SIGLEC-8 binding proteins and uses thereof

Abstract

Provided herein are Siglec-8 binding proteins and methods of using the Siglec-8 binding proteins to modulate the biological activity of Siglec-8.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 333,952, filed on April 22, 2022; the contents of which are incorporated by reference in their entirety for all purposes.

[0003] Incorporation by reference into the sequence listing

[0004] This application incorporates by reference the sequence listing submitted in electronic format with this application, which is named PXS-002WO_SL.xml, created on April 19, 2023, and is 163,709 bytes in size. Technical Field

[0005] The present invention relates to Siglec-8 binding proteins and methods of using such proteins to modulate the biological activity of Siglec-8. Such methods include, but are not limited to, methods for treating eosinophilic disorders or mast cell disorders. Background Art

[0006] Mast cells are tissue-resident cells that regulate acute and chronic tissue inflammation. Abnormal accumulation and activation of mast cells are known to mediate allergic diseases, such as eosinophilic asthma, atopic dermatitis, and eosinophilic gastroenteropathy (Schanin et al. Mucosal Immunology 2021, 14, 366-376). Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is an inhibitory cell surface receptor selectively expressed on these mast cells and mature eosinophils and basophils, and has therefore been used as a target for the treatment of allergic and inflammatory diseases. Cross-linking Siglec-8 has been shown to induce eosinophil death, wherein the process depends on the release of reactive oxygen species (ROS) and the activation of β2-integrin (Legrand et al. J Allergy Clin Immunol 2009, 143, 2227-2237). The selective expression of Siglec-8 makes it a favorable target, which can reduce side effects and improve drug safety. Summary of the invention

[0007] Provided herein are VHH domains, polypeptides and proteins that bind to Siglec-8, and methods of medical treatment using VHH domains, polypeptides and proteins that bind to Siglec-8. For example, the VHH domains, polypeptides and proteins disclosed herein can be used to treat eosinophilic disorders, including eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorders, eosinophilic gastritis (EoG), eosinophilic enteritis, Chag-Strauss syndrome, and hypereosinophilic syndrome. It can also be used to treat mast cell disorders, including but not limited to mast cell leukemia, aggressive systemic mastocytosis and indolent mastocytosis. In addition, it can be used to treat general allergic disorders, such as allergic disorders caused by food allergies, environmental allergies, venom allergies and / or companion animal allergies. In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain. Some embodiments are provided below.

[0008] In one aspect, the present disclosure provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2), and a complementarity determining region 3 (CDR3) sequence of a VHH, wherein the VHH comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164.

[0009] In another aspect, the present disclosure provides a VHH domain that binds Siglec-8 (e.g., human Siglec-8), comprising CDR1, CDR2 and CDR3 sequences as set forth below: (a) SEQ ID NOs: 46, 47 and 48, respectively, as defined according to the AbM numbering system; (b) SEQ ID NOs: 149, 150 and 151, respectively, as defined according to the Chothia numbering system; (c) SEQ ID NOs: 152, 153 and 154, respectively, as defined according to the Kabat numbering system; (d) SEQ ID NOs: 155, 156 and 157, respectively, as defined according to the Contact numbering system; or (e) SEQ ID NOs: 158, 159 and 160, respectively, as defined according to the IMGT numbering system. As defined according to the AbM numbering system, CDR1 may comprise the amino acid sequence of SEQ ID NO: 18, 2, or 13; CDR2 may comprise the amino acid sequence of SEQ ID NO: 29 or 3; and CDR3 may comprise the amino acid sequence of SEQ ID NO: 8. As defined according to the Chothia numbering system, CDR1 may comprise the amino acid sequence of SEQ ID NO: 74, 75, or 76; CDR2 may comprise the amino acid sequence of SEQ ID NO: 80 or 81; and CDR3 may comprise the amino acid sequence of SEQ ID NO: 8. As defined according to the Kabat numbering system, CDR1 may comprise the amino acid sequence of SEQ ID NO: 85, 86, or 87; CDR2 may comprise the amino acid sequence of SEQ ID NO: 92, 93, or 94; and CDR3 may comprise the amino acid sequence of SEQ ID NO: 8. As defined according to the Contact numbering system, CDR1 may comprise the amino acid sequence of SEQ ID NO: 98, 99 or 100; CDR2 may comprise the amino acid sequence of SEQ ID NO: 104 or 105; and CDR3 may comprise the amino acid sequence of SEQ ID NO: 110. As defined according to the IMGT numbering system, CDR1 may comprise the amino acid sequence of SEQ ID NO: 114, 115 or 116; CDR2 may comprise the amino acid sequence of SEQ ID NO: 120 or 121; and CDR3 may comprise the amino acid sequence of SEQ ID NO: 126.

[0010] In certain embodiments of any of the above aspects, the CDR1, CDR2, and CDR3 sequences are set forth in SEQ ID NOs: 18, 29, and 8; 2, 3, and 8; 13, 3, and 8; or 18, 3, and 8, respectively, as defined according to the AbM numbering system; SEQ ID NOs: 74, 80, and 8; 75, 80, and 8; 76, 80, and 8; or 76, 81, and 8, respectively, as defined according to the Chothia numbering system; SEQ ID NOs: 85, 92, and 8; 86, 92, and 8; 87, 93, and 8; 87, 92, and 8; or 87, 94, and 8, respectively, as defined according to the Kabat numbering system; SEQ ID NOs: 98, 104, and 110; 99, 104, and 110; 100, 104, and 110; or 100, 105, and 110, respectively, as defined according to the Contact numbering system; or SEQ ID NOs: 109, 110, and 111, respectively, as defined according to the IMGT numbering system. NO: 114, 120 and 126; 115, 120 and 126; 116, 120 and 126; or 116, 121 and 126.

[0011] In certain embodiments, the VHH domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164. In certain embodiments, the VHH domain is humanized.

[0012] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), comprising CDR1, CDR2, and CDR3 sequences of a VHH, wherein the VHH comprises the amino acid sequence SEQ ID NO: 1. In certain embodiments, CDR1, CDR2, and CDR3 comprise amino acid sequences as set forth below: (a) SEQ ID NOs: 2, 3, and 4, respectively, as defined according to the AbM numbering system; (b) SEQ ID NOs: 74, 80, and 4, respectively, as defined according to the Chothia numbering system; (c) SEQ ID NOs: 85, 91, and 4, respectively, as defined according to the Kabat numbering system; (d) SEQ ID NOs: 98, 104, and 109, respectively, as defined according to the Contact numbering system; or (e) SEQ ID NOs: 114, 120, and 125, respectively, as defined according to the IMGT numbering system. In certain embodiments, the VHH domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0013] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), comprising CDR1, CDR2, and CDR3 sequences of a VHH, wherein the VHH comprises the amino acid sequence SEQ ID NO: 49. In certain embodiments, CDR1, CDR2, and CDR3 comprise amino acid sequences as set forth below: (a) SEQ ID NOs: 52, 53, 54, respectively, as defined according to the AbM numbering system; (b) SEQ ID NOs: 77, 82, 54, respectively, as defined according to the Chothia numbering system; (c) SEQ ID NOs: 88, 95, 54, respectively, as defined according to the Kabat numbering system; (d) SEQ ID NOs: 101, 106, 111, respectively, as defined according to the Contact numbering system; or (e) SEQ ID NOs: 117, 122, 127, respectively, as defined according to the IMGT numbering system. In certain embodiments, the VHH domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:49.

[0014] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), comprising CDR1, CDR2, and CDR3 sequences of a VHH, wherein the VHH comprises the amino acid sequence SEQ ID NO: 55. In certain embodiments, CDR1, CDR2, and CDR3 comprise amino acid sequences as set forth below: (a) SEQ ID NOs: 58, 59, 60, respectively, as defined according to the AbM numbering system; (b) SEQ ID NOs: 78, 83, 60, respectively, as defined according to the Chothia numbering system; (c) SEQ ID NOs: 89, 96, 60, respectively, as defined according to the Kabat numbering system; (d) SEQ ID NOs: 102, 107, 112, respectively, as defined according to the Contact numbering system; or (e) SEQ ID NOs: 118, 123, 128, respectively, as defined according to the IMGT numbering system. In certain embodiments, the VHH domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:55.

[0015] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), comprising the CDR1, CDR2, and CDR3 sequences of a VHH, wherein the VHH comprises the amino acid sequence of SEQ ID NO:61. In certain embodiments, CDR1, CDR2 and CDR3 comprise: (a) the amino acid sequence of SEQ ID NO:63, the amino acid sequence of SEQ ID NO:64, and the amino acid sequence of Gly-Ala-Tyr(GAY), respectively, as defined according to the AbM numbering system; (b) the amino acid sequence of SEQ ID NO:79, the amino acid sequence of SEQ ID NO:84, and the amino acid sequence of Gly-Ala-Tyr(GAY), respectively, as defined according to the Chothia numbering system; (c) the amino acid sequence of SEQ ID NO:90, the amino acid sequence of SEQ ID NO:97, and the amino acid sequence of Gly-Ala-Tyr(GAY), respectively, as defined according to the Kabat numbering system; (d) the amino acid sequences of SEQ ID NOs:103, 108, 113, respectively, as defined according to the Contact numbering system; or (e) the amino acid sequences of SEQ ID NOs:119, 124, 129, respectively, as defined according to the IMGT numbering system. In certain embodiments, the VHH domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:61.

[0016] In another aspect, the present disclosure provides a polypeptide comprising a VHH domain disclosed herein. The polypeptide may comprise at least two of such VHH domains. In certain embodiments, the two VHH domains are operably connected to each other via a peptide linker, e.g., a peptide linker comprising or consisting of the amino acid sequence SEQ ID NO: 69. In certain embodiments, the VHH domains in the polypeptide comprise the same CDR1, CDR2, and CDR3 amino acid sequences. In certain embodiments, the VHH domains in the polypeptide comprise the same VHH amino acid sequences.

[0017] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% identical to the amino acid sequence of SEQ ID NO:9, 10, 12, 17, 22, 27, 32, 37, 50, 51, 56, 57, 62 or 63.

[0018] The polypeptides disclosed herein may further comprise a multimerization domain.

[0019] In another aspect, the present disclosure provides a polypeptide comprising at least two (eg, two or three) antigen binding domains that bind Siglec-8 (eg, human Siglec-8) and a multimerization domain.

[0020] In another aspect, the present disclosure provides a polypeptide that binds to Siglec-8 (eg, human Siglec-8), comprising at least three antigen-binding domains that bind to Siglec-8 (eg, human Siglec-8).

[0021] In certain embodiments of the above two aspects, each of the antigen binding domains is a VHH domain. In other embodiments of these aspects, each of the antigen binding domains comprises a heavy chain variable region and a light chain variable region, for example in the form of a Fab or scFv.

[0022] In certain embodiments of any of the above applicable aspects, the multimerization domain is a dimerization domain, for example an antibody Fc region, such as a human IgG1 Fc region. The antibody Fc region may comprise the amino acid sequence SEQ ID NO: 44, 45, 65, 66, 67 or 68.

[0023] The antibody Fc region can be operably linked to at least one antigen binding domain that binds Siglec-8 via an amino acid linker, for example, an amino acid linker comprising or consisting of the amino acid sequence of SEQ ID NO: 70.

[0024] The polypeptide can take a variety of structural forms. In certain embodiments, the polypeptide comprises the structure VHH-VHH-Fc. Such polypeptides may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence SEQ ID NO: 71. In certain embodiments, the polypeptide comprises the structure VHH-Fc-VHH. In certain embodiments, the polypeptide comprises the structure VHH-VHH-VHH-Fc or VHH-VHH-Fc-VHH.

[0025] The present disclosure also provides a protein comprising two or more polypeptides disclosed herein that are multimerized via a multimerization domain under physiological conditions. For example, the two or more polypeptides may form homomultimers. In the case where the multimerization domain is a dimerization domain, the protein may comprise two polypeptides disclosed herein that are dimerized via a dimerization domain under physiological conditions. For example, the two polypeptides may form homodimers.

[0026] In the case where the VHH, polypeptide or protein disclosed herein binds to human Siglec-8, the amino acid sequence of human Siglec-8 can be set forth in SEQ ID NO:43.

[0027] In certain embodiments, the polypeptides or proteins disclosed herein mediate eosinophil killing in the presence of IL-5. Eosinophil killing can be determined in an in vitro assay. It is contemplated that in the case where the protein comprises at least two VHH domains that bind Siglec-8 in each polypeptide, the EC50 of the homodimeric protein for mediating eosinophil killing in the presence of IL-5 is lower than that of homodimers of polypeptides each having the structure VHH-Fc and comprising only one of the VHH domains that bind Siglec-8.

[0028] In certain embodiments, the polypeptide or protein mediates eosinophil killing in the absence of IL-5. In certain embodiments, the polypeptide mediates eosinophil killing by antibody-dependent cell-mediated cytotoxicity (ADCC).

[0029] The present disclosure also provides a pharmaceutical composition comprising the polypeptide or protein disclosed herein and a pharmaceutically acceptable carrier.

[0030] In addition, the present disclosure provides an isolated nucleic acid encoding the polypeptide disclosed herein, a vector comprising the nucleic acid, a host cell comprising the nucleic acid or the vector, and a host cell expressing the polypeptide or protein.

[0031] The present disclosure also provides a method for producing a polypeptide or protein, the method comprising incubating a host cell under conditions that express the polypeptide or protein. The method may further comprise isolating the polypeptide or protein.

