Nebulization of Fab fragments

Abstract

The present invention relates to a method of treating a disease or disorder associated with the presence or formation of galectin-10 crystals, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is administered as an aerosol. Also included in the invention is a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, and a nebulizer comprising the pharmaceutical composition. The pharmaceutical composition may be useful for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, such as asthma or cystic fibrosis.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is formulated for aerosolization. The present invention also relates to a method of treating a disease or disorder associated with the presence or formation of galectin-10 crystals, and a pharmaceutical composition for use in the method. The method comprises administering the pharmaceutical composition as an aerosol, for example, by using a nebulizer. The present invention also relates to a nebulizer comprising the pharmaceutical composition. [Background technology]

[0002] BACKGROUND OF THEINVENTION Asthma is a chronic inflammatory disease of the conducting airways that affects 300 million people worldwide. Pathologically, the disease is characterized by airway eosinophilia and overproduction of thick mucus that can lead to a fatal eosinophilic inflammatory response in some patients, including irreversible obstruction of small airways.

[0003] In eosinophilic asthma, eosinophils are highly abundant in the airway epithelium and can undergo eosinophil extracellular trap cell death (EETosis). During the process of EETosis, eosinophils release nuclear chromatin along with galectin-10 protein. At high concentrations, extracellular galectin-10 begins to crystallize, forming characteristic colorless crystals called Charcot-Leyden crystals (CLCs). These CLCs have been shown to enhance type 2 immunity in asthmatics and contribute to increased mucus viscoelasticity and anchoring of the mucus plug to the airway epithelium. CLCs have also been observed in human tissues and secretions associated with eosinophilic inflammatory responses, such as myeloid leukemia, allergic and parasitic diseases, and eosinophilic cystic fibrosis.

[0004] The inventors have previously shown that anti-galectin-10 antibodies can solubilize CLCs (WO2019 / 197675). It has been reported how galectin-10 crystals can induce a proinflammatory response in vivo, and how this response can be suppressed by administration of a galectin-10 antibody that can disrupt galectin-10 crystallization; importantly, the galectin-10 antibody was able to dissolve CLCs from patient mucus samples. Taken together, this shows how drugs that target galectin-10 crystallization can be used to treat diseases and disorders whose pathology is related to the presence of CLCs.

[0005] CLCs are present in the mucus of these patients and therefore in the lumen of the airways. Therefore, the only effective way to reach these CLCs is by local delivery to the airways. Aerosolization is routinely used to deliver small molecule drugs; however, administration of proteins by inhalation is rare. Many challenges remain with aerosol technology and drug formulation, especially for monoclonal antibodies (mAbs). Aerosolization likely imposes physical stress on mAbs, inducing changes in protein conformation, potentially reducing their biological activity and rendering them immunogenic.

[0006] Nebulizers are the most widely used inhalation device for generating aerosols from protein solutions; however, nebulizers have not been completely successful, as lower activity levels, partial proteolysis, and higher aggregation levels have been observed (Respaud et al., 2014; mAbs; 6(5): 1347-1355). These aggregates can cause loss of therapeutic activity, and as a result, formulations may need to be optimized in an attempt to reduce aggregate formation. Thus, there is a need for further improvements in the delivery of protein therapeutics by inhalation. Summary of the Invention

[0007] (Summary of the invention) The development of antibody therapeutics suitable for delivery by inhalation has proven challenging for drug formulators. Aerosolization imposes significant physical stress on antibodies, often resulting in the formation of aggregates. This may contribute to reduced biological activity and increased immunogenicity caused by anti-drug antibody (ADA) responses. The inventors have confirmed that aerosolization of Fab fragments results in little, and in many cases no, formation of protein aggregates. In particular, it has been found that Fab fragments derived from full-length IgG can be successfully aerosolized, whereas aerosolization of the full-length IgG counterpart results in aggregation. It is reported herein that aerosolized Fab fragments that bind to galectin-10 not only produce little aggregates, but also retain their biological activity and efficacy. Specifically, these aerosolized Fab fragments retain binding to galectin-10 and the ability to solubilize galectin-10 crystals (CLCs). As a result, it is possible to prepare pharmaceutical formulations containing Fab fragments without the need for, or with the addition of only low concentrations of, surfactants.

[0008] In a first aspect, the present invention provides a pharmaceutical composition comprising a Fab fragment that binds to galectin-10 for use in a method for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition, wherein the composition is administered as an aerosol.

[0009] In a second aspect, the present invention provides a method for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is administered as an aerosol.

[0010] In certain embodiments, the disease or disorder associated with the presence or formation of galectin-10 crystals is selected from the group consisting of asthma, chronic rhinosinusitis, celiac disease, helminth infection, gastrointestinal eosinophilic inflammation, cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA), Churg-Strauss vasculitis, chronic eosinophilic pneumonia, and acute myeloid leukemia. In certain preferred embodiments, the disease or disorder associated with the presence or formation of galectin-10 crystals is asthma or cystic fibrosis (CF).

[0011] In certain embodiments, the Fab fragment, when bound to soluble galectin-10, inhibits crystallization of galectin-10 and / or when bound to crystalline galectin-10, promotes dissolution of crystalline galectin-10. In certain embodiments, the crystalline galectin-10 is a Charcot-Leyden crystal (CLC).

[0012] In one embodiment, the Fab fragment binds to human galectin-10. In one preferred embodiment, the Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:4 [ka] HCDR3 comprising or consisting of: SEQ ID NO:3 [ka] HCDR2 comprising or consisting of: SEQ ID NO:2 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:8 [ka] LCDR3 comprising or consisting of: SEQ ID NO:7 [ka] LCDR2 comprising or consisting of: SEQ ID NO:6 [ka] LCDR1 comprising or consisting of Contains :.

[0013] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0014] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light chain (VL), wherein: The VH domain is represented by SEQ ID NO:1 [ka] and The VL domain is represented by SEQ ID NO:5 [ka] The amino acid sequence of

[0015] In certain embodiments, the Fab fragment is humanized, germlined, or deimmunized.

[0016] In some embodiments, the pharmaceutical composition is administered to a subject via inhalation.In some embodiments, the pharmaceutical composition is administered as an aerosol using a nebulizer, a pressurized metered dose inhaler (pMDI), a dry powder inhaler, a soft mist inhaler, or a smart inhaler.In some preferred embodiments, the pharmaceutical composition is administered as an aerosol using a nebulizer.In some embodiments, the pharmaceutical composition is administered as an aerosol using a jet nebulizer, an ultrasonic nebulizer, or a mesh nebulizer.

[0017] In some embodiments, prior to administration, the pharmaceutical composition is an aqueous liquid formulation. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier or excipient. In some embodiments, the excipient is a liquid excipient, optionally a solvent or an aqueous solvent.

[0018] In some embodiments, the composition comprises a surfactant in an amount of less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.02%, or less than 0.01%. In some alternative embodiments, the composition does not contain a surfactant.

[0019] In certain embodiments, the concentration of the Fab fragment in the composition is about 1 to about 20 mg / ml, about 1 to about 15 mg / ml, about 1 to about 10 mg / ml, about 1 to about 5 mg / ml, about 2 to about 10 mg / ml, about 3 to about 10 mg / ml, about 4 to about 10 mg / ml, or about 5 to about 10 mg / ml. In certain embodiments, the concentration of the Fab fragment in the composition is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / ml.

[0020] In some embodiments, the particle size of the particles in the aerosol is less than 30 μm, less than 25 μm, less than 10 μm, less than 2 μm, less than 1 μm, less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm. In some preferred embodiments, the particle size of the particles in the aerosol is less than 2 μm.

[0021] In one embodiment, the concentration of particles in the aerosol is greater than or equal to 2×10 5 Less than 1×10 5 Less than 2 x 10 4 Less than 1 x 10 4 The total particles are less than 100 / ml.

[0022] In one embodiment, the concentration of particles greater than 2 μm in the aerosol is greater than or equal to 1×10 5 Less than 5 x 10 4 Less than 2 x 10 4 Less than 1×10 4 Less than 1×10 3 Less than 1 x 10 2 Fewer than 10 particles >2 μm / ml.

[0023] In a third aspect, the present invention provides a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is formulated for aerosolization.

[0024] The embodiments described above relating to the pharmaceutical compositions of the first and second aspects of the invention apply equally to the pharmaceutical composition of the third aspect.

[0025] In a fourth aspect, the present invention provides a nebulizer comprising any of the pharmaceutical compositions described herein. In certain embodiments, a nebulizer is provided comprising a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is formulated for aerosolization. [Brief description of the drawings]

[0026] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1 shows the aerosol droplet size analysis by laser diffraction. Raw data is presented in Figure 1A. Each sample was tested at two concentrations (3 mg / ml and 10 mg / ml) and aerosolization was performed using two different devices, Aerogen® Solo and Pari eFlow®. Experiments were performed in triplicate using three Aerogen® Solo and three Pari eFlow® devices, identified by their serial numbers. The bar graph in Figure 1B shows the median range of nebulized volume mean diameter (VMD) for each sample at two concentrations (3 mg / ml and 10 mg / ml). [Diagram 2] Figure 2 shows protein sample concentrations before (3 mg / ml and 10 mg / ml) and after nebulization using two different devices, Aerogen® Solo and Pari eFlow®. Raw data is presented in Figure 2A. Experiments were performed in triplicate using three Aerogen® Solo and three Pari eFlow® devices, identified by their serial numbers. The bar graph in Figure 2B shows the median range (%) of concentration variation after nebulization for the antibodies / fragments tested. [Diagram 3] Figure 3 shows the effect of nebulization on aggregate formation. Aggregates were detected by flow cell microscopy (FCM) and expressed as median particle counts / ml after nebulization with the Aerogen® Solo or Pari eFlow® devices. The average particle counts of buffer only were subtracted from the sample values. Particle counts per mL were expressed as total particles and as particles with equivalent diameters of >2 μm, >10 μm, and >25 μm. In Figure 3B, filled and open circles represent data after nebulization for concentrations of 10 mg / mL and 3 mg / mL, respectively. [Figure 4] Figure 4 shows the dynamic light scattering (DLS) analysis of the samples before and after nebulization. The parameters of the main peak are presented for each sample. If necessary (in case of multimodality), the remaining sample was filtered before a new analysis. [Diagram 5]Figure 5 shows the percentage of monomer analyzed by SEC-HPLC for samples before and after nebulization. The results in Figure 5A are expressed as the average mass percentage of monomer and high molecular weight (HMW) species. The bar graph in Figure 5B shows the percentage of high molecular weight species. Samples were analyzed before nebulization (1) and after nebulization with the Aerogen® Solo device (2) and after nebulization with the Pari eFlow® (3). [Figure 6] Figure 6 shows the protein concentration of each of the tested Fab clones before and after nebulization with an Aerogen Solo vibrating mesh nebulizer. Experiments were performed in triplicate using three different Aerogen Solo devices. Figures 6A and 6B provide two independent experiments. [Figure 7] Figure 7 provides an overview of the dynamic light scattering (DLS) results of all Fab clones before and after mesh spraying at TO and after 4 weeks of storage (4W) at +5°C. All samples were subjected to a pre-filtration step (0.2 μm). [Figure 8] Figure 8 shows subvisible particle counts by flow cell microscopy (FCM) for all sprayed Fabs (top left panel, total particles / mL; top right panel, >2 μm particles / mL; bottom left panel, >10 μm particles / mL; bottom right panel, >25 μm particles / mL; circles - pre-spray; squares - post-spray). [Figure 9] Figure 9 shows the binding activity assessed by SPR (Biacore 3000) for all tested Fabs after nebulization. All clones were found to be fully functional with no loss in percent relative activity to the unstressed reference material (before nebulization) for each clone. [Figure 10] FIG. 10 shows the percent purity as assessed by SEC-HPLC for all nebulized Fabs. [Figure 11] Figure 11 shows the percent purity assessed by capillary gel electrophoresis (cGE) for all nebulized Fabs. Figure 11A shows the % purity of intact Fabs under non-reducing conditions, and Figure 11B shows the % total Fab purity under reducing conditions. [Figure 12] FIG. 12 shows the potency of the tested Fabs, 18C06, 20H09, 23H09, and 24F02_N53A (and 7B07_N53A) against GAL10 crystal lysis. [Figure 13] FIG. 13 shows the efficacy of the tested Fabs, 18C06, 20H09, and 23H09 (as well as 7B07_N53A) against GAL10 crystal dissolution after nebulization before and after 4 weeks of storage at +5° C. (Assay 1). [Figure 14] FIG. 14 shows the efficacy of the tested Fabs, 23H09 and 24F02_N53A, against GAL10 crystal dissolution after nebulization before and after 4 weeks of storage at +5° C. (assay 2). [Figure 15] Figure 15 shows the overall DRB1 risk score for a set of 44 marketed therapeutic antibodies. Human antibodies are indicated by medium grey bars, humanized antibodies by light grey bars, and chimeric antibodies by dark grey bars. Scores are shown for the tested Fabs, 18C06, 20H09, 23H09, and 24F02_N53A. [Figure 16] FIG. 16 shows the percentage of donors with an IFNγ response (left panel) and an IL-5 response (right panel) (DFR2x and DFReq represent the statistical analysis used by Lonza). [Figure 17] FIG. 17 shows the IFNγ response (upper panel) and IL-5 response (lower panel) rankings of the four molecules tested in a 31 donor test population (DFR2x and DFReq represent the statistical analysis used at Lonza). [Figure 18] FIG. 18 provides a summary of the tested Fab attributes after spraying. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] (Detailed Description) (A.Definition) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Without limiting any term, the following provides further clarification of some of the terms used herein.

[0028] As used herein, the term galectin-10 (or Gal10 or Gal-10) refers to a small hydrophobic glycan-binding protein that self-crystallizes to form Charcot-Leyden crystals. Galectin-10 is also known as Charcot-Leyden crystal protein (CLCP), eosinophil lysophospholipase, and lysolecithin acylhydrolase. The term "galectin-10" is broad enough to encompass the human protein and any species homologues. Gal10 is a member of the galectin family characterized by a galactose-binding domain. Gal10 is a marker of inflammation whose various members have been shown to positively or negatively regulate multiple stages of inflammation. This small, autocrystallizing, hydrophobic protein (only 16.5 kDa) is localized in the cytoplasm of eosinophils. The protein is one of the most abundant components of eosinophils, representing 7-10% of the total cellular protein. Importantly, the Gal10 gene is found only in humans and non-human primates. Gal10 lacks the secretory peptide signal and transmembrane domain and is released during eosinophil extracellular trap cell death (EETosis).

