Siglec-9ECD fusion molecules and methods of use thereof

Abstract

The present disclosure is generally directed to Siglec-9ECD and Siglec-9ECD fusion molecules, and methods of treatment using Siglec-9ECD and Siglec-9ECD fusion molecules.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 930,227, filed November 4, 2019, No. 63 / 014,940, filed April 24, 2020, and No. 63 / 092,753, filed October 16, 2020, all of which are incorporated by reference herein for all purposes.

[0002] Field The present disclosure relates to Siglec-9ECD fusion molecules and therapeutic uses of such fusion proteins. [Background technology]

[0003] background Sialic acid-binding Ig-like lectin-9 (Siglec-9) is a type 1 immunoglobulin-like transmembrane protein expressed on immune and hematopoietic cells, including immature and mature myeloid cells such as monocytes, macrophages, dendritic cells, neutrophils, and microglial cells, as well as lymphoid cells such as natural killer cells and a subset of T cells (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; O'Reilly and Paulson (2009) Trends in Pharm. Sci. 30:5:240-248; and Macauley et al. (2014) Nat. Rev. Imm. 14:653-666). Siglec-9 is a member of the Siglec family of lectins that bind to sialic acid residues on glycoproteins and glycolipids. Potential ligands for Siglec proteins are gangliosides, which are glycolipids that contain ceramides linked to sialylated glycans. Diversity in Siglec ligands arises through the addition of other natural sugars and sialic acids at various linkages, either branched or terminal, as well as modification of the sialic acid itself.

[0004] Fourteen Siglec proteins have been identified in humans and nine in mice, which are composed of 2-17 extracellular Ig domains including an amino-terminal V-set Ig-like (IgV) domain that contains a sialic acid-binding site. The IgV domain contains two aromatic residues and an arginine in a motif that is highly conserved in all Siglecs (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; Von Gunten and Bochner (2008) Ann NY Acad Sci. 1143:61-82; May et al. (1998) Mol Cell. 1:719-728; Crocker et al. (1999) Biochem J. 341:355-361; and Crocker and Varki (2001) Trends Immunol. 2:337-342). Ligand-binding sites have been mapped by ligand-bound and unliganded crystal structures (Attrill et al., (2006) J. Biol. Chem.281 32774-32783; Alphey et al. (2003) J. Biol. Chem. 278:5 3372-3377; Varki et al., Glycobiology, 16 pp. 1R-27R; and May et al. (1998) Mol. Cell 1:5:719-728). Since cell membranes are rich in sialic acid, ligand binding by Siglecs can occur in cis and trans, influencing their functional properties. Each Siglec has distinct preferences for binding to the diverse types of sialylated glycans found on the surface of mammalian cells (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; and Crocker et al. (2007) Nat Rev Immunol. 7:255-266).

[0005] Many Siglec proteins, including Siglec-9, are inhibitory receptors that contain one or more immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in their cytoplasmic domains. Inhibitory Siglecs act as negative regulators of immune function (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; and Von Gunten and Bochner (2008) Ann NY Acad Sci. 1143:61-82). Other Siglecs are activating receptors that contain immunoreceptor tyrosine-based activation motif (ITAM) sequences in their cytoplasmic domains. These Siglecs act as positive regulators of immune function (Macauley SM. et al., (2014) Nature Reviews Immunology 14, 653-666).

[0006] The Siglec protein family plays a role in tumor pathogenesis. Many human tumors strongly upregulate sialic acid ligands that bind to Siglec-9, which may enable immune evasion and cancer progression (Jandus et al. (2014) J. Clinic. Invest. 124:1810-1820). In contrast, tumors lacking sialic acid biosynthesis have reduced growth in mice (Stanczak et al. (2018) J Clin Invest.128:4912-4923). Certain SNPs in Siglec-3, 7, and 9 are associated with a reduced risk of colorectal and lung cancer (ibid.).

[0007] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the Invention

[0008] The present disclosure is generally directed to Siglec-9 extracellular domain (ECD) fusion proteins and methods of using Siglec-9ECD fusion proteins to treat cancer and neurodegenerative diseases.

[0009] In some embodiments, the isolated polypeptide comprises a Siglec-9 IgV domain comprising an amino acid sequence selected from any one of SEQ ID NOs: 109-137 and 214-226. In some embodiments, the polypeptide comprises a Siglec-9 extracellular domain (ECD) comprising a Siglec-9 IgV domain, a C2 type 1 (C2T1) domain, and a C2 type 2 (C2T2) domain. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 79-107 and 194-206. In some embodiments, the Siglec-9 IgV domain polypeptide does not comprise the membrane proximal region of Siglec-9 as set forth in SEQ ID NO: 147 (MPR).

[0010] In some embodiments, the polypeptide further comprises an Fc domain. In some such embodiments, the Fc domain is located at the C-terminus of the polypeptide. In some embodiments, the Fc domain has an IgG1 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 142 or 143. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the isolated polypeptide comprises an Fc domain having a human IgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ADCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), relative to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc domain has an IgG4 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs:145-146.

[0011] In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 11-39, 148-160, and 168-170. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 49-77, 171-183, and 191-193. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 49-77 and 171-193, without its signal peptide.

[0012] In some embodiments, an isolated polypeptide is provided comprising a Siglec-9IgV domain comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, an isolated polypeptide is provided comprising a Siglec-9IgV domain comprising the amino acid sequence of SEQ ID NO: 138, optionally further comprising an Fc domain located at the C-terminus of the polypeptide. Optionally, the Fc domain has a human IgG1 isotype. Optionally, the polypeptide further comprises a linker sequence. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 142-144. In some embodiments, the Fc domain comprises an amino acid sequence of SEQ ID NO: 142 or 143. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 139. In some embodiments, the Fc domain has an IgG4 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 145-146.

[0013] In some embodiments, an isolated polypeptide is provided that comprises a Siglec-9 IgV domain comprising the amino acid sequence of SEQ ID NO: 78 attached at its C-terminus to an Fc domain. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 227. In some embodiments, the Fc domain has an IgG1 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 142 or 143. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the isolated polypeptide comprises an Fc domain having a human IgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ADCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), relative to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc domain has an IgG4 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NO: 145-146. In some embodiments, an isolated polypeptide is provided comprising a Siglec-9 IgV domain comprising an amino acid sequence selected from any one of SEQ ID NO: 45-48 and 228-233, and not including its associated signal peptide. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 45-48 and 228-233. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 45, and not including its associated signal peptide. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 48, and not including its associated signal peptide.

[0014] In some embodiments, an isolated polypeptide is provided that comprises a Siglec-9 IgV domain, comprising an amino acid sequence of any one of SEQ ID NOs: 207-213 and an Fc domain disposed at the C-terminus of the polypeptide. In some embodiments, the Fc domain has an IgG1 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc domain comprises an amino acid sequence of SEQ ID NO: 142 or 143. In some embodiments, the Fc domain comprises an amino acid sequence of SEQ ID NO: 142. In some embodiments, the isolated polypeptide comprises an Fc domain having a human IgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ADCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), relative to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises an amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc domain has an IgG4 isotype. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 145-146. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 161-167. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 184-190 and does not include a signal peptide. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 184-190.

[0015] In any of the embodiments of the isolated polypeptide comprising the Siglec-9IgV domain provided herein, the polypeptide can bind to sialic acid on the surface of a cell. In some such embodiments, the cell is a tumor cell. In some embodiments, the cell expresses an FcR, e.g., FcRγIIA. In some embodiments, the cell is a bone marrow cell. In some embodiments, the bone marrow cell is selected from a monocyte, a macrophage, a dendritic cell, a microglial cell, and a myeloid-derived suppressor cell (MDSC).

[0016] In any of the embodiments of the isolated polypeptide comprising a Siglec-9IgV domain provided herein, the polypeptide comprises: a) blocking cellular binding of any one or more Siglec family members selected from Siglec-3, Siglec-5, Siglec-7, Siglec-9, Siglec-10, and Siglec-15; b) optionally relieving MDSC-mediated suppression of T cells, as determined by measuring increased IFNγ expression or increased T cell proliferation; c) repolarizing MDSCs towards a proinflammatory phenotype; d) increasing the expression of CD86 on MDSCs, increasing the expression of CD11b on MDSCs, and / or decreasing the expression of CD163 on MDSCs; e) repolarizing tumor macrophages away from the M2 phenotype; f) reducing CD163+ and / or CD206+ macrophages; g) inducing expression of one or more chemokines selected from CCL3, CCL4, CCL5, CCL17, CXCL1, CXCL9, and IL-8 in MDSCs; h) reducing the recruitment of myeloid cells to the tumor microenvironment; i) binds to MDSCs with an affinity of less than 100 nM, less than 50 nM, less than 25 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, 1-50 nM, 1-25 nM, 1-20 nM, 1-10 nM, 1-5 nM, or 1-2 nM; or j) Do one or more of (a) to (i). In some such embodiments, the MDSCs are human MDSCs and / or the macrophages are human macrophages.

[0017] In some embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding an isolated polypeptide comprising a Siglec-9 IgV domain provided herein. In some embodiments, the isolated nucleic acid encodes an amino acid sequence selected from any one of SEQ ID NOs: 48-77, 171-193, and 228-233. In some embodiments, the isolated nucleic acid encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 10-39, 148-170, and 227. In some embodiments, an expression vector is provided comprising the isolated nucleic acid.

[0018] In some embodiments, a host cell is provided that comprises the isolated nucleic acid or expression vector provided herein. In some embodiments, a host cell is provided that expresses an isolated polypeptide comprising the Siglec-9IgV domain provided herein. In some embodiments, a method for producing a polypeptide is provided, comprising culturing a host cell. In some such embodiments, the polypeptide is isolated.

[0019] In various embodiments, a pharmaceutical composition is provided that includes an isolated polypeptide comprising the Siglec-9IgV domain provided herein and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition may include (i) a polypeptide as described herein, including its signal peptide, or (ii) a polypeptide without its signal peptide; and a pharma- ceutically acceptable carrier.

[0020] In some embodiments, a method of treating cancer is provided comprising administering to a subject having cancer an isolated polypeptide comprising a Siglec-9IgV domain provided herein or a pharmaceutical composition comprising the polypeptide. In some embodiments, the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells. In some embodiments, the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer, and uterine cancer. In some embodiments, the method further comprises administering an antagonist of PD-1 or PD-L1, optionally wherein the antagonist of PD-1 or PD-L1 is an antibody that binds to PD-1 or PD-L1, respectively. In some embodiments, the method further comprises administering a chemotherapeutic agent.

[0021] In some embodiments, a method for treating a neurological or neurodegenerative disease is provided, comprising administering to a subject having a neurological or neurodegenerative disease an isolated polypeptide comprising the Siglec-9IgV domain provided herein or a pharmaceutical composition comprising the polypeptide. In some embodiments, the neurological or neurodegenerative disease is characterized by dysfunctional or defective microglial cells. In some embodiments, the neurological or neurodegenerative disease is selected from dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, and mild cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, taupathy disease, multiple sclerosis, immune-mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, childhood-onset leukoencephalopathy, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

[0022] In some embodiments, a method is provided for repolarizing myeloid-derived suppressor cells (MDSCs) to a proinflammatory phenotype in a subject, comprising administering to a subject having a neurological or neurodegenerative disease an isolated polypeptide comprising a Siglec-9IgV domain as provided herein or a pharmaceutical composition comprising the polypeptide. In some such embodiments, the subject has cancer. In some embodiments, the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells. In some embodiments, the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer, and uterine cancer. In some cases, the cancer is metastatic. In some embodiments, the subject has a neurological or neurodegenerative disease. In some embodiments, the neurological or neurodegenerative disease is characterized by dysfunctional or defective microglial cells. In some embodiments, the neurodegenerative disease is selected from dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, and mild cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, tauopathy, multiple sclerosis, immune-mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, childhood-onset leukoencephalopathy, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

[0023] In some embodiments, a method of repolarizing tumor macrophages from an M2 phenotype in a subject having cancer is provided, comprising administering to the subject an isolated polypeptide comprising a Siglec-9IgV domain provided herein or a pharmaceutical composition comprising the polypeptide. In some embodiments, the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells. In some embodiments, the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer, and uterine cancer. In some cases, the cancer is metastatic.

[0024] In some embodiments, a method of activating myeloid cells in a subject is provided, comprising administering to the subject an isolated polypeptide comprising a Siglec-9IgV domain provided herein or a pharmaceutical composition comprising the polypeptide. Optionally, the myeloid cells are microglial cells. In some embodiments, the subject has cancer. In some embodiments, the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells. In some embodiments, the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer, and uterine cancer. Optionally, the cancer is metastatic. In some embodiments, the subject has a neurological or neurodegenerative disease. In some embodiments, the neurological or neurodegenerative disease is characterized by dysfunctional or defective microglial cells. In some embodiments, the neurodegenerative disease is selected from dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, and mild cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, tauopathy, multiple sclerosis, immune-mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, childhood-onset leukoencephalopathy, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). [Brief description of the drawings]

