Galectin-10 Antibody

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that bind to the protein galectin-10, in particular human galectin-10. The galectin-10 antibodies and antigen-binding fragments of the invention disrupt the crystallization of 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 (CLCs).

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibodies and antigen-binding fragments thereof that bind to the protein galectin-10, in particular human galectin-10. The galectin-10 antibodies and antigen-binding fragments of the invention disrupt the crystallization of 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 (CLCs). [Background technology]

[0002] BACKGROUND OF THEINVENTION Charcot-Leyden crystals (CLCs), first described in 1853, are microscopic colorless crystals found in patients with certain diseases, including allergic asthma and parasitic infections. CLCs are often observed in human tissues and secretions associated with an eosinophilic inflammatory response. In addition to asthma and parasitic infections, these crystals are found in patients with cancer, e.g., myeloid leukemia. Structurally, CLCs accumulate as extracellular hexagonal bipyramidal crystals with lengths of 20-40 μm and widths of 2-4 μm. The protein that forms these crystals has been identified as galectin-10.

[0003] Galectin-10 (also known as Charcot-Leyden crystal protein) is a small (16.5 kDa), self-crystallizing, hydrophobic glycan-binding protein that is expressed in bone marrow primarily by eosinophils (Chua et al. (2012) PLoS One. 7(8): e42549). Galectin-10 is also produced, to a lesser extent, by basophils and Foxp3+ Tregs (Kubach et al. (2007) Blood 110(5): 1550-8). The protein is one of the most abundant eosinophil components, representing 7%-10% of the total cellular protein. Galectin-10 is found only in humans and non-human primates, where it lacks a secretory peptide signal and a transmembrane domain, and is secreted under certain conditions by a non-classical and novel apocrine mechanism.

[0004] Despite many reports showing the appearance of CLCs in tissues from patients with eosinophilic disorders, the prevailing view has been that these crystals are merely a marker of eosinophil disappearance. This view was eventually challenged by a study showing that CLCs enhance type 2 immunity in a mouse model of house dust mites (HDM)-induced asthma (Persson et al., Science (2019)). Furthermore, CLCs have been observed to be abundant in the sticky mucus of patients with aspergillosis and CRSwNP, suggesting that CLCs contribute to mucus viscoelasticity (Su J. et al., Molecules (2018)). Summary of the Invention

[0005] (Summary of the invention) The recent findings that galectin-10 and CLC formation are involved in disease indicate that it is a target for therapeutic agents. It is reported herein that galectin-10 crystals can be dissolved by administration of a galectin-10 antibody. Importantly, the galectin-10 antibody reported herein remains active and stable even when stored at elevated temperatures, such as 37° C., for up to 4 weeks. Taken together, this indicates that the galectin-10 antibody reported herein can be used to treat diseases and disorders whose pathology is related to the presence of CLCs.

[0006] In a first aspect, the present invention provides an antibody or antigen-binding fragment that binds to Galectin-10, the antibody or antigen-binding fragment comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: HCDR3 comprising or consisting of SEQ ID NO:2; HCDR2 comprising or consisting of SEQ ID NO:3; HCDR1 comprising or consisting of SEQ ID NO:1; and (ii) the VL domain comprises the CDR sequences of: LCDR3 comprising or consisting of SEQ ID NO:8; LCDR2 comprising or consisting of SEQ ID NO:9; LCDR1 comprising or consisting of SEQ ID NO:7, Antibodies or antigen-binding fragments are provided.

[0007] In one embodiment, the VH domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0008] In one embodiment, the VH domain comprises the amino acid sequence of SEQ ID NO:4; and the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0009] In some embodiments, the antigen-binding fragment is selected from the group consisting of: a single chain antibody (scFv); a F(ab')2 fragment; a Fab fragment; a Fd fragment; a Fv fragment; a single-arm (monovalent) antibody; a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by combination, association, or conjugation of such antigen-binding fragments. In a preferred embodiment, the antigen-binding fragment is a Fab fragment.

[0010] In a further aspect, the present invention provides an isolated polynucleotide or polynucleotides encoding the antibodies or antigen-binding fragments described herein, including polynucleotides encoding the VH and / or VL domains of the antibodies and antigen-binding fragments described herein.

[0011] In another aspect, provided herein is an expression vector comprising the polynucleotide or polynucleotides described herein operably linked to a regulatory sequence enabling expression of the antibody, antigen-binding fragment, variable heavy chain domain, or variable light chain domain in a host cell or a cell-free expression system.

[0012] In a further aspect, the present invention provides a host cell or a cell-free expression system containing an expression vector described herein.

[0013] Also provided herein is a method of producing a recombinant antibody or antigen-binding fragment described herein, comprising culturing a host cell or cell-free expression system described herein under conditions that allow expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

[0014] In another aspect of the invention, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment described herein and at least one pharma- ceutical acceptable carrier or excipient.

[0015] In a further aspect, the antibody or antigen-binding fragment described herein, or the pharmaceutical composition described herein, is for use as a medicament. In a further aspect, a method of treating a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment described herein, or the pharmaceutical composition described herein.

[0016] The antibody, antigen-binding fragment, or pharmaceutical composition can be administered to prevent or treat a disease or condition associated with the presence or formation of galectin-10 crystals. Suitably, the disease or condition 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 (AML). In a preferred embodiment, the disease or condition is asthma. In another preferred embodiment, the disease or condition is cystic fibrosis.

[0017] The present invention also provides the use of an antibody or antigen-binding fragment described herein for the detection of Galectin-10 in a sample obtained from a patient. Suitably, the patient sample may be a mucus sample or a sputum sample.

[0018] The present invention also provides kits comprising the antibodies or antigen-binding fragments described herein. The kits may further comprise instructions for use. [Brief description of the drawings]

[0019] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the results of two duplicate experiments. They show how the average Charcot-Leyden crystal (CLC) area changes as a function of time when incubated with humanized Fab clones g18C06, g18E04, g18G07, g18G12, g20H09, and g23H09. Dissolution of CLC as a function of time was recorded. Clones were tested at a concentration of 250 μg / mL in two independent experiments (n=4), and images were captured of CLC in samples 2, 5, 7, and 16 hours after antibody addition. Images were segmented using an algorithm that detects individual crystals, and the total crystal area per well was determined. In this assay, 1 μL of antibody sample was diluted to 1.5 mg / mL and then incubated with recombinant CLC formed with 1 μg of Gal10. The y-axis used is the average crystal area percentage dissolved per well after incubation with each antibody sample. The samples labeled "T0" correspond to the reference samples of each clone (samples stored at -80°C before analysis). Samples labeled "T2W" were stored at 37°C for 2 weeks before analysis. [Diagram 2]Figure 2 shows a graph depicting the rate of dissolution of recombinant Charcot-Leyden crystals (CLCs) by g7B07 and g24F02_N53A (hFab) as determined by spinning disk confocal microscopy. Samples containing only PBS were used as negative controls. Figure 2A shows a schematic of the recombinant CLC dissolution assay; Figure 2B shows the results of the assay. The initial area covered by CLCs at the start of the experiment was defined as 1, and the surface occupied by CLCs was determined using the software. Samples labeled "T0" correspond to the reference samples of each clone (samples stored at -80 °C before analysis). Samples labeled "T2W" were stored at a temperature of 37 °C for 2 weeks before analysis. [Diagram 3] Figure 3 shows the protein concentrations of clones g18C06, g20H09, g23H09, g24F02_N53A, and g7B07 as determined by measuring absorbance (A) at a wavelength of 280 nm using a Nanodrop. To avoid misunderstanding, the sample names in the figure do not include the prefix "g", but all clones tested are germlined clones. Samples labeled "T0" correspond to the reference sample of each clone (samples stored at -80°C before analysis). Samples labeled "TxW+y°C" were stored at a temperature of y°C for x weeks before analysis, e.g., samples labeled "T1W+5°C" were stored at +5°C for 1 week before analysis, samples labeled "T1W+25°C" were stored at +25°C for 1 week before analysis, etc. Samples labeled "1F / FT" were subjected to one freeze-thaw cycle before analysis; samples labeled "10F / FT" were subjected to ten freeze-thaw cycles before analysis; and samples labeled "Low pH" were subjected to pH 3.7 for 2 hours before analysis. [Figure 4]Figure 4 shows the relative activity percentages of clones g18C06, g20H09, g23H09, g24F02_N53A, and g7B07 as determined by surface plasmon referencing (SPR). To avoid misunderstanding, the sample names in the figure do not include the prefix "g", but all clones tested are germlined clones. Samples labeled "T0" correspond to the reference sample of each clone (samples stored at -80°C before SPR analysis). Samples labeled "TxW+y°C" were stored at a temperature of y°C for x weeks before SPR analysis, e.g., samples labeled "T1W+5°C" were stored at +5°C for 1 week before SPR analysis, samples labeled "T1W+25°C" were stored at +25°C for 1 week before SPR analysis, etc. Samples labeled "1F / FT" were subjected to one freeze-thaw cycle before SPR analysis; samples labeled "10F / FT" were subjected to 10 freeze-thaw cycles before SPR analysis; and samples labeled "Low pH" were subjected to pH 3.7 for 2 hours before analysis. [Diagram 5]Figure 5 shows the purity percentages of clones g18C06, g20H09, g23H09, g24F02_N53A, and g7B07 as determined by SE-HPLC. To avoid misunderstanding, the sample names in the figure do not include the prefix "g", but all clones tested are germlined clones. The top graph shows the percentage of monomers in each sample, the middle graph shows the percentage of total aggregates in each sample, and the bottom graph shows the percentage of total fragments in each sample. The samples labeled "T0" correspond to the reference samples of each clone (samples stored at -80°C before SE-HPLC). Samples labeled "TxW+y°C" were stored at a temperature of y°C for x weeks before SE-HPLC analysis, e.g., samples labeled "T1W+5°C" were stored at +5°C for 1 week before SE-HPLC analysis, samples labeled "T1W+25°C" were stored at +25°C for 1 week before SE-HPLC analysis, etc. Samples labeled "1F / FT" were subjected to 1 freeze-thaw cycle before SE-HPLC; samples labeled "10F / FT" were subjected to 10 freeze-thaw cycles before SE-HPLC; and samples labeled "low pH" were subjected to pH 3.7 for 2 hours before analysis. [Figure 6]Figure 6 shows the percentage of clonal purity assessed by capillary gel electrophoresis (cGE). To evaluate the effect of stress on the purity of the samples, clonal samples were also subjected to various stresses prior to purity determination. The top graph shows the percentage of intact Fab purity under non-reducing conditions, and the bottom graph shows the percentage of total Fab under reducing conditions. Samples labeled "T0" correspond to the reference sample of each clone (samples stored at -80°C prior to cGE); samples labeled "T4W+5°C" were stored at +5°C for 4 weeks prior to cGE; samples labeled "T4W+25°C" were stored at +25°C for 4 weeks prior to cGE; samples labeled "T4W+37°C" were stored at +37°C for 4 weeks prior to cGE; samples labeled "1F / FT" were subjected to one freeze-thaw cycle prior to cGE; and samples labeled "10F / FT" were subjected to 10 freeze-thaw cycles prior to cGE analysis. To avoid any misunderstanding, the sample names in the figures do not include the "g" prefix, but all clones tested are germlined clones. [Figure 7] Figure 7 shows that clones g18C06, g20H09, g23H09, and g24F02_N53A dissolve GAL10 crystals at a rate similar to that of clone g7B07_N53A. To avoid misunderstanding, the sample names in the figure do not include the "g" prefix, but all clones tested are germlined clones. [Figure 8] Figure 8 shows that clones g18C06, g20H09, and g23H09 are able to dissolve GAL10 crystals. These clones were able to dissolve GAL10 crystals after 4 weeks of storage under different conditions and after spraying. Further details of the assays performed can be found in the Examples section entitled "Materials and Protocols Used in Examples 1-4" (see Assay 1 described therein). To avoid any misunderstanding, the sample names in the figures do not include the "g" prefix, but all the clones tested are germlined clones. [Figure 9]Figure 9 shows that clones g23H09 and g24F02_N53A dissolve GAL10 crystals of different sizes. These clones were able to dissolve GAL10 crystals after 4 weeks of storage at 5°C and after nebulization (sample annotated "solo 0125"). Further details of the assays performed can be found in the Examples section entitled "Materials and Protocols Used in Examples 1-4" (see Assay 2 described therein). [Figure 10] Figure 10 shows the overall DR beta 1 (DRB1) risk score of 44 marketed therapeutic antibodies, as well as the risk scores of clones g20H09, g23H09, g18C06, and g24F02_N53A. Human antibodies are indicated by medium grey bars, humanized antibodies by grey bars, and chimeric antibodies by dark grey bars. [Figure 11] Figure 11 shows the percentage of donors with IFNγ (left graph) and IL-5 (right graph) responses to clones g20H09, g23H09, g18C06, and g24F02_N53A. Distribution-free resampling (DFR2x) algorithm and DFReq were used for statistical analysis (Moodie et al., Cancer Immunol Immunother 59, 1489-1501 (2010)). KLH samples were used as positive controls. [Figure 12] FIG. 12 shows IFNγ (left panel) and IL-5 (right panel) responses in a donor test population (n=31) of clones g20H09, g23H09, g18C06, and g24F02_N53A (DFR2x algorithm was used for statistical analysis (Moodie et al., Cancer Immunol Immunother 59, 1489-1501 (2010)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] (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 of this invention.

[0021] "Antibody" or "Immunoglobulin" - As used herein, 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 (herein, 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, species homologs thereof. Antibodies and immunoglobulins comprise a light chain and a heavy chain, with or without interchain covalent bonds between them. Basic immunoglobulin structure in vertebrate systems is relatively well understood.

[0022] The general term "immunoglobulin" includes five different classes of antibodies that can be biochemically distinguished. All five classes of antibodies are within the scope of the present invention. The following discussion is generally directed to 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.

[0023] 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, 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 the skilled artisan in light of the instant disclosure and, accordingly, are within the scope of the instant invention.

[0024] As shown above, 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-element antibody structure forms an antigen-binding site present 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.

[0025] "Galectin-10" - As used herein, the term "galectin-10" (or Gal10 or Gal-10, which are used interchangeably herein) refers to a small hydrophobic glycan-binding protein that self-crystallizes to form Charcot-Leyden crystals. Galectin-10 is also called Charcot-Leyden crystal protein (CLCP), eosinophil lysophospholipase, and lysolecithin acyl hydrolase. The term "galectin-10" is broad enough to encompass the human protein and any species homologues. The amino acid sequence of full-length human galectin-10 is represented by SEQ ID NO:25 (see below). This sequence corresponds to the sequence deposited in the UniProt database as 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:25 [ka]

[0026] "Galectin-10 Crystals" or "Charcot-Leyden Crystals" - The terms "Galectin-10 Crystals", "Charcot-Leyden Crystals", and "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 2-4 μm in width. These crystals have been associated with eosinophilic inflammatory disorders.

[0027] "Epitope" - As used herein, the term "epitope" refers to a region of a Galectin-10 protein to which an antagonist binds. Antagonists typically bind to their respective Galectin-10 epitopes via complementary binding sites on the antagonist. The epitope to which an antagonist binds typically comprises one or more amino acids from a full-length Galectin-10 protein. An epitope may comprise amino acids that are contiguous in the Galectin-10 protein, i.e., a linear epitope, or may comprise amino acids that are not contiguous in the Galectin-10 protein, i.e., a conformational epitope.

[0028] "Binding Site" - As used herein, the term "binding site" includes a region of a polypeptide that is involved in selective binding to a target antigen of interest (e.g., Galectin-10). A binding domain comprises at least one binding site. Exemplary binding domains include antibody variable domains. An antibody molecule of the invention can comprise a single binding site or multiple (e.g., two, three, or four) binding sites.

[0029] "Derived from" - As used herein, the term "derived from" a specified protein (e.g., a camelid antibody or antigen-binding fragment thereof) refers to the source of a polypeptide or amino acid sequence. In one embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide is a CDR sequence or a sequence related thereto. In one embodiment, an amino acid sequence derived from a particular starting polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from the starting antibody. In one embodiment, a polypeptide or amino acid sequence derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to the amino acid sequence of the starting sequence or a portion thereof (wherein the portion consists of at least 3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids) or is otherwise identifiable to one of skill in the art as having its source in the starting sequence. In one embodiment, one or more CDR sequences from a starting antibody are modified to generate variant CDR sequences, e.g. affinity variants, where the variant CDR sequences retain the target antigen binding activity.

[0030] "Camelid-derived" - In certain preferred embodiments, the antibodies of the invention comprise framework and / or CDR amino acid sequences derived from conventional antibodies of camelids or VHH antibodies raised by active immunization of camelids. However, antibodies of the invention comprising camelid-derived amino acid sequences can be engineered to comprise human amino acid sequences (i.e., human antibodies) or framework and / or constant region sequences derived from other non-camelid mammalian species. For example, human or non-human primate framework regions, heavy chain portions, and / or hinge portions can be included in a Galectin-10 antibody. In one embodiment, one or more non-camelid amino acids may be present in the framework regions of a "camelid-derived" antibody, for example, the camelid framework amino acid sequence may comprise one or more amino acid mutations in which a corresponding human or non-human primate amino acid residue is present. Furthermore, camelid-derived VH and VL domains, or humanized variants thereof, can be linked to constant domains of human antibodies, as described elsewhere herein, to produce chimeric molecules.

[0031] "VHH antibody" - As used herein, the term "VHH antibody" or "heavy chain only antibody" refers to a type of antibody that is produced only 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.

[0032] "Conservative Amino Acid Substitution" - A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide can be replaced with another amino acid residue from the same side chain family. In another embodiment, the string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0033] "Heavy chain portion" - As used herein, the term "heavy chain portion" includes an amino acid sequence derived from a constant domain of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion includes at least one of: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, an antibody or antigen-binding fragment of the invention may include an Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, an antibody or antigen-binding fragment of the invention may lack at least a portion of a constant domain (e.g., all or a portion of a CH2 domain). In certain embodiments, at least one, preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain portion includes a fully human hinge domain. In another preferred embodiment, the heavy chain portion includes a fully human Fc portion (e.g., hinge, CH2, and CH3 domain sequences from a human immunoglobulin).

[0034] In certain embodiments, the constitutive constant domains of the heavy chain portion are derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide can comprise a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain comprising portions of different immunoglobulin molecules. For example, the hinge can comprise a first portion derived from an IgG1 molecule and a second portion derived from an IgG3 or IgG4 molecule. As indicated above, it will be understood by those skilled in the art that the constant domains of the heavy chain portion can be modified to differ in amino acid sequence from a native (wild-type) immunoglobulin molecule. That is, the polypeptides of the invention disclosed herein can comprise alterations or modifications to one or more of the heavy chain constant domains (CH1, hinge, CH2, or CH3) and / or to the light chain constant region domain (CL). Exemplary modifications include addition, deletion, or substitution of one or more amino acids in one or more domains.

[0035] "Chimeric" - A "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. The amino acid sequences can normally be in separate proteins that become one in the fusion polypeptide, or the amino acid sequences can normally be in the same protein, but are arranged in a new arrangement in the fusion polypeptide. Chimeric proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Exemplary chimeric antibodies of the invention include fusion proteins comprising camelid-derived VH and VL domains, or humanized variants thereof, fused to the constant domains of a human antibody, e.g., human IgG1, IgG2, IgG3, or IgG4.

[0036] "Variable Region" or "Variable Domain" - 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 among antibodies and are used in the binding and specificity of each particular antibody for 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)).

[0041] "CDR" - As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within 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 comparisons. 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] "Framework Region" - The term "framework region" or "FR region" as used herein includes amino acid residues that are part of a 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] "Hinge Region" - As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al., J. Immunol. 161:4083-90 1998). Antibodies of the invention that comprise a "fully human" hinge region can contain one of the hinge region sequences shown in Table 2 below. Table 2: Human hinge sequences [Table 2] "CH2 domain" - As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule that extends from about residue 244 to residue 360 ​​of an antibody, using conventional numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system, Kabat EA et al., Sequences of Proteins of Immunological Interest, Bethesda, US Department of Health and Human Services, NIH. 1991). The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It is also well established that the CH3 domain extends from the CH2 domain to the C-terminus of an IgG molecule and comprises about 108 residues.