[0029] The amino acid sequence of full-length human galectin-10 is represented by SEQ ID NO: 9 (see below). This sequence corresponds to the sequence deposited in the UniProt database under human galectin-10, accession number Q05315. Naturally occurring variants of the human sequence, such as the Ala→Val variant at position 28, are also encompassed by the term "galectin-10." SEQ ID NO:9 [ka]

[0030] As used herein, the terms galectin-10 crystals or Charcot-Leyden crystals (CLCs) are used interchangeably herein to refer to crystals formed from galectin-10. Crystals formed by galectin-10 are typically hexagonal bipyramidal crystals, approximately 20-40 μm in length and approximately 2-4 μm in width. These crystals have been associated with eosinophilic inflammatory disorders.

[0031] As used herein, the term antibody or immunoglobulin refers to a structure comprising a light chain and a heavy chain, with or without an interchain covalent bond therebetween. The term "immunoglobulin" includes a polypeptide having a combination of two heavy chains and two light chains, whether or not it possesses any relevant specific immunoreactivity. "Antibody" refers to such an association having significant known specific immunoreactive activity against an antigen of interest, here Galectin-10. The term "Galectin-10 antibody" is used herein to refer to an antibody exhibiting immunological specificity against Galectin-10 protein, including human Galectin-10, and, in some cases, its species homologs.

[0032] The general term "immunoglobulin" includes five different classes of antibodies that can be biochemically distinguished. The following discussion is generally for the IgG class of immunoglobulin molecules. With respect to IgG, immunoglobulins contain two identical light polypeptide chains of molecular weight approximately 23,000 daltons and two identical heavy chains of molecular weight 53,000-70,000. These four chains are connected by disulfide bonds in a "Y" configuration, where the light chains support the heavy chains, which begin at the mouth of the "Y" and continue through the variable region.

[0033] The light chains of an antibody are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked end of the Y to the C-terminus at the bottom end of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, or ε), with several subclasses within them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those of skill in the art in light of the present disclosure.

[0034] The variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This four-component antibody structure forms an antigen-binding site at the end of each arm of a Y. More specifically, the antigen-binding site is defined by three complementarity determining regions (CDRs) on each of the VH and VL chains.

[0035] As used herein, the term VHH antibody or heavy chain-only antibody refers to a type of antibody that is only produced by species of the Camelidae family, which includes camels, llamas, and alpacas. Heavy chain-only antibodies or VHH antibodies are composed of two heavy chains and lack light chains. Each heavy chain has a variable domain at the N-terminus, and these variable domains are referred to as "VHH" domains to distinguish them from the variable domains of the heavy chains of conventional heterotetrameric antibodies, i.e., VH domains, described above.

[0036] As used herein, the terms variable region and variable domain are used interchangeably herein and are intended to have equivalent meanings. The term "variable" refers to the fact that certain portions of the variable domains VH and VL differ extensively in sequence between antibodies and are used in the binding and specificity of each particular antibody to its target antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains that form part of the antigen-binding site. The first, second, and third hypervariable loops of a Vlamda light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as comprising residues 24-33 (L1(λ) of 9, 10, or 11 amino acid residues), 49-53 (L2(λ) of 3 residues), and 90-96 (L3(λ) of 5 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of the V kappa light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and can be defined as comprising residues 25-33 (L1(κ) of 6, 7, 8, 11, 12, or 13 residues), 49-53 (L2(κ) of 3 residues), and 90-97 (L3(κ) of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second, and third hypervariable loops of a VH domain are referred to herein as H1, H2, and H3, and can be defined as comprising residues 25-33 (H1 consisting of 7, 8, or 9 residues), 52-56 (H2 consisting of 3 or 4 residues), and 91-105 (H3, which varies greatly in length) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

[0037] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops, respectively, of the VL domain and include hypervariable loops from both the Vkappa and Vlambda isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops, respectively, of the VH domain and include hypervariable loops from any of the known heavy chain isotypes, including gamma, epsilon, delta, alpha, or mu.

[0038] Hypervariable loops L1, L2, L3, H1, H2, and H3 may each comprise a portion of a "complementarity determining region" or "CDR" as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, since hypervariable loops (HV) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined on the basis of sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983), and the boundaries of HV and CDR may differ in some VH and VL domains.

[0039] The CDRs of the VL and VH domains can generally be defined as comprising the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Thus, HVs can be contained within the corresponding CDRs, and unless otherwise indicated, references herein to the "hypervariable loops" of the VH and VL domains should be construed as encompassing the corresponding CDRs, and vice versa.

[0040] The more highly conserved parts of the variable domains are called framework regions (FRs), as defined below. Natural heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) that are connected by three hypervariable loops and are primarily in a β-sheet configuration. The hypervariable loops of each chain are held in close proximity by the FRs and, together with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of the antibody. Structural analysis of antibodies has revealed the relationship between the sequence and shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt a small repertoire of main-chain conformations, called "canonical structures". These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at specific positions in the loops and in the framework regions that determine the conformation by their packing, hydrogen bonding, or ability to adopt unusual main-chain conformations.

[0041] As used herein, the term CDR or complementarity determining region refers to the non-contiguous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest (1991), as well as Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where these definitions include overlapping or subsets of amino acid residues when compared with each other. The amino acid residues encompassing the CDRs defined by each of the above cited references are provided for comparison. Preferably, the term "CDR" refers to the CDRs defined by Kabat based on sequence comparison. Table 1: CDR definitions [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al. (supra). 2 Residue numbering follows the nomenclature of Chothia et al. (supra). 3 Residue numbering follows the nomenclature of MacCallum et al. (supra).

[0042] As used herein, the term framework region or FR region includes amino acid residues that are part of the variable region (e.g., using the Kabat definition of CDR) but are not part of the CDRs. Thus, the variable region framework is about 100-120 amino acids in length, but includes only amino acids outside the CDRs. For the specific example of a heavy chain variable domain, and for the CDRs defined by Kabat et al., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acid 103 to the end of the variable region. The framework regions of the light chain are similarly separated by each of the light chain variable region CDRs. Similarly, using the CDR definitions by Chothia et al. or McCallum et al., the framework region boundaries are separated by the respective CDR ends as described above. In a preferred embodiment, the CDRs are as defined by Kabat.

[0043] In natural antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically arranged to form an antigen-binding site when the antibody assumes its three-dimensional shape in an aqueous environment. The remaining parts of the heavy and light variable domains show less intermolecular variability in terms of amino acid sequence and are called framework regions. The framework regions are primarily in a β-sheet configuration, and the CDRs form loops that connect to and, in some cases, form part of the β-sheet structure. These framework regions thus act to form a scaffold that orients the six CDRs by interchain non-covalent interactions. The antigen-binding site formed by the arranged CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The location of the CDRs can be easily identified by one skilled in the art.

[0044] As used herein, the term Fab or Fab fragment refers to a molecule composed of a heavy and a light chain, where the light chain consists of a VL domain and one constant domain (CL, Cκ, or Cλ) and the heavy chain consists of only a VH domain and a CH1 domain. A Fab fragment is one arm of an immunoglobulin molecule, usually in a Y shape. A Fab fragment can be generated from an immunoglobulin molecule by the action of the enzyme papain. Papain cleaves the immunoglobulin molecule in the region of the hinge to produce two Fab fragments and a separate Fc region. A Fab fragment is not to be construed as a full-length antibody molecule.

[0045] As used herein, the term humanizing substitution refers to an amino acid substitution in which an amino acid residue present at a particular position in a VH or VL domain of an antibody or fragment (e.g., a camelid-derived galectin-10 antibody or fragment) is replaced with an amino acid residue occurring at the equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a VH or VL domain encoded by human germline. Humanizing substitutions may be made in the framework regions and / or CDRs of an antibody or fragment defined herein.

[0046] As used herein, the term humanized variant refers to a variant antibody that contains one or more "humanizing substitutions" compared to a reference antibody, where a portion of the reference antibody (e.g., the VH domain and / or VL domain or portion thereof containing at least one CDR) has amino acids derived from a non-human species, and where the "humanizing substitutions" occur within the amino acid sequence derived from the non-human species.

[0047] As used herein, the term germlined variant specifically refers to a "humanized variant" in which a "humanizing substitution" results in the replacement of one or more amino acid residues present at a particular position in a VH or VL domain of an antibody or fragment (e.g., a camelid-derived galectin-10 antibody or fragment) with an amino acid residue occurring at an equivalent position in a reference human VH or VL domain encoded by the human germline. For any given "germlined variant", the substituted amino acid residues substituted into the germlined variant are generally obtained exclusively or preferentially from a single VH or VL domain encoded by the human germline. The terms "humanized variant" and "germlined variant" are often used interchangeably herein. The introduction of one or more "humanizing substitutions" into a camelid-derived (e.g., llama-derived) VH or VL domain results in the production of a "humanized variant" of the camelid (llama)-derived VH or VL domain. Where the substituted amino acid residues are derived from a single VH or VL domain sequence that is preferentially or exclusively encoded by the human germline, this can result in a "human germlined variant" of the camelid (llama)-derived VH or VL domain.

[0048] As used herein, the term deimmunized or deimmunization refers to the identification and subsequent removal of T cell epitopes in a binding molecule. Usually, but not necessarily, this is done in the variable regions of the antibody or fragment. Often, but not necessarily, this is done in the framework regions and therefore outside the CDR regions. Removal of the T cell epitopes is usually achieved by replacing one or more amino acids that code for the T cell epitopes. The sequence is thereby changed to a sequence different from the T cell epitopes. A deimmunized variable region usually contains 1-5 amino acid substitutions. The substituted amino acids are selected so that the tertiary structure of the variable region is not significantly altered. Thus, the substituted amino acids are usually selected from the same amino acid group (i.e., neutral, positively charged, negatively charged, lipophilic). When utilized within the same group, amino acids are substituted with amino acids in the same or similar positions in the variable region of a structurally similar human antibody.

[0049] Deimmunization of binding molecules for use in humans can usually be achieved by modifying the heavy chain at up to 5 positions, the light chain at up to 5 positions, or both. Deimmunization of the variable regions is a well-established technique and consistently results in binding molecules with a reduced probability of eliciting an immune response in humans. The number of amino acid substitutions required to achieve this result is usually less than 5 in each chain. In many cases, substitutions of 1, 2, or 3 amino acids in each chain are sufficient to obtain a binding molecule with a reduced probability of eliciting an immune response in humans when compared to the unmodified sequence. The Fab fragment of the present invention is preferably a humanized or deimmunized Fab fragment.

[0050] As used herein, the term % sequence identity refers to the degree of similarity between two sequences. The percent sequence identity between two amino acid sequences can be determined by comparing these two sequences aligned in an optimal manner, and the amino acid sequence to be compared can include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions where the amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of identity between these two sequences. For example, it is possible to use the BLAST program "BLAST 2 sequences" available at the site http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), the parameters used being those given by default (in particular for the parameters "open gap penalty": 5 and "extension gap penalty": 2; the matrix chosen is for example the matrix "BLOSUM 62" proposed by the program), and the percentage of identity between the two sequences to be compared is calculated directly by the program.

[0051] As used herein, the term aerosolization refers to dispersing a substance (e.g., a drug) into an aerosol, where the aerosol is a suspension of fine solid particles or liquid droplets in air or another gas. Therapeutic delivery of aerosols to the lungs can be provided via nebulizers, pressurized metered dose inhalers (pMDIs), as well as other devices (e.g., dry powder inhalers, soft mist inhalers, and smart inhalers).

[0052] As used herein, the term nebulization refers to the process of administering a drug directly by inhalation with the aid of a nebulizer, which converts liquid medicine into an aerosol that the patient can inhale through a breathing mask or mouthpiece. A nebulizer is a device used to convert a liquid into a fine spray of aerosol by oxygen, compressed air, or ultrasonic vibrations. There are three main types of nebulizers: jet nebulizers, which use compressed gas to create an aerosol; ultrasonic nebulizers, which use high frequency vibrations to create an aerosol; and mesh nebulizers (also known as vibrating mesh nebulizers), in which a vibrating element pushes a liquid through a very fine mesh to create an aerosol.

[0053] As used herein, the term surfactant refers to a substance that adsorbs to a surface or interface to reduce surface or interfacial tension. These agents aid in the wetting and dispersion of hydrophobic pharmaceutical active ingredients, and they usually act by reducing the interfacial tension between solids and liquids in suspension. Surfactants are often used to prevent the formation of aggregates in biological preparations. Polysorbates are the most widely used surfactants in the pharmaceutical industry. For example, they are added during the formulation of inhaled corticoids (fluticasone and budesonide) to facilitate the dispersion or dissolution of the drug during nebulization. However, the use of surfactants as emulsifiers, solubilizers, suspension stabilizers, and wetting agents in preparations intended for administration to human subjects or to animals can result in significant changes in the biological activity of the active agent in the formulation. Surfactant molecules incorporated into the formulation can exert their effect in several ways, for example, by affecting the disaggregation and dissolution of solid dosage forms, by controlling the rate of precipitation of drugs administered in solution form, by increasing membrane permeability and affecting membrane integrity, by altering drug metabolizing enzyme activity, and by affecting the binding of drugs to receptor sites.