[0032] Another aspect of the present disclosure provides a method for treating an eosinophilic disorder or a mast cell disorder, the method comprising administering a therapeutically effective amount of a polypeptide, protein, or pharmaceutical composition disclosed herein to a subject in need thereof. The eosinophilic disorder may be eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorder, eosinophilic gastritis (EoG), eosinophilic enteritis, Chag-Strauss syndrome, hypereosinophilic syndrome, eosinophilic leukemia (e.g., chronic eosinophilic leukemia), asthma with eosinophilic phenotype, allergic bronchopulmonary aspergillosis (ABPA), chronic sinusitis with nasal polyps (CRSwNP), or eosinophilic granulomatosis with polyangiitis (EGPA). The mast cell disorder may be systemic mastocytosis, hereditary alpha trypsinemia (HAT), or mast cell activation syndrome (MCAS). Systemic mastocytosis (SM) can be advanced SM (eg, mast cell leukemia, aggressive SM, or SM with associated hematological neoplasms (SM-AHN)) or non-advanced SM (eg, indolent SM).

[0033] The present disclosure also provides a method for treating an inflammatory disease or disorder, the method comprising administering a therapeutically effective amount of the polypeptide, protein or pharmaceutical composition disclosed herein to a subject in need thereof.

[0034] In addition, the present disclosure provides a method for treating or preventing allergic diseases, the method comprising administering a therapeutically effective amount of the polypeptide, protein or pharmaceutical composition disclosed herein to a subject in need thereof. The allergic disease may be food allergy, environmental allergy, companion animal allergy and / or venom allergy.

[0035] Additionally, the present disclosure provides a method of depleting eosinophils in a subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, protein, or pharmaceutical composition disclosed herein.

[0036] In addition, the present disclosure provides a method for killing eosinophils, the method comprising contacting eosinophils with natural killer (NK) cells in the presence of a polypeptide, protein or pharmaceutical composition disclosed herein. The present disclosure also provides a method for killing eosinophils, the method comprising contacting eosinophils with macrophages in the presence of a polypeptide, protein or pharmaceutical composition disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figures 1A to 1D Shown is the binding of the parental anti-human Siglec8-B12 (cx8982; Figure 1A ), Siglec8-D1(cx8983; Figure 1B ), Siglec8-E2(cx8984; Figure 1C ) or Siglec8-G2 (cx10382; Figure 1D ) and the mean fluorescence intensity of binding to CHO cells that were not transfected (UT-CHO) or expressed human Siglec-8.

[0038] FIG. 2A to FIG. 2D Shown are bivalent, tetravalent, and hexavalent anti-Siglec8-B12 antibodies (cx8982, cx8467, cx8466; Figure 2A ), anti-Siglec8-D1 antibodies (cx8983, cx8469, cx8523; Figure 2B ), anti-Siglec8-E2 antibodies (cx8984, cx8463, cx8522; Figure 2C ) or anti-Siglec8-G2v14 antibodies (cx11422, cx11580, cx11579; Figure 2D ) and human Siglec-8 on eosinophils ( FIG. 2A to FIG. 2C ) or the mean fluorescence intensity of the binding to human Siglec-8 transfected CHO cells ( Figure 2D ) is the mean fluorescence intensity of the combination.

[0039] FIG. 3A to FIG. 3E The incorporation of titrated amounts of bivalent, tetravalent, and hexavalent anti-Siglec8-B12 antibodies (cx8982, cx8467, cx8466; Figure 3A ), anti-Siglec8-D1 antibodies (cx8983, cx8469, cx8523; Figure 3B ), anti-Siglec8-E2 antibodies (cx8984, cx8463, cx8522; Figure 3C ) for 24 h, or in the presence of IL-5 ( Figure 3D ) or in the absence of IL-5 ( Figure 3E ) Total red object integrated intensity (RCU x μm2 / image) of the dead cell marker cytotox red in purified human eosinophils after 18 h of treatment with anti-Siglec8-G2v14 (cx11422, cx11580, cx11579) agonist antibodies.

[0040] FIG. 4A to FIG. 4C All tetravalent Siglec-8 agonist antibodies comprising hzG2v52 (cx11913) or hzG2v53 (cx11914) and the comparator analog 2E2 Afuc were shown to bind to freshly isolated human eosinophils ( Figure 4A ) or tetravalent Siglec-8 agonist antibodies containing hzG2v52 (cx11913), hzG2v53 (cx11914), or hzG2v14 (cx11769) and CHO cells transfected with human Siglec-2, 3, 6, 7, 8, 9, 12, and 15 ( FIG. 4B to FIG. 4C ) is the mean fluorescence intensity of the combination.

[0041] FIG. 5A to FIG. 5H The binding of parental anti-human Siglec8-G2 (lmG2) and its humanized variants forming monomeric VHH-hIgG1 fusions to CHO293 cells expressing human Siglec-8 ( FIG. 5A to FIG. 5D ) or untransfected 293 cells ( FIG. 5E to FIG. 5H )'s median fluorescence intensity.

[0042] FIG. 6A to FIG. 6B Direct ( Fig. 6A ) and ADCC-mediated ( Figure 6B )'s killing activity. Fig. 6A Shown is the total red object integrated intensity (RCU x μm2 / image) of the dead cell marker cytotox red spiked into purified human eosinophils in the presence of IL-5. Figure 6B Shown is the percentage of apoptotic apotracker green-positive eosinophils killed by NK cell-mediated ADCC in the absence of IL-5.

[0043] Figure 7 Depicted are exemplary formats of polypeptides that target multivalent Siglec-8 compared to polypeptides that target bivalent Siglec-8. DETAILED DESCRIPTION

[0044] Embodiments provided herein relate to Siglec-8 binding polypeptides and their use in various methods of treating Siglec-8 associated diseases or disorders.

[0045] Definition and various implementations

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] All references, including patent applications, patent publications, and GenBank Accession Numbers, cited herein are incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0048] The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methods, such as, for example, the widely used methods described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al., eds., (2003)); Series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJGait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) PlenumPress; Cell and Tissue Culture Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (JEColigan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty. Ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993); and updated versions thereof.

[0049] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires or clearly indicates, singular terms shall include the plural and plural terms shall include the singular. For any conflict in definitions between various sources or references, the definitions provided herein shall prevail.

[0050] Typically, the numbering of residues in immunoglobulin heavy chains is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0051] It should be understood that the embodiments of the invention described herein include "comprising an embodiment," "consisting of an embodiment," and / or "consisting essentially of an embodiment." Unless otherwise indicated, as used herein, the singular forms "a," "an," and "the" include plural referents. The use of the term "or" herein is not intended to imply that alternatives are mutually exclusive.

[0052] In this application, the use of "or" means "and / or" unless clearly stated or understood by one skilled in the art. In the context of multiple dependent claims, the use of "or" refers back to more than one preceding independent claim or dependent claim.

[0053] The phrase "reference sample", "reference cell" or "reference tissue" means a sample with at least one known characteristic that can be used as a comparison with a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to determine the level of a protein and / or mRNA present in, for example, a healthy tissue, in contrast to the level of a protein and / or mRNA present in a sample with an unknown characteristic. In some embodiments, the reference sample is from the same subject, but from a site different from the tested site of the subject. In some embodiments, the reference sample is from a tissue region surrounding or adjacent to a cancer. In some embodiments, the reference sample is not from the tested subject, but from a sample from a subject known to have or known not to have the disorder in question (e.g., a Siglec-8-related disorder). In some embodiments, the reference sample is from the same subject, but from a time point before the subject develops the disorder. In some embodiments, the reference sample is from the same or different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample will indicate a level understood by those skilled in the art, in view of the present disclosure, the absence and / or presence of low levels of the molecule. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will be indicative of the level to which one of skill in the art would understand, in light of the present disclosure, that a certain level of the molecule is present.

[0054] As used herein in the context of benefiting from or responding to the administration of a therapeutic agent, the terms "benefit", "clinical benefit", "responsiveness" and "therapeutic responsiveness" can be measured by assessing various endpoints, such as inhibiting disease progression to some extent, including slowing and complete arrest; reducing the number of disease and / or symptom attacks; reducing the size of lesions; inhibiting (i.e., reducing, slowing or completely stopping) disease cell infiltration to adjacent peripheral organs and / or tissues; inhibiting (i.e., reducing, slowing or completely stopping) disease spread; alleviating to some extent one or more symptoms associated with the disorder; prolonging the time after treatment without disease manifestation, such as progression-free survival; improving overall survival; higher response rate; and / or reducing the mortality rate at a given time point after treatment. "Non-responsive" or "failed to respond" subjects or cancers are subjects or cancers that fail to meet the above-mentioned qualifying conditions of "responsive".

[0055] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to polymers of nucleotides. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0056] The term "polypeptide" refers to an amino acid residue polymer, and is not limited to a minimum length. Such amino acid residue polymers may contain natural or non-natural amino acid residues, and include, but are not limited to, full-length forms and fragments of peptides, oligopeptides, and natural polypeptides. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, a "polypeptide" may include modifications to the native sequence, such as deletions, additions, and substitutions (usually conservative in nature), as long as it maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0057] The term "protein" refers to a macromolecule comprising one or more polypeptides. For example, a protein may be a multimer (eg, a dimer, trimer, or tetramer) formed by a plurality of identical or different polypeptides.

[0058] As used herein, "Siglec-8" refers to any naturally mature Siglec-8 produced by processing of Siglec-8 precursors in cells. Unless otherwise indicated, the term includes Siglec-8 from any mammalian source, including primates (e.g., humans and cynomolgus macaques or rhesus monkeys) and rodents (e.g., mice and rats). A non-limiting exemplary human Siglec-8 amino acid sequence is shown, for example, in UniProtKB / Swiss-Prot: Q9NYZ4.2. See SEQ ID NO.42. A non-limiting exemplary mature human Siglec-8 amino acid sequence is shown, for example, in SEQ ID NO: 43.

[0059] The term "specific binding" to an antigen or epitope is a well-known term in the art, and methods for determining such specific binding are also well-known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer period of time, and / or with a greater affinity than the molecule reacts or associates with an alternative cell or substance. A single domain antibody (sdAb) or a VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other substances. For example, an sdAb or a VHH-containing polypeptide that specifically or preferentially binds to a Siglec-8 epitope is an sdAb or a VHH-containing polypeptide that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other Siglec-8 epitopes or non-Siglec-8 epitopes. It should also be understood by reading this definition that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, "binding" means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0060] The term "inhibition" refers to the reduction, interruption or containment of any phenotypic characteristic, or the reduction, interruption or containment of the incidence, degree or likelihood of the characteristic. "Reduce" or "inhibit" refers to reducing, reducing, containing or inhibiting an activity, function, amount, abundance or rate of increase compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amount is inhibited or reduced over a certain period of time relative to a control over the same period of time.

[0061] The term "agonize" or "activate" refers to an increase, activation or induction of any phenotypic characteristic, or an increase, activation or induction of the probability of occurrence of the characteristic. "Agitation" or "activation" refers to an increase, activation or induction of activity, function, amount, abundance or rate of increase compared to a reference. In some embodiments, "agonize" or "activation" means the ability to cause an overall increase of 10% or more. In some embodiments, "agonize" or "activation" means the ability to cause an overall increase of 50% or more. In some embodiments, "agonize" or "activation" means the ability to cause an overall increase of 75%, 85%, 90%, 95% or more. In some embodiments, the above amount is increased over a certain period of time relative to a control over the same period of time. As used herein, the term "agonize" with respect to Siglec-8 activity refers to an increase in Siglec-8 activity, which is an inhibitory receptor on mast cells and eosinophils that mediates cell death of eosinophils in the presence of IL-5 and negatively regulates mast cell activation. Thus, in some embodiments, an agonist of Siglec-8 increases eosinophil cell death in the presence of IL-5, and / or increases inhibition of mast cell activation. In some embodiments, "agonism" refers to an increase in Siglec-8 activity compared to Siglec-8 activity in the absence of a modulator.

[0062] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) that binds to an antigen binding molecule (e.g., sdAb or a VHH-containing polypeptide). Epitopes typically include a group of chemically active surface molecules (such as amino acids, polypeptides, or sugar side chains) and have specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed by adjacent and / or juxtaposed non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed by adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent. An epitope may include, but is not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, the length of an epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen. In some embodiments, epitopes can be identified by a certain minimum distance with the CDR residues on the antigen binding molecules. In some embodiments, epitopes can be identified by the above distances, and are further limited to those residues that participate in the bond (e.g., hydrogen bond) between the residues of the antigen binding molecules and the antigen residues. Epitopes can also be identified by various scans, such as alanine scanning or arginine scanning can indicate one or more residues that the antigen binding molecules can interact with. Unless explicitly stated, a group of residues as an epitope does not exclude other residues as a part of the epitope of a specific antigen binding molecule. On the contrary, the presence of this group specifies the epitope of the minimum series (or species group). Therefore, in some embodiments, a group of residues identified as an epitope specifies the minimum epitope associated with an antigen, rather than a unique residue list of an epitope on an antigen.

[0063] A "non-linear epitope" or "conformational epitope" comprises non-adjacent polypeptides, amino acids, and / or sugars within an antigenic protein that bind to an antigen binding molecule that is specific for the epitope. In some embodiments, at least one of the residues will not be adjacent to other indicated residues of the epitope; however, one or more of the residues may also be adjacent to other residues.

[0064] A "linear epitope" comprises adjacent polypeptides, amino acids, and / or sugars within an antigenic protein that bind to an antigen binding molecule that is specific for the epitope. It should be noted that in some embodiments, not every residue within a linear epitope needs to be directly bound (or participate in bonding) by an antigen binding molecule. In some embodiments, a linear epitope may be derived from immunization with a peptide that effectively consists of the sequence of a linear epitope, or from a structural portion of a protein that is relatively isolated from the rest of the protein (such that an antigen binding molecule can interact, at least primarily, with only that sequence portion).

