Pharmacodynamic Effects of Type 2-Targeted Biologics on Small Airway Function and Air Trapping in Severe Asthma

1. Introduction

Asthma is a heterogeneous chronic inflammatory airway disease affecting more than 300 million individuals worldwide and remains a major cause of respiratory morbidity despite substantial therapeutic advances [1,2].

Approximately 5–10% of patients suffer from severe asthma, a condition characterized by persistent symptoms, recurrent exacerbations, impaired quality of life, and high healthcare utilization despite treatment with high-dose inhaled corticosteroids (ICS), long-acting bronchodilators, and optimization of modifiable risk factors. Patients with severe asthma account for a disproportionate share of the disease burden and frequently require systemic corticosteroids, exposing them to significant treatment-related adverse effects. Recent Global Initiative for Asthma (GINA) recommendations emphasize the importance of biologic therapies targeting specific inflammatory pathways in patients with uncontrolled disease, enabling a precision medicine approach based on clinical phenotype and inflammatory endotype [3].

The development of monoclonal antibodies targeting type 2 (T2) inflammation has revolutionized the management of severe asthma. [4,5].

Mepolizumab and benralizumab inhibit the interleukin-5 (IL-5) pathway, reducing eosinophilic inflammation through distinct mechanisms, whereas tezepelumab targets thymic stromal lymphopoietin (TSLP), an upstream epithelial alarmin involved in the initiation and amplification of multiple inflammatory cascades [6,7,8].

These biologics consistently improve symptom control, reduce exacerbation frequency, decrease oral corticosteroid exposure, and improve lung function in both randomized controlled trials and real-world studies. Nevertheless, despite these clinical benefits, important questions remain regarding their effects on distal airway physiology and structural manifestations of disease that are not adequately captured by conventional spirometric measurements [9].

Increasing evidence suggests that the small airways, generally defined as airways measuring less than 2 mm in internal diameter, play a pivotal role in asthma pathophysiology. Historically considered the “silent zone” of the lung because of their limited contribution to total airway resistance, small airways are now recognized as a major site of inflammation, remodeling, mucus accumulation, and airflow limitation, particularly in severe disease [10,11]. Pathological studies have demonstrated that eosinophilic infiltration, goblet cell hyperplasia, mucus plugging, and airway wall thickening are often more pronounced in peripheral airways than in larger conducting bronchi). Dysfunction of these distal airways contributes to ventilation heterogeneity, progressive airflow limitation, poor symptom control, impaired quality of life, and increased risk of exacerbations [12]. Importantly, small airway abnormalities may persist even when conventional spirometry appears relatively preserved, suggesting that standard pulmonary function testing may underestimate the true extent of disease involvement [13,14,15].

One of the most important physiological consequences of small airway dysfunction is air trapping [11,12]. Inflammation, mucus plugging, and structural remodeling of peripheral airways promote expiratory flow limitation and premature airway closure, leading to gas retention and hyperinflation. RV and RV/TLC, measured by body plethysmography, are therefore considered clinically relevant physiological markers of distal airway dysfunction and air trapping in asthma [12]. Previous studies have demonstrated that air trapping is closely associated with asthma severity, exercise intolerance, increased symptom burden, and poor disease control [16].

Moreover, RV/TLC may provide information that complements traditional spirometric indices such as forced expiratory volume in one second (FEV1), potentially identifying subtle physiological improvements before they become detectable through conventional airflow measurements [14,17].

In parallel with the growing recognition of small airway disease, increasing interest has focused on identifying reliable biomarkers for monitoring the biological activity of targeted treatments. Fractional exhaled nitric oxide (FeNO) is widely recognized as a non-invasive biomarker of T2 airway inflammation and has been extensively used to assess treatment response in patients receiving biologic therapies [9]. Reductions in FeNO following biologic treatment reflect suppression of inflammatory pathways and have been associated with improved clinical outcomes. However, while FeNO provides information about inflammatory activity, it does not directly assess functional consequences occurring at the level of the peripheral airways. Consequently, integrating inflammatory biomarkers with physiological measures may provide a more comprehensive evaluation of treatment response. In this context, RV/TLC may represent a functional pharmacodynamic biomarker, reflecting the physiological impact of biologic-induced changes in distal airway inflammation and mucus-related airflow obstruction [18].

Although several studies have confirmed that biologics improve asthma control and reduce exacerbation rates, their effects on small airway dysfunction remain incompletely characterized. Recent investigations employing oscillometry, plethysmography, and peripheral airway indices have suggested that biologics may improve distal airway function, but available evidence remains limited and often derives from small cohorts or studies focused on a single therapeutic agent. Furthermore, data regarding changes in static lung volumes and air trapping parameters during treatment are still scarce, particularly in real-world settings where patients frequently present with heterogeneous clinical and inflammatory profiles. Whether improvements in air trapping represent a common effect of different T2-targeted biologics and whether such changes occur in parallel with reductions in T2 inflammatory activity remain important unanswered questions [15,17,19].