[0054] As used herein, the term aerosol droplets refers to droplets produced after aerosolization of a pharmaceutical composition. The size of the aerosol droplets can be measured, for example, by laser diffraction, to determine whether the majority of the produced aerosol droplets are within the required respirable size range. The size of the aerosol droplets is an important characteristic of aerosolizer performance. Droplets that are too large will not reach the lower respiratory tract, while droplets that are too small will be exhaled. The nebulized solution must produce aerosol droplets with a diameter of, for example, 1-5 μm, which is the optimal aerosol droplet size range for deposition in the lower respiratory tract. Analytical devices provide the ability to measure and report the droplet size distribution of a sample. Aerosol droplet size is often expressed in terms of mass median aerodynamic diameter (MMAD). MMAD is defined as the diameter at which 50% of the droplets are larger and 50% are smaller by mass. The size distribution can also be expressed as the volume mean diameter (VMD), which is the droplet size value that divides the population in half. VMD is directly related to the mass median diameter (MMD) by the density of the droplets. VMD refers to the midpoint droplet size (median) where half of the volume of the aerosol is from droplets smaller than the median and half of the volume is from droplets larger than the median. A VMD of 50 μm (DV0.5), for example, indicates that half of the volume is from droplets smaller than 50 μm and half of the volume is from droplets larger than 50 μm.

[0055] As used herein, the terms particle size and particle concentration are used to refer to the size and quantity of protein particles or protein aggregates present in a protein-containing solution or aerosol. Particle size and particle concentration can be measured in a liquid sample before aerosolization, and can also be measured in a sample that has been aerosolized, collected, and then concentrated. Ideally, a pharmaceutical composition for use in treatment contains little or no protein particles or protein aggregates, both before and after aerosolization. These particles may or may not be visible (i.e., visible to the naked eye). A variety of methods are available for determining both particle size and particle concentration, including, but not limited to, dynamic light scattering and flow cell microscopy. Particle concentration can be reported as the total number of particles per ml of sample. Particle concentration can also be reported as the number of particles of a particular size per ml of sample or the number of particles larger than a particular size per ml of sample.

[0056] B. Galectin-10-binding Fab fragment The present invention relates to a pharmaceutical composition comprising a Fab fragment that binds to galectin-10. The inventors have determined that the Fab fragment is a preferred format for aerosolization. It is reported herein that the Fab fragment can be subjected to aerosolization with little or even no generation of aggregates.

[0057] Any Fab fragment that binds to galectin-10 is contemplated for use in the present invention. The Fab fragment preferentially binds to human galectin-10. In certain embodiments, the Fab fragment binds to human galectin-10. The amino acid sequence of full-length human galectin-10 is represented by SEQ ID NO:9. Thus, in certain embodiments, the Fab fragment binds to the amino acid sequence of SEQ ID NO:9. In certain embodiments, the Fab fragment binds to naturally occurring variants of human galectin-10. In certain embodiments, the Fab fragment binds to naturally occurring variants of the amino acid sequence of SEQ ID NO:9. In certain embodiments, the Fab fragment binds to human galectin-10 and cynomolgus galectin-10.

[0058] In one embodiment, the Fab fragment that binds to galectin-10 binds to an epitope that includes one or more amino acids of the crystal packing interface of galectin-10, preferably human galectin-10, the epitope including one or more amino acids of Ser2, Leu3, Leu4, Tyr8, Thr9, Glu10, Ala11, Ala12, Ser13, Thr16, Thr42, Glu43, Met44, Lys45, Asp49, Ile50, Glu68, Tyr69, Gly70, Ala71, Lys73, Gln74, Gln75, Val76, Glu77, Ser78, Lys79, Asn80, Met81, Leu96, Pro97, Asp98, Lys99, Gln101, Met103, Gly106, Gln107, Ser108, ​​Ser109, Tyr110, Thr111 , Asp113, His114, Arg115, Ile116, Lys117, Ala120, Gln125, Thr133, Lys134, Phe135, Asn136, Val137, Ser138, Tyr139, Leu140, and Lys141. In certain embodiments, the Fab fragment binds to an epitope that includes Tyr69 or an epitope that includes amino acids adjacent to Tyr69. In preferred embodiments, the Fab fragment binds to an epitope that includes Tyr69. Alternatively, or in addition, the Fab fragment can bind to an epitope that includes one or more amino acids from the dimerization interface of galectin-10. In such embodiments, the Fab fragment is capable of binding to an epitope comprising one or more amino acids selected from the group consisting of: Pro5, Pro7, Leu27, Ala28, Cys29, Leu31, Asn32, Glu33, Pro34, Tyr35, Gln37, His41, Glu46, Glu47, Gln55, Arg60, Arg61, Arg67, Trp72, Gln75, Trp127, Arg128, and Asp129. The amino acid residues or positions of Galectin-10 are defined herein with reference to the human protein sequence identified as SEQ ID NO:9.

[0059] After aerosolization, the Fab fragment retains the ability to bind to galectin-10. In certain embodiments, the Fab fragment binds to galectin-10 after aerosolization with the same or similar binding affinity as the Fab fragment before aerosolization. In certain embodiments, the Fab fragment binds to galectin-10 50%, 60%, 70%, 80%, 90%, 95%, or 100% after aerosolization compared to the binding affinity of the Fab fragment before aerosolization. In certain embodiments, the Fab fragment binds to galectin-10 90% or more after aerosolization compared to the binding affinity of the Fab fragment before aerosolization.

[0060] The Fab fragment binds to an epitope of galectin-10, thereby shielding the crystal packing interface of galectin-10. The crystal packing interface of galectin-10 is a surface patch of amino acids that contacts one or more adjacent galectin-10 molecules in the crystal lattice. By binding to an epitope of galectin-10 that serves to shield the crystal packing interface of galectin-10, the Fab fragment disrupts the crystallization of galectin-10. Thus, if the Fab fragment disrupts the crystallization of galectin-10, it follows that the Fab fragment can completely or partially shield the crystal packing interface. In certain embodiments, the Fab fragment promotes the dissolution of galectin-10 when bound to crystalline galectin-10. In certain embodiments, the Fab fragment inhibits the crystallization of galectin-10 when bound to soluble galectin-10. In certain embodiments, the Fab fragment, when bound to crystalline galectin-10, promotes the dissolution of crystalline galectin-10 and, when bound to soluble galectin-10, inhibits the crystallization of galectin-10.

[0061] The antagonistic properties of the galectin-10 Fab fragments described herein can be measured according to the assays described herein. For example, a Fab fragment that binds to galectin-10 can be incubated with soluble galectin-10 under experimental conditions that favor crystallization of galectin-10, and the ability of the Fab fragment to inhibit this process can be measured. The inhibitory activity of the Fab fragment that binds to galectin-10 can be measured relative to a control, for example, a Fab fragment that does not bind to galectin-10. The inhibitory activity of the Fab fragment that binds to galectin-10 can also be measured relative to a control that is a galectin-10 binding molecule that does not have crystallization inhibitory activity. The Fab fragment that binds to galectin-10 can inhibit the crystallization of galectin-10 by 100% relative to the control, by 90% relative to the control, by 80% relative to the control, or by 70% relative to the control.

[0062] Alternatively, a Fab fragment that binds to galectin-10 can be incubated with preformed galectin-10 crystals, and the ability of the Fab fragment to dissolve the crystals can be measured over a suitable time course. The galectin-10 crystals can be recombinant crystals formed from recombinant galectin-10 produced in vitro. Alternatively, the galectin-10 crystals can be crystals obtained from a patient sample, for example, a polyp in the patient's nasal cavity or paranasal sinuses. In certain embodiments, the Fab fragment that binds to galectin-10 can dissolve preformed galectin-10 crystals for a period of up to 10 hours, up to 12 hours, up to 14 hours, up to 16 hours, up to 18 hours, up to 20 hours. The Fab fragment that binds to galectin-10 can completely dissolve the crystals, i.e., 100%. Alternatively, a Fab fragment that binds to galectin-10 can dissolve the crystals such that greater than 50% of the crystals dissolve, greater than 60% of the crystals dissolve, greater than 70% of the crystals dissolve, greater than 80% of the crystals dissolve, or greater than 90% of the crystals dissolve over time.

[0063] After aerosolization, the Fab fragment that binds galectin-10 retains the ability to promote dissolution of crystalline galectin-10 when bound to crystalline galectin-10 and / or the ability to inhibit crystallization of galectin-10 when bound to soluble galectin-10. In certain embodiments, the Fab fragment promotes dissolution of crystalline galectin-10 after aerosolization to the same or a similar extent as the Fab fragment before aerosolization. In certain embodiments, the Fab fragment promotes dissolution of crystalline galectin-10 after aerosolization by 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the Fab fragment before aerosolization. In certain embodiments, the Fab fragment inhibits crystallization of soluble galectin-10 after aerosolization to the same or a similar extent as the Fab fragment before aerosolization. In certain embodiments, the Fab fragment inhibits crystallization of soluble galectin-10 after aerosolization by 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the Fab fragment before aerosolization.

[0064] C. Exemplary Fab Fragments that Bind Galectin-10 Described herein are exemplary Fab fragments that bind to Galectin-10 and are contemplated for use in the present invention. These Fab fragments serve as preferred Fab fragments for use in accordance with the present invention. Exemplary Fab fragments that bind to Galectin-10 can be defined solely with respect to their structural characteristics, as described below.

[0065] A preferred Fab fragment that binds galectin-10 and is contemplated for use in the present invention is the 24F02_N53A clone described herein.

[0066] Thus, in one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:4 [ka] HCDR3 comprising or consisting of: SEQ ID NO:3 [ka] HCDR2 comprising or consisting of: SEQ ID NO:2 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:8 [ka] LCDR3 comprising or consisting of: SEQ ID NO:7 [ka] LCDR2 comprising or consisting of: SEQ ID NO:6 [ka] LCDR1 comprising or consisting of Contains :.

[0067] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0068] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light chain (VL), wherein: The VH domain is represented by SEQ ID NO:1 [ka] and The VL domain is represented by SEQ ID NO:5 [ka] The amino acid sequence of

[0069] A preferred Fab fragment that binds galectin-10 and is contemplated for use in the present invention is the 24F02 clone described herein.

[0070] Thus, in one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:4 [ka] HCDR3 comprising or consisting of: SEQ ID NO:25 [ka] HCDR2 comprising or consisting of: SEQ ID NO:2 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:8 [ka] LCDR3 comprising or consisting of: SEQ ID NO:7 [ka] LCDR2 comprising or consisting of: SEQ ID NO:6 [ka] LCDR1 comprising or consisting of Contains :.

[0071] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0072] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light chain (VL), wherein: The VH domain is represented by SEQ ID NO:24 [ka] and The VL domain is represented by SEQ ID NO:5 [ka] The amino acid sequence of

[0073] A preferred Fab fragment that binds galectin-10 and is contemplated for use in the present invention is the 18C06 clone described herein.

[0074] Thus, in one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:13 [ka] HCDR3 comprising or consisting of: SEQ ID NO:12 [ka] HCDR2 comprising or consisting of: SEQ ID NO:11 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:17 [ka] LCDR3 comprising or consisting of: SEQ ID NO:16 [ka] LCDR2 comprising or consisting of: SEQ ID NO:15 [ka] LCDR1 comprising or consisting of Contains :.

[0075] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0076] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light chain (VL), wherein: The VH domain is represented by SEQ ID NO: 10 [ka] and The VL domain is represented by SEQ ID NO: 14 [ka] The amino acid sequence of

[0077] A preferred Fab fragment that binds galectin-10 and is contemplated for use in the present invention is the 20H09 clone described herein.

[0078] Thus, in one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:21 [ka] HCDR3 comprising or consisting of SEQ ID NO:20 [ka] HCDR2 comprising or consisting of: SEQ ID NO:19 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:8 [ka] LCDR3 comprising or consisting of: SEQ ID NO:7 [ka] LCDR2 comprising or consisting of: SEQ ID NO:6 [ka] LCDR1 comprising or consisting of Contains :.

[0079] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0080] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light chain (VL), wherein: The VH domain is represented by SEQ ID NO: 18 [ka] and The VL domain is represented by SEQ ID NO:5 [ka] The amino acid sequence of

[0081] A preferred Fab fragment that binds galectin-10 and is contemplated for use in the present invention is the 23H09 clone described herein.

[0082] Thus, in one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:21 [ka] HCDR3 comprising or consisting of: SEQ ID NO:23 [ka] HCDR2 comprising or consisting of: SEQ ID NO:19 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:8 [ka] LCDR3 comprising or consisting of: SEQ ID NO:7 [ka] LCDR2 comprising or consisting of: SEQ ID NO:6 [ka] LCDR1 comprising or consisting of Contains :.

[0083] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0084] In one embodiment, a Fab fragment comprises a variable heavy (VH) domain and a variable light chain (VL), wherein: The VH domain is represented by SEQ ID NO:22 [ka] and The VL domain is represented by SEQ ID NO: [ka] The amino acid sequence of

[0085] For embodiments in which the domains of the Fab fragment are defined by a specific percent sequence identity to a reference sequence, the VH and / or VL domains may retain identical CDR sequences as present in the reference sequence, such that variations exist only within the framework regions.

[0086] In certain embodiments, the Galectin-10 Fab fragments described herein may be derived from a camelid. For example, the Galectin-10 Fab fragments may be selected from an immune library obtained by a method comprising immunizing a camelid with a target of interest, i.e. Galectin-10. The camelid may be immunized with a target protein or a polypeptide fragment thereof, or with an mRNA or cDNA molecule expressing said protein or a polypeptide fragment thereof. Methods for producing antibodies or fragments in camelid species and selecting antibodies or fragments against preferred targets from camelid immune libraries are described, for example, in International Patent Application WO2010 / 001251, which is incorporated herein by reference.

[0087] In certain embodiments, the Galectin-10 Fab fragment may be derived from a Camelid in that it comprises at least one hypervariable (HV) loop or complementarity determining region obtained from a VH or VL domain of a species of the Camelidae family. In particular, the Galectin-10 Fab fragment may comprise a VH and / or VL domain, or CDRs thereof, obtained by active immunization of an outbred Camelid, e.g., a llama, with Galectin-10.

[0088] The term "derived from" in this context refers to a structural relatedness in that the HV or CDRs of the Fab fragment embody an amino acid sequence (or a minor variant thereof) originally encoded by a Camelidae immunoglobulin gene, however, this does not necessarily imply a particular relatedness with respect to the production process used to prepare the Fab fragment.

[0089] The camelid-derived Fab fragment may be derived from any camelid species, including inter alia, llama, dromedary, alpaca, vicuna, guanaco, or camel.

