Developmental Progress and Future Potential for Inhaled Biologics in the Treatment of Respiratory Diseases

Abstract

Inhaled biologics to treat lung diseases have an extensive history. This is counterbalanced by the limited products that have reached the commercialization stage of development despite the numerous disease states and targets that may be suitable for the delivery of proteins, peptides, or nucleic acid therapeutics. These opportunities are reviewed alongside current and past clinical and preclinical studies that highlight both successful and discontinued programs, providing key lessons for future development. Although there are physical, biological, and technical barriers that must be overcome to successfully deliver these relatively fragile moieties to the lung, there are commercial and clinical reasons why success has not always been realized. This review captures the current landscape of inhaled biologics, and as the field advances, we expect the inhaled route to expand rapidly given its distinct pharmacokinetic advantages for local lung diseases.

Key Points

Inhaled biologics (proteins, peptides, and nucleic acids) have been explored for decades, but only a handful have reached commercialization.

The inhaled route offers distinct pharmacokinetic advantages for lung diseases, potentially improving efficacy and safety while reducing systemic toxicity.

Failures of many inhaled biologics are often driven not only by target biology or molecular design but also by challenges in formulation stability, delivery, immunogenicity, commercial hurdles, and clinical trial design.

Other disease states with promising inhaled biologic candidates include for example, infectious diseases, genetic disorders, lung cancer, pulmonary hypertension, and pulmonary fibrosis.

With increasing understanding of barriers and rapid advances in biologics (monoclonal antibodies, nanobodies, messenger RNA, and gene therapies), the pipeline is expected to expand significantly over the next decade.

Introduction

Understanding the future potential for inhaled biologics for the treatment of local lung diseases can be best achieved through reflecting on the past successes, failures, and advances made in the science and technology of inhaled biologics. Certainly, many candidate biologics (defined here as proteins, peptides, or nucleic acids) have been identified for treating a wide range of lung diseases. Through these product development efforts, our understanding of the barriers to developing these molecules has improved substantially, including physicochemical, physiological, pharmacological, and commercial challenges.

Inhaled biologics represent a complex therapeutic modality, and because of the complex molecular limitations of the active pharmaceutical ingredient (API), one must be especially careful to integrate formulation design, processing, aerosolization, deposition, and the subsequent distribution/release events in the lung environment. Feasibility is contingent on ensuring that the biologic remains stable throughout manufacturing, storage, and delivery, that excipients are acceptable for inhalation, and that the target dose can be efficiently administered to and deposited at the disease site in the respiratory tract. As evident from the clinical inhaled biologic programs described below, delivery of the biologic to the lung alone does not guarantee success. Disease complexity and commercial factors must also be considered. With this in mind, we review those local lung disease states with potential targets for inhaled biological therapeutics. We then provide an up-to-date inventory of biologics that were/are in clinical studies. These include ongoing trials, programs that did not progress, and approved therapeutics. This retrospective analysis is used to identify key challenges specific to inhaled biologics, including stability, immunogenicity, formulation–device integration, manufacturing, dose requirements, pulmonary physiological barriers, and pharmacokinetic assessment, and to review strategies for overcoming these barriers to enable successful clinical translation.

Methods

A comprehensive literature search was conducted between 1 October, 2024 and 15 August, 2025, to identify clinical and preclinical studies of biologics for lung diseases. Primary databases included PubMed, Web of Science, Semantic Scholar, Google Scholar, ClinicalTrials.gov, FDA Drugs@FDA/EMA databases, and news and company websites consulted for trial updates. Titles and abstracts were screened for relevance, followed by a full-text review to confirm eligibility. Inclusion criteria encompassed phase I–IV trials, peer-reviewed articles, regulatory reports, and company disclosures; non-English, abstract-only, and pre-2000 studies (except landmark studies or therapies such as Pulmozyme^®) were excluded. Risk of bias was not formally assessed; instead, US Food and Drug Administration (FDA) labels and registered trials were treated as high-quality evidence, with clinical trials appraised using CONSORT registration details.

Inhaled Biologics in the Clinic

The successful development of inhaled biologics is influenced by multiple factors. As of 2025, there are five biologics for treating respiratory diseases approved for direct delivery to the human airways: two from the US FDA, one from Japan’s PMDA, one from China’s NMPA, and one from India’s CDSCO [1–5]. There is a growing pipeline of investigational proteins at various stages of development, including recombinant proteins, non-recombinant proteins, and antibody fragments. As for delivery devices, most candidates have been formulated for nebulization, while the others utilize dry powder inhaler (DPI) systems. Inhalable biologics in development are summarized in Table 1.

Table 1.

Clinical studies of inhaled biologics

Product	API (size)	Indications	Formulation	Delivery device	Company	Year	Status	NCT no.	Ref.
Pulmozyme® (dornase alfa)	rhDNase I (37 kDa)	CF	Inhaled Sol.	eRapid™ Nebulizer System	Genentech	2015	Ph. IV/Appr.	NCT01712334	[12, 14, 326–333]
FluMist® (Live Influenza Vac.)	LAIV (~ 80–120 nm)	Influenza A and B	Intranasal Susp.	Single-dose intranasal sprayer	AstraZeneca	2025	Ph. IV/Appr.	NCT00112112 NCT00344305 NCT00461981 NCT03143101	[334–341]
Activase® (alteplase)	rtPA (70 kDa)	Acute plastic bronchitis	Inhaled Sol.	Nebulizer	Univ. of Michigan and Genentech	2017	In development	NCT02315898	[71, 342–344]
Fludase® (DAS181)	rhSialidase fusion (46 kDa)	Parainfluenza infection	Dry powder	Cyclohaler® DPI	Ansun BioPharma	2011	Ph. II	NCT00527865 NCT01651494 NCT01924793 NCT01037205 NCT01644877	[40, 42, 44, 345–347]
Fludase® (DAS181)	rhSialidase fusion (46 kDa)	COVID-19	Inhaled Sol.	Nebulizer	Ansun BioPharma/Wuhan University	2020	In development	NCT04324489 NCT03808922	[348, 349]
Kamada-AAT	rhAAT (52 kDa)	AATD	Inhaled Sol.	PARI eFlow® Nebulizer	Kamada	2017	Ph. III	NCT04204252 NCT02001688	[350–352]
VR942/CDP7766	Anti-IL-13 mAb fragment (46 kDa)	Asthma	Dry powder	Multidose F1P DPI	UCB Pharma	2018	Ph. I	NCT02473939	[49, 51]
CSJ117 (ecleralimab)	Anti-TSLP mAb fragment (46 kDa)	Asthma	PulmoSolTM engineered dry powder	Single-dose Concept DPI	Novartis	2020	Ph. II	NCT03138811 NCT04410523	[353–355]
HH-120	ACE2-IgM fusion (1000 kDa)	SARS-CoV-2	Inhaled Sol.	Aerogen Solo®	Huahui Health	2022	Ph. III	NCT05116865 NCT05787418	[46, 356, 357]
SNG001	IFN-B 1a (22 kDa)	RVI in asthma	Inhaled Sol.	Nebulizer	Synairgen	2022	Ph. II/III	NCT04385095 NCT01126177 NCT04732949	[63, 358, 359]
Alpha-1 HC	pdAAT (52 kDa)	CF	Inhaled Sol.	AKITA2 APIXNEB® Nebulizer System	Grifols Therapeutics	2013	Ph. II/Disc.	NCT01684410	[360, 361]
ALX-0171	Anti-RSV F Nanobody (42 kDa)	RSV	Inhaled Sol.	FOX-Flamingo inhalation system	Ablynx	2017	Ph. II/Disc.	NCT02979431 NCT03418571	[84, 362, 363]
PRX-110 (AIR Dnase™)	Alidornase alfa (37 kDa)	CF	Inhaled Sol.	I-neb AAD inhaler system	Protalix BioTherapeutics	2017	Ph. II/Disc.	NCT02605590	[88, 364, 365]
ALX-009	OSCN-/bLF (80 kDa)	P.aeruginosa and BCC infection in CF	Inhaled Sol.	Nebulizer	Alaxia SAS	2018	Ph. I/Disc.	NCT02598999	[94, 366]
AZD1402/(PRS- 060)	IL-4Rα antagonist (18 kDa)	Asthma	Inhaled Sol.	InnoSpire Go mesh Nebulizer	AstraZeneca and Pieris	2019	Ph. I/Disc.	NCT03384290 NCT03574805 NCT04643158	[99, 102, 367, 368]

AAT alpha-1 antitrypsin, AATD alpha-1 antitrypsin deficiency, ACE2 angiotensin-converting enzyme 2, Appr. approved, BCC Burkholderia cenocepacia complex, bLF bovine lactoferrin, CF cystic fibrosis, Disc. discontinued, DP dry powder, DPI dry powder inhaler, IFN-β1a interferon beta-1a, IL-13 interleukin-13, IL-4Rα interleukin-4 receptor alpha, kDa kilodalton, LAIV live attenuated influenza vaccine, mAb monoclonal antibody, NCT national clinical trial, nm nanometer, OSCN- hypothiocyanite, P. aeruginosa Pseudomonas aeruginosa, pdAAT plasma-derived alpha-1 antitrypsin, Ph. phase, Refs. references, rhDNase I recombinant human deoxyribonuclease I, rhSialidase recombinant human sialidase, RSV respiratory syncytial virus, rtPA recombinant tissue plasminogen activator, RVI respiratory viral infection, Sol. solution, Susp., suspension, TSLP thymic stromal lymphopoietin

Clinical Success

Dornase Alfa (Pulmozyme^®)

An example of successful development of an inhaled biologic is dornase alfa, a recombinant human deoxyribonuclease I, which is approved for the treatment of cystic fibrosis (CF) in the USA [6]. Cystic fibrosis is a genetic disease caused by mutations in the CF transmembrane conductance regulator gene, leading to a build-up of thick sticky mucus lining the airways that causes chronic infections, inflammation, and progressive loss of lung function [7, 8]. Recombinant human deoxyribonuclease I enzymatically cleaves extracellular DNA in mucus, thereby reducing its viscosity and improving airway clearance. The Pulmozyme^® formulation is preservative free, including 2.5 mg of dornase alfa, 8.77 mg of sodium chloride, and 0.15 mg of calcium chloride dihydrate at pH 6.3 [9]. The calcium ions are essential for stabilizing the enzyme’s conformation and preserving enzymatic activity [10]. The solution maintains ionic strength and isotonicity through the inclusion of sodium chloride, supporting compatibility with the airway epithelium [11]. In clinical studies, patients were given 2.5 mg of dornase alfa once daily via either an eRapid^™ Nebulizer System or PARI LC^® jet nebulizer system [12]. A reduced administration time using vibrating mesh nebulization was observed. Additionally, a multicenter, randomized, open-label clinical study across 26 cities in the USA demonstrated comparable improvements in pulmonary function across three different delivery devices: the DeVilbiss Pulmo-Aide compressor with the Marquest Acorn II nebulizer, the Pari LC Jet Plus nebulizer with the Inhaler Boy compressor, and the Hudson T-Up drafter nebulizer [13]. On average, patients showed an increase in forced expiratory volume in 1 second from 13.2 to 14.1%, and in forced vital capacity from 10.9 to 11.8% [13]. Thus, the recommended dosage for most patients with CF is one 2.5 mg once daily using a recommended jet nebulizer/compressor system or eRapid^TM Nebulizer System [14].

Sargramostim (Sargmalin^®)

Sargramostim is a recombinant, human, granulocyte macrophage-colony-stimulating factor approved for autoimmune pulmonary alveolar proteinosis in Japan [15]. Autoimmune pulmonary alveolar proteinosis is a rare lung disorder characterized by the accumulation of surfactant in the pulmonary alveoli and the production of granulocyte macrophage-colony-stimulating factor autoantibodies, which can result in reduced oxygen levels, progressive pulmonary fibrosis, and potentially respiratory failure [16, 17]. Sargramostim improves lung function by binding to alveolar macrophages and stimulating their maturation and activation, enabling them to clear excess surfactant [18, 19]. Sargmalin^®, developed by Partner Therapeutics and Nobelpharma Co., Ltd. formerly referred to as Leukine^®, contains 264 mcg of sargramostim, 42 mg of mannitol, 10.5 mg of sucrose, and 1.27 mg of tromethamine with a pH of approximately 7.1–7.7 [20, 21]. Mannitol and sucrose are essential for stabilizing the lyophilized protein and ensuring the physical integrity of the lyophilized cake [22]. Sucrose is the primary molecular-stabilizing agent owing to its ability to replace water molecules and form a protective amorphous glass matrix that preserves the native conformation of the protein during freezing, drying, and storage [23]. Mannitol functions primarily as a crystalline bulking agent, which ensures the physical integrity of the lyophilized cake and consistent reconstitution prior to administration [24]. Sargmalin^® is reconstituted with 4 mL of sterile saline, and a 125 mcg/dose is administered twice daily for 7 days via a nebulizer and then discontinued for 7 days for 24 weeks [25]. Inhaled sargramostim demonstrated a reduction in alveolar-arterial oxygen difference_, leading to an overall improvement in oxygenation and pulmonary function, which ultimately reduced the need for whole-lung lavage, the historical standard of care [26].

Ad5-nCoV-IH (Convidecia Air^TM)

Ad5-nCoV-IH is an inhaled coronavirus disease 2019 (COVID-19) vaccine developed by CanSino Biologics [27]. Convidecia Air™ is a recombinant adenovirus type-5 viral vector that encodes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein [28]. Severe acute respiratory syndrome coronavirus 2 is an enveloped, positive-sense, single-stranded RNA virus that infects human epithelial cells by using the S protein to bind to the angiotensin-converting enzyme 2 on host cells [29, 30]. The vaccine is orally inhaled via a nebulizer and deposited in the respiratory tract [31, 32]. After the Ad5 vector deposits on the respiratory epithelial cells, it delivers the DNA encoding the S protein, enabling the host cells to express this protein and trigger T-cell responses as well as the production of immunoglobulin A and immunoglobulin G (IgG) [31, 32]. The Convidecia Air™ formulation is a 0.1-mL dose of approximately 1.0 × 10¹⁰ viral particles/mL at a pH of 7.0–8.0 [31, 32]. The formulation also contains mannitol, sucrose, sodium chloride, magnesium chloride, polysorbate 80, glycerin, N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) [HEPES], and water [31, 32]. Mannitol and sucrose stabilize the viral vector during manufacturing and storage, while magnesium chloride helps maintain buffer ionic strength and structural stability of the virus particles [33]. Sodium chloride and glycerin adjust the osmolality and tonicity to the physiological range [34]. Polysorbate 80 prevents aggregation and adsorption to the vial or delivery device surfaces during administration [35]. HEPES buffer, with a pKa of 7.55, is used to maintain physiological pH and preserve viral vector stability during storage and aerosol administration [36]. Clinical trials of aerosolized Ad5-nCoV-IH demonstrated its safety and immunogenicity when administered as a booster in adults after primary immunization [37].