[0065] The term "antibody" is used in the broadest sense and encompasses various polypeptides comprising antibody-like antigen-binding domains, including but not limited to conventional antibodies (usually comprising at least one heavy chain and at least one light chain, more typically comprising two heavy chains and two light chains forming two antigen-binding domains), single domain antibodies (sdAbs, which comprise, for example, at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the foregoing, as long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to heavy chain constant domains (comprising CH1, hinge, CH2, and CH3, wherein CH1 is usually paired with light chain constant domain CL to mediate dimerization) and Fc regions (comprising hinge, CH2, and CH3, wherein hinge and CH3 mediate dimerization). The term antibody also includes but is not limited to chimeric antibodies, humanized antibodies, and antibodies of various species, such as camelids (including llamas), sharks, mice, humans, cynomolgus monkeys, and the like.

[0066] As used herein, the term "antigen binding domain" refers to an antibody portion sufficient to bind to an antigen. In some embodiments, the antigen binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Therefore, in some embodiments, the antigen binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining antigen binding, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining antigen binding. In some embodiments, the antigen binding domain comprises a heavy chain variable region and a light chain variable region. Non-limiting such antigen binding domains include Fab and scFv. In some embodiments, the antigen binding domain comprises a VHH domain. In some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises three CDRs of a VHH domain. Thus, in some embodiments, an antigen binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain antigen binding.

[0067] As used herein, the term "VHH" or "VHH domain" refers to the antigen binding portion of a single domain antibody, such as a camelid antibody. In some embodiments, the VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH may be truncated at the N-terminus or C-terminus so that it comprises only a portion of FR1 and / or FR4, or lacks one or two of those framework regions, as long as the VHH substantially retains antigen binding and specificity.

[0068] The terms "single domain antibody" and "sdAb" are used interchangeably herein and refer to an antibody comprising at least one monomeric domain (such as a VHH domain and without a light chain) and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each of which comprises at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, such as polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different.

[0069] The term "VHH polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three or four or more VHH domains, wherein each VHH domain may be the same or different. In some embodiments, the VHH polypeptide comprises an Fc region. In some such embodiments, the VHH polypeptide may be referred to as an sdAb. In addition, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures containing VHH polypeptides (also sdAb) include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be connected to another VHH via a linker, or one VHH may be connected to the Fc region via a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids mainly consisting of glycine and optionally serine. Non-limiting linkers are presented in SEQ ID NOs: 69 and 70. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered tetravalent if it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered hexavalent if it forms a dimer (i.e., the dimer has six VHH domains). In some embodiments, the VHH-containing polypeptide comprising an Fc region forms a homodimer, for example, by associating a hinge of the Fc region.

[0070] The term "monoclonal antibody" refers to antibodies (including sdAb or VHH polypeptides) of a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. Therefore, a sample of a monoclonal antibody can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by a recombinant DNA method such as described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from a phage library produced using, for example, McCafferty et al., 1990, Nature 348:552-554.

[0071] The term "CDR" means a complementarity determining region as defined by one skilled in the art by at least one identification means. In some embodiments, the CDRs may be defined according to any of the Chothia numbering schemes (see Chothia and Lesk, J Mol Biol, 1987, 196:901-917), the Kabat numbering scheme (see Kabat et al., 1992, Sequences of Proteins of Immunological Interest, DIANE Publishing: 2719), a combination of Kabat and Chothia, the AbM definition (see Whitelegg & Rees, Protein Eng. 2000, 13:819-824; Whitelegg & Rees, Methods Mol Biol. 2004, 248:51-91), the IMGT definition (see Lefranc, (1999) The Immunologist, 7, 132-136), and / or the Contact definition (see MacCallum et al., J. Mol. Biol. 1996, 262:732-745). A VHH comprises three CDRs, designated CDR1, CDR2 and CDR3.

[0072] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, CH1, hinge, CH2, and CH3. Of course, unless otherwise specified, non-functional changes in the domain are missing and changes are included within the scope of the term "heavy chain constant region". Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. In addition, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2. For all heavy chain constant region amino acid positions discussed herein, numbering is according to the Eu index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, which describes the amino acid sequence of myeloma protein Eu (which was the first human IgG1 sequenced). The Eu index of Edelman et al. is also described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrases "EU index as set forth in Kabat" or "EU index of Kabat" and "position according to the EU index as set forth in Kabat..." and grammatical variations thereof refer to the residue numbering system based on the human IgG1 Eu antibody of Edelman et al. as set forth in Kabat 1991.

[0073] As used herein, "Fc region" refers to a portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge and CH3 mediate dimerization between two Fc-containing polypeptides. The Fc region may have any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is from IgG1, IgG2, IgG3, or IgG4.

[0074] As used herein, an "acceptor human framework" is a heavy chain variable domain (V H ) framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise an amino acid sequence identical thereto, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks in a single antigen binding domain (such as a VHH).

[0075] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody such as an sdAb or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K) and the affinity of a molecule X for its partner Y, respectively. d ) or K d-表观 Affinity can be measured by common methods known in the art (e.g., ELISA K d , KinExA, flow cytometry and / or surface plasmon resonance devices), including those described herein. Such methods include, but are not limited to, In some embodiments, the exemplary polypeptide has a chromatin level of less than or equal to about 90, 85, 80, 75, 70, 68, 65, 60, 55, 56, or 50 x 10 -12 M of K D In some embodiments, the exemplary polypeptides have a binding affinity of less than or equal to about 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 68, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 x 10 -12 M of K D Binds to Siglec-8.

[0076] As used herein, the term "K d ” refers to the equilibrium dissociation constant of the antigen binding molecule / antigen interaction. When the term “K d ", it includes K d and K d-表观 .

[0077] In some embodiments, the K of the antigen binding molecule is dK is measured by flow cytometry using a cell line expressing the antigen and fitting the mean fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (Prism Software graphpad). In some such embodiments, K d K d-表观 .

[0078] The term "biological activity" refers to any one or more biological properties of a molecule (whether occurring naturally in vivo or provided or enabled by recombinant means).

[0079] "Agonist" or "activating" antibodies are antibodies that increase and / or activate the biological activity of a target antigen. In some embodiments, an agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0080] An "antagonist," "blocking," or "neutralizing" antibody is an antibody that inhibits the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99%, or more.

[0081] An "affinity matured" sdAb or VHH-containing polypeptide refers to an sdAb or VHH-containing polypeptide having alterations in one or more CDRs, compared to a parent sdAb or VHH-containing polypeptide that does not have such alterations, such alterations resulting in an improvement in the affinity of the sdAb or VHH-containing polypeptide for an antigen.

[0082] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may include residues that are neither present in the original VHH nor in the human framework sequence, but are included to further improve and optimize the performance of sdAb-containing VHH polypeptides. In some embodiments, humanized sdAbs or VHH-containing polypeptides include human Fc regions. It should be understood that humanized sequences can be identified by their primary sequence and do not necessarily represent the process of producing antibodies.

[0083] An "effector-positive Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, among others. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be assessed using a variety of assays.

[0084] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0085] A "variant Fc region" comprises an amino acid sequence that is different from a native sequence Fc region due to at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that is different from a native sequence Fc region due to at least one amino acid modification, but retains at least one effector function of a native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, such as about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions in the native sequence Fc region or the parent polypeptide Fc region compared to the native sequence Fc region or the parent polypeptide Fc region. In some embodiments, the variant Fc region herein will have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of the parent polypeptide, at least about 90% sequence identity with it, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

[0086] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a natural human FcR. In some embodiments, FcR is a receptor that binds to an IgG antibody (gamma receptor) and includes receptors of FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences with the main difference being their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain, and the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). For example, FcRs are summarized in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994) and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0087] As used herein, the term "substantially similar" or "substantially identical" refers to a sufficiently high degree of similarity between two or more values ​​that one skilled in the art would consider the difference between the two or more values ​​to be of little or no biological and / or statistical significance over the range of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than about any of 5%, 10%, 15%, 20%, 25%, or 50%.

[0088] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with the native sequence polypeptide.

[0089] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0090] Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into an antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0091] Table 1

[0092] Original residue Exemplary substitutions Ala(A) Val; Leu; Ile Arg(R) Lys; Gln; Asn Asn(N) Gln; His; Asp, Lys; Arg Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn;Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln; Lys; Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg; Gln; Asn Met(M) Leu; Phe; Ile Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ser(S) Thr Thr(T) Val; Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe; Thr; Ser Val(V) Ile; Leu; Met; Phe; Ala; norleucine

[0093] Amino acids can be grouped according to common side chain properties:

[0094] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0095] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0096] (3) Acidic: Asp, Glu;

[0097] (4) Basic: His, Lys, Arg;

[0098] (5) Residues that affect chain orientation: Gly, Pro;

[0099] (6) Aromatic: Trp, Tyr, Phe.

[0100] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0101] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate the expression of a polypeptide of interest (such as, for example, a promoter and / or enhancer), and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0102] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Host cells include 293 and CHO cells and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include the progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not be completely identical to the original parent cell (in morphology or in genomic DNA complementation). Host cells include cells transfected in vivo with the polynucleotides provided herein.

[0103] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, the supernatant containing the polypeptide is physically separated from the cell that produced it, and is considered to be "isolated" the polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide that is typically found in nature (such as in the case of a DNA polynucleotide, such as genomic DNA or mitochondrial DNA), or, for example in the case of an RNA polynucleotide, is separated from at least some of the components of the cell that produced the polynucleotide. Thus, a DNA polynucleotide contained in a vector inside a host cell can be referred to as "isolated".

[0104] The terms "individual" and "subject" are used interchangeably herein and refer to an animal; for example, a mammal. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, simians, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. In some instances, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or condition. In some embodiments, a subject receiving treatment may be a patient, specifying the fact that the subject has been identified as suffering from a condition relevant to the treatment or is at sufficient risk of developing a condition.

[0105] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0106] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be clear to those skilled in the art, "modulating" may also involve achieving a change (which may be an increase or decrease) in affinity, avidity, specificity and / or selectivity of a target or antigen for one or more of its ligands, binding partners, partners or substrates that associate into homomultimer or heteromultimer forms; achieving a change (which may be an increase or decrease) in the sensitivity of a target or antigen to one or more conditions (such as pH, ionic strength, cofactors present, etc.) in the medium or environment in which the target or antigen is present; and / or cell proliferation or cytokine production compared to the same conditions but without the presence of a test agent. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein (depending on the target involved).

[0107] As used herein, "immune response" is intended to encompass cellular and / or humoral immune responses sufficient to inhibit or prevent the onset of a disease (e.g., cancer or cancer metastasis) or to ameliorate its symptoms. "Immune response" may encompass aspects of the innate immune system and the adaptive immune system.

[0108] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. As used herein, "treatment" encompasses the administration or application of any therapeutic agent for a mammalian (including human) disease. For the purposes of this disclosure, a beneficial or desired clinical result includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, weakening the extent of the disease, preventing disease progression, preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, inhibiting disease or disease progression, and inhibiting or slowing disease or its progression. "Treatment" also encompasses the pathological results of reducing the disease. The method provided herein takes into account any one or more of these treatment aspects. Consistent with the above, the term treatment does not require 100% removal of all aspects of the disease.

[0109] "Ameliorating" means alleviating or improving one or more symptoms compared to not administering the therapeutic agent. "Ameliorating" also includes shortening or reducing the duration of symptoms.

[0110] The term "biological sample" means a quantity of a substance from a living or formerly living organism. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, bronchoalveolar lavage fluid, sputum, nasal lavage fluid, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0111] In the context of an experiment or comparison, the term "control" or "reference" refers to a composition known to contain the analyte (a "negative control") or to contain the analyte (a "positive control"). A positive control may contain a known concentration of the analyte. A control or reference may also refer to a control agent known to lack the activity of a tested agent, such as an antibody.

[0112] As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not imply or require complete prevention at all times, but only during the measured period of time.

[0113] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount that is effective to achieve the desired therapeutic and / or preventive result within the necessary dosage and time period.

[0114] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional components that are unacceptably toxic to a subject to which the preparation is administered. Such preparations may be sterile.

[0115] "Pharmaceutically acceptable carrier" refers to a conventional non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier used with a therapeutic agent in the art, which together constitute a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed.

[0116] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0117] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents wherein at least some of the administration overlaps in time, or wherein the administration of one therapeutic agent falls within a short period of time relative to the administration of another therapeutic agent, or wherein the therapeutic effects of the two agents overlap for at least a period of time.

[0118] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time, or where the therapeutic effects of the agents do not overlap.

[0119] As used herein, "in conjunction with" refers to administering one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" refers to administering one therapeutic modality before, during, or after another therapeutic modality is administered to a subject.

[0120] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0121] An "article of manufacture" is any article (e.g., a package or container) or kit comprising at least one reagent, such as a drug for treating a disease or disorder (e.g., cancer) or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0122] The terms "label" and "detectable label" mean a moiety that is attached to, for example, an antibody or antigen to allow a reaction (e.g., binding) between members of a detectable specific binding pair. Labeled members of a specific binding pair are considered to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that has an incorporated label that provides for identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a signal that can be detected by visual or instrumental means, such as a polypeptide that incorporates a radiolabeled amino acid or is attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of polypeptide labels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho or 153Sm); chromogens, fluorescent tags (e.g., FITC, rose bengal, lanthanide fluorophores), enzyme tags (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as acridine compounds, and fluorescence-generating moieties, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable after reacting with another moiety.