[0090] Camelid-derived VH and VL domains, or Fab fragments comprising the CDRs thereof, are usually recombinantly expressed polypeptides and may be chimeric polypeptides. The term "chimeric polypeptide" refers to an artificial (non-natural) polypeptide created by the juxtaposition of two or more peptide fragments that do not otherwise occur contiguous. Included in this definition are "species" chimeric polypeptides created by the juxtaposition of peptide fragments encoded by two or more species, e.g., camelid and human.

[0091] In certain embodiments, the entire VH domain and / or the entire VL domain can be obtained from a species of the Camelidae family. The Camelid-derived VH domain and / or the Camelid-derived VL domain can then be subjected to protein engineering, in which one or more amino acid substitutions, insertions or deletions are introduced into the Camelid amino acid sequence. These artificially created changes preferably include amino acid substitutions relative to the Camelid sequence. Such changes include "humanization" or "germlining", in which one or more amino acid residues in a Camelid-encoded VH or VL domain are replaced with the equivalent residue from a homologous human-encoded VH or VL domain.

[0092] Isolated camelid VH and VL domains obtained by active immunization of camelids (e.g. llamas) with galectin-10 can be used as a basis for artificially generating galectin-10 Fab fragments according to the present invention. Starting from intact camelid VH and VL domains, it is possible to artificially generate one or more amino acid substitutions, insertions or deletions which deviate from the starting camelid sequence. In certain embodiments, such substitutions, insertions or deletions may be present in the framework regions of the VH and / or VL domains.

[0093] In other embodiments, "chimeric" molecules are provided that comprise camelid-derived VH and VL domains (or artificially created variants thereof) and one or more constant domains from a non-camelid antibody, such as a human-encoded constant domain (or artificially created variants thereof). In such embodiments, it is preferred that both the VH and VL domains are derived from the same species of camelid, for example (prior to the introduction of artificially created amino acid sequence mutations), both the VH and VL may be derived from a llama (Llama glama), or both the VH and VL may be derived from an alpaca (Llama pacos). In such embodiments, both the VH and VL domains may be derived from a single animal, in particular a single animal that has been actively immunized with an antigen of interest.

[0094] As an alternative to artificially creating changes in the primary amino acid sequences of camelid VH and / or VL domains, individual camelid-derived hypervariable loops or CDRs, or combinations thereof, can be isolated from camelid VH / VL domains and transferred by CDR grafting into an alternative (i.e. non-camelid) framework, for example a human VH / VL framework.

[0095] In non-limiting embodiments, an exemplary Galectin-10 Fab fragment having the CDR, VH and / or VL sequences described herein can comprise a CH1 domain and / or a CL domain (from the heavy and light chain, respectively), the amino acid sequence of which is fully or substantially human. For molecules intended for therapeutic use in humans, it is typical that the entire constant region, or at least a portion thereof, has a fully or substantially human amino acid sequence. Thus, one or both of the CH1 and CL domains of the Fab fragments described herein may be fully or substantially human in terms of their amino acid sequence. In the context of the constant region of a humanized Fab fragment, the term "substantially human" refers to at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% amino acid sequence identity with the human constant region.

[0096] (D. Therapeutic use) The Fab fragments described herein can promote dissolution of crystalline galectin-10 and / or inhibit crystallization of soluble galectin-10, and are therefore useful in methods of preventing and treating diseases and disorders whose pathology is associated with the formation / presence of Charcot-Leyden crystals (CLC). When used as medicines, the Fab fragments that bind to galectin-10 are typically formulated as pharmaceutical compositions. Thus, the pharmaceutical compositions of the present invention, comprising a Fab fragment that binds to galectin-10, can be used in methods of treating diseases or disorders associated with the presence or formation of galectin-10 crystals.

[0097] In a first aspect of the present invention there is provided a pharmaceutical composition comprising a Fab fragment that binds to galectin-10 for use in a method for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition, wherein the composition is administered as an aerosol.

[0098] In a second related aspect of the present invention, there is provided a method for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is administered as an aerosol.

[0099] Importantly, all embodiments described with respect to therapeutic applications are equally applicable to both the first and second aspects of the invention.

[0100] In a preferred embodiment, the pharmaceutical composition comprising a Fab fragment that binds to Galectin-10 is a pharmaceutical composition described elsewhere herein.

[0101] The disease or condition to be treated may be any disease or condition associated with the presence or formation of galectin-10 crystals.The disease or condition to be treated may be any disease or condition associated with the presence or formation of CLCs.

[0102] In some embodiments, the subject in need thereof may be a patient diagnosed with a disease or disorder associated with the presence or formation of galectin-10 crystals. In other embodiments, the subject in need thereof may be a patient identified as being "at risk" of developing a disease or disorder associated with the presence or formation of galectin-10 crystals. For patients with a disease or disorder characterized by the presence of galectin-10 crystals, the treatment method typically involves administration of a pharmaceutical composition comprising a Fab fragment capable of binding to galectin-10 and dissolving galectin-10 crystals in the patient's tissue. For patients identified as being "at risk" of developing a disease or disorder characterized by the formation of galectin-10 crystals, the treatment method may involve administration of a pharmaceutical composition comprising a Fab fragment capable of binding to galectin-10 and inhibiting crystallization of galectin-10 crystals.

[0103] Galectin-10 crystals or CLCs have been observed in patients with various diseases and disorders. In certain non-limiting embodiments, the disease or disorder associated with the presence or formation of galectin-10 crystals can be selected from the group consisting of asthma; chronic rhinosinusitis; celiac disease; helminth infection; gastrointestinal eosinophilic inflammation; cystic fibrosis (CF); allergic bronchopulmonary aspergillosis (ABPA); Churg-Strauss vasculitis; chronic eosinophilic pneumonia; and acute myeloid leukemia. In certain preferred embodiments, the disease or disorder associated with the presence or formation of galectin-10 crystals is asthma or cystic fibrosis (CF).

[0104] As mentioned above, galectin-10 crystals or CLCs are particularly associated with diseases or conditions characterized by eosinophilic inflammation. In a preferred embodiment, the pharmaceutical compositions described herein are used to treat diseases or conditions associated with eosinophilic inflammation.

[0105] As used herein, a method of "treating" a disease or disorder means curing the disease or disorder and / or alleviating or eradicating symptoms associated with the disease or disorder such that the patient's suffering is alleviated.

[0106] As used herein, the term "therapeutically effective amount" is intended to mean the amount or dose of the Fab fragment present in the pharmaceutical composition that is sufficient to produce a therapeutic effect, e.g., the amount or dose of the Fab fragment required to eradicate or at least alleviate symptoms associated with a disease or disorder. The appropriate amount or dose can be determined by a physician as necessary. For example, the dose can be adjusted based on factors such as the size or weight of the subject to be treated, the age of the subject to be treated, the general health of the subject to be treated, the disease to be treated, and the route of administration.

[0107] For clinical use, in certain embodiments, the pharmaceutical composition is administered to a subject in need thereof in one or more doses with a concentration of the Fab fragment of about 0.1 mg / kg body weight to about 20 mg / kg body weight. In certain embodiments, the pharmaceutical composition is administered to a subject in need thereof in one or more doses with a concentration of the Fab fragment of about 0.1 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the pharmaceutical composition is administered to a subject in need thereof in one or more doses with a concentration of the Fab fragment of about 0.5 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the pharmaceutical composition is administered to a subject in need thereof in one or more doses with a concentration of the Fab fragment of about 1 mg / kg body weight to about 10 mg / kg body weight.

[0108] (E. Pharmaceutical Preparations) When used as a pharmaceutical, the Fab fragment that binds to galectin-10 is usually formulated as a pharmaceutical composition. Thus, the scope of the present invention includes pharmaceutical compositions comprising a Fab fragment that binds to galectin-10.

[0109] In a first aspect of the present invention, there is provided a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, for use in a method for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, as previously described elsewhere herein, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition, wherein the composition is administered as an aerosol.

[0110] In a related third aspect, there is provided a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is formulated for aerosolization.

[0111] Importantly, all of the embodiments described with respect to the pharmaceutical formulation are equally applicable to both the first and third aspects of the invention.

[0112] As used herein, the term "formulated for aerosolization" means that the pharmaceutical composition is suitable for aerosolization, e.g., by nebulization. The active ingredient present in the pharmaceutical composition, e.g., a Fab fragment that binds to galectin-10, must be medicamentously active after aerosolization, must be delivered to the lungs to the appropriate site of action while avoiding pulmonary clearance mechanisms, and must remain there until the desired pharmacological effect occurs. Pharmaceutical compositions formulated for aerosolization can have a physiological pH and osmolality to avoid irritation, coughing, and pulmonary bronchoconstriction upon inhalation. The physical properties of the drug formulation can affect the aerosolization rate and aerosol droplet size. Pharmaceutical compositions can have a viscosity, ionic strength, and / or surface tension that is particularly suitable for aerosolization. For example, pharmaceutical compositions can contain viscosity enhancing agents and / or electrolytes that reduce the effect of surface charge resulting in smaller atomized droplet sizes for efficient targeting to the lungs. Pharmaceutical compositions can contain agents that protect active proteins from proteolysis in the lungs.

[0113] In some embodiments, prior to aerosolization, the pharmaceutical composition is an aqueous liquid formulation. In some preferred embodiments, prior to aerosolization, the pharmaceutical composition is an aqueous liquid formulation comprising phosphate buffered saline. In some embodiments, prior to aerosolization, the pharmaceutical composition is a non-aqueous liquid formulation. In some embodiments, prior to aerosolization, the pharmaceutical composition is a suspension. In some embodiments, prior to aerosolization, the pharmaceutical composition is a powder.

[0114] The pharmaceutical composition may contain a surfactant. Surfactants aid in wetting and dispersing hydrophobic medicament active ingredients, and they usually act by lowering the interfacial tension between solids and liquids in a suspension. Surfactants are often used to prevent the formation of aggregates in biological preparations. In some embodiments, the pharmaceutical composition comprises one or more surfactants. In some embodiments, the pharmaceutical composition comprises one or more surfactants at a concentration of 0.001% to 0.1% w / v. In some embodiments, the pharmaceutical composition comprises one or more surfactants at a concentration of about 0.02% w / v. In some embodiments, the pharmaceutical composition comprises one or more surfactants at a concentration of less than 0.05% w / v. Those skilled in the art are aware of commercially available surfactants for use in pharmaceutical formulations, including, but not limited to, polysorbates, sorbitan esters, oleic acid, soy lecithin, sodium lauryl sulfate, sodium docusate, phosphatidylcholine, polyoxyethylene 15 hydroxystearate, polyoxyethylene alkyl ether, Brij-35®, Tween-20, Tween-80, polysorbate 20, and polysorbate 80. In some embodiments, the pharmaceutical composition comprises polysorbate 20. In some embodiments, the pharmaceutical composition comprises less than 0.05% polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 0.02% polysorbate 20. In some embodiments, the pharmaceutical composition comprises Tween 80. In some embodiments, the pharmaceutical composition comprises less than 0.05% Tween 80. In some embodiments, the pharmaceutical composition comprises about 0.02% Tween 80.

[0115] As shown in the examples herein, when aerosolized, the pharmaceutical composition of the present invention produces few protein particles and therefore shows less susceptibility to aggregation.As a result, the pharmaceutical composition of the present invention may not require the addition of surfactants or may require the addition of only small amounts of surfactants.This may be particularly beneficial since evidence suggests that the presence of surfactants may adversely affect the molecular integrity of antibodies in therapeutic formulations (Maillet et al., 2007; Pharmaceutical Research; 25(6): 1318-1326).

[0116] Thus, in some embodiments, the pharmaceutical composition does not contain a surfactant, hi other embodiments, the composition comprises a surfactant in an amount of less than 0.05% w / v, less than 0.01% w / v, less than 0.002% w / v, or less than 0.001% w / v.

[0117] In some embodiments, the pharmaceutical composition comprises a Fab fragment. In some preferred embodiments, the pharmaceutical composition comprises a Fab fragment that binds to Galectin-10. The pharmaceutical composition may comprise any of the Fab fragments described herein. It is particularly preferred that the pharmaceutical composition comprises the Fab fragment described herein as 24F02_N53A.

[0118] Thus, in one preferred embodiment, the pharmaceutical composition comprises a Fab fragment comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain is SEQ ID NO:4 [ka] HCDR3 comprising or consisting of: SEQ ID NO:3 [ka] HCDR2 comprising or consisting of: SEQ ID NO:2 [ka] HCDR1 comprising or consisting of Contains; and wherein the VL domain is SEQ ID NO:8 [ka] LCDR3 comprising or consisting of: SEQ ID NO:7 [ka] LCDR2 comprising or consisting of: SEQ ID NO:6 [ka] LCDR1 comprising or consisting of Contains :.

[0119] In one embodiment, the pharmaceutical composition comprises a Fab fragment comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises an amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and The VL domain comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto.

[0120] In one embodiment, the pharmaceutical composition comprises a Fab fragment comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain is represented by SEQ ID NO:1 [ka] and The VL domain is represented by SEQ ID NO:5 [ka] The amino acid sequence of

[0121] Fab fragments must be present in a suitable concentration in pharmaceutical compositions to be pharmacologically effective. If the drug concentration is too high, foaming may occur in some nebulizers, making aerosolization inefficient or even impossible. Therefore, the concentration of Fab fragments should be sufficient to limit protein aggregation both before and after aerosolization, and to prevent foaming or bubbling of the composition in aerosolization devices.

[0122] In certain embodiments of the pharmaceutical composition, the concentration of the Fab fragment in the composition is about 1 to about 20 mg / ml, about 1 to about 15 mg / ml, about 1 to about 10 mg / ml, about 1 to about 5 mg / ml, about 2 to about 10 mg / ml, about 3 to about 10 mg / ml, about 4 to about 10 mg / ml, or about 5 to about 10 mg / ml.

[0123] In certain embodiments of the pharmaceutical composition, the concentration of the Fab fragment in the composition is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / ml.