Insulin

Inhaled insulin products Exubera^® and Afrezza^® illustrate key challenges and lessons for inhaled biologics across formulation, regulation, and commercialization. Formulation was critical for both systems and used dry powder insulin with stabilizing excipients to ensure protein stability at room temperature and achieve respirable particle sizes for deep lung delivery (designed for systemic absorption). The Technosphere technology (insulin adsorbed onto fumaryl diketopiperazine microparticles) of Afrezza^® and its small breath-powered inhaler improved dose consistency and patient convenience compared with Exubera’s bulky inhaler and milligram-dose blister system. Regulatory hurdles were significant as developers had to demonstrate equivalent glycemic control to injections and address pulmonary safety concerns. Both products showed a modest decline in lung function over time and rare pulmonary risks, prompting contraindications for smokers or those with lung disease and stringent lung monitoring requirements. The FDA imposed risk mitigation measures (e.g., a boxed warning for bronchospasm with Afrezza^®) and post-marketing surveillance for long-term risks after a few cases of lung cancer in inhaled insulin trials raised concern, though causality was not established. Commercially, Exubera^® was discontinued approximately a year post-launch because of several debated reasons including poor uptake attributed to high cost, complex dosing, and an inconvenient device, as well as the need for pulmonary testing that deterred patients and providers. The adoption of Afrezza^® has also been limited but remained marketed. A great deal of learning from these programs informs future inhaled therapies (e.g., monoclonal antibodies [mAbs]) by underscoring the need for a robust formulation (to maintain protein integrity and efficient lung delivery) and rigorous safety evaluation (focusing on respiratory effects and immunogenicity), as well as a clearly defined clinical advantage to justify inhalation.

Investigational Inhaled Biologics in the Clinic

Fusion Proteins

DAS181 (also known as Fludase^®) is an inhaled recombinant sialidase fusion protein (46 kDa) with a catalytic sialidase domain and a heparin-binding anchoring domain from human amphiregulin developed by Ansun Biopharma, Inc. [38, 39]. The sialidase domain prevents influenza and parainfluenza viruses from binding to host cells by removing sialic acid from the airway epithelium, blocking entry and replication [39]. DAS181 is formulated into dry powder and aqueous solutions for nebulization [40, 41]. Liquid formulations included 10 mg of DAS181, histidine, trehalose, citric acid, and water [40]. The powder formulation contains 20 mg and is loaded into a capsule to be administered via a Cyclohaler [40]. Several clinical trials evaluated these formulations (NCT00527865, NCT01651494). Based on these studies, a phase II trial was designed to limit exposure to a short 3-day window [40, 42]. A phase II trial of DAS181 for influenza (NCT01037205) investigated inhaled DAS181-F02 in single or multiple doses versus placebo in 294 patients with laboratory-confirmed influenza (H1N1, H5N1, or H274Y) [42, 43]. Here, patients were given the 10-mg dose daily for 3 days, a single 10-mg dose, or a placebo. The protein showed no serious treatment-related adverse events. Multiple doses significantly reduced the viral load compared with placebo [42]. Patients who received the single dose had a significant reduction in viral load compared with placebo after the first day of treatment. A second phase II trial (NCT01644877) investigated delivering 4.5 mg of DAS181-F02 once daily for 10 days as a nebulized reconstituted dry powder [44]. Participants received DAS181-F02 via Aeroneb Solo nebulizers. Pharmacokinetic analyses displayed minimal systemic exposure, indicating that DAS181 is contained within the lungs. Chemaly et al. confirmed DAS181-F02 preserved enzymatic activity as a dry powder during storage and nebulization. Clinically, DAS181-F02 demonstrated an upward trend in the severely immunocompromised patient subgroup. Ansun Biopharma, Inc. has an ongoing phase III trial to investigate DAS181 in immunocompromised patients with lower tract parainfluenza and severe COVID-19 [45].

HH-120, an inhalable, IgM-like, angiotensin-converting enzyme 2 fusion protein (~ 1000 kDa), is currently under clinical evaluation as a host-directed therapeutic strategy against SARS-CoV-2. Designed to act as a soluble decoy receptor, HH-120 neutralizes the virus by competitively binding to the viral spike protein, thereby preventing host cell entry [46]. The angiotensin-converting enzyme 2 fusion protein demonstrated efficacy in preclinical models [46]. A pharmacokinetic analysis established that HH-120 remained localized in the lungs, nasal cavity, and trachea. HH-120 was also well tolerated preclinically. Hence, Huahui Health investigated HH-120 further in a phase I clinical trial [47]. The product was well tolerated across all dose levels, with no reported dose-limiting toxicities, supporting its further clinical development. [48].

Antibody Fragments

VR942 is a dry-powder formulation containing CDP7766, a high-affinity anti-IL-13 mAb fragment. CDP7766 specifically binds to IL-13 and neutralizes the cytokine to downregulate inflammation and pathogenesis of asthma [49]. The VR942 formulation contains CDP-7766, L-leucine, trehalose, sodium chloride, and sodium phosphate and was produced via spray drying [50]. In a phase I (NCT02473939) study, VR942 was administered to healthy adults and patients with mild asthma. Patients were given either a single dose (0.5, 1, 5, 10, or 20 mg) or multiple doses (0.5, 10, or 20 mg) for 10 days via a Vectura DPI [51, 52]. The treatment-emergent adverse events were mostly mild or moderate, and no serious adverse events or deaths were observed [51].

CSJ117, also known as ecleralimab, is an inhaled antibody fragment (Fab) targeting thymic stromal lymphopoietin (TSLP), a key cytokine in asthma pathogenesis [53]. Ecleralimab binds and neutralizes soluble TSLP, which prevents its interaction with the TSLP receptor complex on immune cells [54]. As mentioned previously, the Fab is formulated as a PulmoSol™ powder with amorphous particles with core-shell structures [55, 56]. In a phase I study, NCT03138811, patients (n = 28) were treated with 4 mg of CSJ117 or placebo for 12 weeks [53]. CSJ117 was shown to be safe and effectively reduced late asthmatic responses and sputum eosinophil counts in adults with mild atopic asthma. Thus, Fab demonstrated a significant effective reduction in airway inflammation [53, 57]. In the first phase II trial (NCT04882124), the pharmacodynamics and safety of CSJ117 were assessed in patients with chronic obstructive pulmonary disorder (COPD) [58]. Patients (n = 38) received either 4 or 8 mg once daily over a 12-week period. However, because of the small patient pool, no meaningful conclusions could be drawn [59]. The second phase II clinical trial (NCT04410523) focused on evaluating the efficacy and safety of repeated doses in patients with severe uncontrolled asthma [60]. In this study, patients were given 0.5, 1, 2, 4, or 8 mg of CSJ117 daily over a 12-week period [61]. Although the drug was safe and well tolerated, with no major safety concerns or significant differences in adverse events compared to the placebo group, there are currently no further studies planned for CSJ117 owing to a strategic decision made by Novartis [59].

Cytokines/Interferons (IFNs)

SNG001 is an inhaled formulation of recombinant interferon (IFN)-β1a, an antiviral therapy [62]. Delivering IFN-β1a directly into the lungs was proposed to boost the lungs’ innate antiviral response and limit viral replication. The SNG001 formulation is an aqueous solution containing IFN-β1a, trisodium citrate dihydrate, disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, DL-methionine, and water [63]. The phase III SPRINTER trial (NCT04732949) assessed the efficacy and safety of inhaled SNG001 in adults hospitalized with COVID-19 requiring supplemental oxygen (n = 623) [64]. Despite successes in phase II studies, this trial did not meet its primary or secondary endpoints because of patient variability and the placebo group improving faster than anticipated, reducing the power and statistical significance of the study [64]. However, patients with baseline compromised respiratory function experienced a significant reduction in the risk of progression to severe disease or death [64].

Inhibitory Proteins

Kamada-AAT for Inhalation, developed by Kamada Ltd., is a recombinant human AAT formulation designed for direct pulmonary delivery [65, 66]. Clinical development has included a phase II study, NCT02001688, demonstrating that inhaled AAT is safe, well tolerated, and capable of achieving therapeutic levels in the epithelial lining fluid while reducing neutrophil elastase activity [67]. Patients were administered 80 mg of AAT once daily or twice daily via nebulization for 12 weeks [68]. Currently, the ongoing phase III trial (NCT04204252) is evaluating the long-term efficacy of daily inhaled AAT in adults with severe AATD, with primary endpoints focused on lung function preservation and disease progression [69].

Alteplase (Activase^®)

Alteplase, a recombinant tissue plasminogen activator, is FDA approved for intravenous (IV) administration for acute ischemic stroke, acute myocardial infarction, and pulmonary embolism [70]. However, it is also used off-label, for inhalation to treat acute plastic bronchitis in pediatric patients [71, 72]. In the phase II study NCT02315898, nebulized Alteplase showed evidence of efficacy [73]. Eight pediatric patients were treated with 5 mg (1-mg/mL solution) of a recombinant tissue plasminogen activator via nebulizer every 6 hours for about 72 hours.

Discontinued Investigational Inhaled Biologics

Importantly, the discontinuation of clinical programs for several inhaled solution-based candidates, such as Alpha-1 HC (alpha-1 proteinase inhibitor), ALX-0171 (anti-F nanobody for respiratory syncytial virus [RSV]), and PRX-110 (modified alpha-1 antitrypsin [AAT]), emphasizes the need for not only preclinical efficacy but also robust clinical trial design, strategic indication selection, and formulation strategies that support stability and efficient aerosol delivery (Table 1) [74–76]. The following section examines the clinical trial outcomes for each candidate, highlighting key factors that contributed to their early termination.

The phase IIa clinical trial NCT01684410 evaluated the safety and tolerability of inhaled Alpha-1 HC (hydrophobic chromatography purified AAT) in adults with CF [74]. Alpha-1 HC is an inhaled alpha-1 proteinase inhibitor (also known as AAT), which binds and inhibits neutrophil elastase, a proteolytic enzyme released by activated neutrophils during inflammation [77]. A build-up of neutrophil elastase can degrade elastin and other structural proteins inherent in the lung tissue, leading to tissue destruction and emphysema [78]. Subjects were treated with 100 mg or 200 mg of Alpha-1 HC via the AKITA² nebulizer once daily for 3 weeks, compared to the placebo group. A dose-dependent increase in sputum AAT levels was observed. The study demonstrated that Alpha-1 HC was generally safe and well tolerated, though it did not show significant improvements in pulmonary function over the short 3-week treatment period. Alpha-1 HC is designed to prevent further lung destruction rather than reverse damage, so larger sample sizes, and longer treatment periods may have been needed to demonstrate clinical efficacy.

In two other phase IIb trials, NCT03418571 (n = 15) and NCT02979431 (n = 180), infants and children (aged 28 days to less than 2 years) hospitalized with RSV lower respiratory tract infections (LRTI) were administered a novel inhaled antiviral biologic [79, 80]. The drug, ALX-0171, was composed of a trivalent nanobody (42 kDa), engineered to target the RSV fusion protein [81–83]. Patients received doses of 3.0, 6.0, or 9.0 mg/kg once daily for 3 days versus placebo. The study demonstrated an accelerated reduction in RSV viral load in nasal swabs but did not contribute to a meaningful difference in symptoms or recovery time [84]. Development was subsequently discontinued [80]. Most children in the study were hospitalized and enrolled 3 days after symptom onset, long after lung inflammation was established. Since most antiviral medications are most effective before inflammation occurs [85–87], better efficacy may have been observed if administration was earlier [84].

In another phase 1 clinical study of AIR DNase (alidornase alfa, PRX-110) [NCT02605590], the safety, tolerability, and pharmacokinetics were studied in healthy adult male individuals [88]. PRX-110 is an inhaled, recombinant, chemically modified DNase I. Patients received 1.25, 2.5, or 5 mg daily for 5 days [89]. This study concluded that AIR DNase is generally safe and well tolerated in healthy volunteers [90]. The subsequent phase II study (NCT02722122) examined the safety and efficacy of AIR DNase in patients with CF who were previously treated with Pulmozyme^® [91]. Patients (n = 16) received 2.5 mg of alidornase alfa once a day for 4 weeks [92]. All patients completed the treatment regimen, and lung function was improved by a mean absolute increase of 4.1 ± 2.5 points from baseline [91]. This did not demonstrate a significant improvement in clinical outcomes over Pulmozyme^® [93] and, as a result, PRX-110 lacked the clinical differentiation needed for further development in CF.

ALX-009 is an inhaled antimicrobial therapy that combines hypothiocyanite (OSCN-) and bovine lactoferrin to treat patients with CF. The OSCN/bovine lactoferrin combination therapy proved to be effective against multidrug-resistant Gram-negative pathogens, Pseudomonas aeruginosa and Burkholderia cepacian, across in-vitro and in-vivo models [94, 95]. A phase I trial (ALX-009-CL-038/NCT02598999) aimed to assess the safety, tolerability, and pharmacokinetics of ALX-009 in healthy volunteers and patients with CF and non-CF bronchiectasis. However, the program was terminated early because of financial issues raised by the formulation’s complexity [96]. As OSCN- is a highly reactive ion and has a half-life of approximately 1 hour, ALX-009 could not be kept stable during storage [97, 98]. Thus, the formulation required on-site preparation.

AZD1402, also known as PRS-060 (elarekibep, AZD1402), is an inhaled biologic therapy developed by Pieris Pharmaceuticals and AstraZeneca. Elarekibep is an engineered protein derived from human lipocalins, which are small proteins [99]. PRS-060 specifically targets IL-4 receptor alpha (IL-4Rα), which is a critical component in the signaling pathways of IL-4 and IL-13 cytokines involved in the inflammation [100–102]. In a phase I, randomized, double-blind, placebo-controlled study (NCT03384290), the safety, tolerability, and pharmacokinetics of AZD1402 were assessed in healthy male volunteers [103]. Patients were administered 0.1, 0.5, 2, 8, 60, or 160 mg of PRS-060 via inhalation versus 1 or 2 mg of PRS-060 administered intravenously [104]. The study demonstrated that AZD1402 was well tolerated at all dose levels tested, with no observed serious adverse events. The pharmacokinetic profile showed dose-proportional exposure, and the inhaled delivery results in minimal systemic absorption, indicating targeted and controlled pulmonary delivery [104]. Thus, NCT03384290 showed that the human lipocalin elarekibep is safe and bioavailable in humans. A follow-up phase IIa study (NCT04643158) investigated for efficacy and safety of inhaled corticosteroids in patients with asthma (n = 72) [105]. The drug remained in the lungs while limiting systemic absorption. Despite promising results from the previous trial, the program was terminated because of findings from a non-clinical toxicology study [105]. Although these findings were not observed in human clinical trials, they raised concerns about the long-term safety of the therapy, leading AstraZeneca to discontinue further development [106].

