Strategies for overcoming the biological barriers associated with the administration of inhaled monoclonal antibodies for lung diseases

ABSTRACT

Introduction

Monoclonal antibodies (mAbs) should be administered by inhalation rather than parenterally to improve their efficiency in lung diseases. However, the pulmonary administration of mAbs in terms of aerosol technology and the formulation for inhalation is difficult.

Areas covered

The feasible or suitable strategies for overcoming the barriers associated with administering mAbs are described.

Expert opinion

Providing mAbs via inhalation to individuals with lung disorders is still difficult. However, inhalation is a desirable method for mAb delivery. Inhaled mAb production needs to be well thought out. The illness, the patient group(s), the therapeutic molecule selected, its interaction with the biological barriers in the lungs, the formulation, excipients, and administration systems must all be thoroughly investigated. Therefore, to create inhaled mAbs that are stable and efficacious, it will be essential to thoroughly examine the problems linked to instability and protein aggregation. More excipients will also need to be manufactured, expanding the range of formulation design choices. Another crucial requirement is for novel carriers for topical delivery to the lungs since carriers might significantly enhance proteins’ stability and pharmacokinetic profile.

KEYWORDS:

• Monoclonal antibodies
• inhalation route
• biological barriers
• inhalers
• formulation

1. Introduction

The treatment of severe asthma has dramatically benefited from the availability of several monoclonal antibodies (mAbs) [1], and there is also growing evidence that mAbs, which are mostly immunoglobulins G (IgG) with a molecular weight of ∼150 kDa, might be the future of personalized COPD treatment [2]. Furthermore, mAb therapy is considered an effective way to treat and manage respiratory infections [3], and some new classes of mAbs are emerging as promising treatments for lung cancer [4,5]. However, mAbs are large molecules delivered systemically, and only a small portion of the supplied amount reaches the lung [6]. Typically, the mAb concentration in the lung is 500–2000 times lower than the concentration in the systemic circulation [7].

The consequence of this substantial difference between the mAb concentration in the lung and that in the bloodstream is that it is necessary to administer massive doses systemically to achieve therapeutically effective concentrations in the lung with the risk of exposing the rest of the body to possible toxicity and significant adverse effects [7]. Indeed, injectable mAbs are often associated with detrimental effects such as allergic, immunological and other undesirable responses [8].

In contrast, direct administration of the drug to the airways by inhalation ensures that a higher percentage of the dosed mAb reaches the target site in the lung. This means that a much lower dose can be administered by inhalation than if the systemic route is used, with the advantage of achieving therapeutic equivalence to this considerably higher dose [7].

A xenogeneic murine model documented that the pulmonary route enhances mAb concentration at the target while limiting its passage into the bloodstream and aerosolised mAb produces an effective therapeutic response [9]. Furthermore, administering mAbs via the airways generates persistent and consistent accumulation in the lungs of molecules that preserve their physical and immunological features, at least in the experimental context in non-human primates [10]. The dual interleukin (IL)-4/IL-13 antagonist, pitrakinra, achieved a better therapeutic response when given via the pulmonary route at nominal b.i.d. doses of 3–100 mg than when supplied systemically at b.i.d. subcutaneously doses of 0.05–0.5 mg/kg in an Ascaris suum-sensitive cynomolgus monkey model of asthma [11].

Because it is usually assumed that the concentration of a biomolecule at its site of action influences its pulmonary efficacy, inhalation may also allow for a faster initiation of action (within minutes to hours) on respiratory system organs compared to other routes of administration (days) [12]. Furthermore, mAbs enter the bloodstream slowly and in small amounts after aerosol delivery [13].

This pharmacokinetic (PK)/pharmacodynamic (PD) behavior suggests that mAbs should be administered by inhalation to reduce the risk of adverse events related to the high systemic bioavailability and increase the therapeutic efficacy associated with the local concentration of the biologic [10,14].

However, the pulmonary administration of mAbs in terms of aerosol technology and the formulation for inhalation is problematic because, as already mentioned, they are macromolecules [15]. Several anatomical, physiological, and immunological factors influence the effectiveness of inhaled biologics [16]. These factors include the highly branched airway structure, mucociliary clearance, macrophage uptake, pulmonary surfactant, alveolar epithelial permeation, and enzymatic metabolism. The post-inhalation cough reflex, low delivery efficiency to specific lung target regions, and the inability of inhaled therapies with intracellular targets to sufficiently penetrate cellular phospholipid membranes and maintain therapeutic concentrations at the intracellular level are other biological barriers that may limit the use of inhaled mAbs [17]. All this explains why administering consistent, homogeneous, and safe amounts of inhaled particles remains a critical issue [18].

In this article, we describe the feasible or suitable strategies for overcoming the biological barriers associated with administering mAbs by the inhalation route, focusing on those biologics that could be used in treating severe asthma.