[0124] In a preferred embodiment of the pharmaceutical composition, the concentration of the Fab fragment in the composition is about 3 mg / ml. In a preferred embodiment of the pharmaceutical composition, the concentration of the Fab fragment in the composition is about 10 mg / ml.

[0125] The scope of the present invention includes pharmaceutical compositions containing Fab fragments that bind to Galectin-10 as described herein, formulated with one or more pharma- ceutically acceptable carriers or excipients. Thus, in certain embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier or excipient. In certain embodiments, the excipient is a liquid excipient, optionally a solvent or an aqueous solvent.

[0126] Pharmaceutically acceptable excipients that may be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, sodium citrate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances such as sodium carboxymethylcellulose, polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, glycerol, solubilizers such as ethanol, cyclodextrins, stabilizers such as polysorbate 20, polysorbate 80, PVP K30, Solutol®, and viscosity enhancers such as sucrose.

[0127] The examples described herein utilize a base formulation of either 3 mg / ml or 10 mg / ml of Fab fragments in (Dulbecco's) phosphate buffered saline with 0.02% polysorbate 20 or Tween 80. Thus, in certain embodiments, the pharmaceutical composition formulated for aerosolization comprises a Fab fragment and a surfactant, e.g., polysorbate 20. In certain embodiments, the pharmaceutical composition formulated for aerosolization comprises a Fab fragment and a surfactant, e.g., Tween 80. In certain embodiments, the pharmaceutical composition formulated for aerosolization comprises a Fab fragment and a surfactant, e.g., polysorbate 20 in phosphate buffered saline. In certain embodiments, the pharmaceutical composition formulated for aerosolization comprises a Fab fragment and a surfactant, e.g., Tween 80 in phosphate buffered saline. In a specific embodiment, the pharmaceutical composition formulated for aerosolization comprises a Fab fragment at 1-10 mg / ml and a surfactant, e.g., polysorbate 20, at a concentration of 0.01-0.05%. In a specific embodiment, the pharmaceutical composition formulated for aerosolization comprises 1-10 mg / ml of Fab fragments and a surfactant at a concentration of 0.01-0.05%, such as Tween 80. In a specific embodiment, the pharmaceutical composition formulated for aerosolization comprises 1-10 mg / ml of Fab fragments in phosphate buffered saline and a surfactant at a concentration of 0.01-0.05%, such as polysorbate 20. In a specific embodiment, the pharmaceutical composition formulated for aerosolization comprises 1-10 mg / ml of Fab fragments in phosphate buffered saline and a surfactant at a concentration of 0.01-0.05%, such as Tween 80.

[0128] F. Route of Administration The pharmaceutical composition of the present invention is intended to be delivered as an aerosol to the conducting airways. The conducting airways include the nose, pharynx, larynx, trachea, bronchi, and bronchioles. These structures form a continuous passageway through which air enters and leaves the lungs. The therapeutic effect of aerosolization therapy depends on the dose of drug deposited in the lungs and its distribution. Various inhalation devices are available for the delivery of inhaled drugs. Most inhaled drugs are delivered by pressurized metered dose inhalers (pMDIs), dry powder inhalers, or nebulizers. However, other devices exist, such as soft mist inhalers and smart inhalers.

[0129] Metered dose inhalers (MDIs) are the mainstay of asthma and COPD therapy worldwide. MDIs are pocket-sized, portable drug delivery devices that utilize the energy of compressed propellants to generate an aerosol. They have the drug dissolved or suspended in a propellant or a mixture of propellant and cosolvent. MDIs deliver small drug doses directly to the patient or via add-on devices such as spacers or valved holding chambers (VHCs). In some embodiments, the pharmaceutical composition is administered as an aerosol using a metered dose inhaler, e.g., a pressurized metered dose inhaler (pMDI).

[0130] A dry powder inhaler (DPI) is a device that delivers medication to the lungs in the form of a dry powder. DPIs are commonly used to treat respiratory diseases such as asthma, bronchitis, emphysema, and COPD. Dry powder aerosols are created by passing air through an aliquot of free-flowing powder. DPIs are actuated by breathing, eliminating the need to synchronize inhalation with actuation. However, dispersion of the powder into respirable fine particles relies on the generation of turbulence within the inhaler. Most powder dispensing systems require the use of a carrier substance. When this vehicle substance is mixed with the drug, it allows the powder to be more easily expelled from the device. Commonly used carriers include lactose and glucose. In some embodiments, where the pharmaceutical composition is in powder form, the pharmaceutical composition is administered as an aerosol using a dry powder inhaler (DPI).

[0131] A nebulizer changes a drug from a liquid into a mist so that it can be inhaled into the lungs. A nebulizer consists of a base that holds an air compressor, a small container for the liquid drug, and a tube that connects the air compressor to the drug container. On top of the drug container is a mouthpiece or mask that is used to inhale the mist. There are three main types of nebulizers: jet nebulizers, which use compressed gas to create an aerosol; ultrasonic nebulizers, which create an aerosol through high frequency vibrations; and mesh nebulizers, in which a liquid passes through a very fine mesh to form an aerosol. In some embodiments, the pharmaceutical composition is administered as an aerosol using a nebulizer. In some embodiments, the pharmaceutical composition is administered as an aerosol using a jet nebulizer. In some embodiments, the pharmaceutical composition is administered as an aerosol using an ultrasonic nebulizer. In some embodiments, the pharmaceutical composition is administered as an aerosol using a mesh nebulizer.

[0132] According to a fourth aspect of the present invention, there is provided a nebulizer comprising a pharmaceutical composition as described elsewhere herein. In certain embodiments, there is provided a nebulizer comprising a pharmaceutical composition comprising a Fab fragment that binds to galectin-10, wherein the composition is formulated for aerosolization.

[0133] Each aerosolization device generates its drug aerosol differently, resulting in, for example, different aerosol droplet sizes, drug yields, total lung deposition and distribution, and therefore, it may be necessary to match the pharmaceutical composition to the performance of the particular inhalation device being used.

[0134] The aerosol droplets produced during aerosolization must be in the size range required for pulmonary deposition. Typically, an aerosol droplet size of 1-5 μm is required for deposition in the smaller airways and alveoli. Furthermore, the aerosol droplets should be as close to monodisperse as possible to increase deposition at the desired site of action and to increase efficacy of the treatment. Aerosol droplet size can be measured using laser diffraction and is usually reported as either volume mean diameter (VMD) or mass median aerodynamic diameter (MMAD). As shown in Example 1 (FIG. 1), aerosolization of the pharmaceutical compositions described herein generated aerosol droplets within the size range required for pulmonary deposition. In particular, the VMD after nebulization of the antibody formulation with the Aerogen® Solo device was 4.8 μm, and the VMD after nebulization with the Pari eFlow® device was 4.3 μm.

[0135] The process of aerosolization exposes the proteins in the pharmaceutical composition to physical stresses such as shear forces and heat, as well as large air-liquid interfaces that can alter the conformation and / or structure of the protein through denaturation, chemical modification (oxidation, deamidation), and aggregation. Inherent limitations of the device, such as shear forces generated by jet nebulizers and elevated temperatures generated by ultrasonic nebulizers, can also lead to protein degradation and increased undesirable aggregates. The presence of protein aggregates can have a significant impact on the quality of the product in terms of biological activity and immunogenicity. Adverse immune reactions, such as anti-drug antibody (ADA) responses, can have clinical consequences, such as anaphylaxis, reduced drug half-life, and neutralization of therapeutic proteins. It is therefore important to be able to monitor the level of protein aggregates after aerosolization and limit their number.

[0136] Protein aggregates can range in size from microscopic to macroscopic. These subvisible and visible particles can be detected in protein-containing solutions using a variety of recognized industry techniques. Biochemical assays for monitoring protein aggregates often rely on ultracentrifugation, size exclusion chromatography, gel electrophoresis, dynamic light scattering, flow cell microscopy, or turbidity measurements. As described in the examples herein, pharmaceutical compositions can be nebulized and the aerosol droplets can be collected and condensed, so that particle analysis can be performed on the nebulized solution. In the examples herein, particle size and concentration are determined using a combination of dynamic light scattering and flow cell microscopy; however, it is understood that other well-known techniques can be utilized to detect and quantify protein aggregates after aerosolization. What is important is that the number of visible and subvisible particles should be as low as possible. In particular, the number of particles greater than 2 μm in diameter should be as low as possible, since particles of this size are often associated with immunogenicity and severe ADA responses.

[0137] Thus, in some embodiments, the particle size of the particles in the aerosol is less than 30 μm, less than 25 μm, less than 10 μm, less than 2 μm, less than 1 μm, less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm. Preferably, the particle size of the particles in the aerosol is less than 2 μm.

[0138] Particle size in this case may refer to the size of all of the particles (protein aggregates) present in the aerosolized sample, or may refer to the size of the majority of the particles (protein aggregates) present in the aerosolized sample. For example, an aerosolized composition having a particle size of less than 2 μm may mean that all of the particles in the aerosol have a diameter of less than 2 μm. Alternatively, an aerosolized composition having a particle size of less than 2 μm may mean that the majority of the particles in the aerosol have a diameter of less than 2 μm, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of all particles in the aerosol have a diameter of less than 2 μm.

[0139] The concentration of particles (aggregates) can be quantified as the total number of particles per ml. Preferably, the total number of particles produced after aerosolization is greater than 1×10 per ml. 5 The concentration of particles can also be quantified as the number of particles of a certain size, for example, the number of particles greater than 2 μm in size per ml, or the number of particles greater than 10 μm in size per ml, or the number of particles greater than 25 μm in size per ml.

[0140] Thus, in one embodiment, the concentration of particles in the aerosol is greater than or equal to 2×10 5 Less than 1×10 5 Less than 2 x 10 4 Less than 1 x 10 4 The total particles are less than 100 / ml.

[0141] In one embodiment, the concentration of particles greater than 2 μm in the aerosol is greater than or equal to 1×10 5 Less than 5 x 10 4 Less than 2 x 10 4 Less than 1×10 4 Less than 1×10 3 Less than 1 x 10 2 Fewer than 10 particles >2 μm / ml.

[0142] The invention will be further understood with reference to the following non-limiting examples. EXAMPLES

[0143] (Example) Example 1: Effect of Nebulization on Aggregate Formation - Comparison of Fab with IgG and VHH One of the problems with nebulizing biologics such as antibodies is the formation of aggregates. These aggregates have various functional characteristics and can induce anti-drug antibodies. Therefore, in Example 1, the effect of nebulization on aggregate formation is evaluated.

[0144] The objective of this study was to evaluate the stability and biological activity of five different full-length antibodies or antibody fragments formulated in PBS containing 0.02% Tween 80 at two different concentrations after nebulization with two mesh nebulizers (commercially available).

[0145] This experiment compared the number of aggregates after nebulization of three IgGs, one Fab, and one VHH. Each sample was tested at concentrations of 3 mg / ml and 10 mg / ml. Included in the experiment were the 7B07 clone in Fab format, the corresponding 7B07 clone in IgG format, two more IgGs (10A06 clone and 08H11 clone), and a VHH (1D06 clone).

[0146] Upon arrival, frozen samples were kept at -20°C. Prior to nebulization, aliquots (of the same antibody) were pooled into 15mL polypropylene conical tubes and the solutions were stored at 4°C. Protein concentrations were determined and adjusted to 3 or 10mg / mL with PBS 0.02% Tween 80 as necessary. Samples were filtered using 0.22μm Minisart® High Flow syringe filters (Sartorius). Protein concentration, visual inspection, dynamic light scattering (DLS), flow microscopy (FCM), and size exclusion chromatography (SEC) were performed on samples before and after nebulization.

[0147] Nebulization was performed using two different commercially available vibrating mesh nebulizers in the base formulation: the Aerogen® Solo nebulizer and the PARI eFlow® electronic nebulizer. Both devices are active vibrating mesh nebulizers that convert the drug solution into small aerosol droplets. As with other active devices, the aperture plate vibrates at high frequency, drawing in solution through openings in the plate and generating aerosol droplets. Each sample was nebulized in triplicate using three different Pari eFlow® mesh generators. Prior to antibody nebulization, each mesh nebulizer was characterized by laser diffraction (no inhalation chamber and no closed cell) with 0.9% NaCl solution and found to be equivalent (volume mean diameter = 5.6 μm). Similarly, three different Aerogen® Solo aerosol generator devices were used in triplicate. Prior to nebulization of the formulation, the three mesh nebulizers were characterized by laser diffraction (without closed cell) in 0.9% NaCl solution and found to be equivalent (volume mean diameter for devices s0087 and s0094 = 4.8 μm, volume mean diameter for device s0210 = 4.9 μm).

[0148] Each sample was nebulized and the time of nebulization was monitored. Aerosol droplets were collected using a 15 mL polypropylene conical tube (ref: 352097, Dominique Dutscher) placed just before a vibrating mesh, leaving enough space for aerosol formation and allowing condensation of the nebulized solution. This further condensation step on the surface of the tube could be stressful for the protein and thus represents a worst case compared to what could happen in vivo. Aerosol collection was performed under laminar flow.

[0149] The cleaning and spraying process was similar for the two devices. Process: 1. Immerse the mesh and other parts of the nebulizer in a bath of hot tap water containing 5 mL / L of Surfanios Premium, mix briefly, and wait 5 to 10 minutes. 2. Rinse the mesh and other parts of the nebulizer thoroughly with hot tap water. 3. Spray with 3mL of hot tap water. 4. Nebulize with 1.2 mL (for Aerogen® Solo) or 2 x 1.2 mL (for Pari eFlow®) of 0.22 μm filtered PBS pH 7.2. 5. Nebulize with 1.2 mL of 0.22 μm filtered demineralized water and collect the aerosol. 6. Verify by FCM that particle counts are within laboratory defined limits (<200 particles for Aerogen® Solo and <300 particles for Pari eFlow®). Repeat from step 4 or step 1 if necessary. 7. Spray with 1.2 mL of 0.22 μm filtered PBS 0.02% Tween 80 buffer. 8. Spray 1.2 mL of sample. 9. Start from step 1 with another sample.

[0150] All nebulizations were achieved with the same controller: USB Aerogen or eBase Controller. These controllers provide continuous nebulization. Nebulizations were timed to determine the flow rate for each device / sample (flow rate = time of nebulization / 1.2 mL).