Therefore, the present prospective real-world study aimed to evaluate the longitudinal effects of tezepelumab, mepolizumab, and benralizumab on lung function, inflammatory activity, and air trapping in patients with severe asthma. Particular attention was devoted to changes in RV/TLC, considered a marker of distal airway dysfunction and the primary endpoint of the study, and to their relationship with FeNO as an established biomarker of T2 inflammation. We hypothesized that biologic therapy would be associated with progressive reductions in air trapping alongside improvements in clinical outcomes and inflammatory control, supporting the concept that RV/TLC may serve as a clinically useful functional pharmacodynamic biomarker of response to anti-T2 biologic treatment.

2. Materials and Methods

2.1. Study Design and Setting

This prospective, single-center observational study was conducted at the Respiratory and Allergy Unit of S. Andrea Hospital, (Department of Translational Medicine, University of Eastern Piedmont) Vercelli, Italy. Adult patients with severe asthma who initiated biologic therapy according to current GINA recommendations were consecutively enrolled and followed for nine months. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of A.O. “SS. Antonio e Biagio e Cesare Arrigo” of Alessandria (protocol n. 6651; Ethics Committee meeting of 08 March 2025). All participants provided written informed consent before enrolment and data collection.

2.2. Study Population

Eligible participants were adults (≥18 years) with a diagnosis of severe asthma according to GINA 2025 criteria, requiring high-dose inhaled corticosteroid/long-acting β2-agonist therapy, with or without additional controller medications, and remaining inadequately controlled despite optimized treatment. All patients were receiving inhaled triple therapy consisting of inhaled corticosteroids (ICS), long-acting β2-agonists (LABA), and long-acting muscarinic antagonists (LAMA) at baseline. Patients were prescribed benralizumab, mepolizumab, or tezepelumab according to routine clinical practice. The choice of biologic therapy was based on the treating pulmonologist’s assessment, considering clinical characteristics, biomarker profile, comorbidities, and current prescription criteria. Exclusion criteria included age below 18 years, concomitant respiratory diseases other than asthma, and poor adherence to prescribed treatment. A total of 43 patients were enrolled and allocated to one of three treatment groups: tezepelumab (n = 6), mepolizumab (n = 21), and benralizumab (n = 16).

2.3. Data Collection and Procedures

At baseline, demographic and clinical data were collected through a dedicated questionnaire, including age, sex, body mass index (BMI), smoking status, current therapy, and relevant comorbidities. Clinical assessment included the Asthma Control Test (ACT) and the annual exacerbation rate recorded during the previous 12 months. Asthma exacerbations were defined according to GINA recommendations as worsening of asthma symptoms requiring systemic corticosteroids, emergency department attendance, or hospitalization [3]. Respiratory assessment consisted of complete pulmonary function testing, including spirometry, body plethysmography, and fractional exhaled nitric oxide (FeNO) measurement. At the 3-month follow-up visit, pulmonary function and FeNO measurements were repeated. At the 9-month visit, both clinical and functional assessments were repeated using the same procedures applied at baseline.

2.3.1. Pulmonary Function Testing

Pulmonary function testing was performed using a Platinum Elite™ body plethysmograph (MGC Diagnostics, St. Paul, MN, USA). Measurements included FEV₁, FVC, FEV₁/FVC ratio, and static lung volumes, with particular assessment of residual volume (RV), total lung capacity (TLC), and the RV/TLC ratio as markers of air trapping and hyperinflation. Results were expressed as percentages of predicted values based on established reference equations, and all measurements were performed and interpreted according to current ATS/ERS technical standards [20,21].

Fractional exhaled nitric oxide (FeNO) was assessed using a FeNO+® analyzer (MediSoft, Sorinnes, Belgium) employing an online single-breath technique with a constant expiratory flow of 50 mL/s. For each subject, at least three technically acceptable measurements were obtained and averaged for analysis. FeNO measurements were performed before spirometry. Ambient nitric oxide concentration was checked before each session, and measurements were accepted only when environmental NO levels were below 10 ppb [22].

2.4. Endpoint and Statistical Analysis

The primary endpoint of the study was the longitudinal change in the RV/TLC ratio (T3 and T9), considered a physiological marker of air trapping and distal airway dysfunction. RV/TLC was selected as the primary outcome because it reflects air trapping and small-airway dysfunction, and was therefore considered a candidate functional pharmacodynamic biomarker of biologic response.

Secondary endpoints included changes in: residual volume (RV); total lung capacity (TLC); FEV₁ (% predicted); FEV₁/FVC ratio; FeNO; Asthma Control Test (ACT) score; annual exacerbation rate.

