SIGLEC-8 binding proteins and methods of use thereof

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 333,952, filed April 22, 2022, the contents of which are incorporated by reference herein in their entirety and for all purposes.

[0002] Incorporation by reference of sequence listing This application incorporates by reference a Sequence Listing submitted herewith in electronic format entitled PXS-002WO_SL.xml, created on April 19, 2023, which is 163,709 bytes in size.

[0003] Field The present invention relates to Siglec-8 binding proteins and methods of using such proteins to modulate the biological activity of Siglec-8, including, but not limited to, methods for treating eosinophilic or mast cell disorders. [Background technology]

[0004] background Mast cells are tissue-resident cells that regulate acute and chronic tissue inflammation. Abnormal accumulation and activation of mast cells are known to play a role in mediating allergic diseases such as eosinophilic asthma, atopic dermatitis, and eosinophilic gastrointestinal disease (Schanin et al. Mucosal Immunology 2021,14,366-376 (Non-Patent Document 1)). Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is an inhibitory cell surface receptor selectively expressed on these mast cells as well as mature eosinophils and basophils, and has therefore attracted attention as a target for treating allergic and inflammatory diseases. Cross-linking of Siglec-8 has been shown to induce the death of eosinophils, a process that depends on the release of reactive oxygen species (ROS) and activation of beta2-integrin (Legrand et al. J Allergy Clin Immunol 2009,143,2227-2237 (Non-Patent Document 2)). Selective expression of Siglec-8 makes it an advantageous target that may reduce side effects and increase drug safety. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Schanin et al.Mucosal Immunology 2021,14,366-376 [Non-Patent Document 2] Legrand et al.J Allergy Clin Immunol 2009,143,2227-2237 Summary of the Invention

[0006] overview Provided herein are Siglec-8 binding VHH domains, polypeptides, and proteins, and methods of using said Siglec-8 binding VHH domains, polypeptides, and proteins in medical treatment. For example, the VHH domains, polypeptides, and proteins disclosed herein are useful for treating eosinophilic disorders, including eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorders, eosinophilic gastritis (EoG), eosinophilic enteritis, Churg-Strauss syndrome, and hypereosinophilic syndrome. They are also useful for treating mast cell disorders, including, but not limited to, mast cell leukemia, invasive systemic mastocytosis, and indolent mastocytosis. In addition, they are useful for treating common allergic conditions, such as those induced 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.

[0007] In one aspect, the present disclosure provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), the VHH domain comprising VHH complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164.

[0008] In another aspect, the disclosure provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences as set forth in (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. When 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. When 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. When 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. When 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. When 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.

[0009] 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; SEQ ID NOs: 2, 3, and 8; SEQ ID NOs: 13, 3, and 8; or SEQ ID NOs: 18, 3, and 8, respectively, as defined according to the AbM numbering system; or set forth in SEQ ID NOs: 74, 80, and 8; SEQ ID NOs: 75, 80, and 8; SEQ ID NOs: 76, 80, and 8; or SEQ ID NOs: 76, 81, and 8, respectively, as defined according to the Chothia numbering system; or set forth in SEQ ID NOs: 85, 92, and 8; SEQ ID NOs: 86, 92, and 8; SEQ ID NOs: 87, 93, and 8; SEQ ID NOs: 87, 92, and 8; or SEQ ID NOs: 87, 94, and 8, or as set forth in SEQ ID NOs: 98, 104, and 110, respectively, defined according to the Contact numbering system; SEQ ID NOs: 99, 104, and 110; SEQ ID NOs: 100, 104, and 110; or SEQ ID NOs: 100, 105, and 110, respectively, or as set forth in SEQ ID NOs: 114, 120, and 126, respectively, defined according to the IMGT numbering system; SEQ ID NOs: 115, 120, and 126; SEQ ID NOs: 116, 120, and 126; or SEQ ID NOs: 116, 121, and 126.

[0010] 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 NOs: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164. In certain embodiments, the VHH domain is humanized.

[0011] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), the VHH domain comprising a VHH CDR1, CDR2, and CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in (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.

[0012] The disclosure also provides a VHH domain that binds Siglec-8 (e.g., human Siglec-8), wherein the VHH domain comprises a VHH CDR1, CDR2, and CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 49. In certain embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in (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.

[0013] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), wherein the VHH domain comprises a VHH CDR1, CDR2, and CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 55. In certain embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in (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.

[0014] The present disclosure also provides a VHH domain that binds to Siglec-8 (e.g., human Siglec-8), the VHH domain comprising CDR1, CDR2, and CDR3 sequences of a VHH comprising 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, and 113, respectively, as defined according to the Contact numbering system; or (e) the amino acid sequences of SEQ ID NOs: 119, 124, and 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.

[0015] In another aspect, the 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 linked to each other via a peptide linker, e.g., a peptide linker comprising or consisting of the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the VHH domains of the polypeptide comprise the same CDR1, CDR2, and CDR3 amino acid sequences. In certain embodiments, the VHH domains of the polypeptide comprise the same VHH amino acid sequence.

[0016] 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.

[0017] The polypeptides disclosed herein may further comprise a multimerization domain.

[0018] In another aspect, the disclosure provides a polypeptide comprising at least two (eg, two or three) antigen-binding domains that bind to Siglec-8 (eg, human Siglec-8), and a multimerization domain.

[0019] In another aspect, the 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).

[0020] 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 format of a Fab or scFv.

[0021] In certain embodiments of any of the above applicable aspects, the multimerization domain is a dimerization domain, e.g., an antibody Fc region, such as a human IgG1 Fc region. The antibody Fc region can comprise the amino acid sequence of SEQ ID NO: 44, 45, 65, 66, 67, or 68.

[0022] The antibody Fc region can be operably linked to at least one of the antigen-binding domains that binds to 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.

[0023] The polypeptide can take on a variety of structural forms. In certain embodiments, the polypeptide comprises the structure VHH-VHH-Fc. Such a polypeptide 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: 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.

[0024] The present disclosure also provides a protein comprising two or more of the polypeptides disclosed herein that are multimerized under physiological conditions via multimerization domain.For example, two or more of the polypeptides can form homomultimers.When the multimerization domain is a dimerization domain, the protein can comprise two of the polypeptides disclosed herein that are dimerized under physiological conditions via dimerization domain.For example, two of the polypeptides can form homodimers.

[0025] Where the VHH, polypeptide, or protein disclosed herein binds to human Siglec-8, the amino acid sequence of human Siglec-8 may be set forth in SEQ ID NO:43.

[0026] In certain embodiments, the polypeptide or protein disclosed herein mediates eosinophil killing in the presence of IL-5.Eosinophil killing can be determined by in vitro assay.It is contemplated that when the protein comprises at least two VHH domains that bind to Siglec-8 in each polypeptide, the homodimeric protein can mediate eosinophil killing in the presence of IL-5 with a lower EC50 than the homodimeric polypeptide, and each of the homodimeric polypeptides has the structure of VHH-Fc and comprises only one of the VHH domains that bind to Siglec-8.

[0027] In certain embodiments, the polypeptide or protein mediates eosinophil killing in the absence of IL-5. In certain embodiments, the polypeptide mediates eosinophil killing via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0028] The present disclosure also provides a pharmaceutical composition comprising a combination of a polypeptide or protein disclosed herein and a pharma- ceutically acceptable carrier.

[0029] Additionally, the disclosure provides isolated nucleic acids encoding the polypeptides disclosed herein, vectors containing the nucleic acids, host cells containing the nucleic acids or vectors, and host cells expressing the polypeptides or proteins.

[0030] The disclosure also provides a method of producing a polypeptide or protein, the method comprising incubating a host cell under conditions for expressing the polypeptide or protein. The method may further comprise isolating the polypeptide or protein.

[0031] Another aspect of the disclosure provides a method of treating an eosinophilic disorder or a mast cell disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide, protein, or pharmaceutical composition disclosed herein. The eosinophilic disorder can be eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorder, eosinophilic gastritis (EoG), eosinophilic enteritis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic leukemia (e.g., chronic eosinophilic leukemia), asthma with an eosinophilic phenotype, allergic bronchopulmonary aspergillosis (ABPA), chronic rhinosinusitis with nasal polyposis (CRSwNP), or eosinophilic granulomatosis with polyangiitis (EGPA). The mast cell disorder may be systemic mastocytosis, hereditary alpha tryptasemia (HAT), or mast cell activation syndrome (MCAS). Systemic mastocytosis (SM) may be progressive SM (e.g., mast cell leukemia, aggressive SM, or SM associated with hematologic neoplasms (SM-AHN)) or non-progressive SM (e.g., indolent SM).

[0032] The present disclosure also provides 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, protein, or pharmaceutical composition disclosed herein.

[0033] The present disclosure further provides a method of treating or preventing an allergic condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide, protein, or pharmaceutical composition disclosed herein. The allergic condition can be a food allergy, an environmental allergy, a companion animal allergy, and / or a venom allergy.

[0034] 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.