[0151] (1.1 Particle size distribution) Laser diffraction experiments were carried out with a Spraytec™ instrument (Malvern Instruments Ltd.). This technique was used to determine the size of the aerosol droplets generated by the nebulizer. Laser diffraction allows the measurement of the VMD (Volume Mean Diameter), which is the median size of the spherical aerosol particles. 1 mL of NaCl 0.9% or 0.5 mL of antibody solution for device characterization was used. The experiments were carried out at room temperature (20-25°C) and with or without a closed cell for samples or qualification, respectively. In the closed cell, the aerosol droplets were sucked in by a constant suction pump with an airflow of 30 L / min on the opposite side of the aerosol beam.

[0152] Aerosols were analyzed by laser diffraction. Device qualification in NaCl 0.9% was performed without a closed cell and gave VMD results of 4.8 and 5.6 for the Aerogen® Solo and Pari eFlow®, respectively.

[0153] For the Ab formulation, laser diffraction was performed with a closed cell, which had no effect on the VMD after nebulization with the Aerogen® Solo device and reduced the VMD to 4.3 μm with the Pari eFlow® (Figure 1).

[0154] No significant differences in aerosol droplet size were observed between the different Ab formulations and saline solution (with closed cells).

[0155] (1.2 Protein Concentration) Protein concentration was determined before and after nebulization by measuring absorbance at 280 nm on 2 μl of solution using a Nanodrop 2000 from Thermo Fisher Scientific.

[0156] Samples were filtered before spraying and protein concentration was measured using a spectrophotometer. If necessary, the concentration was adjusted to 3 or 10 mg / mL before spraying. After spraying, protein concentration was measured to determine the loss of biological material during spraying. The raw data and the concentration variation before and after spraying are shown in Figure 2. The concentration changed from -12% to +12% before and after spraying.

[0157] (1.3 Visual inspection of particles) The antibody solutions before and after nebulization were observed in 15 mL polypropylene conical tubes under natural light for visible particles.

[0158] No particles were observed in the vials before nebulization and after filtration. Visible particles were observed in most samples after nebulization (Table 2). Notably, no particles were seen with 7B07 Fab (as opposed to full-length 7B07 IgG) after nebulization.

[0159] It is noteworthy that nebulization of 1D06 VHH resulted in the formation of many visible particles and the solution was cloudy compared to the other samples. In some samples and in the case of buffer only, bubbles were observed in the reservoir of the device. Table 2: Visual inspection of sprayed antibody solutions [Table 2]

[0160] (1.4 Subvisible particles by flow cell microscopy (FCM)) Flow cell microscopy (Flow-Imaging FC200S instrument, Occhio) is an imaging technique applied to detect and measure subvisible and visible particles in protein-containing solutions.

[0161] Before each analysis, the device was washed with demineralized filtered water. To proceed further, the number of measured particles had to be <100. 250 μL of each sample (buffer only and antibody solution) was analyzed by FCM before and after nebulization. The average number of particles with buffer on the mesh was subtracted from the sample value. The number of particles per mL was expressed for particles with equivalent diameters >2 μm, >10 μm, and >25 μm.

[0162] Samples were analyzed by FCM before and immediately after nebulization. The results are presented in Figure 3. The total number of particles should be as low as possible. Preferably, the total number of particles is less than 1 x 10 per ml. 5 This data provides the first evidence that a Fab format biologic has reduced susceptibility to aggregation upon nebulization.

[0163] Prior to analysis, the background was assessed + / - nebulized buffer (PBS 0.02% Tween 80). Results showed a mean of 1208 total particles before nebulization. After nebulization, the mean total particles increased to 3918 and 10203 for the Aerogen® Solo and Pari eFlow® devices, respectively.

[0164] Before nebulization, samples had few particles. After nebulization, the number of particles (>2 μm) usually increased and was sample dependent. Overall, there were more particles at 10 mg / mL than at 3 mg / mL, except for 1D06 VHH.

[0165] For full-length IgG: 10A06 showed the most particles (average of 104381 ​​and 97476 at 3 mg / mL for the Aerogen® Solo and Pari eFlow® devices, respectively). 8H11 IgG produced the fewest particles (average of 1,160 and 1,737 at 3 mg / mL and 9.744 and 776 at 10 mg / mL after nebulization for the Aerogen® Solo and Pari eFlow® devices, respectively).

[0166] Compared to 7B07 IgG, the Fab format produced fewer particles (>2 μm) after nebulization (2349 and 3183 at 3 mg / mL and 6040 and 20353 at 10 mg / mL for the Aerogen® Solo and Pari eFlow® devices, respectively). The worst sample was the 1D06 VHH, which produced the most particles (>2 μm) (2,149,534 and 1,874,236 particles at 10 mg / mL for the Aerogen® Solo and Pari eFlow® devices, respectively).

[0167] In most cases, particle levels were higher following nebulization with the Pari eFlow® mesh compared to the Aerogen® Solo device.

[0168] (1.5 Submicron Particles by Dynamic Light Scattering (DLS)) Dynamic light scattering experiments were performed at 25 °C on a Dynapro Nanostar® instrument (Wyatt Technology, Europe GmbH) using a 659 nm / 100 mW laser and a detection angle of 90° to determine the size distribution profile of the solutions before and after nebulization. 100 μL of sample was placed in a single disposable cuvette (UVette 80, Eppendorf). For each sample, we performed 10 acquisitions every 7 seconds. The results were analyzed with Dynamics® (7.1.5.6) software. Samples with less than 70% acquisition success were considered "unassessable" as recommended by the manufacturer. In this case, the solutions were filtered at 0.45 μm. The dynamic data filter was set as follows: baseline limit ± 0.01 and maximum SOS 100. The results were expressed as mass percentage of monomer and other species. The analysis yielded several parameters including the hydrodynamic radius of the molecules, polydispersity, and polydispersity index. Polydispersity refers to the level of uniformity of the size of particles. If the polydispersity percentage is less than 15%, the level of uniformity is considered high. The polydispersity index is based on cumulant analysis. It is equivalent to the distribution width divided by the mean.

[0169] Samples were analyzed by DLS before and immediately after spraying. The results are presented in Figure 4. Before spraying, the mass percentage of the major species (main peak) in solution was similar for all formulations. The radius of the main peak was consistent with the size of the protein.

[0170] After nebulization, most samples could not be analyzed by DLS, likely due to the amount of large particles (as demonstrated by FCM). Samples that could be analyzed were 08H11 IgG at 3 mg / ml and 10 mg / ml for the two devices, 7B07 Fab at 3 mg / ml, and 7B07 Fab at 10 mg / ml after nebulization with the Aerogen® Solo device.

[0171] In cases where multimodal results were obtained without filtration, the samples were analyzed after 0.45 μm filtration. However, applying filtration did not improve the DLS results, as most of the samples remained multimodal even after filtration. No significant variation was observed between the percentage of polydispersity and the PDI. Overall, the DLS results were consistent with those of the FCM.

[0172] 1.6 Size Purity by SEC-HPLC After dilution of samples to 1 mg / ml and 0.22 μm filtration, size exclusion chromatography (SEC) analysis was performed on an Ultimate™ 3000 UHPLC system equipped with an in-line. For each sample, 50 μg was injected by an Ultimate™ autosampler onto a Bio SEC-3 column (3 μm, 300 Å, 7.8×300 mm) from Agilent Technologies (Santa Clara, CA) and separated at a flow rate of 1 mL / min. The elution buffer was PBS pH 7.2 and UV detection was performed at 280 nm using an Ultimate™ diode array detector (Thermo Fisher Scientific). Results are expressed as percentages of monomer and high molecular weight species (HMWS).

[0173] Samples were analyzed by size exclusion chromatography before and after nebulization. Results are presented in Figure 5. Before nebulization, the mass percentage of the major species (main peak) in solution varied for all formulations and concentrations. 7B07 IgG showed a monomer peak below 95%. After nebulization, the percentage of monomer decreased in some samples. For full-length IgG, nebulization of 7B07 showed the most significant loss, especially at 3 mg / mL. In most cases, the equipment had no effect.

[0174] (1.7 Summary) The aim of this study was to evaluate the stability of different anti-galectin-10 Abs upon nebulization. Three IgGs, one Fab, and one VHH were examined. Results showed that after nebulization, the aggregation profile was antibody dependent. 8H11 IgG and 7B07 Fab were the most stable upon nebulization and induced less aggregation. 1D06 VHH was the least stable and resulted in high aggregation levels.

[0175] Samples were nebulized with two mesh nebulizers: the Aerogen® Solo device and the Pari eFlow®. No significant differences in aggregation profiles were observed between the two systems, although the Aerogen® Solo device tended to produce fewer aggregates. Nebulization of samples showed that the Aerogen® Solo device induced a mild decrease in protein concentration and had a lower flow rate and slightly larger droplet size than the Pari eFlow®.

[0176] Samples were nebulized at two concentrations: 3mg / ml and 10mg / ml. Typically, more subvisible aggregates (FCM) were observed at 10mg / ml than at 3mg / ml. Overall, the data showed that, among the five molecules tested, the 7B07 Fab form was the most stable upon nebulization. In fact, nebulization of this antibody fragment induced very few particles after recovery using a method that scanned the different aggregate populations.

[0177] Example 2: Effect of nebulization on the stability of various Fab fragments This study evaluated some physicochemical properties of four anti-galectin-10 Fab clones capable of solubilizing CLCs. The molecules evaluated in Fab format were clones: 18C06, 20H09, 23H09, and 24F02_N53A (see Example 3 below for further details on Fab production and characterization).

[0178] The Fab format clone 7B07_N53A was also included for comparison. Fab 7B07_N53A is related to Fab 7B07 tested in Example 1 above, but was engineered to contain an N53A mutation to reduce deamidation propensity. Fab 7B07_N53A was found to be less stable than Fab 7B07, but was found to be sufficiently potent to lyse CLCs in vitro. Therefore, 7B07_N53A was included for selected conditions and specific time points for comparison purposes.

[0179] All four clones described in this study share a common VH+CH1, CH2, and CH3 portion, and three clones (20H09, 23H09, and 24F02_N53A) share a common light chain that is the parental LC of 7B07_N53A, while one clone (18C06) has a unique light chain.

[0180] Endotoxin-free germlined Fabs were produced by U-Protein Express (UPX), Utrecht, The Netherlands. Anti-galectin-10 Fab molecules were purified by UPX in Dulbecco's PBS buffer containing 0.02% polysorbate 20 and delivered to argenx. Protein and formulation buffer were delivered as 0.22 μm filtered material. Produced material was stored at 2-8 °C at UPX and shipped to argenx at 2-8 °C. Upon arrival, purified material of all clones was stored at 2-8 °C for up to 48 h and protein concentration was adjusted to 10 mg / mL in the original formulation buffer under sterile conditions.

[0181] All four Fabs were evaluated for their stability after nebulization. Nebulization stress and its effect on the physicochemical properties of the clones were evaluated using triplicate mesh nebulizers before storage (T0) and after 4 weeks of storage at +5° C. (T4W). All aerosolized solutions (including formulation solutions) were fully characterized at both time points.

[0182] Each clone was nebulized with an Aerogen Solo, an active vibrating mesh nebulizer that converts the drug solution into an inhalable aerosol. Three different devices were used per clone for each time point. Results are reported by device (nebulizer) serial number. In the event of device fouling, indicated by longer than normal nebulization times for a given size aliquot, a spare device of the same type was available to complete nebulization with triplicate devices.

[0183] Clones 18C06, 20H09, 23H09, and 7B07_N53A were nebulized with Aerogen Solo devices with serial numbers #1690, #0125, and #0259. Clone 24F02_N53A, nebulized later, was evaluated after being nebulized with the same devices, however, an additional device with number #1512 was used due to some fouling observed with #0125. Aliquots from clones 20H09 and 23H09 were also nebulized with this fourth device along with clone 24F02_N53A to bridge the results of aerosolized protein with this fourth device used in the study. Results are presented as the average of triplicate measurements per device unless otherwise noted.

[0184] A tabular summary showing some core characteristics of the tested Fabs (before any type of stress was applied) is provided in Table 3. Table 3: Summary of anti-galectin-10 Fab properties. [Table 3] NT: Not tested

[0185] 2.1 Protein Concentration Protein concentrations were assessed by A280nm (Nanodrop). Protein concentrations of all aerosolized solutions showed a clear decrease for all clones. As shown in Figure 6, this was the same for all nebulizers used at both T0 and T4W time points after storage under refrigerated conditions. This trend was confirmed by independent measurements.

[0186] (2.2 Visual Inspection of Particles) Visual inspection of samples was performed in duplicate for all clones at both T0 and T4W time points before and after nebulization (0.5 mL filled into 1.5 mL clear glass vials). Before nebulization, solutions were inspected for the presence of visible particles, but no particles could be identified for any of the clones inspected. The T0 aerosolized solutions showed no visible particles, except for clone 23H09. For the 4W time point, white particles were observed in post-nebulization aliquots of 23H09 and 24F02_N53A, while 18C06 and 20H09 were found to be particle-free. Aerosolization induced effervescence upstream of the mesh vibrating module in the reservoir (Table 4). Table 4: Visual inspection of clones before and after mesh spraying [Table 4] TIFF2025503701000063.tif114170+: 2–5 aggregates; ++: 5–15 aggregates; Solo 0125 is the device in which anomalous results were observed.

[0187] (2.3 Subvisible particles by dynamic light scattering (DLS)) DLS analysis was performed on all clone and formulation samples at both time points T0 and T4W before and after nebulization. Measurements were performed on triplicate preparations using a DynaPro Nanostart instrument. Mass percent, molecular hydrodynamic radius, percent polydispersity (%PD), and polydispersity index (PDI) were used to monitor the distribution profile of submicron particles in the solution. To perform the analysis, all sample solutions at both time points were filtered (0.2 μm) before and after nebulization.