[0045] "Fragment" - The term "fragment" as used in the context of an antibody of the invention refers to a part or portion of an antibody or antibody chain that comprises fewer amino acid residues than an intact or complete antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody (i.e., with the intact antibody from which it was derived) for antigen binding (i.e., specific binding to Galectin-10). As used herein, the term "fragment" of an antibody molecule includes antigen-binding fragments of an antibody, such as an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, a Fd fragment, a Fv fragment, a single-arm (monovalent) antibody, a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by combination, association, or conjugation of such antigen-binding fragments. The term "antigen-binding fragment" as used herein is further intended to encompass an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody. Fragments can be obtained, for example, by chemical or enzymatic treatment of an intact or complete antibody or antibody chain, or by recombinant means.

[0046] "Fab" - "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. Fab fragments can be generated from immunoglobulin molecules 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.

[0047] "scFv" or "scFv fragment" - By scFv or scFv fragment is meant a single chain variable fragment. An scFv is a fusion protein of the VH and VL domains of an antibody connected via a linker.

[0048] "Valency" - As used herein, the term "valency" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. When a polypeptide contains multiple target binding sites, each target binding site can specifically bind to the same or different molecules (e.g., can bind to different ligands or different antigens, or different epitopes on the same antigen).

[0049] "Specificity" - The term "specificity" refers to the ability to bind (e.g., immunoreact with) a given target, e.g., Galectin-10. A polypeptide may be monospecific and contain one or more binding sites that specifically bind to a target, or a polypeptide may be multispecific and contain two or more binding sites that specifically bind to the same or different targets.

[0050] "Synthetic" - As used herein, the term "synthetic" in reference to a polypeptide includes polypeptides that contain an amino acid sequence that is not naturally occurring, e.g., a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (including, for example, mutations such as additions, substitutions, or deletions) or that contains a first amino acid sequence (which may or may not be naturally occurring) linked in the linear sequence of amino acids to a second amino acid sequence (which may or may not be naturally occurring) to which it is not naturally linked in nature.

[0051] "Artificially produced" - As used herein, the term "artificially produced" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, by in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or by some combination of these techniques). Preferably, the antibodies of the invention are artificially produced, and include, for example, humanized and / or chimeric antibodies, as well as antibodies that have been artificially produced to improve one or more characteristics, such as antigen binding, stability / half-life, immunogenicity, or effector function.

[0052] "Modified antibodies" - As used herein, the term "modified antibodies" includes synthetic forms of antibodies that have been modified to be non-natural, such as antibodies that contain at least two heavy chain portions, but do not contain two complete heavy chains (e.g., domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been modified to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules connected to scFv molecules, etc. scFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. Additionally, the term "modified antibodies" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc. antibodies that bind to three or more copies of the same antigen). In another embodiment, the modified antibodies of the invention are fusion proteins that contain at least one heavy chain portion that lacks a CH2 domain and that contain a binding domain of a polypeptide that contains the binding portion of one member of a receptor-ligand pair.

[0053] The term "modified antibody" can also be used herein to refer to amino acid sequence variants of the antibodies of the invention as structurally defined herein. It will be understood by those skilled in the art that antibodies can be modified to produce variant antibodies that differ in amino acid sequence compared to the antibody from which it was derived. For example, nucleotide or amino acid substitutions that result in conservative substitutions or changes in "non-essential" amino acid residues can be made (e.g., in CDR and / or framework residues). Amino acid substitutions can include the replacement of one or more amino acids with natural or non-natural amino acids.

[0054] "Humanizing substitution" - 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 (e.g., a camelid-derived galectin-10 antibody) 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 defined herein.

[0055] "Humanized variant" - 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.

[0056] "Germlined variant" - The term "germlined variant" is used herein to specifically refer to a "humanized variant" in which "humanizing substitutions" result in the replacement of one or more amino acid residues present at a particular position in a VH or VL domain of an antibody (e.g., a camelid-derived galectin-10 antibody) 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 typically taken 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.

[0057] "% identity" - as used herein, is intended to describe the sequence similarity between two sequences, such as amino acid sequences and nucleotide sequences. It can be determined by comparing 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 residues are 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.

[0058] "Affinity variant" - As used herein, the term "affinity variant" refers to a variant antibody that exhibits one or more changes in amino acid sequence compared to a reference antibody, where the affinity variant exhibits an altered affinity for a target antigen compared to the reference antibody. For example, the affinity variant exhibits an altered affinity for Galectin-10 when compared to a reference Galectin-10 antibody. Preferably, the affinity variant exhibits an improved affinity for a target antigen, e.g., Galectin-10, when compared to the reference antibody. Affinity variants usually exhibit one or more changes in amino acid sequence in the CDRs when compared to the reference antibody. Such substitutions can result in the replacement of the original amino acid present at a given position in the CDR with a different amino acid residue, which can be a natural amino acid residue or a non-natural amino acid residue. The amino acid substitutions can be conservative or non-conservative.

[0059] "High human homology" - An antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) can be considered to have high human homology if the VH and VL domains taken together exhibit at least 90% amino acid sequence identity with the closest matching human germline VH and VL sequences. Antibodies with high human homology can include antibodies comprising the VH and VL domains of naturally occurring non-human antibodies that exhibit sufficiently high % sequence identity with human germline sequences, including, for example, antibodies comprising the VH and VL domains of conventional antibodies from camelids, as well as artificially created, in particular humanized or germlined, variants of such antibodies, and also "fully human" antibodies.

[0060] In one embodiment, the VH domain of an antibody with high human homology may exhibit 80% or more amino acid sequence identity or sequence homology with one or more human VH domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments, the amino acid sequence identity or sequence homology between the VH domain of a polypeptide of the invention and the closest corresponding human germline VH domain sequence may be 85% or more, 90% or more, 95% or more, 97% or more, or up to 99%, or even 100%.

[0061] In one embodiment, the VH domain of an antibody with high human homology may contain one or more (e.g., 1 to 10) amino acid sequence mismatches across the framework regions FR1, FR2, FR3, and FR4 compared to the closest matching human VH sequence.

[0062] In another embodiment, the VL domain of an antibody with high human homology may exhibit 80% or more sequence identity or sequence homology with one or more human VL domains over the framework regions FR1, FR2, FR3, and FR4. In other embodiments, the amino acid sequence identity or sequence homology between the VL domain of a polypeptide of the invention and the closest corresponding human germline VL domain sequence may be 85% or more, 90% or more, 95% or more, 97% or more, or up to 99%, or even 100%.

[0063] In one embodiment, the VL domain of an antibody with high human homology may contain one or more (e.g., 1 to 10) amino acid sequence mismatches across the framework regions FR1, FR2, FR3, and FR4 compared to the closest matching human VL sequence.

[0064] B. Antibodies and Antigen-Binding Fragments that Bind Galectin-10 As noted above, the present invention is directed to antibodies or antigen-binding fragments that bind to Galectin-10. The term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (i.e., bispecific antibodies), so long as they exhibit the appropriate immunological specificity for Galectin-10 protein. The antibodies and antigen-binding fragments that bind to Galectin-10 described herein may exhibit immunological specificity for any Galectin-10 epitope.

[0065] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population that are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which usually contain different antibodies directed against different determinants (epitopes) on the antigen, each monoclonal antibody is directed against a single determinant or epitope on the antigen. An "antibody fragment" or "antigen-binding fragment" comprises a portion of a full-length antibody, usually the antigen-binding or variable domain thereof. Antibody fragments are described elsewhere herein, and examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab', and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (see Holliger and Hudson, Nature Biotechnol. 23:1126-36 (2005), the contents of which are incorporated herein by reference).

[0066] The antibodies and antigen-binding fragments that bind Galectin-10 described herein are intended for therapeutic use in humans and therefore are typically immunoglobulins of the IgA, IgD, IgE, IgG, IgM type, and often of the IgG type, which may belong to any of the four subclasses IgG1, IgG2a and b, IgG3, or IgG4. In a preferred embodiment, the antibody is an IgG antibody. Monoclonal antibodies are preferred, as they are highly specific and directed against a single antigenic site. In a preferred embodiment, the antigen-binding fragment that binds Galectin-10 is a Fab fragment or "Fab".

[0067] Antibodies and antigen-binding fragments that bind to Galectin-10 can exhibit high human homology as defined elsewhere herein. Such antibody molecules with high human homology can include antibodies that comprise the VH and VL domains of a naturally occurring non-human antibody that exhibits a sufficiently high percentage sequence identity to human germline sequences. In certain embodiments, the antibody or antigen-binding fragment thereof is a humanized or germline variant of a non-human antibody.

[0068] In some embodiments, the antibodies and antigen-binding fragments that bind to galectin-10 described herein may be derived from a camelid. The camelid-derived antibodies may be heavy chain only antibodies, i.e. VHH antibodies, or may be conventional heterotetrameric antibodies. In a preferred embodiment, the galectin-10 antibodies and antigen-binding fragments are derived from camelid heterotetrameric antibodies. In a further preferred embodiment, the galectin-10 antibodies are derived from VHH antibodies.

[0069] For example, the antibodies and antigen-binding fragments described herein can be selected from immune libraries obtained by a method comprising immunizing a camelid with a target of interest, i.e., galectin-10. The camelid can be immunized with the 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 in camelid species and selecting antibodies against preferred targets from camelid immune libraries are described, for example, in International Patent Application WO2010 / 001251, which is incorporated herein by reference.

[0070] In certain embodiments, the antibodies and antigen-binding fragments may be derived from a camelid in that they comprise at least one hypervariable (HV) loop or complementarity determining region (CDR) obtained from a VH or VL domain of a species of the Camelidae family. In particular, the antibodies and antigen-binding fragments may comprise a VH and / or VL domain, or CDRs thereof, obtained by active immunization of an outbred camelid, i.e., a llama, with galectin-10.

[0071] The term "derived from" in this context refers to a structural relatedness in that the HV or CDRs of the antibody embody an amino acid sequence (or a minor variant thereof) originally encoded by a Camelidae immunoglobulin gene, but does not necessarily imply a particular relatedness with respect to the production process used to prepare the antibody or antigen-binding fragment thereof.

[0072] Camelid-derived antibodies or antigen-binding fragments thereof may be derived from any camelid species, including, inter alia, llama, dromedary, alpaca, vicuna, guanaco, or camel.

[0073] Antibody molecules comprising camelid-derived VH and VL domains, or 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, i.e., camelids and humans.

[0074] In certain embodiments the entire VH domain and / or the entire VL domain may be obtained from a species of the Camelidae family. The Camelid-derived VH domain and / or the Camelid-derived VL domain may 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.

[0075] These artificially created changes preferably involve amino acid substitutions to camelid sequences, including "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.

[0076] Isolated camelid VH and VL domains obtained by active immunization of camelids (i.e. llamas) with galectin-10 can be used as a basis for artificially generating antibodies and antigen-binding fragments that bind galectin-10 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 that 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.

[0077] In other embodiments, "chimeric" antibody molecules are provided that comprise camelid-derived VH and VL domains (or artificial variants thereof) and one or more constant domains from a non-camelid antibody, e.g., a human-encoded constant domain (or artificial variants thereof). In such embodiments, both the VH and VL domains are preferably obtained from the same species of camelid, e.g., (prior to the introduction of artificially created amino acid sequence mutations), both the VH and VL may be derived from a Lama glama, or both the VH and VL may be derived from an alpaca. 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.

[0078] 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.

[0079] In non-limiting embodiments, the antibodies described herein may comprise a CH1 domain and / or a CL domain (from the heavy and light chains, respectively), the amino acid sequence of which is fully or substantially human. For antibody molecules intended for therapeutic use in humans, it is typical that the entire constant region of the antibody, or at least a part thereof, has a fully or substantially human amino acid sequence. Thus, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may be fully or substantially human in terms of its amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain, and / or CL domain (and / or CH4 domain, if present) may be derived from a human antibody, preferably a human IgG antibody, more preferably a human IgG1 antibody of subtype IgG1, IgG2, IgG3, or IgG4.

[0080] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may all have fully or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody 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. The term "human amino acid sequence" in this context refers to the amino acid sequence encoded by the human immunoglobulin gene, including rearranged and somatically mutated germline genes. The present invention also contemplates polypeptides comprising constant domains of "human" sequence that have been modified by one or more amino acid additions, deletions, or substitutions with respect to the human sequence, except in embodiments where the presence of a "fully human" hinge region is explicitly required.

[0081] Antibodies that bind Galectin-10 can have one or more amino acid substitutions, insertions, or deletions in the heavy and / or light chain constant regions, particularly in the Fc region. The amino acid substitutions can result in the replacement of the substituted amino acid with a different natural amino acid or with a non-natural or modified amino acid. Other structural modifications are also permissible, such as changes in the glycosylation pattern (e.g., by addition or deletion of N-linked or O-linked glycosylation sites).

[0082] Antibodies can be modified in the Fc region to increase their binding affinity to the neonatal receptor FcRn. The increased binding affinity can be measurable at acidic pH (e.g., about pH 5.5 to about pH 6.0). The increased binding affinity can also be measurable at neutral pH (e.g., about pH 6.9 to about pH 7.4). "Increased binding affinity" refers to increased binding affinity to FcRn compared to an unmodified Fc region. Typically, an unmodified Fc region retains the wild-type amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. In such embodiments, the increased FcRn binding affinity of an antibody molecule having a modified Fc region is measured compared to the binding affinity of wild-type IgG1, IgG2, IgG3, or IgG4 to FcRn.

[0083] In some embodiments, one or more amino acid residues in the Fc region can be replaced with different amino acids to increase binding to FcRn. Several Fc substitutions have been reported that increase FcRn binding, thereby improving antibody pharmacokinetics. Such substitutions are reported, for example, in Zalevsky et al. (2010) Nat. Biotechnol. 28(2):157-9; Hinton et al. (2006) J Immunol. 176:346-356; Yeung et al. (2009) J Immunol. 182:7663-7671; Presta LG (2008) Curr. Op. Immunol. 20:460-470; and Vaccaro et al. (2005) Nat. Biotechnol. 23(10):1283-88, the contents of which are fully incorporated herein.

[0084] In certain embodiments, the antibody comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, where the Fc domain numbering is according to the EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat, EA; National Institutes of Health (US) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA,(1991)). In further embodiments, the antibody described herein comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K, and N434F, where the Fc domain numbering is according to the EU numbering. In a preferred embodiment, the present invention provides antibodies that bind to galectin-10 (i.e., anti-galectin-10 antibodies), wherein the antibodies comprise at least one variant Fc domain incorporating ABDEG™ technology. ABDEG™ antibodies and FcRn antagonists incorporating ABDEG™ technology have been described for the treatment of antibody-mediated diseases, such as autoimmune diseases (see WO2006 / 130834 and WO2015 / 100299, which are incorporated herein by reference).

[0085] Additional Fc domain modifications that may be incorporated into a variant Fc domain or FcRn-binding fragment include, but are not limited to, those described in Ghetie et al., 1997, Nat. Biotech. 15:637-40; Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol., 147:2657-2662; Lund et al., 1992, Mol. Immunol., 29:53-59; Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA, 92:11980-11984; Jefferis et al., 1995, Immunol Lett., 20:11980-11984; 44:111-117; Lund et al., 1995, Faseb J., 9:115-119; Jefferis et al., 1996, Immunol Lett., 54:101-104; Lund et al., 1996, J. Immunol., 157:4963-4969; Armour et al., 1999, Eur J Immunol. 29:2613-2624; Idusogie et al., 2000, J. Immunol., 164:4178-4184; Reddy et al., 2000, J. Immunol., 164:1925-1933; Xu et al., 2000, Cell Immunol., 200:16-26; Idusogie et al., 2001, J. Immunol., 166:2571-2575; Shields et al., 2001, J Biol. Chem., 276:6591-6604; Jefferis et al., 2002, Immunol Lett., 82:57-65; Presta et al., 2002, Biochem Soc Trans., 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Publication No. 2004 / 0002587 and PCT Publication Nos. WO 94 / 29351; WO 99 / 58572; WO 00 / 42072; WO This includes those disclosed in WO 02 / 060919; WO 04 / 029207; WO 04 / 099249; WO 04 / 063351.

[0086] In certain embodiments, the antibodies described herein comprise a modified human IgG Fc domain that consists of up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 20 substitutions compared to the corresponding wild-type IgG sequence.

[0087] Any of the galectin-10 antibodies described herein can exhibit pH-dependent antigen binding, ie, pH-dependent binding to galectin-10.

[0088] Antibodies that are bound to an antigen are internalized and transported to the endosomal-lysosomal degradation pathway. Antibodies that can dissociate from their antigen in early endosomes can be recycled back to the cell surface. Antibodies that bind with high affinity to their antigen in endosomal compartments are usually transported to lysosomes for degradation. It has been previously shown that antibodies are more efficiently recycled to the cell surface if they have pH-dependent antigen binding activity such that they have lower binding affinity to their antigen at early endosomal pH compared to plasma pH. This can extend the antibody plasma half-life and allow the same antibody to bind to multiple antigens. For these reasons, it is advantageous that the anti-galectin-10 antibodies described herein exhibit pH-dependent antigen binding. The pH-dependent anti-galectin-10 antibodies according to the present invention have the ability to eliminate galectin-10 by binding to this protein. Galectin-10 can then be released in acidic endosomal compartments and transported to lysosomes for degradation. The free anti-galectin-10 antibodies of the invention can then be recycled back to the cell surface so that they can bind and internalize additional galectin-10.

[0089] The anti-galectin-10 antibody of the present invention may possess an intrinsic pH-dependent antigen-binding activity, i.e., it may have been selected for this property. Alternatively or additionally, the anti-galectin-10 antibody described herein may be artificially engineered to exhibit pH-dependent target binding. Methods for artificially engineer pH-dependent antigen-binding activity in antibody molecules are described, for example, in EP2275443, which is incorporated herein by reference. Methods for artificially engineer pH-dependent antigen-binding in antibody molecules are also described in WO2018 / 206748, which is incorporated herein by reference. The antibodies described herein may be modified by any technique to achieve pH-dependent binding. For example, the antibodies may be modified according to the methods described in EP2275443 or WO2018 / 206748 to exhibit pH-dependent antigen-binding.

[0090] For pH-dependent embodiments of the anti-galectin-10 antibodies described herein, the antigen-binding activity is lower at endosomal pH compared to the antigen-binding activity at plasma pH. Endosomal pH is usually an acidic pH, whereas plasma pH is usually a neutral pH. Thus, the antibodies described herein may exhibit pH-dependent antigen-binding such that their antigen-binding activity is lower at acidic pH compared to the antigen-binding activity at neutral pH. Endosomal pH or "acidic pH" may be a pH of about pH 4.0 to about pH 6.5, preferably about pH 5.5 to about pH 6.5, preferably about pH 5.5 to about pH 6.0, preferably pH 5.5, pH 5.6, pH 5.7, or pH 5.8. Plasma pH or "neutral pH" can be a pH of about pH 6.9 to about pH 8.0, preferably about pH 7.0 to about pH 8.0, preferably about pH 7.0 to about pH 7.4, preferably a pH of pH 7.0 or pH 7.4.

[0091] In certain embodiments, the anti-galectin-10 antibody exhibits pH-dependent binding such that the antigen-binding activity at pH 5.8 is lower compared to the antigen-binding activity at pH 7.4. The pH-dependent anti-galectin-10 antibody may be characterized by a higher dissociation constant (KD) of the antibody-antigen interaction at acidic pH or pH 5.8 than the dissociation constant (KD) of the antibody-antigen interaction at neutral pH or pH 7.4. In certain embodiments, the anti-galectin-10 antibody exhibits pH-dependent binding such that the ratio of the KD for the antigen at pH 5.8 to the KD for the antigen at pH 7.4 (KD(pH5.8) / KD(pH7.4)) is 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, or 12 or more.

[0092] The pH-dependent antigen-binding activity of an antibody molecule can be artificially created by modifying the antibody molecule to impair the antigen-binding ability at acidic pH and / or increase the antigen-binding ability at neutral pH. For example, the antibody molecule can be modified by substituting at least one amino acid of the antibody molecule with histidine or by inserting at least one histidine into the antibody molecule. The site of such histidine mutation (substitution or insertion) is not particularly limited, and any site is acceptable as long as the antigen-binding activity at endosomal pH (e.g., pH 5.8) is lower than the antigen-binding activity at plasma pH (e.g., pH 7.4) compared to before the mutation or insertion.