[0035] Further, the present disclosure provides a method of killing eosinophils, 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 of killing eosinophils, comprising contacting eosinophils with macrophages in the presence of a polypeptide, protein, or pharmaceutical composition disclosed herein. [Brief description of the drawings]

[0036] [Figure 1A] Figures 1A-1D show the mean fluorescence intensity of binding of parental anti-human Siglec8-B12 (cx8982, Figure 1A), Siglec8-D1 (cx8983, Figure 1B), Siglec8-E2 (cx8984, Figure 1C), or Siglec8-G2 (cx10382, Figure 1D) to either non-transfected (UT-CHO) or human Siglec-8 expressing CHO cells, as measured by binding of A647-conjugated anti-human Fcγ-specific secondary antibodies using flow cytometry. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2A] Figures 2A-2D show the mean fluorescence intensity of binding to human Siglec-8 on eosinophils (Figures 2A-2C) or the median fluorescence intensity of binding to Siglec-8-transfected CHO cells (Figure 2D) by bivalent, tetravalent, and hexavalent anti-Siglec8-B12 (cx8982, cx8467, cx8466; Figure 2A), anti-Siglec8-D1 (cx8983, cx8469, cx8523; Figure 2B), anti-Siglec8-E2 (cx8984, cx8463, cx8522; Figure 2C), or anti-Siglec8-G2v14 (cx11422, cx1158, cx11579; Figure 2D) antibodies, as measured by binding of an A647-conjugated anti-human Fcγ-specific secondary antibody using flow cytometry. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3A] 3A-3E show titrated amounts of bivalent, tetravalent, or hexavalent anti-Siglec8-B12 antibodies (cx8982, cx8467, cx8466; FIG. 3A), anti-Siglec8-D1 antibodies (cx8983, cx8469, cx8523; FIG. 3B), anti-Siglec8-E2 antibodies (cx8984, cx8463, cx8522; FIG. 3C), and anti-Siglec8-P12 antibodies (cx8984, cx8463, cx8522; FIG. 3D) in the presence of IL-5 after 24 hours. 3C) or after 18 hours with anti-Siglec8-G2v14 (cx11422, cx11580, cx11579) agonist antibodies in the presence (Fig. 3D) or absence (Fig. 3E) of IL-5. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A] Figures 4A-4C show the median fluorescence intensity of binding of tetravalent Siglec-8 agonist antibodies including hzG2v52 (cx11913) or hzG2v53 (cx11914) and comparison analog 2E2 Afuc to freshly isolated human eosinophils (Figure 4A), or the mean fluorescence intensity of binding of tetravalent Siglec-8 agonist antibodies including hzG2v52 (cx11913), hzG2v53 (cx11914), or hzG2v14 (cx11769) to CHO cells transfected with human Siglec-2, 3, 6, 7, 8, 9, 12, and 15, all measured by binding of A647-conjugated anti-human Fcγ-specific secondary antibodies using flow cytometry (Figure 4B-4C). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A] Figures 5A-5H show the median fluorescence intensity of parental anti-human Siglec8-G2 (lmG2) and its humanized variants formatted as monomeric VHH-hIgG1 fusions binding to either human Siglec-8 expressing CHO293 cells (Figures 5A-5D) or non-transfected 293 cells (Figures 5E-5H) as measured by binding of A647-conjugated anti-human Fcγ-specific secondary antibody using flow cytometry. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 5G] See legend to Figure 5A. [Figure 5H] See legend to Figure 5A. [Figure 6A] Figures 6A-6B show the direct (Figure 6A) and ADCC-mediated (Figure 6B) killing activity of multivalent Siglec-8 agonist antibodies, including hzG2v53 with engineered Fc regions (cx12562 and / or cx11914), or tetravalent with wild-type Fc region (cx12532), and the comparison analog 2E2 Afuc. Figure 6A shows the total red object integrated intensity (RCU x μm2 / image) of the dead cell marker Cytotox Red incorporated into purified human eosinophils in the presence of IL-5. Figure 6B shows the percent of apoptotic ApoTracker Green positive eosinophils killed by NK cell-mediated ADCC in the absence of IL-5. [Figure 6B] See legend to Figure 6A. [Figure 7] 1 illustrates exemplary formats of multivalent Siglec-8 targeting polypeptides compared to bivalent Siglec-8 targeting polypeptides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description Embodiments provided herein relate to Siglec-8 binding polypeptides and their uses in various methods of treating Siglec-8-associated diseases or disorders.

[0038] Definitions and Various Embodiments The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0039] All references cited in this specification, including patent applications, patent publications, and GenBank accession numbers, are incorporated by reference into this specification as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0040] The techniques and procedures described or referenced herein are generally employed using methodologies well known and conventional to those of skill in the art, and widely used methodologies include those described in, for example, 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 (FMA Usubel, et al. eds., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR2: 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(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture Laboratory Procedures (A.Doyle, JBGriffiths, and DGNewell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987);PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(JEColigan et al.,eds.,1991);Short Protocols in Biocular Bicolology(Wiley et al.,eds.,1991); Sons,1999);Immunobiology(CAJaneway and P.Travers,1997);Antibodies(P.Finch,1997);Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989);Monoclonal Antibodies:A Practical Approach(Pal.Shepherd. C.Dean,eds.,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 JDCapra,eds.,Harwood Academic Publishers,1995); Oncology(VTDeVita et al.,eds.,JBLippincott Company,1993);

[0041] Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required or indicated by the context, singular terms shall include plural terms and plural terms shall include the singular terms. In the event of discrepancies in definitions among various sources or references, the definitions provided herein shall prevail.

[0042] Generally, 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). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0043] It is understood that embodiments of the invention described herein include "comprising," "consisting," and / or "consisting essentially of" embodiments. As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated. Use of the term "or" herein is not intended to imply that alternatives are mutually exclusive.

[0044] In this application, the use of "or" means "and / or" unless expressly stated otherwise or understood by one of ordinary skill in the art. In the context of multiple dependent claims, the use of "or" refers back to multiple preceding independent or dependent claims.

[0045] The phrases "reference sample", "reference cell", or "reference tissue" refer to 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, the reference sample can be used as a positive or negative indicator. A reference sample can be used to establish the level of protein and / or mRNA present in, for example, healthy tissue as opposed to the level of protein and / or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is from the same subject, but from a different part of the subject being tested. In some embodiments, the reference sample is from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but is a sample from a subject known to have or not have the disorder of interest (e.g., a Siglec-8-associated 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 a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample will indicate an absence and / or low level of the molecule, as would be understood by one of skill in the art in light of the present disclosure. When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample will indicate the level of the molecule at the molecular level, as one of skill in the art would understand in light of the present disclosure.

[0046] In the context of benefit from or response to administration of a therapeutic agent, the terms "benefit", "clinical benefit", "responsiveness" and "therapeutic response" as used herein can be measured by assessing various endpoints, such as, for example, inhibition of disease progression to some extent, including slowing down and complete prevention; reduction in the number of attacks and / or symptoms of disease; reduction in lesion size; inhibition of disease cell infiltration into adjacent peripheral organs and / or tissues (i.e., reduction, slowing down, or complete halt); inhibition of disease spread (i.e., reduction, slowing down, or complete halt); reduction in autoimmune response, which may lead to regression or elimination of disease lesions, but does not necessarily lead to some alleviation of one or more symptoms associated with the disorder; increase in the length of disease-free presentation, e.g., progression-free survival, after treatment; increased overall survival; higher response rate; and / or reduced mortality at a given time point after treatment. A "non-responsive" or "non-responsive" subject or cancer is one that does not meet the above qualifications for being "responsive".

[0047] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides can contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0048] The term "polypeptide" refers to a polymer of amino acid residues and is not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, and full-length forms and fragments of natural polypeptides. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, sialylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, a "polypeptide" may include modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the desired activity is maintained. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through errors due to, for example, mutations of the host producing the protein or PCR amplification.

[0049] The term "protein" refers to a macromolecule that includes one or more polypeptides. For example, a protein can be a multimer (e.g., a dimer, trimer, or tetramer) formed by multiple identical or different polypeptides.

[0050] As used herein, "Siglec-8" refers to any naturally occurring mature Siglec-8 resulting from processing of a Siglec-8 precursor in a cell. The term includes Siglec-8 from any mammalian source, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise specified. 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.

[0051] The term "specifically binds" to an antigen or epitope is a term well understood 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 duration, and / or with a higher affinity than it does with alternative cells or substances. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, affinity, more readily, and / or for a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a Siglec-8 epitope is an sdAb or VHH-containing polypeptide that binds this epitope with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other Siglec-8 epitopes or non-Siglec-8 epitopes. It is also 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 can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0052] The term "inhibition" or "inhibiting" refers to the reduction, cessation, or suppression of any phenotypic characteristic, or the reduction, cessation, or suppression of the incidence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, reduction, suppression, or cessation of an activity, function, amount, abundance, or rate of increase compared to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is inhibited or reduced over a period of time compared to a control over the same period of time.

[0053] The term "agonize" or "activate" refers to the increase, activation, or induction of any phenotypic characteristic, or the increase, activation, or induction of the incidence of the potential of that characteristic. "Agonize" or "activate" refers to increasing, activating, or inducing an activity, function, amount, abundance, or rate of increase compared to a reference. In some embodiments, "agonize" or "activate" refers to the ability to cause an overall increase of 10% or more. In some embodiments, "agonize" or "activate" refers to the ability to cause an overall increase of 50% or more. In some embodiments, "agonize" or "activate" refers to the ability to cause an overall increase of 75%, 85%, 90%, 95%, or more. In some embodiments, the amount is increased over a period of time compared to a control over the same period of time. As used herein, the term "agonize" with respect to the activity of Siglec-8 refers to increasing the activity of Siglec-8, an inhibitory receptor on mast cells and eosinophils that mediates eosinophil cell death 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 and / or increases inhibition of mast cell activation in the presence of IL-5. In some embodiments, "agonize" refers to an increase in Siglec-8 activity compared to Siglec-8 activity in the absence of a modulator.

[0054] As used herein, the term "epitope" refers to a site to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often consist of chemically active surface groupings of molecules, such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, epitopes are less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. If two antibodies show competitive binding to an antigen, they may bind to the same epitope in the antigen. In some embodiments, an epitope may be identified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope may be identified by the above distances and may be further limited to residues involved in binding (e.g., hydrogen bonds) between the antigen-binding molecule residues and the antigen residues. An epitope may also be identified by a variety of scans, for example, an alanine or arginine scan may indicate one or more residues with which the antigen-binding molecule can interact. Unless explicitly indicated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the existence of such a set specifies a minimal series (or species set) of epitopes. Thus, in some embodiments, a set of residues identified as an epitope specifies a minimal epitope associated with the antigen, rather than an exclusive list of residues for the epitope on the antigen.