[0188] The evaluation was performed by comparing the sprayed protein before and immediately after spraying (Figure 7). Before spraying, the percentage by mass of the major species (monomer) in solution for all clones was greater than 99.9%. Similarly, for clones 18C06 and 24F02_N53A (together with 7B07_N53A), the percentage by mass of the major species was greater than 99.9% after spraying, with negligible presence of non-monomer species. Data for clones 20H09 and 23H09 after spraying were not available due to elevated particle levels. The radius of the main peak was consistent with the expected protein size for all Fabs.

[0189] (2.4 Sub-visible particles by flow cell microscopy (FCM)) Flow cell microscopy (FCM) was performed using an Occhio Flow-Imaging FC200S instrument to detect and measure particles between 1 and 100 μm in solution. Before each analysis, the instrument was thoroughly washed with an aliquot of demineralized filtered water. As a system suitability test, each analysis was performed when the number of particles in these aliquots was determined to be <100 total. The number of particles per mL is expressed as total particles and as particles with equivalent diameters of >2 μm, >10 μm, and >25 μm.

[0190] All clones were measured before and after nebulization for time points TO and after 4 weeks of storage at 5°C (T4W). As above, all samples were filtered (0.2 μm) to complete the evaluation. Buffer samples for both time points were similarly filtered (before and after nebulization). All FCM values ​​herein represent the average value obtained from the three devices (triplicate measurements per device) used after subtracting the average value of the particles (total and per category) of the formulation buffer for all devices (triplicate measurements per device).

[0191] Fab clones 18C06, 20H09, 23H09, and 7B07_N53A were sprayed all together in the first wave, while Fab clone 24F02_N53A was sprayed at a later stage after replacing a fouled apparatus (#0125). To bridge the spray waves and results, solutions of clones from the first wave were sprayed with the new apparatus used for clone 24F02_N53A (#01215), when available.

[0192] As shown in Figure 8, Fabs 18C06 and 24F02_N53A (along with 7B07_N53A) showed lower particle counts in all size categories after nebulization compared to other samples at both TO and T4W. The subvisible counts obtained from the nebulization experiments are shown in Table 5, focusing on total counts / mL and >2 μm particles / mL, as this last category contains particles that may potentially induce immunogenicity upon drug administration in an inhaled pharmaceutical.

[0193] Subvisible and submicron particle testing indicated the need for formulation optimization. Clone 24F02_N53A showed lower and more stable particle counts for all categories after 4 weeks of storage and nebulization. Clone 23H09 showed higher counts in all categories after nebulization when compared to clone 24F02_N53A, including particles >2 μm in size, a category often associated with immunogenicity and severe ADA responses that may compromise drug safety, efficacy, and pharmacokinetics. Table 5: Summary of results for subvisible particles expressed as total particles / mL and particles >2 μm / mL. * Asterisks indicate cumulative numbers including device #0125 that exhibited fouling leading to anomalous results [Table 5]

[0194] 2.5 Galectin-10 binding activity by surface plasmon resonance (SPR) The functional activity concentration of the nebulized solutions was assessed by SPR on a Biacore 3000 instrument. All samples were assessed by methods that met standard qualification criteria. Aliquots were tested at predefined test concentrations against a titration curve from a reference sample (T0) and in the presence of quality control (QC) samples.

[0195] All sprayed clones were found to be fully functional without compromising the percent activity of each clone relative to the non-stressed reference material, as shown in Figure 9. Spray stress did not affect the activity of the clones either at T0 or after 4 weeks of storage at +5°C.

[0196] 2.6 Size Purity by SE-HPLC Purity by SE-HPLC was assessed on an Agilent 1260 Infinity II chromatography system equipped with a quaternary pump, autoinjector, online degasser and DAD detector, column thermostatted compartment (21° C.), and autosampler set at 6° C. The detector was set simultaneously at wavelengths of 280 nm and 214 nm to monitor size changes. Samples were analyzed before and after nebulization for both time points T0 and T4W.

[0197] As shown in Figure 10, all four Fab clones were measured with a purity of greater than 97% after nebulization, regardless of the time point or device used. The percentage of total aggregation (% HMW species) and the percentage of total fragmentation (% LMW species) remained at very low levels in all cases, i.e., less than 3% as a percentage of total non-monomers.

[0198] All aliquots were filtered prior to spraying, for both time points T0 and T4W. This was performed because the solutions showed increased particle counts by DLS measurements (section 2.3). As a result, the SEC results presented herein for the sprayed clones should be interpreted with a primarily qualitative approach. The figure shows the relative stability of all clones after mesh spraying, even after 4 weeks of storage under refrigerated conditions.

[0199] (2.7 cGE purity) Purity by cGE was assessed by lab-on-a-chip analysis using an Expert 2100 Bioanalyzer instrument (Agilent). Samples were analyzed under reducing and non-reducing conditions before and after nebulization.

[0200] As shown in FIG. 11, the applied nebulization stress did not affect the purity of the Fab clones, except for clone 18C06, which had the lowest purity measurement. This is consistent with all previous analyses, even for non-stressed material. This clone did not show a main peak purity above 90% under non-reducing conditions, but was not further affected by nebulization. For the remaining clones, purity was above 90% under non-reducing conditions, regardless of the time point of nebulization (FIG. 11, top panel). Under reducing conditions, all molecules showed a purity above 95% (FIG. 11, bottom panel).

[0201] 2.8 Post-translational Modifications (PTMs) Structural characterization of all Fab clones was performed at protein and peptide level using several analytical techniques (icIEF, online desalting MS, RPLC-UV-MS on reduced proteins, peptide map using RPLC-MS after trypsin digestion). Clones were analyzed for PTMs both at TO and after 4 weeks of storage at +5°C (T4W) post-nebulization. In all cases, analysis was performed for each clone side-by-side with a control non-stressed reference material (before nebulization).

[0202] No major problems were found with the clones after spraying. The main results are summarized below: The amino acid sequences of the four Fab clones were confirmed at the protein level based on the molecular weight of each intact Fab (LC and VH+CH1), and the peptide sequence coverage was 100%. The structural integrity of the Fab before and after stress remained unchanged and was confirmed by intact protein and peptide mapping analysis demonstrating and confirming the presence of the expected disulfide bridges (inter- and intra-chain). Only in 18C06 (all samples including reference) was a free cysteine ​​in the LC detected instead of the expected bridge with the VH+CH1 part, and instead an alternative formation of a disulfide bridge between two closely located cysteines in the LC was detected. Post-spray oxidation remained <1% for all clones. Site-specific events such as isomerization and deamidation remained largely unaffected after spraying for all clones.

[0203] 2.9 Potency - Crystal Dissolution Assay In this study, all Fab clones were evaluated in vitro for their ability to dissolve GAL10 crystals. For this purpose, a crystal dissolution assay (CDA) was developed and standardized at Charles Rivers Laboratories (CRL), Leiden, The Netherlands, to evaluate the biological activity of the Fab clones before and after nebulization. The aim was to evaluate whether pre- / post-storage nebulization influenced the (biological) activity of the clones, leading to a decrease in their ability to dissolve GAL10 crystals.

[0204] For all Fabs, the analysis was performed in two independent experiments with the presence of appropriate assay controls. For clones aerosolized with triplicate devices, aerosolized protein from one common device was analyzed as an 8 concentration point curve (Solo #0125) and the remaining two were analyzed at preselected fixed concentrations, always in two independent assays. Samples after 4 weeks of storage at +5°C were analyzed as 8 concentration point curves for all clones. The results presented here are from assays where the size distribution of crystals was above 10 μm (10-15 μm).

[0205] Figure 12 shows the potency of unstressed samples of the four Fab clones to dissolve GAL10 crystals. Along with the unstressed material, all clones were tested for potency after undergoing the stress conditions described above to assess whether this potency could be retained after storage and / or nebulization. Clones 18C06, 20H09, and 23H09 were tested in parallel in the first wave (assay 1, Figure 13) and clone 7B07_N53A was used for comparison. Clone 24F02_N53A was analyzed alongside clone 23H09 in the second wave (assay 2, Figure 14).

[0206] As shown in Figures 12, 13, and 14, all clones have potency to completely dissolve galectin-10 crystals throughout the analytical run, independent of the applied stress. Therefore, the comparison of clones was mainly focused on the first time point up to 5 hours, where potency differences could be found and evaluated. Clones 23H09 and 24F02_N53A were the most potent molecules among the four clones in the assays performed. Figure 14 shows the results from assay 2 with a side-by-side comparison of these two most potent clones. For all independent runs, clone 24F02_N53A always appeared to be more potent in dissolving crystals in vitro, regardless of the size distribution of the plated crystals. Temperature or spray stress did not affect its dissolving ability when compared to non-stressed material. Based on this assay monitoring crystal dissolving potency, the clones can be ranked from most potent to least potent: 24F02_N53>23H09>20C06>18C06

[0207] Clone 24F02_N53A showed several advantages over 23H09; both clones were able to dissolve crystals in vitro, but when the two clones were tested side-by-side on independent occasions, 24F02_N53A showed a faster dissolution rate and dissolution of crystallized protein.

[0208] (2.10 Immunogenicity) Endotoxin-free material of all clones was sent to Lonza, Slough, UK for assessment of immunogenicity risk using Lonza Epibase®, which includes in silico and cell-based in vitro assessments.

[0209] Lonza Epibase® in silico evaluation uses an algorithm to screen the amino acid sequences of clones for potential immunogenic epitopes, including potentially affected allotypes and major histocompatibility complexes, by determining the HLA-DRB1 score.

[0210] The assay screened these allotypes against their global population frequencies (Figure 15) and ranked the clones from least to most immunogenic based on their immunogenicity score: ·g24F02_N53<[g23H09, g18C06] <g20H09

[0211] These results were also confirmed in the Lonza Epibase in vitro assay. Briefly, clones were evaluated for T cell responses induced in PBMCs from 31 healthy donors. Screening was evaluated upon detection and enumeration of stimulated IFNγ and IL-5 cells to determine the number of donors eliciting T cell responses as a risk of unwanted immune responses (Figure 16), and the magnitude across the study population (Figure 17).

[0212] As shown in the corresponding figures (Figures 16, 17), all Fabs showed a low capacity to induce immune responses. Of the four, clones 23H09 and 18C06 showed the highest frequency for IFNγ responses, while Fabs 18C06 and 20H09 showed the highest frequency for IL-5 responses. In all cases and for all statistical approaches, clone g24F02_N53A was the molecule that induced the least frequent unwanted T cell responses, and therefore this clone is considered the lowest risk clone.

[0213] (2.11 Summary of results) Four germlined Fab clones were evaluated and all four clones showed improved properties compared to the derived 7B07_N53A clone. A summary of the results is provided in Figure 18.

[0214] Nebulization of all clones had no significant effect on protein concentration, purity by SE-HPLC and cGE (reduced and non-reduced), binding activity by SPR, post-translational modifications, and in vitro galactin-10 crystal dissolution. Based on the stability data obtained for nebulization before and after a 4-week storage period, all clones showed comparable attributes without significant deviation from the set target criteria.

[0215] Concerning the immunogenicity of the anti-GAL10 Fab clones, the relative risk assessment showed an advantage for clone 24F02_N53A, which is also favorable in terms of a lower immunogenicity score, but with limited differences from 23H09. When these two clones were compared for their human identity (Table 3), the same picture was obtained, with clone 24F02_N53A showing 100%.

[0216] Subvisible and submicron particle analysis (by DLS and FCM) indicated the need for formulation optimization, as even non-stressed samples of clone 23H09 (and 20H09) were found to be highly heterogeneous, multimodal, and unusable (Figure 7). Nebulization amplified these phenomena for all particle categories, with clone 24F02_N53A showing relatively lower and more stable counts after 4 weeks of storage and nebulization. Clone 23H09 showed higher counts in all categories after nebulization when compared to 24F02_N53A (Table 5). Although there is a need to optimize aerosolized formulations for drug delivery by inhalation, overall clone 24F02_N53A was found to have lower particle counts and be more desirable, especially for the category of particles >2 μm in size, a category that is often associated with immunogenicity and severe ADA responses that can compromise drug safety, efficacy, and pharmacokinetics.

[0217] Clone 24F02_N53A had an advantage over 23H09 from potency results obtained before any nebulization stress (FIG. 12) and after storage and aerosolization with a vibrating mesh nebulizer (FIG. 14). Regardless of the assay, for all samples tested side-by-side, all clones were able to clear crystallized protein in vitro, but 24F02_N53A showed faster potency than 23H09 in dissolving galectin-10 crystals.

[0218] Example 3: Generation and characterization of anti-galectin-10 Fab fragments In a previous discovery campaign, clone 7B07 was identified. This clone showed the best potency to lyse recombinant CLCs and displayed suitable binding properties. However, further stability studies highlighted a deamidation site (N53G54) in CDR2 of the heavy chain that caused a dramatic decrease in binding and potency after incubation at 37°C. Mutants of the germlined 7B07 (g7B07) clone in which N53 and G54 were randomized were generated, but all mutations, including the 7B07_N53A clone, resulted in a clear decrease in binding properties. However, the potency of these mutant 7B07 clones to lyse recombinant CLCs was maintained.

[0219] Therefore, three discovery campaigns were initiated to identify anti-Gal10 clones with good potency, affinity, and stability. The campaigns aimed to find mutant clones that bind to a similar epitope as 7B07, since this clone was the most potent clone and was expected to bind to a unique epitope. The premise was that if a clone binds to a similar epitope as 7B07, it will have a similar potency. The first campaign aimed to find clones that bind to a similar epitope in the available immune library ("7B07 epitope campaign"). In the second campaign, the sequence of CDR2_VH was randomized at up to six positions, including N53 and G54, to identify clones with good binding properties and without deamidation sites ("g7B07_CDR2_VH randomization campaign"). In the third campaign, a heavy chain shuffling approach was performed to find clones that paired with the 7B07 light chain and allowed good affinity for Gal10 and improved stability ("heavy chain shuffling campaign").

[0220] Following these discovery campaigns, four clones were identified for further testing: 18C06, 24F02_N53A, 23H09, and 20H09.

