SIGLEC-8 BINDING PROTEINS AND USES THEREOF

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

2 . An VHH domain that binds Siglec-8, comprising CDR1, CDR2, and CDR3 sequences 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.

3 . The VHH domain of claim 2 , wherein:

a) the CDR1 comprises the amino acid sequence of SEQ ID NO: 18, 2, or 13; the CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or 3; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 8, as defined according to the AbM numbering system;

b) the CDR1 comprises the amino acid sequence of SEQ ID NO: 74, 75, or 76; the CDR2 comprises the amino acid sequence of SEQ ID NO: 80 or 81; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 8, as defined according to the Chothia numbering system;

c) the CDR1 comprises the amino acid sequence of SEQ ID NO: 85, 86, or 87; the CDR2 comprises the amino acid sequence of SEQ ID NO: 92, 93, or 94; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 8, as defined according to the Kabat numbering system;

d) the CDR1 comprises the amino acid sequence of SEQ ID NO: 98, 99, or 100; the CDR2 comprises the amino acid sequence of SEQ ID NO: 104 or 105; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 110, as defined according to the Contact numbering system; or

e) the CDR1 comprises the amino acid sequence of SEQ ID NO: 114, 115, or 116;

the CDR2 comprises the amino acid sequence of SEQ ID NO: 120 or 121; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 126, as defined according to the IMGT numbering system.

4 . The VHH domain of claim 2 , wherein the CDR1, CDR2, and CDR3 sequences are set forth in:

a) SEQ ID NOs: 18, 29, and 8; 2, 3, and 8; 13, 3, and 8; or 18, 3, and 8, respectively, as defined according to the AbM numbering system;

b) SEQ ID NOs: 74, 80, and 8; 75, 80, and 8; 76, 80, and 8; or 76, 81, and 8, respectively, as defined according to the Chothia numbering system;

c) SEQ ID NOs: 85, 92, and 8; 86, 92, and 8; 87, 93, and 8; 87, 92, and 8; or 87, 94, and 8, respectively, as defined according to the Kabat numbering system;

d) SEQ ID NOs: 98, 104, and 110; 99, 104, and 110; 100, 104, and 110; or 100, 105, and 110, respectively, as defined according to the Contact numbering system; or

e) SEQ ID NOs: 114, 120, and 126; 115, 120, and 126; 116, 120, and 126; or 116, 121, and 126, respectively, as defined according to the IMGT numbering system.

5 - 8 . (canceled)

9 . The VHH domain of claim 2 , comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 5, 11, 16, 21, 31, 36, 130-138, 140-148, 161, 163, and 164.

10 . The VHH domain of claim 2 , wherein the VHH domain is humanized.

11 . An VHH domain that binds Siglec-8, comprising CDR1, CDR2, and CDR3 sequences of a VHH that comprises the amino acid sequence of SEQ ID NO: 1, 49 or 61.

12 - 22 . (canceled)

23 . A polypeptide comprising the VHH domain of claim 2 .

24 . The polypeptide of claim 23 , comprising at least two VHH domains of claim 2 .

25 . The polypeptide of claim 24 , wherein the two VHH domains are operably linked to each other via a peptide linker, a peptide linker comprising the amino acid sequence of SEQ ID NO: 69, or a peptide linker consisting of the amino acid sequence of SEQ ID NO: 69.

26 - 27 . (canceled)

28 . The polypeptide of claim 24 , wherein the VHH domains comprise the same VHH amino acid sequence.

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

30 . The polypeptide of claim 23 , further comprising a multimerization domain.

31 . A polypeptide comprising:

(i) at least two antigen-binding domains that bind Siglec-8 and a multimerization domain; or

(ii) at least three antigen-binding domains that bind Siglec-8.

32 - 37 . (canceled)

38 . The polypeptide of claim 30 , wherein the multimerization domain is an antibody Fc region.

39 . (canceled)

40 . The polypeptide of claim 38 , wherein the antibody Fc region is a human IgG1 Fc region or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44, 45, 65, 66, 67, or 68.

41 - 43 . (canceled)

44 . The polypeptide of claim 38 , wherein the polypeptide comprises the structure VHH-VHH-Fc, VHH-Fc-VHH, VHH-VHH-VHH-Fc, or VHH-VHH-Fc-VHH.

45 . The polypeptide of claim 44 , comprising an amino acid sequence 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 - 47 . (canceled)

48 . A protein comprising two or more polypeptides of claim 30 multimerized under physiological conditions via the multimerization domain.

49 . (canceled)

50 . A protein comprising two polypeptides of claim 38 dimerized under physiological conditions via the dimerization domain.

51 - 58 . (canceled)

59 . A pharmaceutical composition comprising the protein of claim 50 , and a pharmaceutically acceptable carrier.

60 . An isolated nucleic acid that encodes the polypeptide of claim 23 .

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

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

63 . A host cell that expresses the polypeptide of claim 23 .

64 . A method of producing a polypeptide or protein, the method comprising incubating the host cell of claim 62 under conditions for expression of the polypeptide or protein.

65 . (canceled)

66 . A method of treating an eosinophilic disorder or a mast cell disorder, an inflammatory disease or condition, or an allergic condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 59 .

67 - 73 . (canceled)

74 . A method of depleting eosinophils in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein of claim 50 .

75 . A method of killing an eosinophil, the method comprising contacting the eosinophil with a natural killer (NK) cell in the presence of the polypeptide or protein of claim 50 .

76 . A method of killing an eosinophil, the method comprising contacting the eosinophil with a macrophage in the presence of the protein of claim 50 .