2. Barriers to inhaled drug therapy

The pulmonary deposition of any mAb is conditioned by the fact that the lungs comprise a complex network of interconnected branched airways with variable dimensions and specialized morphological structures, including conducting airways, bronchioles and alveoli [12]. Bronchial epithelial cells and the epithelial layer goblet cells are found in the bronchioles. They present mucus on the surface. Dendritic cells, smooth muscle cells, lymphocytes and fibroblasts are in the lamina propria. Alveolar epithelial cells, coated with surfactant, cover the surface of the alveoli. They are essential in the immunological response of the lung in addition to functioning as a physical barrier. Although alveolar epithelial cells I and alveolar epithelial cells II are equally required for airway defense mechanisms [19], alveolar epithelial cells II are more immunologically active [20]. Alveolar epithelial cells generate cytokines and chemokines responsible for immune cell activation and differentiation and act as antigen-presenting cells for particular T cells [19]. Macrophages are also implicated in several chronic and acute diseases and contribute to normal physiological activities of the lung [21] Lower respiratory tract macrophages are classified into alveolar macrophages and interstitial macrophages [22]. Alveolar macrophages patrol the surfactant layer on the surface. They are thought to act as an immunological barrier to recombinant Abs since they are known to play a significant part in IgG transport, biodistribution, or metabolism [23,24]. Endothelial cells lie below the alveolar epithelium.

The delivery efficiency to specific target lung regions is greatly influenced by particle size [25,26]. Gravity deposition places particles between 1 to 5 μm in diameter in the middle and distal ends of the lung, which is thought to be the ideal aerodynamic diameter. Aerodynamic diameters of less than 5 μm are needed to carry medications to the whole lung. In contrast, less than 3 μm of aerodynamic diameters are required to deliver medicines to the alveolar epithelium. Particles larger than 10 µm are deposited in the oropharyngeal region, and those smaller than 1 µm are exhaled during current respiration [16].

However, there is still uncertainty about the best location in the lung for the deposition of inhaled biologicals [16]. The optimal place for IgG absorption may likely be in the upper and middle airways, where the neonatal fragment crystallizable (Fc) receptor, also termed Brambell receptor or neonatal Fc receptor (FcRn), an IgG-binding receptor that is critical in the recycling of IgG into the circulation [27], is more highly expressed in their epithelial cells than in the alveolar epithelial cells [28].

When drug particles are deposited in the lungs, they interact with the fluids composing the lining layer, a barrier to protect the underlying epithelium. The mucosal fluid sink for dissolution is the lung lining fluid with a small total volume (15–70 mL in humans). However, the thickness of the lining layer, and consequently the volume of the lung lining fluid, differs between the central to the peripheral lungs [29]. Particles deposited in the upper airways might dissolve faster due to a larger solid-liquid interface than in the alveoli [30]. The dissolution of particles is critical because the aerosol particles must dissolve to release the active drug for subsequent pharmacological action and absorption. However, focusing only on the trachea-bronchial or the alveolar areas is very challenging due to the simultaneous influence on the distribution of the inhaled drug exerted by airway constriction, excess mucus, and/or edema that can lower the delivery of the mAb to the lung target regions [17]. The mucus layer of conducting airways is 5 to 55 µm thick, creating a mesh with hydrophobic and negatively charged areas [31].

In any case, imaging offers a great deal of promise to help improve our knowledge of pulmonary deposition of mAbs by providing quantitative, real-time biodistribution data [32]. The destiny of cetuximab, a mAb that binds to the epidermal growth factor receptors with high affinity, was studied in various animal models and imaging techniques after aerosol particles were deposited in the lungs [10]. This mAb accumulated rapidly in lung tissue. The accumulation of cetuximab in the bronchial and alveolar area following administration into the airways was the main difference compared to intravenous administration, following which there was no evidence of cetuximab in the normal lung. In the healthy mouse lung, cetuximab was found in immune cells and the lumen around bronchial epithelial cells.

Good lung deposition is only useful if the mAb can resist the clearance mechanisms of the lung. Three mechanisms are involved in the clearance of inhaled mAbs [33]. The first mechanism is mucociliary clearance, the second is macrophage uptake (the predominant clearance mechanism in the alveoli), which is important for large proteins with moderate absorption rates, such as IgG, that might remain in the alveolar space for hours providing enough residence time for macrophage uptake [16], and the third is absorption into the systemic circulation.

When designing a medicine for local delivery in the lung, a residence period in the lung of at least a few hours is ideal, and minimal absorption into circulation is typically chosen to have less systemic adverse effects [34]. However, inhaled mAbs are quickly removed from the airways [35]. This rapid removal seems mostly unaffected by antibody type [36] and still be lower for higher molecular weight molecules, which are, therefore, likely to be a better alternative for using inhaled mAbs to treat local diseases [34].