[0093] In certain embodiments, anti-galectin-10 antibodies can be engineered to exhibit pH-dependent antigen binding by the introduction of one or more substitutions into the variable domain. In a preferred embodiment, anti-galectin-10 antibodies can be engineered to exhibit pH-dependent antigen binding by the introduction of one or more substitutions into one or more CDRs of the antibody. The substitutions can be to introduce one or more His residues into the variable domain, preferably into one or more sites in the heavy and / or light chain CDRs, to confer pH-dependent antigen binding.

[0094] For embodiments of the invention in which an antibody comprises three heavy chain CDR sequences and three light chain CDR sequences, the six combined CDRs can consist of a total of 1-10 His substitutions, optionally 1-5 His substitutions, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 His substitutions. Anti-galectin-10 antibodies can be engineered according to the methods described in WO2018 / 206748, which is incorporated herein by reference. Non-histidine substitutions can also be incorporated into the variable domains, particularly the CDRs, of the pH-dependent antibodies described herein.

[0095] In a preferred embodiment, an exemplary anti-galectin-10 antibody having the specific CDR, VH, and / or VL domain sequences listed herein is engineered such that it exhibits pH-dependent antigen binding. For example, the CDR sequences of the exemplary anti-galectin-10 antibodies described herein can be modified by the introduction of one or more histidine substitutions to generate an antibody that exhibits pH-dependent antigen binding.

[0096] The antibodies described herein can also be modified to form immunoconjugates comprising an antibody conjugated to a cytotoxic agent, e.g., a chemotherapeutic agent, a toxin (i.e., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope (i.e., a radioconjugate). The Fc region can also be engineered to extend half-life, as described by Chan and Carter (2010) Nature Reviews: Immunology 10:301-316, incorporated herein by reference.

[0097] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids.

[0098] In certain embodiments, Fc region can be artificially created to have no effector function.In some embodiments, the antibody molecule of the present invention can have an Fc region derived from a natural IgG isotype, such as IgG4, with reduced effector function.The Fc region derived from IgG4 can be further modified to increase therapeutic utility, for example, by introducing modifications that minimize arm exchange between IgG4 molecules in vivo.The Fc region derived from IgG4 can be modified to include S228P substitution.

[0099] In certain embodiments, the antibody molecule is modified with respect to glycosylation. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of the antibody to the target antigen. Such carbohydrate modification can be achieved, for example, by modifying one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the abolishment of one or more variable region framework glycosylation sites, thereby abolishing glycosylation at that site. Such aglycosylation can increase the affinity of the antibody to the antigen.

[0100] Mutant antibodies that bind to galectin-10 with modified types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or fully or partially defucosylated antibodies (as described in Natsume et al., Drug Design Development and Therapy, Vol. 3, pp7-16, 2009) or antibodies with increased bisecting GlcNac structures, are also envisioned. Such modified glycosylation patterns have been shown to increase the ADCC activity of antibodies, typically resulting in a 10-fold ADCC enhancement compared to equivalent antibodies containing a "native" human Fc domain. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation enzyme machinery (as described by Yamane-Ohnuki and Satoh, mAbs 1:3, 230-236, 2009). An example of a non-fucosylated antibody with enhanced ADCC function is one produced using BioWa's Potelligent™ technology.

[0101] Exemplary Antibodies that Bind Galectin-10 The present invention provides exemplary antibodies and antigen-binding fragments that bind to Galectin-10. The antibodies and antigen-binding fragments of the present invention can be defined solely with respect to their structural characteristics, as described below.

[0102] [Clone g24F02_N53A] Provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: HCDR3 comprising or consisting of SEQ ID NO:2; HCDR2 comprising or consisting of SEQ ID NO:3; HCDR1 comprising or consisting of SEQ ID NO:1; and (ii) the VL domain comprises the CDR sequences of LCDR3 comprising or consisting of SEQ ID NO:8; LCDR2 comprising or consisting of SEQ ID NO:9; LCDR1 comprising or consisting of SEQ ID NO:7.

[0103] Also provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0104] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0105] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0106] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0107] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0108] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0109] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:10.

[0110] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:10.

[0111] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:10.

[0112] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:10.

[0113] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:10.

[0114] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises the amino acid sequence of SEQ ID NO:4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0115] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain consists of the amino acid sequence of SEQ ID NO:4; and (ii) the VL domain consists of the amino acid sequence of SEQ ID NO:10.

[0116] [Clone g24F02] Further provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: HCDR3 comprising or consisting of SEQ ID NO:2; HCDR2 comprising or consisting of SEQ ID NO:5; HCDR1 comprising or consisting of SEQ ID NO:1; and (ii) the VL domain comprises the CDR sequences of LCDR3 comprising or consisting of SEQ ID NO:8; LCDR2 comprising or consisting of SEQ ID NO:9; LCDR1 comprising or consisting of SEQ ID NO:7.

[0117] Also provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0118] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:6; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0119] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:6; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0120] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:6; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0121] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:6; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0122] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:6; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0123] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:10.

[0124] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:10.

[0125] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:10.

[0126] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:10.

[0127] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:10.

[0128] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises the amino acid sequence of SEQ ID NO:6; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0129] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain consists of the amino acid sequence of SEQ ID NO:6; and (ii) the VL domain consists of the amino acid sequence of SEQ ID NO:10.

[0130] [Clone g23H09] Further provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: HCDR3 comprising or consisting of SEQ ID NO: 12; HCDR2 comprising or consisting of SEQ ID NO: 13; HCDR1 comprising or consisting of SEQ ID NO: 11; and (ii) the VL domain comprises the CDR sequences of LCDR3 comprising or consisting of SEQ ID NO:8; LCDR2 comprising or consisting of SEQ ID NO:9; LCDR1 comprising or consisting of SEQ ID NO:7.

[0131] Also provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0132] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 14; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0133] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0134] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 14; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0135] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 14; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0136] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 14; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0137] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:10.

[0138] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:10.

[0139] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:10.

[0140] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:10.

[0141] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:10.

[0142] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises the amino acid sequence of SEQ ID NO: 14; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0143] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain consists of the amino acid sequence of SEQ ID NO: 14; and (ii) the VL domain consists of the amino acid sequence of SEQ ID NO:10.

[0144] [Clone g18C06] Further provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: HCDR3 comprising or consisting of SEQ ID NO: 16; HCDR2 comprising or consisting of SEQ ID NO: 17; HCDR1 comprising or consisting of SEQ ID NO: 15; and (ii) the VL domain comprises the CDR sequences of: LCDR3 comprising or consisting of SEQ ID NO:20; LCDR2 comprising or consisting of SEQ ID NO:21; LCDR1 comprising or consisting of SEQ ID NO:19.

[0145] Also provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0146] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0147] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 18; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0148] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 18; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0149] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 18; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0150] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 18; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0151] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:22.

[0152] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:22.

[0153] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:22.

[0154] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:22.

[0155] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:22.

[0156] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises the amino acid sequence of SEQ ID NO: 18; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO:22.

[0157] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain consists of the amino acid sequence of SEQ ID NO: 18; and (ii) the VL domain consists of the amino acid sequence of SEQ ID NO:22.

[0158] [Clone g20H09] Further provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: HCDR3 comprising or consisting of SEQ ID NO: 12; HCDR2 comprising or consisting of SEQ ID NO: 23; HCDR1 comprising or consisting of SEQ ID NO: 11; and (ii) the VL domain comprises the CDR sequences of LCDR3 comprising or consisting of SEQ ID NO:8; LCDR2 comprising or consisting of SEQ ID NO:9; LCDR1 comprising or consisting of SEQ ID NO:7.

[0159] Also provided herein is an antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0160] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 24; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0161] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 24; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0162] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 24; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0163] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 24; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0164] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 24; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto.

[0165] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:10.

[0166] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:10.

[0167] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 97% identity to SEQ ID NO:10.

[0168] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 98% identity to SEQ ID NO:10.

[0169] In some embodiments, the antibody or antigen-binding fragment that binds to Galectin-10 comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO:10.

[0170] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain comprises the amino acid sequence of SEQ ID NO: 24; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO:10.

[0171] In some embodiments, the antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: (i) the VH domain consists of the amino acid sequence of SEQ ID NO: 24; and (ii) the VL domain consists of the amino acid sequence of SEQ ID NO:10.

[0172] For embodiments in which antibody or antigen-binding fragment domains are defined by a particular percentage 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 mutations are present only within the framework regions. In alternative embodiments, the CDR sequences may also include amino acid substitutions (e.g., conservative substitutions, humanizing substitutions, or affinity variants) relative to the reference sequence.

[0173] The present invention also provides antibodies or antigen-binding fragments thereof that bind to the same epitope as the galectin-10 antibodies exemplified herein.

[0174] In certain embodiments, exemplary antibodies and antigen-binding fragments defined as having the CDR sequences listed above or defined as having a particular percentage identity with a particular VH / VL domain amino acid sequence listed above are humanized, germlined, or affinity variants of the antibody or antigen-binding fragment thereof from which the CDR, VH, and / or VL sequences are derived.

[0175] In a preferred embodiment, for example, exemplary antibody molecules having the CDR sequences listed above that exhibit high human homology are humanized or germline variants of the antibody or antigen-binding fragment thereof from which the CDR sequences are derived.

[0176] For antibody molecules intended for therapeutic use in humans, it is typical that the entire constant region of the antibody, or at least a portion thereof, has a fully or substantially human amino acid sequence. Thus, in one embodiment, the Fc region may be fully or substantially human with respect to its amino acid sequence. In the context of the constant region of a humanized or chimeric antibody or antibody 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. The term "human amino acid sequence" in this context refers to the amino acid sequence encoded by the human immunoglobulin gene, including rearranged and somatically mutated germline genes. The present invention also contemplates polypeptides comprising a constant domain of "human" sequence that has been modified by one or more amino acid additions, deletions, or substitutions with respect to the human sequence, except in embodiments where the presence of a "fully human" hinge region is explicitly required. Any of the exemplary Fc region modifications described herein may be incorporated into an antibody having the CDR and / or VH / VL domain sequences listed above. In certain embodiments, an antibody having the CDR and / or VH / VL domain sequences listed above comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions H433K and N434F, where the Fc domain numbering is according to EU numbering. In certain embodiments, an antibody having the CDR and / or VH / VL domain sequences listed above comprises a modified human IgG Fc domain comprising or consisting of the amino acid substitutions M252Y, S254T, T256E, H433K, and N434F.

[0177] D. Polynucleotides Encoding Antibodies that Bind Galectin-10 The present invention also provides polynucleotide molecules encoding the Galectin-10 antibodies of the invention or fragments thereof. Polynucleotide molecules encoding full-length Galectin-10 antibodies are provided, along with polynucleotide molecules encoding fragments of Galectin-10 antibodies described herein, such as the VH and / or VL domains. Also provided are expression vectors containing the nucleotide sequences of the invention operably linked to regulatory sequences enabling expression of the antibodies or fragments thereof in a host cell or cell-free expression system, and host cells or cell-free expression systems containing the expression vectors.

[0178] Polynucleotide molecules encoding Galectin-10 antibodies of the invention include, for example, recombinant DNA molecules. The terms "nucleic acid", "polynucleotide", or "polynucleotide molecule" are used interchangeably herein and refer to any DNA or RNA molecule, either single-stranded or double-stranded, and, if single-stranded, to the molecule of its complementary sequence. When discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule may be described herein according to the usual convention of providing the sequence in the 5' to 3' direction. In some embodiments of the invention, a nucleic acid or polynucleotide is "isolated". This term, when applied to a nucleic acid molecule, refers to a nucleic acid molecule that is separated from sequences with which it is immediately adjacent in the naturally occurring genome of the organism from which it was derived. For example, an "isolated nucleic acid" can include a DNA molecule that is inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or a non-human host organism. When applied to RNA, an "isolated polynucleotide" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been purified / separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated polynucleotide (either DNA or RNA) can further refer to a molecule that has been produced directly by biological or synthetic means and that has been separated from other components present during its production.

[0179] For recombinant production of a Galectin-10 antibody according to the invention, a recombinant polynucleotide encoding it, or encoding the different chains or domains, can be prepared (using standard molecular biology techniques) and inserted into a replicable vector for expression in a chosen host cell or cell-free expression system. Suitable host cells can be prokaryotes, yeast, higher eukaryotic cells, in particular mammalian cells. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL 8287) or NS0 (HPA Culture Collection No. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM's PERC-6 cell line. Suitable expression vectors for use in each of these host cells are also generally known in the art.

[0180] It should be noted that the term "host cell" generally refers to a cultured cell line. A whole human into which an expression vector encoding an antigen-binding polypeptide according to the invention has been introduced is expressly excluded from the definition of a "host cell".

[0181] (E. Antibody Production) In a further aspect, the present invention also provides a method of producing an antibody of the invention, comprising culturing a host cell (or a cell-free expression system) containing a polynucleotide (e.g., an expression vector) encoding the antibody under conditions allowing expression of the antibody, and recovering the expressed antibody. This recombinant expression process can be used for large-scale production of antibodies, including Galectin-10 antibodies according to the invention, including monoclonal antibodies intended for therapeutic use in humans. Suitable vectors, cell lines, and production processes for large-scale production of recombinant antibodies suitable for in vivo therapeutic use are generally available in the art and well known to the skilled artisan.

[0182] F. Pharmaceutical Compositions The present invention includes pharmaceutical compositions containing one or a combination of Galectin-10 antibodies or antigen-binding fragments thereof, formulated with one or more pharma- ceutically acceptable carriers or excipients. Such compositions may contain one or a combination (i.e., two or more different) Galectin-10 antibodies. Techniques for formulating monoclonal antibodies for therapeutic use in humans are well known in the art and are reviewed, for example, in Wang et al., Journal of Pharmaceutical Sciences, Vol. 96, pp1-26, 2007, the contents of which are incorporated herein in their entirety.

[0183] The pharmaceutical compositions according to the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially.

[0184] 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, 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 (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0185] In certain embodiments, the composition is formulated for administration to a subject via any suitable route of administration, including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, buccal, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration.

[0186] In a preferred embodiment, the route of administration is by inhalation.Preferably, the composition of the present invention can be formulated as a powder for inhalation or as an aerosolized liquid for inhalation.Preferably, the composition according to the present invention can be formulated as a dry powder.Alternatively, the composition according to the present invention can be formulated as a liquid aerosol or liquid spray that is nebulized.

[0187] Means and devices for inhalation administration of compositions are well known in the art. Inhalation administration of compositions can be achieved, for example, through a nebulizer. A nebulizer is a drug delivery device that is used to administer a drug as a mist that is inhaled into the lungs. In an alternative method, an inhaler can be used to administer the composition of the present invention. An inhaler is a drug delivery device that delivers a drug to the lungs via inhalation. Several types of inhalers are well known in the art, including, for example, metered dose inhalers (MDI), dry powder inhalers (DPI) and soft mist inhalers (SMI).

[0188] (G.Treatment method) The antibodies and antigen-binding fragments that bind to galectin-10 described herein can be used in methods of treatment. Accordingly, the present invention provides antibodies and antigen-binding fragments that bind to galectin-10 for use as medicaments. Alternatively, provided herein are antibodies and antigen-binding fragments that bind to galectin-10 for use in methods of treatment. The antibodies and antigen-binding fragments of the present invention that are intended for use as medicaments are typically formulated as pharmaceutical compositions.

[0189] Importantly, all of the embodiments described above with respect to antibodies and antigen-binding fragments are equally applicable to the methods described herein.

[0190] The present invention also provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment as described elsewhere herein. In such treatment methods, the antibodies and antigen-binding fragments are typically formulated as pharmaceutical compositions. As used herein, the term "therapeutically effective amount" is intended to mean an amount or dose of a Galectin-10 antibody that is sufficient to produce a therapeutic effect, e.g., an amount or dose of an antagonist 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.

[0191] For clinical use, in certain embodiments, the galectin-10 antibody or antigen-binding fragment described elsewhere herein is administered to the subject in one or more doses of about 0.1 mg / kg body weight to about 20 mg / kg body weight. In certain embodiments, the antibody or antigen-binding fragment described elsewhere herein is administered to the subject in a dose of about 0.1 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the antibody or antigen-binding fragment described elsewhere herein is administered to the subject in a dose of about 0.5 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, the antibody or antigen-binding fragment described elsewhere herein is administered to the subject in a dose of about 1 mg / kg body weight to about 10 mg / kg body weight.

[0192] Antibodies and antigen-binding fragments that bind to galectin-10 are useful in therapeutic methods because they can disrupt galectin-10 crystallization. As described elsewhere herein, the antibodies of the invention bind to an epitope on galectin-10, thereby disrupting crystallization of galectin-10. In certain embodiments, the antibodies and antigen-binding fragments inhibit crystallization of galectin-10. In certain embodiments, the antibodies and antigen-binding fragments promote dissolution of crystalline galectin-10.

[0193] Galectin-10 antibodies and antigen-binding fragments thereof can be for use in preventing or treating a disease or disorder associated with the presence or formation of galectin-10 crystals or CLCs. Provided herein are methods of preventing or treating a disease or disorder associated with the presence or formation of galectin-10 crystals or CLCs in a patient or subject in need thereof by administering an effective amount of a Galectin-10 antibody or antigen-binding fragment thereof.

[0194] As used herein, a method of "preventing" a disease or disorder means preventing the onset of the disease, preventing the worsening of symptoms, preventing the progression of the disease or disorder, or reducing a subject's risk of developing the disease or disorder. 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 reduced.

[0195] For patients with a disease or disorder characterized by the presence of galectin-10 crystals, treatment methods typically involve administration of a galectin-10 antibody or antigen-binding fragment thereof capable of dissolving galectin-10 crystals in the patient's tissues. For patients identified as "at risk" of developing a disease or disorder characterized by the formation of galectin-10 crystals, prevention methods can involve administration of a galectin-10 antibody or antigen-binding fragment thereof capable of inhibiting crystallization of galectin-10.

[0196] Galectin-10 crystals or CLCs have been observed in patients with various diseases and disorders. It follows that the galectin-10 antagonists described herein can be used to prevent or treat a disease or condition selected from the group consisting of asthma; chronic sinusitis; celiac disease; helminth infection; gastrointestinal eosinophilic inflammation; cystic fibrosis (CF); allergic bronchopulmonary aspergillosis (ABPA); Churg-Strauss vasculitis; chronic eosinophilic pneumonia; and acute myeloid leukemia (AML). In a preferred embodiment, the galectin-10 antibody or antigen-binding fragment thereof is used to prevent or treat a disease or condition selected from the group consisting of asthma; chronic sinusitis; celiac disease; helminth infection; gastrointestinal eosinophilic inflammation; cystic fibrosis (CF); allergic bronchopulmonary aspergillosis (ABPA); Churg-Strauss vasculitis; chronic eosinophilic pneumonia; and acute myeloid leukemia (AML).

[0197] As mentioned above, galectin-10 crystals or CLCs are particularly associated with disorders or diseases characterized by eosinophilic inflammation. Thus, in a preferred embodiment, the galectin-10 antibodies or antigen-binding fragments thereof described herein are used to treat disorders or diseases associated with eosinophilic inflammation.

[0198] In certain preferred embodiments, the galectin-10 antibodies or antigen-binding fragments thereof described herein are used to prevent or treat asthma. Analysis of the airways and lungs of asthmatic patients has shown the presence of CLCs (Persson EK, Verstraete K, Heyndrickx I et al. Protein crystallization promotes type 2 immunity and is reversible by antibody treatment. Science. 2019;364(6442)). Thus, the antibodies of the invention bind to an epitope on galectin-10, thereby disrupting galectin-10 crystallization. This in turn prevents CLC formation in the airways and lungs of asthmatic patients.

[0199] Clinically, asthma is characterized by reversible airway obstruction and airway hyperresponsiveness that results in shortness of breath and wheezing. Although it is often treatable with inhaled steroids and bronchodilators, a subgroup of patients has severe, treatment-resistant disease that requires frequent hospitalizations that can lead to fatal attacks (Braido F. Failure in asthma control: reasons and consequences. Scientifica (Cairo) 2013;2013:549252). Pathologically, the disease is characterized by airway eosinophilia and excessive production of thick mucus that can lead to irreversible obstruction of small airways (Zhang L, He L, Gong J, Liu C, Risk Factors Associated with Irreversible Airway Obstruction in Asthma: A Systematic Review and Meta-Analysis. Biomed Res Int. 2016;2016:9868704). In most cases, the disease is caused by an immune response of type 2 immune cells (CD4 Th2 lymphocytes and type 2 innate lymphoid cells (ILC2)), resulting in the production of IL-4 (which stimulates goblet cell metaplasia and IgE synthesis), IL-5 (which promotes tissue eosinophilia), and IL-13 (which causes bronchial hyperresponsiveness and goblet cell metaplasia) (Lambrecht BN, Hammad H. The immunology of asthma. Nat Immunol. 2015;16(1):45-56).