[0055] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids, and / or sugars within an antigenic protein to which an epitope-specific antigen-binding molecule binds. In some embodiments, at least one of the residues is non-contiguous with other listed residues of the epitope, however, one or more of the residues can be contiguous with other residues.

[0056] A "linear epitope" includes contiguous polypeptides, amino acids and / or sugars in an antigenic protein to which an antigen-binding molecule specific for the epitope binds. Note that in some embodiments, not all of the residues in a linear epitope need to be directly bound (or involved in binding) by an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that is effective from the sequence of the linear epitope, or from a structural section of a protein that is relatively isolated from the rest of the protein (so that the antigen-binding molecule can at least temporarily interact with only that sequence section).

[0057] The term "antibody" is used in the broadest sense and encompasses a variety of polypeptides that contain antibody-like antigen-binding domains, including, but not limited to, conventional antibodies (typically comprising at least one heavy chain and at least one light chain, more typically comprising two heavy chains and two light chains that form two antigen-binding domains), single domain antibodies (e.g., sdAbs comprising 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, so long as they exhibit the desired antigen-binding activity. In some embodiments, an antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain, CL, to mediate dimerization) and an Fc region (comprising hinge, CH2, and CH3, where 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, and cynomolgus monkeys.

[0058] As used herein, the term "antigen binding domain" refers to a portion of an antibody 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. Thus, in some embodiments, the antigen binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen. In some aspects, 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, the antigen binding domain of an sdAb or VHH-containing polypeptide comprises three CDRs of a VHH domain. Thus, in some embodiments the 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 necessary to maintain binding to the antigen.

[0059] The term "VHH" or "VHH domain" as used herein refers to the antigen-binding portion of a single domain antibody, such as a camelid antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N- or C-terminus to include only a portion of FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0060] 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 without a light chain, and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising 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, for example, polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different.

[0061] The term "VHH-containing polypeptide" refers to a polypeptide that includes at least one VHH domain. In some embodiments, a VHH polypeptide includes two, three, or four or more VHH domains, and each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide includes an Fc region. In some such embodiments, a VHH-containing polypeptide may be referred to as an sdAb. Furthermore, in some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide that is also an sdAb include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be connected to another VHH by a linker, or one VHH may be connected to an Fc region by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids composed primarily of glycine and optionally serine. Non-limiting linkers are provided 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, when it forms a dimer, is considered to be tetravalent (i.e., the dimer has four VHH domains). Similarly, the structure VHH1 -VHH2-VHH3-Fc is considered hexavalent when it forms a dimer (i.e., the dimer has six VHH domains). In some embodiments, VHH-containing polypeptides that include an Fc region form homodimers, for example, via association of hinges of the Fc regions.

[0062] The term "monoclonal antibody" refers to an antibody (sdAb or VHH-containing polypeptide) of a substantially homogenous population of antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made using the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0063] The term "CDR" refers to a complementarity determining region defined by at least one method of identification to one skilled in the art. In some embodiments, the CDRs can be defined according to any of the Chothia numbering scheme (see Chothia and Lek, 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, called CDR1, CDR2 and CDR3.

[0064] The term "heavy chain constant region" as used herein refers to a region that includes at least three heavy chain constant domains, CH1, hinge, CH2, and CH3. Of course, non-functional deletions and modifications within the domains are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, and an antibody that includes an alpha constant region is an IgA antibody. Furthermore, an antibody that includes a mu constant region is an IgM antibody, and an antibody that includes an epsilon constant region is an IgE antibody. A particular isotype may be further divided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region), and IgA2 (containing an alpha 2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2. For all heavy chain constant region amino acid positions discussed in this invention, the 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 the myeloma protein Eu, the first sequenced human IgG1. The Eu index of Edelman et al. is also described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., 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.

[0065] As used herein, "Fc region" refers to a portion of a 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 can be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[0066] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as described herein. H ) framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence, 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 of the human frameworks in a single antigen-binding domain such as a VHH.

[0067] "Affinity" refers to the strength of the total 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 is generally expressed as the dissociation constant (K d ) or K d見かけAffinity can be expressed by any of the common methods known in the art, including those described herein (e.g., ELISA K d , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc. Such methods include, but are not limited to, methods involving BIAcore®, Octet®, or flow cytometry. In some embodiments, exemplary polypeptides have a molecular mass of about 90, 85, 80, 75, 70, 68, 65, 60, 55, 56, or 50×10 as measured by a surface plasmon resonance (SPR) assay. -12 K below M D In some embodiments, exemplary polypeptides bind to Siglec-8 at 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 10×10 as measured by a surface plasmon resonance (SPR) assay. -12 K below M D It binds to Siglec-8.

[0068] As used herein, "K d The term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. d As used herein, the term " d and K d-見かけ Includes.

[0069] In some embodiments, the K d is measured by flow cytometry using antigen-expressing cell lines and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Prism Software GraphPad). In some such embodiments, K d is K d-見かけ It is.

[0070] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or enabled by recombinant means).

[0071] An "agonist" or "activating" antibody is one that increases and / or activates 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.

[0072] An "antagonist," "blocking," or "neutralizing" antibody is one 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.

[0073] An "affinity matured" sdAb or VHH containing polypeptide refers to an sdAb or VHH containing polypeptide which has one or more changes in one or more CDRs compared to a parent sdAb or VHH containing polypeptide which does not have the changes, which changes result in improved affinity of the sdAb or VHH containing polypeptide for antigen.

[0074] "Humanized VHH" as used herein refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further refine and optimize the performance of the sdAb VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be understood, a humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was created.

[0075] An "effector positive Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activity. Effector functions such as these 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.

[0076] A "native sequence Fc region" comprises an amino acid sequence 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.

[0077] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, and further retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions in the native sequence Fc region or in the Fc region of a parent polypeptide, and preferably about 1 to about 5 amino acid substitutions. In some embodiments, the variant Fc region herein will retain at least about 80% sequence identity with, at least about 90% sequence identity with, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with, a native sequence Fc region and / or the Fc region of a parent polypeptide.

[0078] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or splice forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting 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)). FcRs are reviewed, for example, 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 identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also encompasses the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus and for regulating immunoglobulin homeostasis (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). 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.)).

[0079] As used herein, the terms "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two or more numerical values ​​such that one of skill in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance in the context of the biological attribute measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0080] By polypeptide "variant" is meant a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, not considering conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of 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 peptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0081] As used herein, with respect to peptide, polypeptide, or antibody sequences, "percent (%) amino acid sequence identity" and "homology" are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0082] Amino acid substitutions can 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 can be introduced into the antibody of interest and the product screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0083] (Table 1) TIFF2025515304000002.tif158128

[0084] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0085] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0086] The term "vector" is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide(s) that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more control sequences (such as, for example, a promoter and / or enhancer) that control the expression of a polypeptide of interest, and / or a more selectable marker gene (e.g., 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.

[0087] "Host cell" refers to a cell that can be or has been the 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 animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S cells, and CHO-DS cells. A host cell includes the progeny of a single host cell, and the progeny need not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. A host cell includes cells transfected in vivo with a polynucleotide(s) provided herein.

[0088] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components in which it is typically found or produced in nature. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) in which it is normally found in nature or, for example, in the case of an RNA polynucleotide, separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector in a host cell can be referred to as "isolated".

[0089] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to be treated may be an identified patient who presents with facts that the subject has or is at sufficient risk of developing a disorder relevant to the treatment.

[0090] As used herein, "disease" or "disorder" refers to a condition for which treatment is necessary and / or desirable.

[0091] 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" also involves causing a change (which can be either an increase or a decrease) in the affinity, avidity, specificity, and / or selectivity of a target or antigen to one or more of its ligands, binding partners, partners for association into homo- or hetero-multimeric forms, or substrates; causing a change (which can be either an increase or a decrease) in the sensitivity of a target or antigen to one or more conditions (e.g., pH, ionic strength, cofactor presence, 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 in the absence of the 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.

[0092] As used herein, "immune response" is meant to encompass a cellular and / or humoral immune response sufficient to inhibit, prevent the onset of, or ameliorate a symptom of a disease (e.g., cancer or cancer metastasis). An "immune response" can include aspects of both the innate and adaptive immune systems.

[0093] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of a disease, prevention of disease progression, prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of pathology, inhibition of a disease or disease progression, and inhibition or slowing of a disease or its progression. Relief of pathological consequences of a disease is also encompassed by "treatment". Any one or more of those aspects provided herein are contemplated. In line with the above, the term treatment does not require 100% elimination of all aspects of a disorder.

[0094] By "ameliorate" is meant that one or more symptoms are lessened or improved compared to when the therapeutic agent is not administered. "Ameliorate" also includes a shortening or reduction in the duration of the symptoms.

[0095] The term "biological sample" refers to a quantity of a living or formerly living substance, including, but not limited to, blood (e.g., whole blood), plasma, serum, bronchoalveolar lavage fluid, swallow, nasal wash, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocyte lavage, cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0096] The term "control" or "reference" in an experimental or comparative context refers to a composition known to not contain the analyte (a "negative control") or a composition known 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 activity of the agent being tested, e.g., an antibody.

[0097] As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject susceptible to the disease but not yet diagnosed with the disease. Unless otherwise specified, the terms "reducing," "inhibiting," or "preventing" do not indicate or require complete prevention over the entire period of time, but rather over the period of time being measured.

[0098] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh the toxic or detrimental effects of the substance / molecule, agonist, or antagonist. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic and / or prophylactic result.