Pulmonary surfactant was thought to be a barrier to medication delivery in the peripheral lung, where mucus is absent in healthy individuals [37], and large proteins may interact with its components, causing aggregation and macrophage destruction [38]. However, natural pulmonary surfactant and its most abundant phospholipid, dipalmitoylphosphatidylcholine, are potential peptide and protein absorption enhancers [39].

The membrane of epithelial cells below the acellular layers (mucus, surfactant) is the initial barrier that inhibits biologically active molecules from being transported directly into the cell, preventing effective intracellular delivery [40]. MAbs can enter the epithelium by transcytosis (through the cells, such as passive diffusion or carrier-mediated), paracellular (between cells), and large transient pores in the epithelial layer. The paracellular pathway can only absorb molecules of a specific size. The intercellular tight junctions of respiratory epithelium have a 0.5–0.9 nm cutoff, but the underlying endothelium permits molecules up to 6.5–7.5 nm in diameter to pass through [35]. Cellular uptake is affected by the interaction between the chemical entity and the cell membrane. Evidence shows that FcRn-transcytosis is important in transporting IgG and Fc-Fusion proteins through the lung epithelium [16,27]. However, mAbs are absorbed very slowly with limited bioavailability [41] because their size influences their transport across the respiratory epithelium. Poor permeability restricts their distribution to the appropriate sites and, as a result, their bioavailability for treatment. Without external aid, cellular absorption of macromolecules and drug carriers is inefficient.

The pulmonary epithelium is the main barrier for inhaled mAbs to act systemically [35]. The 20–25 nm thick anionic-charged basement membrane is positioned under the respiratory epithelium but is not a substantial barrier to the transit of the biologic. However, biologics can bind to other macromolecules in the interstitium below the basement membrane, be phagocytosed by interstitial macrophages, or be transferred into lymphatic arteries [42].

Finally, the effectiveness of aerosolised mAbs may be compromised because of the activity of several proteases and peptidases that are present in the airspace and epithelial cells and increase when the lung is inflamed [43]. However, proteins having greater tertiary and quaternary structures may be able to suppress peptidase hydrolysis [44]. The degradation of proteins plays only a minor role, as more than 95% of them are absorbed intact from the lung periphery [45]. Nevertheless, most lung diseases probably cause increased biological deterioration [46]. Another troubling finding is that the rise in inflammatory cells (mostly neutrophilic granulocytes and macrophages) might result in immunological reactions directed against the therapeutic proteins [46].

Inhalation therapies may induce the onset of coughing mainly by activation of afferent nerves with stimulation of extrapulmonary Widdicombe cough receptors and/or the intrapulmonary bronchopulmonary C-fibers, due to extreme pH, mechanical stimulation of environmental irritants or extreme local osmolarity of airway surface fluid in the vicinity of dissolving inhaled particles [17]. Post-inhalation cough is more frequent in women [47] and pediatric patients [48] and, in any case, in young patients compared to elderly patients [49].

3. Aerosol technology to deliver mAbs

The pulmonary delivery of mAbs is challenging regarding aerosol technology and the formulation of biological agents for inhalation [9]. Like other therapeutic proteins, mAbs can suffer structural changes, aggregation, oxidation, deamidation, or glycation, reducing biological activity and making them immunogenic [25,41]. The tertiary structure may be impaired and elicit antidrug-antibody production that may neutralize the Ab and lead to side effects, such as hypersensitivity and anaphylactic reactions. In any case, because of the rate of retention of macromolecules in the lungs following administration of the mAb via the airways, it is critical to address the pathophysiological mechanisms of action of the antigen and to target only antigens active within the lung to produce a therapeutic impact [50].

The device must efficiently and consistently deposit a pharmacologically active and safe mAb in the lung area of interest. Several inhalation devices administer mAb directly to the airways. The three most often used inhalation devices are nebulizers (jet, ultrasonic, and mesh), dry powder inhalers (DPIs), and metered-dose inhalers (MDIs).

Most inhaled mAbs in clinical development have been developed as liquids for nebulization [15]. Nebulized formulations avoid protein drying and can provide high dosages [51]. The efficient and reliable deposition of sufficient numbers of particles in the lung region of interest is required for successful inhalation therapy [25].

However, the air-liquid interface quickly grows when the bulk liquid is atomized [52]. It has been calculated that nebulizing a 10 mL solution with a jet nebulizer produces an air-liquid interface larger than 24 m² over 20 min [53]. However, the estimate is a gross underestimate of the true surface area generated. If all fluid were atomized into 10 µm droplets, the surface exposure would rise to at least 35%. It has been estimated that some 1500 m² of surface will be produced over 10 min with particles having 10 µm median droplet size and an aspiration rate of 100 ml/min [54]. The extensive formation of air-liquid interface during nebulization significantly negatively influences protein stability with the possibility of protein adsorption, unfolding, or aggregation [52].