These setbacks highlight some of the issues experienced when translating promising preclinical data into clinically meaningful outcomes for inhaled biologics. They underscore how challenges such as insufficient design, suboptimal timing of administration, and limited clinical differentiation can derail programs even when early safety and pharmacokinetic data appear favorable. In several trials, the pharmacological rationale was strong, but the complexity of the disease pathology, variability in patient populations, and the difficulty of achieving adequate drug exposure at the site of action limited the ability to demonstrate therapeutic benefit. Additionally, practical barriers, such as formulation instability, device-dependent delivery efficiency, and the cost of manufacturing inhaled biologics, further complicate development. Collectively, these examples illustrate that successful clinical translation requires not only preclinical efficacy but also careful attention to trial design, patient selection, dosing strategy, and formulation strategy to ensure that inhaled biologics can achieve the consistency, potency, and clinical differentiation needed for commercial viability.

Challenges in the Development of Inhaled Biologic Formulations

Although late-stage failures of inhaled biologics are often driven by clinical or commercial factors (as discussed in the preceding sections), early development must focus on maximizing the likelihood of success through ensuring molecular stability and effective pulmonary delivery. Biologic therapeutics are inherently fragile and may be exposed to harsh conditions during manufacturing, storage, and aerosolization, making inhaled delivery particularly challenging. As a result, key barriers to successful development span multiple domains, including manufacturing, physicochemical and biological stability, immunogenicity, formulation design, and device compatibility, Fig. 1. This section focuses primarily on stability and immunogenicity, while also highlighting formulation and device-related considerations that directly influence these risks.

Fig. 1.

Schematic of the key challenges in the development of inhalable biologic therapeutics across four domains, including physical and chemical stability, immunogenicity, formulation considerations, and device and manufacturing technologies. DPI dry powder inhaler, pMDI pressurized metered-dose inhaler, RH relative humidity

Most inhaled biologics investigated in preclinical and early clinical trials are delivered via nebulization [107, 108], largely because biologics are typically formulated during initial feasibility studies as dilute aqueous solutions [109]. In many cases, these programs follow a systemic-to-local development strategy, in which molecules originally designed for systemic administration are repurposed for inhalations. This approach leverages existing proof-of-concept, including validated biological targets and established toxicology profiles, thereby reducing regulatory uncertainty and potentially shortening development timelines and costs [110]. For example, ATT, initially developed for IV augmentation therapy, has been repurposed for inhalation to improve dose efficiency. Recent clinical studies demonstrate that repurposed inhaled AAT therapy can successfully restore pulmonary homeostasis; in contrast, systemic AAT augmentation through IV administration typically results in only 5–10% of serum AAT reaching the lungs [111].

A lung-first development strategy involves engineering molecules de novo to withstand pulmonary clearance mechanisms, the local microenvironment, and the mechanical stresses of aerosol generation, while optimizing pharmacokinetics to maximize local retention. For example, the success of dornase alfa shows that this design for an inhalation approach can also be a viable route to clinical success. Ultimately, the chosen strategy can depend on factors such as the developer’s strategic objective, the physicochemical properties of the molecule, the disease pathophysiology, and the desired therapeutic index.

Stability

Biologics typically rely on intricate three-dimensional structures to enable specific interactions with their biological targets. Unlike most small molecules, biologics are more labile, making disruption of this three-dimensional structure more likely during processing, shelf life, and administration [112].

Proteins such as biologics may degrade through physical or chemical pathways, which are often inter-related [113]. Physical instability includes changes to the protein’s higher-order structure, such as unfolding, aggregation, denaturation, surface adsorption, and interfacial stress, while chemical degradation includes deamidation, oxidation, hydrolysis, covalent aggregation, and reactions with excipients. Changes to a higher-order structure often lead to the irreversible loss of a protein’s biological activity. [114].

Dry powder manufacturing methods, such as spray drying, can lead to protein’s instability due to exposure to thermal and interfacial stresses during processing [115]. For example, Tao et al. reported the formation of insoluble aggregates in spray-dried Fab molecules (Fab-1 and Fab-2) [115]. In this case, increased conformational dynamics in the heavy-chain CDR-1 exposed a previously buried Phe side chain, which accelerated aggregation through hydrophobic interactions. Sécher and colleagues reported that IgG aggregates generated during nebulization caused immunotoxic events in the lungs of C57BL/6 mice post-inhalation [116].

Stability challenges extend beyond proteins to nucleic acid-based therapeutics. A major hurdle is that the messenger RNA (mRNA)-based vaccine products necessitate ultra-cold chain storage conditions for the entire product shelf life (e.g., − 90 to − 60 °C for 12–18 months for Comirnaty^®), which are not only costly but also challenging to implement in clinical settings. The physical stability of mRNA-lipid nanoparticle (LNP) vaccines is primarily affected by aggregation, fusion, and leakage of the encapsulated mRNA [117]. These effects are amplified during the freeze-thaw cycle, but use of appropriate lyo-/cryo protectants can reduce freeze-induced instability. In addition, chemical impurities arising from ionizable lipid oxidation and hydrolysis can inhibit mRNA translation. By modifying the ionizable lipid amine structure, Hashiba et al. showed that N-methyl piperidine head groups maintained mRNA-LNPs activity at 4 °C due to reduced levels of impurities [118].

Following pulmonary administration, additional stability challenges for biologics arise from the lung microenvironment. Interactions with mucins and other charged or hydrophobic domains of the pulmonary mucosa can induce aggregation or premature release of nucleic acid cargo. Surface modification strategies, for example, using polyethylene glycol components or multi-layered polymer particles, may enhance mucosal stability by repelling mucin components [119–121].

Immunogenicity

Protein therapeutics inherently pose an intrinsic risk of immunogenicity, primarily by eliciting adaptive immune responses that result in the formation of anti-drug antibodies, which can compromise therapeutic efficacy and trigger hypersensitivity reactions [122]. In contrast, nucleic acid-based therapeutics can trigger both innate and adaptive immune pathways, including cytokine release, T-cell activation, and complex antibody responses [123]. The immunogenicity profiles of inhaled biologics are further influenced by the distinct lung microenvironment.

Prior studies have demonstrated that the pulmonary administration of biologics is generally more immunogenic than the SC route [124]. For example, increased anti-drug antibody formation was reported in patients with type 1 and 2 diabetes when transitioning from the SC to the pulmonary route [125, 126], and in non-human primate studies [125–127]. The lung’s extensive alveolar surface areas with abundant immune cells within the lung lining fluid, particularly alveolar macrophages, increase immunogenicity risk.

The immunogenicity of inhaled biologics is influenced by formulation and delivery-related factors, including dosage, dosing frequency, deposition site, and the presence and characteristics of aggregates [108]. While submicron aggregates (100–1000 nm) have been reported to increase immunogenic potential [128], other studies suggest that aggregate attributes, such as chemical and physical modifications, conformational changes, and impurity content, may have a similar or greater effect on immunogenicity than size alone [128, 129]. For example, Schellekens suggests that the immune responses are less influenced by the administration and more by the characteristics of the active pharmaceutical ingredient itself, including its structure, impurities, formulation, aggregate, and patient-specific factors [130].

Several studies have investigated the effect of inhalation-induced aggregation on immune responses. Mahri et al. investigated the immunogenicity of spray-dried rhod-mAb formulations containing varying levels and found higher immune responses following pulmonary administration compared with SC dosing in mice [131]. Alveolar macrophages were identified as the main immune cells in the lungs, while differences in aggregate content between formulations did not significantly alter immunogenic outcomes. Similarly, Sécher et al. looked into the effect of aggregates formed during nebulization and how they affect the immune system [116]. Nebulization-generated protein (IgG) aggregates led to a dose-dependent activation of monocyte-derived dendritic cells and reduced immune cells. This effect was higher when administered locally in the airways compared with systemic administration, which was later resolved by improving IgG stability through the addition of polysorbate in the formulation [116].

Immunogenicity considerations are crucial for inhaled nucleic acid-based therapeutics. The first-in-human inhaled mRNA-LNP system, MRT5005, nebulized in patients with CF, was generally well tolerated, with just one pulmonary exacerbation reported within 1 month of treatment [132]. However, numerous individuals experienced febrile responses hours post-dosage, which were not predicted by preclinical toxicological studies in rats and non-human primates. These reactions were mild to moderate, and subsided within 1–2 days. Though it is unclear whether these febrile responses are caused by mRNA or lipids, prior studies have found similar unwanted responses using gene therapy-based LNP systems in patients with CF [133, 134].

From a formulation perspective, the immunogenicity of inhaled mRNA-LNPs is primarily affected by the physicochemical properties of both the nucleic acid cargo and the delivery vehicle [123]. Both immune and non-immune cells can trigger recognition of nucleic acid through pattern recognition receptors. It is known that unmodified or impurity-containing mRNA can also activate the innate immune system through Toll-like receptors [135]. Simultaneously, the lipid composition, particularly ionizable lipid chemistry; polyethylene glycol-lipid content, size, and chemistry, nitrogen-to-phosphate molar ratio, and particle size and surface properties can independently influence immune activation [136]; therefore, caution should be taken while evaluating the new lipids, especially those intended for airway deposition. For example, cationic lipids can trigger Toll-like receptor 2 and Toll-like receptor 4 [137], and even empty lipid nanoparticles can induce IL-1β release. Endosomal uptake and disruption are critical for delivering nucleic acids to the cytosol [138]; however, endosomal disruption also acts as a danger signal, promoting cytokine release [139]. Additionally, anti-polyethylene glycol antibodies can accelerate phagocytosis-mediated LNP’s clearance, affecting therapeutic efficacy and even triggering anaphylactic reactions [140]. Replacing cationic with ionizable lipids can reduce immunogenicity risks [141]. Furthermore, using biodegradable ionizable lipids facilitates the formation of smaller reparable endosomal apertures, yielding increased RNA expression with minimal inflammation [142]. Overall, these insights guide the rational formulation design to reduce the immunogenicity of inhaled nucleic acid therapeutics.

Formulation Considerations

Formulation development and manufacturing process selection are governed by critical quality attributes , including molecular stability, aerosol properties, dose, and solid-state properties (Fig. 1) [113, 143–147]. Excipients, including non-reducing sugars, sugar alcohols, polymers, amino acids, surfactants, and buffers, are commonly used to stabilize biologics through vitrification, hydrogen bonding, and interfacial stress [144, 148, 149]. Lactose, glucose, maltose, and maltodextrins can effectively inhibit protein unfolding during the drying cycle; however, their reducing properties restrict their applicability for proteins and nucleic acids [143, 150]. Consequently, non-reducing sugars and sugar alcohols (such as trehalose, sucrose, and mannitol), polymers, amino acids, surfactants, and buffers are frequently utilized. The rationale for excipient selection is influenced by the API, the processing method, and the delivery device. Higher glass transition temperature (Tg) generally correlates with improved physical stability of the dry powder [151], although excessive drying or moisture uptake can negatively affect protein integrity and aerosol performance [152, 153]. In addition, buffers with minimal pH changes during phase transitions are preferred. Non-ionic surfactants such as polysorbates 20/80 can be added to mitigate interfacial aggregation, although they can reduce the final formulation Tg, thereby compromising physical stability [113]. Overall, the formulation development requires careful optimization of excipient composition, solid content, aerosol performance, residual moisture, and storage conditions.

Device and Manufacturing Considerations

A liquid nebulized formulation can be used in early development and clinical studies as initial proof-of-concept [108, 109, 154] or when APIs are limited, with the possibility of switching to a solid form later if the development aligns with the target product profile. From a manufacturing perspective, liquid formulations for nebulization are generally less constrained by particle engineering requirements; DPIs require additional processing steps to produce respirable stable particles. Increasing interest in developing solid DPIs is mainly driven by improved solid-state stability, higher dose delivery, and most importantly, enhanced patient compliance [155]. Dry powders for biologics are commonly developed through spray drying, spray-freeze drying, lyophilization, and jet milling, among others [156–158]. However, conventional technologies such as jet milling and spray drying can compromise biologic stability via high mechanical shear and air-liquid interfacial stress. To mitigate these issues, emerging technologies such as thin-film freeze drying, which employ larger droplets and reduced shear forces, leverage an ultra-rapid-freezing process (100–1000 K/s) to minimize ice crystal growth and biomolecule aggregation [159, 160]. In addition, other specialized platforms, such as Pulmosphere™, PulmoSol™, and Technosphere™ formulations, are also widely reported for inhaled biologics.

Beyond manufacturing, device selection plays a critical role in formulation performance and biologic stability. Nebulizers can impart varying degrees of shear, thermal, and air-liquid interfacial stress on biologics depending on the device architectures, such as jet, ultrasonic, and next-generation systems like the surface acoustic wave. Typically, conventional nebulization exposes biologics to a high mechanical shear force, heat, and air-liquid interfacial stresses that can potentially trigger irreversible denaturation or aggregation of proteins or loss of cargo and size disruption with nanoparticles. Next-generation systems, such as vibrating mesh or surface acoustic wave nebulizers, offer gentler aerosolization to preserve cargo integrity. In contrast, DPIs avoid liquid-phase-related stresses during administration; however, they introduce other challenges related to aerosolizable powder engineering, hygroscopicity, solid-state stability, and efficient powder dispersion and lung deposition. Additionally, human factors, including inspiratory capacity and inhalation technique, remain crucial, as deposition efficiency depends on the patient’s inhalation effort to disperse the powder [161, 162].

Physiological and Disease Barriers to Inhaled Delivery of Biologics

Physiological and disease-related barriers governing the inhaled delivery of biologics are well established in the literature. This section provides a concise overview of the main barriers that impact pulmonary delivery performance. The macromolecular nature of biologics makes them particularly vulnerable to enzymatic and immune-mediated degradation, often resulting in reduced lung deposition, absorption, and therapeutic efficacy [163]. Regional lung deposition is governed by physical and anatomical factors, including airway geometry, breathing patterns, disease state, and aerodynamic particle size (Fig. 2). Optimal pulmonary deposition is generally achieved with particles in the 1–5 µm aerodynamic range, although density, shape, and humidity can significantly influence deposition efficiency (Fig. 2) [164–168].

Fig. 2.

Schematic summarizing the key barriers for the pulmonary delivery of inhaled biologics. These interconnected mechanisms collectively determine the fate of inhaled biologics. Understanding these multifaceted challenges is essential for developing effective formulation strategies and delivery systems that can overcome or circumvent these barriers to achieve optimal therapeutic outcomes in respiratory diseases. ROS reactive oxygen species

After reaching the lungs, mucus-associated barriers dominate the post-deposition fate of inhaled biologics. The dense and viscous mucus network can limit diffusion of biologics, while coordinated ciliary transport rapidly clears deposited material toward the gastrointestinal tract, removing most inhaled particles within 24 hours [169–172]. Interactions with pulmonary surfactants can destabilize inhaled biologics or enhance immune recognition, though surfactant-mimetic approaches may improve delivery of biologics [173]. Enzyme-rich lung environments, especially in diseased states, accelerate the breakdown of inhaled biologics [174, 175]. Immune surveillance, particularly macrophage-mediated phagocytosis, is a major determinant of particle clearance from the alveolar region [176–178].

