in clinical trials.

Background: Induced sputum is a non-invasive technique used to assess airway inflammation. Sputum supernatant biomarker measurements may be prone to variability due to multiple factors, including matrix interference and poor analyte recovery. It is important to comprehensively validate sputum supernatant assays for use in clinical studies. Aims and Objectives: We validated commonly used COPD sputum supernatant biomarkers as per recommendations in current international guidelines. The assays were used to evaluate differences between COPD patients and controls, and changes during exacerbations. Methods: Assay validation involved assessment of immunological similarities between recombinant reference material and endogenous biomarker, diluent suitability, effect of dilution on analyte recovery, calibration curve establishment, assay selectivity and reproducibility, limits of quantification and stability. Sputum MPO, IL-1β, IL-6, IL-8 and TNF-α were measured in 34 COPD subjects, 14 paired exacerbation samples, 20 healthy smokers and non-smokers. Results: All assays demonstrated acceptable precision and reproducibility. All cytokines were increased in COPD vs controls. IL-1β, IL-6 and TNF-α were significantly increased during exacerbations (Table 1). Conclusion: We successfully validated a panel of sputum biomarkers, able to differentiate between COPD and controls and were changed during exacerbation.