[0123] Exemplary Siglec-8 Binding Polypeptides and Proteins

[0124] Siglec-8 binding polypeptides and proteins are provided herein. In various embodiments, the Siglec-8 binding polypeptide or protein is a Siglec-8 agonist. In some embodiments, a Siglec-8 binding polypeptide comprising at least two antigen binding domains that bind to Siglec-8 is provided. In some embodiments, a Siglec-8 binding polypeptide or protein comprising at least three antigen binding domains that bind to Siglec-8 is provided. In some embodiments, a Siglec-8 binding polypeptide comprising at least two antigen binding domains that bind to Siglec-8 and a dimerization domain is provided. In some embodiments, a Siglec-8 binding polypeptide comprising more than three antigen binding domains that bind to Siglec-8 and a dimerization domain is provided. In some embodiments, the dimerization domain is an Fc region. In some embodiments, the EC50 of the polypeptide mediating eosinophil killing in the presence of IL-5 is lower than that of a polypeptide comprising two antigen binding domains that bind to Siglec-8 under physiological conditions, such as a conventional antibody. In some embodiments, two or more antigen binding domains are identical. In some embodiments, two or more antigen binding domains are different. In some embodiments, all antigen binding domains are the same. In some embodiments, each antigen binding domain comprises a heavy chain variable region and a light chain variable region. In some embodiments, each antigen binding domain is a Fab or scFv.

[0125] In various embodiments, the Siglec-8 binding polypeptide or protein comprises at least one VHH domain that binds to Siglec-8. In some embodiments, the Siglec-8 binding polypeptide or protein binds to human Siglec-8. In some embodiments, the Siglec-8 binding polypeptide or protein binds to Siglec-8 and excites Siglec-8 (i.e., increases its activity). In some embodiments, the Siglec-8 binding polypeptide or protein excites Siglec-8, resulting in the killing of eosinophils in the presence of IL-5. In some embodiments, the Siglec-8 binding polypeptide or protein depletes eosinophils in vitro or in vivo in the presence of IL-5.

[0126] In some embodiments, the Siglec-8 binding polypeptide provided herein comprises one, two, three, four, five, six, seven or eight VHH domains that bind to Siglec-8. In some embodiments, the Siglec-8 binding polypeptide provided herein comprises one, two, three or four VHH domains that bind to Siglec-8. In some embodiments, the Siglec-8 binding polypeptide provided herein comprises two or three VHH domains that bind to Siglec-8. The Siglec-8 binding polypeptide may comprise one or more VHH domains that bind to one or more target proteins other than Siglec-8. Such polypeptides may be referred to as "multi-specific" polypeptides.

[0127] In various embodiments, the Siglec-8 binding polypeptide comprises one, two, three or four VHH domains that bind to Siglec-8. In various embodiments, the Siglec-8 binding polypeptide comprises two or three VHH domains that bind to Siglec-8. In some embodiments, the Siglec-8 binding polypeptide comprises two, three or four VHH domains that bind to Siglec-8, which are operably connected to each other via a peptide linker. Non-limiting examples of linkers that can be used to connect VHH domains are presented in SEQ ID NO: 69.

[0128] In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain that binds to Siglec-8 and an Fc region. In some embodiments, the Siglec-8 binding polypeptide provided herein comprises one, two, three or four VHH domains that bind to Siglec-8 and an Fc region. In some embodiments, the Siglec-8 binding polypeptide provided herein comprises two or three VHH domains that bind to Siglec-8 and an Fc region. In some embodiments, the Fc region mediates dimerization of the Siglec-8 binding polypeptide under physiological conditions, thereby forming a dimer that doubles the number of Siglec-8 binding sites in the protein. For example, a Siglec-8 binding polypeptide comprising three VHH domains that bind to Siglec-8 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization to form a Siglec-8 binding protein that is a hexavalent dimer under such conditions. In some embodiments, the Fc region is a human IgG1 Fc region variant, such as any human IgG1 Fc region variant described herein.

[0129] In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain described herein fused to an Fc region. In some embodiments, the Fc region comprises the sequence SEQ ID NO: 44, 45, 65, 66, 67 or 68. In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain described herein operably linked to the Fc region via a peptide linker. Non-limiting examples of linkers that can be used to connect the VHH domain and the Fc region are presented in SEQ ID NO: 70.

[0130] In various embodiments, the Fc region included in the Siglec-8 binding polypeptide is a human Fc region, or is derived from a human Fc region. In some embodiments, the Fc region is an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype. In some embodiments, the Fc region is a wild-type IgG1 isotype (see, e.g., SEQ ID NOs: 45 and 66).

[0131] In some embodiments, the Fc region included in the Siglec-8 binding polypeptide is derived from a human Fc region and comprises mutations designed for heterodimerization.

[0132] In some embodiments, the Fc region included in the Siglec-8 binding polypeptide is derived from a human Fc region and comprises mutations designed to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), such as amino acid modifications described in WO 00 / 42072, WO 04 / 029207, WO 04 / 099249, and WO 04 / 063351. In some embodiments, the Fc region included in the Siglec-8 binding polypeptide comprises a deletion of Glu233, Leu234, Leu235, or any combination thereof according to the EU index as set forth in Kabat. In some embodiments, the Fc region included in the Siglec-8 binding polypeptide comprises a deletion of Glu233, Leu234, and Leu235 (xELL) according to the EU index as set forth in Kabat to reduce or eliminate ADCC (see, e.g., SEQ ID NOs: 45 and 67). In some embodiments, the Fc region included in the Siglec-8 binding polypeptide includes modifications at Ser239 and / or Ile332 according to the EU index as set forth in Kabat. In one embodiment, the Fc region included in the Siglec-8 binding polypeptide comprises the following modifications: Ser239Asp and Ile332Glu (S239D, I332E) according to the EU index as set forth in Kabat to enhance ADCC (see, e.g., SEQ ID NOs: 65 and 68). In some embodiments, the Fc region lacks Lys447 according to the EU index as set forth in Kabat (see, e.g., SEQ ID NOs: 66, 67, and 68).

[0133] Fc regions that can be used for Siglec-8 binding polypeptides include Fc regions comprising the amino acid sequence of SEQ ID NO: 44, 45, 65, 66, 67 or 68.

[0134] The Siglec-8 binding proteins disclosed herein can take various forms, for example, Figure 7In the form depicted in. In some embodiments, the protein is a homodimer comprising two identical polypeptide chains, which comprises one or more, two or more, three or more, two, three or two or three VHH domains that bind to Siglec-8 and an Fc region. In some embodiments, the polypeptide chain comprises a VHH domain and an Fc region that binds Siglec-8 from the N-terminus to the C-terminus. In some embodiments, the polypeptide chain comprises a first VHH domain that binds Siglec-8, a second VHH domain that binds Siglec-8, and an Fc region from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain may be the same or different. In some embodiments, the polypeptide chain comprises a first VHH domain that binds Siglec-8, an Fc region, and a second VHH domain that binds Siglec-8 from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain may be the same or different. In some embodiments, the polypeptide chain comprises from N-terminus to C-terminus a first VHH domain that binds to Siglec-8, a second VHH domain that binds to Siglec-8, a third VHH domain that binds to Siglec-8, and an Fc region, wherein the first VHH domain, the second VHH domain, and the third VHH domain may be the same or different. In some embodiments, the polypeptide chain comprises from N-terminus to C-terminus a first VHH domain that binds to Siglec-8, a second VHH domain that binds to Siglec-8, an Fc region, and a third VHH domain that binds to Siglec-8, wherein the first VHH domain, the second VHH domain, and the third VHH domain may be the same or different.

[0135] In some embodiments, when the Siglec-8 binding polypeptide or protein contains an Fc domain with ADCC effector function, the Siglec-8 binding polypeptide or protein increases the killing of eosinophils by NK cells in the absence of IL-5.

[0136] In some embodiments, the Siglec-8 binding polypeptide comprises at least two VHH domains, wherein the first VHH domain binds Siglec-8 and the second VHH domain binds an antigen other than Siglec-8. Such polypeptides may be referred to as "bispecific" or "multispecific".

[0137] Non-limiting exemplary Siglec-8 binding VHH domains are described below in the subsection “Siglec-8 binding VHH domains”.

[0138] VHH domain binding to Siglec-8

[0139] The present disclosure provides VHH domains that bind to Siglec-8. Table 2 lists exemplary VHH domains and CDR sequences in these VHH domains under various CDR definitions. VHH names beginning with "hz" indicate that they are humanized versions of the corresponding parent polypeptides.

[0140] Table 2: Polypeptides comprising VHHs that bind to Siglec-8

[0141]

[0142] Where multiple sets of CDR sequence identifiers are provided, such as for hzG2v14 under the AbM CDR definition, the multiple sets of CDR sequences refer to the same sequence. In other words, in the hzG2v14 example, SEQ ID NOs: 2 and 6 represent the same sequence, and SEQ ID NOs: 3 and 7 represent the same sequence.

[0143] The amino acid sequences of additional exemplary humanized VHH domains are set forth in SEQ ID NOs: 130-138, 140-148, 161, 163 and 164. Although their CDR sequences are not provided in the sequence listing, the sequences can be identified by methods known in the art, such as using the AbYsis server (www.abysis.org / abysis / ). These VHH domains are variants of hzG2v14, hzG2v47, hzG2v51, hzG2v52, hzG2v53, hzG2v54 and hzG2v55 of Table 2. Therefore, their CDR sequences can also be identified by aligning the VHH amino acid sequence with one or more of those reference sequences (e.g., hzG2v14) and isolating the regions corresponding to the CDRs of the reference sequences.

[0144] In certain embodiments, the Siglec-8 binding VHH domain comprises a CDR1, CDR2, and CDR3 sequence of a VHH disclosed herein, e.g., as set forth in a sequence selected from SEQ ID NOs: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164. In certain embodiments, the VHH domain comprises a CDR1, CDR2, and CDR3 sequence of a VHH disclosed herein under the AbM definition, e.g., a set of CDR sequences disclosed in the "AbM" column of Table 2. In certain embodiments, the VHH domain comprises a CDR1, CDR2, and CDR3 sequence of a VHH disclosed herein under the Chothia definition, e.g., a set of CDR sequences disclosed in the "Chothia" column of Table 2. In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the Kabat definition, e.g., a set of CDR sequences disclosed in the "Kabat" column of Table 2. In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the Contact definition, e.g., a set of CDR sequences disclosed in the "Contact" column of Table 2. In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the IMGT definition, e.g., a set of CDR sequences disclosed in the "IMGT" column of Table 2.

[0145] In certain embodiments, under the AbM definition, the VHH domain that binds Siglec-8 comprises the consensus hzG2 CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively. In certain embodiments, X1 in SEQ ID NO: 48 is Q, S, or T. In certain embodiments, X2 in SEQ ID NO: 48 is T. In various embodiments, the VHH domain comprises a CDR1 sequence of SEQ ID NOs: 2, 6, 13, 18, 23, 28, 33, or 38; a CDR2 sequence of SEQ ID NOs: 3, 7, 14, 19, 24, 29, 34, or 39; and a CDR3 sequence of SEQ ID NOs: 4, 8, 15, 20, 25, 30, 35, or 40. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 13, 14, and 15. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 18, 19, and 20. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 23, 24, and 25. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 28, 29, and 30. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 33, 34, and 35. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 38, 39, and 40.

[0146] In certain embodiments, under the Chothia definition, the VHH domain that binds to Siglec-8 comprises the consensus hzG2CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively. In certain embodiments, X1 in SEQ ID NO: 151 is Q, S, or T. In certain embodiments, X2 in SEQ ID NO: 151 is T. In various embodiments, the VHH domain comprises a CDR1 sequence of SEQ ID NO: 74, 75, or 76; a CDR2 sequence of SEQ ID NO: 80 or 81; and a CDR3 sequence of SEQ ID NO: 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 74, 80, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75, 80, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 76, 80, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 76, 81, and 8.

[0147] In certain embodiments, under the Kabat definition, the VHH domain that binds to Siglec-8 comprises the consensus hzG2CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 152, 153, and 154, respectively. In certain embodiments, X1 in SEQ ID NO: 154 is Q, S, or T. In certain embodiments, X2 in SEQ ID NO: 154 is T. In various embodiments, the VHH domain comprises the CDR1 sequence of SEQ ID NOs: 85, 86, or 87; the CDR2 sequence of SEQ ID NOs: 92, 93, or 94; and the CDR3 sequence of SEQ ID NO: 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 85, 92, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 86, 92, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 87, 93, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 87, 92, and 8. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 87, 94, and 8.

[0148] In certain embodiments, under the Contact definition, the VHH domain that binds to Siglec-8 comprises the consensus hzG2CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 155, 156, and 157, respectively. In certain embodiments, X1 in SEQ ID NO: 157 is Q, S, or T. In certain embodiments, X2 in SEQ ID NO: 157 is T. In various embodiments, the VHH domain comprises a CDR1 sequence of SEQ ID NOs: 98, 99, or 100; a CDR2 sequence of SEQ ID NOs: 104 or 105; and a CDR3 sequence of SEQ ID NOs: 110. In some embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 98, 104, and 110. In some embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 99, 104, and 110. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 100, 104, and 110. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 100, 105, and 110.