Pharmacokinetic Considerations for Inhaled Biologics Used in Treating Lung Diseases

Systemic Delivery Versus Localized Delivery

Different models and considerations are used when evaluating the pharmacokinetics of different types of biologics depending on their route of administration. For systemic administration via injection, a one-compartment model may be utilized to monitor patient exposure; however, it may not be representative of exposure at the active site. Because of their poor absorption, most biologics are administered intravenously, subcutaneously, or intramuscularly. Researchers have employed multi-compartment models for studying drug disposition of locally delivered biologics to the lung. In some cases, researchers have opted for a three- or four-compartment model that includes a compartment for the lung epithelial cells as well as a compartment for a macrophage pool [179]. It is important to note that the decision to select a model is highly dependent on the local residence time, which is dictated by the rate-limiting step for clearance from the airway. The rate-limiting step for clearance from the airway is largely variable depending on the class of molecule and needs to be investigated on a case-by-case basis. Here, we illustrate a few examples of actives from each biologics class, but focus on the pharmacokinetics of inhaled mAbs.

Absorption

Because of the complexity and diversity of pulmonary tissues, absorption through this route is highly site specific, with differing regions offering different physiological/anatomical barriers as well as available surface area for absorption. Depending on the targeted site for delivery (systemic or local), these regions can be more or less favorable depending on the physicochemical properties of the molecule. The upper airways represent the largest barrier with an epithelial thickness of about 50 µm.The thickness of the tissue decreases moving down the airway to the small bronchi and bronchioles (~ 10 µm) and into the terminal regions of the lung, the alveoli, which can be as small as 0.1 um [180]. In studies collected by Patton, it was found that as the molecular weight (MW) of the peptide/protein increased, there was a decrease in the rate and extent of absorption through alveolar epithelia [181]. While the exact mechanism for absorption of macromolecules is not completely understood, some believe that the process is diffusion limited and the molecules either pass through tight junctions in endothelial cells or through endocytitic vesicles [182]. This diffusion-based concept takes into account the lower diffusivity of larger molecules to make their way to ‘pores’ at a lower rate than smaller molecules. In contrast, others contend larger immunoglobulins have a receptor-mediated transport mechanism, which allows for passage across mucosal barriers, and therefore are bottlenecked by access to the binding site [183, 184]. It has been widely reported that mAbs have low and slow absorption through the respiratory tract, but some scientists attribute this to the fact that tertiary and quaternary protein structures have a lower susceptibility to degradation from extracellular proteases and therefore have a longer residence time at the site of action [185]. Furthermore, it is believed by some that alveolar macrophages are the primary barrier to absorption [186]. Combined with the ability to deliver larger payloads to the active site, all of these factors combined likely contribute to the ability to maintain higher levels of larger MW molecules at the site of action for prolonged periods of time [76].

Distribution

Distribution of biologics takes a similar theme to that of the properties of molecules that affect their absorption (MW, charge, and hydrophobicity). For mAbs, because of their large size they commonly have a very low volume of distribution. Once in the systemic circulation, most mAbs have a volume of distribution that is approaching the plasma volume of 3 5 L or 0.04–0.07 L/kg [187]. Independent of size, these drug molecules must pass across or between the endothelial cells of the vasculature and tissue. Because mAbs commonly have their lipophilic chains sequestered to the interior portions of their structures, it is not believed that they can pass readily across endothelial cells [188]. Instead, it is conventionally believed that they pass through convection (diffusion between endothelial cells) [189]. This is consistent with the observations of a slightly increased volume of distribution at steady state of 5–10 L, which signifies some level of escape from the central compartment [187]. In a study by Jagdale et al., while looking at systemic administration of a mAb (trastuzumab) and several smaller molecular-weight fragments, they demonstrated that intact mAb can reach lung tissue, but the smaller fragment (~ 50 kDa) could reach the endothelial cell surface and be detectable in the bronchial alveolar lavage fluid [190]. For smaller peptides, the diffusion rate is greater than mAbs and allows them to move more passively into the interstitial space, depending on the degree of vascularization and capillary fenestration variety present in that tissue.

Metabolism

While all of the cytochrome p450 enzymes that are used in hepatic metabolism of small molecules are present in lung tissues, they are present to a far lower degree, and biologics are not usually metabolized by such enzymes. Rather, a majority of protein-based biologics are subject to hydrolysis via a variety of proteases and peptidases in pulmonary epithelial cells, as described above in the barriers section. While there are lower exposures to these enzymes in the lower respiratory tract compared with an SC or IM injection, smaller peptides are still vulnerable to degradation before they can be absorbed and can be protected by the use of protease inhibitors [191]. The rate and degree to which these molecules are catabolized appears to be largely dependent on the MW and the degree of protein folding, with peptides having the fastest rate of hydrolysis and larger proteins (6–50 kDa) being more resistant [181]. Larger protein-based biologics, such as mAbs, are primarily metabolized by proteolysis after being phagocytized in the reticuloendothelial system, where they can be taken up by lysosomes and degraded. Alternatively, they can bind FcRn and be recycled, thus contributing to their frequently observed prolonged half-lives.

Elimination/Renal Clearance

The upper airways are lined with either ciliated columnar cells or mucus-producing goblet cells, which together form what is known as the mucociliary escalator. Macromolecules that are deposited in these conducting airways are subject to these clearance mechanisms as described above. Material entrapped in this region has a residence half-life of about 1.5 hours [191, 192]. Particles that reach the respiratory region are available to undergo phagocytosis by alveolar macrophages (usually in the 1-3 µm range). Macromolecules < 40 kDa are cleared relatively rapidly by macrophages, and larger molecules > 40 kDa, as mentioned, are slowly endocytosed. After phagocytosis, the macrophages travel along the epithelial surface towards the mucociliary escalator or are cleared by tracheobronchial lymph.

Pharmacokinetics/Pharmacodynamics and Target-Mediated Drug Disposition

Traditional pharmacokinetic/pharmacodynamic modeling for small molecules has focused on exposure and how it relates to a pharmacodynamic effect. While the pharmacokinetic and exposure data yield useful information for targeting therapeutic performance and tracking safety metrics, biologics typically exhibit target-mediated drug disposition, which complex biological processes govern efficacy.

Regulatory Considerations

The FDA recognizes the challenges associated with traditional pharmacokinetic assessments for biologics, where additional factors not seen with small molecules can have an effect on efficacy, such as protein folding, denaturation, and dimerization, and thus has taken a more mechanistic approach to developing first-in-human dosing guidelines and encourages early conversations about developing informative models that inform decision making in development [193].

While there is currently no formal guidance for inhaled biologics, considerations can be made across the guidance for inhaled small-molecule drugs and biologics as a whole. For product development, similar to small molecules intended for inhalation, biologics should also meet the rigorous standards for Chemistry, Manufacturing, and Controls. The guidance varies depending on the final dosage form, but generally quality tests for inhaled products include critical quality attributes such as: potency, emitted dose, aerodynamic particle size distribution, fine particle fraction (FPF) to ensure the proper dose is consistently delivered to the appropriate respiratory tissue [194]. Furthermore, depending on the dosage form and delivery device (pMDIs, DPIs, nebulizers, or soft mist aerosol devices), additional performance testing should be considered around dispensing characteristics related to that device and dosage form combination. Drug-device combinations should be assessed for potential leachable and extractable compounds, but for biologics, additional guidance has been provided to ensure product stability and quality are not compromised over time because of packaging incompatibility [195]. In addition, owing to their complex structures and potentially labile nature, stability is a crucial consideration when developing inhaled biologics. Stability assessments largely follow ICH Q5C guidelines around ensuring the maintenance of molecular conformation and biological activity. For additional information regarding protocols and data reporting, refer to the FDA guidance [196]. Finally, immunogenicity from biologics is another concern, whether it be from the innate parent molecule or generated from partially degraded or aggregated biomolecules. The FDA has released guidance for establishing proper assays to assess immunogenicity and limits for particulates/aggregates [197–199]. However, when considering the aerosolization of biologics, additional concerns have arisen because of the potential for the biologic drug to be degraded or aggregated during this process of aerosolization. Scientists have begun developing in-vitro assays to assess post-aerosolization aggregation and have acknowledged the potential for immunogenicity within lung tissues, which warrants investigation during development [108, 200]. While scientists are still in the early stages of understanding the complex nature of the pharmacokinetics of inhaled biologics, important next steps involve creating more sensitive analytical methods for tracking actives as they are modified and/or move through the different compartments of the body.

The Opportunity: Respiratory Diseases with Targets Suitable for Biological Therapies

In this section, we review potential biological targets with suitability for treatment using inhaled biologics. Tables 2, 3, 4, 5, 6, 7 and 8 summarize biologic therapeutics across all routes of administration that are either approved or in clinical development for respiratory and pulmonary disease states.

Asthma

Despite standard treatments that include high-dose inhaled corticosteroids combined with long-acting bronchodilators, approximately 5–10% of patients with asthma remain poorly controlled and continue experiencing significant symptoms [201]. Advances in understanding the underlying mechanisms of asthma have led to the development of biologic therapies, now routinely considered for patients with severe uncontrolled asthma [202]. In general, these biologics are generally administered systemically and have shown efficacy through this route; they are discussed here to highlight established biologic mechanisms and therapeutic targets in asthma.

Currently available biologic therapies for asthma primarily target type 2 inflammation mediators, including IgE and cytokines IL-4, IL-5, IL-13, and TSLP [203, 204]. Available injectable biologics include omalizumab, mepolizumab, reslizumab, benralizumab, dupilumab, and tezepelumab [205]. Omalizumab is a mAb that binds to IgE, reducing asthma exacerbations [206]. Mepolizumab, reslizumab, and benralizumab target the IL-5 receptor, effectively reducing eosinophilic inflammation [207]. Dupilumab inhibits IL-4 and IL-13 signaling by targeting IL-4Rα, improving lung function and reducing asthma attacks [208]. Tezepelumab, a novel mAb, targets TSLP, an epithelial cytokine involved in initiating asthma inflammation [209]. Table 2 provides a summary of approved injectable biologics.

Table 2.

Summary of approved biologic therapeutics approved or in development for asthma/COPD

Product/API	Size (kDa)	Biological target/class	Indication	Company	RoA	Year	Status	Ref./NCT no.
Xolair® (omalizumab)	149	IgE	Moderate-to-severe persistent asthma	Genentech/Novartis	SC	2003	Appr.	[224]
Nucala® (mepolizumab)	149	IL-5	Severe eosinophilic asthma/COPD	GlaxoSmithKline	SC	2015	Appr.	[369]
Cinqair® (reslizumab)	147	IL-5	Severe eosinophilic asthma	Teva Pharmaceuticals	IV	2016	Appr.	[224, 370]
Fasenra® (benralizumab)	150	IL-5Rα	Severe eosinophilic asthma	AstraZeneca	SC	2017	Appr.	[371]
Dupixent® (dupilumab)	147	IL-5	Severe eosinophilic asthma	GSK	SC	2025	Appr.	[372]
Tezspire® (tezepelumab)	147	TSLP	Severe asthma	Amgen/AstraZeneca	SC	2021	Appr.	[373]
Tralokinumab	147	IL-13	Severe asthma	AstraZeneca	SC	2017	Disc.	[374]
Lebrikizumab	145	IL-13	Severe asthma	Genentech	SC	2016	Disc.	[375]
Secukinumab	151	IL-17A	Severe asthma	Novartis	IV	2014	Disc.	[376]
Brodalumab	144	IL-17RA	MSPA	Amgen	SC	2015	Disc.	[377]
Anakinra	17.3	rhIL-1Ra and E. coli	Acute asthma exacerbation/treatment for allergic airway inflammation	UNC Chapel Hill	SC	2021	Disc.	[378]
Astegolimab	145	IL-33/ST2-targeted	Uncontrolled severe asthma	Genentech/Roche	SC	2021	Disc.	[379]
Ecleralimab	46.6	TSLP	Atopic asthma	Novartis Pharmaceuticals	Inhalation	2022	Disc.	[380]
Lunsekimig	69.7	TSLP/IL-13	Mild-to-moderate asthma, high-risk asthma	Sanofi	SC	2025	Ph. II	[381], NCT06676319 NCT06102005

Appr. approved, COPD chronic obstructive pulmonary disease, Disc. discontinued clinical trial, IgE immunoglobulin E, IL interleukin, IL-5Rα interleukin-5 receptor alpha, IL-17RA interleukin-17 receptor A, IV intravenous, kDa kilodalton, Mkt. marketed, NCT national clinical trial, Ph phase, Refs. references, rhIL-1Ra recombinant human interleukin-1 receptor antagonist, RoA route of administration, SC subcutaneous, TSLP thymic stromal lymphopoietin, UNC University of North Carolina

Among the approved asthma biologics, omalizumab, dupilumab, and tezepelumab can influence airway smooth muscle contractility and airway hyperresponsiveness. The observed effect is mediated by the interaction with receptors located on the airway smooth muscle, as well as their effect on eosinophils and inflammatory cells, leading to a reduction of the inflammatory cascade [210]. This highlights the potential benefits of delivering biologics directly to lung tissue through inhalation [210].

Several injectable biologic therapies for asthma have been discontinued during clinical trials because of inadequate clinical efficacy or safety issues. For instance, tralokinumab, administered via a SC injection, despite having a safety profile comparable to placebo, showed only minor physiological improvements without meaningful clinical benefits, as it produced only modest improvements in forced expiratory volume in one second and forced vital capacity in patients with moderate-to-severe disease [211]. These physiological enhancements did not translate into meaningful clinical benefits; thus, tralokinumab failed to improve asthma-related quality of life and did not reduce exacerbation frequency [212]. Similarly, another mAb, secukinumab, administered via an IV infusion and targeting IL-17A, failed to show consistent clinical benefits [213]. Additional biologic candidates whose trials have been terminated are summarized in Table 2. The discontinued biologics highlight the complexity and heterogeneity of asthma, emphasizing, in some cases, the importance of patient selection using reliable biomarkers.

Recent research efforts in asthma therapeutics have broadened the focus beyond classical cytokine signaling to include a wider range of biological mechanisms. Among these, intracellular kinases have emerged as promising targets. For example, inhibiting Bruton’s tyrosine kinase may reduce autoantibody production and inhibit key processes such as macrophage activity, mast cell degranulation, granulocyte migration, and the release of proinflammatory mediators, addressing multiple pathways involved in inflammation and immune regulation [214]. Similarly, targeting Janus kinases, which are intracellular tyrosine kinases involved in IL receptor signaling, may be particularly beneficial in patients with elevated IL-6 levels [215]. Additional areas of interest include antagonism of the D prostanoid 2 receptor, also known as the chemoattractant receptor-homologous molecule expressed on T helper 2 (Th2) cells, which plays a central role in recruiting Th2 cells and eosinophils to inflamed airways. Other novel targets under investigation include CC-chemokine receptor 4 antagonists and inhibition of the transcription factor GATA3, which regulates the expression of multiple Th2-associated cytokines [214]. Future biologic treatments may also focus on better understanding non-Th2 inflammatory mechanisms and developing targeted antibodies to address these pathways [216]. Moreover, bispecific antibodies that simultaneously target two mediators could enhance treatment effectiveness and reduce development costs compared with using two separate mAbs [205]. All available injectable biologics used in asthma are characterized by large MW, hydrophilicity, and relative structural instability. Another limitation is that available asthma biologics are injectables, which can lower patient adherence to the treatment [217]. Systemic delivery is associated with several challenges, including decreased pulmonary bioavailability [218]. Furthermore, systemic exposure raises the likelihood of adverse effects, such as hypersensitivity reactions, and possible toxicity. Systemically administered biologics may also demonstrate a delayed onset [219]. These restrictions emphasize the opportunity for direct targeted delivery as an inhalation route, which could potentially improve patient compliance by providing a non-invasive dosage form.