[0200] In some embodiments, the asthma is characterized as allergic asthma, which is a chronic inflammatory disease of the conducting airways that affects 8-12% of people in Europe (Selroos O, Kupczyk M, Kuna P, et al. National and regional asthma programmes in Europe. Eur Respir Rev. 2015;24(137):474-483).

[0201] In other certain preferred embodiments, the Galectin-10 antibodies, or antigen-binding fragments thereof, described herein are used to prevent or treat cystic fibrosis (CF).

[0202] The present invention also provides the use of a galectin-10 antibody or an antigen-binding fragment thereof for the detection of galectin-10 in a sample obtained from a patient. The antibody or antigen-binding fragment thereof is typically used to detect crystalline galectin-10. As mentioned above, galectin-10 crystals or CLC crystals have been observed in patients with several different diseases and disorders. It therefore follows that a patient sample can be obtained from a subject having or suspected of having any one of the following diseases or disorders: asthma, chronic sinusitis, celiac disease, helminth infection, gastrointestinal eosinophilic inflammation, cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA), Churg-Strauss vasculitis, chronic eosinophilic pneumonia, or acute myeloid leukemia (AML). The detection of crystalline galectin-10 in a patient sample can be used to diagnose the disease or disorder of the subject from which the sample was obtained. The sample can be any suitable patient sample, for example any fluid or tissue in which CLC is observed in a disease state. In some embodiments, the sample is a tissue sample obtained from a polyp, e.g., a nasal polyp. In some embodiments, the sample is a mucus sample. In such embodiments, detection of crystalline galectin-10 in a mucus sample using the antibody or antigen-binding fragment thereof of the present invention can be used to detect or diagnose chronic sinusitis. In a preferred embodiment, the patient sample is a sputum sample. In such embodiments, detection of crystalline galectin-10 in a sputum sample using the antibody or antigen-binding fragment thereof of the present invention can be used to detect or diagnose asthma.

[0203] (H.Kit) Any of the antibodies or antigen-binding fragments described herein can be packaged as a kit, and optionally include instructions for use. EXAMPLES

[0204] (Example) The invention will be further understood with reference to the following non-limiting examples.

[0205] (Background and Objectives) Clone 7B07 was described in WO 2019 / 197675. This clone was observed to bind and dissolve recombinant Charcot-Leyden crystals (CLCs, also known as galectin-10 (GAL10) crystals). The process of germlining by complementarity determining region (CDR) grafting did not affect the binding and potency of this clone. However, stability studies identified a deamidation site (N53G54) in the CDR2 of the heavy chain, which caused a decrease in binding and potency at incubation temperatures of 25°C and 37°C. To overcome this issue, mutants of the germlined 7B07 (g7B07) clone were generated that carried point mutations at N53 and G54 in the CDR2 of the heavy chain. The potency of these g7B07 mutants to dissolve recombinant CLCs was maintained, but all mutations resulted in a decrease in binding properties.

[0206] As a result, three discovery campaigns were initiated to further identify anti-galectin-10 (anti-Gal10) compounds with more favorable properties compared to clone 7B07.

[0207] Example 1. Selection of recombinant antibodies that bind to Gal10 (1.1 7B07 Epitope Campaign) (Selection of clones that bind to the 7B07 epitope on Gal10 by phage display) A phage panning approach was used to select scFv clones with suitable binding ability to human Gal10. A competitive setting was used to select clones that bind to a region on Gal10 similar to clone 7B07.

[0208] In this setup, the anti-human specific clone 1D11, which targets the tyrosine 69 residue, was coated onto Maxisorp plates to capture Gal10-His. Capturing Gal10 with 1D11 had two advantages in selecting clones that bind to the 7B07 epitope. The first advantage was that the 7B07 epitope was available to phage expressing an scFv against Gal10, since 1D11 binds to a site on Gal10 opposite to 7B07. The second advantage was that by capturing Gal10-His with a clone that binds to tyrosine 69 (clone 1D11), this epitope was masked. This is relevant because during the selection campaign, most clones appeared to bind close to the 1D11 epitope. Elution of bound phage was performed by trypsin (non-specific elution) or by competitive elution with high concentrations of 7B07 IgG (specific elution of phage expressing scFvs that bind to the same binding region as clone 7B07).

[0209] Two llama-derived scFv libraries (lambda and kappa) were used to select for scFv clones with binding activity to Gal10. Two rounds of selection clearly enriched phages expressing scFvs specific for human Gal10. Similar enrichment (up to 100-fold) was observed compared to the PBS control.

[0210] (Screening of Gal10-specific binders) Two master plates were generated after the second round of selection against the 7B07 epitope of Gal10, where both trypsin and competitive elution with 7B07 were used. Master plate 18 (MP18) was generated from the second round of selection of the lambda library, where competitive elution was performed from the first round. Master plate 19 (MP19) was generated from all other conditions, where elution was performed with trypsin or 7B07 (Table 3). From these master plates, periplasmic extracts (scFv) were generated and their binding ability to Gal10 was analyzed by ELISA and Biacore. [Table 3] Table 3: Overview of Master Plates (MP) generated after the selection campaign against the 7B07 epitope on Gal10

[0211] (Screening of scFv periplasmic extracts) The binding capacity of the periplasmic extracts was analyzed by ELISA (binding and competition with 7B07) and surface plasmon resonance (SPR).

[0212] (ELISA binding screening) The binding ability of scFv (periplasmic extract) to human Gal10 was analyzed by ELISA. In this experiment, clones with an OD of 0.3 or higher were classified as Gal10 binders. In total, 48 Gal10-specific clones were identified.

[0213] (Competitive screening by ELISA) Next, the target binding region of the scFv on Gal10 was analyzed by an additional ELISA examining the competition of the clones against clone 7B07. In this setup, Gal10 was captured on a Maxisorp plate coated with 7B07. Therefore, it was expected that clones with a similar binding position to clone 7B07 on Gal10 would not be able to bind and would show low OD values, whereas clones binding to other regions would show high OD values.

[0214] Analysis of binding and competition ELISA experiments revealed that 25 clones competed with clone 7B07 for Gal10 binding, with OD values ​​>0.3 in the binding ELISA and <0.1 in the competition ELISA.

[0215] (Off-rate screening with SPR (Biacore 3000)) The off-rates of the remaining 25 clones from the ELISA experiment were determined by SPR on a Biacore 3000 instrument. Periplasmic extracts were injected over a 2500 RU Gal10-His coated CM5 sensor chip. Eleven clones showed at least a 2-fold improvement in off-rate compared to clone 7B07 (2.18E-03 1 / s) and were selected for further characterization (Table 4). [Table 4] Table 4: Off-rates of scFv periplasmic extracts. This table shows the amplitude of binding (Rmax), dissociation (off-rate) and fold change in off-rate compared to the control (g7B07) for each clone.

[0216] (1.2 Heavy chain shuffling campaign) A heavy chain shuffling approach was performed to find clones that could pair with the 7B07 light chain and allow good affinity for Gal10 and improved stability.

[0217] (Library Construction (Fab VH Shuffling)) For the construction of the shuffled heavy chain library, a two-step PCR was used. First, non-tagged primers were used directly against the cDNA of two immune llamas (obtained in the previous selection campaign) to amplify the VH-CH1. The resulting PCR product was then purified and used in a second PCR with tagged primers to amplify the VH. As clone 7B07 was isolated from llama Montoyo, the VL of clone 7B07 was shuffled with the PCR amplified VH repertoire from llama Montoyo. The size of the final Fab library was 1.5E+07 VH / VL combinations, and the correct insertion rate of VL and VH was 94% as determined by colony PCR.

[0218] A phage panning approach was used to select new Fabs with binding capacity to human Gal10 equal to or greater than that of the parent g7B07 Fab. To this end, the first and second rounds of selection were performed against human Gal10-His and an unrelated His-tagged protein (as a control). The third and fourth rounds of selection were performed against soluble non-His-tagged human Gal10.

[0219] The first two rounds of selection were performed against 1 and 10 μg / mL coated human Gal10-His and 10 μg / mL of an irrelevant His-tagged protein. For both the first and second rounds of selection, eluted phage from the 10 μg / mL Gal10-His condition were used for the subsequent third and fourth rounds of selection.

[0220] (Screening of Gal10-specific binders) Production of Fab as a periplasmic extract From trypsin eluted phage from round 3 and round 4, single clones were generated and two master plates were generated (Table 5). Master plate 24 (MP24) was generated from the third round of selection using colonies selected from the different conditions (Gal10, no off-rate wash, and off-rate wash). Master plate 26 (MP26) was generated from the fourth round of selection using colonies selected from both the no off-rate and off-rate washes. [Table 5] Table 5: Overview of master plates generated following the selection campaign against human Gal10.

[0221] (Sequence analysis) The results of sequencing Master Plate 24 (MP24) revealed that there were only four groups of VH families that were different from 7B07_VH based on the CDR3 sequences. Further analysis showed that two of these four VH families were camelid single domain antibodies. From the remaining two VH families, representative clones-clone 24A04 and clone 24F02 were selected for further analysis.

[0222] (Gal10 binding of two selected clones using BLI technique) Periplasmic extracts of two selected clones were tested for binding to captured Gal10-His using an Octet RED96 instrument (Bio-Layer Interferometry (BLI) technology).

[0223] In this analysis, a germlined clone of clone 7B07 (g7B07) was included as a reference. A lower response to Gal10-His was measured compared to the reference clone. Only clone 24F02 showed a better (128-fold) off-rate compared to clone g7B07 (Table 6). [Table 6] Table 6: Calculated off-rates kd (1 / s)

[0224] (Competitive ELISA for 7B07 epitope) To confirm that the selected clones targeted the same region on Gal10 as clone g7B07, a competitive ELISA was performed. Briefly, 96-well Maxisorp plates were coated with 7B07_hIgG1 to capture Gal10-His. Then, periplasmic extracts containing Fab-Myc were incubated and bound Fab was detected with anti-Myc-HRP antibody. Clones with OD values ​​<0.1 were defined as sharing the 7B07 epitope. A positive control sample (clone 18C06) was used as a reference sample.

[0225] Clone 24F02 showed no binding, suggesting that 24F02 binds to the same epitope as clone 7B07. Similar data were obtained for the control antibody 18C06, which is known to compete with 7B07. In contrast, clone 24A04 showed an OD value >0.1, indicating that it binds to a separate epitope from 7B07. [Table 7] Table 7: Mean OD 450 nm values.

[0226] (1.3 g7B07_CDR2_VH Randomization Campaign) (Library Construction (Fab)) Randomization of the deamidation site within CDR2-N53G54- of g7B07 failed to provide g7B07 variants without a deamidation site and good binding affinity to Gal10. CDR2 residues were randomized to find sequences without a deamidation site and good binding affinity. Structural modeling of the portion of the 7B07 Fab complexed with Gal10 led to the creation of a randomized CDR2 library for further studies. Flexible tip of the CDR2 loop [ka] Four libraries were constructed: one in which only the 7B07 VH-CDR2 residues at the flexible tip of the CDR2 loop were randomized, leaving the antiparallel beta-sheet intact; in the library designated X6, all six residues at the flexible tip of the CDR2 loop were randomized (IXXXXXXT, where X represents a single amino acid that was randomized). As this entailed the risk of revisiting the original 7B07 VH-CDR2 sequence, three further libraries were constructed that were one amino acid shorter: [ka] ;where "X" represents a randomized position.

[0227] In this crystal structure, residues 54-56 (GGG) collide with the Gal10 molecule, bending G55 back, and it was therefore suggested that shortening this sequence by one amino acid would allow a better fit and binding to Gal10.

[0228] A specific primer set was made for each library to randomize six residues in CDR2 of the heavy chain variable domain of clone g7B07. After two-step nested PCR using the heavy chain DNA of germline clone 7B07 as template, the amplicon was digested with restriction enzymes and then ligated into PCB13 phagemid vector containing the light chain variable domain of g7B07.

[0229] As a result of library construction, four libraries were obtained that showed library sizes that were 5-1224 times larger than the theoretical library size (Table 8). [Table 8] Table 8: Sizes of the four different libraries created.

[0230] (Selection by phage display) To identify stable g7B07 variants with good binding affinity to Gal10, phages (input) from four different libraries were bound to Gal10-His coated on Maxisorp plates for 24 h in the presence or absence of an off-rate wash with 10-fold excess of Gal10-His. To assess specific enrichment for Gal10, but not the His-tag, an irrelevant His-tagged protein was coated as a negative control. This process resulted in clear enrichment after the first round of selection. The output titers of the irrelevant His-tagged protein panning were clearly higher than the PBS control. Furthermore, a 24-h off-rate wash performed with 10-fold excess of Gal10 resulted in lower enrichment (10-100-fold) compared to the elution performed on day 0, but similar to the elution control performed after 24 h (data not shown), suggesting that phages expressing Fab likely have a higher affinity for human Gal10.

[0231] Eluted phages from the round 1 direct trypsin elution condition at 10 μg / mL were further selected against two concentrations of coated Gal10-His (10 and 1 μg / mL). The same process, with off-rate washes, was applied during the second round of selection.

[0232] The results of the second round of selection showed similar or up to 10-fold lower enrichment compared to the first round of selection. The 24 hour off-rate wash resulted in 10-fold lower output titers compared to the no off-rate wash condition, except in the case of the X3 library. One master plate containing clones from the first and second round selections with and without off-rate selection was prepared for each library.

[0233] (Screening of binding of CDR2 randomized clones) (Production of Fab periplasmic extracts) Single clones were generated from the eluted phages from the first and second round of selection (with and without off-rate washes) and a total of four master plates (one master plate per library containing colonies selected from different conditions as shown in Table 9) were generated. [Table 9] Table 9: Summary table of master plates (MP) generated after the selection campaign against human Gal10-His (CDR2 randomization campaign).

[0234] A total of four master plates were generated using the trypsin elution method after first and second round selection against 10 μg / mL human Gal10, with or without an off-rate wash performed with a 10-fold excess of antigen.

[0235] From these master plates, periplasmic extracts were prepared (Fab) and their binding ability to human Gal10 was analyzed by SPR.

[0236] (Screening of Fab periplasmic extracts generated from a CDR2 randomization campaign) The binding properties of Fab (periplasmic extract) to human Gal10-His were analyzed by SPR using a Biacore 3000 instrument. For this purpose, diluted periplasmic extracts were applied onto a CM5 chip coated with human Gal10-His. As a positive control, injections of 20 nM of purified g7B07 clone in human Fab backbone were included at the beginning and end of the run. During the screening, only the dissociation (off-rate) of Fab could be determined, since the effective concentration of Fab was unknown and could vary widely from clone to clone.

[0237] Of the 376 clones screened by SPR, most of the periplasmic extracts screened showed higher off-rates than clone g7B07 (off-rate = 6.9E-03 1 / s). [Table 10] Table 10: Screening of Fab periplasmic extracts generated from the CDR2 randomization campaign using SPR technology. Clones with similar or better off-rates compared to the control g7B07 are highlighted in bold (column "Fold off-rate"). The randomized portion of the CDR2 sequence is underlined.

[0238] The binding ability of Fab periplasmic extracts was analyzed by SPR technology using Biacore 3000. Briefly, diluted periplasmic extracts were injected onto a CM5 chip coated with 2500 RU of Gal10-His. The amplitude of binding (Rmax), dissociation (off-rate), and off-rate fold change of each clone compared to the control (g7B07 and g7B07_N53A) are shown. Clones g20H09 and g23H09 had similar or better off-rates compared to the control g7B07 (Table 10). The randomized portion of the CDR2 sequence is underlined (Table 10).

[0239] A limited number of clones isolated from four different CDR2 randomized libraries showed suitable binding properties (Table 10). Among this panel, clone g23H09 isolated from the X6 library was a unique clone that showed a better off-rate (2.5-fold better off-rate) than the parent clone 7B07. Clone 20H09 isolated from the X3 library showed a similar off-rate compared to clone g7B07, with an off-rate equal to 8.8E-03 1 / s. However, the randomized part of CDR2 of clone g20H09 (WHR) and the randomized part of CDR2 of g23H09 (AQFQHW) showed one possible downside represented by the presence of one tryptophan, which can potentially be oxidized under the influence of light.

[0240] Finally, clones 23E05 (X6 library) and 20F04 (X3 library) showed binding properties close to those of the parent clones, with off-rates 1.8-2.3 times higher compared to g7B07. None of the clones isolated from the X4 and X5 libraries showed suitable binding properties.

[0241] (Summary of Results of Example 1) Seven clones from Example 1 were selected for further characterization (Table 11). These clones were selected based on binding ability (BLI or SPR binding), epitope characterization (competitive ELISA), and VH and VL sequences (Table 11). [Table 11] Table 11: Characteristics of scFv / Fab clones selected for germlining and reformatting in human Fab

[0242] Example 2: Germlining, reformatting, and production of selected clones in a human Fab backbone. Seven selected clones were humanized and recloned into a human Fab backbone before being further characterized.

[0243] (Germlinization of selected clones by CDR grafting approach) To reduce the immunogenicity of the selected anti-Gal10 clones, we initiated germlining of the variable regions (VH and VL) by grafting the complementarity determining regions (CDRs) into the closest human germline frameworks (FWs). Human germline sequences with the highest identity to the V-regions of the selected clones were identified.

[0244] Mutant 24F02 was further engineered to remove a potential deamidation site (pos53_CDR2_VH) ​​at exactly the same position as g7B07: N position 53 was mutated to A.

[0245] Since the CDR2 randomized library was constructed using DNA of the heavy chain variable domain of germlined mutants of 7B07, all of the clones generated (g20H09 and g23H09) were already humanized.

[0246] Example 3: Characterization of Binding of Germlined Clones in a Human Fab Backbone SPR characterization of the binding properties of selected Fab clones from three discovery campaigns. The binding properties of the selected Fab clones to human Gal10 were analyzed by the established capture method on a Biacore 3000 instrument. Two concentrations of the selected Fab clones were tested against human Gal10-His immobilized on a CM5 chip coated with monoclonal anti-His. As controls, clones g7B07 and g7B07_N53A were injected at the beginning and end of the run. Briefly, a CM5 chip was coated with monoclonal anti-His antibody (4000 RU) and then a fixed concentration of human Gal10-His (25 μg / mL) was captured on the anti-His chip before receiving two concentrations of the clones in the human Fab backbone.

[0247] Germlined variants of the clones identified in Table 11 showed similar or better off-rates and affinities than clone g7B07 (see Table 12). Within this panel, clones isolated from the 7B07 epitope campaign showed the best off-rates, with off-rates ranging from 6.2 to 9.8 times better than clone g7B07.

[0248] The germlined (g24F02) and engineered version (g24F02_N53A) of the initial clone 24F02 showed higher off-rates compared to the initial off-rate (5.86E-05 1 / s) observed during a previous screening campaign at BLI. These two Fabs showed very similar binding activity, indicating that removal of the deamidation site found at position 53 does not affect the binding properties of g24F02, unlike g7B07, which showed a marked decrease in binding activity in the engineered variant g7B07_N53A.

[0249] 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 exhibited 1.6-1.9-fold better on-rates, 2.8-fold better off-rates, and 4.7-fold better affinities than clone g7B07.

[0250] Finally, the two clones 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 off-rate but similar affinity.

[0251] 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 12] Table 12: Binding characteristics of selected germline clones from Table 11.

[0252] Example 4: Stability testing of selected clones After stress testing, the stability of selected clones was analyzed to investigate similar issues to those of clone g7B07, where a deamidation site in CDR2 of the heavy chain variable domain induced a clear decrease in binding and potency.

[0253] For this purpose, a temperature stress test was performed. In this setup, stressed samples were incubated at 37 °C for 2 weeks and then analyzed side-by-side with non-stressed samples (T0) for binding, potency (CLC lysis) and post-translational modifications (focusing on the CDR2 sequence). The clones tested presented different starting concentrations (3.2-7.3 mg / mL).