[0149] In certain embodiments, under the IMGT definition, the VHH domain that binds to Siglec-8 comprises the consensus hzG2CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively. In certain embodiments, X1 in SEQ ID NO: 160 is Q, S, or T. In certain embodiments, X2 in SEQ ID NO: 160 is T. In various embodiments, the VHH domain comprises a CDR1 sequence of SEQ ID NO: 114, 115, or 116; a CDR2 sequence of SEQ ID NO: 120 or 121; and a CDR3 sequence of SEQ ID NO: 126. In some embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 114, 120, and 126. In some embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 115, 120, and 126. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 116, 120, and 126. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 116, 121, and 126.

[0150] In some embodiments, under the AbM definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 3, and 4, respectively. In some embodiments, under the AbM definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 52, 53, and 54, respectively. In some embodiments, under the AbM definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 58, 59, and 60, respectively. In some embodiments, under the AbM definition, the VHH domain comprises the CDR1 and CDR2 sequences of SEQ ID NOs: 63 and 64, respectively, and a CDR3 comprising the amino acid sequence "GAY". In various embodiments, the VHH domain is humanized.

[0151] In some embodiments, under the Chothia definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 74, 80, and 4, respectively. In some embodiments, under the Chothia definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 77, 82, and 54, respectively. In some embodiments, under the Chothia definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 78, 83, and 60, respectively. In some embodiments, under the Chothia definition, the VHH domain comprises the CDR1 and CDR2 sequences of SEQ ID NOs: 79 and 84, respectively, and a CDR3 comprising the amino acid sequence "GAY". In various embodiments, the VHH domain is humanized.

[0152] In some embodiments, under the Kabat definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 85, 91 and 4, respectively. In some embodiments, under the Kabat definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 88, 95 and 54, respectively. In some embodiments, under the Kabat definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 89, 96 and 60, respectively. In some embodiments, under the Kabat definition, the VHH domain comprises the CDR1 and CDR2 sequences of SEQ ID NOs: 90 and 97, respectively, and a CDR3 comprising the amino acid sequence "GAY". In various embodiments, the VHH domain is humanized.

[0153] In some embodiments, under the Contact definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 98, 104 and 109, respectively. In some embodiments, under the Contact definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 101, 106 and 111, respectively. In some embodiments, under the Contact definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 102, 107 and 112, respectively. In some embodiments, under the Contact definition, the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 103, 108 and 113, respectively. In various embodiments, the VHH domain is humanized.

[0154] In some embodiments, under the IMGT definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 114, 120, and 125, respectively. In some embodiments, under the IMGT definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 117, 122, and 127, respectively. In some embodiments, under the IMGT definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 118, 123, and 128, respectively. In some embodiments, under the IMGT definition, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 119, 124, and 129, respectively. In various embodiments, the VHH domain is humanized.

[0155] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VHH domain selected from SEQ ID NO: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, or 61. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VHH domain selected from SEQ ID NO: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, or 61. In some embodiments, the antibodies provided herein comprise three CDRs of a VHH domain selected from SEQ ID NO: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, or 61. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0156] In some embodiments, the CDR is a CDR that has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to a CDR provided herein. In some embodiments, CDR1 is selected from SEQ ID NO: 2, 6, 13, 18, 23, 28, 33, 38, 52, 58, 63, 74, 75, 76, 77, 78, 79, 85, 86, 87, 88, 89, 90, 98, 99, 100, 101, 102, 103, 114, 115, 116, 117, 118 or 119 having at most 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, CDR2 is selected from SEQ ID NO: 3, 7, 14, 19, 24, 29, 34, 39, 53, 58, 64, 80, 81, 82, 83, 84, 91, 92, 93, 94, 95, 96, 97, 104, 105, 106, 107, 108, 120, 121, 122, 123, or 124 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR3 is selected from SEQ ID NO:4, 8, 15, 20, 25, 30, 35, 40, 54, 60, "GAY", 109, 110, 111, 112, 113, 125, 126, 127, 128 or 129 with at most 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, such variants are derived from sequences provided herein, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from sequences provided herein, and can be isolated de novo, for example, according to methods for obtaining antibodies provided herein.

[0157] In some embodiments, the VHH domain that binds to Siglec-8 comprises a modification at Leu 11 within framework 1. For example, the VHH domain may comprise Leu 11 Glu (L11E), Leu 11 Asp (L11D), Leu 11 Arg (L11R), or Leu 11 Lys (L11K). In some embodiments, the VHH domain that binds to Siglec-8 comprises a modified C-terminal sequence, such as TVKP (SEQ ID NO: 51), TVKPG (SEQ ID NO: 52), TVKPGG (SEQ ID NO: 50), TVRP (SEQ ID NO: 53), TVRPG (SEQ ID NO: 54), TVRPGG (SEQ ID NO: 55), TVEP (SEQ ID NO: 56), TVEPG (SEQ ID NO: 57), TVEPGG (SEQ ID NO: 58), TVDP (SEQ ID NO: 59), TVDPG (SEQ ID NO: 60), or TVDPGG (SEQ ID NO: 61). In some embodiments, the modified C-terminal sequence is LVTVKPGG (SEQ ID NO: 49). In some embodiments, the VHH domain that binds to Siglec-8 comprises a modification at Leu 11 and a modified C-terminal sequence. The L11E mutation and the modified C-terminal sequence provide improved properties for the Siglec-8 binding polypeptide, such as reduced immunogenicity. See, for example, WO 2016 / 118733.

[0158] In some such embodiments, the VHH domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, 61, 130-138, 140-148, 161, 163, and 164 (e.g., an amino acid sequence herein that comprises the CDR1, CDR2, and CDR3 sequences of the VHH domain). In some embodiments, the VHH domain that binds to Siglec-8 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, 61, 130-138, 140-148, 161, 163, and 164.

[0159] In various embodiments, the Siglec-8 binding polypeptide comprises one, two, three, or four VHH domains that bind Siglec-8. In various embodiments, the Siglec-8 binding polypeptide comprises two or three VHH domains that bind Siglec-8.

[0160] In some embodiments, the Siglec-8 binding polypeptide comprises two, three or four Siglec-8 binding VHH domains, which are operably linked to each other via a peptide linker. Non-limiting examples of linkers that can be used to link VHH domains are presented in SEQ ID NO:69.

[0161] In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain described herein fused to an Fc region. In some embodiments, the Fc region comprises the sequence SEQ ID NO: 44, 45, 65, 66, 67 or 68.

[0162] In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain described herein, which is operably linked to an Fc region via a peptide linker. Non-limiting examples of linkers that can be used to connect the VHH domain and the Fc region are presented in SEQ ID NO: 70.

[0163] In some embodiments, the VHH domain that binds Siglec-8 is humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) can be used as therapeutic molecules because humanized antibodies reduce or eliminate human immune responses to non-human antibodies, which may lead to immune responses to antibody therapeutics and reduce the effectiveness of therapeutics. Typically, humanized antibodies comprise one or more variable domains, wherein CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0164] Humanized antibodies and methods of making them are summarized in, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, and further described in, e.g., Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"); Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing the "guided selection" method of FR shuffling).

[0165] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (see, e.g., Carte r et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, the FR regions of VHH are replaced by human FR regions to prepare humanized VHH. In some embodiments, certain FR residues of human FR are replaced to improve one or more properties of humanized VHH. VHH domains with such substituted residues are still referred to herein as "humanized".

[0166] Expression and production of peptides and proteins

[0167] Nucleic acid molecules comprising polynucleotides encoding Siglec-8 binding polypeptides or proteins are provided. In some embodiments, the nucleic acid molecule may also encode a guide sequence that directs the secretion of the Siglec-8 binding polypeptide or protein, which is typically cleaved so that it is not present in the secreted polypeptide or protein. The guide sequence may be a native heavy chain (or VHH) guide sequence, or may be another heterologous guide sequence.

[0168] Nucleic acid molecules can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0169] Vectors comprising nucleic acids encoding Siglec-8 binding polypeptides and proteins described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors optimized for expression of polypeptides or proteins in desired cell types, such as CHO or CHO-derived cells or NSO cells, are selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004).

[0170] In some embodiments, Siglec-8 binding polypeptides or proteins can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides and proteins include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; cells (Crucell); and NSO cells. In some embodiments, Siglec-8 binding polypeptides or proteins can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform a desired post-translational modification on a polypeptide or protein. For example, in some embodiments, the sialylation level of a polypeptide or protein produced by CHO cells is higher than that of the same polypeptide or protein produced in HEK 293 cells.

[0171] One or more nucleic acids (such as vectors) can be introduced into a desired host cell by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into a desired host cell according to any suitable method.

[0172] A host cell comprising any of the nucleic acids or vectors described herein is also provided. In some embodiments, a host cell expressing the Siglec-8 binding polypeptide or protein described herein is provided. The Siglec-8 binding polypeptide or protein expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and an agent that binds to the Fc region. For example, protein A, protein G, protein A / G, or an antibody affinity column can be used to bind to the Fc region and purify the Siglec-8 binding polypeptide or protein containing the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be applicable to the purification of some polypeptides or proteins, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be applicable to the purification of some polypeptides or proteins, such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be applicable to the purification of some polypeptides or proteins, such as antibodies. Many methods for purifying polypeptides or proteins are known in the art.

[0173] In some embodiments, the Siglec-8 binding polypeptide or protein is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, e.g., Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0174] In some embodiments, a Siglec-8 binding polypeptide or protein prepared by the method described above is provided. In some embodiments, the Siglec-8 binding polypeptide or protein is prepared in a host cell. In some embodiments, the Siglec-8 binding polypeptide or protein is prepared in a cell-free system. In some embodiments, the Siglec-8 binding polypeptide or protein is purified. In some embodiments, a cell culture medium containing the Siglec-8 binding polypeptide or protein is provided.

[0175] In some embodiments, a composition comprising an antibody prepared by the methods described above is provided. In some embodiments, the composition comprises a Siglec-8 binding polypeptide or protein prepared in a host cell. In some embodiments, the composition comprises a Siglec-8 binding polypeptide or protein prepared in a cell-free system. In some embodiments, the composition comprises a purified Siglec-8 binding polypeptide or protein.

[0176] Exemplary Methods of Treating Disease Using Siglec-8 Binding Polypeptides or Proteins

[0177] In some embodiments, a method for treating a disease in an individual is provided, comprising administering a Siglec-8 binding polypeptide or protein. In some embodiments, a method for treating a disease associated with Siglec-8 (e.g., a disease associated with increased expression of Siglec-8 on certain cells relative to normal reference cells of the same type and / or the same tissue). Such diseases include eosinophilic disorders, including eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorders, eosinophilic gastritis (EoG), eosinophilic enteritis, Chag-Strauss syndrome, hypereosinophilic syndrome (HES) (e.g., familial HES, idiopathic HES, primary (neoplastic) HES, secondary (reactive) HES, or lymphoid variants of HES), Gleich Syndrome, Eosinophilia myalgia syndrome (Eosinophilia myalgia syndrome, EMS), IgG4-related disease (IgG4-RD), eosinophilic leukemia (e.g., chronic eosinophilic leukemia), asthma with eosinophilic phenotype, allergic bronchopulmonary aspergillosis (ABPA), chronic sinusitis with nasal polyps (CRSwNP) or eosinophilic granulomatosis with polyangiitis (EGPA). Additional exemplary eosinophilic disorders and related syndromes are described in Valent et al., Allergy (2023) 78 (1): 47-59. Such diseases also include mast cell disorders, including systemic mastocytosis, hereditary alpha trypsinemia (HAT), mast cell activation syndrome (MCAS) or general allergic disorders. Systemic mastocytosis (SM) can be advanced SM (e.g., mast cell leukemia, aggressive SM or SM with associated hematological tumors (SM-AHN)) or non-advanced SM (e.g., inert SM). In certain embodiments, the subject with SM comprises a D816V mutation in c-KIT. In certain embodiments, increased expression of CD25 and / or CD2 is observed on mast cells of subjects with SM. General allergic disorders can be induced by allergies, such as food allergies (e.g., peanut, sesame, milk, egg, fish or tree nut allergies), environmental allergies (e.g., dust mites, pollen, mold or latex allergies), allergies to companion animals (e.g., cats, dogs, birds, horses or cattle allergies) and venom allergies (e.g., bees, wasps or spiders allergies). In some embodiments, a method for treating an allergic disorder is provided. The method comprises administering to an individual an effective amount of a Siglec-8 binding polypeptide or protein provided herein. In some embodiments, the Siglec-8 binding polypeptide or protein induces eosinophil cell death in the presence of IL-5.In some embodiments, the Siglec-8 binding polypeptide or protein inhibits the activation of mast cells. In some embodiments, the Siglec-8 binding polypeptide or protein inhibits an inflammatory response.

[0178] In some embodiments, a Siglec-8 binding polypeptide or protein is linked to an additional agent, such as an additional therapeutic agent, to form an immunoconjugate.

[0179] In some embodiments, the methods of treatment can be performed in humans or animals. In some embodiments, methods of treating humans are provided.

[0180] Siglec-8 binding polypeptides and proteins can be administered to the subject as needed. The frequency of administration can be determined by a person skilled in the art, such as an attending physician, based on considerations of the disease being treated, the age of the subject being treated, the severity of the disease being treated, the general health of the subject being treated, and the like. In some embodiments, one or more effective doses of Siglec-8 binding polypeptides or proteins are administered to the subject. In some embodiments, an effective dose of Siglec-8 binding polypeptides or proteins is administered to the subject daily, semi-weekly, weekly, biweekly, monthly, and the like. An effective dose of Siglec-8 binding polypeptides or proteins is administered to the subject at least once. In some embodiments, multiple effective doses of Siglec-8 binding polypeptides or proteins are administered.