Targeting TSLP with tezepelumab is effective but requires monthly injections, [220] whereas inhaled anti-TSLP therapies may enhance local delivery and simplify dosing. Gauvreau et al. studied ecleralimab, the first inhaled anti-TSLP potential candidate [53, 221]. The IgG1/λ Fab fragment, ecleralimab, lacks an Fc region and therefore has a smaller MW (46.6 kDa) compared with a full-length antibody (~ 150 kDa). With no Fc region, Fab fragments are small enough to penetrate the airway tissue and improve lung distribution and tissue penetration while maintaining antigen-binding specificity, which is advantageous for targeted lung administration [53, 221]. Ecleralimab, formulated as dry-powder capsules for DPI administration, was investigated in atopic asthma. The trial demonstrated significant decreases in allergen-induced bronchoconstriction and airway inflammation, along with a positive safety profile [53]. Inhalation administration could further improve localized effectiveness and patient outcomes, suggesting that there must be continued innovation in inhaled biologic therapies for asthma.

Chronic Obstructive Pulmonary Disease (COPD)

Targets under investigation for COPD biologics substantially overlap with those in asthma, including IL-4/IL-13, IL-5/IL-5Rα, IL-33/ST2, and TSLP pathways discussed in the section above, with several asthma-approved agents now being evaluated in COPD populations. However, the path to drug approval in COPD has been challenging because of differences in underlying pathophysiology and inflammatory responses compared with asthma [222]. Asthma is largely driven by T helper 2 T-cell cytokine-mediated eosinophilic inflammation, whereas COPD is predominantly characterized by neutrophilic inflammation [223]. These differences may explain why only two asthma biologics have gained additional approval for COPD: dupilumab and mepolizumab [224, 225]. For instance, while benralizumab, which targets the interleukin (IL)-5 receptor, is approved for treating severe eosinophilic asthma, it did not demonstrate a statistically significant reduction in the annualized rates of COPD exacerbations compared to placebo during the 56-week GALATHEA and TERRANOVA trials. Although benralizumab effectively reduces eosinophil levels, it was insufficient in controlling COPD exacerbations [226].

Recent studies suggest that IL-6, a 26-kD inflammatory cytokine, contributes to asthma and COPD pathogenesis, with COPD showing a distinct dysregulation characterized by reduced IL-6 gene expression but elevated IL-6ST (signal transducer) levels in induced sputum compared with healthy controls [227]. IL-6ST levels were directly correlated with deteriorating lung function and increased COPD severity, indicating its potential as a biomarker for disease severity [228]. These findings highlight the complex nature of IL-6 signaling disruption in COPD and support the development of targeted therapies that modulate specific components of this pathway rather than broad anti-inflammatory approaches. Several anti-IL-6 mAbs have been developed [229], but few have been studied in clinical settings for COPD. Recent preclinical studies have shown that nebulized atlizumab (tocilizumab) is well tolerated and effective compared with the IV route in a murine model [230]. Nebulized tocilizumab showed no systemic toxicity and exhibited fewer pulmonary hemorrhages and reduced intra-alveolar edema compared with saline controls [230]. Additionally, nebulized tocilizumab induced a significant increase in alveolar macrophage infiltration (60–100% vs 0% in controls, p < 0.001), suggesting enhanced local immunomodulatory activity [230]. Despite preclinical evidence and genetic co-relationships supporting the role of IL-6 in asthma, a randomized, double-blind, placebo-controlled clinical trial found no significant difference in allergen-induced bronchoconstriction between tocilizumab and placebo groups [231] [232–234].

Infectious Diseases

Respiratory infections caused by viral and bacterial pathogens, such as RSV, SARS-CoV-2, Staphylococcus aureus, and Pseudomonas aeruginosa, are leading causes of global morbidity and mortality [235]. Each pathogen presents unique therapeutic challenges; for example, SARS-CoV-2 invades airway epithelial cells and triggers intense local inflammation, necessitating therapies that simultaneously block viral replication and modulate the immune response [236], whereas P. aeruginosa establishes protective polymeric biofilms that act as diffusive barriers, shielding bacterial cells from therapeutic agents and promoting antibiotic resistance [237]. Given the complexity of pulmonary infections and their frequent resistance to conventional therapies, recent efforts have focused on developing biologics that specifically target microbial virulence factors. These biologics include mAbs, nanobodies, and nucleic acid-based therapies. Table 3 summarizes biologics in clinical use or development for respiratory infections given by any route of administration. For SARS-CoV-2, mAbs such as casirivimab/imdevimab and tixagevimab/cilgavimab target the receptor-binding domain of the spike protein, preventing viral entry [238, 239]. For RSV, palivizumab and nirsevimab are clinically approved mAbs that inhibit viral fusion [239–242]. In bacterial infections, biologic strategies are similarly pathogen specific. [243, 244] In S. aureus, investigational mAbs such as AR-301 and AR-320 neutralize alpha-toxins, a virulence factor responsible for host cell lysis [245, 246]. Panobacumab and MEDI3902 target P. aeruginosa lipopolysaccarides O-polysaccharide and PcrV (a type III secretion protein), respectively, impairing biofilm formation and adhesion [247, 248].

Table 3.

Summary of biologic therapeutics in clinical use and development for respiratory infectious diseases

Product/API	Biological class (size)	Biological target	Indication/pathogen	Company/research group	RoA	Year	Status	Ref./NCT no.
SPIKEVAX®	mRNA Vac.	SCV-2 spike (S)	COVID-19	Moderna	IM	2022	Appr.	[382]
Actemra® (tocilizumab)	mAb (148 kDa)	IL-6 receptor	COVID-19	Genentech	SC/IV	2010	Appr.	[383]
COMIRNATY®	mRNA Vac.	SCV-2 spike (S)	COVID-19	Pfizer/BioNTech	IM	2021	Appr.	[384]
Synagis® (palivizumab)	mAb (148 kDa)	RSV F protein	RSV	MedImmune	IM	1998	Appr.	[385]
BEYFORTUS® (nirsevimab-alip)	mAb (146.3 kDa)	RSV F protein (site A)	RSV	Sanofi/AstraZeneca	IM	2023	Appr.	[386–388]
MRESVIATM®	mRNA Vac.	RSV pre-F	RSV	Moderna	IM	2024	Appr.	[389]
ABRYSVO®	Recomb. protein Vac.	RSV pe-F (A/B)	RSV	Pfizer	IM	2023	Appr.	[390]
AREXVY®	Recomb. protein Vac.	RSV Pre-F3	RSV	GSK	IM	2023	Appr.	[391]
PREVNAR 20®	Conj. Vac.	S. pneumoniae capsular polysacc. (20-valent)	S. pneumoniae	Pfizer/Wyeth	IM	2021	Appr.	[392]
PREVNAR 13®	Conj. Vac.	S. pneumoniae capsular polysacc. (13-valent)	S. pneumoniae	Pfizer/Wyeth	IM	2010	Appr.	[393]
ENFLONSIA™	mAb (149 kDa)	RSV F protein	RSV	Merck	IM	2025	Appr.	[394]
Sotrovimab	mAb (149 kDa)	SCV-2 spike RBD	COVID-19	GSK	IV	2021	EUA revoked	[395]
J&J COVID Vaccine	Adenoviral vector Vac.	SCV-2 spike (S)	COVID-19	Janssen	IM	2021	EUA revoked	[396]
REGEN-COV™	mAb cocktail (casirivimab, 145.23 kDa) and (imevimab, 144.14 kDa)	SCV-2 spike RBD	COVID-19	Regeneron	IC/IV	2020	EUA revoked	[397]
EVUSHELD™	mAb cocktail (tixagevimab, 149 kDa), and (cilgavimab, 152 kDa)	SCV-2 spike RBD	COVID-19	AstraZeneca	IM	2021	EUA revoked	[398]
Bamlanivimab	mAb (140 kDa)	SCV-2 spike RBD	COVID-20	Eli Lilly	IV	2020	EUA revoked	[399, 400]
Regdanvimab	mAB	SCV-2 spike RBD	COVID-19	Celltrion	IV	2021	Withdrawn	[258, 401]
DS-Cav1	Subunit protein Vac.	RSV pre-F	RSV	Vaccine Research Center	IM	2017	Ph. I	[402]
AR-301 (tosatoxumab)	mAb	S. aureus alpha-toxin	S. aureus	Aridis	IV	2019	Ph. III	[403]
AR-320 (suvratoxumab)	mAb	S. aureus alpha-toxin	S. aureus	Aridis	IV	2022	Ph. III/Disc	[245], NCT05331885
Panobacumab	mAb	P. aeruginosa O11 LPS	P. aeruginosa	Kenta Biotech	IV	2008	Ph. II	[247], NCT00851435
AR-105 (Aerucin®)	mAb	P. aeruginosa alginate	P. aeruginosa	Aridis	IV	2017	Ph. II	[404], NCT03027609
MEDI3902 (gremubamab)	mAb	P .aeruginosa PcrV and Psl	P. aeruginosa	AstraZeneca/MedImmune	IV	2016	Ph. II	[248], NCT02696902
IN-006 (regdanvimab)	mAb	SCV-2 spike RBD	COVID-19	Inhalon Bipharma	Inhalation	2021	Ph. I	[258, 405], NCT06287450
ALN-RSV01	siRNA	RSV nucleoside (N) mRNA	RSV	Alynlan	Inhalation	2007	Ph. II/Disc.	[257, 406], NCT01065935
MEDI-524 (Numax®, motavizumab)	mAb (148.4 kDa)	RSV F protein (site A)	RSV	AstraZeneca	IM	2008	Ph. III/Disc.	[407, 408]
REGN2222 (suptavumab)	mAb (146.1 kDa)	RSV pre-F	RSV	Regeneron/MedImmune	IM	2015	Ph. III/Disc.	[409–411]
KB001-A	mAb	P. aeruginosa (PcrV)	P. aeruginosa	KaloBios	IV	2012	Ph. II/Disc.	[363, 412]
ALX-0171	Antibody fragment	RSV F protein (site II)	RSV	Ablynx	Inhalation/intranasal	2018	Ph. II/Disc.	[79, 81]
REG	G-protein Vac.	RSV G attachment protein	RSV	CBER	IM	2015	Preclin.	[413]
AR-701 (COVID-19 mAb)	mAb	SCV-2 envelop proteins	COVID-19	Aridis	-	-	Preclin.	[414]

Appr. approved, CBER Center for Biologics Evaluation and Research, Conj. conjugate, Disc. discontinued, EUA emergency use authorization, IM intramuscular, IV intravenous, kDa kilodalton, LPS lipopolysaccharide, mAb monoclonal antibody, NCT national clinical trial, P. aeruginosa Pseudomonas aeruginosa, Ph. phase, Preclin. preclinical, RBD receptor-binding domain, Recomb. recombinant, Refs. references, RoA route of administration, RSV respiratory syncytial virus, S. aureus Staphylococcus aureus, S. pneumoniae Streptococcus pneumoniae, SC subcutaneous, SCV-2 severe acute respiratory syndrome coronavirus 2, siRNA small interfering RNA, Vac. vaccine

Despite their therapeutic potential, many of these biologics express significant limitations, such as the risk of systemic side effects and poor tissue penetration. For example, tocilizumab, administered intravenously or subcutaneously, shows significant dose-related systemic side effects, such as elevated liver enzymes, neutropenia, and thrombocytopenia, sometimes necessitating dose interruption when given at high doses [249]. Palivizumab, delivered intramuscularly, reaches serum concentrations that are roughly 2000 times higher than those found in bronchoalveolar lavage fluid [250]. This disparity suggests that only a small fraction of the antibody reaches the lungs where RSV replication occurs, which may help explain the modest reductions in hospitalization rates observed in treated infants [250]. More generally, IgG antibodies delivered systemically have been shown to exhibit bronchoalveolar lavage concentrations approximately 1000-fold lower than corresponding serum levels [251].

Most biologics currently approved or under investigation for infectious diseases are formulated for intravenous (IV), intramuscular (IM), or subcutaneous (SC) administration, as shown in Table 3. However, because these agents are designed to target microbial components such as viral surface proteins, bacterial toxins, or biofilm structures, they are also well suited for direct delivery to the lungs, the primary site of infection. Inhaled delivery provides an alternative approach by enabling direct deposition in the lungs, thereby mitigating limitations of systemic IV, IM, or SC formulations, such as low pulmonary exposure and off-target effects. This strategy has already proven effective with antibiotics such as aztreonam and tobramycin [252]. The feasibility of inhaled biologics for respiratory infections is illustrated by examples including palivizumab, ALX-0171, ALN-RSV01, and IN-006, which are discussed below.

In a study conducted by Rajapaksa et al., the pulmonary delivery of aerosolized palivizumab was investigated in neonatal lambs [253]. Using two nebulization devices, the investigational HYDRA and the AeroNeb Go^®, the researchers generated aerosols in the respirable 1–5 µm range, with comparable median droplet diameters (1.84 ± 1.40 µm for HYDRA and 3.07 ± 1.56 µm for AeroNeb Go^®) [253]. Both devices achieved efficient lung targeting, delivering approximately 89–94% of the aerosolized dose to the lungs [253]. These results highlight the feasibility of delivering mAbs such as palivizumab directly to the lungs via inhalation. Compared with conventional IM administration, which can take 3–5 days to reach therapeutic serum levels, this route offers more rapid and localized drug exposure, potentially improving therapeutic outcomes in high-risk infants [253].

Among recently developed inhaled biologics is ALX-0171, a trimeric nanobody delivered by inhalation for the treatment of RSV [81]. This trimeric nanobody targets the antigenic site II of the RSV fusion (F) protein and exhibits stronger in vitro neutralization than palivizumab, a systemic mAb, across both RSV-A and B strains, blocking replication in 87% of clinical isolates [81]. Its lower MW (42.2 kDa) compared with the full-length palivizumab (148 kDa) may contribute to its enhanced potency and more efficient viral neutralization. In preclinical studies, Detalle et al. demonstrated that ALX-0171 significantly reduced viral titers in both the nose and lungs of cotton rats following prophylactic and therapeutic administration [81]. Additionally, they showed that the nanobody maintained its physicochemical integrity following nebulization with the Akita² Apixneb nebulizer [81]. In the phase IIb clinical study, ALX-0171 formulated as a 50-mg/mL solution in 10 mM of phosphate buffer containing 130 mM of sodium chloride (pH 7.0) and administered using the FOX-Flamingo inhalation system with a pediatric face mask [79, 84, 254]. Despite demonstrating strong antiviral effects in phase II trials, Sanofi discontinued the program after a strategic pipeline prioritization review [255].

