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Type-2 biomarkers and related cytokines (IL-5, IL-13), lung function and asthma symptoms were measured in 44 poorly-controlled severe oral corticosteroid (OCS)-dependent asthmatics for up to 88 days after a 7-day prednisolone boost (0.5 mg/kg). High-dose OCS reduced median blood eosinophils (−60 cells/µl; 95% CI −140 to 10), periostin (−8.4 ng/mL; −11.6 to –2.8), FeNO (−19.0 ppb; −28.5 to –4.0), IL-5 (−0.17 pg/mL; −0.28 to –0.08) and IL-13 (−0.15 pg/mL; −0.27 to –0.03). There were small improvements in mean FEV1 (0.16 L; 0.05 to 0.27) and (Asthma Control Questionnaire) ACQ-7 score (0.3; 0.0 to 0.7). Study measures returned to baseline 1-month postintervention. Following rescue OCS, 1 month is sufficient before using type-2 biomarkers to guide long-term treatment.Trial registration numberNCT01948401.

BACKGROUND Allergic rhinitis is characterized by rhinorrhea, nasal congestion, sneezing and nasal pruritus. Group 2 innate lymphoid cells (ILC2s), CD4+ T cells and eosinophils in nasal mucosa are increased significantly after nasal allergen challenge (NAC). Effects of intranasal corticosteroids (INCS) on ILC2s remain to be investigated. METHODS Subjects (n = 10) with allergic rhinitis and mild asthma were enrolled in a single-blind, placebo-controlled, sequential treatment study and treated twice daily with intranasal triamcinolone acetonide (220 µg) or placebo for 14 days, separated by a 7-day washout period. Following treatment, subjects underwent NAC and upper airway function was assessed. Cells from the nasal mucosa and blood, sampled 24 h post-NAC, underwent flow cytometric enumeration for ILC2s, CD4+ T and eosinophil progenitor (EoPs) levels. Cell differentials and cytokine levels were assessed in nasal lavage. RESULTS Treatment with INCS significantly attenuated ILC2s, IL-5+ /IL-13+ ILC2s, HLA-DR+ ILC2s and CD4+ T cells in the nasal mucosa, 24 h post-NAC. EoP in nasal mucosa was significantly increased, while mature eosinophils were significantly decreased, 24 h post-NAC in INCS versus placebo treatment arm. Following INCS treatment, IL-2, IL-4, IL-5 and IL-13 were significantly attenuated 24 h post-NAC accompanied by significant improvement in upper airway function. CONCLUSION Pre-treatment with INCS attenuates allergen-induced increases in ILC2s, CD4+ T cells and terminal differentiation of EoPs in the nasal mucosa of allergic rhinitis patients with mild asthma, with little systemic effect. Attenuation of HLA-DR expression by ILC2s may be an additional mechanism by which steroids modulate adaptive immune responses in the upper airways.

Background The IL-33/ST2 pathway is linked with asthma susceptibility. Inhaled allergens, pollutants, and respiratory viruses, which trigger asthma exacerbations, induce release of IL-33, an epithelial-derived “alarmin.” Astegolimab, a human IgG2 mAb, selectively inhibits the IL-33 receptor, ST2. Approved biologic therapies for severe asthma mainly benefit patients with elevated blood eosinophils (type 2-high), but limited options are available for patients with low blood eosinophils (type 2-low). Inhibiting IL-33 signaling may target pathogenic pathways in a wider spectrum of asthmatics. Objectives This study evaluated astegolimab efficacy and safety in patients with severe asthma. Methods This double-blind, placebo-controlled, dose-ranging study (ZENYATTA [A Study to Assess the Efficacy and Safety of MSTT1041A in Participants With Uncontrolled Severe Asthma]) randomized 502 adults with severe asthma to subcutaneous placebo or 70-mg, 210-mg, or 490-mg doses of astegolimab every 4 weeks. The primary endpoint was the annualized asthma exacerbation rate (AER) at week 54. Enrollment caps ensured ∼30 patients who were eosinophil-high (≥300 cells/μL) and ∼95 patients who were eosinophil-low ( Results Overall, adjusted AER reductions relative to placebo were 43% (P = .005), 22% (P = .18), and 37% (P = .01) for 490-mg, 210-mg, and 70-mg doses of astegolimab, respectively. Adjusted AER reductions for patients who were eosinophil-low were comparable to reductions in the overall population: 54% (P = .002), 14% (P = .48), and 35% (P = .05) for 490-mg, 210-mg, and 70-mg doses of astegolimab. Adverse events were similar in astegolimab- and placebo-treated groups. Conclusions Astegolimab reduced AER in a broad population of patients, including those who were eosinophil-low, with inadequately controlled, severe asthma. Astegolimab was safe and well tolerated.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.