[0099] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation in which the biological activity of the active ingredient is effective and which does not contain any ingredients that are toxic to an extent that they are unacceptable to the subject to which the formulation is administered. Such formulations may be sterile.

[0100] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation being used.

[0101] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0102] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent is within a short period of time relative to the administration of the other therapeutic agent, or where the therapeutic effects of both agents overlap for at least some period of time.

[0103] 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.

[0104] As used herein, "in conjunction with" refers to the administration of one therapy in addition to another. Thus, "in conjunction with" refers to the administration of one therapy before, during, or after administration of another therapy to an individual.

[0105] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic agents that contain information about the indications, uses, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic agent.

[0106] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., a pharmaceutical agent for the treatment of 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.

[0107] The terms "label" and "detectable label" refer to a moiety that, for example, binds to an antibody or antigen, making the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that incorporates a label that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a detectable signal by visual or instrumental means, for example, the incorporation of a radioactively labeled amino acid, or the binding of a biotinyl moiety to the polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods). Examples of labels for polypeptides include 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 153 Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. In this regard, the moiety itself need not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0108] Exemplary Siglec-8 Binding Polypeptides and Proteins Provided herein are Siglec-8 binding polypeptides and proteins. In various embodiments, the Siglec-8 binding polypeptide or protein is a Siglec-8 agonist. In some embodiments, a Siglec-8 binding polypeptide is provided that comprises at least two antigen binding domains that bind to Siglec-8. In some embodiments, a Siglec-8 binding polypeptide or protein is provided that comprises at least three antigen binding domains that bind to Siglec-8. In some embodiments, a Siglec-8 binding polypeptide is provided that comprises at least two antigen binding domains that bind to Siglec-8 and a dimerization domain. In some embodiments, a Siglec-8 binding polypeptide is provided that comprises at least three antigen binding domains that bind to Siglec-8 and a dimerization domain. In some embodiments, the dimerization domain is an Fc region. In some embodiments, the polypeptide mediates eosinophil killing in the presence of IL-5 at a lower EC50 than a polypeptide comprising two antigen binding domains that bind to Siglec-8 under physiological conditions, e.g., than a conventional antibody. In some embodiments, the two or more antigen binding domains are the same. In some embodiments, the two or more antigen binding domains are different. In some embodiments, all of the antigen-binding domains are the same. In some aspects, each antigen-binding antibody fragment comprises a heavy chain variable region and a light chain variable region. In some embodiments, each antigen-binding domain is a Fab or scFv.

[0109] 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 agonizes (i.e., increases the activity of) Siglec-8. In some embodiments, the Siglec-8 binding polypeptide or protein agonizes Siglec-8 and results in eosinophil killing 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.

[0110] In some embodiments, the Siglec-8 binding polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that bind to Siglec-8. In some embodiments, the Siglec-8 binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind to Siglec-8. In some embodiments, the Siglec-8 binding polypeptides provided herein comprise two or three VHH domains that bind to Siglec-8. The Siglec-8 binding polypeptides 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 "multispecific" polypeptides.

[0111] 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 that are operably linked to each other via a peptide linker. A non-limiting example of a linker that can be used to link VHH domains is provided in SEQ ID NO:69.

[0112] 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 in physiological conditions such that dimers are formed that double 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 in physiological conditions, the Fc region mediates dimerization to form the Siglec-8 protein as a hexavalent dimer under such conditions. In some embodiments, the Fc region is a human IgG1 Fc region variant, such as any of the human IgG1 Fc region variants described herein.

[0113] 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 of 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 an Fc region via a peptide linker. A non-limiting example of a linker that may be used to link the VHH domain and the Fc region is provided in SEQ ID NO: 70.

[0114] 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, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc region is a wild-type IgG1 isotype (see, e.g., SEQ ID NOs: 45 and 66).

[0115] In some embodiments, the Fc region included in the Siglec-8 binding polypeptide is derived from a human Fc region and contains mutations designed for heterodimerization.

[0116] In some embodiments, the Fc region of the Siglec-8 binding polypeptide is derived from a human Fc region and includes mutations designed to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., amino acid modifications described in WO00 / 42072, WO04 / 029207, WO04 / 099249, and WO04 / 063351. In some embodiments, the Fc region of the Siglec-8 binding polypeptide includes 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 of the Siglec-8 binding polypeptide includes a deletion of Glu233, Leu234, 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 comprised in the Siglec-8 binding polypeptide comprises modifications at Ser239 and / or Ile332 according to the EU index set forth in Kabat. In one embodiment, the Fc region comprised in the Siglec-8 binding polypeptide comprises modifications at Ser239Asp and Ile332Glu (S239D, I332E) according to the EU index set forth in Kabat to enhance ADCC (e.g., SEQ ID NOs: 65 and 68). In some embodiments, the Fc region lacks Lys447 according to the EU index set forth in Kabat (see, e.g., SEQ ID NOs: 66, 67, and 68).

[0117] Fc regions that may be used in Siglec-8 binding polypeptides include Fc regions comprising the amino acid sequence of SEQ ID NO:44, 45, 65, 66, 67, or 68.

[0118] The Siglec-8 binding proteins disclosed herein can take a variety of formats, for example, those depicted in FIG. 7. In some embodiments, the protein is a homodimer comprising two identical polypeptide chains, comprising one or more, two or more, three or more, two, three, or two or three VHH domains that bind Siglec-8, and an Fc region. In some embodiments, the polypeptide chain comprises, from N-terminus to C-terminus, a VHH domain that binds Siglec-8, and an Fc region. In some embodiments, the polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain that binds Siglec-8, a second VHH domain that binds Siglec-8, and an Fc region, where the first and second VHH domains can be the same or different. In some embodiments, the polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain that binds Siglec-8, an Fc region, and a second VHH domain that binds Siglec-8, where the first and second VHH domains can be the same or different. In some embodiments, the polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain that binds Siglec-8, a second VHH domain that binds Siglec-8, a third VHH domain that binds Siglec-8, and an Fc region, where the first, second, and third VHH domains 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 Siglec-8, a second VHH domain that binds Siglec-8, an Fc region, and a third VHH domain that binds Siglec-8, where the first, second, and third VHH domains may be the same or different.

[0119] In some embodiments, in the case of a Siglec-8 binding polypeptide or protein comprising an Fc domain with ADCC effector function, the Siglec-8 binding polypeptide or protein increases eosinophil killing by NK cells in the absence of IL-5.

[0120] In some embodiments, a Siglec-8 binding polypeptide comprises at least two VHH domains, a first VHH domain that binds to Siglec-8 and a second VHH domain that binds to an antigen other than Siglec-8. Such polypeptides may be referred to as "bispecific" or "multispecific."

[0121] Non-limiting exemplary Siglec-8-binding VHH domains are described below in the "Siglec-8-binding VHH domains" subsection.

[0122] Siglec-8 binding VHH domain The present disclosure provides VHH domains that bind to Siglec-8. Table 2 lists exemplary VHH domains based on various CDR definitions and the CDR sequences within these VHH domains. VHH names beginning with "hz" indicate that they are humanized versions of the corresponding parent polypeptide.

[0123] Table 2 Polypeptides containing VHH domains that bind to Siglec-8 TIFF2025515304000003.tif125165 For example, in the case of hzG2v14 under the AbM CDR definition, where multiple sets of CDR sequence identifiers are provided, the multiple sets of CDR sequences refer to the same sequence. In other words, in the example of hzG2v14, SEQ ID NOs: 2 and 6 represent the same sequence, and SEQ ID NOs: 3 and 7 represent the same sequence.

[0124] Additional exemplary amino acid sequences of humanized VHH domains are set forth in SEQ ID NOs: 130-138, 140-148, 161, 163, and 164. Their CDR sequences are not provided in the sequence listing, but they can be identified by methods well known in the art, for example, using the AbYsis server (www.abysis.org / abysis / ). These VHH domains are variants of hzG2v14, hzG2v47, hzG2v51, hzG2v52, hzG2v53, hzG2v54, and hzG2v55 in Table 2. Thus, their CDR sequences can also be identified by aligning the VHH amino acid sequence with one or more of their reference sequences (e.g., hzG2v14) and isolating the regions corresponding to the CDRs of the reference sequence.

[0125] In certain embodiments, the Siglec-8 binding VHH domain comprises the CDR1, CDR2, and CDR3 sequences 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 the CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the AbM definition, e.g., the set of CDR sequences disclosed in the "AbM" column of Table 2. In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the Chothia definition, e.g., the set of CDR sequences disclosed in the "Chothia" column of Table 2. In certain embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the Kabat definition, e.g., the set of CDR sequences disclosed in the "Kabat" column of Table 2. In certain embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the Contact definition, e.g., the set of CDR sequences disclosed in the "Contact" column of Table 2. In certain embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 sequences of a VHH disclosed herein under the IMGT definition, e.g., the set of CDR sequences disclosed in the "IMGT" column of Table 2.

[0126] In certain embodiments, under the AbM definition, the Siglec-8 binding VHH domain comprises the consensus hzG2 CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 46, 47, and 48, respectively. In certain embodiments, X1 of SEQ ID NO: 48 is Q, S, or T. In certain embodiments, X2 of SEQ ID NO: 48 is T. In various embodiments, the VHH domain comprises the CDR1 sequence of SEQ ID NO: 2, 6, 13, 18, 23, 28, 33, or 38, the CDR2 sequence of SEQ ID NO: 3, 7, 14, 19, 24, 29, 34, or 39, and the CDR3 sequence of SEQ ID NO: 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.

[0127] In certain embodiments, under the Chothia definition, the Siglec-8 binding VHH domain comprises the consensus hzG2 CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 149, 150, and 151, respectively. In certain embodiments, X1 of SEQ ID NO: 151 is Q, S, or T. In certain embodiments, X2 of SEQ ID NO: 151 is T. In various embodiments, the VHH domain comprises the CDR1 sequence of SEQ ID NO: 74, 75, or 76; the CDR2 sequence of SEQ ID NO: 80 or 81; and the CDR3 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.