[0025] [Figure 1] Shown is surface expression of Siglec-9 on tumor-infiltrating T cells, macrophages, and granulocytes from representative lung adenocarcinoma samples. [Diagram 2]1 shows the amino acid sequence of human Siglec-9 (SEQ ID NO:1). From N-terminus to C-terminus, the signal peptide sequence is in bold; the IgV ligand binding domain is underlined with the conserved Arg shown in shaded bold; the intervening sequence is in bold italics (ALTHR; SEQ ID NO:3); the C2 type 1 domain is in italics; the intervening sequence is in bold italics (LNVSYP; SEQ ID NO:4); and the C2 type 2 domain is underlined and italics. The ITIM motif (LQYASL; SEQ ID NO:5) and the SLAM-like (TEYSEI; SEQ ID NO:6) motif are underlined and shaded. The transmembrane domain is predicted to occur at amino acids 349-369 of SEQ ID NO:1. [Diagram 3] 1 shows the in silico calculated properties of certain engineered Siglec9-IgV variants at pH 7.4, 100 mM NaCl concentration, and 298 K, as described in Example 5. [Figure 4] 1 shows IFNγ expression by T cells contacted with S9.1-hIgG1 (S9-hIgG1) and co-cultured alone or with myeloid-derived suppressor cells (MDSCs) as described in Example 9. [Diagram 5] 1 shows IFNγ expression by T cells contacted with antibodies against Siglec-3 (aS3), Siglec-7 (aS7), Siglec-9 (aS9-1 and aS9-2), a combination of aS3, aS7, and aS9-2, or S9.1-hIgG1 (S9-hIgG1), either alone or co-cultured with MDSCs, as described in Example 10. [Figure 6] FIG. 14 shows IFNγ expression by T cells co-cultured with MDSCs in the presence of increasing concentrations of S9.1-hIgG1 (S9-hIgG1) or S9.A-hIgG1 LALAPS (S9-hIgG1 LALAPS), as described in Example 11. [Figure 7]As shown in Example 12, CCL5 (top) and CCL17 (bottom) expression from MDSCs contacted with S9.A-hIgG1 (S9-hIgG1) or S9.A-hIgG1 NSLF (S9-hIgG1 NSLF) is shown. [Figure 8] Shown are CD86 (top) and CD163 (bottom) expression from MDSCs contacted with S9.A-hIgG1 (S9-hIgG1) or S9.A-hIgG1 NSLF (S9-hIgG1 NSLF) as described in Example 13. [Figure 9A] 1 shows the percentage of CD14+CD163+ macrophages relative to total CD45+ cells in mice treated with S9.1-hIgG1 (S9-IgG1), S9.A-hIgG1 NSLF (S9-hIgG1 NSLF), or hIgG1 isotype control, as described in Example 14. [Figure 9B] 1 shows the percentage of CD14+CD206+ macrophages relative to total CD45+ cells in mice treated with S9.1-hIgG1 (S9-IgG1), S9.A-hIgG1 NSLF (S9-hIgG1 NSLF), or hIgG1 isotype control, as described in Example 14. [Figure 9C] As shown in Example 14, FIG. 1 shows surface expression of CD206 on CD14+ macrophages in mice treated with S9.1-hIgG1 (S9-IgG1), S9.A-hIgG1 NSLF (S9-hIgG1 NSLF), or hIgG1 isotype control. [Figure 10] 1 shows the number of platelets, neutrophils, lymphocytes, and monocytes per microliter of blood in mice treated with S9.1-hIgG1 (S9-hIgG1), S9.A-hIgG1 NSLF (S9-hIgG1 NSLF), or hIgG1 isotype control, as described in Example 15. [Figure 11]As shown in Example 16, shows tumor growth in transgenic C57BL / 6 mice expressing human Siglec-3, Siglec-7, and Siglec-9 (S3 / 7 / 9BAC) implanted with MC38 cells and treated with anti-PD-L1 antibody. [Figure 12] As shown in Example 17, tumor growth in transgenic C57BL / 6 mice expressing human Siglec-3, Siglec-7, and Siglec-9 implanted with MC38 cells and treated with S9.B-mIgG2a (S9-mIgG2a) is shown. [Figure 13] As shown in Example 18, shows tumor growth in transgenic C57BL / 6 mice expressing human Siglec-3, Siglec-7, and Siglec-9 implanted with MC38 cells and treated with anti-PD-L1 antibody or a combination of S9.B-mIgG2a (S9-mIgG2a) and anti-PD-L1 antibody. [Figure 14] As shown in Example 19, FIG. 1 shows binding of Fc fusions containing the extracellular domains (ECDs) of Siglec-3 (S3-mIgG1), Siglec-5 (S5-mIgG1), Siglec-7 (S7-mIgG1), Siglec-9 (S9-mIgG1), and Siglec-10 (S10-mIgG1) to the surface of MDSCs in the presence of increasing concentrations of S9.1-hIgG1. [Figure 15]An exemplary model of the mechanism of action of Siglec-9-ECD-Fc fusion molecule (Siglec-9-Fc) is shown. Siglec-9-Fc binds to a ligand (sialic acid) on cancer cells via its Siglec-9ECD portion (left panel). Based on the studies herein, and without being bound by theory, it is believed that Siglec-9-Fc binds to both FcR (e.g., FcγRIIA) and a ligand (sialic acid) expressed on myeloid cells (right panel). Binding occurs via the Fc portion and the Siglec-9ECD portion of the Siglec-9-Fc molecule, respectively, by cooperative binding (or cis) interactions. As a result, Siglec-9-Fc binds to myeloid cells with higher affinity than cells that do not express FcR, resulting in preferential targeting to myeloid cells in vivo and activation of myeloid cells. [Figure 16A] As shown in Example 19, S9.A-mIgG1 (S9-mIgG1) has been shown to uniquely repolarize MDSCs compared to other Siglec-Fc fusions. Each set of bars, from left to right, is S3-mIgG1, S5-mIgG1, S7-mIgG1, S9-mIgG1, S10-mIgG1, and mIgG1. [Figure 16B] As shown in Example 19, S9.A-mIgG1 (S9-mIgG1) has been shown to uniquely repolarize MDSCs compared to other Siglec-Fc fusions. Each set of bars, from left to right, is S3-mIgG1, S5-mIgG1, S7-mIgG1, S9-mIgG1, S10-mIgG1, and mIgG1. [Figure 17] 1 shows detection of sialic acid expression on tumor samples by immunohistochemistry (IHC). [Figure 18] Binding of various Siglec-9-Fc variants to A375 tumor cells and repolarization of myeloid-derived suppressor cells (MDSCs) as measured by CD86 upregulation and CD163 downregulation are shown. Also shown are production yield and stability as measured by melting temperature and percent monomer for each variant. [Figure 19]Correlation between CD86 induction in MDSC assay and A375 tumor cell binding (19A), correlation between production yield and A375 tumor cell binding (19B), and correlation between stability and A375 tumor cell binding (19C) are shown for various Siglec-9-Fc variants. [Figure 20] 1 shows a reduction in pulmonary nodules in S3 / 7 / 9BAC mice injected intravenously with B16F10 murine melanoma cells and treated with S9.B-mIgG2a (S9-Fc) compared to S3 / 7 / 9BAC mice injected intravenously with B16F10 murine melanoma cells and treated with an isotype control. [Figure 21] 14 shows that S9.B-mIgG2a monotherapy inhibits tumor growth in the E0771 syngeneic breast cancer model compared to isotype control. [Figure 22A] The results of flow cytometry experiments to determine the effect of Fcγ receptor engagement on the binding of Siglec-9-Fc to myeloid-derived suppressor cells (MDSCs) are shown. Binding of Siglec-9-hIgG1 (S9-hIgG1) NSLF (diamonds) to MDSCs is shown to be in the low nM range. Binding curves with isotype controls (triangles) are also shown in each figure panel. [Figure 22B] The results of flow cytometry experiments to determine the effect of Fcγ receptor engagement on the binding of Siglec-9-Fc to myeloid-derived suppressor cells (MDSCs) are shown. The binding of the Fc-silent Siglec-9-hIgG1 LALAPS is approximately 75-fold weaker than that of Siglec-9-hIgG1 NSLF. Binding curves with isotype controls (triangles) are also shown in each figure panel. [Figure 22C]To determine the effect of Fcγ receptor engagement on the binding of Siglec-9-Fc to myeloid-derived suppressor cells (MDSC), the results of flow cytometry experiments are shown. Binding of Siglec-9-hIgG1 (SEQ ID NO: 40) to a reference cancer cell line A549 that does not express any Fcγ receptors is shown. Binding curves with isotype controls (triangles) are also shown in each panel of the figure. [Figure 23] The results of exposure of a panel of sialic acid-containing glycans to various Siglec Fc fusion molecules, including Siglec-9-hIgG1, are shown. Darker shading indicates a higher degree of binding. As the figure shows, the Siglec-9-hIgG1 molecule binds to a variety of sialic acid moieties in contrast to other Siglec fusion molecules. [Figure 24A] Binding of Siglec-9-hIgG1 and Siglec-9-hIgG1 NSLF to blood cells is compared. Binding of the two molecules to blood monocytes is compared based on mean fluorescence intensity (MFI). [Figure 24B] Binding of Siglec-9-hIgG1 and Siglec-9-hIgG1 NSLF to blood cells is compared, showing that MFI correlates with binding to several blood cell types. [Figure 25A] Figure 1 shows the effect of Siglec-9-hIgG1 NSLF on T cell proliferation, showing that the presence of MDSCs inhibited T cell proliferation in two donor samples, which was restored in each sample by Siglec-9-hIgG1 NSLF. [Figure 25B] FIG. 1 shows the effect of Siglec-9-hIgG1 NSLF on T cell proliferation. A dose-response curve is presented to determine the EC50 of Siglec-9-hIgG1 NSLF in restoring T cell proliferation, which is approximately 1-2 nM. [Figure 26]These results show that when Siglec-9-hIgG NSLFs were incubated with MDSCs and T cells, Siglec-9-hIgG1 NSLFs demonstrated approximately 10-fold enhanced potency in inducing interferon gamma (IFN-g) compared to Siglec-9-hIgG1. [Figure 27] We show that when incubated with MDSCs, Siglec-9-hIgG1 NSLF induces a robust gene expression profile, and that this profile is consistent with macrophage repolarization. [Figure 28A] We show that Siglec-9-hIgG1 NSLF induces increased Ml polarization (upregulation of CD86) compared to anti-Siglec15, anti-PD-L1, and anti-LILRB2 antibodies. [Figure 28B] We show that Siglec-9-hIgG1 NSLF causes decreased M2 polarization (downregulation of CD206) compared to anti-Siglec15, anti-PD-L1, and anti-LILRB2 antibodies. [Figure 29] Siglec-9-mIgG2a, in combination with anti-PD-L1 antibody, has been shown to reduce the growth of implanted E0771 breast tumor cells in mice more than isotype control or either Siglec-9-mIgG2a or anti-PD-L1 antibody alone. [Diagram 30] The effect of Siglec-9-mIgG2a (upward pointing triangles) or isotype control (mIgG2a) (squares) on CD86 (A) or CDllb (B) expression from splenic bone marrow cells is shown. [Diagram 31] Several additional Siglec-9-Fc variants are characterized. [Figure 32A] Variants S9.36, S9.37 and S9.38 were shown to behave similarly to Siglec-9-Fc-hIgG1 (black bars, second from the left) and show reduced CD163 expression compared to the isotype control (leftmost hatched bar). [Figure 32B]Variants S9.36, S9.37 and S9.38 were shown to behave similarly to Siglec-9-Fc-hIgG1 (black bar, second from the left) and show reduced CD206 expression compared to the isotype control (leftmost hatched bar). [Figure 32C] Variants S9.36, S9.37 and S9.38 were shown to behave similarly to Siglec-9-Fc-hIgG1 (black bars, second from the left) and show increased CD86 expression compared to the isotype control (leftmost hatched bar). [Diagram 33] 1 shows the mean concentration-time profiles of Siglec-9-hIgG1 (filled circles) and Siglec-9-hIgG1 NSLF (open squares) in cynomolgus monkey serum. [Diagram 34] 1 shows the kinetic profiles of multiple Siglec-9-Fc variants following intravenous bolus injection into Siglec3 / 7 / 9BAC transgenic mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the following detailed description.

[0027] Detailed Description Provided herein is a polypeptide comprising the extracellular domain of Siglec-9 and a fusion partner, e.g., an Fc domain. Unexpectedly, Siglec-9ECD-Fc fusion molecules exhibit cooperative binding to myeloid cells, resulting in potent activation of these innate immune cells, as compared to antibodies against Siglec-9 or other Siglec proteins. Such activation is useful, for example, in the treatment of cancer, neurodegenerative disorders, and other diseases and disorders in which the immune system may otherwise be inappropriately suppressed. Further provided herein is a polypeptide comprising variants of the Siglec-9 extracellular domain, particularly in the IgV domain, that have been engineered to improve stability, solubility, ligand binding, and / or other properties. Such variants are useful in fusion molecules to activate immune responses, as described above. Other inventions and embodiments are further described herein.

[0028] definition The terms "Siglec-9 extracellular domain" and "Siglec-9ECD" refer to an extracellular domain polypeptide of Siglec-9 or a fragment thereof that binds to sialic acid on the surface of a cell. The terms include naturally occurring and engineered variants thereof. In some embodiments, Siglec-9ECD comprises an IgV domain of Siglec-9. In some embodiments, Siglec-9ECD comprises an IgV domain of Siglec-9 as well as a C2 type 1 (C2T1) domain and a C2 type 2 (C2T2) domain. Non-limiting exemplary Siglec-9ECDs are set forth in SEQ ID NOs: 78-138.

[0029] The term "Siglec-9ECD fusion molecule" refers to a molecule comprising Siglec-9ECD and a covalently linked fusion partner, such as an Fc domain, albumin, or polyethylene glycol (PEG). In some embodiments, the fusion partner is attached to the C-terminus of Siglec-9ECD. Siglec-9ECD fusion molecules in which the fusion partner is an Fc domain may also be referred to herein as "Siglec-9ECD-Fc fusion molecule," "Siglec-9ECD-Fc," or "Siglec-9-Fc." Non-limiting exemplary Siglec-9ECD-Fc fusion molecules are set forth in the amino acid sequences of SEQ ID NOs: 10-77 and 139, including sequences with or without their associated signal peptides.

[0030] The term "specific binding" or "specifically binds" or "specific for" a target moiety means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a test molecule for a target moiety compared to the binding of the test molecule for a control moiety. A test molecule specifically binds to a target moiety if the binding affinity for the target moiety is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold higher than the binding affinity for the control moiety. For the avoidance of doubt, specific binding does not require that the test molecule does not bind to any other moiety.

[0031] For example, an "amino acid modification" at a specified position of Siglec-9ECD of the present disclosure refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a specified residue means an insertion of up to 1-2 residues therefrom. The insertion may be on the N-terminal or C-terminal side of the specified residue. A preferred amino acid modification herein is a substitution.

[0032] The term "Fc region" is used herein to mean the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined to include the polypeptide from an amino acid residue at position Cys226, or Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the Fc region-containing polypeptide, or by recombinantly engineering a nucleic acid encoding the Fc region-containing polypeptide. Suitable native sequence Fc regions for use in the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0033] 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.

[0034] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution, e.g., from about 1 to about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions, in the native sequence Fc region compared to the native sequence Fc region. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region, most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0035] "Fc receptor" or "FcR" describes a receptor that binds to an Fc region. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds to an IgG Fc region (gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, with FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but 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. Other FcRs are encompassed by the term "FcR" herein. FcRs can also increase the serum half-life of molecules that contain an Fc region.

[0036] The in vivo FcR binding and serum half-life of human FcR high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcR, or in primates administered with polypeptides having variant Fc regions. WO2004 / 42072 (Presta) describes Fc region variants with improved or reduced binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).

[0037] As used herein, "percent amino acid sequence identity" and "homology" with respect to a reference polypeptide sequence refer to the percentage of amino acid residues in a query sequence that are identical to those in the reference 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 to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms known in the art that are required to achieve maximum alignment over the entire length of the sequences being compared.

[0038] An "isolated" nucleic acid molecule encoding a polypeptide, such as a polypeptide comprising Siglec-9ECD of the present disclosure, is a nucleic acid molecule that is identified and separated from at least one contaminant molecule that is normally associated with the environment in which it occurs. Preferably, an isolated nucleic acid is free from association with most or substantially all components associated with the production environment. An isolated nucleic acid molecule encoding a polypeptide herein is distinguished from a nucleic acid that is naturally present in a cell.

[0039] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A "plasmid" is a type of vector and refers to a circular double stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors", or simply "expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector.

[0040] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0041] A "host cell" includes, for example, an individual cell or cell culture that can contain a vector(s) or other exogenous nucleic acid incorporating a polynucleotide insert(s). In some embodiments, the vector or other exogenous nucleic acid is integrated into the genome of the host cell. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells that contain (e.g., are transfected with) a polynucleotide(s) of the invention.

[0042] "Carrier" as used herein includes pharma- ceutically acceptable carriers, additives, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. In many cases, the physiologically acceptable carrier is a pH-buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0043] As used herein, the term "preventing" includes providing prophylaxis with respect to the occurrence or recurrence of a particular disease, disorder, or condition in an individual who is predisposed to, susceptible to, or at risk of developing a particular disease, disorder, or condition, but has not yet been diagnosed as having the disease, disorder, or condition.