[0254] (SPR analysis of binding ability of temperature-stressed samples) The binding properties of seven selected clones after two weeks of temperature stress were analyzed using an optimized capture method on a Biacore 3000 instrument. The binding properties of the stressed samples were determined, compared to the calibration points, and expressed as a percentage of relative activity (%RA).

[0255] Briefly, the capture method was set up on a Biacore3000 instrument. For this purpose, a CM5 chip was coated with a monoclonal anti-His antibody (4000 RU) and then a constant concentration of human Gal10-His (25 μg / mL) was captured on the anti-His chip, after which it received duplicate injections of calibrators, QC samples, and temperature-stressed samples of each clone. The slope of each injected sample was then calculated using the BIA evaluation software. The concentrations of the QC and temperature-stressed samples were back-calculated by interpolating the obtained slopes of these samples onto the calibrator curve. The values ​​obtained were within ±20% of the nominal concentrations of the tested samples. This is expressed as the average relative precision (average %RA) (Table 13). [Table 13] Table 13: Analysis of the binding properties of temperature stressed samples of selected clones isolated from the three discovery campaigns.

[0256] Clones g18G12, g18C06, and g24F02_N53A showed the best stability after 2 weeks at 37° C., with binding capacities similar to non-temperature stressed samples (95%, 106%, and 99% RA, respectively) (Table 13). Furthermore, non-artificial mutants of g24F02 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.

[0257] Clones g23H09 and g20H09 showed a decrease in binding capacity after incubation at 37° C., resulting in 78% and 86% RA, respectively, after 2 weeks, however, the 14% and 22% decrease in binding could be attributed to assay variability.

[0258] Temperature stress of samples containing g18G07 and g18E04 showed a clear decrease in binding (33% and 74% RA), highlighting their instability after 2 weeks at 37°C.

[0259] (Analysis of post-translational modifications (focus on CDR2_VH)) After two weeks of temperature stress, the generated samples were analyzed for post-translational modifications focusing on CDR2 of the heavy chain of these clones.

[0260] Most of the clones tested showed a relatively low percentage of modifications after 2 weeks of incubation at 37° C. Clones g18C06, g20H09, and g23H09 showed the least amount of modifications, with 3.1%, 2.4%, and 8.4% modifications, respectively, in the analyzed sequences (Table 14).

[0261] Consistent with the decrease in binding activity after incubation at 37° C., clone g18G07, and to a lesser extent clone g18E04, showed post-translational modifications (Table 14). Clone g18E04 showed 11.7% modification after 2 weeks of incubation at 37° C., mainly due to deamidation of Asn at position 52, which may explain its decreased binding capacity (Tables 13 and 14). Clone g18G07, the non-stressed sample, showed 20.2% modification, which increased to 63% after 2 weeks of incubation at 37° C., mainly due to deamidation of Asn at position 53 (58.8%), which may explain the decreased binding capacity of the temperature-stressed sample.

[0262] Clone g18G12, an unstressed sample, showed 30% deamidation (Asn at position 54), which was not affected by incubation for 2 weeks at 37° C. Deamidation in this clone most likely occurred during production (6 days at 37° C.). [Table 14] TIFF2025503707000019.tif41170 Table 14: Post-translational modifications of temperature stress samples of selected anti-Gal10 clones (PM focuses on CDR2_VH). Only FW2 and part of CDR2 sequences of VH of selected clones are shown in the table, since post-translational modifications were observed in FW2, mostly with W at position 47. Underlined residues represent CDR2 (according to Kabat numbering).

[0263] (Analysis of the effectiveness of temperature stress samples to lyse recombinant CLC) The ability of temperature stress preparations of selected clones in a human Fab backbone to lyse recombinant CLC was tested.

[0264] Clone 1D11 (anti-human specific) was included as a reference to correct for inter-assay variability. Dissolution of CLCs was monitored over time using an InCell 2200 analyzer after 2, 5, 7, and 16 hours of incubation (Figure 1). Briefly, antibodies were tested at 250 μg / mL in two independent experiments (n=4) and images were taken at 2, 5, 7, and 16 hours after antibody addition. The images were segmented using an algorithm developed to detect individual crystals and total crystal area per well. In this setup, 1 μL of sample (T0 and T2W) diluted at 1.5 mg / mL was applied to recombinant CLCs formed with 1 μg Gal10. [Table 15] Table 15: Mean % lysed crystal area / well after incubation with each clone.

[0265] Assays were performed with CLCs of various sizes, ranging from 5-10 μm (run 2) up to 10-20 μm (run 1). It was observed that the crystal size influences the efficacy of the Fabs. In particular, CLCs with a size between 10 and 20 μm allowed the best discrimination between clones.

[0266] Clones g20H09 and g23H09 were the most potent clones in the panel (Figure 1 and Table 15). These clones were able to lyse 59.6% and 68.6% of the recombinant CLCs within 2 h (Run 1). In contrast, the other clones showed 30.6%-37.9% lysis after 2 h (Run 1). Overall, after 5 h of incubation, most clones (except g18G07 and 1D11) were able to solubilize ~90% of the CLCs.

[0267] The positive control, clone 1D11, showed the least potency in dissolving crystals, with 20% and 36% CLC lysis after 2 and 5 hours of incubation, respectively.

[0268] Consistent with other stability results (binding and post-translational modifications), temperature-stressed samples of clones g18C06, g20H09, g23H09 and g18G12 showed similar potency as non-stressed samples. Consistent with the reduced binding capacity of temperature-stressed samples of clones g18G07 and g18E04, these samples showed reduced efficacy in lysing recombinant CLCs.

[0269] The potency of g24F02_N53A to lyse recombinant CLC was analyzed using spinning disk confocal microscopy. Briefly, humanized Fab fragments were incubated with preformed CLC and the dissolution of crystals was monitored over time.

[0270] Consistent with the binding data, temperature stressed samples (2 weeks at 37°C) showed similar potency when compared to non-stressed samples (Figure 2 and Table 16). This highlights that this clone is stable for 2 weeks at 37°C. In fact, tested samples of this clone showed 50% CLC lysis within 43-46 minutes (Table 16). Clone g24F02 showed similar, but slightly lower potency compared to g7B07 in both non-stressed and temperature samples. [Table 16] Table 16: EC50 and EC90 values ​​of temperature stress samples (T0 and T2W, 37°C). Calculations were performed using non-linear regression (variable slope of log(agonist) vs. response (4 parameters)) and reported in the table. Results are a combination of one experiment in which each well was monitored in six replicates.

[0271] Characterization of the binding sites of selected clones by epitope binning using ELISA Epitope binning analysis of the humanized Fab clones was performed in comparison with the humanized Fab of g7B07.

[0272] For this experiment, a suboptimal concentration of biotinylated clone g7B07 humanized Fab was added to Gal10-coated plates and pre-incubated with the selected clones. The percentage of competition with g7B07 was then determined (Table 17). Motavizumab (Mota) in a human Fab backbone was used as a negative control (0% competition). Clone g7B07 was used as a positive control for competition and was therefore set as a 100% competition value. Clone 1D11 is known to bind to the opposite side of Gal10 (including tyrosine 69), so anti-human specific clone 1D11 (binding to tyrosine 69 residue) was included in the test panel as a negative control to compete with 7B07 for Gal10 binding. [Table 17] Table 17: Epitope binning of selected anti-Gal10 clones against g7B07-biotinylated human Fab in ELISA. The percentage of competition against g7B07-hFab-Biot was determined by using the OD value of the negative control Motavizumab as 0% competition and the OD value of the non-biotinylated clone g7B07 as 100% competition.

[0273] These results confirmed that the selected clones competed with g7B07 for Gal10 binding (Table 17).

[0274] SPR characterization of the binding properties of selected Fab clones from three discovery campaigns. The binding properties of the selected clones to human and cynomolgus Gal10 were analyzed by the established capture method on a Biacore 3000 instrument.

[0275] For this purpose, two approaches were used: first, two concentrations of selected clones were applied to a cynomolgus (WGS isoform) Gal10-His capture on a CM5 chip coated with a monoclonal anti-His antibody, and the second approach entailed serial dilutions applied in the same setup to human or cynomolgus (WGS isoform) Gal10-His.

[0276] In a first step, the cynomolgus cross-reactivity of the panel was tested by injection of two concentrations against captured cynomolgus Gal10 (WGS isoform).

[0277] Clone g7B07 and its artificial variant g7B07_N53A showed weak binding to the WGS isoform of cynomolgus Gal10 (KD of 69 nM for g7B07-hFab vs. 1.5 nM for g7B07-mIgG1) (Table 18).

[0278] Clone g20H09 showed poor binding ability to the cynomolgus monkey antigen, whereas clone g18C06 showed no binding at all (Table 18). However, clones g23H09, g24F02 and their artificial mutant g24F02_N53A showed good cynomolgus monkey cross-reactivity (Table 18).

[0279] 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, and similar affinities and off-rates were observed.

[0280] Consistent with the screening data, clone g18C06 showed the best affinity (1.27 nM) and off-rate (4.9E-04 1 / s) among the panel tested for human Gal10, but did not bind to its cynomolgus monkey counterpart.

[0281] Randomization of six amino acids in CDR2 of g7B07_VH to generate g23H09 showed increased cynomolgus 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, indicating that key amino acids were introduced into CDR2 of clone g23H09 resulting in increased binding to cynomolgus Gal10. [Table 18] Table 18: Affinity determinations determined by SPR

[0282] (Summary of Results of Example 4) Considering all the parameters analyzed, four clones were selected for further characterization:

[0283] g18C06: Identified in the "7B07 epitope campaign" and showed the best off-rate on human Gal10 (4.9E-04 1 / s), good potency to lyse recombinant CLCs (76.8% after 5 hours) and adequate stability (3.1% post-translational modification, no change in binding and potency after 2 weeks at 37°C). Despite the similarity of the binding region on Gal10 to g7B07, this clone showed no binding to cynomolgus Gal10.

[0284] g23H09: This variant of g7B07, in which six residues in the CDR2 of the heavy chain are randomized, showed similar binding properties to the parent clone. In addition to favorable stability (binding and potency unaffected after 2 weeks of incubation at 37°C), this clone showed the second best efficacy in lysing recombinant CLCs (84.1% after 5 hours). It also showed clear cynomolgus cross-reactivity, with an affinity for the WGS isoform of 9.9 nM, which is 7-fold better than g7B07, allowing the use of g23H09 for toxicity testing in cynomolgus monkeys.

[0285] g24F02_N53A: A unique clone identified from "heavy chain" shuffling of g7B07 fulfilled all acceptance criteria. Potential deamidation sites similar to those found in g7B07 were mutated to alanine. Potency and binding capacity were unchanged after 2 weeks at 37°C. With an affinity of 8 nM for cynomolgus Gal10, this clone showed good cynomolgus cross-reactivity enabling toxicity testing in cynomolgus monkeys.

[0286] g20H09: This variant of g7B07, in which three residues in CDR2 of the heavy chain were randomized, clearly showed a lower binding capacity than the parent clone. Despite favorable stability and the best efficiency in dissolving Gal10 crystals (91.4% after 5 h), this clone showed weak cross-reactivity to cynomolgus Gal10. [Table 19] TIFF2025503707000025.tif66170Table 19: Summary of characteristics of clones: g23H09, g24F02_N53A, g18C06, and g20H09.

[0287] Materials and protocols used in Examples 1 to 4 (Charcot-Leyden Crystal (CLC) lysis assay) Assay 1: Purified monoclonal Fab was supplied in PBS and stored at 4° C. Protein concentration was determined by measuring absorbance at 280 nm using theoretical extinction coefficients. Human CLC crystals were prepared according to the method described in Persson et al. 5 CLC crystals were produced as described in. Briefly, 5 mL of purified N-terminally His-tagged human Gal10 in PBS at a concentration of 4 mg / mL was incubated with TEV protease at a protease:target ratio of 1% (g / g) overnight at room temperature. The next day, crystallization was induced by vortexing the solution (15 s). CLC crystals appeared within 30 min to 1 h, resulting in a cloudy solution. This crystallization solution was then stored at 4 °C until further use.

[0288] Assay 2: Purified monoclonal Fab was supplied in PBS and stored at 4° C. Protein concentration was determined by measuring absorbance at 280 nm using theoretical extinction coefficients. Antibodies were tested at 250μg / mL in two independent experiments (n=4) and images were taken 2, 5, 7 and 16 hours after antibody addition. A 1D11 concentration response curve (CRC) was included as an additional positive control (n=3). Images were segmented using a proprietary algorithm to detect individual crystals and calculate total crystal area per well.

[0289] (protocol) 1.1 Library construction for CDR2 randomization campaign (Nested PCR) To generate a library for the CDR2 randomization campaign, two-step nested PCR was performed to isolate the V Hwas amplified to introduce 3-4-5 residues (-1 residue) and a control 6 residue randomization. After digestion with NcoI and NheI, the PCR product was ligated into PCB13 (NheI / NcoI and BsteIII (to avoid self-ligation)) vector containing the light chain of g7B07 and then electroporated into TG1 ECC.

[0290] (Two-step nested PCR:) (First PCR:) g7B07_V H _PCB13 was used as a template and a solution of 50 ng / μL was prepared in 2×MQ water. [Table 20] Table 20: Primers

[0291] Preparation of First Sample Reaction (see table below): [Table 21] Table 21: PCR sample components

[0292] The plate was then sealed and loaded into the PCR machine. [Table 22] Table 22: PCR cycling conditions

[0293] Finally, the DNA products from the first PCR were isolated by agarose gel electrophoresis.

[0294] (DNA isolation by agarose gel electrophoresis:) 1. Prepare a 0.8% agarose gel (10-well comb (120 μL per well)). 2. Identical PCR products were pooled (8 replicates in a total volume of 400 μL) and 80 μL of 6× Orange loading dye (catalog R0631) was added to the mixture. 3. After polymerization, the gel was transferred to the electrophoresis system and the tank was filled with fresh 1x TEA. 4. Loader (Gene Ruler Mix, 20 μL) was loaded into one well. 5. Then 120 μL of cDNA was transferred into each well (3 wells in total). 6. The separation was carried out at 200 volts for at least 70 minutes. 7. Before continuing with the extraction of the expected DNA products, adequate separation of the products was assessed (15 mL tubes).

[0295] (PCR cleanup:) Clean-up of the PCR products isolated from the agarose gel was performed according to the protocol provided with the NucleoSpin Gel and PCR Clean-up kit: 1. After weighing the PCR product, NTI buffer was added (2x 100mg gel, 20-30min at 65℃). The solution was left on the bench (cooled) for 15min, then loaded onto a PCR Clean-up column and centrifuged at 11,000g for 1min. 2. The flow-through was discarded, 700 μL of NT3 was added to wash the membrane, and the column was centrifuged at 11,000 g for 1 minute. This step was performed twice. 3. The flow-through was discarded and the column was centrifuged at 11,000 g for 3 minutes. 4. Finally, the PCR Clean-up column was transferred to a 1.5 mL tube and elution was carried out by adding 20 μL of warm MQ water (70° C.). 5. After an incubation step (1 min) at room temperature, the column was centrifuged at 11,000 g for 1 min and the eluate DNA was measured in a Nanodrop (260 nm).

[0296] (Second PCR:) For the second nested PCR, the DNA product was adjusted to 10 ng / μL from the first nested PCR. [Table 23] Table 23: Primers

[0297] 1. Preparation for the second PCR: [Table 24] Table 24: PCR sample components 2. The plate was then sealed and loaded into the PCR machine. [Table 25] Table 25: PCR cycling conditions 3. While the PCR program was running, a 0.8% agarose gel was prepared. 4. At the end of the program, purification of the DNA products was performed by electrophoresis (0.8% agarose gel) and PCR cleanup was performed as described above. Finally, the DNA concentration was measured by Nanodrop.

[0298] (PCR products and g7B07_VL L Digestion of vector containing 1. The PCR product from the second nested PCR was diluted to 10ng / μL. [Table 26] Table 26: Digestion of PCR products 2. Distribute this solution into PCR tubes at 50 μL (total of 6 tubes (300 μL)). 3. The digestion was carried out in a PCR machine (Thermocycler) at 37°C for 4 hours. 4. At the end of the incubation period, the PCR reactions for each library were pooled into a 1.5 mL tube. 5. Purification was performed on Nucleospin columns (see protocol above, one column for each library). 6. Elution was performed with 75 μL of 2× warm MQ water per column (total of 150 μL for 10 μg). 7.Then, DNA concentration was measured using Nanodrop.

[0299] (Ligation of digested VH to digested PCB13 containing VL) g7B07_V digested with Ncol / Nhel / Bstell of VH (N3-4-5 or 6) digested with NcoI / NheI L Ligation to PCB13 was performed.

[0300] The mixture was prepared in 1.5 mL tubes (one tube per library). [Table 27] Table 27: Ligation reaction conditions 1. The ligation product was incubated at room temperature for 2 hours or at 16°C overnight. 2. After this incubation period, additional DNA ligase (2.5 μL T4 DNA ligase, 5 μL buffer T4 DNA ligase in 50 μL MQ water) was added for a total volume of 250 μL. 3. Finally, the ligation was continued for 2 hours at 37°C (water bath). 4. The DNA was then purified on Nucleospin columns. For this purpose, 500 μL of NTI buffer was added to the ligation solution, which was then followed by the washing steps described above. To terminate, elution was carried out with 30 μL of warm MiliQ water per column.

[0301] Electroporation for the final library Day -1: The harvest medium was transferred from -80°C to 4°C. Day 0: Four hours before the start of the experiment, the recovery medium was warmed to 37°C (incubator) and the Gene Pulser / MicroPulser Electroporation Cuvettes were placed on ice. 1. Electrocompetent cells (ECC, TG1) were transferred to ice for 15 minutes. 2. Then, 50 μL of ECC was added to 30 μL of purified ligation products (library). 3. An additional 50 μL of ECC was added on top of the previous volume to reach a total volume of 127 μL. 4. This volume was then divided into three fractions and 42 μL was transferred into three pre-chilled BioRad cuvettes. 5. For the negative control, a 1 / 10 dilution was prepared. From this predilution, 10 μL was transferred to a new 1.5 mL tube. Then, 20 μL of ECC was added on top of the previous volume to reach a total volume of 30 μL. 6. Electroporation was performed using the EC1 program (values ​​should be above 4.6, BioRad Micropulser). 7. The ECC was harvested by adding 4 mL of pre-warmed harvest buffer (1 mL per cuvette + 1 mL used to rinse the three cuvettes). This solution was then transferred to a 15 mL tube (4 mL total per library). 8. The culture was incubated at 37°C for 30 minutes at 180 rpm. 9. After incubation, a 1 / 1000 dilution was performed for each library in 2TY medium / ampicillin / glucose (fresh 15 mL tubes). Then, a 1 / 10 serial dilution was performed in a 96-well plate (F-bottom) to obtain 10 -07 Dilution was reached. 10. Finally, 5 μL of each dilution was spotted onto a dedicated Petri dish. The plate was then incubated overnight at 37°C. 11. The remainder of the harvested culture (4 mL in a 15 mL tube) was used to inoculate 300 mL of pre-warmed 2TY medium containing 2% glucose and ampicillin. The culture was then incubated with shaking (110 rpm) at 37° C. for 9 hours.

[0302] (Phage preparation) (Phage infection:) From an overnight culture of the saturated library: 1. Add 4 mL to 400 mL of 2TY / ampicillin / glucose (OD should be less than 0.1) and incubate at 37° C., 120 rpm until an OD of 0.5 is reached (approximately 2 hours). 2. 100 mL of this culture (100 mL per library) was transferred to a new 500 mL Erlenmeyer. 3. Add 20 µL of helper phage VCSM (stock 1 x 10 13 Phage / mL) was added and the culture was incubated at 37° C. for 30 min without shaking, then with shaking (110 rpm) for 30 min. 4. 100 mL of this culture was transferred into two 50 mL Falcon tubes (2 per library) and then centrifuged at 3500 g for 10 minutes at room temperature. 5. Each bacterial pellet (2 pellets for each library) was resuspended in 200 mL of 2TY / ampicillin / kanamycin (dilution 1 / 1000). 6. The cultures were then incubated overnight at 28° C. with shaking (110 rpm).

[0303] (Phage display selection of scFv library (Montoyo+Ynigo, kappa+lambda) against g7B07 epitope) Day 1: (coating:) 1. Maxisorp plates were coated with 100 μL per well of 25 μg / mL clone 1D11 (binding to the Gal10 region opposite the g7B07 epitope). A PBS control was included as a negative control. 2. The plate was covered with sealing tape and incubated at 4°C overnight.