Background: Tryptase is the most abundant secretory granule protein in human lung mast cells and plays an important role in asthma pathogenesis. MTPS9579A is a novel monoclonal antibody that selectively inhibits tryptase activity by dissociating active tetramers into inactive monomers. The safety, tolerability, pharmacokinetics (PK), and airway pharmacodynamics (PD) of MTPS9579A were assessed in healthy volunteers. Methods: The Phase I studies were single-center, randomized, observer-blinded, and placebo-controlled. Single and multiple ascending doses of MTPS9579A were administered subcutaneously (SC) or intravenously (IV) in healthy volunteers. An activity based probe immunoassay was developed and used to specifically measure active tryptase in nasosorption (non-invasive sampling of nasal mucosal lining fluid). Results: This study included 106 healthy volunteers (82 on active). Overall, MTPS9579A was well tolerated with adverse events being predominantly mild or moderate. There were no serious adverse events reported. MTPS9579A showed dose-proportional increase in Cmax values and a half-life of 30-34 days. Rapid and dose dependent reduction in nasosorption active tryptase was observed post-dose, confirming activity of MTPS9579A in the airway. Conclusions: A novel immunoassay was used to demonstrate for the first time that an investigative antibody therapeutic (MTPS9579A) can inhibit tryptase activity in the upper airway. A favorable safety and tolerability profile supports further assessment of MTPS9579A in asthma. PKPD relationship may help inform future trials.

Here we review how biomarkers have been used in the design, execution and interpretation of recent clinical studies of therapeutic candidates targeting cytokine-mediated inflammatory pathways in asthma. This review focuses on type 2 inflammation, as there are multiple therapeutics and/or clinical studies that can be compared within that specific pathway. Comparative analyses of data from these clinical studies illustrate the utility of biomarkers to quantify pharmacodynamic effects, clarify mechanism of action and stratify patients, which may facilitate the interpretation of outcomes in the development of molecularly targeted therapies. These case examples provide a basis for biomarker considerations in the design of future studies in the asthma setting.

Introduction: Genome-wide association studies (GWAS) have discovered >30 loci associated with asthma risk. Despite the characterization of asthma as a heterogeneous disorder, most GWAS evaluate asthmatics as a single population. To explore whether genetics are associated with this phenotypic heterogeneity, we whole-genome sequenced (WGS) >5,000 severe, uncontrolled asthmatics who were clinically well characterized. Methods: After quality-control we had WGS data for 3,223 asthmatics of European ancestry. We performed common (minor allele frequency, MAF, ≥1%) variant and rare (MAF Results: Our risk analysis replicated ten previously reported asthma risk loci and identified a novel SNP in THSD4 that was significantly associated with asthma risk (P=4.43x10-8). This locus has been previously associated with both COPD risk and FEV1. In the rare variant burden GWAS, loss-of-function variants in IL33 were associated with decreased risk. We observed no overlap among the top genetic association signals for asthma risk and the other phenotypes. However, we found significant enrichment of genes implicated in Primary Ciliary Dyskinesia, PCD, as well as motile cilia genes, associated with asthma risk in the low-eosinophil asthmatics (P Conclusions: We found a new genome-wide significant association with asthma risk at a SNP in THSD4. We also identified enrichment of PCD genes with asthma risk among eosinophil-low patients. Replication of these findings in an independent cohort is needed to confirm sharing of asthma risk factors with COPD and PCD.