[0128] In certain embodiments, under the Kabat definition, the Siglec-8 binding VHH domain comprises the CDR1, CDR2, and CDR3 sequences of consensus hzG2 as set forth in SEQ ID NOs: 152, 153, and 154, respectively. In certain embodiments, X1 of SEQ ID NO: 154 is Q, S, or T. In certain embodiments, X2 of SEQ ID NO: 154 is T. In various embodiments, the VHH domain comprises the CDR1 sequence of SEQ ID NO: 85, 86, or 87; the CDR2 sequence of SEQ ID NO: 92, 93, or 94; and the CDR3 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.

[0129] In certain embodiments, under the definition of Contact, the Siglec-8-binding VHH domain comprises the CDR1, CDR2, and CDR3 sequences of consensus hzG2 as set forth in SEQ ID NOs: 155, 156, and 157, respectively. In certain embodiments, X1 of SEQ ID NO: 157 is Q, S, or T. In certain embodiments, X2 of SEQ ID NO: 157 is T. In various embodiments, the VHH domain comprises the CDR1 sequence of SEQ ID NO: 98, 99, or 100; the CDR2 sequence of SEQ ID NO: 104 or 105; and the CDR3 sequence of SEQ ID NO: 110. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 98, 104, and 110. In some embodiments, the VHH domain comprises the 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.

[0130] In certain embodiments, under the IMGT definition, the Siglec-8 binding VHH domain comprises the consensus hzG2 CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 158, 159, and 160, respectively. In certain embodiments, X1 of SEQ ID NO: 160 is Q, S, or T. In certain embodiments, X2 of SEQ ID NO: 160 is T. In various embodiments, the VHH domain comprises the CDR1 sequence of SEQ ID NO: 114, 115, or 116; the CDR2 sequence of SEQ ID NO: 120 or 121; and the CDR3 sequence of SEQ ID NO: 126. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 114, 120, and 126. In some embodiments, the VHH domain comprises the 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.

[0131] In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 3, and 4, respectively, under the AbM definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 52, 53, and 54, respectively, under the AbM definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 58, 59, and 60, respectively, under the AbM definition. In some embodiments, the VHH domain comprises the CDR1 and CDR2 sequences of SEQ ID NOs: 63 and 64, respectively, under the AbM definition, and a CDR3 comprising the amino acid sequence "GAY". In various embodiments, the VHH domain is humanized.

[0132] In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 74, 80, and 4, respectively, under the Chothia definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 77, 82, and 54, respectively, under the Chothia definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 78, 83, and 60, respectively, under the Chothia definition. In some embodiments, the VHH domain comprises the CDR1 and CDR2 sequences of SEQ ID NOs: 79 and 84, respectively, under the Chothia definition, and a CDR3 comprising the amino acid sequence "GAY". In various embodiments, the VHH domain is humanized.

[0133] In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 85, 91, and 4, respectively, under the Kabat definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 88, 95, and 54, respectively, under the Kabat definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 89, 96, and 60, respectively, under the Kabat definition. In some embodiments, the VHH domain comprises the CDR1 and CDR2 sequences of SEQ ID NOs: 90 and 97, respectively, under the Kabat definition, and a CDR3 comprising the amino acid sequence "GAY". In various embodiments, the VHH domain is humanized.

[0134] In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 98, 104, and 109, respectively, under the Contact definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 101, 106, and 111, respectively, under the Contact definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 102, 107, and 112, respectively, under the Contact definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 103, 108, and 113, respectively, under the Contact definition. In various embodiments, the VHH domain is humanized.

[0135] In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 114, 120, and 125, respectively, under the IMGT definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 117, 122, and 127, respectively, under the IMGT definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 118, 123, and 128, respectively, under the IMGT definition. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 119, 124, and 129, respectively, under the IMGT definition. In various embodiments, the VHH domain is humanized.

[0136] 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 or 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.

[0137] In some embodiments, the CDRs are those having at least about 50%, 75%, 80%, 85%, 90%, or 95% identity with the CDRs 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, with up to 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, and has up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the 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 up to 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 the 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 the sequences provided herein, but may be de novo isolated, for example, according to the methods provided herein for obtaining antibodies.

[0138] In some embodiments, the VHH domain that binds Siglec-8 comprises a modification at Leu 11 in framework 1. For example, the VHH domain may comprise Leu11Glu (L11E), Leu11Asp (L11D), Leu11Arg (L11R) or Leu11Lys. (L11K). In some embodiments, the VHH domain that binds 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), TVTVRPGG (SEQ ID NO:55), TVEP (SEQ ID NO:56), TVEPG (SEQ ID NO:57), TVEPGG (SEQ ID NO:57)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 Siglec-8 binding VHH domain comprises a Leu11 mutation and a modified C-terminal sequence. The L11E mutation and the modified C-terminal sequence provide the Siglec-8 binding polypeptide with improved properties, such as reduced immunogenicity. See, e.g., WO2016 / 118733.

[0139] 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 (e.g., an amino acid sequence herein including the CDR1, CDR2, and CDR3 sequences of the VHH domain) selected from SEQ ID NOs: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, 61, 130-138, 140-148, 161, 163, and 164. In some embodiments, the VHH domain that binds Siglec-8 comprises an amino acid sequence selected from SEQ ID NOs: 1, 5, 11, 16, 21, 26, 31, 36, 49, 55, 61, 130-138, 140-148, 161, 163, and 164.

[0140] 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.

[0141] In some embodiments, the Siglec-8 binding polypeptide comprises two, three, or four VHH domains that bind to Siglec-8 operably linked to each other via a peptide linker. A non-limiting example of a linker that can be used to link the VHH domains is provided in SEQ ID NO:69.

[0142] 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 of SEQ ID NO: 44, 45, 65, 66, 67, or 68.

[0143] In some embodiments, the Siglec-8 binding polypeptide comprises at least one VHH domain as described herein operably linked to an Fc region via a peptide linker. A non-limiting example of a linker that can be used to link the VHH domain and the Fc region is provided in SEQ ID NO:70.

[0144] In some embodiments, the VHH domain that binds Siglec-8 is humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies, which results in an immune response to antibody therapeutics and reduces the efficacy of the treatment. Generally, a humanized antibody comprises one or more variable domains, in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues were derived), e.g., to restore or improve antibody specificity or affinity.

[0145] Humanized antibodies and methods for making them are described, for example, in Almagro and Fransson, (2008) Biosci. 13:1619-1633, and further described, for example, in 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. al., (2005) Methods 36:43-60 (describing "FR shuffling"); as well as Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0146] 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 consensus sequences of human antibodies of particular subpopulations of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol., 151:2623), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (2008) J. Immunol. 13:1623). (see Rosok et al., (1997) J. Biol. Chem. 272:10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Usually, the FR regions of the VHH are replaced with human FR regions to generate a humanized VHH. In some embodiments, specific FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. VHH domains with such residue replacements are still referred to as "humanized" herein.

[0147] Polypeptide and Protein Expression and Production Provided is a nucleic acid molecule comprising a polynucleotide encoding a Siglec-8 binding polypeptide or protein. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the Siglec-8 binding polypeptide or protein, and this leader sequence is typically cleaved so that it is not present in the secreted polypeptide or protein. The leader sequence may be the native heavy chain (or VHH) leader sequence or another heterologous leader sequence.

[0148] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0149] Vectors are provided that include nucleic acids encoding the Siglec-8 binding polypeptides and proteins described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector is selected that is optimized for expression of the polypeptide or protein in a desired cell type, such as a CHO or CHO-derived cell, or an NSO cell. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol.Prog.20:880-889 (2004).

[0150] 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, mammalian cells, etc. Such expression can be carried out, for example, according to procedures well 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 COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, Siglec-8 binding polypeptides or proteins can be expressed in yeast. See U.S. Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on a polypeptide or protein. For example, in some embodiments, CHO cells produce a polypeptide or protein that has a higher sialylation level than the same polypeptide or protein produced in HEK293 cells.

[0151] Introduction of one or more nucleic acids (such as vectors) into a desired host cell can be accomplished 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, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press, 2001. The nucleic acids may be transiently or stably transfected into the desired host cells according to any suitable method.

[0152] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells expressing the Siglec-8 binding polypeptides or proteins described herein are provided. The Siglec-8 binding polypeptides or proteins expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to bind to the Fc region and purify Siglec-8 binding polypeptides or proteins that include an Fc region. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can be suitable for purifying some polypeptides or proteins, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) can also be suitable for purifying some polypeptides or proteins, such as antibodies. Mixed-mode chromatography (e.g., reversed phase / anion exchange, reversed phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides or proteins, such as antibodies. Many methods of purifying polypeptides or proteins are well known in the art.

[0153] 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, for example, in 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).

[0154] In some embodiments, a Siglec-8 binding polypeptide or protein prepared by the above-mentioned method 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 comprising a Siglec-8 binding polypeptide or protein is provided.

[0155] In some embodiments, a composition is provided comprising an antibody prepared by the above-described method. 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.