ALN-RSV01, an inhaled small interfering RNA has also been developed to treat RSV infections by targeting and silencing viral nucleocapsid mRNA [82]. The therapeutic small interfering RNA, when formulated in phosphate-buffered saline at a concentration of 150 mg/mL and aerosolized using the PARI eFlow^® electronic mesh nebulizer, was characterized by a mass median aerodynamic diameter (MMAD) of 3.1 µm and a respirable fraction of 85% (particles <5 µm), supporting its suitability for airway delivery. Anion exchange chromatography confirmed that the small interfering RNA maintained its integrity following nebulization, with no evidence of degradation [256]. In a randomized, placebo-controlled, phase IIb trial involving lung transplant recipients with RSV, nebulized administration of ALN-RSV01 at a dose of 0.6 mg/kg for 5 days resulted in a 55–65% reduction in the incidence of new or worsening bronchiolitis obliterans syndrome at 180 days [257].

IN-006 is an inhaled formulation of regdanvimab, an mAb that targets the receptor-binding domain of the SARS-CoV-2 spike protein. In preclinical studies by McSweeney et al., IN-006 demonstrated stable nebulization using multiple, commercially available, vibrating mesh nebulizers across a wide range of concentrations [258]. Aerosol characterization for 20 and 30 mg/mL nebulized IN-006, using InnoSpire Go VMS, showed an MMAD of 5.7 µm and 5.3 µm, with FPFs of 47% and 51%, respectively [258]. These characteristics support its suitability for lung deposition. The antibody retained its biophysical properties post-nebulization, without significant aggregation or loss of function [258]. Moench et. al. evaluated the safety and pharmacokinetics of nebulized IN-006 in healthy volunteers [259]. Participants received either a single 30- or 90-mg dose or seven consecutive daily 90-mg doses using the InnoSpire Go handheld nebulizer, during which they were instructed to inhale slowly through the mouthpiece and exhale through the nose [259]. IN-006 was well tolerated across all cohorts, with only mild transient adverse events [259]. In the multiple-dose group, geometric mean nasal fluid concentrations reached 739.8 µg/mL at 30 minutes post-dose and remained above 1.2 µg/mL at 22 hours, levels several orders of magnitude above typical antiviral half-maximal inhibitory concentration [259]. These findings support the potential of inhaled IN-006 for achieving high local antibody concentrations in the respiratory tract, offering a promising alternative to systemic mAb therapies for COVID-19.

Genetic Diseases

Respiratory genetic disorders, such as CF, primary ciliary dyskinesia, AAT deficiency (AATD), and surfactant protein (SP) deficiency, can also be suitable targets for inhaled biologics (Fig. 3, Table 4).

Fig. 3.

Illustration of biologic therapeutics in development for respiratory genetic diseases, including cystic fibrosis, primary ciliary dyskinesia, alpha-1-antitrypsin (AAT) deficiency, and surfactant deficiencies. Readers may refer to Table 4 for comprehensive details on the drug products, target proteins, delivery systems, administration routes, clinical trial identifiers, and phases. The information is current as of August 2025. cDNA complementary DNA, mRNA messenger RNA, LNPs lipid nanoparticles

Table 4.

Summary of biologic therapeutics approved or in development or discontinued for respiratory genetic diseases. The information is obtained from the updated prescribing label through Drugs@FDA: FDA-Approved Drugs and clinical trial registry data, current as of August 2025

Indication	Drug product/candidates	Delivery system	Target protein	RoA	NCT no.	Clinical phase (timeline; status)	Company
CF	Pulmozyme®	rhDNase I	–	Nebulization	NCT00179998	Ph. II (2008–16; Appr.	Genentech
CF	BI 3720931 (LENTICLAIR™)	Lentiviral vector	CFTR	Inhalation	NCT06515002	Ph. I/II (2024–ongoing)	Boehringer Ingelheim
CF	ARCT-032	mRNA-LNPs	CFTR	Nebulization	NCT05712538 NCT06747858	Ph. Ib, II (2024–ongoing)	Arcturus
CF	4D-710	AAV vector	CFTRΔR	Inhalation	NCT05248230	Ph. I/II (2022–ongoing)	4D Molecular Therapeutics
CF	VX-522	mRNA-LNPs	CFTR	Nebulization	NCT05668741	Ph. I/II (2023–ongoing)	Vertex
CF	SP-101	AAV vector	CFTR	Nebulization	NCT06526923	Ph. I/II (2024-ongoing)	Spirovant
CF	RCT2100	mRNA-SORT LNPs	CFTR	Nebulization	NCT06237335	Ph. I (2024–ongoing)	ReCode
CF	pGM169/GL67A	pDNA-cationic liposomes	CFTR	Nebulization	NCT01621867	Ph. IIb (2012–15; completed)	Imperial College London
CF	MRT5005	mRNA-LNPs	CFTR	Intranasal	NCT03375047	Ph. I/II (2018–21; Disc.)	Translate Bio
CF	QR-011	ASO	CFTR	Intranasal	NCT02532764	Ph. Ib (2015–17; Disc.)	ProQR
CF	Eluforsen (QR-010)	ASO	CFTR	Intranasal	NCT02564354	Ph. I (2015–16; Disc.)	ProQR
CF	KB407	Viral vector (HSV-1)	CFTR	Nebulization	NCT05095246	Ph. I (2022–24; Disc.)	Krystal
PCD	RCT1100	mRNA-LNPs	DNAI1	Nebulization	NCT05737485 NCT06600425	Ph. I, Ib (2024–ongoing)	Recode
PCD	ETH42 and ETH43	mRNA-LNPs SNIM®RNA	CCDC40	–	Preclinical	–	Ethris
PCD	MRT-DNAI1-LNP	mRNA-LNPs	DNAI1	–	Preclinical	–	Translate-Bio
AATD	AAT glycoprotein (~52 kDa)	–	AAT augmentation	Nebulization	NCT04204252	Ph. III (2019–ongoing)	Kamada
AATD	SAR447537 (INBRX-101)	–	AAT augmentation	IV	NCT03815396 NCT05856331	Ph. II (2023–25; Disc.)	Sanofi
AATD	Beam-302	Base-editing LNPs	AAT augmentation	IV	NCT06389877	Ph. I/II (2024–ongoing)	Beam Therapeutics
AATD	KRRO-110	OPERA®-LNPs	AAT augmentation	IV	NCT06677307	Ph. I/II (2025–ongoing)	Korro Bio
AATD	WVE-006 (AIMer)	GalNAc-ASO	M-AAT augmentation	SC	NCT06405633 NCT06186492	Ph. I and Ib/IIa (2025–ongoing)	Wave Life Sciences
AATD	AIR-001 RESTORE+ and AIR-100	ASO	Repair PiZZ mutation	SC	–	Preclin.	AIRNA
AATD	N/A	SC AAT infusion	AAT augmentation	SC	NCT04722887	Ph. I and II (2021–25)	GRIFOLS

AAT alpha-1 antitrypsin, AATD alpha-1 antitrypsin deficiency, AAV adeno-associated virus, Appr. Approved, ASO antisense oligonucleotide, CCDC40 coiled-coil domain containing 40, CF cystic fibrosis, CFTR cystic fibrosis transmembrane conductance regulator, Disc. discontinued, DNAI1 dynein axonemal intermediate chain 1, HSV-1 herpes simplex virus type 1, IV intravenous, LNP lipid nanoparticle, M-AAT mutant alpha-1 antitrypsin, NCT national clinical trial, pDNA plasmid DNA, Ph. phase, Preclin. preclinical, PCD primary ciliary dyskinesia, rhAAT recombinant human alpha-1 antitrypsin, rhDNase I recombinant human deoxyribonuclease I, RoA route of administration, SC subcutaneous

Cystic Fibrosis (CF)

Cystic fibrosis is an autosomal recessive monogenic disorder characterized by mutations in a 230-kb gene located on chromosome 7, which encodes a 1480-amino-acid polypeptide known as the CF transmembrane conductance regulator (CFTR). Cystic fibrosis varies in severity from the absence of functional protein production to reduced synthesis, stability, or functionality of the CFTR protein [260]. Over 2100 distinct CFTR variants have been identified [261]. The extensive range of mutations complicates the identification of a universal drug for the treatment of all patients with CF.

Novel biologic treatment therapies, including protein replacement therapy utilizing DNA, protein, or mRNA (Fig. 3), present significant potential as a universal approach for patients with CF. Such therapies can enhance CFTR expression on the cell surface, increase the number of functional channels, or target alternative ion channels to mitigate the effects of CFTR impairment. Prior studies have shown that mRNA represents a promising approach for restoring sufficient CFTR expression in the lungs, as merely 10% of normal levels may prevent detrimental effects in CF [262]. However, many previous attempts to administer gene therapy for CF through inhalation have failed to achieve therapeutically relevant levels of expression. Lipid nanoparticles have been proven to be immunogenic with low airway epithelial transduction. For example, the mRNA-LNP system, MRT5005 from Translate Bio, has demonstrated a 1500-fold increase in transgenic expression over endogenous CFTR in preclinical models. However, when evaluated in patients with CF with class I/II CFTR mutations via nebulization, it did not result in a clear efficacy signal compared to placebo [132]. Several other mRNA therapies are currently being evaluated in phase I/II trials using, for example, RCT2100, ARCT-032, VX-522, 4D-710, BI 3720931, and SP-101 (Table 4, Fig. 3). These trials involve healthy volunteers or both healthy volunteers and patients with CF and patients ineligible for CFTR modulator therapy. Despite significant transduction and gene expression in preclinical animal species, translation of these gene therapies into a commercial product continues to remain a bottleneck. These can be primarily linked to several factors, such as low transduction efficiency, inadequate potency, CF mucosal and immune barriers, lack of cell specificity, disease severity, patient heterogenicity (wide phenotypic variation in patients homozygous for F508Δ) [263], formulation, and clinical study sample size, among others [262]. Additionally, future development of bioanalytical tools for directly assessing the expression and function of the targeted protein in target cells may aid in demonstrating mechanistic proof-of-concept and determining appropriate dosages for inhaled biologic therapies. While inhaled gene delivery specifically targets the lungs, the primary site of morbidity and mortality, it is inherently limited by CF’s multi-specific nature. Extrapulmonary manifestations, such as pancreatic insufficiency, CF-related diabetes, and gastrointestinal malignancies, remain unaddressed by local therapy and will represent the cause of growing morbidity as pulmonary outcomes improve [264]. Consequently, a dual therapeutic strategy by coupling localized inhaled interventions with systemic management is required to treat both pulmonary and extrapulmonary manifestations [265].

Other biologic therapies such as recombinant human deoxyribonuclease I, dornase alfa (Pulmozyme^®) are available as inhaled therapies to treat and manage CF. Dornase alfa hydrolyzes extracellular long-chain DNA in the sputum and reduces mucus viscosity and prevents airway infections, thereby improving lung function in patients with CF [266]. Additional detail on dornase alfa is provided in Sect. 3.1.1. Table 4 illustrates advancements in using biologics, specifically mRNA-LNPs, lentiviral, and adeno-associated virus -based gene therapies, to deliver therapeutic payloads to treat CF.

Primary Ciliary Dyskinesia

Primary ciliary dyskinesia, also called immotile-cilia syndrome, is a rare congenital disorder characterized by impaired mucociliary clearance. The condition affects approximately 1 in every 15,000–20,000 newborns worldwide [267]; however, the prevalence of undiagnosed cases indicates that the actual number can be higher [268]. Mutations in genes responsible for cilia structure and its components can lead to impaired ciliary movement [269–271]. More than 50 distinct genetic variants associated with primary ciliary dyskinesia have been identified [269]. As primary ciliary dyskinesia is caused by a genetic mutation, inhaled biologics that use viral or non-viral vectors, such as mRNA therapy that can induce functional ciliary proteins, could be a promising treatment option. As illustrated in Table 4 and Fig. 3, there are currently no approved treatments, and most ongoing studies are either in preclinical or early clinical stages, mainly focusing on dynein axonemal intermediate chain 1.

Alpha-1-Antitrypsin Deficiency (AATD)

Alpha-1-antitrypsin deficiency, commonly referred to as “genetic chronic obstructive pulmonary disease (COPD),” is a rare inherited genetic disorder marked by the misfolding and polymerization of the AAT protein in hepatocytes [272, 273]. More than 150 mutations have been documented, with the predominant defective alleles identified as protease inhibitor PiZ and PiS [147, 274, 275]. However, in dominant cases, AATD results from a G-to-A point mutation in the SERPINA1 gene, which governs the expression of the AAT protein [276].

Currently, there are five active trials within The Alpha 1 Project (TAP) aimed at developing novel treatments for the liver and lung manifestations of AAT deficiency [277]. INBRX-101, a recombinant human AAT-Fc fusion protein, is currently being developed as an IV-administered biologics for AATD (ClinicalTrials.gov identifier, NCT03815396, Table 4) [278]. Additionally, Beam-302 LNPs (Fig. 3) are now undergoing a phase I/II clinical trial (ClinicalTrials.gov identifier, NCT06389877) aimed at the PiZ mutation in adult patients with AATD-associated lung disease and/or liver disease. Furthermore, Wave Life Sciences (WVE-006) [see Fig. 3] is intended to correct the single base mutation in mRNA coded by the SERPINA1 Z allele, thereby enabling restoration and circulation of functional M-AAT protein (NCT06405633, Table 4). A well-established method for the treatment of AATD is through plasma AAT substitution by IV administration. However, the amount of AAT entering the lung via IV administration was only 2–3% [279]. A study by Kropp and colleagues demonstrated that nebulization of radiolabeled AAT did not degrade and maintained its biological activity. Additionally, patients with severe AATD well tolerated the treatment and showed significantly higher AAT deposition in the lungs post-inhalation compared with the IV delivery (14.6% vs 2%, respectively) [280]. In addition to local delivery, the inhalation route can offer other advantages to such individuals, such as convenience, reduced cost, improved patient compliance, and quality of life [281].

Surfactant Protein (SP) Deficiencies

Surfactant protein deficiencies are genetic disorders that impair the synthesis of pulmonary surfactants (PS). The PSs are composed of phospholipids and proteins: 80–85% phospholipids, 5–10% neutral lipids, and 8–10% protein (w/w). Within proteins, 5–6% is attributed to four specific SPs: SP-A, SP-B, SP-C, and SP-D, with respective distributions of 5.3%, 0.7%, 0.4%, and 0.6% [282, 283]. Congenital SP-B deficiency is a rare (~ 1/million) [284], fatal autosomal recessive disorder, primarily caused by loss-of-function mutations in bi-allelic SFTPB alleles [285]. More than 40 pathogenic variants have been found, all with poor prognosis and increased mortality within the first few months after birth. Moreover, the absence of SP-C caused by mutations in the SFTPC gene leads to lung inflammation and pneumonia. The ATP-binding cassette 3 protein is essential for surfactant production, facilitating the transport of phospholipids into lamellar bodies, where they assemble with SP-B and SP-C to form a functional PS [286]. Mutations in the SFTPB and SFTPC genes, which encode SPs B and C, as well as mutations in the ATP-binding cassette 3 gene, result in PS deficiency that contributes to childhood interstitial lung disease [286, 287].