[0181] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat a disease associated with Siglec-8. The therapeutically effective amount generally depends on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the disorder to be treated, or the age of the subject being treated.

[0182] In some embodiments, the Siglec-8 binding polypeptide or protein can be administered in vivo by various routes, including but not limited to intravenous, intraarterial, parenteral, intraperitoneal or subcutaneous. Appropriate formulations and administration routes can be selected according to the intended application.

[0183] Pharmaceutical composition

[0184] In some embodiments, the composition comprising the Siglec-8 binding polypeptide or protein is provided in the form of a formulation with a variety of pharmaceutically acceptable carriers (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers can be used, including vehicles, adjuvants, and diluents. In addition, various pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc. can also be used.

[0185] Combination therapy

[0186] The Siglec-8 binding polypeptides and proteins of the present disclosure can be administered alone or in combination with other treatment modalities (such as additional therapeutic agents). The Siglec-8 binding polypeptides or proteins can be provided before, substantially simultaneously with, or after (i.e., concurrently or sequentially) one or more other treatment modalities.

[0187] Non-limiting exemplary diagnostic methods

[0188] In some embodiments, the methods described herein can be used to evaluate a subject and / or a sample from a subject. In some embodiments, the evaluation is one or more of a diagnosis, a prognosis, and / or a response to treatment.

[0189] In some embodiments, the methods described herein include assessing the presence, absence, or level of proteins or other molecules, such as Siglec-8, histamine, beta-tryptase, or other allergic mediators. In some embodiments, the methods described herein include assessing the presence, absence, or level of nucleic acid expression. The compositions described herein can be used for these measurements. For example, in some embodiments, the methods described herein include contacting a sample, serum, or cells from a sample with a therapeutic agent as described herein.

[0190] Pill Box

[0191] Also provided are articles of manufacture and kits comprising any of the Siglec-8 binding polypeptides and proteins described herein, and suitable packaging. In some embodiments, the invention includes a kit having (i) a Siglec-8 binding polypeptide or protein, and (ii) instructions for administering the Siglec-8 binding polypeptide or protein to an individual using the kit.

[0192] Suitable packaging for compositions, such as pharmaceutical compositions, comprising polypeptides or proteins described herein are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, cans, flexible packages (e.g., sealed Mylar or plastic bags), etc. These articles may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in single or multiple unit doses, and may also be further sterilized and sealed. The instructions supplied in the kits of the present invention are typically written instructions on a label or drug insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic storage disk or optical storage disk) are also acceptable. Instructions related to the use of the antibody typically include information about the dosage, dosing regimen, and route of administration for the intended treatment or industrial use.

[0193] The container may be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. For example, a kit may also be provided that contains a sufficient dose of a polypeptide or protein disclosed herein to provide effective treatment for an individual over an extended period of time. The kit may also include multiple unit doses of a polypeptide or protein and instructions for use, and the number of packages may be sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy and a compounding pharmacy).

[0194] Example

[0195] The examples discussed below are intended to be examples of the present invention only and should not be considered to limit the present invention in any way. The embodiments are not intended to represent that the following experiments are all experiments performed or are the only experiments performed. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., amount, temperature, etc.), but some experimental errors and deviations should be allowed. Unless otherwise stated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0196] Example 1: Development of a single domain antibody that specifically binds to human Siglec-8

[0197] Siglec-8 is an inhibitory receptor expressed on eosinophils, mast cells, and basophils. Single domain antibodies targeting human Siglec8 were generated by immunizing llamas and alpacas with a recombinant version of the human Siglec-8 extracellular domain (ECD: amino acids 17-363 of human Siglec-8).

[0198] Following the development of specific anti-Siglec-8 antibody titers, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from immunized animals and total mRNA was isolated using the Qiagen RNeasy Maxi kit and subsequently converted to first-strand cDNA using Thermo Superscript IV reverse transcriptase and oligo-dT priming. Single domain antibody (sdAb) sequences were specifically amplified via PCR using the cDNA as a template and cloned into the phage display vector pADL.

[0199] Phage libraries displaying llama-derived sdAbs (3.7 × 10 10 Diversity). DNA from the enriched Siglec8 phage library was transformed into BL21 competent cells, which were plated and separated into 96-well blocks. IPTG-induced medium allowed sdAb to be secreted into the supernatant, which was applied to Siglec-8 positive and negative cells. Cells were washed, treated with fluorophore-labeled anti-Myc secondary antibodies, and analyzed by 96-well flow cytometer.

[0200] The nucleic acid sequence encoding the sdAb that binds to Siglec-8 positive cells but not to Siglec-8 negative cells was cloned into a mammalian expression vector together with the human Fc coding region and expressed by transient transfection using polyethyleneimine in HEK293 freestyle cells (293F cells) or CHO cells. The supernatant was collected after 3-7 days, the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated by the absorbance at 280 nm and the extinction coefficient.

[0201] Anti-human Siglec-8 VHH, B12, D1, E2 and G2 as bivalent VHH-hIgG1-xELL-Fc (VHH of SEQ ID NO: 49, 55, 61 and 1, which are connected to Fc xELL via linker 2, respectively, and these constructs are also named cx8982, cx8983, cx8984 and cx10382, respectively) were determined by flow cytometry using CHO cells transiently transfected with full-length human Siglec-8. Binding specificity was assessed using untransfected (UT) CHO cells as a control. For binding assays, 50,000 Siglec-8 CHO or UT CHO were seeded in 96-well plates, and titrated anti-Siglec8-VHH-xELL was added in FACS buffer (PBS 1% BSA, 0.1% NaN3 pH7.4) and incubated at 4°C for 30 minutes. After washing, the plates were incubated with A647-conjugated anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch) diluted 1:2000 in FACS buffer for 30 minutes at 4° C. The plates were washed and binding was determined by flow cytometry using Intellicyt iQue Plus to quantify anti-human A647 mean fluorescence intensity.

[0202] The binding of each anti-human Siglec-8 VHH-xELL Fc construct is shown in Figures 1A to 1D Anti-Siglec8-B12-xELL( Figure 1A ) specifically binds to CHO cells expressing human Siglec-8 with a calculated affinity of 0.367 nM, and anti-Siglec8-D1-xELL ( Figure 1B ) specifically binds to CHO cells expressing human Siglec-8 with a calculated affinity of 0.427 nM, and anti-Siglec8-E2-xELL ( Figure 1C ) specifically binds to CHO cells expressing human Siglec-8 with a calculated affinity of 0.331 nM, and anti-Siglec8-G2-xELL ( Figure 1D ) specifically binds to CHO cells expressing human Siglec-8 with a calculated affinity of 0.268 nM. Binding is specific for Siglec-8, as there is no detectable binding to untransfected CHO cells for any of the constructs tested.

[0203] Example 2: Binding of multivalent anti-Siglec8-Fc antibodies to cells expressing human Siglec-8

[0204] To determine whether increasing the valency of Siglec-8 antibodies could enhance agonist activity, parental llama B12, D1, and E2 VHH sequences were used to generate tetravalent (Tet-B12 (cx8467; SEQ ID NO:50 linked to Fc), Tet-D1 (cx8469; SEQ ID NO:56 linked to Fc), and Tet-E2 (cx8463; SEQ ID NO:62 linked to Fc)) and hexavalent (Hex-B12 (cx8466; SEQ ID NO:51 linked to Fc), Hex-D1 (cx8523; SEQ ID NO:57 linked to Fc), and Hex-E2 (cx8522; SEQ ID NO:165 linked to Fc)) Siglec8-VHH-Fc antibodies, which were linked to the xELL human Fc region with reduced effector function via Linker 2. The expression of bivalent (Bi-B12 (cx8982), Bi-D1 (cx8983), and Bi-E2 (cx8984)), tetravalent, and hexavalent Siglec8-VHH-Fc antibodies in eosinophils (CD16 - CCR3 + ) binding. Cells were separated from whole blood by centrifugation, washed twice with 2% FBS PBS, and resuspended in 1×RBC lysis buffer at room temperature (RT) for 10 minutes, washed once in 2% FBS PBS and treated with the second RBC lysis step, and washed twice with 2% FBS. Cells were inoculated at 200k / well and resuspended in human FC blocker at RT for 10 minutes. The cell spin was slowed down, then resuspended in the titrated test article starting with 200nM 1:4, and incubated at 4°C for 30 minutes, then washed twice and resuspended in a secondary mixture (2*; anti-CCR3 FITC, anti-human AF647, PI, anti-CD16 BV605), and incubated at 4°C for 30 minutes, washed twice, and the bound antibodies were detected via flow cytometry (Novocyte).

[0205] like FIG. 2A to FIG. 2C As shown in Figure , the bivalent, tetravalent and hexavalent Siglec8-VHH-Fc antibody B12 ( Figure 2A )、D1( Figure 2B ) and E2( Figure 2C ) binds to eosinophils. The calculated affinities are similar between bivalent, tetravalent, and hexavalent hzSiglec8-VHH-Fc antibodies. As expected, the observed Bmax decreases with increasing valence, because the Fc portion of the antibody is detected in the assay, and the ratio of the Fc region to the anti-Siglec-8 binding domain is inversely proportional to the valence. In other granulocyte populations (CD16 + CCR3- ) No binding was observed.

[0206] In other studies, the parental llama G2 sequence was humanized and the multivalent hzSiglec8-G2v14-Fc antibody was cloned to have a human Fc with reduced effector function xELL. Binding of the bivalent (cx11422; SEQ ID NO: 5 linked to Fc xELL via linker 2), tetravalent (cx11580; SEQ ID NO: 9 linked to Fc xELL via linker 2) and hexavalent (cx11579; SEQ ID NO: 10 linked to FcxELL via linker 2) hzSiglec8-G2v14-Fc antibodies was evaluated by flow cytometry using CHO cells transfected with human Siglec-8. A titration series of the fusion proteins were incubated with CHO cells expressing Siglec8 (5×10 4 Cells / well) were incubated together in a 96-well plate in FACS buffer (PBS 1% BSA, 0.1% NaN3 pH 7.4) for 30 minutes. After washing once with 150 μL of FACS buffer, APC-conjugated anti-human Fcγ-specific secondary antibody (Jackson ImmunoResearch) was added and the cells were incubated at 4°C for 30 minutes. After washing once more in FACS buffer, bound antibodies were detected by flow cytometry (iQue Intellicyte).

[0207] like Figure 2D As shown in, bivalent, tetravalent, and hexavalent hzSiglec8-G2v14-Fc antibodies bind to CHO cells expressing human Siglec-8. The calculated affinities are similar between bivalent, tetravalent, and hexavalent hzSiglec8-G2v14-Fc antibodies. As expected, the observed Bmax decreases with increasing valence because the Fc portion of the antibody is detected in the assay, and the ratio of the Fc region to the anti-Siglec-8 binding domain is inversely proportional to the valence.

[0208] Example 3: Increased valency of anti-Siglec-8 antibodies leads to enhanced eosinophil killing activity

[0209] Siglec-8 agonists promote direct killing of eosinophils in the presence of IL-5. To evaluate the effect of valence on Siglec-8-mediated killing activity, StemCell EasySep TMThe Direct Human Eosinophil Isolation Kit isolates eosinophils from whole blood of healthy donors and treats them with bivalent (Bi-B12 (cx8982), Bi-D1 (cx8983), and Bi-E2 (cx8984)), tetravalent (Tet-B12 (cx8467), Tet-D1 (cx8469), and Tet-E2 (cx8463)), and hexavalent (Hex-B12 (cx8466), Hex-D1 (cx8523), and Hex-E2 (cx8522)) Siglec8-VHH-Fc antibodies. Antibodies were titrated across the plate starting at 50 nM (3 point titration, 1:10 dilution), isolated eosinophils were resuspended to 160k / mL in 10% FBS RPMI + Cytotox red and added to 48-well plates at 80k / well in the presence of IL-5. The Cytotox Red incorporation was monitored over time by the Cytoanalyzer, and the total red object integrated intensity (RCU×μm 2 / image). For cells treated with 5 nM of each test antibody, the killing observed at 24 hours is plotted on FIG. 3A to FIG. 3C Increasing the valency from divalent to tetravalent resulted in increased eosinophil killing potency for all Siglec8-VHH-Fc antibodies tested. Further increasing the valency to hexavalent resulted in similar or slightly increased potency relative to tetravalent valency.

[0210] In other studies, isolated eosinophils were added to 96-well plates at 30,000 cells / well and treated with bivalent (cx11422), tetravalent (cx11580) or hexavalent (cx11579) hzSiglec8-G2v14-Fc antibodies and incubated with the test article titrated across the plate starting at 10 nM in the presence of IL-5. The killing activity was compared to an in-house derived defucosylated analog of AK002 (lirentelimab) titrated in the same manner. To monitor eosinophil killing, Cytotox Red was added to each well at a final concentration of 250 nM. Using The Cytotox Red incorporation was monitored over time by the Cytoanalyzer, and the total red object integrated intensity (RCU×μm 2 / image).

[0211] like Figure 3DAs shown in, the bivalent hzSiglec8-G2v14-Fc (cx11422) more effectively enhanced the killing of eosinophils in the presence of IL-5 than the defucosylated AK002 (lilentinib) analog antibody. Increasing the valency of hzSiglec8-G2v14-Fc to tetravalent (cx11580) resulted in a significant increase in eosinophil killing potency, resulting in a nearly 30-fold increase relative to the defucosylated AK002 analog antibody and a 10-fold increase compared to the bivalent hzSiglec8-G2v14-Fc. Further increasing the valency to hexavalent (cx11579) did not result in more potent eosinophil killing relative to the tetravalent hzSiglec8-G2v14-Fc antibody, but maintained superior killing activity compared to the defucosylated AK002 analog and the bivalent hzSiglec8-G2v14-Fc. No killing was observed in the absence of IL-5 ( Figure 3E ).