Furthermore, prolonged storage of proteins in liquid solutions can result in protein instability and conformational changes via degradation pathways (e.g. deamination and hydrolysis), temperature and pH changes, and aggregation (via aqueous carrier agitation) [55].

Mesh nebulizers are now commonly utilized for therapeutic protein administration [56]. They employ a perforated membrane (mesh) or a plate with numerous pores to create an aerosol [57]. Mesh nebulizers enable the administration of large quantities of medication (typically necessary for mAbs) while preserving the molecular integrity of the proteins by being less strict regarding chemical and physical limitations [50]. Mesh nebulizers are classified into two types based on their aerosol-generating principle [58,59]. Active mesh nebulizers are made of a thin perforated membrane (mesh) containing microscopic holes or plate with multiple apertures. A vibrating element (piezo crystal) pushes the drug through the narrow holes in the membrane upon the application of electric current. This generates an aerosol composed of tiny droplets. Passive mesh nebulizers, suitable for low fill volumes, use a transducer horn that induces passive vibrations in the perforated plate with 6000 tapered holes to produce an aerosol. However, the holes on the mesh have been known to clog readily due to drug particle precipitation and crystallization [58]. This obstruction causes inefficiency in delivering aerosol medication to patients, yet it might be difficult to detect and remove the blockage. However, surfactants in Ab formulations can preserve mAb molecular integrity and pharmacological activity during vibrating-mesh nebulization [25].

DPIs are a more patient-friendly delivery approach because of their short administration time, manageability, powder formulations, and inherent stability and shelf-life advantages. However, the traditional lactose-based formulation cannot deliver mAbs as it requires potent, low-dose therapeutic agents [60]. Spray-dried particles with stabilizing substances in each particle provide high dosage administration relevant to Ab treatments [61]. However, the particle engineering methods necessary to generate respirable dry powders for DPI administration entail several potential stressors for mAbs [62]. Pure Ab solutions agglomerate substantially during this procedure [63]. Techniques like spray drying, spray freeze drying, and thin film freezing unavoidably expose molecules to various types of atomization- or drying-induced stresses like shearing stress in the nozzle, heat stress during drying, and contaminant surface adsorption [64,65], and, in any case, denaturation remains a concern [66]. Protecting and stabilizing excipients (usually disaccharides such as trehalose, which has a high glass transition temperature, low hygroscopicity, and great water replacement efficacy, sucrose, amino acids and surfactants) and controlling drying conditions are critical to minimizing molecular damage [62,66].

There is the possibility of cohesive and adhesive forces appearing due to interactions between individual particles when dispensing spray-dried particles, the surface of which may be a heterogeneous mixture of antibody and excipient(s) [67]. Typically, these forces are inadequate to induce instability during aerosolisation. However, the particle composition employed to stabilize the mAb may need to be optimized to allow dispersion under the flow parameters imposed by the device [68]. In addition to the common concerns about the chemical (structural and functional) stability of the Ab, the physical stability of the powder must be considered, as variations in particle size or delivered dose impact potential safety and efficacy outcomes [68]. Furthermore, the possible infiltration of residues or moisture into DPI formulations is a source of instability [66]. Nonetheless, the main possible advantage of the solid dose form is the superior chemical and colloidal stability of the protein, which allows formulations to have a longer shelf-life and avoid cold-chain logistics [69].

There is currently no approved pMDI product for inhaled biologics therapy. MAbs are generally hydrophilic and poorly soluble in nonpolar hydrofluoroalkane (HFA) propellants [69]. Furthermore, the denaturation of mAbs when interacting with the propellants limits the use of pMDIs for mAbs administration [67]. Poor solubility of biologics in the propellants also limits the dose range delivered per actuation [70]. To enhance the stability, therapeutic proteins could be incorporated in a particulate carrier to suspend in a propellant. There is evidence that co-spray drying proteins with polyvinyl alcohol and sodium carboxymethylcellulose can improve the physical stability of mAbs in surfactant-free HFA propellant [71,72].

4. Formulations

As previously stated, the instability of Ab during aerosolisation creates both pharmacological and safety concerns because it results in Ab aggregation. Inhaled Ab instability is caused by the general vulnerability of proteins that require precise regulation of tertiary structure for function and, as a result, necessitate a protective formulation method [67]. Therefore, a critical objective of manufacturing therapeutic mAbs is to control the rate of degradation of Ab to ensure sufficient shelf life for transit and storage worldwide [73]. However, the stability of the Ab throughout its existence as a pharmaceutical product is an obstacle also experienced during the creation of intravenous Ab [74].

Selecting appropriate formulation excipients and other formulation parameters is a fundamental need. The protein formulation approach minimizes aggregation and degradation in aqueous media and creates amorphous rather than crystalline solid structures that disperse freely in aqueous media or biological fluids [75].