[0304] (Phage preparation:) 1. Round 1: 50 OD (1 OD = 2 × 10 8 A solution containing 1000 ng / mL glycerol stock (TG1 cells electroporated with the final library) was added to 650 mL of 2TY / 2% glucose / ampicillin. Round 2: Overnight infections (rescued from previous round of selection) were diluted 1 / 100 in 15 mL LB / ampicillin / glucose and incubated at 37°C with shaking (110 rpm) until an A600 of approximately 0.5 (± 2 hours) was reached. 2. The helper phage step was started when the OD value reached 0.5-0.6 (at this stage, maximum expression of pili on the bacterial surface allows good infection by the phage). 3. Add 10 µL of helper phage (VCSM13 (10 13 )) was added to each Falcon tube (10 mL R1-TG1). No mixing is necessary (the phage:bacteria ratio should be 10:1, the stock is 1 x 10 13 / mL). 4. The tubes were incubated at 37°C for 30 min without shaking (infection) and then centrifuged at 4800g for 15 min at room temperature (supernatant removed). 5. The pellet was resuspended in 50 mL 2TY / ampicillin / kanamycin (diluted 1 / 1000) (without glucose) in a 250 mL Erlenmeyer (50 mL tube) and then incubated overnight at 28° C. (110 rpm).

[0305] (TG1 inoculation:) 1. 10 mL of LB medium was inoculated with a single colony of TG1 grown on a minimal salts agar plate in a 50 mL Erlenmeyer. 2. The culture was incubated overnight at 37°C (100 rpm).

[0306] Day 2: (blocking) 1. After overnight incubation, the coated plates were washed three times with 300 μL per well of 1× PBS-0.05% Tween using a multistepper pipette. 2. A blocking step was performed by adding 200 μL of 1×PBS 2% MARVEL per well using a multichannel pipette from a disposable reservoir. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker.

[0307] (Phage Precipitation:) 1. From the overnight growth (TG1 infected with helper phage), 50 mL was transferred to a new 50 mL tube and centrifuged (4800 g) for 15 min at 4°C (Round 1: 2 × 50 mL). 2. The supernatant was collected and 40 mL was transferred to a new 50 mL tube containing 10 mL of cold 20% PEG6000 / 2.5M NaCl (precipitation of phage). 3.The tubes were then incubated on ice for 30 minutes. 4. Phage precipitation was performed by a centrifugation step at 4800 g for 15 min. 5. The supernatant was removed and the pellet was resuspended in 1 mL of sterile PBS and transferred to a new sterile 1.5 mL tube. 6. The tubes were then centrifuged at maximum speed for 3 minutes (tabletop centrifuge). 7. The supernatant was collected and added to a new Eppendorf tube containing 250 μL of 20% PEG / 2.5M NaCl, then incubated on ice for 15 minutes. 8. The tubes were centrifuged at maximum speed for 3 minutes and the supernatant was removed. 9. Pellets were resuspended in PBS (Round 1: 500 μL per tube finally pooled; Round 2: 1 mL). 10. As an additional step, the solution was centrifuged at top speed for 3 minutes to pellet remaining cell debris. The supernatant was collected in a new 1.5 mL tube (phage) to generate the input. 11. From this stock solution, a glycerol stock was made (800 μL of phage in 400 μL of 60% glycerol).

[0308] (Gal10 capture on Maxisorp-coated plates coated with clone 1D11:) 1. Blocking buffer was removed from the coated plate by inverting the plate and tapping it on a piece of paper. 2. Added 100 μL per well of 5 μg / mL Gal10-His in 0.2% MARVEL. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker.

[0309] (Phage Selection:) 1. Round 1: 10 μL phage / selection (90 μL PBS 0.2% MARVEL + 10 μL phage). 2. Round 2: 1 μL phage / selection (99 μL PBS 0.2% MARVEL + 1 μL phage). 3. The blocking buffer was removed from the 96-well plate by inverting the plate and tapping it on a piece of paper, and 100 μL of diluted phage was added. 4. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker. 5. After the 2 hour incubation, the supernatant was removed from the plate (by pipetting). 6. Wells were washed thoroughly (200 μL / well) (total 25 times). a. 5 times with 200 μL PBS / Tween 0.05%. It is then sealed and incubated for 5 minutes at room temperature with shaking at 450 rpm on a platform shaker. This process was carried out four times. b. The wells are then washed 3 times with 200 μL of PBS before proceeding with the elution step.

[0310] (Elution: Trypsin:) 1. The PBS was removed and elution was performed with 150 μL of trypsin (10 mg / mL) per well. 2. The plate was sealed and incubated at room temperature for 15 minutes with shaking at 450 rpm on a platform shaker. 3. After this incubation period, 150 μL of the elution solution was transferred to the corresponding wells of a 96-well V-bottom plate containing 7.5 μL of AEBSF (an inhibitor of trypsin) in each well. The solution was then mixed 5 times to properly neutralize the trypsin (by pipetting up and down).

[0311] (Elution: Competitive elution:) 1. Competitive elution was performed by adding 100 μL per well of 2.5 mg / mL 7B07 or Motavizumab (isotype control) in 0.2% MARVEL. 2. The plate was sealed and incubated overnight at room temperature with shaking at 450 rpm on a platform shaker. 3. After this incubation period, the elution solution was transferred to a new 96-well plate.

[0312] (Infection / Rescue:) Rescue (50mL tube): 1. 300 μL of TG1 cells (OD value of approximately 0.5) were added to a 50 mL tube containing 625 μL of 2TY and 75 μL of eluted phage (trypsin elution or competitive elution). 2. The tubes were incubated at 37°C for 30 minutes without shaking. 3. At the end of this incubation period, 10 mL of LB / ampicillin / 2% glucose was added to each 50 mL tube containing TG1 / phage. 4. The tubes were then incubated overnight at 37° C. with shaking (110 rpm).

[0313] (Spotting:) 1. Dilutions for spotting: a. Input: A dilution series of phage (input) was prepared in 2TY for spotting (LB agar plates). A 12-point dilution series (1 / 10) was prepared in 2TY (5 μL phage in 45 μL 2TY) (10 -1 From 10 -12 to). b. Output: A dilution series of phage (output) was prepared in 2TY for spotting (LB agar plates). A six-point dilution series (1 / 10) was prepared in 2TY (5 μL phage in 45 μL 2TY) (10 -1 From 10 -6 to). 2. Then, 50 μL of TG1 cells (OD value of about 0.5) was added to 50 μL of phage in the dilution plate (input and output). 3. The plate was sealed and incubated without shaking at 37°C for 30 minutes. 4. 5 μL of each dilution (Input: 10 -6 From 10 -12 up to;Output: 10 -1 From 10 -6 (up to 100 ml) were spotted onto dry Petri dishes (LB / ampicillin / glucose).

[0314] Day 3: 1. The presence of single colonies on the spotting plates was controlled. 2. Glycerol stocks were made from the overnight rescue (1 mL culture + 500 μL 60% glycerol in a cryovial and stored at -80°C).

[0315] (Master plate preparation:) 1. From the appropriate rescues, a dilution series in 2TY was prepared. A six-point dilution series (1 / 10) was prepared in 2TY (5 μL phage in 45 μL 2TY) (10 -1 From 10 -6 to). 2. 50 μL of the selected dilution (10 -3 / 10 -4 ) was transferred to a Petri dish containing solid LB agar medium with 2% glucose and ampicillin. 3. TG1 cells were distributed evenly by shaking with glass beads. 4. The beads were removed and the Petri dish was incubated overnight at 37°C.

[0316] Day 4: 1. After overnight incubation, single colonies were picked with a p10 tip and transferred to flat-bottom 96-well plates containing 100 μL of LB medium supplemented with 2% glucose and ampicillin. 2. The plate was sealed with breathable sealing tape and incubated at 37°C and 120 rpm for 5 hours.

[0317] (Phage display of heavy chain shuffling and CDR2 randomization campaign) Day 1: (Coating (heavy chain shuffling):) 1. Maxisorp plates were coated with 100 μL of human Gal10-His or non-tagged human Gal10 diluted in 1×PBS: Round 1 and Round 2: 10 μg / mL and 1 μg / mL human Gal10-His. Round 3 and Round 4: human Gal10-His and Gal10 at 5 μg / mL and 0.5 μg / mL. Controls included non-coating conditions (PBS) and an irrelevant His-tagged protein (mCD11c-His R&D Cat. No. 7987 AX). 2. The plate was covered with sealing tape and incubated at 4°C overnight.

[0318] (Coding (CDR2 Randomization):) 1. Maxisorp plates were coated with 100 μL of human Gal10-His diluted in 1x PBS: Round 1 and Round 2: human Gal10-His at 10 μg / mL and 1 μg / mL, diluted in 1×PBS; Controls included non-coating conditions (PBS) and an irrelevant His-tagged protein (mCD11c-His R&D Cat. No. 7987 AX). 2. The plate was covered with sealing tape and incubated at 4°C overnight.

[0319] (Phage preparation:) 1. Overnight infections (rescued from previous round of selection) were diluted 1 / 100 into 15 mL LB / ampicillin / glucose and incubated at 37°C with shaking (110 rpm) until an OD (A600) of approximately 0.5 (+ / - 2 hours) was reached. 2. The helper phage step was started when the OD value reached 0.5-0.6 (at this stage, maximum expression of pili on the bacterial surface allows good infection by the phage). 3. Add 10 µL of helper phage (VCSM13 (10 13 )) was added to each Falcon tube (10 mL R1-TG1). No mixing is necessary (the phage:bacteria ratio should be 10:1, the stock is 1 x 10 13 / mL). 4. The tubes were incubated at 37°C for 30 min without shaking (infection) and then centrifuged at 4800g for 15 min at room temperature (supernatant removed). 5. The pellet was resuspended in 50 mL 2TY / ampicillin / kanamycin (diluted 1 / 1000) (without glucose) in a 250 mL Erlenmeyer (50 mL tube) and then incubated overnight at 28° C. (110 rpm).

[0320] (TG1 inoculation:) 1. 10 mL of LB medium was inoculated with a single colony of TG1 grown on a minimal salts agar plate in a 50 mL Erlenmeyer. 2. The culture was incubated overnight at 37°C (100 rpm).

[0321] Day 2: (blocking) 1. After overnight incubation, the coated plates were washed three times with 300 μL per well of 1× PBS-0.05% Tween using a multistepper pipette. 2. A blocking step was performed by adding 200 μL of 1×PBS 2% MARVEL per well using a multichannel pipette from a disposable reservoir. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker.

[0322] (Phage Precipitation:) 1. From the overnight growth (TG1 infected with helper phage), 50 mL was transferred to a new 50 mL tube and centrifuged (4800 g) for 15 min at 4°C (Round 1: 2 × 50 mL). 2. The supernatant was collected and 40 mL was transferred to a new 50 mL tube containing 10 mL of cold 20% PEG6000 / 2.5M NaCl (precipitation of phage). 3.The tubes were then incubated on ice for 30 minutes. 4. Phage precipitation was performed by a centrifugation step at 4800 g for 15 min. 5. The supernatant was removed and the pellet was resuspended in 1 mL of sterile PBS and transferred to a new sterile 1.5 mL tube. 6. The tubes were then centrifuged at maximum speed for 3 minutes (tabletop centrifuge). 7. The supernatant was collected and added to a new Eppendorf tube containing 250 μL of 20% PEG / 2.5M NaCl, then incubated on ice for 15 minutes. 8. The tubes were centrifuged at maximum speed for 3 minutes and the supernatant was removed. 9. Pellets were resuspended in PBS (Round 1: 500 μL per tube finally pooled, Round 2: 1 mL). 10. As an additional step, the solution was centrifuged at top speed for 3 minutes to pellet remaining cell debris. The supernatant was collected in a new 1.5 mL tube (phage) to generate the input. 11. From this stock solution, a glycerol stock was made (800 μL of phage in 400 μL of 60% glycerol).

[0323] (Phage Selection:) 1. 1 μL of phage diluted in MARVEL from the input of the second, third and fourth round selections (495 μL PBS 2% MARVEL + 5 μL phage) was used. 2. The blocking buffer was removed from the 96-well plate by inverting the plate and tapping it on a piece of paper, and 100 μL of diluted phage was added. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker. 4. After the 2 hour incubation, the supernatant was removed from the plate (by pipetting). 5. Wells were washed thoroughly (200 μL / well) (total 25 times). a. 5 times with 200 μL PBS / Tween 0.05%. It is then sealed and incubated for 5 minutes at room temperature with shaking at 450 rpm on a platform shaker. This process was carried out four times. b. The wells are then washed 3 times with 200 μL of PBS before proceeding with the elution step.

[0324] (For off-rate cleaning conditions:) 1. 150 μL of 50-100 μg / mL Gal10-His or soluble Gal10 was added to each selected well. 2. Plates were incubated at 37° C. overnight or up to 2 days with shaking at 450 rpm on a platform shaker. 3. After the incubation period, the wells were washed 5 times with 200 μL of PBS.

[0325] (Elution:) 1. The PBS was removed and elution was performed with 150 μL of trypsin (10 mg / mL) per well. 2. The plate was sealed and incubated at room temperature for 15 minutes with shaking at 450 rpm on a platform shaker. 3. After this incubation period, 150 μL of the elution solution was transferred to the corresponding wells of a 96-well V-bottom plate containing 7.5 μL of AEBSF (an inhibitor of trypsin) in each well. The solution was then mixed 5 times to properly neutralize the trypsin (by pipetting up and down).

[0326] (Infection / Rescue:) Rescue (50mL tube): 1. 300 μL of TG1 cells (OD value of approximately 0.5) were added to a 50 mL tube containing 625 μL of 2TY and 75 μL of eluted phage (trypsin elution or competitive elution). 2. The tubes were incubated at 37°C for 30 minutes without shaking. 3. At the end of this incubation period, 10 mL of LB / ampicillin / 2% glucose was added to each 50 mL tube containing TG1 / phage. 4. The tubes were then incubated overnight at 37° C. with shaking (110 rpm).

[0327] (Spotting:) 1. Dilutions for spotting: a. Input: A dilution series of phage (input) was prepared in 2TY for spotting (LB agar plates). A 12-point dilution series (1 / 10) was prepared in 2TY (5 μL phage in 45 μL 2TY) (10 -1 From 10 -12 to). b. Output: A dilution series of phage (output) was prepared in 2TY for spotting (LB agar plates). A six-point dilution series (1 / 10) was prepared in 2TY (5 μL phage in 45 μL 2TY) (10 -1 From 10 -6 to). 2. Then, 50 μL of TG1 cells (OD value of about 0.5) was added to 50 μL of phage in the dilution plate (input and output). 3. The plate was sealed and incubated without shaking at 37°C for 30 minutes. 4. 5 μL of each dilution (Input: 10 -6 From 10 -12 up to;Output: 10 -1 From 10 -6 (up to 100 ml) were spotted onto dry Petri dishes (LB / ampicillin / glucose).

[0328] Day 3: 1. The presence of single colonies on the spotting plates was controlled. 2. Glycerol stocks were made from the overnight rescue (1 mL culture + 500 μL 60% glycerol in a cryovial and stored at -80°C).

[0329] (Master plate preparation:) 1. From the appropriate rescues, a dilution series in 2TY was prepared. A six-point dilution series (1 / 10) was prepared in 2TY (5 μL phage in 45 μL 2TY) (10 -1 From 10 -6 to). 2. 50 μL of the selected dilution (10 -3 / 10 -4 ) was transferred to a Petri dish containing solid LB medium with 2% glucose and ampicillin. 3. TG1 cells were distributed evenly by shaking with glass beads. 4. The beads were removed and the Petri dish was incubated overnight at 37°C.

[0330] (Production and purification of anti-Gal10 lead (Fab)) Production of periplasmic extracts Day 1: (Inoculation:) 1. The master plate was incubated at 37° C. with shaking (110 rpm). 2. 1 mL of 2TY / ampicillin / 0.1% glucose was added to each well of a V-bottom 96 deep-well plate and then incubated for 15 minutes at 37° C. with shaking (110 rpm). 3. Then, 10 μL of bacteria from the master plate was added to 1 mL of medium in the deep-well plate. 4. The plates were then incubated at 37°C with shaking (110 rpm) until an OD600 value of approximately 0.8-1.0 was reached (6-7 hours).

[0331] (Induction of production) 1. To induce scFv or Fab production, 100 μL of IPTG was added per well (10 mM IPTG in 2TY+ampicillin; final concentration in well=+ / -1 mM IPTG). 2. The plates were incubated overnight at 26° C. with shaking (110 rpm).

[0332] Day 2: 1. The next day, the 96-deep well plate was centrifuged at 4800 g for 15 minutes in a centrifuge cooled to 4° C. to pellet the bacteria. 2. The medium was removed (poured off and allowed to dry on paper). 3. The plates were sealed and stored at -20°C overnight.

[0333] Day 3: 1. The plates were transferred from -20°C to -80°C for at least 1 hour. 2. The bacterial pellet was thawed at room temperature for 30 minutes. 3. The pellet was resuspended in 110 μL of PBS per well and then vortexed for 1 minute. 4. The plate was incubated for 90 minutes at room temperature on a shaker (900 rpm). 5. After this incubation period, the bacteria were then centrifuged (4800 g, 15 min, 4° C.). 6. The supernatant (scFv or Fab peri) was transferred to a new V-bottom 96 well plate. 7. Periplasmic fractions containing scFv or Fab clones were stored at -20°C.

[0334] Recloning of VH and VL sequences into a human Fab fragment backbone (DNA string design) 1. Using AbAligner software (no version number), H and V L were aligned and compared to the closest human germline variants. 2. The CDR regions (+1 residue before CDR1 and 3) were grafted into the FW regions of the germline variant. 3. Optimization of the nucleotide sequence for human production was performed using GeneArt® tools (Life Technologies™). 4. BsmBI restriction sites were then added 5' and 3' of the optimized amino acid sequence for recloning into the pUPEX vector. 5. Finally, DNA strings were ordered from Life Technologies™ (Thermo Fisher Scientific™).

[0335] (Digest of DNA string with BsmBI) 1. For each clone, 200 ng of V H and V L was digested with BsmBI in a final volume of 20 μL of 10× Tango buffer. 2. Mixtures were prepared according to the table below and 10 μL was dispensed into each well according to the plate layout of the PCR plate. 3. The PCR plate was then incubated at 37°C for 2 hours (in a Thermocycler). [Table 28] Table 28: PCR cycling conditions

[0336] (PCR cleanup of digested DNA) Cleanup of the digested strings was performed according to the protocol provided with the NucleoSpin Gel and PCR Clean up kit: 1. After 2 hours of incubation at 37°C, 80 μL of MQ water was added to each digested DNA string, followed by 200 μL of NTI buffer, then transferred to a PCR Clean up column and centrifuged at 11,000 g for 1 minute. 2. The flow-through was discarded, 700 μL of NT3 was added to wash the membrane, and the column was centrifuged at 11,000 g for 1 minute. This step was performed twice. 3. The flow-through was discarded and the column was centrifuged at 11,000 g for 3 minutes. 4. Finally, the PCR Clean-up column was transferred to a 1.5 mL tube and elution was carried out by adding 25 μL of warm MQ water (70° C.). 5. After an incubation step (1 min) at room temperature, the column was centrifuged at 11,000 g for 1 min and the DNA in the eluate was measured in a Nanodrop (260 nm).

[0337] (Ligation of digested DNA with heavy and light chain vectors) 1. Digested VH / VL (BsmBI) a. Human CH1 domain BsmBI digested vector for VH (pUPEX86) b. Human lambda BsmBI digested vector for VL (pUPEX116.9) Inserted in :. 2. Prepare the ligation mixture in a 1.5 mL tube at a ratio of 1:5 (vector:insert) according to the table below. [Table 29] Table 29: Ligation conditions 3. The ligation mixture was incubated at room temperature for 1 hour.