[0044] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable disease or disease symptoms, and may or may not show detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" indicates that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a particular disease, disorder, or condition, as known in the art. Individuals who have one or more of these risk factors are more likely to develop a particular disease, disorder, or condition than individuals who do not have one or more of these risk factors.

[0045] As used herein, the terms "treat", "treatment", "treating" and the like refer to a clinical intervention designed to alter the natural course of a clinical pathology in the individual being treated. Desirable effects of treatment include slowing the rate of progression, improving or remission of the pathological condition, remission or improvement of prognosis, and / or alleviating or reducing the symptoms of a particular disease, disorder, or condition. For example, an individual is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated. In certain embodiments, a patient is successfully "treated" for cancer by the methods of the present invention if the patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; an inhibition or absence of cancer cell invasion into peripheral organs, including, for example, the spread of cancer to soft tissue and bone; an inhibition or absence of tumor metastasis; an inhibition or absence of tumor growth; a reduction in one or more symptoms associated with a specific cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumorigenicity, tumor frequency, or tumorigenic potential of a tumor; a reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival rate (DFS), overall survival (OS), complete response (CR), partial response (PR), or stable disease (SD); a reduction in progressive disease (PD); a reduction in time to progression (TTP); or any combination thereof.

[0046] The terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of therapeutic agents, such as Siglec-9ECD fusion molecules (e.g., Siglec-9ECD-Fc fusion molecules). Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0047] An "effective amount" refers to at least an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. The effective amount herein can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual. An effective amount is also an amount in which any toxic or detrimental effects of the treatment are outweighed by the therapeutic beneficial effects. In prophylactic use, advantageous or desired results include results such as eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes during disease development. In therapeutic applications, advantageous or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug via targeting, etc., delaying the progression of the disease, and / or prolonging survival. An effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment either directly or indirectly.As understood in clinical context, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition.Therefore, "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if it can or does achieve the desired result in combination with one or more other agents.

[0048] An "individual" or "subject" or "patient" for the purposes of treating, preventing, or reducing risk refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual is a human.

[0049] The terms "cancer" and "cancerous" refer to and describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell proliferation. A cancer may be a primary tumor or an advanced or metastatic cancer. A "refractory" cancer is one that progresses despite anti-tumor treatment being administered to a cancer patient. A "recurrent" cancer, or a cancer that is "recurring," is one that is regrowing at the original site or at a distant site after a response to an initial treatment. A "recurrent" patient is one that has signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant treatment.

[0050] As used herein, administration of an agent or composition "in conjunction with" or "in combination with" another agent or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a simultaneous formulation or as separate compositions, including at different dosing frequencies or intervals and using the same or different routes of administration. In some embodiments, concurrent administration refers to administration as part of the same treatment regimen. In some embodiments, administration of an agent in combination with another agent results in a "synergistic" or "synergistic effect", i.e., the effect achieved when the agents are used together is greater than the sum of the effects resulting from using the agents separately. In some embodiments, administration of an agent in combination with another agent results in an "additive" effect, i.e., the effect achieved when the agents are used together is equal to the sum of the effects resulting from using the agents separately.

[0051] The term "about" as used herein refers to a normal error range for each numerical value, which is readily apparent to one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to the numerical value or parameter itself.

[0052] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise.

[0053] It is understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0054] Polypeptides Containing the Siglec-9 Extracellular Domain In some embodiments, a Siglec-9ECD or Siglec-9ECD fusion molecule according to any of the embodiments herein may incorporate any of the features as described herein, singly or in combination.

[0055] Provided herein is a polypeptide comprising a Siglec-9 IgV domain. In certain embodiments, the Siglec-9 IgV domain comprises amino acids 20-140 of human Siglec-9 of SEQ ID NO: 1. See FIG. 2. As shown in Example 2 herein, the IgV domain of Siglec-9 is sufficient to bind sialic acid on the surface of a cell. In some embodiments, a polypeptide is provided comprising a Siglec-9 extracellular domain (ECD) comprising an IgV domain, a C2 type 1 (C2T1) domain, and a C2 type 2 (C2T2) domain. The Siglec-9 C2T1 domain comprises amino acids 146-229 of human Siglec-9 of SEQ ID NO: 1, and the Siglec-9 C2T2 domain comprises amino acids 236-336 of human Siglec-9 of SEQ ID NO: 1. In some embodiments, the Siglec-9 ECD comprises amino acids 20-336 of SEQ ID NO: 1, optionally with one or more amino acid modifications. In some embodiments, the Siglec-9 ECD comprises amino acids 20-336 of SEQ ID NO: 1, optionally with one or more amino acid modifications, and optionally with 1-5 amino acid deletions or additions at the N-terminus and / or C-terminus. In some embodiments, the Siglec-9 ECD may comprise the IgV, C2T1 and C2T2 domains, but may not comprise, for example, the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 C-terminal (membrane proximal) amino acids of the ECD. Those 12 C-terminal (membrane proximal) amino acids of the ECD are set forth in SEQ ID NO: 147. One example is SEQ ID NO: 78, which, for example, comprises the IgV, C2T1 and C2T2 domains, and does not comprise the C-terminal membrane proximal region.

[0056] In some embodiments, the polypeptide comprises a Siglec-9IgV domain comprising one or more amino acid substitutions that improve the stability of the polypeptide, improve the binding affinity for sialic acid, improve the function of the polypeptide, improve the pharmacokinetic properties (e.g., half-life, Cmax, or AUC) of the polypeptide, or any combination of the foregoing. In some embodiments, the polypeptide comprises a Siglec-9IgV domain having an amino acid sequence selected from any one of SEQ ID NOs: 108-137 and 214-226. In some embodiments, the polypeptide comprises a Siglec-9IgV domain having an amino acid sequence selected from any one of SEQ ID NOs: 109-137 and 214-226. In some embodiments, the polypeptide comprises a Siglec-9IgV domain having an amino acid sequence selected from any one of SEQ ID NOs: 108-137 and 214-226, optionally with 1-5 amino acid deletions or additions at the N-terminus and / or C-terminus. In some embodiments, the polypeptide comprises a Siglec-9 IgV domain having an amino acid sequence selected from any one of SEQ ID NOs: 109-137 and 214-226, optionally with 1-5 amino acid deletions or additions at the N-terminus and / or C-terminus. In some embodiments, the polypeptide comprises a Siglec-9 ECD with one or more substitutions C-terminal to the IgV domain. For example, in some embodiments, the polypeptide comprises a Siglec-9 ECD of any one of SEQ ID NOs: 207-213. The sequence table below shows sequences corresponding to the SEQ ID NOs listed herein. In many cases, the location of the amino acid substitution is indicated in the table by underlining the mutated residue or by bolding and underlining it.

[0057] In some embodiments, the polypeptide comprises a Siglec-9 ECD comprising one or more amino acid substitutions that improve the stability of the polypeptide, improve the binding affinity for sialic acid, improve the function of the polypeptide, improve the pharmacokinetic properties of the polypeptide, or any combination of the foregoing. In some embodiments, the polypeptide comprises a Siglec-9 ECD having an amino acid sequence selected from any one of SEQ ID NOs: 78-107, 138, and 194-206. In some embodiments, the polypeptide comprises a Siglec-9 ECD having an amino acid sequence selected from any one of SEQ ID NOs: 78-107, 138, 194-206, optionally with 1-5 amino acid deletions or additions at the N-terminus and / or C-terminus.

[0058] In any of the embodiments provided herein, the polypeptide may further comprise a fusion partner. Non-limiting exemplary fusion partners include an Fc domain, albumin, and polyethylene glycol (PEG). In some embodiments, the fusion partner is covalently linked to the C-terminus of Siglec-9ECD. In some aspects, the fusion partner comprises an Fc domain. In some embodiments, a polypeptide comprising Siglec-9ECD and an Fc domain is provided herein, where the Fc domain is optionally fused to the C-terminus of Siglec-9ECD with or without an intervening linker sequence. "Linker sequence" as used herein refers to a polypeptide sequence not found in native Siglec-9ECD or its fusion partner (e.g., Fc domain), where such a polypeptide sequence is disposed between Siglec-9ECD and its fusion partner. In some embodiments, the linker sequence may be between about 4 and 25 amino acids. In some embodiments, the Fc domain is fused to the C-terminus without a linker sequence. In various embodiments, the polypeptide comprises a Siglec-9ECD and an IgG1 Fc domain, e.g., an IgG1 Fc domain of SEQ ID NO: 142. In some embodiments, the polypeptide comprising Siglec-9ECD comprises an IgG1 Fc domain that includes an NSLF substitution, e.g., SEQ ID NO: 143. In some embodiments, the polypeptide comprising Siglec-9ECD comprises an IgG1 Fc domain that includes a K322A substitution, e.g., SEQ ID NO: 144. In some embodiments, the polypeptide comprising Siglec-9ECD comprises an IgG4 Fc domain, e.g., as set forth in SEQ ID NOs: 145 or 146, respectively, or an IgG4 Fc domain that includes a S228P substitution.

[0059] In some embodiments, a Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 10-39, 148-160, and 168-170. In some embodiments, a Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 40-77, 171-183, and 191-193, optionally without a signal sequence.

[0060] In some embodiments, the Siglec-9ECD IgV domain of the Siglec-9ECD or Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 109-137 and 214-226. Optionally, the Siglec-9ECD comprises an IgV, C2T1, and C2T2 domain. In some embodiments, the Siglec-9ECD does not comprise a membrane proximal region (MPR) of SEQ ID NO: 147. In some embodiments, the Siglec-9ECD comprises an IgV, C2T1, and C2T2 domain and does not comprise an MPR. In some embodiments, the Siglec-9ECD comprises an amino acid sequence selected from any one of SEQ ID NOs: 79-107 and 194-206. In some embodiments, the Siglec-9ECD comprises an amino acid sequence selected from any one of SEQ ID NOs: 79-107 and 194-206 and does not comprise an MPR of SEQ ID NO: 147. In some embodiments, the Siglec-9ECD consists of an amino acid sequence selected from any one of SEQ ID NOs: 79-107 and 194-206. In some aspects, the Siglec-9ECD is part of a Siglec-9ECD fusion molecule comprising an ECD and a fusion partner. In some embodiments, the fusion partner is Fc, albumin, or PEG. In some embodiments, the fusion partner is Fc. In some embodiments, the fusion partner is Fc and is disposed at the C-terminus of the molecule (i.e., Fc is attached to the C-terminus of Siglec-9ECD directly or via a linker). In some embodiments, the Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype, having (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), relative to the IgG1 polypeptide of SEQ ID NO: 142.In some embodiments, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is a human IgG4 with or without a S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146.

[0061] In some embodiments, the Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 49-77 and 171-193, and does not include a signal sequence. In some embodiments, the Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 49-77 and 171-193, and does not include a signal sequence. In some embodiments, the Siglec-9ECD fusion molecule consists of an amino acid sequence selected from any one of SEQ ID NOs: 49-77 and 171-193, and does not include a signal sequence. In some embodiments, the Siglec-9ECD fusion molecule consists of an amino acid sequence selected from any one of SEQ ID NOs: 49-77 and 171-193, and does not include a signal sequence.

[0062] In some embodiments, Siglec-9ECD or Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, Siglec-9ECD does not comprise the membrane proximal region (MPR) sequence of SEQ ID NO: 147. Optionally, Siglec-9ECD consists of the amino acid sequence of SEQ ID NO: 138. Optionally, Siglec-9ECD comprises or consists of the amino acid sequence of SEQ ID NO: 138, without a signal sequence, where Siglec-9ECD is expressed from a nucleic acid encoding SEQ ID NO: 138, including a signal sequence. Optionally, Siglec-9ECD is a Siglec-9ECD fusion molecule comprising an ECD and a fusion partner. In some such embodiments, the fusion partner may be Fc, albumin, or PEG. In some embodiments, the fusion partner is Fc. In some embodiments, the fusion partner is Fc and is located at the C-terminus of the molecule (i.e., Fc is attached directly or via a linker to the C-terminus of Siglec-9ECD). In some embodiments, the Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), compared to the IgG1 polypeptide of SEQ ID NO: 142. Optionally, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is human IgG4 with or without a S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO:139.

[0063] In some embodiments, the Siglec-9ECD or Siglec-9ECD fusion molecule comprises the sequence of SEQ ID NO: 78. In some embodiments, the Siglec-9ECD does not comprise the membrane proximal region sequence of SEQ ID NO: 147 (MPR). Optionally, the Siglec-9ECD consists of the amino acid sequence of SEQ ID NO: 78. Optionally, the Siglec-9ECD is a Siglec-9ECD fusion molecule comprising an ECD and a fusion partner. In some such embodiments, the fusion partner may be Fc, albumin, or PEG. In some embodiments, the fusion partner is Fc. In some embodiments, the fusion partner is F and is disposed at the C-terminus of the molecule (i.e., Fc is attached to the C-terminus of the Siglec-9ECD directly or via a linker). In some embodiments, the Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c) compared to the IgG1 polypeptide of SEQ ID NO: 142. Optionally, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is a human IgG4 with or without a S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146.

[0064] In some cases, a Siglec-9ECD fusion molecule comprises a Siglec-9ECD of SEQ ID NO: 78 linked at its C-terminus, optionally via a linker or directly, to an Fc domain or another fusion partner such as albumin or PEG. In some embodiments, SEQ ID NO: 78 is linked directly to an Fc domain at its C-terminus. In some embodiments, SEQ ID NO: 78 is linked at its C-terminus via a linker to an Fc domain. In some embodiments, a Siglec-9ECD fusion molecule comprises the sequence of SEQ ID NO: 78 linked at its C-terminus to a human IgG1 or IgG4 isotype Fc domain, such as an Fc comprising any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype having: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c) compared to the IgG1 polypeptide of SEQ ID NO: 142. Optionally, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc domain comprises SEQ ID NO: 143. In some embodiments, the Fc is human IgG4 with or without a S228P substitution. In some embodiments, the Fc domain comprises SEQ ID NO: 145. In some embodiments, the Fc domain comprises SEQ ID NO: 146. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 227. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO:227.

[0065] In some embodiments, a Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 78 attached at its C-terminus to an Fc domain, wherein the molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 45-48 and 228-233, and does not include its associated signal peptide. In some embodiments, a Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 78 attached at its C-terminus to an Fc domain, wherein the molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 45-48 and 228-233, and does not include its associated signal peptide. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 45. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 48. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 228. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 228. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 229. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 229. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 230. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 231. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 231. In some embodiments, the molecule comprises the amino acid sequence of SEQ ID NO: 232 ...3. In some embodiments, the molecule consists of the amino acid sequence of SEQ ID NO: 233.

[0066] In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 218. In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 198. In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 218 or 198 and does not comprise the membrane proximal region (MPR) sequence of SEQ ID NO: 147. In some embodiments, Siglec-9ECD consists of the amino acid sequence of SEQ ID NO: 198. In some embodiments, Siglec-9ECD is a Siglec-9ECD fusion molecule comprising an ECD and a fusion partner. In some such embodiments, the fusion partner may be Fc, albumin, or PEG. In some embodiments, the fusion partner is Fc. In some embodiments, the fusion partner is Fc, which is located at the C-terminus of the molecule (i.e., Fc is attached directly or via a linker to the C-terminus of Siglec-9ECD). In some embodiments, Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), compared to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is a human IgG4 with or without a S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146. In some embodiments, the Siglec-9ECD or Siglec-9ECD fusion molecule comprises a signal sequence. In other embodiments, it does not. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 152.In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 152. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 168. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 175. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 191.