Surfactants have been extensively investigated and employed in the formulation of Abs for parenteral administration, and as already mentioned, they may be effective in stabilizing Ab during nebulization [76]. Surfactants, which act by replacing proteins at the air-liquid interface during nebulization, may inhibit Ab aggregation by preventing protein adsorption at the massive air-liquid interface created during mesh-nebulization without compromising aerosol aerodynamic qualities [77].

Sugars, polyols, and small amino acids are other excipients that stabilize molecules against air-liquid interface generation [50]. Sugars (sucrose, trehalose, mannitol) and polyols stabilize proteins in the liquid state via a single mechanism: preferential hydration of proteins through the steric exclusion of sugars and polyols from native proteins. Sugars and polyols may also sterically inhibit monomer-monomer interactions at the air-water interface, inhibiting further aggregation. Although the processes that support amino acid-dependent stabilization remain unknown, amino acids (glycine, lysine, isoleucine, alanine, histidine, and arginine) are routinely utilized as stabilizers.

The stability against proteases and mucus penetration may be improved by conjugating proteins to polyethylene glycol (PEG) [16].

Small Ab fragments, such as Fab, domain Abs, such as nanobodies [46], and small synthetic proteins, such as anticalins, engineered versions of lipocalins [78], have often been used to address the stability issues with inhaled Abs. Ab fragments provide the advantages of improved tissue penetration and simple and low-cost manufacture [16]. They decay rapidly and have a short serum half-life [77]. Because of their smaller size and improved stability, engineered Fab are appropriate for inhalation and are thus simpler to manufacture as aerosols. The pharmacologically active antigen-binding portion of the Fab is retained after its administration to the lungs by inhalation to neutralize inflammatory cytokines. Nanobodies, which are recombinant single-domain Abs produced from Camelidae heavy chain-only Abs [79], combine the benefits of small-molecule medications, such as their reduced size, good stability, and simplicity of synthesis, with the traits of traditional Abs, such as their high selectivity and affinity [80]. They can readily interact with biomolecules on the cell surface and within the cell [81]. Lipocalins and immunoglobulins exhibit structural flexibility inside structurally varied loops maintained by a rigid framework, demonstrating similarities in their protein design [77].

PEGylation, the covalent and non-covalent attachment or amalgamation of PEG polymer chains to molecules and macrostructures, can enhance the molecular mass of conjugated molecules while also providing a shielding effect [16]. It protects proteins against renal clearance and proteolytic enzyme breakdown, allowing them to stay in the body longer. It should be an effective method to extend the retention time of therapeutic proteins in the lung [82].

5. Pharmacokinetic/Pharmacodynamic of inhaled mAbs

To be therapeutically effective, inhaled mAbs must achieve sufficient unbound concentrations at the target site in the lung to interact with their PD characteristics and elicit the pharmacological action over an acceptable dosage interval [83]. As a result, assessing the PK/PD relationship is crucial for predicting their potential therapeutic efficacy. However, characterizing the PK of inhaled drugs is highly complex.

As mentioned, size, molecular weight, lipophilicity, structural complexity, and how it is generated (particle and molecular engineering) impact lung absorption of any biopharmaceutical and, consequently, its PK [84]. Large proteins might take hours or days to reach the maximum concentration [34]. However, macromolecules taken by receptor-mediated active transport might be absorbed more quickly than predicted based on their molecular weight [44]. The most common therapeutic mAbs are IgG. FcRn facilitates their pulmonary absorption and, by controlling the natural recycling process, allows an extended serum half-life of 10–21 days in humans [16].

Regrettably, the concentration profile of a drug in the bloodstream reflects its fate after it has been absorbed and removed from the airways and may not indicate the true kinetics of inhaled mAbs in the lungs. Actually, their pulmonary interstitial concentration is expected to be higher than that of the blood compartment [83]. Therefore, correctly assessing the PK profile of an inhaled mAb involves simultaneously assessing its pulmonary and systemic PK [12].

Much information on the PK of mAbs has been generated from animal studies [85]. In nonhuman primates, inhaled mAbs demonstrated a two-phase non-linear elimination and/or distribution with lung exposition greater than the systemic one over 33 hours and above mAb affinity for its target [86]. This PK behavior, however, was examined over many days by continuous sampling in lung parenchyma using microdialysis. Such a strategy is impracticable in humans. Furthermore, it is challenging to extrapolate animal data on inhaled protein therapeutics to humans due to the physiological, anatomical, biological, and respiratory differences between species. There are considerable changes in FcRn-Ab binding between humans and animals [86], which may lead to discrepancies in Ab distribution and clearance. The architecture and respiratory physiology of the animal species that heavily influence medication delivery is another important issue that must be considered. Drug deposition in animals is further influenced by aerosol-generating systems, which, even using human-designed technologies, do not accurately represent the aerosol dispersion observed in humans [85]. All this indicates that it is theoretically incorrect to extrapolate PK data from animals to humans.