[0156] Exemplary Methods of Treating Disease Using Siglec-8 Binding Polypeptides or Proteins In some embodiments, methods are provided for treating a disease in an individual comprising administering a Siglec-8 binding polypeptide or protein, in some embodiments, for treating a Siglec-8 associated disease (e.g., a disease associated with increased Siglec-8 expression in certain cells compared to normal reference cells of the same type and / or tissue). Such diseases include eosinophilic cystitis, eosinophilic fasciitis, eosinophilic gastrointestinal disorders, eosinophilic gastritis (EoG), eosinophilic enteritis, Churg-Strauss syndrome, eosinophilic syndromes (HES) (e.g., familial HES, idiopathic HES, primary (neoplastic) HES, secondary (reactive) HES, or lymphocytic variants of HES), Gleich syndrome, eosinophilia-myalgia syndrome (EMS), IgG4-related disease (IgG4-RD), eosinophilic leukemia (e.g., chronic eosinophilic leukemia), eosinophilic phenotype asthma, allergic bronchopulmonary aspergillosis (ABPA), chronic rhinosinusitis with nasal polyposis (CRSwNP), or eosinophilic disorders including eosinophilic granulomatosis with polyangiitis (EGPA). Additional exemplary eosinophilic disorders and associated 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 tryptasia (HAT), mast cell activation syndrome (MCAS), or general allergic pathology. Systemic mastocytosis (SM) can be progressive SM (e.g., mast cell leukemia, aggressive SM, or SM with hematologic neoplasms (SM-AHN)) or non-progressive SM (e.g., indolent 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 in mast cells of subjects with SM. Common allergic conditions may be triggered by allergies such as food allergies (e.g., peanut, sesame, milk, egg, fish, or nut allergies), environmental allergies (e.g., dust mite, pollen, mold, or latex allergies), allergies to companion animals (e.g., cat, dog, gizzard, horse allergies, etc.), and venom allergies (e.g., bee, wasp allergies, etc.).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 mast cell activation. In some embodiments, the Siglec-8 binding polypeptide or protein inhibits inflammatory responses.

[0157] In some embodiments, the Siglec-8 binding polypeptide or protein is linked to an additional agent, such as an additional therapeutic agent, to form an immunoconjugate.

[0158] In some embodiments, the methods of treatment can be in humans or animals. In some embodiments, methods of treating humans are provided.

[0159] Siglec-8 binding polypeptides and proteins may be administered to a subject as needed. The frequency of administration may be determined by a person skilled in the art, such as a physician, based on consideration of the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health of the patient. In some embodiments, an effective dose of a Siglec-8 binding polypeptide or protein is administered to a subject one or more times. In some embodiments, an effective dose of a Siglec-8 binding polypeptide or protein is administered to a subject daily, twice weekly, weekly, biweekly, monthly, etc. An effective dose of a Siglec-8 binding polypeptide or protein is administered to a subject at least once. In some embodiments, an effective dose of a Siglec-8 binding polypeptide or protein may be administered multiple times.

[0160] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat a disease associated with Siglec-8. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated.

[0161] In some embodiments, the Siglec-8 binding polypeptide or protein may be administered in vivo by a variety of routes, including, but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Appropriate formulations and administration routes may be selected according to the intended use.

[0162] Pharmaceutical Compositions In some embodiments, compositions comprising Siglec-8 binding polypeptides or proteins are provided in formulations comprising a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (2004)). th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd (See, e.g., J.D., Pharmaceutical Press (2000)). A variety of pharma- ceutically acceptable carriers, including vehicles, adjuvants, and diluents, are available. In addition, a variety of pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, isotonicity adjusting agents, stabilizers, wetting agents, and the like, are also available.

[0163] Combination therapy The Siglec-8 binding polypeptides and proteins of the present disclosure may be administered alone or in combination with other therapeutic modalities, such as additional therapeutic agents. The Siglec-8 binding polypeptide or protein may be provided prior to, substantially simultaneously with, or following (i.e., simultaneously or sequentially) one or more other therapeutic modalities.

[0164] Non-Limiting Exemplary Diagnostic Methods In some embodiments, the methods described herein are useful for evaluating a subject and / or a sample from a subject, hi some embodiments, the evaluation is one or more of a diagnosis, a prognosis, and / or a response to treatment.

[0165] In some embodiments, the methods described herein include assessing the presence, absence, or level of a protein or other molecule, such as Siglec-8, histamine, b-tryptase, or other allergy mediator. In some embodiments, the methods described herein include assessing the presence, absence, or level of expression of a nucleic acid. 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 the sample with a therapeutic agent described herein.

[0166] kit 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 comprising (i) a Siglec-8 binding polypeptide or protein, and (ii) instructions for using the kit to administer the Siglec-8 binding polypeptide or protein to an individual.

[0167] Suitable packaging for compositions, such as pharmaceutical compositions comprising the polypeptides or proteins described herein, is well known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These products may be further sterilized and / or sealed. Also provided are unit dosage forms comprising the compositions described herein. These unit dosage forms may be stored in suitable packaging in single or multiple unit dosage forms, which may also be further sterilized and sealed. Instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions delivered on a magnetic or optical storage disk) are also acceptable. Instructions for use of the antibody generally include information regarding the dosage, dosing schedule, and route of administration for the intended therapeutic or industrial application.

[0168] The containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. For example, kits may also be provided that contain a sufficient dosage of the polypeptide or protein disclosed herein to provide effective treatment to an individual over an extended period of time. The kits may also include multiple unit doses of the polypeptide or protein, and instructions for use, packaged in sufficient quantities for storage and use in pharmacies, e.g., hospital pharmacies and compounding pharmacies. EXAMPLES

[0169] The examples discussed below are intended to be merely illustrative of the present invention and should not be considered as limiting the present invention in any way. The examples are not intended to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0170] Example 1: Development of a single domain antibody that specifically binds to human Siglec-8 Siglec-8 is an inhibitory receptor expressed on eosinophils, mast cells, and basophils. Single-domain antibodies targeting human Siglec-8 were generated by immunizing llamas and alpacas with a recombinant form of the human Siglec-8 extracellular domain (ECD: amino acids 17-363 of human Siglec-8).

[0171] After 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 Qiagen RNeasy Maxi Kit and then converted to first strand cDNA using Thermo Superscript IV reverse transcriptase and oligo dT priming. Single domain antibody (sdAb) sequences were specifically amplified by PCR using cDNA as template and cloned into the phage display vector pADL.

[0172] Phage library displaying llama-derived sdAb (3.7x10 10 The total diversity of Siglec8 phage libraries was enriched by cell panning on free-style HEK293 or CHO cells transfected to surface express a recombinant form of Siglec8-ECD. DNA from the enriched Siglec8 phage library was transformed into BL21 competent cells, which were plated and isolated in 96-well blocks. IPTG induction medium allowed for secretion of sdAb into the supernatant, which was applied to Siglec-8 positive and negative cells. Cells were washed, treated with a fluorophore-labeled anti-Myc secondary antibody, and analyzed by 96-well flow cytometry.

[0173] Nucleic acid sequences encoding sdAbs that bind to Siglec-8 positive cells but not to Siglec-8 negative cells were cloned in frame with the human Fc coding region into a mammalian expression vector and expressed by transient transfection in HEK293 freestyle cells (293F cells) or CHO cells using polyethylenimine. Supernatants were harvested after 3-7 days, and secreted recombinant proteins were purified by protein A chromatography, and concentrations were calculated from the absorbance at 280 nm and extinction coefficient.

[0174] Binding of anti-human Siglec-8 VHHs, B12, D1, E2, and G2, as bivalent VHH-hIgG1-xELL-Fc (VHHs of SEQ ID NOs: 49, 55, 61, and 1 linked to Fc xELL via linker 2, respectively, these constructs are also called cx8982, cx8983, cx8984, and cx10382, respectively) was measured 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 a 96-well plate and anti-Siglec8-VHH-xELL was added dropwise in FACS buffer (PBS 1% BSA, 0.1% NaN3 pH 7.4) and incubated at 4° C. for 30 min. After washing, plates were incubated with A647-conjugated anti-human Fcγ-specific secondary antibody (JacksonImmunoResearch) diluted 1:2000 in FACS buffer for 30 minutes at 4° C. The plates were washed and binding was measured by flow cytometry using Intellicyt iQue Plus to quantify the mean fluorescence intensity of anti-human A647.

[0175] The binding of each anti-human Siglec-8 VHH-xELL Fc construct is shown in Figures 1A-1D. Anti-Siglec8-B12-xELL (Figure 1A) specifically binds to human Siglec-8 expressing CHO cells with a calculated affinity of 0.367 nM, anti-Siglec8-D1-xELL (Figure 1B) specifically binds to human Siglec-8 expressing CHO cells with a calculated affinity of 0.427 nM, anti-Siglec8-E2-xELL (Figure 1C) specifically binds to human Siglec-8 expressing CHO cells with a calculated affinity of 0.331 nM, and anti-Siglec8-G2-xELL (Figure 1D) specifically binds to human Siglec-8 expressing CHO cells with a calculated affinity of 0.268 nM. Binding was specific to Siglec-8 as there was no detectable binding to untransfected CHO cells for any of the constructs tested.

[0176] Example 2: Multivalent anti-Siglec8-Fc antibodies that bind to cells expressing human Siglec-8 To determine whether increasing the valency of Siglec-8 antibodies could enhance agonist activity, the parent llama B12, D1, and E2 VHH sequences were used to construct 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 (cx8467; SEQ ID NO: 52 linked to Fc), and Hex-D2 (cx8468; SEQ ID NO: 53 linked to Fc)) VHHs. We generated Siglec8-VHH-Fc antibodies linked to the low effector function xELL human Fc region via linker 2. Bivalent (Bi-B12 (cx8982), Bi-D1 (cx8983), and Bi-E2 (cx8984)), tetravalent, and hexavalent Siglec8-VHH-Fc antibodies were analyzed by flow cytometry for binding to eosinophils (CD16 - CCR3 +). Cells were isolated from whole blood by centrifugation, washed twice with 2% FBS PBS, resuspended in 1x RBC lysis buffer for 10 min at room temperature (RT), washed once with 2% FBS PBS, treated with a second RBC lysis step, washed twice with 2% FBS. Cells were plated at 200k / well and resuspended in human FC block for 10 min at RT. Cells were spun down and then resuspended in titrated test articles starting at 200nM 1:4 and incubated for 30 min at 4°C before being washed twice and resuspended in secondary mixture (2*; anti-CCR3 FITC, anti-human AF647, PI, anti-CD16 BV605)), incubated for 30 min at 4°C, washed twice, and bound antibodies were detected by flow cytometry (Novocyte).