The available therapeutic interventions include the administration of surfactants, corticosteroids, and other immunosuppressants, lung lavage, and, eventually, lung transplantation [286]. Gene or mRNA therapies could represent treatment options for congenital SP deficiencies. For example, a study has demonstrated the clinical potential of a gene therapy utilizing a viral vector-mediated approach (adeno-associated virus 6) in a mouse model with SP-B deficiency, prolonging its survival over 200 days [146]. The company, Ethris, utilized chemically modified mRNA technology, which has since shifted focus to other lung indications beyond SP-B deficiency (Table 4). Hence, there is a clear unmet medical need for a drug product to treat individuals with surfactant deficiencies.

Lung Cancer

Lung cancer remains one of the most difficult malignancies globally, responsible for nearly one in five cancer-related deaths and impacting millions of patients every year [145]. Biologic therapies have emerged as important in the management of lung cancer by targeting specific molecular pathways involved in tumor progression and immune evasion. These agents include inhibitors of cell surface receptors, immune checkpoint regulators, angiogenesis inhibitors, immune cell activators, cytokine-based therapies, and bispecific antibodies or antibody-drug conjugates. For example, antibodies or antibody-drug conjugates targeting antigens on lung tumor cells, such as delta-like ligand 3 on small cell lung cancer or trophoblast cell surface antigen 2 on non-small cell lung cancer, as recently approved by the FDA. Other well-known cellular targets such as programmed death ligand-1, programmed death-1, G protein-coupled receptors, and lymphocyte-activation gene 3 are primarily expressed on immune cells and play a central role in modulating immune responses against tumors, including lung cancer. Over the last decade, substantial progress has been made in the development of biological treatment with antibodies that inhibit these checkpoints to restore immune surveillance and enhance tumor clearance [288–294]. Table 5 summarizes FDA-approved immune checkpoint inhibitors used in lung cancer. Tumor-intrinsic targets expressed on lung epithelial tumor cells include human epidermal growth factor receptor 3, epidermal growth factor receptor, and carcinoembryonic antigen cell adhesion molecule 5, but systemically delivered biologics against broadly expressed epithelial targets have often been constrained by toxicity or limited efficacy [295, 296]. Other systemic immunotherapy modalities, including therapeutic vaccines, have shown acceptable safety and immune activation yet modest clinical benefit, reinforcing the need to improve how these agents are delivered to the lung tumor microenvironment [297].

Table 5.

Summary of approved biologics for the treatment of lung cancer

Product/PI	Biological class	Size (kDa)	Biological target	Indication	RoA	Year	Status	Company/research group	Ref.
Rybrevant® (amivantamab)	BsAb (EGFR-MET)	~148	EGFR and MET	EGFR ex20 mut. NSCLC	IV	2021	Appr.	Janssen	[415]
Yervoy® (ipilimumab)	mAb (anti-CTLA-4)	~148	CTLA-4	Adv. NSCLC	IV	2020	Appr.	BMS	[416]
Keytruda® (pembrolizumab)	mAb (anti-PD-1)	~149	PD-1	Adv. NSCLC	IV	2014	Appr.	Merck	[417]
Opdivo® (nivolumab)	mAb (anti-PD-1)	~146	PD-1	Adv. NSCLC	IV	2015	Appr.	BMS	[418]
Libtayo® (cemiplimab)	mAb (anti-PD-1)	~149	PD-1	Adv. NSCLC	IV	2021	Appr.	Regeneron	[419]
Tecentriq® (atezolizumab)	mAb (anti-PD-L1)	~145	PD-L1	Adv. NSCLC	IV	2016	Appr.	Roche	[420]
Imfinzi® (durvalumab )	mAb (anti-PD-L1)	~147	PD-L1	Unresectable stage III NSCLC	IV	2018	Appr.	AstraZeneca	[421]
Cyramza® (ramucirumab)	mAb (anti-VEGFR-2)	~147	VEGFR-2	Adv. NSCLC	IV	2014	Appr.	Eli Lilly	[422]
Alymsys® (bevacizumab-maly)	mAb (anti-VEGF-A)	~147	VEGFA	NSCLC in combination	IV	2022	Appr.	Amneal	[423]
Avastin® (bevacizumab)	mAb (anti-VEGF-A)	~147	VEGFA	NSCLC in combination	IV	2004	Appr.	Genentech	[424]
Mvasi® (bevacizumab-awwb)	mAb (anti-VEGF-A)	~147	VEGFA	NSCLC in combination	IV	2017	Appr.	Amgen	[425]
Zirabev® (bevacizumab-bvzr)	mAb (anti-VEGF-A)	~147	VEGFA	NSCLC in combination	IV	2019	Appr.	Pfizer	[426]
Portrazza® (necitumumab)	mAb (anti-EGFR)	~148	EGFR	Metastatic squamous NSCLC	IV	2015	Appr.	Eli Lilly	[427]
Imjudo® (tremelimumab-actl)	mAb (anti-CTLA-4)	~148	CTLA-4	NSCLC	IV	2022	Appr.	AstraZeneca	[428]
Enhertu® (fam-trastuzumab deruxtecan-nxki)	ADC	~148	HER2 (topoisomerase I)	HER2+ NSCLC	IV	2020	Appr.	Daiichi Sankyo/AstraZeneca	[429]
Imdelltra® (tarlatamab-dlle)	BiTE	~105	DLL3	SCLC + DLL3	IV	2024	Appr.	Amgen	[430]
Datroway® (datopotamab deruxtecan-dlnk)	ADC	~145	TROP2 protein	EGFR mut. Adv./Met. NSCLC	IV	2025	Appr.	Daiichi Sankyo	[431]

ADC antibody-drug conjugate, Adv. advanced, Appr. approved, BiTE bispecific T-cell engager, BMS Bristol-Myers Squibb, BsAb bispecific antibody, CTLA-4 cytotoxic T-lymphocyte-associated protein 4, DLL3 delta-like ligand 3, EGFR epidermal growth factor receptor, HER2 human epidermal growth factor receptor 2, IV intravenous, kDa kilodalton, LAG-3 lymphocyte-activation gene 3, mAb monoclonal antibody, Met. metastatic, mut. mutated, NSCLC non-small cell lung cancer, PD-1 programmed cell death protein 1, PD-L1 programmed death-ligand 1, RoA route of administration, SCLC small cell lung cancer, TROP2 trophoblast cell-surface antigen 2, VEGF-A vascular endothelial growth factor A, VEGFR-2 vascular endothelial growth factor receptor 2​​

A major limitation of systemic biologic administration is the lack of selectivity, as target antigens are often expressed on both tumors and healthy tissues. Checkpoint inhibitors and growth factor receptors are often present on immune or epithelial cells in non-malignant tissues, increasing the probability of systemic toxicity. This issue underscores the need for targeted delivery methods that enhance tumor specificity. High-dose IL-2 immunotherapy is known for systemic toxicity when administered intravenously; the majority of the patients had developed vascular leak syndrome, and 75% of patients had stopped the treatment for other side effects [298]. In contrast, inhaled IL-2 has shown reduced adverse events with mild toxicities such as cough and respiratory tract irritation in 16% of patients after receiving inhalation delivery. Most side effects were mild and localized to the respiratory tract [298]. A recent preclinical study of nebulized anti-programmed death-1 antibodies showed preferential accumulation in tumor-associated macrophages and CD103+ dendritic cells, thereby enhancing CD8+ T-cell-mediated antitumor activity compared with IV administration [299]. Table 6 summarizes the biologic therapeutics in development or discontinued for lung cancer.

Table 6.

Summary of biologic therapeutics in development or discontinued for lung cancer

Product/API	Biological class (size)	Biological target	Indication	Company/research group	RoA	Year	NCT no.	Status
Envafolimab (KN035)	Anti-PD-L1 mAb (~ 80 kDa)	PD-L1	Adv. NSCLC	Alphamab	SC	2015	NCT05414630	Ph. I
Tiragolumab	Anti-TIGIT mAb (~ 150 kDa)	T-cell and NK cells	Adv. NSCLC	Hoffmann-La Roche	IV	2022	NCT04294810	Ph. III
Carotuximab (TRC105)	Anti-CD105 (~ 144.8 kDa)	CD105	EGFR-mutated	Karen Reckamp, MD, MS	IV	2023	NCT05401110	Ph. I
MRNA-4157	mRNA Vac.	Neoantigens	Adv. NSCLC	Moderna/Merck	IM	2017	NCT03313778	Ph. I
BNT111	mRNA Vac.	Tumor antigens	Adv. NSCLC	BioNTech	IM	2024	NCT04526899	Ph. I/II
SurVaxM (SVN53-67/M57-KLH)	Peptide Vac. (~ 16.5 kDa)	Survivin	Lung tumor	Roswell Park Cancer Institute	SC	2025	NCT03879694	Ph. I
Patritumab deruxtecan	ADC (~ 158 kDa)	HER3	EGFR-mutated NSCLC	Daiichi Sankyoa	IV	2021	NCT04619004	Ph. II
Rovalpituzumab tesirine (Rova-T)	ADC (~ 145 kDa)	DLL3	SCLC	AbbVie	IV	2013	NCT03061812	Ph. III/Disc.
Tusamitamab ravtansine (SAR408701)	ADC (~ 150 kDa)	CEACAM5	CEACAM5+ adv. NSCLC	Sanofi	IV	2022	NCT05245071	Ph. II/Disc.
Datopotamab deruxtecan	ADC (158 kDa)	TROP2	TROP2+ adv. NSCLC	Daiichi Sankyo/AstraZeneca	IV	2021	NCT04619004	Ph. II
SHR-1701	BsAb (174.61 kDa)	PD-L1/TGF-βRII	Stage III NSCLC	Hengrui Medicine	IV	2022	NCT05179239	Ph. III/Disc.
LY2510924	Peptide (~ 1.19 kDa)	CXCR4	Adv. NSCLC	Eli Lilly	SC	2017	NCT02737072	Ph. I/Disc.
Bempegaldesleukin (NKTR-214)	Cytokine	IL pathway	Adv. NSCLC	Nektar/BMS	IV	2018	NCT03729245	Ph. III/Disc.
Nemvaleukin alfa	Cytokine (~ 20 kDa)	IL pathway	Adv. NSCLC	Alkermes	IV/SC	2019	NCT03861793	Ph. I/II
Ociperlimab + tislelizumab	mAb (~ 145 kDa)	TIGIT	Adv. NSCLC	BeiGene	IV	2021	NCT04746924	Ph. III
GSK1572932A	Recomb. Protein Vac. (~ 4–6 kDa)	MAGE-A3	Adv. NSCLC	GSK	IM	2020	NCT00480025	Ph. III/Disc.
Patritumab deruxtecan	ADC	HER3-DXd	Adv. NSCLC	Daiichi Sankyo	IV	2021	NCT04619004	Ph. II
BI1361849 (CV9202)	mRNA Vac.	CV9202 mRNA	Adv. NSCLC	Boehringer Ingelheim	ID	2016	NCT01915524	Ph. I/II

ADC antibody-drug conjugate, Adv. advanced, BMS Bristol-Myers Squibb, BsAb bispecific antibody, CD105 cluster of differentiation 105, CEACAM5 carcinoembryonic antigen-related cell adhesion molecule 5, CXCR4 C-X-C chemokine receptor type 4, Disc. discontinued, DLL3 delta-like ligand 3, HER3 human epidermal growth factor receptor 3, ID intradermal, IL-2 interleukin-2, IM intramuscular, IV intravenous, kDa kilodalton, mAb monoclonal antibody, MAGE-A3 melanoma-associated antigen 3, NCT national clinical trial, NSCLC non-small cell lung cancer, PD-L1 programmed death-ligand 1, Ph. phase; Recomb. Recombinant, Ref. reference, RoA route of administration, SC subcutaneous, SCLC small cell lung cancer, TGF-βRII transforming growth factor beta receptor II, TIGIT T-cell immunoreceptor with Ig and ITIM domains, TROP2 trophoblast cell-surface antigen 2, Vac. vaccine

Administering biologics through the inhalation route in lung cancer offers the possibility of depositing therapeutics precisely at the site of disease, potentially resulting in higher local concentrations and substantially lower systemic exposure. In preclinical models, this hypothesis has shown promise [300]. Using a C57BL/6 murine model of B16 melanoma lung metastases, the study demonstrated that inhaled delivery of 15 mg of polyinosinic-polycytidylic acid, 1.5 mg of CpG-oligodeoxynucleotides, and 4×10⁶ IU of IFN-α every 72 hours over 2weeks resulted in up to a 79% reduction in metastatic burden and significantly enhanced natural killer cell activation, marked by elevated CD69 and natural killer group 2 member D expression, providing quantitative evidence that localized pulmonary immunotherapy can overcome the tumor penetration barriers and off-target toxicities associated with systemic biologic therapies [300]. It has been extensively discussed in this study that inhaled drug delivery offers a promising approach for lung cancer by increasing local drug concentration while minimizing systemic side effects [301]. In a rat model of orthotopic lung tumors, inhaled carboplatin at 10 mg/kg resulted in lung tissue platinum levels of 11.96 ± 2.50 µg/g, compared with 3.36 ± 0.68 µg/g following IV administration. The inhaled route also delivered 0.45 ± 0.08 µg/g to mediastinal lymph nodes and 0.28 ± 0.05 µg/mL to plasma, showing effective regional and systemic distribution with reduced off-target exposure [302]. These results highlight the efficiency of inhalation for targeted drug delivery in lung cancer. Based on this evidence, inhaled biologics could offer similar advantages, but further improvements in formulation and delivery systems are needed. Continued clinical studies will be essential to confirm their safety, effectiveness, and long-term benefit in patients.

Systemic biologics have reshaped lung cancer care, yet they still help only a fraction of patients and carry risks of immune and other off-target toxicities. Increasing pulmonary exposure of biologics while limiting systemic effects is an obvious approach facilitated by inhaled therapies. Preclinical studies have confirmed higher lung concentrations, reduced metastatic burden in some models, and more active local immunity. Converting these preclinical signals into the clinic will require formulations that stay stable during aerosolization, payloads and doses patients can realistically complete, and trials that document safety and benefit with rigor. The near absence of late-stage clinical trials of inhaled biologics in lung cancer is more likely to reflect developmental and regulatory hurdles, including challenges in achieving stable aerosolizable formulations and in designing feasible dosing regimens, than intrinsic unsuitability of the pulmonary route, as preclinical studies with inhaled cytokines, mRNA-loaded exosomes, and inhaled anti-programmed death-1 antibodies have shown enhanced local immune activation, tumor regression, and reduced systemic toxicity compared with IV administration. If those pieces come together, inhaled biologics could broaden the reach of immunotherapy while easing toxicity.