[0067] In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 219. In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 199. In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 219 or 199 and does not include the membrane proximal region (MPR) sequence of SEQ ID NO: 147. In some embodiments, Siglec-9ECD consists of the amino acid sequence of SEQ ID NO: 199. In some embodiments, Siglec-9ECD is a Siglec-9ECD fusion molecule comprising an ECD and a fusion partner. In some such embodiments, the fusion partner may be Fc, albumin, or PEG. In some embodiments, the fusion partner is Fc. In some embodiments, the fusion partner is Fc and is located at the C-terminus of the molecule (i.e., Fc is attached to the C-terminus of Siglec-9ECD directly or via a linker). In some embodiments, Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), compared to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is a human IgG4 with or without a S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146. In some embodiments, the Siglec-9ECD or Siglec-9ECD fusion molecule comprises a signal sequence. In other embodiments, it does not. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 153.In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 153. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 169. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 192. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 192.

[0068] In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 220. In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 200. In some embodiments, Siglec-9ECD comprises the sequence of SEQ ID NO: 220 or 200 and does not comprise the membrane proximal region (MPR) sequence of SEQ ID NO: 147. In some embodiments, Siglec-9ECD consists of the amino acid sequence of SEQ ID NO: 200. In some embodiments, Siglec-9ECD is a Siglec-9ECD fusion molecule comprising an ECD and a fusion partner. In some such embodiments, the fusion partner may be Fc, albumin, or PEG. In some embodiments, the fusion partner is Fc. In some embodiments, the fusion partner is Fc and is located at the C-terminus of the molecule (i.e., Fc is attached to the C-terminus of Siglec-9ECD directly or via a linker). In some embodiments, Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype with: (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), compared to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is a human IgG4 with or without a S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146. In some embodiments, the Siglec-9ECD or Siglec-9ECD fusion molecule comprises a signal sequence. In other embodiments, it does not. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 154.In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 154. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 170. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, the Siglec-9ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, the Siglec-9ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 193.

[0069] In some embodiments, the Siglec-9ECD fusion molecule comprises an amino acid sequence of any one of SEQ ID NOs: 207-213 linked at its C-terminus to an Fc domain. In some embodiments, the linkage is direct. In other cases, it is via a linker. In some embodiments, the Fc is human IgG1 (hIgG1). In some embodiments, the Fc comprises an amino acid sequence of any one of SEQ ID NOs: 142-144 and 234-239. In some embodiments, the Fc comprises an amino acid sequence of SEQ ID NO: 142. In some embodiments, the Fc domain has a hIgG1 isotype with (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), relative to the IgG1 polypeptide of SEQ ID NO: 142. In some embodiments, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the Fc is human IgG4 with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 145 or 146. In some embodiments, the Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 161-167. In some embodiments, the Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 184-190, and does not include its associated signal peptide. In some embodiments, the Siglec-9ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 184-190, and does not include its associated signal peptide.

[0070] Exemplary Fc Domains In some embodiments of any of the Siglec-9ECD fusion molecules provided herein, the fusion molecule may comprise an Fc domain, hi some embodiments, the Fc domain is of the human IgG1, IgG2, IgG3, and / or IgG4 isotype.

[0071] In certain embodiments of any of the Siglec-9ECD fusion molecules provided herein, the Fc domain has an IgG1 isotype. In some embodiments, the Siglec-9ECD fusion molecule contains a mouse IgG1 Fc domain. In some embodiments, the Siglec-9ECD fusion molecule comprises a human IgG1 Fc domain (hIgG1), for example, as set forth in SEQ ID NO: 142. In some embodiments, the human IgG1 Fc domain of the Siglec-9ECD fusion molecule binds to an activating Fc receptor. In certain embodiments, the activating Fc receptor is selected from any one or more of FcγRI, FcγRIIa and IIc, and FcγRIIIa and IIIb.

[0072] In some embodiments, the human IgG1 Fc domain of the Siglec-9ECD fusion molecule does not bind or has reduced binding to FcγRIII (CD16) and / or Clq. In some embodiments, the human IgG1 Fc domain of the Siglec-9ECD fusion molecule has reduced antibody-dependent cellular cytotoxicity (ADCC) and / or complement fixation activity, respectively, which in each case may reduce undesired killing of cells to which the Siglec-9ECD fusion molecule binds, e.g., myeloid cells. This effect may be achieved by certain amino acid modifications, e.g., "NSLF" mutations, in which the IgG1 Fc domain contains the mutations N325S and L328F (according to EU numbering of the IgG1 Fc domain), e.g., as shown in SEQ ID NO: 143. In another embodiment, the human IgG1 Fc domain contains a mutation corresponding to K322A (EU numbering), e.g., as presented in SEQ ID NO: 144.

[0073] Exemplary modifications to the IgG1 Fc domain are listed in Table A below. [Table 1]

[0074] For example, in some embodiments, the Fc domain has a hIgG1 isotype with (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), relative to the IgG1 polypeptide of SEQ ID NO: 142. Optionally, the Fc domain comprises SEQ ID NO: 143. Optionally, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions.

[0075] In some embodiments, substitutions and changes may also be made in the Fc region of Siglec-9-hIgG1 NSLF (see, e.g., SEQ ID NO: 45), for example, to improve its binding to FcRn in vitro and thus potentially improve its ability to be recycled in vivo. Exemplary substitutions and changes include "YTE" and "LS" substitutions, as well as cysteine-containing loop insertions, as described in Dall' Acqua et al. (2002) J. Immunol. 169:5171-5180; Zalevsky et al. (2010) Nat. Biotechnol. 28:157-159; and U.S. Patent No. 9,688,756, each of which is incorporated herein by reference in their entirety. In some embodiments, the Fc domain may comprise a sequence as set forth in SEQ ID NOs: 228-230 (substitutions and changes are indicated by double underlining in the sequence listing herein). The modified constructs can be tested in vitro for improved binding to FcRn, for example by surface plasmon resonance, and then studied in vivo for pharmacokinetics (PK) and pharmacodynamics (PD). The modified Fc constructs may contain "YTE" or "LS" substitutions or cysteine-containing loop insertions in the Fc rather than NSLF substitutions. Such constructs are shown in SEQ ID NOs: 231-233.

[0076] In certain embodiments of any of the Siglec-9ECD fusion molecules provided herein, the Fc domain has an IgG2 isotype. In some embodiments, the Siglec-9ECD fusion molecule contains a mouse IgG2 Fc domain, such as mouse IgG2a (mIgG2a). In some embodiments, the Siglec-9ECD fusion molecule contains a human IgG2 Fc domain (hIgG2). In some embodiments, the human IgG2 Fc domain of the Siglec-9ECD fusion molecule binds to an activating Fc receptor. In certain embodiments, the activating Fc receptor is selected from any one or more of FcγRI, FcγRIIa and Ile, and FcγRIIIa and IIIb.

[0077] In certain embodiments of any of the Siglec-9ECD fusion molecules provided herein, the Fc domain has an IgG4 isotype. In some embodiments, the Siglec-9ECD fusion molecule comprises a human IgG4 Fc domain (hIgG4), e.g., as set forth in SEQ ID NO: 145. In some embodiments, the human IgG4 Fc region of the Siglec-9ECD fusion molecule binds to an activating Fc receptor. In certain embodiments, the activating Fc receptor is selected from any one or more of FcγRI, FcγRIIa and IIe, and FcγRIIIa and IIIb. In certain embodiments, the human IgG4 Fc region comprises a mutation corresponding to S228P (according to EU numbering), e.g., as set forth in SEQ ID NO: 146.

[0078] Polypeptide variants In some embodiments of any of the polypeptides provided herein, amino acid sequence variants are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide.

[0079] Substitution, insertion, and deletion variants In some embodiments of any of the polypeptides provided herein, polypeptide variants are provided that have one or more amino acid substitutions.The amino acid sequence variants of a polypeptide can be prepared by introducing appropriate modifications into the nucleotide sequence that codes for the polypeptide or by peptide synthesis.Such modifications include, for example, deletion of and / or insertion of and / or substitution of residues in the amino acid sequence of a polypeptide. [Table 2]

[0080] Modification of the biological properties of a polypeptide can be achieved by selecting substitutions that differ in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are selected based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe They can be classified into the following groups.

[0081] For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member from another class. Such substituted residues can be introduced, for example, into regions of the human polypeptide that are homologous with the non-human polypeptide, or into the non-homologous regions of the molecule.

[0082] In altering the polypeptides described herein, in certain embodiments, the hydrophobicity index of amino acids can be taken into consideration. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0083] The importance of the hydrophobic amino acid index in conferring interactive biological function to a protein is understood in the art. Kyte et al. J. Mol. Biol., 157:105-131(1982). It is known that certain amino acids can be substituted for other amino acids with similar hydrophobicity index or score and still retain similar biological activity. When making changes based on hydrophobicity index, certain embodiments include substitution of amino acids with hydrophobicity index within ±2. Certain embodiments include within ±1, and certain embodiments include within ±0.5.

[0084] It is also understood in the art that substitutions of like amino acids can be effectively made on the basis of hydrophilicity, particularly with the biologically functional proteins or peptides so produced being contemplated for use in immunological embodiments, as in this case. In certain embodiments, the greatest local average hydrophilicity of a protein, as influenced by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0085] These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0±1); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, certain embodiments include substitutions of amino acids with hydrophilicity values ​​within ±2, certain embodiments include those within ±1, and certain embodiments include those within ±0.5.

[0086] Amino acid sequence insertions include amino- and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0087] Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability.

[0088] Other Polypeptide Modifications In some embodiments of any of the polypeptides, the polypeptide is a derivative. The term "derivative" refers to a molecule that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative contains a covalent modification, including, but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified polypeptide may have a longer circulating half-life than a polypeptide that is not chemically modified. In certain embodiments, a chemically modified antigen binding protein may have improved targeting capabilities for a desired cell, tissue, and / or organ. In some embodiments, a derivative polypeptide is covalently modified to contain one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In certain embodiments, the derivative polypeptide comprises one or more polymers including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers.

[0089] In certain embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits.In certain embodiments, one or more water-soluble polymers are attached to one or more specific positions of the derivative, for example, the amino terminus.In certain embodiments, one or more water-soluble polymers are attached randomly to one or more side chains of the derivative.In certain embodiments, PEG is used to improve the therapeutic ability of polypeptide.A certain such method is discussed, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0090] Nucleic Acids, Vectors, and Host Cells The Siglec-9ECD fusion molecules of the present disclosure can be produced using recombinant methods and compositions. In some embodiments, an isolated nucleic acid having a nucleotide sequence encoding any of the Siglec-9ECD fusion molecules of the present disclosure is provided. For example, the nucleic acid herein can encode a polypeptide of any one of SEQ ID NOs: 10-39, 78, 138, 148-170, and 227. The nucleic acid herein can encode an amino acid sequence selected from any one of SEQ ID NOs: 45-77, 171-193, and 228-233.

[0091] In some embodiments, the nucleic acid encodes a Siglec-9ECD fusion molecule comprising a signal sequence. In some embodiments, the signal sequence is a native signal sequence. A native human Siglec-9 signal sequence is set forth in SEQ ID NO: 140. In some embodiments, the signal sequence is a non-native signal sequence. Those skilled in the art will appreciate that any signal sequence that adequately provides for intracellular transport of the encoded polypeptide, cleavage of the signal sequence, and secretion of the encoded polypeptide from the cell can be used. In some such embodiments, the nucleic acid encodes a Siglec-9ECD fusion molecule comprising a signal sequence that improves intracellular transport of the encoded polypeptide, cleavage of the signal sequence, and / or secretion (efficiency and / or yield) of the encoded polypeptide compared to the native human Siglec-9 signal sequence. In some such embodiments, the nucleic acid encodes a Siglec-9ECD fusion molecule comprising a signal sequence, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the signal sequence of SEQ ID NO: 141 improves production of the Siglec-9ECD fusion molecule.

[0092] In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 10. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 45. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 48. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 138. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 139. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 227. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 228. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 229. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 230. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 231. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 232. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:233. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:48. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:198. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:199. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:200. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:218. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:219. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:220.In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 152. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 153. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 154. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 168. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 169. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 170. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 175. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 176. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 177. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 191. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 192. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO:193.

[0093] In some embodiments, one or more vectors (e.g., expression vectors) are provided that contain any of the aforementioned nucleic acids. In some embodiments, host cells that contain such nucleic acids are also provided. In some embodiments, the host cell contains (e.g., is transduced with) a vector that contains a nucleic acid encoding a Siglec-9ECD fusion molecule. In some embodiments, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

[0094] The present disclosure provides a method for producing a Siglec-9ECD fusion molecule. In some embodiments, the method comprises culturing a host cell of the present disclosure that comprises a nucleic acid encoding a Siglec-9ECD fusion molecule under conditions suitable for expression of the Siglec-9ECD fusion molecule. In some embodiments, the Siglec-9ECD fusion molecule is then recovered from the host cell (or host cell culture medium).

[0095] For recombinant production of the Siglec-9ECD fusion molecules of the present disclosure, nucleic acids encoding the Siglec-9ECD fusion molecules are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures.

[0096] Suitable vectors comprising a nucleic acid sequence encoding any of the disclosed Siglec-9ECD fusion molecules include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed by standard techniques or can be selected from a large number of cloning vectors available in the art. The cloning vector selected can vary depending on the host cell intended to be used, but useful cloning vectors generally have the ability to replicate autonomously, may have a single target for a specific restriction endonuclease, and / or may carry a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0097] Suitable host cells for cloning or expressing vectors encoding Siglec-9ECD fusion molecules include prokaryotic or eukaryotic cells. For example, the Siglec-9ECD fusion molecules of the present disclosure can be produced in eukaryotes, particularly where glycosylation and Fc effector functions contribute to the activity of the molecule.

[0098] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of Siglec-9ECD fusion molecules with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for vectors encoding Siglec-9ECD fusion molecules (e.g., Gerngross Nat. Biotech. 22:1409-1414 (2004); and Li et al. Nat. Biotech. 24:210-215 (2006)).

[0099] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line used herein to recombinantly produce the Siglec-9ECD fusion molecules of the Examples (293 cells, or, e.g., 293 cells as described in Graham et al. J. Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); e.g., Mather et al. Annals NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al. Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0.