[0177] As shown in Figures 2A-2C, bivalent, tetravalent, and hexavalent Siglec8-VHH-Fc antibodies B12 (Figure 2A), D1 (Figure 2B), and E2 (Figure 2C) bound to eosinophils. The calculated affinities were similar between bivalent, tetravalent, and hexavalent hzSiglec8-VHH-Fc antibodies. The observed Bmax decreased with increasing valency as expected. This is due to the fact that the Fc portion of the antibody is detected in this assay and the ratio of Fc region to anti-Siglec-8 binding domain is inverse to the valency. Other granulocyte populations (CD16 + CCR3 - ) no binding was observed.

[0178] In further studies, the parent llama G2 sequence was humanized and multivalent hzSiglec8-G2v14-Fc antibodies were cloned with low effector function xELL human Fc. 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 Fc xELL via linker 2) hzSiglec8-G2v14-Fc antibodies were evaluated by flow cytometry using CHO cells transfected with human Siglec-8. A titration series of fusion proteins was incubated with Siglec8-expressing CHO cells (5×10 4 The cells were incubated with 100 μL of 100% PBS (cells / well) for 30 min. After one wash with 150 μL of FACS buffer, APC-conjugated anti-human Fcγ-specific secondary antibody (Jackson ImmunoResearch) was added and the cells were incubated for 30 min at 4° C. After one additional wash in FACS buffer, bound antibody was detected via flow cytometry (iQue Intellicyte).

[0179] As shown in Figure 2D, bivalent, tetravalent, and hexavalent hzSiglec8-G2v14-Fc antibodies bound to CHO cells expressing human Siglec-8. The calculated affinities were similar between bivalent, tetravalent, and hexavalent hzSiglec8-G2v14-Fc antibodies. The observed Bmax decreased with increasing valency as expected. This is due to the fact that the Fc portion of the antibody is detected in this assay and the ratio of Fc region to anti-Siglec-8 binding domain is inverse to the valency.

[0180] Example 3: Increasing the valency of anti-Siglec-8 antibodies results in improved eosinophil killing activity Siglec-8 agonism promotes direct eosinophil killing in the presence of IL-5. To evaluate the effect of valency on Siglec-8-mediated killing activity, eosinophils were isolated from healthy donor whole blood using the StemCell EasySep™ Direct Human Eosinophil Isolation Kit and treated 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 tested were titrated across the plate starting at 50 nM (3-point titration, 1:10 dilution) and isolated eosinophils were resuspended at 160 k / mL in 10% FBS RPMI + Cytotox Red and added at 80 k / well in 48-well plates in the presence of IL-5. Cytotox Red uptake was monitored over time using an Incucyte® cell analyzer and total red object intensity (RCU x μm) was measured at 24 h using the on-board software. 2 The observed killing at 24 hours for cells treated with 5 nM of each test antibody is plotted in Figures 3A-3C. Increasing the valency from bivalent to tetravalent resulted in increased potency of eosinophil killing for all Siglec8-VHH-Fc antibodies tested. Further increase in valency to hexavalent resulted in similar or slightly increased potency compared to tetravalent valency.

[0181] In further testing, isolated eosinophils were added to 96-well plates at 30,000 cells / well, treated with bivalent (cx11422), tetravalent (cx11580), or hexavalent (cx11579) hzSiglec8-G2v14-Fc antibodies, and incubated with test articles titrated across the plate starting at 10 nM in the presence of IL-5. Killing activity was compared to a similarly titrated endogenously derived, defucosylated AK002 (Lilentelimab) analog. To observe eosinophil killing, Cytotox Red was added to each well at a final concentration of 250 nM. Cytotox Red uptake was monitored over time using an Incucyte® cell analyzer, and total red object intensity (RCU×μm) was measured at 18 h using the on-board software. 2 / image) was calculated.

[0182] As shown in Figure 3D, bivalent hzSiglec8-G2v14-Fc (cx11422) was more potent than the defucosylated AK002 (lirentelimab) analog antibody in enhancing eosinophil killing in the presence of IL-5. Increasing the valency of hzSiglec8-G2v14-Fc to tetravalent (cx11580) resulted in a dramatic increase in eosinophil killing potency, resulting in a nearly 30-fold improvement over 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 compared 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).

[0183] Example 4: G2 VHH sequence optimization 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 including hzG2v14 (SEQ ID NO: 9), hzG2v47 (SEQ ID NO: 12), hzG2v51 (SEQ ID NO: 17), hzG2v52 (SEQ ID 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 and compared to that of the defucosylated anti-Siglec-8 2E2 (2E2 Afuc) reference antibody by flow cytometry. Eosinophils were isolated from healthy donor whole blood using the StemCell EasySep™ Direct Human Eosinophil Isolation Kit, diluted in FACS buffer, added at 10k / well to a 96-well assay plate, and incubated for 20 minutes at 4°C with a titration series (10-12 point titration, 1:4 dilution) starting at 10 nM of test article. After one wash in FACS buffer, Alexa Fluor® 647 AffiniPure Donkey Anti-Human Fcγ-specific secondary antibody (Jackson ImmunoResearch) was added and cells were incubated for 20 minutes at 4°C. After one additional wash in FACS buffer, bound antibody was detected via flow cytometry (NovoCyte 3000). SSC 高 / CCR3 + Median fluorescence intensity was calculated using Novoexpress on-board software gating on cells. Dissociation constant (Kd) values ​​from 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 pI of the VHH domains, determined using ExPasy, is shown in Table 3. In general, a higher pI is more favorable for manufacturing.

[0184] Table 3. Humanized tetravalent Siglec-8 antibodies that bind to eosinophils TIFF2025515304000004.tif46128*Determined from 1 to 3 experiments

[0185] As shown in FIG. 4A, the tetravalent anti-Siglec8 antibodies cx11913 and cx11914 inhibited CCR3 in a titration-dependent manner. + Both cx11913 and cx11914 bound to the eosinophil population with approximately 8-fold higher affinity than the 2E2 Afuc reference antibody. As expected, the Bmax of both cx11913 and cx11914 was reduced compared to 2E2 Afuc due to fewer available Fc receptors to be bound by the tetravalent antibody.

[0186] This data demonstrates high affinity binding of further engineered humanized tetravalent anti-Siglec-8 antibodies, including hzG2v47, hzG2v52, hzG2v53, hzG2v54, and hzG2v55, to the target human eosinophil population. In ELISA assays, tetravalent anti-Siglec8 antibodies, including hzG2v47, showed some binding to recombinant Siglec-2 (CD22). The relative cross-reactivity to Siglec-2 was: hzG2v14>hzG2v47≒hzG2v54>hzG2v52≒hzG2v53≒hzG2v55 It was.

[0187] 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 in CHO cells was also assessed by flow cytometry. CHO cells were transiently transfected (by PEI transfection into BalanCD Transfectry CHO cell culture medium (Irvin 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 transmembrane proteins for cell surface expression with co-expression of either Citrine or GFP in the same vector. The next day, a titration series (22 points, 1:3 dilution) starting at 1 μM of test article was incubated with Siglec-expressing cell lines (50k cells / well) in FACS buffer (PBS 1% BSA, 0.1% NaN3 (pH 7.4)) in 96-well plates for 20 min at 4°C. After one wash with FACS buffer, Alexa Fluor® 647 AffiniPure Donkey Anti-Human Fcγ-specific secondary antibody (Jackson ImmunoResearch) was added and cells were incubated for 20 min at 4°C. After one additional wash in FACS buffer, bound antibody was detected via flow cytometry (iQue Intellicyte). Mean fluorescence intensity was calculated using iQue on-board software gating on GFP+ or Citrine+ cells.

[0188] As shown in Figures 4B-4C, the tetravalent anti-human Siglec-8 antibodies cx11913 (containing hzG2v52) and cx11914 (containing hzG2v53) bound with high affinity to Siglec-8 (Figure 4C, top left), but did not bind any other Siglec proteins. At the highest 1 μM concentration tested, cx11913 bound to Siglec-12 (Figure 4C, bottom left), but showed no binding at other concentrations. Both constructs showed a Siglec specificity profile similar to the 2E2 Afuc reference antibody. In contrast, at certain higher concentrations tested, the anti-human Siglec-8 antibody cx11769 (containing hzG2v14) showed cross-reactivity with Siglec-2 and several other Siglec proteins (Figures 4B-4C).

[0189] Example 5: Evaluation of additional humanized G2 VHH constructs The llama anti-Siglec8-G2 VHH domain (lmG2) was humanized to produce additional variants, namely, Siglec8 hzG2v11, Siglec8 hzG2v12, Siglec8 hzG2v13, Siglec8 hzG2v15, Siglec8 hzG2v16, Siglec8 hzG2v17, Siglec8 hzG2v18, Siglec8 hzG2v19, Siglec8 hzG2v20, Siglec8 hzG2v21, Siglec8 hzG2v22, Siglec8 hzG2v24, Siglec8 hzG2v25, Siglec8 hzG2v26, Siglec8 hzG2v28, Siglec8 hzG2v29, 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.