[0338] (Transformation in Top10 competent cells) Each ligation product was transformed into Top10 competent cells by heat shock. 1. After 1 hour incubation, the ligation mixture was transferred to ice for 5 minutes. 2. Top10 competent cells were thawed on ice and 40 μL of Top10 cells were added to 15 μL of each ligation product. 3. The tube was transferred to ice for 5 minutes. 4. Transformation of competent Top10 cells was performed by heat shock at 42°C for 90 seconds in a warm water bath. 5.The tube was then incubated on ice for 1-2 minutes. 6. Finally, the transformed Top10 cells were transferred onto Petri dishes containing solid LB medium with 2% glucose and ampicillin (resistance gene of the vector). 7. Uniform distribution of cells was achieved by shaking with glass beads. 8. The beads were removed and the Petri dish was incubated overnight at 37°C.

[0339] (Colony picking and sequencing) 1. After overnight incubation, the ligation products showed numerous single bacterial colonies, whereas no / few colonies were observed for the negative control (empty vector). 2. Eight single colonies per VH or VL were picked with the tip of a p10 and transferred to a flat-bottom 96-well plate containing 100 μL of LB medium supplemented with 2% glucose and 1 / 1000 ampicillin. 3. The plate was sealed with breathable sealing tape and incubated at 37°C for 5 hours at 120 rpm. 4. Finally, 10 μL of each clone was transferred to a sequencing plate (96-well flat bottom plate containing solid LB medium + 2% glucose and 1 / 1000 ampicillin). 5. The sequencing plate was sent to LGC genomic (primer p90). 6. The DNA sequences obtained from LGC genomic were analyzed using the software Clone Manager version n° 9. The sequences of each clone were aligned and compared with the ordered sequences. 7. Clones showing DNA sequences (DNA strings) similar to the parent sequence were selected.

[0340] (Amplification and MidiPrep:) 1. For each construct, 20 μL of culture was inoculated into 10 mL of LB medium containing 2% glucose and 1 / 1000 ampicillin in the morning. 2. The cultures were incubated at 37°C and 120 rpm. 3. In the evening, cultures were transferred to 100 mL + 1 / 1000 ampicillin (glucose was not included as it may interfere with sequencing of Midiprep DNA; pUPEX vectors do not require glucose). 4. The culture was incubated overnight at 37°C and 120 rpm.

[0341] MidiPrep was performed according to the protocol provided with the kit: 1. 110 mL of culture was pelleted by centrifugation (4800 g, 15 min at 4° C.). 2. The supernatant was removed and the pellet was resuspended (by vortexing or pipetting) in 8 mL RES buffer containing RNAse. 3. 8 mL of LYS buffer was added and each tube was inverted 5 times (do not vortex) until the solution was completely blue, and incubated at room temperature for 5 minutes. 4. 8 mL of NEU buffer was added and mixed until the solution turned white. 5. The NucleonBond Xtra Midi Column and filter were equilibrated with 12 mL of EQU buffer. 6. The bacterial solution was gently and slowly transferred onto the interface of the equilibrated column. 7. The first wash was performed with 5 mL of EQU buffer, and after complete elution, the NucleonBond Xtra column filter was removed and a second wash was performed with 8 mL of WASH buffer. 8.The column was then transferred to a 50 mL tube and elution was carried out with 5 mL of ELU buffer. 9. DNA was precipitated by adding 3.5 mL of isopropanol. The tubes were vortexed and centrifuged at 4800 g for 30 min at 4°C. 10. The supernatant was removed and 2 mL of 70% ethanol was added. 11. The tubes were centrifuged at 4800g for 30 minutes at 4°C. 12. The supernatant was removed and the pellet was dried at room temperature for 1 hour. 13. Finally, 100 μL of 2×MQ water was added to resuspend the pellet and incubated at room temperature with shaking (900 rpm) for 30 min, after which the DNA concentration was measured by Nanodrop.

[0342] (Generation of anti-galectin-10 clones) (Transfection of HEK293E cells) Day 1: 1. HEK293E cells were seeded at 0.3E+06 cells / mL.

[0343] Day 2: For each anti-Gal10 clone, a 15 mL tube containing + / - 7.6 mL of pre-warmed (37°C) Optimem medium was prepared. [Table 30] Table 30: Transformation components

[0344] 1. VH and VL MidiPrep DNA were added at a 1:3 ratio for a final amount of 100 μg (VH 25 μg + VL 75 μg). 2. Finally, 300 μg of PEI was added dropwise to the tube and vortexed briefly. 3. The medium / DNA / PEI mixture was incubated at room temperature for 10 minutes and then added to a 200 mL HEK293E culture. 4. Finally, the cells were returned to the incubator (37°C, 5% CO2, 120 rpm). 5. Four hours after transfection, pre-warmed (37° C.) 1 / 20 Hypep 1510 was added to each culture. 6. After 6 days of production, the entire medium of each anti-Gal10 clone was collected into a 50 mL tube. 7. The tubes were centrifuged at 1000g and 4°C for 10 minutes and the supernatant was transferred to a new 50 mL tube.

[0345] (Purification of produced anti-Gal10 Fab using Capture Select IgG CH1 beads) Purification of the produced Fab was performed using Capture Select IgG CH1 beads.

[0346] Day 1: 1. CaptureSelect IgG CH1 beads were equilibrated in a 50 mL tube by washing 3 times with PBS to make a 50% slurry in PBS. 2. 500 μL of equilibrated 50% slurry beads was added per 50 mL tube containing HEK culture supernatant. 3. The tubes were incubated overnight at 4°C on a rotor (19 rpm).

[0347] Day 2: 1. The beads were spun down (630g, 2 min, 4°C, acceleration = 9, deceleration = 7). 2. The supernatant was collected in a new 50 mL tube. 3. Add 1 mL of 1x PBS to the beads (down the side of the Falcon); resuspend; and transfer to columns (1 column per 100 mL of HEK culture). 4. The tube was washed with 4 mL of 1x PBS and then transferred to the column. 5. The washing step was repeated twice. 6. The column was washed 5 times with 5 mL of 1x PBS. 7. During the washing step, prepare 3 x 2mL tubes per clone and add 100μL of neutralizing solution (1M Tris pH8) to each tube. 8. For elution, the column was transferred to a 2 mL tube (containing neutralization solution) and 1 mL of elution solution (0.1 M glycine, pH 3) was added. After complete elution, the column was transferred to another 2 mL tube and elution was repeated twice. The tube was mixed properly to ensure good neutralization of the elution solution. 9. The protein concentration of each eluted fraction was then determined using a Nanodrop (280 nm). 10. Buffer exchange was performed by loading 4 mL onto an Amicon Ultra-4 centrifugal filter. The column was then centrifuged at 4000 g for 15 min at 4°C, the flow-through was discarded, and 5 mL of 1x PBS was added to the upper chamber. The column was centrifuged again and washed a total of 5 times with 5 mL of 1x PBS. 11. Protein concentrations were then measured using Nanodrop and corrected for the extinction coefficient of each clone.

[0348] Screening and characterization of binding and epitope binning of anti-Gal10 molecules by ELISA Screening of the binding properties of scFv or Fab periplasmic extracts (Binding ELISA) Day 1: 1. Maxisorp plates were coated with 100 μL per well of human Gal10-His (0.5 μg / mL, diluted in 1×PBS). 2. The plate was sealed with sealing tape and incubated at 4°C overnight.

[0349] Day 2: 1. The next day, plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 2. Then, 300 μL of blocking solution (1× PBS 1% casein) was added per well. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker. 4. During the incubation period, periplasmic extracts from the Periplasmic Master Plate (PMP) plate were diluted 1 in 5 into 1× PBS-0.1% casein in Microplate 96-well U-bottom plates. 5. After 2 hours of incubation in blocking solution, plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 6. Using a 200 μL multichannel pipette, 100 μL per well of each diluted scFv-periplasmic extract was transferred from the dilution plate to the coating plate. 7. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 8. During this incubation period, detection antibody (rabbit anti-Myc HRP conjugate, 1 / 2000) was prepared in a sufficient amount of 1x PBS-0.1% casein. 9. After 1 hour incubation, plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 10. Using a 200 μL multichannel pipette, transfer 100 μL per well of detection antibody to the coated plate. 11. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 12. 15 minutes prior to the end of the incubation period, the TMB solution was transferred to room temperature. 13. After incubation, plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 14. Using a 200 μL multichannel pipette, 100 μL of TMB was added per well and incubated for 5 minutes with shaking at 450 rpm, after which the reaction was stopped by adding 100 μL of 0.5M H2SO4 per well. 15. The absorbance was then measured at 450 nm (reference 620 nm) using a Tecan instrument and Magellan software version n° 7.2.

[0350] (Competitive ELISA (binding to 7B07 epitope)) First, Maxisorp plates were coated with 7B07 hIG1 (where hGal10-His is captured). Then, periplasmic extracts diluted at 1 / 5 dilution were added. Detection was performed with rabbit anti-Myc-HRP antibody.

[0351] Day 1: 1. Maxisorp plates were coated with 100 μL per well of 7B07_hIgG1 (3 μg / mL, diluted in 1×PBS). 2. The plate was sealed and incubated at 4°C overnight.

[0352] Day 2: 1. Plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 2. A blocking step was performed by adding 300 μL per well of 1×PBS-1% casein. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker. 4. After this incubation period, the plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 5. Next, 1 μg / mL human Gal10-His was added (100 μL per well, diluted in 1×PBS-0.1% casein). 6. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 7. During the incubation period, 1 in 5 dilutions of the selected periplasmic extracts of scFv / Fab were prepared in 1x PBS-0.1% casein in separate Microplate 96-well U-bottom plates. 8. After 1 hour incubation with Gal10-His, plates were washed 5 times with 300 μL per well of 1× PBS-0.05% Tween. 9. Then, using a 200 μL multichannel pipette, 100 μL of each periplasmic extract was added to the coated plate. 10. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 11. After this incubation period, the plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 12. Detection was performed by adding 100 μL per well of HRP-conjugated rabbit anti-Myc diluted in 1× PBS-0.1% casein. 13. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 14. 15 minutes prior to the end of the incubation period, the TMB was transferred to room temperature. 15. The plates were then washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 16. Finally, 100 μL of TMB was added per well to the plate using a 200 μL multichannel pipette. The reaction was allowed to proceed for 5 minutes with shaking at 450 rpm, and the reaction was stopped by adding 100 μL of 0.5M H2SO4 per well. 17. Absorbance was measured at 450 nm (reference was 620 nm) using a Tecan instrument.

[0353] (Screening of Fab binding properties) (epitope binning) Day 1: 1. Maxisorp plates were coated with 100 μL of human Gal10-His (1.25 μg / mL, diluted in 1×PBS). 2. The plate was sealed and incubated at 4°C overnight.

[0354] Day 2: 1. Plates were washed 3 times with 300 μL per well of 1×PBS-0.05% Tween. 2. A blocking step was performed by adding 300 μL per well of 1×PBS-1% casein. 3. The plate was sealed and incubated for 2 hours at room temperature with shaking at 450 rpm on a platform shaker. 4. During this incubation time, dilutions of the selected Fab were prepared at 40 μg / mL in 50 μL of 1×PBS-0.1% casein in a Microplate 96-well U-bottom plate. The final concentration of the diluted Fab will be 20 μg / mL. 5. After 2 hours of incubation in blocking solution, plates were washed 3 times with 300 μL per well of 1×PBS-0.05% Tween. 6. Using a 200 μL multichannel pipette, 50 μL of each Fab dilution was transferred from the dilution plate to the coating plate. 7. The plate was sealed and incubated at room temperature for 30 minutes with shaking at 450 rpm on a platform shaker. 8. After 30 minutes, 50 μL of 400 ng / mL biotinylated clone 7B07 (hFab) was added to each well to reach a final concentration of 200 ng / mL. 9. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 10. After this incubation, the plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 11. Detection was then performed using 100 μL of peroxidase streptavidin (diluted 1 / 5000 in 1× PBS-0.1% casein). 12. The plate was sealed and incubated for 1 hour at room temperature with shaking at 450 rpm on a platform shaker. 13. 15 minutes prior to the end of the incubation period, the TMB was transferred to room temperature. 14. After this incubation period, the plates were washed 5 times with 300 μL per well of 1×PBS-0.05% Tween. 15. Finally, 100 μL of TMB was added per well. The reaction was carried out for 10 minutes with shaking at 450 rpm, and then stopped by adding 100 μL of 0.5M H2SO4 per well. 16. Absorbance was measured at 450 nm (reference 620 nm) using a Tecan instrument and Magellan software version n° 7.2.

[0355] (Binding screening of Fab periplasmic extracts with OctetRed96) BLI is a label-free technique for measuring biomolecular interactions. It is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized proteins at the biosensor tip and an internal reference layer. Any change in the number of molecules bound to the biosensor tip produces a change in the interference pattern that can be measured in real time.

[0356] The BLI Octet screening assay was used to test the binding capacity of Fabs produced as periplasmic extracts after the selection step. 1. His-tagged human Gal10 was diluted in kinetic buffer (200 μg / mL) and captured onto the tip of anti-Penta His 1K until a capture level of 1 nm was reached (loading step, 30 sec). 2. Periplasmic extracts were diluted 1:5 in kinetic buffer and applied for 120 seconds (association) followed by 120 seconds of dissociation. 3. Regeneration of the tip was performed by two washing steps of 10 seconds in glycine pH 1.5. 4. To assess non-specific binding of periplasmic extracts to the sensor tips, we used reference anti-Penta His-1K tips, which were coated to a level of 1 nm with an irrelevant His-tagged protein (a-CD70 107B8-sortase-His D3 control) and incubated with periplasmic extracts. 5. BLI binding analysis of Gal10-His was performed at 25°C using OctetRed 96 (ForteBio). 6. Double referencing analysis was performed to subtract binding at the reference tip and buffer (on the Gal10-His coated tip) from the signal detected at the coated tip. 7. Kinetic parameters were determined by fitting the binding of Fab on captured Gal10-His with a 1:1 binding model using ForteBio Data Analysis 9.0 software (Y-axis alignment to baseline, step-by-step correction for dissociation, Savitzky-Golay filtering, local / partial curve fitting). Note that one column of anti-Penta His-1K was used per PMP and discarded after 10 regeneration cycles.

[0357] (Binding characteristics of anti-Gal10 molecules on Biacore 3000) (Coating of CM5 chip with Gal10-His) To determine the binding properties of periplasmic extracts, CM5 sensor chips were coated with 2500-3000 RU of Gal10-His. 1. The coating material was buffered in acetic acid pH 5.5 at a concentration of 20 μg / mL. 2. Gal10-His was then immobilized onto the activated NHS ester on the surface of a CM5 chip by reaction with EDC and NHS. 3. After target immobilization, washing and quenching of free NHS esters was performed with ethanolamine.

[0358] (Coating of CM5 chip with anti-His tag antibody) A capture approach was used to determine the binding capacity of anti-Gal10 constructs on the Biacore 3000. For this purpose, BioRad monoclonal mouse anti-histidine tag, clone AD1.1.10, was coated onto four channels of a CM5 chip by amine coupling with a final response of 4000 RU. 1. The coating material was buffered in acetic acid pH 4.5 at a concentration of 20 μg / mL. 2. The monoclonal mAb was then immobilized onto the activated NHS ester on the surface of a CM5 chip by reaction with EDC and NHS. 3. After target immobilization, washing and quenching of free NHS esters was performed with ethanolamine.

[0359] Screening of binding properties of scFv periplasmic extracts (off-rate screening) For screening of the binding properties of periplasmic extracts, 2600 RU Gal10-His coated chips were used. 1. Thaw Peri stored at -20°C in a 96-well plate and dilute 1:5 by adding 10 μL of Peri to 40 μL of HBS-EP running buffer in a new round-bottom 96-well Biacore plate. The plate was sealed. 2. After the priming, normalization, and priming cycles, the coated CM5 chip was equilibrated against the running buffer with 4 cycles of 10 μL HBS-EP buffer pH 7.4. 3. 25 μL of the diluted periplasmic extract was injected using a flow rate of 5 μL / min. 4. After 15 min, the chip was regenerated by injecting 6 μL of 8 mM NaOH (Quickinject at a flow rate of 100 μL / min) followed by injection of a new sample. 5. Data analysis was performed using the "BIAevaluation" software version n° 4.1.1, using 2-1 and 4-3 subtraction. Only the sensorgram buffer was used as a reference and its signal was excluded (Y-transformation). Finally, a fitting model was used to determine the affinity and off-rate of the clones for Gal10-His (Fit Kinetics individual ka / kd / dissociation / Langmuir dissociation).

[0360] (Screening of Fab binding properties (off-rate screening)) For screening of the binding properties of periplasmic extracts and purified anti-Gal10 molecules, chips coated with 4000 RU of anti-His tag antibody were used. 1. Purified Fab was diluted to multiple concentrations, including calibrators and QC samples (70%, 100%, and 130% of the test concentration). 2. After the priming, normalization, and priming cycles, the coated CM5 chip was equilibrated against the running buffer with 2 cycles of 60 μL HBS EP buffer pH 7.4. 3. As a first step, Gal10-His was captured onto one channel of the coated CM5 chip by injecting ("Quickinject", flow rate set at 30 μL / min) 20 μL of diluted Gal10-His until a final capture level of approximately 500 RU was reached. 4. As a second step, a dilution of one of the clones in the human Fab backbone was injected over the captured Gal10-His (2 channels, 60 μL, "Kinject"). 5. After 100 seconds dissociation time, the chip was regenerated with a single injection of 8 μL of 10 mM glycine HCl pH 2.4 ("Quickinject"). 6. Data analysis was performed using the "BIAevaluation" software version n° 4.1.1, using Fc4-Fc3 or Fc2-Fc1 subtraction. Normalization of the sensorgrams was performed after the capture level of Gal10-His (approximately 200 seconds). Only the sensorgram buffer was used as a reference and its signal was excluded (Y-transformation / Curve-Curve 2 (blank run subtraction)). Finally, a fitting model with mass transfer effects was used to determine the affinity and off-rate of the clones for Gal10-His (Fit Kinetics simultaneous ka / kd / binding with mass transfer / local Rmax).

[0361] (Human CLC Lysis (Time Lapse)) 1. Fill the outer wells of a 96-well plate with 300 μL of PBS buffer. 2. Sufficient vacuum grease was applied to the edges of the plate to prevent evaporation. 3. 2 μL of diluted CLC solution droplets (0.7 mg / mL in PBS) were spotted into a 96-well plate. 4. A clear plastic cover was then placed over the plate. 5. The plate was then placed into the 96-well plate holder of a spinning disk confocal microscope, with two positions determined per well. 6. After all positions were chosen, the plate was removed from the holder, the lid was removed, and 2 μL of antibody solution was added to the 2 μL CLC drops in the wells. For the control condition, 2 μL of PBS buffer was added. 7. Each Fab condition was imaged multiple times to ensure reproducibility. 8. The plate was then sealed and placed back into the microscope holder. The plate was then secured with adhesive tape to further ensure there was no stage drift. All positions were then rechecked and refined. The positions were saved in the metadata output by the software. 9. Samples were then imaged every 3–5 min with a Plan ApoChromat 10× dry objective (NA 0.30, DIC) on an Axio Observer.Z1 (Zeiss) equipped with a CSU-X1 Yokogawa spinning disk head (Yokogawa Corporation) and a Zeiss AxioCam Mrm (Zeiss). Images were acquired continuously for up to 3 h (the entire cycle took approximately 72 s). 10. Image reconstruction and data analysis are performed using ImageJ (NIH). Briefly, sum projections were made for each stack and then an edge filter was applied to the images. Thresholding can be applied at this point. Measurements were made on each image to calculate the overall area occupied by the crystals, and the data was then exported to an Excel file where it was normalized to time zero and plotted as a function of time. Data from replicate samples were combined for statistical analysis. The overall size of CLCs was measured over time and plotted in GraphPad Prism 7.01. EC50 (50% lysis) and EC10 (90% lysis) values ​​for each antibody calculated using nonlinear regression (log(agonist) vs. variable slope of response (4 parameters)).

[0362] (Human CLC Lysis (Time Lapse)) 1. The objective was to determine the activity of 14 antibody fragments in a 1536-well High Content assay in the presence of 1 μg Gal10 per well. 2. 3.8 mg / mL Gal10 was incubated overnight (16 hours) at 20° C. in a 1.5 mL tube containing 38 μg / mL TEV protease. 3. 4 mg / mL Gal10 was vortexed for 30 seconds and diluted to 0.2 mg / mL in PBS 30 minutes after vortexing. 4. Assay controls were added to 1536-well plates: 10-point serial dilutions of 1D11 were prepared and 1 μL was added manually to 1536-well plates (n=3). 1 μL of PBS and 1D11 (3 mg / mL) was added (n=64). 5. Antibodies were diluted to 1.5 mg / mL and 1 μL was transferred to a 1536-well plate (n=4). 6. 5 μL of Gal10 (1 μg total) was dispensed into the antibody in the plate using a bulk dispenser (BioTek MultiFlo). 7. Plates were imaged (InCell 2200) 2, 5, 7, and 16 hours after antibody addition.