[0100] Exemplary Activities of Siglec-9ECD Fusion Molecules Provided herein is a polypeptide that comprises Siglec-9ECD, wherein the polypeptide binds to sialic acid on the surface of a cell.The polypeptide that comprises Siglec-9ECD can be a Siglec-9ECD fusion molecule, such as Siglec-9ECD-Fc fusion molecule.The polypeptide that comprises Siglec-9ECD can bind to the cell that comprises sialic acid on the surface with an affinity (Kd) of less than 100nM, or less than 90nM, or less than 80nM, or less than 70nM, or less than 60nM, or less than 50nM, or less than 40nM, or less than 30nM. In some embodiments, the polypeptide binds to cells that contain sialic acid on their surface with an affinity (Kd) of 0.1-100 nM, or 0.1-90 nM, or 0.1-80 nM, or 0.1-70 nM, or 0.1-60 nM, or 0.1-50 nM, or 0.1-40 nM, or 0.1-30 nM. In some embodiments, the Siglec-9ECD or Siglec-9ECD fusion molecule may bind to MDSC with a Kd of, for example, less than 100 nM, or less than 90 nM, or less than 80 nM, or less than 70 nM, or less than 60 nM, or less than 50 nM, or less than 40 nM, or less than 30 nM, or less than 25 nM, or less than 20 nM, or less than 10 nM, or less than 5 nM, or less than 2 nM, or 0.1-50 nM, or 1-50 nM, or 1-25 nM, or 1-20 nM, or 1-10 nM, or 1-5 nM, or 1-2 nM. In various embodiments, the cell is a myeloid-derived suppressor cell (MDSC). In some cases, the MDSC is a human MDSC.

[0101] A non-limiting exemplary assay for determining affinity is as follows: MDSCs, such as human MDSCs, are isolated and incubated with a polypeptide comprising a dose-determined amount of Siglec-9ECD-Fc fusion molecule. A fluorescently tagged anti-Fc domain antibody (e.g., an antibody that binds to IgG1 Fc domain) is used for detection, and binding is evaluated by flow cytometry. In some embodiments, a non-human Fc domain (e.g., mouse IgG1 Fc domain) is used in the fusion molecule to reduce the background binding of fluorescently tagged anti-Fc domain antibody to MDSCs. An exemplary assay is provided in Example 7.

[0102] In some embodiments, the polypeptide comprising Siglec-9ECD repolarizes myeloid-derived suppressor cells (MDSCs). The polypeptide comprising Siglec-9ECD can be a Siglec-9ECD fusion molecule, such as a Siglec-9ECD-Fc fusion molecule. Repolarization of MDSCs can be determined, for example, by measuring an increase in chemokine expression from MDSCs incubated with the polypeptide. Non-limiting exemplary chemokines whose expression can be increased to indicate repolarization of MDSCs include CCL3, CCL4, CCL5, CCL17, CXCL1, CXCL9, and IL-8. Assays for determining repolarization can measure the expression of one, two, three, four, five, or more chemokines. Repolarization of MDSCs can also be determined by measuring the expression of CD86 and / or CD163 expression on MDSCs cultured in the presence of a polypeptide comprising Siglec-9ECD. CD86 is a proinflammatory marker, and an increase in CD86 expression is consistent with repolarization of MDSCs. CD163 is an M2 macrophage marker, and a decrease in CD163 expression is consistent with a repolarization of MDSCs to a proinflammatory phenotype. An exemplary assay is provided in Example 8.

[0103] In some embodiments, the polypeptide comprising Siglec-9ECD relieves MDSC-mediated suppression of T cells. The polypeptide comprising Siglec-9ECD can be a Siglec-9ECD fusion molecule, such as a Siglec-9ECD-Fc fusion molecule. A non-limiting exemplary assay for determining relief of MDSC-mediated suppression of T cells is as follows: MDSCs are isolated and cultured with the polypeptide, for example, for 48 hours. The MDSCs are then co-cultured with isolated T cells (e.g., CD8+ T cells) and a T cell activator, such as Dynabeads® Human T-Activator CD3 / CD28. T cell activation can be determined by measuring IFNγ expression. In some embodiments, when MDSCs are incubated with a polypeptide comprising Siglec-9ECD, IFNγ expression is increased compared to a control polypeptide, indicating T cell activation. An exemplary assay is provided in Example 9.

[0104] In some embodiments, the polypeptide comprising Siglec-9ECD blocks other Siglecs from binding to MDSC. In some such embodiments, the polypeptide blocks Siglec-3, Siglec-5, Siglec-7, Siglec-9, and / or Siglec-10 from binding to MDSC. Binding can be measured, for example, using the flow cytometry assay described herein for measuring Kd. An exemplary assay is provided in Example 19.

[0105] In some embodiments, the Siglec-9ECD fusion molecule may comprise the amino acid sequence of SEQ ID NO: 78, with or without a signal sequence, linked at its C-terminus to an Fc domain, either directly or via a linker molecule, such as the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 227, or any one of SEQ ID NOs: 45-48 and 228-233. In some such cases, the molecule may bind to MDSCs, such as human MDSCs, with a Kd of, for example, less than 100 nM, or less than 90 nM, or less than 80 nM, or less than 70 nM, or less than 60 nM, or less than 50 nM, or less than 40 nM, or less than 30 nM, or less than 25 nM, or less than 20 nM, or less than 10 nM, or less than 5 nM, or less than 2 nM, or 0.1-50 nM, or 1-50 nM, or 1-25 nM, or 1-20 nM, or 1-10 nM, or 1-5 nM, or 1-2 nM.

[0106] For example, in some embodiments, the Fc domain has a human IgG1 isotype with (a) reduced binding to FcγRIII; (b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement fixation activity; (c) increased binding to FcγRIIa; or any combination of a), b), and / or c), compared to the IgG1 polypeptide of SEQ ID NO: 142. Optionally, the Fc domain comprises SEQ ID NO: 143. Optionally, the Fc domain comprises a human IgG1 isotype with N325S and L328F (NSLF) substitutions. In some such cases, such molecules may also have increased potency in inducing IFNγ production in the presence of MDSCs, compared to Siglec-9ECD having the same amino acid sequence but bound at its C-terminus to a hIgG1 wild-type Fc molecule. In some embodiments, the molecule may relieve MDSC-mediated suppression of T cells, for example, as determined by measuring increased IFNγ expression or increased T cell proliferation. In some cases, such molecules may increase the expression of CD86 on MDSCs and / or decrease the expression of CD206 on MDSCs. In some cases, such molecules may also bind to MDSCs, such as human MDSCs, with a Kd lower than that of a molecule containing the same amino acid sequence of Siglec-9ECD but bound to hIgG1 wild type Fc at its C-terminus.

[0107] Pharmaceutical Compositions / Formulations Provided herein is a pharmaceutical composition comprising a Siglec-9ECD fusion molecule, such as a Siglec-9ECD-Fc fusion molecule of the present disclosure, and a pharma- ceutically acceptable carrier. In some embodiments, provided herein is a pharmaceutical composition comprising a Siglec-9ECD fusion molecule of the present disclosure having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed.

[0108] In various embodiments, pharmaceutical compositions comprising Siglec-9ECD fusion molecules are provided in a formulation with a pharma- ceutically acceptable carrier (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., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0109] therapeutic use As disclosed herein, Siglec-9ECD fusion molecules, for example, the Siglec-9ECD-Fc fusion molecules of the present disclosure, can be used to prevent, reduce the risk of, or treat diseases and disorders. In addition, Siglec-9ECD fusion molecules, for example, the Siglec-9ECD-Fc fusion molecules of the present disclosure, can be used in a method of repolarizing myeloid-derived suppressor cells (MDSCs) to a proinflammatory phenotype, for example, where the subject has cancer or a neurological or neurodegenerative disease, as described below. Siglec-9ECD fusion molecules, for example, the Siglec-9ECD-Fc fusion molecules of the present disclosure, can also be used in a method of activating myeloid cells, for example, where the subject has cancer or a neurological or neurodegenerative disease, as described below. Siglec-9ECD fusion molecules, such as the Siglec-9ECD-Fc fusion molecules of the present disclosure, can further be used in methods of repolarizing tumor macrophages away from the M2 phenotype in a subject with cancer as described herein.

[0110] In one embodiment of the invention, a Siglec-9ECD fusion molecule, e.g., a Siglec-9ECD-Fc fusion molecule, is used as a therapeutic agent. The therapeutic regimen is carried out by identifying a subject, e.g., a human patient, suffering from (or at risk of developing) a disease or disorder that would benefit from treatment with a Siglec-9ECD fusion molecule.

[0111] As described in more detail below, Siglec-9ECD fusion molecules, such as Siglec-9ECD-Fc fusion molecules, can be used in combination with additional therapeutic agents used to treat the diseases or conditions presented herein. The terms "in combination" and "in conjunction" are used interchangeably in this disclosure. Additional therapeutic agents administered in combination with Siglec-9ECD fusion molecules can be administered before, after, or simultaneously with the Siglec-9ECD fusion molecules.

[0112] In some embodiments, the disease or disorder to be treated is cancer. In certain embodiments, the cancer is a solid tumor. Solid tumors may be associated with a tumor microenvironment that includes myeloid cells, such as macrophages, monocytes, microglial cells (in the CNS), dendritic cells, neutrophils, and / or granulocytes. In certain embodiments, the tumor microenvironment includes macrophages and monocytes. In certain embodiments, myeloid cells create an immunosuppressive tumor microenvironment that allows tumors to evade the immune system. Treatment with Siglec-9ECD fusion molecules herein can alleviate this suppression by activating myeloid cells and promoting anti-tumor immune responses.

[0113] In certain embodiments, cancers that are prevented or treated by the methods of the present disclosure include, but are not limited to, gastric cancer or stomach cancer, including squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-squamous NSCLC, glioma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, and gastrointestinal stromal cancer. cancer), kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, sarcoma, pancreatic cancer, brain cancer (e.g., astrocytoma such as glioblastoma (glioblastoma multiforme)), cervical cancer, bladder cancer, hepatocellular carcinoma, breast cancer (e.g., triple negative breast cancer), and head and neck cancer (squamous cell carcinoma of the head and neck), melanoma (e.g., metastatic melanoma such as cutaneous or intraocular malignant melanoma), thyroid cancer, bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, vulvar cancer, bile duct cancer, and esophageal cancer. In certain embodiments, the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer, and uterine cancer.

[0114] In certain embodiments, cancers prevented or treated by the methods of the present disclosure include, but are not limited to, hematopoietic cancers, such as leukemia, lymphoma, or myeloma.

[0115] In some embodiments, the cancer may be an early stage cancer or a late stage cancer. In some embodiments, the cancer may be a primary tumor. In some embodiments, the cancer may be a metastatic tumor at a secondary site derived from any of the aforementioned types of cancer.

[0116] In some embodiments, the disclosure provides a method of treating an individual having cancer, where the individual is refractory to checkpoint inhibitor therapy, by administering to the individual an effective amount of a Siglec-9ECD fusion molecule of the disclosure, e.g., a Siglec-9ECD-Fc fusion molecule. In certain embodiments, the individual has a cancer that is refractory to treatment with a PD-1 or PD-L1 antagonist, e.g., a PD-1 or PD-L1 antibody, such as those presented below.

[0117] In some embodiments, the present disclosure provides a method of treating an individual having cancer, the method comprising the steps of: Methods are provided for treating cancer that has recurred after checkpoint inhibitor treatment by administering to an individual a therapeutically effective amount of a Siglec-9ECD fusion molecule of the present disclosure, e.g., a Siglec-9ECD-Fc fusion molecule. In certain embodiments, the individual has cancer that has recurred after treatment with a PD-1 or PD-L1 antagonist, e.g., a PD-1 or PD-L1 antibody, such as those presented below.

[0118] In some embodiments, the Siglec-9ECD fusion molecule of the present disclosure, for example, Siglec-9ECD-Fc fusion molecule, can be administered in conjunction with an antagonist of an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory checkpoint molecule is PD-1 (programmed cell death protein-1) or its ligand PD-L1 (programmed death ligand-1). In some embodiments, the PD-1 antagonist is an antibody against PD-1. PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), MEDI-0680 (AMP-5I4; WO2012 / 145493), camrelizumab (SHR-1210), tislelizumab (BGB-A317), or spartalizumab (NPVPDR001, NVS240118, PDR001). A recombinant protein consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224, can also be used to antagonize the PD-1 receptor. In some embodiments, the PD-L1 antagonist is an antibody against PD-L1. PD-L1 antibodies include, for example, TECENTRIQ (atezolizumab), durvalumab (MEDI4736), BMS-936559 (W02007 / 005874), MSB0010718C (WO2013 / 79174) or rHigM12B7. In some embodiments, the Siglec-9ECD fusion molecules of the invention are administered in combination with radiation therapy and / or chemotherapy.

[0119] In some embodiments, a method is provided for treating a neurological or neurodegenerative disorder by administering a Siglec-9ECD fusion molecule, such as a Siglec-9ECD-Fc fusion molecule, to a patient in need thereof. In some embodiments, the neurological or neurodegenerative disorder is characterized by dysfunctional (e.g., hypofunctioning) or defective microglial cells. Microglial cells are innate immune cells that are specifically present in the brain and function as macrophages to remove debris and dead neurons through the process of phagocytosis and provide other support functions to maintain brain health. Without being bound by theory, activation of microglial cells by Siglec-9ECD fusion molecules will treat the neurological or neurodegenerative disorder. In some embodiments, the patient has symptoms of a neurological or neurodegenerative disorder, and the Siglec-9ECD fusion molecule is administered to treat the neurological or neurodegenerative disorder. In some embodiments, the patient is at risk for a neurological or neurodegenerative disorder, and the Siglec-9ECD fusion molecule is administered to reduce the risk of, delay the onset of, or prevent the neurological or neurodegenerative disorder. In some embodiments, the neurological or neurodegenerative disorder is selected from dementia, including frontotemporal dementia, Alzheimer's disease, vascular dementia, and mild cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, tauopathy, multiple sclerosis, immune-mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, childhood-onset leukoencephalopathy, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

[0120] Dementia Dementia is a non-specific syndrome (i.e., a set of signs and symptoms) that manifests in previously unimpaired individuals as a significant loss of overall cognitive ability beyond that which can be expected from normal aging. Dementia may be static, as a result of inherent global brain damage. Alternatively, dementia may be progressive, with long-term decline resulting from physical injury or disease. Dementia is more common in the elderly population, but may also develop before age 65. Cognitive domains affected by dementia include, but are not limited to, memory, attention span, language, and problem solving. In general, symptoms must be present for at least six months before an individual is diagnosed with dementia.

[0121] Exemplary forms of dementia include, but are not limited to, frontotemporal dementia, Alzheimer's disease, vascular dementia, semantic dementia, and dementia with Lewy bodies.

[0122] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat dementia. In some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with dementia.

[0123] frontotemporal dementia Frontotemporal dementia (FTD) is a condition resulting from progressive degeneration of the frontal lobes of the brain. This degeneration can progress to the temporal lobes over time. In prevalence, FTD accounts for 20% of presenile dementia cases, second only to Alzheimer's disease (AD). Clinical features of FTD include memory deficits, behavioral abnormalities, personality changes, and language disorders (Cruts, M. & Van Broeckhoven, C., Trends Genet. 24:186-194 (2008);Neary, D., et al., Neurology 51:1546-1554 (1998);Ratnavalli, E., Brayne, C., Dawson, K. & Hodges, JR, Neurology 58:1615-1621 (2002)).