Pulmonary Hypertension

Pulmonary hypertension (PH) is characterized by elevated blood pressure in the pulmonary arteries, which can be caused by heart disease, lung disease, genetic factors, or other etiologies. Current treatment for pulmonary arterial hypertension (PAH) focuses on the inhibition of vascular remodeling using endothelin receptor antagonists, phosphodiesterase-5 inhibitors, and prostacyclin analogs, often administered as a combination therapy to enhance efficacy [303].

Several biologic drugs targeting key molecular pathways are under investigation or have been recently approved for treating PAH. Notably, sotatercept (Winrevair^®), KER-012, HS135, and PF-07868489 target the transforming growth factor-β superfamily and bone morphogenetic protein signaling axis, aiming to balance pro- and anti-proliferative pathways that trigger vascular remodeling in PAH. While these biologics can be effective in decreasing the disease progression, their current formulations are administered subcutaneously and intravenously, and none is available as an inhaled therapy [304–308]. The pulmonary route is attractive for PH owing to its capability of delivering drugs directly to the pulmonary vasculature, increasing local concentrations and minimizing systemic side effects [309]. However, the large molecular size and complex structure of fusion proteins and mAbs such as sotatercept (78 kDa), KER-012, and PF-07868489 can present significant formulation and delivery challenges for inhaled administration [305, 310].

Among biologics with anti-inflammatory and vasoactive targets, tocilizumab and satralizumab (IL-6 inhibitors), anakinra (IL-1 receptor antagonist), getagozumab (endothelin receptor A antagonist), and pemziviptadil (vasoactive intestinal peptide analog) [Table 7] are all currently administered via a SC or IV injection. The IL-1 targets in PH, which include endothelial and immune cells within the lung, may be more readily accessible via inhalation [311]. The molecules’ smaller size may allow for more rapid trans-epithelial absorption by specific transporters or via tight junctions and reduce susceptibility to macrophage clearance, while larger proteins have longer residence times in the alveolar luminal space, making them prone to degradation or subject to macrophage clearance [312, 313]. A study demonstrated the viability of developing spray-dried DPI formulations of anakinra, maintaining its structural integrity and pharmacological efficacy in preclinical models [314]. Peptide/fusion proteins, Winrevair™ (sotatercept-csrk), KER-012, and HS135 may be suitable candidates to repurpose for inhalation owing to their smaller size than full-length antibodies, lung-specific mechanism of action, and moderate/high potency. Winrevair (sotatercept) is administered subcutaneously; thus, the therapeutic dose is likely low to moderate, which can be achieved by inhalation to achieve sufficient local concentration in the lungs. This is further supported by a recent study that demonstrated that sotatercept can be nebulized to generate fine aerosol droplets within the respirable size range (< 5 µm) for PH [315]. However, these findings are still preliminary and do not include in vivo studies.

Table 7.

Summary of biologic therapeutics approved or in development or discontinued for pulmonary arterial hypertension

Product/API	Biological class (size)	Biological target	Company/research group	RoA	Year	Status	NCT no.	Ref.
Winrevair™ (sotatercept-csrk)	mAb (78 kDa)	Activin A and other TGF-β ly ligands	Merck	SC	2024	Appr.	NCT03738150 NCT03496207 NCT04576988 NCT05587712 NCT04811092	[308]
Tocilizumab (Actemra®)	mAb (148 kDa)	IL-6R	Papworth Hospital	IV	2018	Ph. II/Disc.	NCT02676947	[432]
Anakinra (Kineret®)	rhIL-1Ra (17.3 kDa)	IL-1R	Virginia Commonwealth Univ.	SC	2018	Ph. I/II/ Disc.	NCT03057028 NCT01479010	[433]
Getagozumab (GMA301)	mAb	ET-A	Gmax Biopharm	IV	2021	Ph. I/Disc.	NCT04503733 NCT04505137	[434]
PF-07868489	Rcomb. protein	BMP9	Pfizer	SC	2023	Ph. I/II	NCT06137742	[435]
Satralizumab (Enspryng®)	mAb (143 kDa)	IL-6R	Int.Univ. of Health and Welfare	SC	2022	Ph. II	NCT05679570	[436]
Cibotercept (KER-012)	Fusion protein	Activins and GDFs	Keros Therapeutics	SC	2023	Ph. II/Disc.	NCT05975905	[307, 437]
HS135	mAb	Activins and GDFs	35Pharma	SC	2024	Ph. Ib/Disc.	NCT06493461 NCT06581159	[304, 438]
Pemziviptadil	VIP analog	VPAC2	PhaseBio	SC	2018	Ph. II/Disc.	NCT03795428 NCT03315507 NCT03556020	[439]
GSK2586881	rhACE2	ACE2	GSK	IV	2017	Ph. II/Disc.	NCT03177603	[440]
Adipose derived MSCs	Cell therapy	Immunomodulation	Liaocheng People’s Hospital	IV	2019	Ph. I/II/Disc.	NCT04055415	[441]
Cardiac progenitor cells	Cell therapy	Tissue repair	Cedars-Sinai Medical Center	IV	2017	Ph. I	NCT03145298	[442, 443]
PLX-PAD	Cell therapy	Tissue repair	United Therapeutics	IV	2013	Ph. I	NCT01795950	[444, 445]

ACE2 angiotensin-converting enzyme 2, Appr. approved, BMP9 bone morphogenetic protein 9, Disc. discontinued, ET_A endothelin receptor type A, GDF growth differentiation factor, IL-1R interleukin-1 receptor, IL-6R interleukin-6 receptor, IV intravenous, kDa kilodalton, mAb monoclonal antibody, MSC mesenchymal stem cell, NCT national clinical trial, PAH pulmonary arterial hypertension, Ph. phase, Recomb. recombinant, Refs. references, rhACE2 recombinant human angiotensin-converting enzyme 2, rhIL-1Ra recombinant human interleukin-1 receptor antagonist, RoA route of administration, SC subcutaneous, TGF-β transforming growth factor beta, VIP vasoactive intestinal peptide, VPAC2 vasoactive intestinal peptide receptor 2

Getagozumab (GMA301) [Table 7] is an mAb against endothelin receptor A, a key mediator of vasoconstriction and vascular proliferation in PAH. Additionally, pemziviptadil, a vasoactive intestinal peptide analog, targets the vasoactive intestinal peptide type II receptor to promote vasodilation and inhibit smooth muscle proliferation. These biologics represent a change from traditional vasodilator therapies toward disease-modifying strategies that address the underlying molecular drivers of PH, with the purpose of offering targeted approaches when treating PAH [305, 306, 310].

Idiopathic Pulmonary Fibrosis (IPF)

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by irreversible scarring of lung tissue, leading to declining pulmonary function and symptoms such as dyspnea, dry cough, and reduced exercise tolerance [316]. Without treatment, IPF carries a median survival of 3–4 years, with acute exacerbations, characterized by rapid respiratory decline, contributing significantly to mortality [317].

Biologic therapies for IPF mainly target profibrotic pathways such as connective tissue growth factor (CTGF/CCN2). PRS-220, an Anticalin protein (Table 8), which inhibits CTGF/CCN2, is administered through a vibrating mesh nebulizer. It achieves 12-fold higher fibrotic tissue exposure compared with systemic administration of pamrevlumab, while minimizing its plasma circulation. Preclinical studies demonstrate its ability to penetrate fibrotic lung regions, reduce collagen deposition in silica-induced fibrosis models, and maintain stability after nebulization [318]. This inhaled biologic is currently in phase I clinical trials, (NCT05473533) as a potential inhaled CTGF inhibitor for IPF treatment (Table 8). Additionally, the aerosol performance of the nebulized formulation (i.e., MMAD: 3.7–4.2 µm and FPF: 65-77%) allows for deep lung deposition [318].

Table 8.

Summary of biologic therapeutics in development or discontinued for idiopathic pulmonary fibrosis

Product/API	Biological class (size)	Biological target	Company/research group	RoA	Year	Status	Clinical Trial no.	Ref.
Pamrevlumab	mAb	CTGF	FibroGen	IV	2023	Ph. III/Disc.	NCT04419558 NCT03955146 NCT01890265	[446, 447]
Atezolizumab	mAb (~150 kDa)	PD-L1	Cedars-Sinai Medical Center	IV	2022	Ph. I	NCT05515627	[448]
Axatilimab	mAb (~150 kDa)	Colony-stimulating factor-1 receptor	Syndax Pharmaceuticals	IV	Ongoing	Ph. II	NCT06132256	[449, 450]
TRK-250	siRNA	TGF-β1 protein	Toray Industries	Inhalation	2022	Ph. I	NCT03727802	[451]
Garadacimab	mAb	Factor XII	CSL Behring	SC	2024	Ph. II	NCT05130970	[452]
SHR-1906	mAb	CTGF	Hengrui	SC	2024	Ph. II	NCT05722964	[453]
PRS-220	Anticalin protein (~18–20 kDa)	CTGF/CCN2	Pieris Australia Pty Ltd	Inhalation (nebulizer)	2023	Ph. I/Disc.	NCT05473533	[318, 454]
Simtuzumab	mAb (~145.5 kDa)	LOXL2	Gilead Sciences	SC	2016	Ph. II/Disc.	NCT01769196 NCT01759511	[319, 455]
Zinpentraxin alfa (PRM-151)	rhPTX-2 (~116 kDa)	Macrophage modulation	Hoffmann-La Roche	IV	2023	Ph. III/Disc.	NCT04594707 NCT04552899	[456, 457]
Human umbilical cord MSCs	Cell therapy	Tissue repair/immunomodulation	Shanghai Life Science & Technology	IV	2022	Ph. I	NCT05468502	[458]
REGEND001 (P63+ cells)	Cell therapy	Tissue repair/regeneration	Regend Therapeutics	IV	2023	Ph. II	NCT05657184 NCT06081621	[324, 459]
VUM02 (human umbilical cord tissue-derived MSCs)	Cell therapy	Tissue repair/imunomodulation	Wuhan Optics Valley Vcanbiopharma Co., Ltd.	IV	Ongoing	Ph. I	NCT06230822	[460]

CTGF connective tissue growth factor, Disc. discontinued, FXII factor XII, Inh. inhalation, IV intravenous, kDa kilodalton, LOXL2 lysyl oxidase-like 2, mAb monoclonal antibody, MSCs mesenchymal stem cells, NCT national clinical trial, PD-L1 programmed death-ligand 1, Ph. phase, Refs. references, rhPTX-2 recombinant human pentraxin-2, RoA route of administration, SC subcutaneous, siRNA small interfering RNA, TGF-β1 transforming growth factor-beta 1, UC-MSC umbilical cord mesenchymal stem cell

Other biologics in development include pamrevlumab (anti-CTGF mAb) and SHR-1906 (anti-CTGF mAb), though neither is currently formulated for inhalation (Table 8). Simtuzumab (anti-LOXL2 mAb, SC) and atezolizumab (anti-PD-L1, IV) represent alternative systemic approaches that target matrix stiffness and immune checkpoints, respectively [319, 320]. While these drugs regulate disease-relevant pathways, their administration routes lack the pulmonary targeting of PRS-220. Additionally, unlike these systemic therapies, inhaled PRS-220 delivers CTGF/CCN2 inhibition directly to the lungs, addressing the limitations of IV pamrevlumab observed in failed phase III trials [321]. Although Pieris discontinued PRS-220 after phase I trials, its small size (~ 18–20 kDa), stable tertiary structure, high-affinity specificity, and reduced off-target effects make it an attractive candidate for inhaled therapy in IPF [99, 318]. A recent study by Neiens et al. demonstrated that PRS-220 maintains structural integrity during vibrating mesh nebulization and achieves efficient aerosol properties (FPF 65–77%, MMAD 3.7–4.2 µm) that lead to a high lung distribution with enhanced tissue exposure and low plasma levels [318]. This highlights the potential of inhaled PRS-220 for IPF inhalation therapy, offering localized fibrotic regulation without compromising systemic safety.

Cell-based therapies for IPF may restore damaged lung tissue and reduce/correct fibrosis. The general mechanism involves delivering stem or progenitor cells, such as mesenchymal stem cells, alveolar epithelial type II cells, or airway basal progenitor cells, directly into the lungs, where they can engraft, differentiate, and secrete bioactive factors. These cells help with lung repair by replacing lost or dysfunctional alveolar epithelial cells, modulating immune responses, reducing inflammation, and inhibiting the deposition of the extracellular matrix [322, 323]. For example, P63+ lung progenitor cells can differentiate into the functional alveolar epithelium, supporting the regeneration of gas-exchanging surfaces and potentially reversing structural damage in the lung [324]. While early clinical studies with REGEND001 (Table 8) suggest improvements in gas exchange and stabilization of lung function, the potential of cell therapies in IPF relies on the capability to address the loss of regenerative capacity and to regulate the fibrotic microenvironment, offering a disease-modifying strategy beyond what current antifibrotic drugs can achieve [323, 325].

Conclusions

The prospects for inhaled biologics for local lung diseases appear promising. Many candidate disease targets have already been identified, and effective treatments have been developed for these by alternative routes (systemic). When systemic administration is limited by poor targeting, off-target side effects, or reduced patient adherence and acceptability, these biologics may represent strong candidates for inhaled delivery. While our analysis demonstrates a strong pipeline of inhaled biologics at the clinical stage, important cautionary lessons can be drawn from prior failures. Discontinued programs illustrate that a multitude of factors beyond biologic aerosol stability and preclinical proof of concept exist that may result in failure. As understanding of chemical, physical, and biological barriers to pulmonary delivery continues to expand, the risk of failure can be reduced. We believe that transformative and impactful therapies can be achieved through the development of biologics as inhaled products.

Funding

No external funding was used in the preparation of this article.

Declarations

Conflicts of interest/competing interests

Hugh D.C. Smyth, an author of this paper, consults for and has equity ownership in Via Therapeutics, Nob Hill Therapeutics, and Cloxero Therapeutics on inhaled product development. The terms of this arrangement have been reviewed and approved by the University of Texas at Austin in accordance with its policy on objectivity in research. Bhupendra Raj Giri, Jamie Erin Spahn, Rawidh Alsaidalani, Beatriz Behrend-Keim, Mohammed Bhia, Jailen Haley Doyle, Matthew Joseph Herpin, Rex Corbin Moore, Nila Sangeetha Murali, and Arifenur Safak have no conflicts of interest that are directly relevant to the content of this article.

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The authors affirm that their work is original, has not been previously published, and is not under consideration for publication elsewhere.

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Not applicable. This is a review article and does not contain any new data. All data discussed in this article are sourced from previously published research, and the original sources are cited in the text.

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Authors’ contributions

All authors contributed to the writing and revision of this manuscript. The conceptualization and design of the review were led by HS, who also provided supervision. Project administration was handled by HS and JS. The tables and figures were prepared by BG, AS, NM, JD, RA, and BB, JS, BG, and RM were also responsible for managing the references and overall manuscript formatting. All authors read and approved the final submitted version.