[0212] Example 4: G2 VHH sequence optimization

[0213] The anti-Siglec8 G2 VHH sequence hzG2v14 was further modified to minimize binding to siglec-2 (CD22) and other siglec proteins (see below), reduce potential immunogenicity and optimize manufacturability. Humanized tetravalent Siglec-8 agonist antibodies comprising hzG2v14 (SEQ ID NO: 9), hzG2v47 (SEQ ID NO: 12), hzG2v51 (SEQ ID NO: 17), hzG2v52 (SEQID NO: 22), hzG2v53 (SEQ ID NO: 27), hzG2v54 (SEQ ID NO: 32) or hzG2v55 (SEQ ID NO: 37) were linked to an engineered Fc SDIE region (further described in Example 5) via Linker 2. Binding to human Siglec-8 on eosinophils was assessed by flow cytometry and compared to binding of afucosylated anti-Siglec-8 2E2 (2E2 Afuc) reference antibody. TM Direct Human Eosinophil Isolation Kit Eosinophils were isolated from whole blood of healthy donors, diluted in FACS buffer, added to 96-well assay plates at 10k / well and incubated with a titration series of test article starting at 10 nM (10-12 point titration, 1:4 dilution) at 4°C for 20 minutes. After washing once with FACS buffer, Alexa Fluor 647 AffiniPure donkey anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch) was added and the cells were incubated at 4°C for 20 minutes. After washing once more in FACS buffer, bound antibodies were detected by flow cytometry (NovoCyte 3000). 高 / CCR3 + The median fluorescence intensity was calculated by Novoexpress onboard software for cell gating. The dissociation constant (Kd) values ​​for these studies are summarized in Table 3, and representative binding data for tetravalent anti-Siglec8 antibodies including hzG2v52 (cx11913) and hzG2v53 (cx11914) are shown in Figure 4A The pis of the VHH domains determined using ExPasy are shown in Table 3, with higher pis generally being preferred for manufacturing.

[0214] Table 3: Binding of humanized tetravalent Siglec-8 antibodies to eosinophils

[0215] name Kd(nM)* pI hzG2v14 0.00812 6.66 hzG2v47 0.00888 5.39 hzG2v51 0.119 6.67 hzG2v52 0.012 5.42 hzG2v53 0.013 6.66 hzG2v54 0.036 6.59 hzG2v55 0.010 6.58 2E2 Afuc 0.112

[0216] *Determined based on 1-3 experiments

[0217] like Figure 4A As shown in Figure 2, the tetravalent anti-Siglec8 antibodies CX11913 and CX11914 bound to CCR3 in a titration-dependent manner. + Eosinophil population binding. Both cx11913 and cx11914 bound with approximately 8-fold higher affinity than the 2E2 Afuc comparator antibody. As expected, due to fewer available Fc receptors for binding to the tetravalent antibody, the Bmax of both cx11913 and cx11914 was reduced compared to 2E2 Afuc.

[0218] This data indicates that further modified humanized tetravalent anti-Siglec-8 antibodies comprising hzG2v47, hzG2v52, hzG2v53, hzG2v54, and hzG2v55 bind with high affinity to the target human eosinophil population. In an ELISA assay, tetravalent anti-Siglec8 antibodies comprising hzG2v47 exhibited some binding to recombinant Siglec-2 (CD22). The relative cross-reactivity with Siglec-2 was:

[0219] hzG2v14>hzG2v47≈hzG2v54>hzG2v52≈hzG2v53≈hzG2v55.

[0220] The binding of tetravalent Siglec-8 agonist antibodies cx11913 and cx11914, cx11769 (hzG2v14-Fc SDIE), and 2E2 Afuc to a panel of human siglec proteins transiently expressed on CHO cells was also evaluated by flow cytometry. CHO cells were transiently transfected (by PEI transfection in BalanCDTransfectory CHO cell culture medium (Irvine Scientific)) with a panel of plasmids encoding the full-length extracellular domains of human Siglec-2, 3, 6, 7, 8, 9, 12, or 15 (linked to a transmembrane protein for cell surface expression, with tartrazine or GFP co-expressed in the same vector). The next day, a titration series of the test article starting at 1 μM (12 points, 1:3 dilution) was incubated with Siglec-expressing cell lines (50k cells / well) in FACS buffer (PBS 1% BSA, 0.1% NaN3 pH 7.4) in a 96-well plate at 4°C for 20 minutes. After washing once with FACS buffer, Alexa Fluor was added. 647AffiniPure donkey anti-human Fcγ specific secondary antibody (Jackson ImmunoResearch), and the cells were incubated at 4°C for 20 minutes. After washing once more in FACS buffer, bound antibodies were detected via flow cytometry (iQue Intellicyte). The mean fluorescence intensity was calculated using iQue onboard software gating on GFP+ or tartrazine+ cells.

[0221] like FIG. 4B to FIG. 4C As shown in , the tetravalent anti-human Siglec-8 antibodies cx11913 (containing hzG2v52) and cx11914 (hzG2v53) bind to Siglec-8 with high affinity ( Figure 4C , upper left), but not other siglec proteins. At the highest tested concentration of 1 μM, cx11913 bound to Siglec-12 ( Figure 4C , lower left), but showed no binding at any other concentration. Both constructs exhibited similar siglec-specific characteristics to the 2E2 Afuc comparator antibody. In contrast, at some of the higher tested concentrations, the anti-human Siglec-8 antibody cx11769 (containing hzG2v14) exhibited cross-reactivity with Siglec-2 as well as several other siglec proteins ( FIG. 4B to FIG. 4C ).

[0222] Example 5: Evaluation of additional humanized G2 VHH constructs

[0223] The llama anti-Siglec8-G2 VHH domain (lmG2) was humanized to generate additional variants, namely Siglec8 hzG2v11, Siglec8 hzG2v12, Siglec8 hzG2v13, Siglec8 hzG2v15, Siglec8 hzG2v16, Siglec8 hzG2v17, Siglec8hzG2v18, Siglec8 hzG2v19, Siglec8 hzG2v20, Siglec8 hzG2v21, Siglec8 hzG2v22, Siglec8 hzG2v24, Siglec8 hzG2v25, Siglec8 hzG2v26, Siglec8 hzG2v27, Siglec8 hzG2v28, Siglec8 hzG2v29, Siglec8 hzG2v30, Siglec8 hzG2v31, Siglec8 hzG2v32, Siglec8 hzG2v33, Siglec8 hzG2v34, Siglec8 hzG2v35, Siglec8 hzG2v36, Siglec8 hzG2v37, hzG2v28, Siglec8hzG2v29, Siglec8 hzG2v30, Siglec8 hzG2v31, Siglec8 hzG2v32, Siglec8 hzG2v33, Siglec8 hzG2v2.1, Siglec8 hzG2v5.1, and Siglec8 hzG2v6.1. The substitution of asparagine (N) at position 2 or serine (S) at position 3 of AbM CDR3 in hzG2v11-hzG2v14 was designed to eliminate potential deamidation sites.

[0224] The binding of lmG2 and humanized versions (further including Siglec8 hzG2v14) formatted as monomeric VHH-hIgG1 fusion proteins using non-dimeric human IgG1 Fc variant regions lacking hinge Fc NNTs was evaluated by flow cytometry using 293 cells that had been transiently transfected with full-length human Siglec-8 and detected with anti-human AF647 secondary antibodies. Untransfected 293 cells were used as controls to evaluate binding specificity. For binding assays, Siglec-8 293 cells or untransformed 293 cells were seeded at 50k cells / well in FACS buffer in 96-well U-bottom plates and incubated with titrated anti-Siglec8-VHH-NNT at 4°C for 30 minutes. After washing, the plate was incubated at 4°C for 20 minutes with an A647-conjugated anti-human Fcγ-specific secondary antibody diluted 1:10,000 in FACS buffer. The plates were washed and binding was determined by flow cytometry quantification of anti-human A647 median fluorescence intensity. Maximum specific binding (Bmax) and affinity (Kd) were determined from flow cytometry binding data using a nonlinear fitting model. The binding curves for each plate are presented in FIG. 5A to FIG. 5H middle.

[0225] The humanized version of G2 has good expression and FIG. 5A to FIG. 5D As shown in Figure , almost all variants, except hzG2v19-hzG2v21 and Siglec8 hzG2v5.1, bound Siglec-8 with similar affinity as the parental clone. No binding was observed on untransfected cells ( FIG. 5E to FIG. 5H ).

[0226] Example 6: Enhanced antibody-dependent cellular cytotoxicity (ADCC) activity of anti-Siglec-8 antibodies leads to enhanced eosinophil killing activity

[0227] As described above, bivalent, tetravalent and hexavalent anti-Siglec-8 antibodies can directly kill eosinophils in the presence of inflammatory mediators such as IL-5, with tetravalent and hexavalent antibodies showing higher relative potency than bivalent antibodies. However, in the absence of IL-5, the killing activity is abolished. In order to evaluate the effect of the Fc region on Siglec-8-mediated killing activity, a tetravalent anti-Siglec8-VHH-Fc antibody was generated, which contained humanized G2 VHH, hzG2v53 and wild-type human IgG1 Fc region (hIgG1) (cx12532; SEQ ID NO: 27 (see SEQ ID NO: 72) attached to Fc) or an engineered IgG1 Fc region with mutations at two residues S239D and I332E (SDIE) (cx11914; SEQ ID NO: 27 (see, SEQ ID NO: 71) attached to Fc) to improve CD16 binding and subsequent antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In addition, a bivalent anti-Siglec8-VHH-Fc antibody comprising hzG2v53 and a SDIE Fc region (cx12562; SEQ ID NO: 26 (see SEQ ID NO: 73) linked to Fc) was generated. A tetravalent anti-Siglec8-VHH-Fc antibody based on hzG2v53 was evaluated in a direct eosinophil killing assay in the presence of IL-5. Using StemCell EasySep TM Direct Human Eosinophil Isolation Kit Eosinophils were isolated from whole blood of healthy donors and added at 30,000 cells / well in 10% FBS RPMI in the presence of IL-5 (final 10 ng / mL) and incubated with a titration series (11 point titration, 1:4 dilution) of the test antibody starting at 100 nM. Killing activity was compared with an in-house derived defucosylated AK002 (lilentinib) analog titrated in the same manner. To monitor eosinophil killing, Cytotox Red was added to each well at a final concentration of 250 nM. Use The Cytotox Red incorporation was monitored over time by the Cytoanalyzer, and the total red object integrated intensity (RCU×μm 2 / image).

[0228] like Fig. 6A As shown in , treatment with a humanized tetravalent anti-Siglec-8 antibody construct exhibited the highest efficacy in the presence of IL-5, demonstrating that Fc modification did not significantly affect direct eosinophil killing activity in the presence of IL-5.

[0229] The ability of hzG2v53-based tetravalent and bivalent anti-Siglec8-VHH-Fc antibodies to promote ADCC of naive human eosinophils in the absence of IL-5 was also evaluated along with defucosylated AK002 (lilentinib) analogs. Eosinophils were isolated as described above and assayed using EasySep TM Human NK cell enrichment kit is separated from the enriched PBMCs by autologous NK cells. Antibodies are titrated in 96-well plates (11-point titration, 1:4 dilution). Separated NK cells (50,000 cells / well) and eosinophils (10,000 cells / well) are seeded (5:1 effector: target ratio) in 100 μL 10% FBS RPMI+IL-2 (final 2ng / mL) for NK cell stimulation without IL-5. The next day, cells were washed and apoptotic target cells were labeled using ApotrackerGreen (Biolegend) for 20 minutes. Next, cells were washed, resuspended in FACS buffer, and analyzed on NovoCyte 3000 flow cytometer (ACEA Biosciences, Inc.).

[0230] like Figure 6B As shown in , treatment with tet-hzG2v53-SDIE resulted in a dose-dependent increase in eosinophil killing, even in the absence of IL-5. The optimal range of ADCC activity was between 1-100 pM, with a gradual decline at higher concentrations. SDIE Fc modification significantly increased ADCC activity compared to tet-hzG2v53-IgG1 with a wild-type effector-enabled Fc. Although the tet-hzG2v53-SDIE antibody showed a slight decrease in maximum ADCC killing activity compared to the bi-hzG2v53-SDIE and 2E2 Afuc antibodies, the tetravalent format showed higher relative potency in the direct killing assay as shown above.

[0231] Without departing from the spirit or essential features of the present disclosure, the present disclosure may be embodied in other specific forms. Therefore, the aforementioned embodiments are considered to be illustrative in all respects, rather than limiting the present disclosure. Therefore, the scope of the present disclosure is indicated by the appended claims rather than the aforementioned embodiments, and therefore all changes falling within the meaning and scope of the equivalents of the claims are intended to be included herein.

[0232] Table of some sequences

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

Claims

1. A VHH domain that binds to Siglec-8, comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3) sequence of a VHH, wherein the VHH comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163 and 164.

2. A VHH domain that binds to Siglec-8, comprising the CDR1, CDR2 and CDR3 sequences described below: a) SEQ ID NOs: 46, 47 and 48, respectively, as defined according to the AbM numbering system; b) SEQ ID NOs: 149, 150 and 151, respectively, as defined according to the Chothia numbering system; c) SEQ ID NOs: 152, 153 and 154, respectively, as defined according to the Kabat numbering system; d) SEQ ID NOs: 155, 156 and 157, respectively, as defined according to the Contact numbering system; or e) SEQ ID NOs: 158, 159 and 160, respectively, as defined according to the IMGT numbering system.