[0124] Although a significant proportion of FTD cases are inherited in an autosomal dominant manner, symptoms can range from FTD with behavioral disorders, to primary progressive aphasia, to corticobasal ganglionic degeneration, even in one family. FTD, like most neurodegenerative diseases, can be characterized by the pathological presence of specific protein aggregates in affected brains. Historically, the first description of FTD recognized the presence of intraneuronal accumulations of hyperphosphorylated tau protein in neurofibrillary tangles or Pick's globules. The causal role of the microtubule-associated protein tau was supported by the identification of mutations in the gene encoding the tau protein in several families (Hutton, M., et al., Nature 393:702-705 (1998). However, the majority of FTD brains do not show accumulation of hyperphosphorylated tau and show immunoreactivity for ubiquitin (Ub) and the TAR DNA-binding protein (TDP43) (Neumann, M., et al., Arch. Neurol. 64:1388-1394 (2007)). The majority of these FTD cases with Ub inclusions (FTD-U) were shown to have mutations in the progranulin gene.

[0125] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat FTD, hi some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with FTD.

[0126] Alzheimer's Disease Alzheimer's disease (AD) is the most common form of dementia. There is no cure for the disease, which worsens as it progresses, eventually leading to death. In most cases, AD is diagnosed in people over the age of 65. However, early-onset Alzheimer's disease, which is less common, can develop earlier. Common symptoms of Alzheimer's disease include behavioral symptoms, such as difficulty recalling recent events; cognitive symptoms, confusion, irritability and aggression, mood swings, speech disorders, and long-term memory loss. As the disease progresses, bodily functions are lost, eventually leading to death. Alzheimer's disease develops in an unknown and variable amount of time before becoming fully evident, and may proceed undiagnosed for years.

[0127] Also reported herein is the observation that the minor allele of rs2075803, a SNP, at the Siglec-9 locus on chromosome 19 is associated with both increased plasma Siglec-9 levels and risk of Alzheimer's disease. Additionally, reported herein is the observation that the minor allele of rs12983058, a SNP, at the Siglec-7 locus on chromosome 19 is associated with both increased plasma Siglec-7 levels and risk of Alzheimer's disease.

[0128] Thus, in some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat Alzheimer's disease, hi some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with Alzheimer's disease.

[0129] Parkinson's disease Parkinson's disease, sometimes called idiopathic or primary parkinsonism hypokinetic rigidity syndrome (HRS), or paralysis agitans, is a neurodegenerative brain disorder that affects motor system control. The progressive death of dopamine-producing cells in the brain causes the main symptoms of Parkinson's disease. In most cases, Parkinson's disease is diagnosed in people over the age of 50. Parkinson's disease is idiopathic (of unknown cause) in most people. However, genetic factors also play a role in the disease.

[0130] Symptoms of Parkinson's disease include, but are not limited to, tremors in the hands, arms, legs, jaw and face, muscle rigidity of the limbs and trunk, slowness of movement (bradykinesia), postural instability, difficulty walking, neuropsychiatric disturbances, speech or behavior changes, depression, anxiety, pain, psychosis, dementia, hallucinations, and sleep problems.

[0131] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat Parkinson's disease. In some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with Parkinson's disease.

[0132] Amyotrophic lateral sclerosis (ALS) As used herein, amyotrophic lateral sclerosis (ALS) or motor neuron disease or Lou Gehrig's disease are used interchangeably and refer to a debilitating disease with various etiologies characterized by rapidly progressive muscle weakness, muscle atrophy and fasciculations, muscle spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (respiratory disorders).

[0133] It has been shown that progranulin plays a role in ALS (Schymick, JC et al., (2007) J

[0343] Neurol Neurosurg Psychiatry.;78:754-6) and reprotects damage caused by ALS caused by proteins such as TDP-43 (Laird,AS et al.,(2010).PLoS ONE 5:e13368). It has also been demonstrated that pro-NGF induces p75-mediated death of oligodendrocytes and corticospinal neurons after spinal cord injury (Beatty et al., Neuron (2002),36, pp. 375-386;Giehl et al, Proc. Natl. Acad. Sci USA (2004), 101, pp 6226-30).

[0134] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat ALS disease. In some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with amyotrophic lateral sclerosis.

[0135] Huntington's disease Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an autosomal dominant mutation in the huntingtin gene (HTT). An increase in the cytokine-adenine-guanine (CAG) triplet repeat in the huntingtin gene leads to the production of a mutant form of the huntingtin protein (Htt) encoded by the gene. This mutant huntingtin protein (mHtt) is toxic and is involved in neuronal death. Symptoms of Huntington's disease most commonly appear between the ages of 35 and 44, but can appear at any age.

[0136] Symptoms of Huntington's disease include, but are not limited to, motor dysfunction, seizure-like irregular movements (chorea), abnormal eye movements, balance problems, seizures, difficulty chewing, difficulty swallowing, cognitive impairment, speech changes, memory problems, difficulty thinking, insomnia, fatigue, dementia, personality changes, depression, anxiety, and obsessive-compulsive behaviors.

[0137] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat Huntington's Disease (HD). In some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with Huntington's Disease.

[0138] Tauopathy disease Tauopathy or tauopathy is a group of neurodegenerative diseases caused by the aggregation of microtubule-associated protein tau in the brain. Alzheimer's disease (AD) is the most well-known tauopathy disease, which involves the accumulation of tau protein in the form of insoluble neurofibrillary tangles (NFTs) in neurons. Other tauopathy diseases and disorders include progressive supranuclear palsy, dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia parkinsonism linked to chromosome 17, Lytico-Bodig disease (Parkinson-dementia complex syndrome of Guam), neurofibrillary tangle-dominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic grain disease (AGD), Huntington's disease, and frontotemporal lobar degeneration.

[0139] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat a taupathic disease. In some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with a taupathic disease.

[0140] Multiple sclerosis Multiple sclerosis (MS) is sometimes called disseminated sclerosis or disseminated encephalomyelitis. MS is an inflammatory disease in which the lipid myelin sheath that covers the axons in the brain and spinal cord is damaged, resulting in demyelination and scarring, as well as a wide range of signs and symptoms. MS affects the ability of nerve cells in the brain and spinal cord to communicate effectively with each other. Nerve cells communicate by sending electrical signals called action potentials along long fibers called axons that are contained within an insulating substance called myelin. In MS, the body's own immune system attacks and damages the myelin. When the myelin is lost, the axons are no longer able to transmit signals effectively. MS usually develops in young adults and is more common in women.

[0141] Symptoms of MS include, but are not limited to, sensory changes such as loss of sensation or tenderness to percussion; tingling or numbness such as hypoesthesia and dysesthesias; muscle weakness; clonus; muscle seizures; difficulty moving; difficulties with coordination and balance such as ataxia; speech disorders such as dysarthria or swallowing difficulties such as dysphagia; vision problems such as nystagmus, optic neuritis including phosphenes, and diplopia; fatigue; acute or chronic pain; and bladder and bowel disorders; varying degrees of cognitive impairment; emotional symptoms of depression or mood lability; Uthof's phenomenon, in which exposure to temperatures higher than normal ambient temperatures aggravates existing symptoms; and Lhermitte's sign, in which there is a sensation of electricity running down the back when bending the neck.

[0142] In some embodiments, administering a Siglec-9ECD fusion molecule of the present disclosure can prevent, reduce the risk of, and / or treat MS, hi some embodiments, administering a Siglec-9ECD fusion molecule can modulate one or more Siglec-9 activities in an individual with MS.

[0143] Administration Siglec-9ECD fusion molecules (and any additional therapeutic agents), such as Siglec-9ECD-Fc fusion molecules provided herein, can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intratumoral, intralesional administration, intracerebrospinal, intracranial, intrathecal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusion includes intramuscular, intravenous administration as a bolus, or continuous infusion over a period of time, intraarterial, intraarticular, perivascular, or subcutaneous administration. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various times, bolus administration, and pulse infusions.

[0144] In preventing or treating disease, the appropriate dosage of a Siglec-9ECD fusion molecule of the invention, such as a Siglec-9ECD-Fc fusion molecule, when used alone or in combination with one or more other additional therapeutic agents will depend on the type of disease being treated, the type of fusion molecule, the severity and course of the disease, whether the fusion molecule is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the fusion molecule, and the discretion of the attending physician. The fusion molecule is suitably administered to the patient at one time or over a series of treatments.

[0145] Diagnostic Uses In some embodiments, the Siglec-9ECD fusion molecule provided herein is useful for detecting the presence of Siglec ligand, such as sialic acid, in a sample or individual.The term "detect" as used herein includes quantitative or qualitative detection.The method of using the Siglec-9ECD fusion molecule of the present disclosure for diagnostic purposes, such as detecting sialic acid in an individual or a tissue sample derived from an individual, is provided herein.In some embodiments, the individual is a human.

[0146] The detection method may involve the quantification of sialic acid-bound Siglec-9ECD fusion molecules. Such detection in biological samples can be carried out by any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micro-positron emission tomography. In certain embodiments, the Siglec-9ECD fusion molecule is radiolabeled, for example, with 18F, and then detected using micro-positron emission tomography analysis. Sialic acid binding can also be quantified in patients by non-invasive techniques, such as positron emission tomography (PET), X-ray computed tomography, single photon emission computed tomography (SPECT), computed tomography (CT), and computed tomography (CAT).

[0147] product Provided herein is an article of manufacture (e.g., a kit) that includes a Siglec-9ECD fusion molecule, e.g., a Siglec-9ECD-Fc fusion molecule, as described herein. The article of manufacture may include one or more containers that include a Siglec-9ECD fusion molecule as described herein. The containers may be any suitable packaging, including, but not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses.

[0148] In some embodiments, the kit may further include a second agent. In some embodiments, the second agent is a pharma- ceutically acceptable buffer or diluent, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. In some embodiments, the second agent is a pharma- ceutically active agent as described above.

[0149] In some embodiments of any of the articles of manufacture, the article of manufacture further comprises instructions for use in accordance with the methods of the present disclosure. The instructions generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. In some embodiments, these instructions include instructions for use in the treatment of squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), squamous non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-squamous NSCLC, glioma, peritoneal carcinoma, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer, or stomach cancer, according to any of the methods of the present disclosure. cancer), kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, colorectal cancer, endometrial cancer, liver cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, brain cancer (e.g., astrocytoma such as glioblastoma (glioblastoma multiforme)), cervical cancer, bladder cancer, hepatocellular carcinoma, breast cancer (e.g., triple negative breast cancer), and head and neck cancer (squamous cell carcinoma of the head and neck), melanoma (e.g., metastatic melanoma such as cutaneous or intraocular malignant melanoma), thyroid cancer, bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, vulvar cancer The instructions include instructions for administering a Siglec-9ECD fusion molecule of the present disclosure to prevent, reduce the risk of, or treat an individual having a disease, disorder, or injury selected from: cholangiocarcinoma, esophageal cancer, dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, and dementia including mild cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, taupathies, multiple sclerosis, immune-mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, childhood-onset leukoencephalopathy, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). In some embodiments, the instructions include instructions for use with the Siglec-9ECD fusion molecule and a second agent (e.g., a second pharmacologic active agent).

[0150] The present disclosure will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the disclosure. All citations throughout this disclosure are expressly incorporated herein by reference. EXAMPLES

[0151] The following examples are offered by way of illustration only and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of parameters that could be changed or modified to yield essentially similar results.

[0152] Example 1: Siglec-9 is expressed on the surface of myeloid cells in human tumors To examine the expression of Siglec-9 on immune cells in human tumors, freshly resected primary human tumors were processed by enzymatic digestion followed by flow cytometry analysis. As shown in Figure 1, in a representative lung adenocarcinoma sample, Siglec-9 was detected on the surface of tumor-infiltrating macrophages and granulocytes, but not on T cells. Similar Siglec-9 expression profiles were observed in samples from colorectal, liver, ovarian, and head and neck cancers. Further characterization of Siglec-9 myeloid cell expression revealed that Siglec-9 expression was highest on HLA-DRlo cells, which could be characterized as myeloid-derived suppressor cells (MDSCs).

[0153] Example 2: Siglec-9 ECD domain exhibits binding and efficient recombinant expression Siglec-9-hIgG1 truncated variants were evaluated for expression and binding efficiency to A375 melanoma cells. The ECD of Siglec-9 consists of three domains: the ligand-binding IgV domain is located at the N-terminus, followed by the C2T1 and C2T2 domains. Of the three domains, the C2T2 domain is located closest to the plasma membrane. As shown in Table 1, only the Siglec-9ECD-Fc variants containing all three domains were efficiently expressed in Expi293 cells. To detect Siglec-9-hIgG1 binding, A375 cells were incubated with 250 μg / ml Siglec-9ECD-Fc variants for 2 hours on ice in the dark, followed by incubation with fluorescently conjugated anti-human IgG (Jackson Immunoresearch) for 30 minutes. Binding was evaluated by flow cytometry using a BD FACS Canto and analyzed using FlowJo software. The data in Table 1 show that the IgV domain is required for binding to A375 cells, consistent with the IgV domain functioning as the major ligand recognition domain in Siglec-9ECD. [Table 3]

[0154] Example 3: Siglec9-Fc fusion protein engineering: homology modeling The Siglec9-Fc fusion protein was engineered by creating a Siglec9-IgV homology model to allow the design of mutations to improve solubility and rebalance charge distribution. Structure-based protein homology modeling and stability calculations were used to design Siglec-9 variants with improved solubility and redistribution of surface charge, utilizing the protein modeling and protein design modules of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada).

[0155] Briefly, a homology model of the Siglec9IgV domain ("HM_S9") was generated using the Protein Modeler application (Molecular Operating Environment (MOE). Montreal (QC, Canada): Chemical Computing Group ULC.; 2019 January) in MOE2019.01. The primary amino acid sequence of Siglec9-IgV (SEQ ID NO: 7) was used as the query sequence, which is shown in FIG. 2. A homology search was performed using PDB_ID 1O7V (high-resolution structure of Siglec-7) from the Protein Data Bank (www(dot)rcsb(dot)org / ) as a template for the query, which is approximately 73% identical to the template. Energy minimization over the protein homology modeling was performed in MOE 2019.01 using the Amber10:EHT force field to generate a refined model.

[0156] The refined HM_S9 model was then used to calculate electrostatic and hydrophobic surface patches to identify residues associated with potentially problematic protein-protein interactions. This in silico analysis can be used to predict reversible aggregation, which typically results from relatively weak non-covalent interactions. Hydrophobic interactions can contribute to high affinity non-specific interactions between macromolecules (Wildman, SA, Crippen, GM; Prediction of Physiochemical Parameters by Atomic Contributions; J. Chem. Inf. Comput. Sci. 39 (1999) 868-873). In addition, electrostatic interactions are involved in the formation of self-associated aggregates in many proteins, including antibodies (Sharp K., Honig B.; Electrostatic Interactions in Macromolecules: Theory and Applications. Ann. Rev. Biophys. Biophys. Chem. 19 (1990) 301-332).

[0157] Several positively charged hydrophobic surface patches were identified. Site-directed mutagenesis was performed in silico using the residue scanning function in MOE to target hydrophobic patches, positively charged patches, or hydrophobic positively charged patches simultaneously in the IGV domain. Single, double, triple, or quadruple mutations were introduced in each variant. Mutations were selected among alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, lysine, serine, threonine, tyrosine, and valine.