The analysis was designed to evaluate the evolution of these parameters during treatment with type 2-targeted biologic therapies in a real-world setting.

Continuous variables are presented as mean ± standard error of the mean (SEM), whereas categorical variables are reported as absolute numbers and percentages. Baseline differences among treatment groups were assessed using one-way analysis of variance (ANOVA) for continuous variables and the chi-square test for categorical variables. Longitudinal changes in pulmonary function parameters and FeNO measured at T0, T3, and T9 were analyzed using linear mixed-effects models, with time and treatment group included as fixed effects and participant included as a random effect. Treatment-by-time interactions were explored to assess whether longitudinal responses differed among biologic therapies. ACT score and annual exacerbation rate were compared between baseline and the 9-month follow-up.

Considering the limited sample size, particularly in the tezepelumab subgroup, the primary analyses focused on the pooled cohort to investigate class-related effects of T2-targeted biologic therapies, whereas subgroup analyses were considered exploratory. A two-sided p-value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA)

3. Results

3.1. Demographic and Clinical Characteristics of the Study Population

A total of 43 patients with severe asthma were included in the study, of whom 23 (54%) were male. Mean age was 49.6 ± 2.8 years and mean BMI was 26.9 ± 1.1 kg/m². Patients were treated with tezepelumab (n = 6), mepolizumab (n = 21), or benralizumab (n = 16). No significant differences were observed among treatment groups regarding age, sex distribution, BMI, smoking status, or prevalence of major comorbidities (Table 1). All participants were receiving inhaled triple therapy at enrolment.

Clinical status was assessed with the ACT and the number of exacerbations, recorded at baseline (first clinical visit) and at the end of the observation period, nine months after starting treatment. Data were analyzed as absolute values rather than percentage changes. The mean ACT score rose from 12.7 ± 0.570 to 23.5 ± 0.337 (p < 0.0001), and the mean number of exacerbations fell from 4.74 ± 0.268 to 0.63 ± 0.185 (p < 0.0001), indicating better symptom control and quality of life over the study period (Figure 1).

Significant improvements in clinical outcomes were observed during follow-up. Mean ACT score increased from 12.7 ± 0.6 at baseline to 23.5 ± 0.3 at 9 months (p < 0.0001), indicating a marked improvement in disease control. Similarly, the annual exacerbation rate (NE) decreased from 4.74 ± 0.27 to 0.63 ± 0.19 events per patient (p < 0.0001), corresponding to an approximate 87% reduction (Figure 1).

3.2. Pulmonary Function Results

Lung function parameters for the overall population are summarized in Table 2, while relative changes from baseline are illustrated in Figure 2.

The most pronounced functional changes were observed in parameters reflecting air trapping and distal airway dysfunction. RV/TLC, the primary endpoint of the study, decreased significantly from 111.15 ± 4.97% predicted at baseline to 91.47 ± 4.06% predicted at T3 (p = 0.0004) and to 83.71 ± 3.91% pred at T9 (p < 0.0001). Similarly, RV decreased from 108.35 ± 6.01% predicted at baseline to 90.47 ± 4.32% predicted at T3 (p = 0.0032) and 82.22 ± 4.08% at T9 (p < 0.0001), indicating a progressive reduction in air trapping during biologic therapy. Improvements were also observed in conventional spirometric parameters. FEV₁ increased from 80.30 ± 3.10% predicted at baseline to 92.10 ± 3.34% predicted at T3 and 96.28 ± 3.42% predicted at T9 (both p < 0.0001). Likewise, the FEV₁/FVC ratio increased from 71.93 ± 1.80% to 75.01 ± 1.76% at T3 (p = 0.0008) and 76.64 ± 1.84% at T9 (p = 0.0059). Consistent with suppression of type 2 airway inflammation, FeNO progressively decreased from 68.75 ± 6.72 ppb at baseline to 40.45 ± 3.98 ppb at T3 and 28.31 ± 2.89 ppb at T9 (both p < 0.0001).

Notably, the mean baseline FEV₁/FVC ratio was close to the threshold commonly used to define airflow obstruction (71.9 ± 1.8%). Despite this relatively preserved baseline value, the FEV₁/FVC ratio improved significantly during follow-up, suggesting a beneficial effect of biologic therapy on airflow limitation in addition to its impact on air trapping.

No significant treatment-by-time interaction was observed for the primary endpoint (RV/TLC), supporting the analysis of the pooled cohort and suggesting a broadly consistent longitudinal response across the different biologic therapies.

3.3. Exploratory Analysis According to Biologic Therapy

Exploratory analyses stratified by biologic treatment are reported in Supplementary Table S1. Improvements in inflammatory, spirometric, and air-trapping indices were observed across all treatment groups. However, given the limited sample size, particularly in the tezepelumab subgroup, these findings should be considered descriptive and hypothesis-generating rather than comparative. Therefore, no formal conclusions regarding differences among biologic agents can be drawn from the present study.