Therefore, introductory PK studies in humans are necessary, but evaluating the PK of inhaled biopharmaceuticals is still an experimentally difficult task. As a result, there are significant difficulties in examining the PK/PD relationship of these medications in humans [83]. It is almost impossible to accurately determine the distribution of inhaled medications to the airways, lung tissue and other organs because practical and ethical aspects make it impracticable to repeat in humans what has been explored in animals.

There is an obvious need for more predictive models, particularly an early prediction of clinical doses [83]. The recent application of promising in vitro models (such as multilayer cell-based models in an aerosol chamber) by incorporating cutting-edge functional research in the first stages of the formulation may make the creation of inhaled drugs simpler [87]. There may be further alternatives created by aerosol delivery in sophisticated organ-mimicking systems, such as ‘lungs-on-a-chip,’ which enables truly matched comparisons of the dynamic tissue-tissue interactions on a microdevice [88].

Better predictive algorithms are required, especially ones anticipating therapeutic dosages in advance. The calculation of the maximum recommended starting dose for pulmonary delivered mAbs must consider the No Observed Adverse Effect Level (NOAEL), the Minimal Anticipated Biologic Effect Level (MABEL), and the Pharmacologically Active Dose (PAD), as well as the NOAEL endpoint scaled for body weight or lung weight, the lung deposition factor, and an additional inter-species safety factor [89]. The NOAEL is ‘the highest dose level that does not produce a significant increase in adverse effects compared to the control group’ [90]. The doses for MABEL and PAD are calculated using information from several sources by combining predicted human PK data with a concentration expected to have minimal biologic effects [90]. However, the dose to be delivered must be characterized since it might be extremely variable and immunogenic, making PK evaluation difficult.

Linking biomimetic microsystems to aerosol delivery systems when examining the PD and PK characteristics of inhaled biopharmaceuticals might solve these problems [85]. There is a platform that allows the transepithelial uptake of aerosolised protein therapeutics to be assessed by combining and improving two easy-to-automate technologies, an optimized aerosol-surrogate assay for protein stability and the ALICE-CLOUD dose-controlled aerosol-cell exposure system [87]. Another option is organ-on-chip technology, which accurately compares dynamic tissue-tissue interactions on a microdevice [91]. The Cloud α AX12, which consists of a cloud-based exposure chamber (VITROCELL) that integrates the breathing ^AXLung-on-chip system (AlveoliX), seems to be a valuable tool for inhalation toxicology and drug safety and efficacy testing [92].

The scarcity of information on the PK/PD interaction of inhaled mAbs makes it necessary to conduct further research by also applying new technologies. In any case, we must have specific information on the PK of each inhaled mAb, as the current knowledge on these inhaled biologics is insufficient to predict accurately the behavior of the inhaled mAb we want to develop clinically [93].

6. Inhaled mAbs already tested in asthmatic patients

Three inhaled mAbs, omalizumab, abrezekimab, and ecleralimab, were tested in patients with severe asthma [94] (Table 1). Omalizumab, which is a humanized mAb that blocks the binding of IgE to cell-membrane receptors, thereby inhibiting the release of mediators, but it does not bind to cell-bound IgE [95], was administered by inhalation in 33 subjects with mild allergic asthma [96]. While this mAb was then measurable in the serum, no significant changes in serum IgE levels were identified, and early-phase allergen-induced airway responses were unaffected. The lack of efficacy was ascribed to insufficient concentrations of omalizumab to neutralize IgE in lung tissue containing IgE effector cells. Furthermore, one patient developed anti-omalizumab serum IgG and IgA Abs at day 28 post-treatment with reduced serum omalizumab concentration. This finding raised concern that inhaled mAbs may be more immunogenic than injectable mAbs. Aggregated proteins, which are highly immunogenic and promote the formation of potentially effective anti-drug antibodies, may be the cause of this increased immunogenicity [41].

Table 1. Clinical studies of inhaled mAbs therapy in asthma.