[0221] 3.1 Germlining, reformatting, and production of clones in human Fab backbones Four clones were selected for further characterization, humanized, and recloned into a human Fab backbone. To reduce the immunogenicity of the llama-derived anti-Gal10 clones, germlining of the variable regions (VH and VL) was initiated by grafting the complementarity determining regions (CDRs) into the closest human germline framework (FW). AbAligner and Antibody extractor software version N°8.1 were used to identify the human germline sequences with the highest identity of the V regions of the selected clones. Mutant 24F02 was engineered to remove a potential deamidation site (pos53_CDR2_VH) ​​at exactly the same position as 7B07. For this, N position 53 was mutated to A.

[0222] 3.2 Confirmation of binding properties of Fab clones The binding properties of the selected Fab clones to human Gal10 were analyzed by the established capture method on a Biacore 3000. Two concentrations of the selected Fab clones were applied to human Gal10-His immobilized on a monoclonal anti-His coated CM5 chip. As controls, clones 7B07 and 7B07_N53A were injected at the beginning and end of the run.

[0223] Selected clones isolated from three different discovery campaigns showed similar or better off-rates and affinities than clone 7B07. Among this panel, the clone isolated from the 7B07 epitope campaign (18C06) showed the best off-rate, with a 9.8-fold better off-rate than clone g7B07. A unique clone (g24F02) and its artificial variant (g24F02_N53A) isolated from the heavy chain shuffling approach showed very similar binding abilities, indicating that removal of the deamidation site found at position 53 does not affect its binding properties. With on-rates of 7.1-8.3 E+05, and off-rates equal to 1.6E-03 1 / s, and affinities of 2.0-2.3 nM, these clones showed 1.6-1.9-fold better on-rates, 2.8-fold better off-rates, and 4.7-fold better affinities than clone g7B07. Finally, two clones (20H09 and 23H09) isolated from the CDR2 randomization campaign showed distinct binding properties, consistent with the screening data. Clone g23H09 showed a similar on-rate, 2.8-fold better affinity (3.9 nM), and 1.8-fold better off-rate compared to clone g7B07. Clone g20H09 showed a 3-fold higher dissociation rate, but similar affinity. As expected, all clones showed better affinity and off-rate compared to the artificial mutant g7B07_N53A (kd 7.8E-02 1 / s, KD 143.5 nM). Table 6: Biacore 3000 characterization of binding properties of selected germline clones [Table 6]

[0224] To confirm that selected clones bind to the 7B07 epitope, epitope binning analysis of clones was performed against 7B07 (human Fab) in human Fab format. For this purpose, a suboptimal concentration of biotinylated clone 7B07 human Fab was added to Gal10-coated plates preincubated with selected clones. The percentage of competition against 7B07 is listed in Table 7. Motavizumab (Mota) in a human Fab backbone was used as a negative control (0% competition). Clone 7B07 was used as a positive control for competition and was therefore set as a 100% competition value. Since the anti-human specific clone 1D11 (binding to the tyrosine 69 residue) is known to bind to the opposite side of Gal10 (including tyrosine 69) compared to where 7B07 binds, 1D11 was included in the test panel as a negative control to compete with 7B07 for Gal10 binding. Consistent with the screening data, all selected clones were found to compete with 7B07 for Gal10 binding. Table 7: Epitope binning of selected clones anti-Gal10 against biotinylated 7B07 human Fab in ELISA. [Table 7]

[0225] The binding properties of the selected clones against human and cynomolgus Gal10 were analyzed by the established capture method on the Biacore 3000. For this purpose, two approaches were used. First, two concentrations of the selected Fab clones were applied against cynomolgus (WGS isoform) Gal10-His capture on a CM5 chip coated with monoclonal anti-His. In the second approach, serial dilutions were applied against human or cynomolgus (WGS isoform) Gal10-His in the same settings.

[0226] In a first step, the cynomolgus cross-reactivity of the clones was tested by injection of two concentrations against captured cynomolgus Gal10 (WGS isoform). Compared to previous data generated with the full antibody format, clone g7B07 and its artificial variant g7B07_N53A showed weak binding to the WGS isoform of cynomolgus Gal10 (KD 69 nM for g7B07-hFab vs. 1.5 nM for g7B07-mIgG1). Among the tested panel, clone g20H09 showed poor binding capacity to the cynomolgus antigen, whereas clone g18C06 showed no binding at all. However, clones g23H09, g24F02 and their artificial variant g24F02_N53A showed good cynomolgus cross-reactivity. Further characterization of the cynomolgus cross-reactivity of these clones highlighted g24F02_N53A and g23H09. Indeed, these two clones showed affinities of 1.4 nM and 5.34 nM for human Gal10 and 8.0 nM and 9.9 nM for cynomolgus Gal10, respectively. Furthermore, these two clones (g23H09 and g24F02_N53A) showed affinities 1.7- and 6.3-fold better than clone g7B07, and off-rates 1.9-fold up to 3.5-fold better. Mutation of Asn at position 53 in CDR2 of g24F02_VH did not lead to a decrease in binding to human or cynomolgus Gal10, with similar affinities and off-rates. Consistent with the screening data, clone g18C06 showed the best affinity (1.27 nM) and off-rate (4.9E-04 1 / s) of the panel tested against human Gal10, but did not bind to its cynomolgus monkey counterpart. Interestingly, randomization of six amino acids in CDR2 of g7B07_VH to generate g23H09 showed increased cynomolgus monkey cross-reactivity compared to the parent clone.However, clone g20H09, isolated from a similar discovery campaign in which three amino acids in CDR2 were randomized, did not show this increased cynomolgus cross-reactivity, and key amino acids were introduced into CDR2 of clone g23H09, showing increased binding to cynomolgus monkeys. Table 8: Biacore characterization of binding properties of selected clones in human Fab backbone to human and cynomolgus Gal10 (WGS isoforms) [Table 8]

[0227] 3.3 Confirmation of Fab clone stability After the stress test, the stability of selected clones was analyzed in order to avoid similar problems as faced with clone 7B07, where a deamidation site in the CDR2 of the variable domain of the heavy chain induced a clear decrease in binding and potency. For this purpose, a short accelerated temperature stress test was performed. In this setup, stressed samples were incubated at 37°C for 2 weeks and then analyzed for binding (Biacore performed at argenx) and potency (CLC lysis performed at VIB) side by side with non-stressed samples (T0).

[0228] The established capture method was optimized on the Biacore 3000 to analyze the binding properties of 2-week temperature stressed samples of selected clones. Briefly, calibrators and QC points were determined before temperature stress. The binding properties of the stressed samples were determined, compared to the calibration points, and expressed as a percentage of relative activity (%RA).

[0229] As shown in Table 9, clones 18C06 and 24F02_N53A showed the best stability after 2 weeks at 37°C, with similar binding capacity (106% and 99% RA, respectively) as the non-temperature stressed sample. Furthermore, the non-artificial mutants of 24F02 showed no loss of binding after 2 weeks of incubation at 37°C, indicating that deamidation at position 53 does not affect its binding properties. Clones 23H09 and 20H09 showed a decrease in binding capacity after incubation at 37°C, resulting in 78% and 86% RA, respectively, after 2 weeks. However, it is believed that this 14% and 22% decrease in binding could either be within the range of assay variability or that a longer temperature stress test is required. Table 9: Analysis of binding properties of selected clones in temperature stress samples [Table 9]

[0230] 3.4 Confirmation of potency of Fab clones The potency of temperature stressed samples of selected clones in a human Fab backbone to lyse recombinant CLCs was tested at Charles River Laboratories. Clone 1D11 was included as a reference to correct for inter-assay variability. Crystal dissolution was monitored over time by an InCell 2200 after 2, 5, 7, and 16 hours of incubation. Crystal generation performed prior to the runs showed CLCs of various sizes, ranging from 5-10 μm (run 2) to 10-20 μm (run 1). Consistent with previous observations, crystal size had a strong effect on compound potency, with CLCs with a size between 10-20 μm allowing the best discrimination between clones. CLC dissolution data generated at Charles River Laboratories showed that clones 20H09 and 23H09, both isolated from the CDR2 randomization campaign, were the most potent clones in the panel (Table 10). Indeed, these clones were able to lyse 59.6% and 68.6% of recombinant CLCs within 2 hours (run 1). Overall, after 5 h of incubation, most clones (except 1D11) were able to solubilize ∼90% of the CLCs. The positive control, clone 1D11, showed the least potency in dissolving crystals, with 20% and 36% CLC dissolution after 2 and 5 h of incubation, respectively.

[0231] Consistent with other stability results (binding), temperature stressed samples of clones 18C06, 20H09, and 23H09 demonstrated similar potency as the non-stressed samples. Table 10: Characterization of the potency of temperature stress preparations of selected clones (human Fab) to lyse recombinant CLCs on the Charles River Laboratories Cell Imaging System. [Table 10]

[0232] Since clone 24F02_N53A appears late in the panel, the potency of this clone to lyse recombinant CLC could not be tested at Charles River Laboratories. However, the potency of 24F02_N53A to lyse recombinant CLC was analyzed using spinning disk confocal microscopy at VIB. For this purpose, human Fab fragments were incubated with preformed CLC and the dissolution of crystals was monitored over time. In agreement with the binding data, temperature stressed samples (2 weeks at 37 °C) showed similar potency compared to non-stressed samples (Table 11), highlighting that this clone is stable for 2 weeks at 37 °C. Indeed, tested samples of this clone showed 50% CLC lysis within 43-46 min. It showed a close, but slightly lower potency compared to 7B07, regardless of whether 24F02 was stressed at 37 °C or not. Table 11: Time lapse of solubilization of recombinant Charcot-Leyden crystals (CLCs) by temperature stress samples of clone 24F02_N53A (hFab) on a spinning disk confocal microscope. [Table 11] * partial curve

[0233] 3.5 Fab clone sequences [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

[0234] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the present invention described herein are considered to be broadly applicable and capable of being combined, where appropriate, with any and all other consistent embodiments, including those taken (including separately) from other aspects of the present invention.

[0235] Various publications and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. 1. A pharmaceutical composition comprising a Fab fragment that binds to galectin-10, the Fab fragment comprising a variable heavy (VH) domain and a variable light (VL) domain, the pharmaceutical composition being formulated for aerosolization; wherein the VH domain is SEQ ID NO:4 【Chemistry 1】 an HCDR3 comprising or consisting of: SEQ ID NO: 3 【Chemistry 2】 HCDR2 comprising or consisting of: SEQ ID NO: 2 【Transformation 3】 HCDR1 comprising or consisting of Contains: wherein the VL domain is SEQ ID NO:8 【Chemistry 4】 LCDR3 comprising or consisting of: SEQ ID NO:7 【Transformation 5】 LCDR2 comprising or consisting of: SEQ ID NO:6 【Transformation 6】 LCDR1 comprising or consisting of The pharmaceutical composition comprising:

2. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is an aqueous liquid formulation.

3. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.02%, or less than 0.01% of a surfactant, or is surfactant-free.

4. 2. The pharmaceutical composition of claim 1, wherein the concentration of the Fab fragment in the pharmaceutical composition is 1 to 20 mg / ml, 1 to 15 mg / ml, 1 to 10 mg / ml, 1 to 5 mg / ml, 2 to 10 mg / ml, 3 to 10 mg / ml, 4 to 10 mg / ml, or 5 to 10 mg / ml, or the concentration of the Fab fragment in the pharmaceutical composition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / ml.

5. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient, optionally wherein the excipient is a liquid excipient (optionally a solvent or an aqueous solvent).

6. The Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: the VH domain comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto; 2. The pharmaceutical composition of claim 1.

7. The Fab fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain is SEQ ID NO: 1 【Transformation 7】 and The VL domain is SEQ ID NO:5 【Transformation 8】 comprising the amino acid sequence 2. The pharmaceutical composition of claim 1.

8. 10. The pharmaceutical composition of claim 1, wherein the Fab fragment is humanized, germlined, or deimmunized.

9. 9. A pharmaceutical composition according to any one of claims 1 to 8, comprising a Fab fragment that binds to galectin-10, for use in a method for treating a disease or disorder associated with the presence or formation of galectin-10 crystals, comprising: The method comprises administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition; the pharmaceutical composition is administered as an aerosol; The pharmaceutical composition, wherein the disease or disorder associated with the presence or formation of galectin-10 crystals is selected from the group consisting of asthma; chronic rhinosinusitis; celiac disease; helminth infection; gastrointestinal eosinophilic inflammation; cystic fibrosis (CF); allergic bronchopulmonary aspergillosis (ABPA); Churg-Strauss vasculitis; chronic eosinophilic pneumonia; and acute myeloid leukemia.

10. 10. The pharmaceutical composition of claim 9, wherein the disease or condition associated with the presence or formation of galectin-10 crystals is asthma or cystic fibrosis (CF).

11. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is administered to the subject via inhalation.

12. The pharmaceutical composition of claim 9, wherein the Fab fragment inhibits crystallization of galectin-10 when bound to soluble galectin-10 and / or promotes dissolution of crystalline galectin-10 when bound to crystalline galectin-10, and optionally the crystalline galectin-10 is a Charcot-Leyden crystal (CLC).

13. 10. The pharmaceutical composition of claim 9, wherein the particle size of the particles in the aerosol is less than 30 μm, less than 25 μm, less than 10 μm, less than 2 μm, less than 1 μm, less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm.

14. (i) the concentration of particles in the aerosol is 2 × 10 5 Less than 1 x 10 5 Less than 2 x 10 4 Less than 1 x 10 4 less than 10 total particles / ml, and / or (ii) the concentration of particles in the aerosol with a size greater than 2 μm is 1 × 10 5 Less than 5 x 10 4 Less than 2 x 10 4 Less than 1 x 10 4 Less than 1 x 10 3 Less than 1 x 10 2 10. The pharmaceutical composition of claim 9, wherein the composition has less than 10 particles >2 μm / ml.

15. 10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is administered as an aerosol using a nebulizer, a pressurized metered dose inhaler (pMDI), a dry powder inhaler, a soft mist inhaler, or a smart inhaler.

16. 16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered as an aerosol using a nebulizer, optionally the nebulizer is a jet nebulizer, an ultrasonic nebulizer, or a mesh nebulizer.

17. A nebulizer comprising the pharmaceutical composition of any one of claims 1 to 8.

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