[0158] Approximately 50,000 mutations were sampled and the stability change was calculated. Based on improved stability, reduction of positively charged patches, and reduction of hydrophobic patches, mutants were selected to be constructed and further tested for function, expression, solubility, and stability. The parent Siglec-9-Fc is shown in SEQ ID NO: 10. The sequence "WIYP" at amino acids 50-53 is replaced in S9.2-S9.7 with the four amino acids shown (SEQ ID NOs: 11-16, respectively; DIEG, SEQ ID NO: 11; SIET, SEQ ID NO: 12; SIEP, SEQ ID NO: 13; DIEP, SEQ ID NO: 14; YQES, SEQ ID NO: 15; THET, SEQ ID NO: 16). In S9.8-S9.22 (SEQ ID NOs: 17-31), the indicated substitutions are made. See the table of specific sequences (numbering at mutated residues has been adjusted as a result of molecular modeling with MOE. The first amino acid (S in SKLL...) of the mature polypeptide sequence is residue number 8).

[0159] Example 4: Siglec9-Fc fusion protein engineering: replacement of loop residues The high-resolution crystal structure of Siglec-7 was compared to the Siglec-9 model (Alpheny, MS, et al; High Resolution Crystal Structures of Siglec-7, Insights into Ligand Specificity in the Siglec Family; J. Biol. Chem. 278 (2003) 3372-3377). The isosteric Siglec-9 loop composed of SHGWIYPG (SEQ ID NO: 9) presents a larger hydrophobic surface when compared to the negatively charged Siglec-7 loop composed of VDSQTDSD (SEQ ID NO: 8). Therefore, a systematic in silico loop-swapping protein engineering was applied; each amino acid in the Siglec-7 loop was replaced by a Siglec-9 residue or up to eight residues were kept with the Siglec-7 residue, resulting in a total of 256 variants, including 255 mutants and one wild-type variant. Stability changes were calculated for all 256 variants. Based on improved stability, reduced positively charged patches, and reduced hydrophobic patches, mutants were selected for further characterization, and the amino acid sequences of these mutants S9.23-S9.39 are shown in SEQ ID NOs: 32-39.

[0160] Example 5: Improved in silico characterization of engineered Siglec9-IgV variants In silico biophysical properties including stability change (more negative values ​​are more stable), area of ​​hydrophobic protein patches, area of ​​positively charged protein patches, area of ​​negatively charged protein patches, isoelectric point, and net charge were calculated at pH 7.4, 100 mM NaCl, and 298 K and compared to the parent construct. The results are shown in Figure 3. Negative values ​​in the "Stability Change" column indicate increased stability. The improvement in stability change is mostly due to charge redistribution and reduced exposed hydrophobic surface area. Such changes usually result in increased protein expression and production.

[0161] Example 6: Siglec-9-Fc binds to FcR-negative cell lines with moderate affinity S9.1-hIgG1 variant (SEQ ID NO: 10) was used to evaluate Siglec-9-Fc binding to a panel of cancer cell lines. S9.1-hIgG1 contains the native sequence Siglec-9ECD, but with a deletion of amino acid residues LQSKATSGVTQG (SEQ ID NO: 147) occurring after the C2T2 domain and before the transmembrane domain, and the signal sequence is cleaved during production. Titrated amounts of S9.1-hIgG1 were incubated with the cancer cell lines listed in Table 2, essentially as shown in Example 2. FACS Kd was calculated essentially as described in Drake and Klakamp, ​​Journal of Immunological Methods, 2007. [Table 4]

[0162] As shown in Table 2, S9.1-hIgG1 bound to K562 leukemia cells with higher affinity compared to other cancer cell lines. Since K562 cells are derived from cells from myeloid lineage, they express FcR on the surface, whereas other cancer cell lines do not. Therefore, without being bound by theory, it is believed that the binding of S9.1-hIgG1 to both FcR and sialic acid on leukemia cells results in a cooperative binding effect (see Example 7) compared to the binding of S9.1-hIgG1 to only sialic acid on cancer cells that do not express FcR. This may partially explain the enhanced affinity of S9.1-hIgG1 for K562 cells compared to other cancer cells tested.

[0163] Example 7: Siglec-9-Fc binds to myeloid-derived suppressor cells with high affinity To examine Siglec-9-Fc binding on primary human cells, S9.A-mIgG1 was used to evaluate the affinity of Siglec-9-Fc to myeloid-derived suppressor cells (MDSCs). S9.A-mIgG1 (SEQ ID NO: 43) contains full-length Siglec-9 ECD (amino acid residues 1-348 of SEQ ID NO: 1) fused to a mouse IgG1 Fc domain via a 7 amino acid linker, where the signal sequence is cleaved during production. MDSCs were generated essentially as follows: CD14+ monocytes were isolated from healthy human donors using the RosetteSep Human Monocyte Enrichment Cocktail kit (StemCell) and differentiated in RPMI medium containing 10 ng / ml hGM-CSF (R&D) and 10 ng / ml hIL-6 (R&D) for 7 days at 37°C and 5% CO2. Cell binding was assessed by incubating MDSCs with a titrated amount of S9.A-mIgG1 for 2 h on ice in the dark, followed by incubation with fluorescently conjugated anti-mouse IgG (Jackson Immunoresearch) for 30 min. Binding was assessed by flow cytometry on a BD FACS Canto and analyzed using FlowJo software. The variant S9.A-mIgG1 with mouse Fc was used because anti-human detection on MDSCs would result in prohibitively high background binding. As shown in Table 3, the calculated FACS Kd on MDSCs for S9.A-mIgG1 was in the low nM range in three independent donors and was approximately 10-100 times weaker on the reference cancer cell line A549, a lung cancer epithelial cell line. These studies indicate that Siglec-9-Fc binds with higher affinity to myeloid cells, such as MDSCs, than to cancer cells. Thus, these studies provide further evidence for a cooperative binding mechanism in which Siglec-9-Fc binds to both FcR and sialic acid on myeloid cells, compared with the binding of Siglec-9-Fc to only sialic acid on cancer cells that do not express FcR, which may explain the enhanced affinity of S9.A-mIgG1 for MDSCs compared with the A549 lung cancer cell line that does not express FcR.

[0164] FIG. 15 shows an exemplary model of the mechanism of action of Siglec-9-ECD-Fc fusion molecule (Siglec-9-Fc). Siglec-9-Fc binds to a ligand (sialic acid) on cancer cells via its Siglec-9ECD portion (left panel). Based on the studies herein, and without being bound by theory, it is believed that Siglec-9-Fc binds to both FcR and a ligand (sialic acid) expressed on myeloid cells (right panel). Binding occurs through cooperative binding (or cis) interactions via the Fc and Siglec-9ECD portions of the Siglec-9-Fc molecule, respectively. As a result, Siglec-9-Fc binds to myeloid cells with higher affinity compared to cells that do not express FcR, resulting in preferential targeting to myeloid cells in vivo. [Table 5]

[0165] Example 8: Siglec-9-Fc potently repolarizes MDSCs. S9.A-hIgG1 (SEQ ID NO: 40) and S9.A-hIgG1 LALAPS (SEQ ID NO: 42) were evaluated for their ability to repolarize MDSCs. S9.A-hIgG1 (SEQ ID NO: 40) and S9.A-hIgG1 LALAPS (SEQ ID NO: 42) contain full-length Siglec-9ECD (amino acid residues 1-348 of SEQ ID NO: 1) fused to a human IgG1 Fc domain via a 7 amino acid linker, where the signal sequence is cleaved during production. In S9.A-hIgG1, the human IgG1 Fc domain is native sequence hIgG1, and in S9.A-hIgG1 LALAPS, the human IgG1 Fc domain contains a "LALAPS" substitution. As previously described, LALAPS substantially eliminates Fc-FcR interactions.

[0166] Human MDSCs were generated from CD14+ monocytes as shown in Example 7, and then incubated with 10 μg / ml S9.A-hIgG1 or S9.A-hIgG1 LALAPS for 48 hours at 37° C. and 5% CO2. Supernatants were harvested and secreted chemokines were analyzed using the LEGENDplex Human Proinflammatory Chemokine Panel kit (Biolegend). As shown in Table 4, S9.A-hIgG1 potently repolarized MDSCs to a proinflammatory phenotype, while the efficacy of S9.A-hIgG1 LALAPS was much lower. This indicates that in addition to ligand (sialic acid) binding, FcR binding significantly enhances the ability of Siglec-9-Fc to repolarize MDSCs. [Table 6] Units in Table 4 are pg / ml. Results are expressed as mean ± SEM pooled from 4 donors. Values ​​in italics indicate p<0.05 when comparing Siglec-9-Fc variants with hIgG1 isotype control by two-tailed t-test.

[0167] Example 9: Siglec-9 relieves MDSC-mediated suppression of T cells The effect of S9.1-hIgG1 (SEQ ID NO: 10) treatment was evaluated in a human MDSC-T cell co-culture system. Briefly, human MDSCs were generated as shown in Example 7. Autologous CD8+ T cells were isolated from blood using RosetteSep™ Human CD8+ T Cell Enrichment Cocktail kit (StemCell). MDSCs were treated with 10 μg / ml S9.1-hIgG1 or IgG control for 48 hours at 37° C. and 5% CO2, and then co-cultured with autologous CD8+ T cells in the presence of Dynabeads® Human T-Activator CD3 / CD28 at a ratio of 1:2:2 MDSC:T cells:Dynabeads®. In some conditions, CD8+ T cells incubated only with CD3 / CD28 Dynabeads® were treated with S9.1-hIgG1. All cell conditions were cultured for 4 days at 37°C and 5% CO2, followed by quantification of IFNγ in the culture supernatants by ELISA (Thermo Fisher).

[0168] As shown in Figure 4, S9.1-hIgG1 (referred to as "S9-hIgG1" in Figure 4) potently relieved MDSC-mediated suppression of T cells. S9.1-hIgG1 also had an effect on CD8+ T cells cultured in the presence of CD3 / CD28 Dynabeads® but not on MDSC. S9.1-hIgG1-treated MDSCs cultured alone produced <20 pg / ml IFNγ (data not shown). Mean ± SEM is shown. These studies indicate that Siglec-9-Fc can enhance T cell activation in the absence of MDSCs, while it can more potently enhance T cell activation in the presence of MDSCs by relieving myeloid cell immunosuppressive signals, e.g., by blocking engagement of Siglec ligands on myeloid cells.

[0169] Example 10: Siglec-9-Fc, but not Siglec antibodies, relieves MDSC-mediated suppression of T cells The ability of S9.A-hIgG1 (SEQ ID NO: 40) to release MDSC-mediated suppression of T cells was directly compared with a panel of functional anti-Siglec antibodies. MDSCs and autologous CD8+ T cells were prepared for co-culture as described in Example 9. MDSCs were treated with 15 μg / ml S9.A-hIgG1 or antibodies directed against Siglec-3, Siglec-7, or Siglec-9 that induce downregulation of the target receptor or block cognate ligand binding for 48 hours, followed by co-culture with CD8+ T cells and CD3 / CD28 Dynabeads® for 4 days. IFNγ was assessed in the culture supernatants by ELISA.

[0170] As shown in FIG. 5, anti-Siglec antibodies alone or in combination were not able to reverse MDSC-mediated suppression of T cells as effectively as S9.A-hIgG1. Mean±SEM is shown. aS9-1 and aS9-2 are two different Siglec-9 antibodies. p-values ​​were determined by comparing S9.A-hIgG1 to the triple antibody combination condition. This study provides further evidence of the cooperative binding mechanism shown in FIG. 15. Siglec-9-Fc is able to achieve this mechanism, whereas anti-Siglec antibodies, including anti-Siglec-9 antibodies, cannot. In addition, a commercially available human Siglec-9-Fc fusion protein was obtained (R&D Systems Catalog #1139-SL-050, "Recombinant Human Siglec-9 Fc Chimera Protein, CF", www(dot)rndsystems(dot)com) and tested in the aforementioned assay. It did not show any significant activity (data not shown).

[0171] Example 11: Siglec-9-Fc with intact FcR binding potently relieves MDSC-mediated suppression of T cells The efficacy of S9.1-hIgG1 (SEQ ID NO: 10) was compared with S9.A-hIgG1 LALAPS (SEQ ID NO: 42) in a human MDSC-T cell co-culture system. MDSCs and autologous CD8+ T cells were prepared for co-culture as described in Example 9. MDSCs were treated with the indicated amounts of S9.1-hIgG1, S9.A-hIgG1 LALAPS, or isotype control for 48 hours, followed by co-culture with CD8+ T cells and CD3 / CD28 Dynabeads® for 4 days. IFNγ was assessed in culture supernatants by ELISA.

[0172] As shown in Figure 6, S9.l-hIgG1 with intact FcR engagement is significantly more potent than the LALAPS variant in relieving MDSC-mediated suppression of T cells. The EC50 for S9.l-hIgG1 in this assay was calculated to be 17.5 nM, which is in the range of the FACS Kd for MDSCs listed in Table 3. Additionally, these results are consistent with the data in Table 4, indicating that Siglec-9-Fc with intact FcR engagement is more potent than the Siglec-9-Fc variant with LALAPS mutation in repolarizing MDSCs as measured by chemokine production.

[0173] Example 12: Siglec-9-hIgG1 and Siglec-9-hIgG1 NSLF equally repolarize MDSCs in a dose-dependent manner Variants of Siglec-9-Fc containing NSLF mutations in the Fc portion of the fusion protein were evaluated for their ability to repolarize human MDSCs. The NSLF mutation disrupts the interaction between human IgG1 Fc, human Clq (complement component 1q), and human CD16 / FcRIII that induces antibody-dependent cellular cytotoxicity (ADCC). MDSCs were generated from CD14+ monocytes as previously described. On day 7, MDSCs were treated with the indicated amounts of S9.A-hIgG1 (SEQ ID NO: 40) or S9.A-hIgG1 NSLF (SEQ ID NO: 41) for 48 hours at 37°C and 5% CO2. Supernatants were harvested and secreted chemokines were analyzed using the LEGENDplex™ Human Proinflammatory Chemokine Panel kit (Biolegend).

[0174] Figure 7 shows that S9.A-hIgG1 and S9.A-hIgG1 NSLF repolarized MDSCs comparably, as demonstrated by similar increases in representative chemokines CCL5 and CCL17. These studies indicate that complement fixation and ADCC are not required for Siglec-9-Fc activity. Furthermore, the possibility of using hIgG1 with NSLF to achieve desired effects in myeloid cells reduces the chance of adverse effects that could otherwise result from activation of complement and ADCC.

[0175] Example 13: Siglec-9-hIgG1 and Siglec-9-hIgG1 NSLF increase CD86 expression and decrease CD163 expression in MDSCs. Human MDSCs were generated from CD14+ monocytes as previously described. On day 7, MDSCs were treated with the indicated amounts of S9.A-hIgG1 (SEQ ID NO: 40) or S9.A-hIgG1 NSLF SEQ ID NO: 41) for 48 hours at 37° C. and 5% CO2, after which expression of CD86, a proinflammatory marker, and CD163, an M2 macrophage marker, was quantified using anti-CD86 (clone IT2.2, Biolegend) and anti-CD163 (clone GHI / 61, BD) antibodies.