4. Discussion

In this prospective real-world study, treatment with T2-targeted biologics was associated with significant improvements in asthma control, exacerbation burden, lung function and airway inflammation. The most relevant finding, however, was the progressive reduction in air trapping, reflected by decreases in RV and RV/TLC. These changes were observed as early as three months and became more pronounced after nine months of treatment, suggesting a sustained effect on distal airway dysfunction [17,23].

Small airway dysfunction is increasingly recognized as a major determinant of disease severity in asthma. Distal airway inflammation, mucus accumulation and airway wall remodeling promote premature airway closure during expiration, leading to gas trapping and hyperinflation [11,24,25]. Interestingly, improvements were observed not only in air-trapping indices but also in the FEV₁/FVC ratio, which increased significantly despite being only mildly reduced at baseline (71.9 ± 1.8%). This finding suggests that biologic therapy may affect both distal airway dysfunction and conventional measures of airflow limitation. RV and RV/TLC are therefore considered, physiological “markers” of peripheral airway impairment. The significant reductions observed in both parameters suggest that biologic therapy may improve pathophysiological processes occurring beyond the central airways and not fully captured by conventional spirometry [11,12,13,14].

An additional finding was the parallel reduction in FeNO and air-trapping indices. FeNO is widely accepted as a biomarker of T2 inflammatory activity and is known to decrease following effective biologic treatment [26,27]. Type 2 inflammation promotes goblet cell hyperplasia, mucus hypersecretion and mucus plugging, all of which contribute to peripheral airway obstruction and air trapping. In particular, mucus plugging has recently emerged as a key mechanism linking type 2 inflammation with airflow limitation, air trapping and disease severity in severe asthma [28,29]. In the present study, improvements in FeNO occurred concurrently with reductions in RV and RV/TLC, suggesting that suppression of T2 inflammation may translate into measurable physiological changes in distal airway function. Although causality cannot be inferred from the present design, these findings support the hypothesis that RV/TLC could represent a functional pharmacodynamic biomarker of treatment response, complementing established inflammatory biomarkers such as FeNO [9,18].

Our findings are consistent with emerging evidence suggesting beneficial effects of biologic therapies on peripheral airway function. Potential effects on airway remodeling may also contribute to the observed reduction in air trapping [30,31]. Recent studies employing oscillometry, plethysmography and small-airway indices have reported improvements following treatment with mepolizumab, benralizumab and tezepelumab. However, most available investigations focused on airflow measurements or oscillometric parameters, whereas data specifically addressing changes in RV/TLC and air trapping remain limited. The present study expands current evidence by demonstrating a progressive reduction in physiologically measured air trapping across a real-world cohort receiving different T2-targeted biologics [12,14,15,17,32].

Several limitations should be acknowledged. First, the relatively small sample size, particularly within the tezepelumab subgroup, limited the ability to draw conclusions regarding individual biologic agents. For this reason, analyses primarily focused on the pooled cohort and should be interpreted as reflecting class-related effects of T2-targeted therapies. Second, the absence of a control group prevents definitive attribution of the observed changes exclusively to biologic treatment. Third, because the study was conducted in a real-world setting, unmeasured confounders and clinical heterogeneity may have influenced outcomes. Finally, although RV/TLC appeared highly responsive to treatment, formal analyses comparing its responsiveness with that of conventional spirometric parameters were not performed and therefore the hypothesis that RV/TLC may provide complementary information on treatment response that is not fully captured by conventional spirometric measures such as FEV1 and requires further validation [12,14,17]. Taken together, our findings support the concept that biologic therapies may exert clinically relevant effects on distal airway physiology in addition to improving symptoms and exacerbations. Monitoring air-trapping indices such as RV/TLC may provide additional information on treatment response beyond conventional spirometric measures. The consistency of the observed trends across different treatment groups may suggest that reduction in air trapping represents a common consequence of T2-targeted biologic therapy, although adequately powered comparative studies are required to confirm this hypothesis.

5. Conclusions

In this prospective real-world study, treatment with type 2-targeted biologics was associated with significant improvements in asthma control, exacerbation burden, lung function, and type 2 inflammatory activity. Beyond these established clinical benefits, a progressive reduction in air trapping was observed, as reflected by significant decreases in RV and RV/TLC, suggesting improvements in distal airway dysfunction. The parallel reduction in FeNO and air-trapping indices supports the hypothesis that suppression of type 2 inflammation may translate into measurable physiological changes within the peripheral airways. In this context, RV/TLC may represent a promising functional biomarker of biologic response, complementing conventional inflammatory biomarkers and spirometric measurements. Although these findings require confirmation in larger controlled studies, they highlight the potential value of incorporating air-trapping assessment into the routine evaluation of patients with severe asthma receiving biologic therapy.