Drug	Pharmacological target	Patients	Study design	Treatment	Key findings	Reference
Omalizumab	Humanised mAb that blocks of the binding of IgE to cell-membrane receptors.	33 with mild allergic asthma.	Randomised, placebo-controlled, parallel-group clinical trial.	8-week once-daily treatment with aerosolised placebo (n = 11), aerosolised omalizumab 1 mg (n = 12), or aerosolised omalizumab 10 mg (n = 10), and 4-week follow-up.	Omalizumab detectable in the serum during aerosol treatment. No significant change in serum IgE. No attenuation of the early phase responses to allergen with both doses of omalizumab. One patient receiving aerosolized omalizumab 10 mg daily had serum IgG and IgA antibodies to omalizumab.	[97]
Abrezekimab	A dry-powder formulation containing CDP7766, a high-affinity anti-human-IL-13 antigen-binding Fab that binds to IL-13, preventing binding to the IL-13 Rα1 subunit.	40 healthy individuals in part 1. 45 patients with mild asthma in part 2.	Part 1, randomized, double-blind, placebo-controlled, ascending-dose study. Part 2, randomized, double-blind, placebo-controlled, repeated doses study.	Part 1, subjects randomized 3:1 to a single inhaled dose of abrezekimab 0.5, 1.0, 5.0, 10, or 20 mg, or placebo. Part 2, patients randomized to once-daily inhaled abrezekimab 0.5 or 10 mg, or placebo (2:2:1), or abrezekimab 20 mg or placebo (3:2), for 10 days.	Abrezekimab well tolerated for up to 10 days in patients with asthma. Fast, persistent, and dose-dependent decreases in FENO. No detectable systemic exposure or clinically significant anti-drug-antibody effects. Few subjects developed bronchospasm and reductions in FEV1 60 min after inhaling abrezekimab.	[101]
Ecleralimab	Potent neutralizing Fab against human TSLP.	28 patients with stable mild atopic asthma.	Randomised, double-blind, placebo-controlled, parallel-design, multicentre allergen bronchoprovocation study.	Patients randomized (1:1) to receive 4 mg once-daily inhaled ecleralimab (n = 15) or placebo (n = 13) for 12 weeks.	LAR AUC3–7 h attenuated by 64%, LAR% by 48%, and allergen-induced sputum eosinophils by 64% at 7 h and by 52% at 24 h post-challenge with ecleralimab compared with placebo. EAR AUC0–2 h and EAR% numerically reduced. FENO levels significantly reduced from baseline throughout the study, except at 24 h post-allergen (day 43 and day 85).	[100]

F_ENO, fractional exhaled nitric oxide; EAR, allergen-induced early asthmatic response; EAR AUC_0–2 h , EAR measured by area under the curve (AUC_0–2 h); EAR%, EAR with maximum percentage decrease in FEV₁; Fab, fragment antigen binding; FEV₁: forced expiratory volume in 1 s; Ig, immunoglobulin; IL, interleukin; LAR, late asthmatic response; LAR AUC_3–7 h, LAR measured by area under the curve (AUC_3–7 h); LAR%, LAR with maximum percentage decrease in FEV₁; TSLP, thymic stromal lymphopoietin.

A 2012 study by Hacha et al. demonstrated a significant antiasthma effect after nebulizing anti-IL-13 Fab in an asthma model driven by allergen exposure compared with saline and nonimmune Fab fragments [97]. The effectiveness of inhaled CDP7766, a humanized, high-affinity, neutralizing, anti-human-IL-13 Fab that binds to IL-13, was tested. CDP7766 was biophysically stable and well tolerated by the animals in a cynomolgus macaque allergic asthma model [98]. There was a substantial decrease in allergen-induced cytokines, chemokines, and eosinophils in the treatment groups’ bronchoalveolar lavage fluid. Abrezekimab is a dry powder formulation that comprises CDP7766, leucine and trehalose. It prevents the binding of IL-13 to the IL-13 Rα1 subunit [99]. It was well tolerated for up to 10 days in patients with asthma who experienced fast, persistent, and dose-dependent decreases in fractional exhaled nitric oxide levels (F_ENO) [99]. There was no detectable systemic exposure or clinically significant anti-drug-antibody effects, although a few subjects developed bronchospasm and reductions in FEV₁ 60 min after inhaling abrezekimab. The researchers interpreted this decline as an artifact of administering 4 doses of the dry powder drug, and symptoms ultimately disappeared spontaneously.

Ecleralimab is a potent neutralizing Fab that belongs to the immunoglobulin G1/λ isotype subclass and is directed against human thymic stromal lymphopoietin [100], an epithelial derived cytokine produced in response to proinflammatory stimuli [101]. It was administrated once daily via a DPI for 12 weeks in patients with stable mild atopic asthma. Ecleralimab reduced the late asthmatic response to the allergen on day 84 by 48%. Furthermore, ecleralimab was shown to reduce airway hyperresponsiveness compared to placebo, as seen by the higher dosage of methacholine required to produce a 20% drop in FEV₁. It also considerably reduced F_ENO from baseline throughout the study, except at 24 h post-allergen (day 43 and day 85) and allergen-induced sputum eosinophils after 3 months of treatment and was safe [100]. Nevertheless, two Phase two trials, the first aiming to determine the efficacy and safety of multiple ecleralimab doses inhaled once daily compared with placebo, when added to standard-of-care (asthma therapy in adult patients with uncontrolled asthma concerning the change from baseline in FEV₁ at the end of 12 weeks of treatment (ClinicalTrials.gov Identifier: NCT04410523), and the second, a 12/24-week extension study conducted to evaluate the safety and tolerability, PK and immunogenicity of 5 dose levels of ecleralimab in adult asthma participants treated with existing standard of care asthma therapy who have completed the prior core study NCT04410523 (ClinicalTrials.gov Identifier: NCT04946318), were discontinued. However, it is unclear whether this was due to the poor efficacy of ecleralimab, its unfavorable PK, insufficient delivery, or changed business priorities.