[0190] Binding of lmG2 and humanized versions (further including Siglec8 hzG2v14) formatted as monomeric VHH-hIgG1 fusion proteins using a non-dimerized human IgG1 Fc variant region lacking the hinge Fc NNT was assessed by flow cytometry using 293 cells transiently transfected with full-length human Siglec-8 and detected with anti-human AF647 secondary antibody. Binding specificity was assessed using untransfected 293 cells as a control. For binding assays, Siglec-8 293 cells or untransformed 293 cells were seeded in 96-well U-bottom plates at 50k cells / well in FACS buffer and incubated with a titration of anti-Siglec8-VHH-NNT for 30 min at 4°C. After washing, the plates were incubated with A647-conjugated anti-human Fcγ-specific secondary antibody diluted 1:10,000 in FACS buffer for 20 min at 4°C. Plates were washed and binding was measured by flow cytometry quantified anti-human A647 median fluorescence intensity. Maximum specific binding (Bmax) and affinity (Kd) were determined from the flow cytometry binding data using a nonlinear fit model. Binding curves for each plate are presented in Figures 5A-5H.

[0191] The humanized forms of G2 were well expressed, and almost all variants bound to Siglec-8 with similar affinity to the parental clones, except for hzG2v19-hzG2v21 and Siglec8 hzG2v5.1, as shown in Figures 5A-5D. No binding was observed in non-transfected cells (Figures 5E-5H).

[0192] Example 6: Enhancement of antibody-dependent cellular cytotoxicity (ADCC) activity of anti-Siglec-8 antibodies results in enhanced eosinophil killing activity As demonstrated 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, killing activity is lost in the absence of IL-5. To evaluate the effect of the Fc region on Siglec-8-mediated killing activity, tetravalent anti-Siglec8-VHH Fc antibodies were generated that included humanized G2 VHH, hzG2v53, and either a native human IgG1 Fc region (hIgG1) (cx12532; SEQ ID NO: 27 linked to Fc (see SEQ ID NO: 72)) or an engineered IgG1 Fc region with mutations at two residues S239D and I332E (SDIE) to improve CD16 binding and subsequent antibody-dependent cell-mediated cytotoxicity (ADCC) activity (cx11914; SEQ ID NO: 27 linked to Fc (see SEQ ID NO: 71)). Additionally, a bivalent anti-Siglec8-VHH-Fc antibody was generated that contains hzG2v53 and an SDIE Fc region (cx12562; SEQ ID NO: 26 (see SEQ ID NO: 73) linked to Fc. The hzG2v53-based tetravalent anti-Siglec8-VHH-Fc antibody was evaluated in a direct eosinophil killing assay in the presence of IL-5. Eosinophils were isolated from healthy donor whole blood using the StemCell EasySep™ Direct Human Eosinophil Isolation Kit and killed in 10% FBS in the presence of IL-5 (10 ng / mL final). 30,000 cells / well were added in RPMI and incubated with a titration series of the tested antibodies (11-point titration, 1:4 dilution) starting at 100 nM. Killing activity was compared to a similarly titrated endogenously derived defucosylated AK002 (Lilentelimab) analog. To observe eosinophil killing, Cytotox Red was added to each well at a final concentration of 250 nM. Cytotox Red uptake was monitored over time using an Incucyte® cell analyzer, and the total red object intensity (RCU×μm2 / image) was calculated at 18 hours using the on-board software.

[0193] As shown in Figure 6A, treatment with the humanized tetravalent anti-Siglec-8 antibody construct showed the highest efficacy in the presence of IL-5, indicating that the Fc modifications do not significantly affect direct eosinophil killing activity in the presence of IL-5.

[0194] hzG2v53-based tetravalent and bivalent anti-Siglec8-VHH-Fc antibodies were also evaluated for their ability to promote ADCC of primary human eosinophils in the absence of IL-5, together with a defucosylated AK002 (Lilentelimab) analog. Eosinophils were isolated as described above, and autologous NK cells were isolated from enriched PBMCs using the EasySep™ Human NK Cell Enrichment Kit. Antibodies were titrated into 96-well plates (11-point titration 1:4 dilution). Isolated NK cells (50,000 cells / well) and eosinophils (10,000 cells / well) were plated in 100 μL 10% FBS RPMI+IL-2 (2 ng / mL final) for NK cell stimulation (effector:target ratio 5:1) without the addition of IL-5. The next day, cells were washed and Apotracker Green (Biolegend) was used to label apoptotic target cells for 20 minutes. Cells were then washed, resuspended in FACS buffer, and analyzed on a NovoCyte 3000 flow cytometer (ACEA Biosciences, Inc.).

[0195] As shown in Figure 6B, 6B 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 1-100 pM, with a gradual decline at higher concentrations. SDIE Fc modification dramatically improved ADCC activity compared to tet-hzG2v53-IgG1 with wild-type effector-enabled Fc. The tet-hzG2v53-SDIE antibody showed a slight decrease in maximum ADCC killing activity compared to the bi-hzG2v53-SDIE and 2E2 Afuc antibodies, but as demonstrated above, the tetravalent format showed higher relative potency in the direct killing assay.

[0196] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

[0197] Table of specific sequences TIFF2025515304000005.tif242162TIFF2025515304000006.tif243162TIFF202 5515304000007.tif246162TIFF2025515304000008.tif252162TIFF20255153040 00009.tif252162TIFF2025515304000010.tif249162TIFF2025515304000011.t if245162TIFF2025515304000012.tif255162TIFF2025515304000013.tif168162

Claims

1. A VHH domain that binds to Siglec-8, comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences of a VHH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164.

2. A VHH domain that binds to Siglec-8, (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. The VHH domain comprises the CDR1, CDR2 and CDR3 sequences shown in

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

4. The VHH domain according to any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 18, 29 and 8; SEQ ID NOs: 2, 3 and 8; SEQ ID NOs: 13, 3 and 8; or SEQ ID NOs: 18, 3 and 8, respectively, as defined according to the AbM numbering system.

5. 4. A VHH domain according to any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 74, 80 and 8; SEQ ID NOs: 75, 80 and 8; SEQ ID NOs: 76, 80 and 8; or SEQ ID NOs: 76, 81 and 8, respectively, as defined according to the Chothia numbering system.

6. The VHH domain according to any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 85, 92 and 8; SEQ ID NOs: 86, 92 and 8; SEQ ID NOs: 87, 93 and 8; SEQ ID NOs: 87, 92 and 8; or SEQ ID NOs: 87, 94 and 8, respectively, as defined according to the Kabat numbering system.

7. The VHH domain according to any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are as shown in SEQ ID NOs: 98, 104 and 110; SEQ ID NOs: 99, 104 and 110; SEQ ID NOs: 100, 104 and 110; or SEQ ID NOs: 100, 105 and 110, respectively, as defined according to the Contact numbering system.

8. The VHH domain according to any one of claims 1 to 3, wherein the CDR1, CDR2 and CDR3 sequences are shown in SEQ ID NOs: 114, 120 and 126; SEQ ID NOs: 115, 120 and 126; SEQ ID NOs: 116, 120 and 126; or SEQ ID NOs: 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 NOs: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164.

10. A VHH domain according to any one of claims 1 to 9, which is humanized.

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

1.

12. the CDR1, CDR2, and CDR3 being (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.

12. The VHH domain of claim 11, comprising the amino acid sequence shown in:

13. 13. A VHH domain according to 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 a VHH comprising the amino acid sequence of SEQ ID NO:

49.

15. the CDR1, CDR2, and CDR3 being (a) SEQ ID NOs: 52, 53, and 54, respectively, as defined according to the AbM numbering system; (b) SEQ ID NOs: 77, 82, and 54, respectively, as defined according to the Chothia numbering system; (c) SEQ ID NOs: 88, 95, and 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, and 127, respectively, as defined according to the IMGT numbering system.

15. The VHH domain of claim 14, comprising the amino acid sequence shown in:

16. 16. A VHH domain according to 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 a VHH comprising the amino acid sequence of SEQ ID NO:

55.

18. the CDR1, CDR2, and CDR3 being (a) SEQ ID NOs: 58, 59, and 60, respectively, as defined according to the AbM numbering system; (b) SEQ ID NOs: 78, 83, and 60, respectively, as defined according to the Chothia numbering system; (c) SEQ ID NOs: 89, 96, and 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, and 128, respectively, as defined according to the IMGT numbering system.

18. The VHH domain of claim 17, comprising the amino acid sequence shown in:

19. 19. A VHH domain according to 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 a VHH comprising the amino acid sequence of SEQ ID NO:

61.

21. the CDR1, CDR2, and CDR3 being (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, and 113, respectively, as defined according to the Contact numbering system; or (e) the amino acid sequences of SEQ ID NOs: 119, 124, and 129, respectively, as defined according to the IMGT numbering system.

21. The VHH domain of claim 20, comprising:

22. 22. A VHH domain according to 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 a VHH domain according to any one of claims 1 to 22.

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

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

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

69.

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

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

29. 29. The polypeptide of any one of claims 24 to 28, comprising 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. 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 to Siglec-8 and a multimerization domain.

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

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

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

35. A polypeptide according to 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. 37. The polypeptide of claim 36, wherein each antigen-binding domain is a Fab or an scFv.

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

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

40. 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 to Siglec-8 via an amino acid linker.

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

70.

44. A polypeptide according to any one of claims 39 to 43, comprising the structure VHH-VHH-Fc.

45. 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. A polypeptide according to any one of claims 39 to 43, comprising the structure VHH-Fc-VHH.

47. 44. A polypeptide according to any one of claims 39 to 43, comprising 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 under physiological conditions via the multimerization domain.

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

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

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

52. A VHH, polypeptide or protein according to any one of claims 1 to 51, which binds to 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. A polypeptide or protein according to any one of claims 23 to 53, which mediates eosinophil killing in the presence of IL-5.

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

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

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

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

59. A pharmaceutical composition comprising a polypeptide or protein according to any one of claims 23 to 58 and a pharma- ceutically acceptable carrier.

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

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

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

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

64. 64. A method for producing a polypeptide or protein, comprising incubating a host cell according to claim 62 or 63 under conditions for expressing said polypeptide or protein.

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

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

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

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

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

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

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

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

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

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

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

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

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