[0363] Images were segmented using an in-house developed algorithm to detect individual crystals and calculate the total crystal area per well.

[0364] Example 5: Further Temperature Stress Stability Test Results All clones were stored at 2-8°C for up to 48 hours and the protein concentration of the samples was adjusted to 10mg / mL in the original formulation buffer under sterile conditions. The clones were then stored under different temperature conditions (+5°C, +25°C, and +37°C) for 4 weeks and tested at weekly intervals for stability. Stability was tested after several freeze-thaw cycles (1, 5, and 10 cycles) as well as after heat stress (thermostability) within different denaturation temperatures spanning the range of 55°C to 80°C.

[0365] (2.1 Temperature stress stability test results) (Appearance and visual inspection) Visual inspection of sample stability at all time points and all temperature stress conditions (0.5 mL filled at 1.5 mL in clear glass vials) was performed by two individuals. Overall, the samples had the same visual appearance (Table 31). [Table 31] A: No visible particles; B: Few visible particles (<5); F: Fibers Table 31: Summary of results of visual inspection of clones during temperature stress testing

[0366] (Turbidity) For qualitative detection of scattering and agglutination, the particles were evaluated by absorption spectrum acquisition in a spectrophotometer at two wavelengths. The results were expressed as an agglutination index (AI) at 340 nm and 500 nm for all samples. The AI ​​is calculated according to the following formula: AI 340nm =A 340nm / (A 280nm -A 340nm ) and AI 500nm =A 500nm / (A 280nm -A 500nm ) was determined according to

[0367] For each sample, the corresponding buffer, negative control (sterile filtered mQ water), and positive control (protein sample known to contain increased aggregates) were also subjected to the same spectrophotometric analysis. No clear differences were observed between clones in terms of the aggregation index when compared to the formulation buffer, or between clones for any of the temperature stress conditions applied (Table 32). [Table 32] ND=Not determined. Table 32. Turbidity and aggregation index AI340 and AI500nm obtained for clones throughout the test for different temperature stress conditions.

[0368] (Detection of submicron particles by dynamic light scattering (DLS)) DLS analysis was performed on all clones and formulation samples at the 4 week time point (T4W) of the study for all storage conditions. Measurements were performed on triplicate preparations using a DynaPro Nanostart instrument. Unstressed control samples and samples kept for 4W under different stress conditions (at T0 and T4W) were analyzed in parallel. Mass percent, molecular hydrodynamic radius, percent polydispersity (%PD), and polydispersity index (PDI) were used to monitor the distribution profile of submicron particles in solution.

[0369] The formulation buffer was not suitable for any of the applied storage conditions before and after filtration (0.2 μm). Aliquots stored at +25° C. and +37° C. for 4W (T4W) were not suitable for any of the candidates. Additionally, solutions of clones 20H09 and 23H09 were not suitable for the unstressed samples at the start of the study (T0). Therefore, all samples for all four candidates were filtered and reanalyzed (triplicate measurements). The DLS profiles in T4W of the samples were relatively similar, with some peaks in intensity, but non-monomeric species were generally negligible, as indicated by the percent mass numbers (Table 33). The radius of the main peak for all candidates was consistent with the expected protein size. The polydispersity of the samples is an indicator of homogeneity. Polydispersity varied between replicates of each clone, but overall, the polydispersity scores indicated a narrow particle size distribution for all samples. Most of the post-filtration samples at the different storage conditions tested were found to be multimodal, indicating the presence of particles with a range of sizes in the samples. Samples from clone 24F02-N53A were the least prone to multimodal scattering, indicating that these samples had a narrower particle size distribution. [Table 33] TIFF2025503707000040.tif242170

[0370] (protein concentration) Protein concentration was assessed by A280nm (Nanodrop) for temperature and freeze-thaw stressed samples (Figure 3). Overall, no protein loss was observed throughout any of the clones tested at any storage temperature.

[0371] For clone 18C06 stored for 4 weeks at +37° C., results were atypical, however analysis of an independent aliquot kept as a reserve sample (under the same experimental conditions) nevertheless measured within the expected range.

[0372] (SPR binding activity) Functional activity concentrations of the candidates were then assessed by SPR on a Biacore 3000 instrument (Figure 4). 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, stored at -80 °C) and in the presence of quality control (QC) samples covering the range of 70-130% relative activity (%RA) against the reference material.

[0373] Nearly all of the temperature-stressed samples from the four candidate clones retained greater than 90% relative activity regardless of the conditions under which they were stored (Figure 4). Similar observations were made for samples subjected to freeze-thaw cycles and low pH (Figure 4). The only atypical result recorded was for clone 18C06, which was stored for 4W at +37°C, but was observed to retain greater than 90% RA when an independent aliquot of the same sample treated under the same conditions was tested (Figure 4).

[0374] (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 variation. Samples were analyzed from all storage conditions and at all time points.

[0375] Temperature stressed, freeze-thawed, and low pH samples of all four clones were observed to have a purity greater than 95% (Figure 5). The percentage of aggregation and fragmentation remained below 1% in the majority of temperature stressed samples (Figure 5). The only exception was the clone 23H09 sample, which showed a temperature-induced increase in aggregate formation (Figure 5).

[0376] Samples subjected to up to 10 freeze-thaw cycles showed similar purity to the reference material for all clones (Figure 5). For samples subjected to low pH stressors, minor aggregation was detected for clone 23H09 sample (Figure 5). All other clones tested showed some fragmentation after application of low pH stress (Figure 5). However, in all cases the percentage of impurities remained below 5% (Figure 5).

[0377] (Purity by capillary gel electrophoresis (cGE)) Purity by cGE was assessed using lab-on-a-chip analysis with an Expert2100 Bioanalyzer instrument (Agilent). Samples were analyzed under reducing and non-reducing conditions at the end of each study.

[0378] The different storage conditions did not affect the purity of the clones, except for clone 18C06 (Figure 6). No sample containing this clone showed a main peak purity above 90% under non-reducing conditions (Figure 6). This was also the case for the unstressed reference sample containing clone 18C06 (Figure 6). For the remaining samples containing the other three clones, the purity was above 90% under non-reducing conditions, regardless of the storage temperature, the time of analysis, or the number of freeze-thaw cycles to which they were subjected (up to 10 times) (Figure 6). Under reducing conditions, all samples tested showed a purity above 95% (Figure 6).

[0379] (Heat resistance) To assess the thermal stability of the candidates, the clones were subjected to a gradient temperature degradation test spanning a temperature range of 55-85°C. After applying this stress in a Biometra Thermocycler programmed to apply several denaturation temperatures, the binding activity of the stressed samples was evaluated in a Biacore 3000 to identify the temperature at which 50% binding activity to Gal10 was lost. 100% binding activity was generated by the non-stressed samples of each clone which were then analyzed in parallel by SPR. Samples for all clones showed 50% activity loss at high temperatures (Table 34). The most stable clone identified following this analytical approach was candidate 24F02_N53A, which, together with the reference clone 7B07_N53A, showed a melting temperature above 70°C. [Table 34] Table 34: Summary of thermal stability of lead candidates

[0380] (Summary table) A tabular summary showing some core properties (before any type of stress) of selected Fabs is provided in Table 35. 7B07_N53A Fab is included for comparison and reference purposes.

[0381] A tabular summary of the attributes of the selected Fabs under different conditions is provided in Table 36. A tabular summary showing some physicochemical properties of individual candidates after freeze-thaw, low pH, and heat stress is provided in Tables 37-40. [Table 35] NT: Not tested; L: Low immunogenicity score Table 35: Summary of lead Fab candidate attributes Table 36: Summary of the attributes of the four Fab clones after several stresses: binding activity, biological activity against CLC, purity by CE-SDS (reduced and non-reduced) and SE-HPLC, post-translational modifications, and particle assessment by DLS and FCM. [Table 36] TIFF2025503707000044.tif235170TIFF2025503707000045.tif236170For comparison / reference 1 Samples correspond to aliquots kept at +37°C for 4 weeks 2 Samples correspond to aliquots kept at +5°C for 4 weeks before nebulization. Briefly, samples were nebulized with the 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 case of device fouling, indicated by longer than normal nebulization times for a given size aliquot, spare devices of the same type were available to complete nebulization with triplicate devices. 3 The %RA of this sample after 4 weeks storage at +37°C was measured at 82% (Eppendorf) and 93% (glass vial) for two independent samples. 4 Samples 20H09 and 23H09 did not qualify without filtration, even in the non-stressed aliquots; the information in this row shows the results after filtration for all clones, all sample types. [Table 37] (1) A low pH stress test was achieved by reaching pH 3.7 with 0.1 M glycine pH 3.0 and after a 2 hour holding step at ambient conditions aliquots were returned to neutralization with 1 M Tris pH 8.0. (2) One aliquot underwent five freeze-thaw cycles, but the 10 freeze-thaw aliquot did not yield any indeterminate results and was therefore not analyzed as per the protocol. NT: not tested; NA: not applicable; ND: not detected Table 37: Summary of some physicochemical properties of Fab clone 18C06. [Table 38] (1) A low pH stress test was achieved by reaching pH 3.7 with 0.1 M glycine pH 3.0 and after a 2 hour holding step at ambient conditions aliquots were returned to neutralization with 1 M Tris pH 8.0. (2) One aliquot underwent five freeze-thaw cycles, but the 10 freeze-thaw aliquot did not yield any indeterminate results and was therefore not analyzed as per the protocol. NT: not tested; NA: not applicable; ND: not detected Table 38: Summary of some physicochemical properties of Fab clone 20H09. [Table 39] (1) A low pH stress test was achieved by reaching pH 3.7 with 0.1 M glycine pH 3.0 and after a 2 hour holding step at ambient conditions aliquots were returned to neutralization with 1 M Tris pH 8.0. (2) One aliquot underwent five freeze-thaw cycles, but the 10 freeze-thaw aliquot did not yield any indeterminate results and was therefore not analyzed as per the protocol. NT: not tested; NA: not applicable; ND: not detected Table 39: Summary of some physicochemical properties of Fab clone 23H09. [Table 40] (1) A low pH stress test was achieved by reaching pH 3.7 with 0.1 M glycine pH 3.0 and after a 2 hour holding step at ambient conditions aliquots were returned to neutralization with 1 M Tris pH 8.0. (2) One aliquot underwent five freeze-thaw cycles, but the 10 freeze-thaw aliquot did not yield any indeterminate results and was therefore not analyzed as per the protocol. NT: not tested; NA: not applicable; ND: not detected Table 40: Summary of some physicochemical properties of Fab clone 24F02_N53A.

[0382] Example 6: Post-translational modification (PTM) analysis Structural characterization of the clones was performed at protein and peptide level using several analytical techniques (icIEF, online desalting MS, RPLC-UV-MS on reduced proteins, peptide map with RPLC-MS after trypsin digestion). Clones were analyzed for PTMs after being subjected to several stress conditions, including 4W storage at different temperatures (+5°C, +25°C, +37°C), spraying before (T0) and after 4W storage at +5°C, and after a low pH hold step (2h / pH 3.7). In each case, the analysis was performed in parallel with control unstressed reference material for each clone.

[0383] What we found can be summarized as follows: The amino acid sequences of four clones were confirmed at the protein level based on the molecular weight of each intact Fab (LC and VH+CH1), while the coverage of peptide sequences was 100%. The structural integrity of the Fab before and after stress remained unchanged, as confirmed by intact protein and peptide mapping analysis which verified and confirmed the presence of the expected disulfide bridges (inter- and intra-chain). Only 18C06 (all samples including reference) was detected with a free cysteine ​​in the LC, rather than the expected bridge with the VH+CH1 portion, instead another disulfide bridge formation between two closely positioned cysteines in the LC was detected. N-terminal cyclization was detected for the common VH+CH1 portion of the stressed clones, which was clearly temperature-induced, with a net increase in pyroglutamic acid (8.5-10.5%) after +37°C storage, and was present but less pronounced (2.0-3.5%) after +25°C storage; this modification in VH+CH1 was not identified for low pH or nebulized samples. Moreover, for g18C06, the LC was found to be fully cyclic after nebulization. Oxidation under all different stress conditions remained <1% for all clones. Site-specific events such as isomerization and deamidation remained largely unaffected after the low pH stress step or spraying for all clones. For temperature stressed samples, clone 18C06 showed a temperature sensitive deamidation spot in one variable region of the LC chain (14.7% after 4 weeks at +37°C). Two moderate deamidation spots were detected at high temperatures (up to 5% each) on the VH+CH1 peptide shared between all pre-lead candidates. For isomerization, one spot was detected in the LC chain, with a slightly more limited increase in isomerization for g20H09, g23H09, and less for g24F02. A second peptide in the LC shared between constructs was also accompanied by a slight increase in isomerization (up to 1.3%). [Table 41] 1 Bold indicates the CDR regions. 2 Clone 18C06 yields a distinct LC from clones 20H09, 23H09, and 24F2_N53A, which share a common LC. Table 41: Major post-translational modifications observed in clones after 4 weeks storage at +37°C.

[0384] Example 7: Crystal dissolution assay results All clones were evaluated for their ability to dissolve GAL10 crystals in vitro. Briefly, a crystal dissolution assay (CDA) was developed and standardized to evaluate the biological activity of clones before and after the application of several stress conditions, including nebulization. The aim of this study was to evaluate whether pre- / post-storage nebulization could affect the (biological) activity of clones, resulting in a loss of their ability to dissolve GAL10 crystals.

[0385] For all clones, the analysis was performed in two independent experiments with appropriate assay controls present. Briefly, samples were nebulized with 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 case 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. For candidates nebulized 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, +25°C, and +37°C were analyzed as 8 concentration point curves for all clones. The results shown here are from assays in which the size distribution of GAL10 crystals was greater than 10 μm (10-15 μm).

[0386] Figure 7 shows the potency of non-stressed samples containing one of the four clones to dissolve GAL10 crystals. All clones were then tested for potency after undergoing the stress conditions described above to assess whether this potency could be maintained after storage and / or after pre- and post-storage nebulization (Figure 8). Clones 18C06, 20H09, and 23H09 were tested in parallel in a first set of assays (Figure 8), and clone 24F02_N53A was tested in parallel with clone 23H09 in a second set of assays (Figure 9). Clone 7B07_N53A was tested as a comparator in the first set of assays.

[0387] All clones were able to completely dissolve GAL10 crystals throughout the entire analytical run, regardless of the stress factors applied to the samples (Figures 7-9). Therefore, further analysis of the clones was primarily focused on the earliest time points up to 5 h, where differences in potency between clones could be observed.

[0388] According to the assay, clones 23H09 and 24F02_N53A were the most potent molecules among the four clones (Figure 9). For all independent runs and regardless of the size distribution of the GAL10 crystals tested, clone 24F02_N53A was more potent in terms of its ability to dissolve GAL10 crystals in vitro. Temperature or spray stress did not affect its dissolving ability when compared to non-stressed material (Figure 9). Based on the above assays, the candidates were ranked from most potent to least potent: ·g24F02_N53 ~g23H09>g20C06>g18C06

[0389] Example 8: Immunogenicity of candidate clones Endotoxin-free material for all clones was assessed for immunogenicity risk using Lonza Epibase®, which includes in silico and cell-based in vitro assessments. Briefly, the in silico assessment utilized an algorithm to screen the amino acid sequence of the clone for potential immunogenic epitopes, including potentially affected allotypes and major histocompatibility complexes, by measuring the HLA-DRB1 score. The assay screened these allotypes relative to their overall population frequency (Figure 10). Based on the immunogenicity score, the clones were ranked from least to most immunogenic: ·g24F02_N53<[g23H09, g18C06] <g20H09

[0390] These results were confirmed using the Lonza Epibase® in vitro assay. Briefly, candidates 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 a T cell response as an unwanted immune response risk (Figure 11), and the magnitude across the study population (Figure 12). The positive control used in this study was clone KLH (Figure 11).

[0391] All clones tested showed a low ability to induce immune responses. Among the four clones, clones 23H09 and 18C06 showed the highest frequency of IFNγ responses, while clones 18C06 and 20H09 showed the highest frequency of IL-5 responses. In all cases and for all statistical approaches, clone g24F02_N53A appeared to be the least likely to induce undesired T cell responses, and therefore this clone appears to have the lowest risk of immunogenicity.

[0392] (Conclusions from the examples) Of the clones tested in detail, the germlined Fab clone 24F02_N53A was considered to be the most promising clone for the following reasons: · Samples remained stable after being subjected to elevated storage temperatures, multiple freeze-thaw cycles, and low pH conditions; ·Showed strong binding to galectin-10; · Recombinant Gal10 crystals could be rapidly dissolved in vitro; · Cross-reacted with cynomolgus Gal10; assumed to have a low immunogenicity risk in humans; and The stability and binding properties of this clone were not affected by spraying. [Table 42] TIFF2025503707000052.tif248170 [Table 43] [Table 44] (References) [Table 45]

Claims

1. 1. An antibody or antigen-binding fragment that binds to Galectin-10, wherein the antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein: (i) the VH domain comprises the CDR sequences of: an HCDR3 comprising or consisting of SEQ ID NO:2; an HCDR2 comprising or consisting of SEQ ID NO:3; and an HCDR1 comprising or consisting of SEQ ID NO:1; and (ii) the VL domain comprises the CDR sequences of: LCDR3 comprising or consisting of SEQ ID NO: 8; LCDR2 comprising or consisting of SEQ ID NO: 9; and LCDR1 comprising or consisting of SEQ ID NO: 7; The antibody or antigen-binding fragment.

2. (i) the VH domain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto: The antibody or antigen-binding fragment of claim 1.

3. (i) the VH domain comprises the amino acid sequence of SEQ ID NO: 4; and (ii) the VL domain comprises the amino acid sequence of SEQ ID NO: 10; The antibody or antigen-binding fragment of claim 1.

4. 2. The antibody or antigen-binding fragment of claim 1, wherein the antigen-binding fragment is selected from the group consisting of: a single-chain antibody (scFv); an F(ab')2 fragment; an Fab fragment; an Fd fragment; an Fv fragment; a single-arm (monovalent) antibody; a diabody, a triabody, a tetrabody, or any antigen-binding molecule formed by a combination, association, or conjugation of such antigen-binding fragments.

5. The antigen-binding fragment of claim 4, wherein the antigen-binding fragment is a Fab fragment.

6. 6. An isolated polynucleotide or polynucleotides encoding the antibody or antigen-binding fragment of any one of claims 1 to 5, or the VH or VL domain thereof.

7. 10. An expression vector comprising the polynucleotide of claim 6 , operably linked to a regulatory sequence that enables expression of the antibody, antigen-binding fragment, variable heavy chain domain, or variable light chain domain in a host cell or a cell-free expression system.

8. A host cell or cell-free expression system containing the expression vector of claim 7.

9. 10. A method for producing a recombinant antibody or antigen-binding fragment of claim 1, comprising culturing a host cell or cell-free expression system of claim 8 under conditions that allow expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 5 and at least one pharmaceutically acceptable carrier or excipient.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 5 for use as a medicament.

12. 12. A pharmaceutical composition comprising an antibody or antigen-binding fragment for use according to claim 11, wherein the pharmaceutical composition is administered to prevent or treat a disease or disorder associated with the presence or formation of galectin-10 crystals.

13. The pharmaceutical composition comprising an antibody or antigen-binding fragment for use according to claim 11, wherein the pharmaceutical composition is administered to prevent or treat a disease or condition 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 (AML).

14. 14. A pharmaceutical composition comprising the antibody or antigen-binding fragment for use according to claim 13, wherein the disease or condition is asthma.

15. 14. A pharmaceutical composition comprising the antibody or antigen-binding fragment for use according to claim 13, wherein the disease or condition is cystic fibrosis.

16. Use of the antibody or antigen-binding fragment of any one of claims 1 to 5 for the detection of galectin-10 in a sample obtained from a patient.

17. 17. The use according to claim 16, wherein the patient sample is a mucus sample or a sputum sample.

18. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 5.

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