7. Conclusion

It is becoming increasingly accepted that mAbs should be administered by inhalation rather than parenterally to improve their efficiency in lung diseases. However, the pulmonary administration of mAbs in terms of aerosol technology and the formulation for inhalation is difficult. Important anatomical, physiological, and immunological barriers influence the effectiveness of inhaled mAb delivery. Furthermore, other obstacles, including heterogeneity, loss of stability, limited loading capacity, susceptibility to enzymatic, mucociliary, and phagocytic clearances, immunogenicity, and toxicity, limit the clinical development of inhaled mAbs. Although progress has been made in this area, several crucial aspects will require additional research to promote the development of more effective inhaled mAbs and preserving their stability will be critical.

8. Expert opinion

The advantages of Abs inhalation to treat severe asthma have not yet been shown in the clinical context, even though airway administration of Abs is viable and promising in preclinical investigations. Data from a systematic review that included these three studies indicated that the efficacy of inhaled mAbs in asthma is controversial [94]. Despite having a certain degree of effectiveness and safety and being linked with a fast beginning of the pharmacological action, increased efficacy at lower doses, limited systemic exposure, and decreased risk of side effects, administering mAbs by inhalation to asthmatic patients is still challenging.

Nevertheless, inhalation is an attractive route for delivering mAbs also because it offers non-hospitalized patients the possibility of self-administering Abs therapy, with a reduction of healthcare expenses while enhancing patient comfort.

However, creating inhaled mAbs must be carefully planned. It requires an extensive investigation of all relevant factors, including the disease, the patient group(s), the therapeutic molecule chosen, its interaction with the biological barriers in the lungs, the formulation, excipients, and delivery methods [17].

It will, therefore, be necessary to examine in depth the issues related to instability and protein aggregation to develop inhaled mAbs that are stable and effective. In addition, more excipients will have to be produced, providing more formulation design options [23]. Novel carrier systems for topical administration to the lungs are another important need, as carrier systems could greatly improve proteins’ stability and PK profile [23]. Inhaled mAbs, like all inhaled therapies, are most effective when created and given in their intended location inside the airways with a well-designed inhalation unit that will provide appropriate therapeutic concentrations in the respiratory system.

Expanding knowledge and technological advances indicate that it is possible to begin implementing viable strategies to overcome biological barriers. Nevertheless, many questions remain that need to be adequately answered before we can be confident that we can develop inhaled mAbs for use in patients with asthma (Table 2). Apart from the need to define for each inhaled mAb the PK/PD ratio in humans, especially in the asthmatic patient, also considering his/her age and gender, it is of paramount importance to find a valid method that allows us to transpose the useful information on inhaled mAbs generated by animal studies to clinical applications and also to produce pulmonary delivery systems for mAbs that can overcome the current limitations. We fully agree with Chow et al. [69] that it will be important to understand the role of the Fc domain of an antibody in asthma, the possible differences in the efficacy of neutralizing antibodies between the nasal cavity and the lower airways, to produce new safe and effective excipients to obtain truly stable formulations of inhaled mAbs, and to understand the potentially toxic and immunogenic risks of neutralizing antibodies when they are localized at high concentrations in the airways. Therefore, it is likely to be some time before effective inhaler mAbs are available for use in the treatment of asthma.

Table 2. Substantial issues that must be considered for adequate clinical development of inhaled mAbs to treat asthma.

To define the PK/PD ratio of each inhaled mAb in humans, especially in asthmatic patients, also considering their age and gender. To find a valid method to translate the useful information on inhaled mAbs generated by animal studies to clinical applications. To understand the role of the Fc domain of a mAb in asthma. To understand the possible differences in the efficacy of neutralizing Abs between the nasal cavity and the lower airways. To produce new safe and effective excipients to obtain truly stable formulations of inhaled mAbs, To understand the potentially toxic and immunogenic risks of neutralizing Abs when localized at high concentrations in the airways. To produce pulmonary delivery systems for mAbs that can overcome the current limitations.

Article highlights

• In several experimental situations, mAbs achieve a better therapeutic response when given via the pulmonary route than when supplied systemically.

• Direct administration of high local doses to the lungs offers the advantage of achieving therapeutic equivalence to considerably higher doses administered systemically by parenteral methods.

• The pulmonary administration of mAbs in terms of aerosol technology and the formulation for inhalation is complex because they are macromolecules.

• Several anatomical, physiological, and immunological barriers influence the effectiveness of inhaled biologics delivery.

• Selecting appropriate formulation excipients and other formulation parameters and the aerosol technology to deliver mAbs is a fundamental need.

• The absorption and disposition of each inhaled mAb being considered for therapeutic use must be studied individually because there is insufficient knowledge to make accurate predictions with the currently available data.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.