3. The VHH domain of claim 2, wherein: a) the CDR1 comprises the amino acid sequence of SEQ ID NO: 18, 2 or 13; the CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or 3; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 8, as defined according to the AbM numbering system; b) the CDR1 comprises the amino acid sequence of SEQ ID NO:74, 75 or 76; the CDR2 comprises the amino acid sequence of SEQ ID NO:80 or 81; and the CDR3 comprises the amino acid sequence of SEQ ID NO:8, as defined according to the Chothia numbering system; c) the CDR1 comprises the amino acid sequence of SEQ ID NO:85, 86 or 87; the CDR2 comprises the amino acid sequence of SEQ ID NO:92, 93 or 94; and the CDR3 comprises the amino acid sequence of SEQ ID NO:8, as defined according to the Kabat numbering system; d) the CDR1 comprises the amino acid sequence of SEQ ID NO:98, 99 or 100; the CDR2 comprises the amino acid sequence of SEQ ID NO:104 or 105; and the CDR3 comprises the amino acid sequence of SEQ ID NO:110, as defined according to the Contact numbering system; or e) the CDR1 comprises the amino acid sequence of SEQ ID NO: 114, 115 or 116; the CDR2 comprises the amino acid sequence of SEQ ID NO: 120 or 121; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 126, as defined according to the IMGT numbering system.

4. The VHH domain of any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NO: 18, 29 and 8; 2, 3 and 8; 13, 3 and 8; or 18, 3 and 8, respectively, as defined according to the AbM numbering system.

5. The VHH domain of any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NO: 74, 80 and 8; 75, 80 and 8; 76, 80 and 8; or 76, 81 and 8, respectively, as defined according to the Chothia numbering system.

6. The VHH domain of any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NO: 85, 92 and 8; 86, 92 and 8; 87, 93 and 8; 87, 92 and 8; or 87, 94 and 8, respectively, as defined according to the Kabat numbering system.

7. The VHH domain of any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NO: 98, 104 and 110; 99, 104 and 110; 100, 104 and 110; or 100, 105 and 110, respectively, as defined according to the Contact numbering system.

8. The VHH domain of any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are set forth in SEQ ID NOs: 114, 120 and 126; 115, 120 and 126; 116, 120 and 126; or 116, 121 and 126, respectively, as defined according to the IMGT numbering system.

9. The VHH domain of any one of claims 1 to 8, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163 and 164.

10. The VHH domain of any one of claims 1 to 9, wherein the VHH domain is humanized.

11. A VHH domain that binds to Siglec-8, comprising CDR1, CDR2 and CDR3 sequences of VHH, wherein the VHH comprises the amino acid sequence SEQ ID NO:

1.

12. The VHH domain of claim 11, wherein the CDR1, CDR2 and CDR3 comprise the amino acid sequence as set forth below: a) SEQ ID NOs: 2, 3 and 4, respectively, as defined according to the AbM numbering system; b) SEQ ID NOs: 74, 80 and 4, respectively, as defined according to the Chothia numbering system; c) SEQ ID NOs: 85, 91 and 4, respectively, as defined according to the Kabat numbering system; d) SEQ ID NO: 98, 104 and 109, respectively, as defined according to the Contact numbering system; or e) SEQ ID NOs: 114, 120 and 125, respectively, as defined according to the IMGT numbering system.

13. The VHH domain of claim 11 or 12, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

1.

14. A VHH domain that binds to Siglec-8, comprising CDR1, CDR2 and CDR3 sequences of VHH, wherein the VHH comprises the amino acid sequence of SEQ ID NO:

49.

15. The VHH domain of claim 14, wherein the CDR1, CDR2 and CDR3 comprise the amino acid sequence as set forth below: a) SEQ ID NO: 52, 53, 54, respectively, as defined according to the AbM numbering system; b) SEQ ID NO: 77, 82, 54, respectively, as defined according to the Chothia numbering system; c) SEQ ID NO: 88, 95, 54, respectively, as defined according to the Kabat numbering system; d) SEQ ID NO: 101, 106, 111, respectively, as defined according to the Contact numbering system; or e) SEQ ID NO: 117, 122, 127, respectively, as defined according to the IMGT numbering system.

16. The VHH domain of claim 14 or 15, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

49.

17. A VHH domain that binds to Siglec-8, comprising CDR1, CDR2 and CDR3 sequences of VHH, wherein the VHH comprises the amino acid sequence of SEQ ID NO:

55.

18. The VHH domain of claim 17, wherein the CDR1, CDR2 and CDR3 comprise the amino acid sequence set forth below: a) SEQ ID NO: 58, 59, 60, respectively, as defined according to the AbM numbering system; b) SEQ ID NO: 78, 83, 60, respectively, as defined according to the Chothia numbering system; c) SEQ ID NO: 89, 96, 60, respectively, as defined according to the Kabat numbering system; d) SEQ ID NO: 102, 107, 112, respectively, as defined according to the Contact numbering system; or e) SEQ ID NO: 118, 123, 128, respectively, as defined according to the IMGT numbering system.

19. The VHH domain of claim 17 or 18, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

55.

20. A VHH domain that binds to Siglec-8, comprising CDR1, CDR2 and CDR3 sequences of VHH, wherein the VHH comprises the amino acid sequence of SEQ ID NO:

61.

21. The VHH domain of claim 20, wherein the CDR1, CDR2 and CDR3 comprise: a) the amino acid sequence SEQ ID NO: 63, the amino acid sequence SEQ ID NO: 64 and the amino acid sequence Gly-Ala-Tyr (GAY), respectively, as defined according to the AbM numbering system; b) the amino acid sequence SEQ ID NO: 79, the amino acid sequence SEQ ID NO: 84 and the amino acid sequence Gly-Ala-Tyr (GAY), respectively, as defined according to the Chothia numbering system; c) the amino acid sequence SEQ ID NO: 90, the amino acid sequence SEQ ID NO: 97 and the amino acid sequence Gly-Ala-Tyr (GAY), respectively, as defined according to the Kabat numbering system; d) the amino acid sequences of SEQ ID NO: 103, 108, 113, respectively, as defined according to the Contact numbering system; or e) the amino acid sequences SEQ ID NO: 119, 124, 129, respectively, as defined according to the IMGT numbering system.

22. The VHH domain of claim 20 or 21, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

61.

23. A polypeptide comprising the VHH domain of any one of claims 1 to 22.

24. The polypeptide of claim 23, comprising at least two VHH domains of any one of claims 1 to 22.

25. The polypeptide of claim 24, wherein the two VHH domains are operably linked to each other via a peptide linker.

26. The polypeptide of claim 25, wherein the peptide linker comprises or consists of the amino acid sequence SEQ ID NO:

69.

27. The polypeptide of any one of claims 24 to 26, wherein the VHH domains comprise identical CDR1, CDR2 and CDR3 amino acid sequences.

28. The polypeptide of claim 27, wherein the VHH domains comprise the same VHH amino acid sequence.

29. A polypeptide as described in any one of claims 24 to 28, wherein the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% identical to the amino acid sequence of SEQ ID NO:9, 10, 12, 17, 22, 27, 32, 37, 50, 51, 56, 57, 62 or 63.

30. The polypeptide of any one of claims 23 to 29, further comprising a multimerization domain.

31. A polypeptide comprising at least two antigen binding domains that bind Siglec-8 and a multimerization domain.

32. The polypeptide of claim 31, comprising two antigen binding domains that bind Siglec-8.

33. The polypeptide of claim 31, comprising three antigen binding domains that bind Siglec-8.

34. A polypeptide that binds to Siglec-8, comprising at least three antigen binding domains that bind to Siglec-8.

35. The polypeptide of any one of claims 31 to 34, wherein each of the antigen binding domains is a VHH domain.

36. The polypeptide of any one of claims 31 to 34, wherein each of the antigen binding domains comprises a heavy chain variable region and a light chain variable region.

37. The polypeptide of claim 36, wherein each antigen binding domain is a Fab or a scFv.

38. The polypeptide of any one of claims 30 to 33 and 35 to 37, wherein the multimerization domain is a dimerization domain.

39. The polypeptide of claim 38, wherein the dimerization domain is an antibody Fc region.

40. The polypeptide of claim 39, wherein the antibody Fc region is a human IgG1 Fc region.

41. The polypeptide of claim 40, wherein the antibody Fc region comprises the amino acid sequence of SEQ ID NO:44, 45, 65, 66, 67 or 68.

42. The polypeptide of any one of claims 39 to 41, wherein the antibody Fc region is operably linked to at least one of the antigen binding domains that binds Siglec-8 via an amino acid linker.

43. The polypeptide of claim 42, wherein the amino acid linker comprises or consists of the amino acid sequence SEQ ID NO:

70.

44. The polypeptide of any one of claims 39 to 43, wherein the polypeptide comprises the structure VHH-VHH-Fc.

45. The polypeptide of claim 44, comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

71.

46. ​​The polypeptide of any one of claims 39 to 43, wherein the polypeptide comprises the structure VHH-Fc-VHH.

47. The polypeptide of any one of claims 39 to 43, wherein the polypeptide comprises the structure VHH-VHH-VHH-Fc or VHH-VHH-Fc-VHH.

48. A protein comprising two or more polypeptides according to any one of claims 30 to 47 which are multimerized via the multimerization domain under physiological conditions.

49. The protein of claim 48, wherein the two or more polypeptides form a homomultimer.

50. A protein comprising two polypeptides according to any one of claims 38 to 47 which dimerize via the dimerization domain under physiological conditions.

51. The protein of claim 50, wherein the two polypeptides form a homodimer.

52. The VHH, polypeptide or protein of any one of claims 1 to 51, wherein the VHH, polypeptide or protein binds human Siglec-8.

53. The VHH, polypeptide or protein of claim 52, wherein the human Siglec-8 comprises the amino acid sequence of SEQ ID NO:

43.

54. The polypeptide or protein of any one of claims 23 to 53, wherein the polypeptide or protein mediates eosinophil killing in the presence of IL-5.

55. The polypeptide or protein of claim 54, wherein eosinophil killing is determined in an in vitro assay.

56. The protein of any one of claims 50 to 55, wherein the protein comprises at least two VHH domains that bind Siglec-8 in each polypeptide, and wherein the protein mediates eosinophil killing in the presence of IL-5 at a lower EC50 than a homodimer of polypeptides each having the structure VHH-Fc and comprising only one of the VHH domains that bind Siglec-8.

57. The polypeptide or protein of any one of claims 23 to 56, wherein the polypeptide or protein mediates eosinophil killing in the absence of IL-5.

58. The polypeptide or protein of any one of claims 23 to 57, wherein the polypeptide mediates eosinophil killing by antibody-dependent cell-mediated cytotoxicity (ADCC).

59. A pharmaceutical composition comprising the polypeptide or protein of any one of claims 23 to 58 and a pharmaceutically acceptable carrier.

60. An isolated nucleic acid encoding the polypeptide of any one of claims 23 to 47 and 52 to 58.

61. A vector comprising the nucleic acid of claim 60.

62. A host cell comprising the nucleic acid of claim 60 or the vector of claim 61.

63. A host cell expressing the polypeptide or protein of any one of claims 23 to 58.

64. A method of producing a polypeptide or protein, the method comprising incubating the host cell of claim 62 or 63 under conditions where the polypeptide or protein is expressed.

65. The method of claim 64, further comprising isolating the polypeptide or protein.

66. A method of treating an eosinophilic disorder or a mast cell disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide or protein as described in any one of claims 23 to 58 or a pharmaceutical composition as described in claim 59.

67. The method of claim 66, wherein the eosinophilic disorder is eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorder, eosinophilic gastritis, eosinophilic enteritis, Tschag-Strauss syndrome, hypereosinophilic syndrome, eosinophilic leukemia (e.g., chronic eosinophilic leukemia), asthma with eosinophilic phenotype, allergic bronchopulmonary aspergillosis, chronic sinusitis with nasal polyps, or eosinophilic granulomatosis with polyangiitis.

68. The method of claim 66, wherein the mast cell disorder is systemic mastocytosis, hereditary alpha trypsinemia, or mast cell activation syndrome.

69. The method of claim 68, wherein the systemic mastocytosis is advanced systemic mastocytosis optionally selected from the group consisting of: aggressive systemic mastocytosis, mast cell leukemia, and systemic mastocytosis with associated hematological neoplasms.

70. The method of claim 68, wherein the systemic mastocytosis is non-advanced systemic mastocytosis, optionally indolent systemic mastocytosis.

71. A method of treating an inflammatory disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide or protein as described in any one of claims 23 to 58 or a pharmaceutical composition as described in claim 59.

72. A method for treating or preventing an allergic disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of the polypeptide or protein of any one of claims 23 to 58 or the pharmaceutical composition of claim 59.

73. The method of claim 72, wherein the allergic disorder is a food allergy, an environmental allergy, a companion animal allergy, and / or a venom allergy.

74. A method of depleting eosinophils in a subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide or protein as described in any one of claims 23 to 58 or a pharmaceutical composition as described in claim 59.

75. A method of killing eosinophils, the method comprising contacting the eosinophils with natural killer (NK) cells in the presence of a polypeptide or protein as described in any one of claims 23 to 58 or a pharmaceutical composition as described in claim 59.

76. A method of killing eosinophils, the method comprising contacting the eosinophils with macrophages in the presence of a polypeptide or protein as described in any one of claims 23 to 58 or a pharmaceutical composition as described in claim 59.

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