Background Inhibition of interleukin (IL)-13, a Type 2 inflammatory mediator in asthma, improves lung function and reduces exacerbations; however, more effective therapies are needed. A subset of asthma patients also exhibits elevated IL-17, which is associated with greater disease severity, neutrophilic inflammation, and steroid resistance. BITS7201A is a novel, humanized bispecific antibody that binds and neutralizes both IL-13 and IL-17. Methods Safety, pharmacokinetics, and immunogenicity of BITS7201A were evaluated in a phase 1 study. Part A was a single ascending-dose design with 5 cohorts: 30-, 90-, and 300-mg subcutaneous (SC), and 300- and 750-mg intravenous (IV). Part B was a multiple ascending-dose design with 3 cohorts: 150-, 300-, and 600-mg SC every 4 weeks × 3 doses. Both parts enrolled approximately 8 healthy volunteers into each cohort (6 active: 2 placebo). Part B included an additional cohort of patients with mild asthma (600-mg SC). Results Forty-one subjects (31 active, 10 placebo) and 26 subjects (20 active, 6 placebo) were enrolled into Parts A and B, respectively. The cohort with mild asthma patients was terminated after enrollment of a single patient. No deaths, serious adverse events, or dose-limiting adverse events occurred. In Part A, 12 active (39%) and 5 placebo subjects (50%), and in Part B, 6 active (30%) and 3 placebo subjects (50%) experienced at least 1 treatment-emergent adverse event (TEAE). The most common AEs were fatigue (n = 3) and influenza-like illness (n = 2). One injection-site reaction was reported. Two subjects with elevated blood eosinophil counts at baseline had transient elevations in blood eosinophils (≥Grade 2, > 1500 cells/μL). In Parts A and B, 16 of 30 (53%) and 16 of 17 (94%) active subjects, respectively, tested positive for anti-drug antibodies (ADAs). No anaphylaxis or hypersensitivity events occurred. BITS7201A exhibited single- and multiple-dose pharmacokinetic characteristics consistent with an IgG monoclonal antibody; exposure generally increased dose-proportionally. Postdose elevations of the serum pharmacodynamic biomarkers, IL-17AA and IL-17FF, occurred, confirming target engagement. Conclusions BITS7201A was well tolerated, but was associated with a high incidence of ADA formation. Trial registration ClinicalTrials.gov, NCT02748642; registered April 6, 2016 (retrospectively registered).

IntroductionAsthma exacerbations spike in the spring and autumn months, yet the seasonal variation of asthma symptoms and lung function is poorly studied.MethodsSeasonal variation of lung function, rescue medication use and patient-reported symptoms was evaluated by posthoc analyses of the Phase III lebrikizumab (anti-IL-13) LAVOLTA I and II studies in 2148 subjects with uncontrolled asthma. Lung function measurements (prebronchodilator FEV1, forced vital capacity (FVC) and peak expiratory flow (PEF)), rescue medication use and Standardised Asthma Quality of Life Questionnaire (AQLQ(S)) were measured every 4 weeks over 52 weeks. By-month estimates normalised by hemispheric season were based on mixed-effect models with repeated measures (MMRM), adjusted by study stratification factors as covariates when appropriate. The dependency of clinical outcomes with seasonal variability was assessed by employing linear contrasts comparing hemisphere normalised December versus July group means from an MMRM regression and presented as the difference in means (adjusted 95% CI).ResultsFEV1, FVC and PEF, rescue medication use and AQLQ(S) progressively worsened towards winter, unlike spring and autumn surges in asthma exacerbations. The December versus July mean differences were: (1) PEF=−6.5 (–8.7 to –4.2) L/min, 2) prebronchodilator FEV1=−42 (–57 to –27) mL, (3) FVC=−41 (−59 to –23) mL and (4) AQLQ(S)=−0.15 (–0.19 to –0.1) units. Among AQLQ questions, discomfort or distress related to cough was most variable with respect to season (−0.33 (−0.42 to –0.24) units).DiscussionInterpretation of interventional studies biased by seasonal exposures may be confounded by seasonal variability.Trials registration numbersNCT01867125 and NCT01868061.