[0176] As shown in Figure 8, treatment with either S9.A-hIgG1 or S9.A-hIgG1 NSLF resulted in a dose-dependent increase in CD86 and a decrease in CD163, consistent with a repolarization of MDSCs towards a proinflammatory phenotype (e.g., from an M2 immunosuppressive phenotype to an M1 activated phenotype). Means ± SEM are shown.

[0177] Example 14: Siglec-9-hIgG1 and Siglec-9-hIgG1 NSLF repolarize tumor macrophages in vivo in humanized mice. A humanized mouse model was used to evaluate the effect of Siglec-9-Fc treatment in vivo. Immunodeficient HuNOG-EXL mice expressing human IL-3 and GM-CSF transgenes were transplanted with human CD34+ hematopoietic progenitor cells (Taconic) to effectively reconstitute a human immune response. Mice were transplanted with 3×10 6 A375 human melanoma cells were implanted subcutaneously in each mouse. After 16 days, the tumors had grown to approximately 300 mm 3 At 1 h, mice were treated with two perivascular (ip) injections of 10 mg / kg S9.1-hIgG1 (SEQ ID NO: 10), S9.A-hIgG1 NSLF (SEQ ID NO: 41), or hIgG1 isotype control, 3 days apart. Tissues were analyzed 24 hours after the second dose.

[0178] As shown in Figures 9A-9C, in vivo treatment with either S9.1-hIgG1 or S9.A-hIgG1 NSLF confirmed the in vitro results observed with human MDSCs. The percentage of M2-like CD14+CD163+ macrophages relative to human CD45+ cells was reduced in tumors of Siglec-9-Fc-treated mice compared to isotype control. See Figure 9A (in the experiment shown in Figure 9A, S9.1-hIgG1 produced a more robust in vivo effect than S9.A-hIgG1 NSLF; however, in a repeat experiment (data not shown), S9.A-hIgG1 NSLF produced a more robust in vivo effect than S9.1-hIgG1). M2 macrophages, identified by the surface marker CD206, were also reduced in tumors of mice treated with Siglec-9-Fc. See Figure 9B. Furthermore, surface expression of CD206 on CD14+ macrophages in tumors was reduced. See Figure 9C. With reference to Figures 9B and 9C, S9.1-hIgG1 produced a more robust in vivo effect than S9.A-hIgG1 NSLF. In each panel of Figure 9, the average is shown.

[0179] Example 15: Siglec-9-Fc does not result in blood cell deficiency in humanized mice in vivo Tumor-bearing HuNOG-EXL mice treated with S9.1-hIgG1 or S9.A-hIgG1 NSLF were also assessed for blood cell depletion using a standard complete blood count. Blood was collected by cardiac puncture on the day of tissue collection (24 hours after the second 10 mg / kg dose) and placed into heparin-containing blood collection tubes.

[0180] Figure 10 shows that neither S9.1-hIgG1 nor S9.A-hIgG1 NSLF produced significant changes in blood cell composition compared to isotype controls (means shown). Although S9-hIgG1 engages CD16 / FcRIII and can therefore induce ADCC, surprisingly no depletion of major blood cell types was observed.

[0181] Example 16: Siglec-3 / 7 / 9BAC transgenic mice respond poorly to anti-PD-L1 treatment in the context of MC38 tumor growth. We generated bacterial artificial chromosome (BAC) transgenic C57BL / 6 mice expressing human Siglec-3, Siglec-7, and Siglec-9, and evaluated MC38 syngeneic tumor growth in these mice. MC38 cells (a murine colon adenocarcinoma cell line) were subcutaneously implanted in S3 / 7 / 9 BAC mice, and tumors grew to an average of 100 mm 3 Once this was reached, mice were treated intraperitoneally with 3 mg / kg (BMI) of anti-PD-L1 antibody twice per week for 3 weeks.

[0182] As shown in Figure 11, S3 / 7 / 9BAC mice had a reduced response to anti-PD-L1 treatment compared to WT controls. Means ± SEM are shown. These studies indicate that blocking Siglec protein function can improve responses to treatments that inhibit PD-1 or PD-L1, such as anti-PD-1 or anti-PD-L1 antibody therapy.

[0183] Example 17: Siglec-9-Fc monotherapy delays MC38 tumor growth. Siglec-9-mIgG2a (S9.B-mIgG2a; SEQ ID NO: 44) was generated to analyze the effect of Siglec-9-Fc in a mouse syngeneic tumor model with an Fc that would maximize Fc-FcR interactions. S3 / 7 / 9BAC mice were implanted subcutaneously with MC38 cells. Tumors grew to an average of 100 mm 3 Once this was reached, mice were treated intraperitoneally with 10 mg / kg S9.B-mIgG2a twice per week for 3 weeks.

[0184] As shown in Figure 12, S9.B-mIgG2a delayed MC38 tumor growth compared to isotype control with 20% tumor growth inhibition at day 22 post-implantation. Means ± SEM are shown.

[0185] Example 18: Siglec-9-Fc, in combination with anti-PD-L1, reduces MC38 tumor growth. MC38 cells were implanted subcutaneously in S3 / 7 / 9BAC mice. Tumors grew to an average of 100 mm 3 Once this was reached, mice were treated intraperitoneally with 10 mg / kg S9.B-mIgG2a and 3 mg / kg anti-PD-L1 antibody twice weekly for 3 weeks.

[0186] As shown in Figure 13, the combination of Siglec-9-Fc and anti-PD-L1 antibody reduced MC38 tumor growth to a greater extent than anti-PD-L1 antibody treatment alone. At day 20 post-implantation, 41% tumor growth inhibition was achieved compared to anti-PD-L1 antibody monotherapy. Mean ± SEM is shown. These studies indicate that the combination of Siglec-9-Fc with PD-1 or PD-L1 inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies, may improve anti-tumor responses.

[0187] Example 19: Siglec-9-Fc can block cellular binding of multiple Siglec family members Human MDSCs were generated from CD14+ monocytes as previously described. On day 7, MDSCs were first incubated with a titrated amount of S9.1-hIgG1 for 20 minutes in the dark on ice, followed by incubation with the Siglec family members represented as mouse IgG1 fusion proteins for an additional 2 hours in the dark on ice. Binding was detected using fluorescently conjugated anti-mouse IgG (Jackson Immunoresearch) and analyzed by flow cytometry. Cell binding of each Siglec family member was normalized to a non-blocking control (no S9.1-hIgG1 added).

[0188] As shown in Figure 14, S9.1-hIgG1 blocked the binding of Siglec-3, Siglec-5, Siglec-7, Siglec-9, and Siglec-10 to the surface of MDSCs. These studies indicate that Siglec-9-Fc is advantageous in that it is a potent inhibitor of the activity of multiple Siglec proteins.

[0189] Similar repolarization experiments as described in Examples 13 and 14 were performed with S.9A-mIgG1 and other Siglec protein-mIgG1 Fc domain fusions. As shown in Figures 16A and 16B, Siglec-9-Fc only repolarizes MDSCs compared to other Siglec-Fc fusions. CD40 and CD86 markers are increased, and CD163 and CD206 are decreased, indicating a repolarization of MDSCs towards a more pro-inflammatory phenotype.

[0190] Example 20: Analysis of Siglec-9-Fc variants for stability, binding affinity, fusion, and pharmacokinetic properties. Siglec-9-Fc variants are evaluated for stability using protein thermal shift assays and long-term incubation at 40°C. In thermal shift assays, melting temperatures are determined using real-time PCR instruments. The binding affinity of Siglec-9-Fc variants is measured by flow cytometry in A375 human melanoma cells, as shown in Example 2. Biological function is evaluated as the ability to relieve MDSC-mediated suppression in MDSC-T cell co-culture assays, as shown in Example 9. Pharmacokinetic (PK) properties are evaluated in vitro using extracellular matrix binding assays using Matrigel plates, or in vivo using standard PK evaluation in mice.

[0191] Example 21: Evaluation of pharmacodynamic markers in humanized mice following treatment with Siglec-9-Fc. Humanized mice are generated as described in Example 14. These mice are injected with 3×10 6A375 human melanoma cells are implanted subcutaneously. After 2–3 weeks, tumors grow to approximately 300 mm 3 At 1 h, mice are treated with two intraperitoneal injections of 10 mg / kg Siglec-9-Fc or hIgG1 isotype control, 3 days apart. Tissues are analyzed 24 h after the second dose. Pharmacodynamic (PD) effects are assessed in serum using LEGENDplex (Biolegend) cytokine and chemokine panel kits or standard sandwich ELISA. Separately, human CD45+ cells in spleen and tumor are isolated using human CD45 MicroBeads (Miltenyi) and transcriptional expression profiles are generated using the Nanostring Myeloid Innate Immunity Panel.

[0192] Example 22: Analysis of the combinatorial effects of Siglec-9-Fc with anti-PD-L1 or anti-TRP1 in syngeneic tumor model studies. Syngeneic tumor cell lines are injected intravenously or implanted subcutaneously in S3 / 7 / 9BAC mice. In the subcutaneous setting, tumors grow to an average of 100 mm 3 Once tumors reach 10 days, mice are treated intraperitoneally with 10 mg / kg Siglec-9-Fc alone or in combination with 3 mg / kg anti-PD-L1 antibody twice per week for 3 weeks. Tumor growth is measured using calipers 2-3 times per week. The experimental endpoint is 50 days or when tumors reach 2000 mm 3 Decreased tumor growth, prolonged survival, greater T cell influx in tumors, and decreased CD163 or CD206 on tumor macrophages are some of the indicators of the anti-cancer effects of Siglec-9-Fc.

[0193] In the intravenous setting, B16F10 mouse melanoma cells are injected via the tail vein. 24 hours after implantation, mice are treated intraperitoneally with anti-TRPI antibodies that recognize tumor antigens highly expressed on B16F10 cells and cause tumor cell death by antibody-dependent cellular phagocytosis (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Mice will be treated intraperitoneally with Siglec-9-Fc alone or in combination with anti-TRPI antibodies twice per week until the end of the study. A typical study duration is approximately 2 weeks. At the end of the study, lungs from mice are harvested and tumor nodules are counted. A reduction in tumor nodules will indicate an anti-cancer effect of Siglec-9-Fc treatment.

[0194] Example 23: Effects of Siglec-9-Fc on macrophage cell surface markers Myeloid cells in both the CNS and peripheral organs are inherently plastic in their phenotype and function. This can be modeled in vitro by macrophages that can differentiate into M1 and M2 type macrophages, exhibiting different phagocytic and inflammatory potential, phenotype, and activity. In peripheral organs, macrophages associated with the M1 phenotype are considered to be more proinflammatory and antimicrobial, while M2-like macrophages are more homeostatic and anti-inflammatory. Within the CNS, microglial cells under homeostatic conditions also express M2 markers such as CD200R, CD163, suggesting a regulatory function in this cell type.

[0195] The effect of Siglec-9-Fc on various M1 and M2 macrophage cell surface markers is examined as follows: Human primary macrophages are treated with Siglec-9-Fc (e.g., 10 μg / ml) in complete RPMI 1640 for 48 hours. Cells are then harvested and subjected to flow cytometry using antibodies specific for M1 markers (CD16, MHC Class II, CD86, etc.), M2 markers (CD200R, Dectin-1, CD163, etc.), and pan-macrophage markers including CD14.

[0196] Example 24: Sialic acid expression on tumor cells The expression of sialic acid in various tumor types Fc was evaluated by immunohistochemistry. Tumor multi-arrays (Pantomics) containing human samples of adrenal, bladder, breast, bone, brain, esophagus, stomach, small intestine, colon, rectum, kidney, liver, lung, lymphoma, ovarian, pancreatic, prostate, skin, testis, thyroid, and uterine cancer were stained with 0.1 μg / ml S9.A-mIgG1 and visualized by colorimetric detection. As shown in FIG. 17, tumor samples were qualitatively scored based on the intensity and frequency of staining (1+ low intensity and / or frequency, 2+ moderate intensity or frequency, and 3+ high intensity or frequency). Scores across tumor types are summarized in Table 5. [Table 7]

[0197] Siglec-9-Fc binding was observed across all tumor types, indicating the presence of cells expressing sialic acid in tumor samples. Therefore, these tumor types can be targeted by Siglec-9-Fc. Tumor types that achieve 2+ or higher staining intensity may indicate more effective targeting of Siglec-9-Fc.

[0198] Example 25: Binding of Siglec-9-Fc variants to tumor cells S9.1 Fc variants were expressed and tested for binding to tumor cells and functional activity on MDSCs using methods similar to those described in Example 6 (binding) and Example 13 (MDSC activity / marker expression). The variants were also tested for monomer content using size exclusion chromatography and for stability in protein thermal shift assays and long-term incubation at 40° C. For thermal shift assays, melting temperatures were determined using real-time PCR.

[0199] The data are summarized in Figure 18. S9.1 Fc variants showed reduced binding to A375 tumor cells and reduced functional activity in MDSCs, as measured by induction of CD86 or downregulation of CD163. Correlation analysis showed that binding to tumor cells was positively correlated with induction of CD86 (Figure 19A). A similar trend was observed for CD163 (data not shown). S9.1 Fc variants generally had a neutral or positive effect on protein production yield and stability. However, increased yield or thermal stability was inversely correlated with binding to tumor cells (Figure 19B, Figure 19C). These data demonstrate that certain variants of S9.1 Fc improve expression or protein stability but reduce pharmacological potency.

[0200] Example 26: Siglec-9-Fc reduces lung nodules in an intravenous tumor model of metastasis To analyze the effect of Siglec-9-mIgG2a in an intravenous tumor setting, B16F10 mouse melanoma cells were injected into S3 / 7 / 9BAC or WT mice via the tail vein. 24 hours after implantation, all mice were treated intraperitoneally with 7 μg of anti-TRPI, which recognizes tumor antigens highly expressed on B16F10 cells and leads to tumor cell death by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In addition, starting 24 hours after implantation, mice were treated intraperitoneally with 10 mg / kg of S9.B-mIgG2a or mIgG2a isotype control once every 3 days until the end of the study. 15 days after implantation, lungs were harvested from mice and tumor nodules were counted.

[0201] As shown in Figure 20, S3 / 7 / 9BAC mice treated with S9.B-mIgG2a (S9-Fc) had a significant reduction in lung nodules compared to S3 / 7 / 9BAC mice treated with isotype control. Means are shown. **p<0.01, two-tailed t-test. These results suggest that Siglec-9-Fc may be effective in treating metastatic cancer. Furthermore, the results also suggest that Siglec-9-Fc enhances the ADCC and / or ADCP activity of anti-TRP1.

[0202] Example 27: Siglec-9-Fc monotherapy significantly inhibits E0771 tumor growth The ability of Siglec-9-Fc to reduce solid tumor growth was tested in the E0771 syngeneic breast cancer model. This tumor model is relatively rich in myeloid cell content. S3 / 7 / 9BAC mice were implanted with E0771 cells subcutaneously. Tumors grew to an average of 100 mm 3 Once this was reached, mice were treated intraperitoneally with 20 mg / kg S9.B-mIgG2a or isotype control, twice per week for 3 weeks.

[0203] As shown in Figure 21, S9.B-mIgG2a monotherapy inhibits tumor growth compared to isotype control. Means ± SEM are shown. * p<0.05, two-tailed t-test for all time points indicated. These data indicate that Siglec-9-Fc has efficacy in treating tumors populated by myeloid cells.

[0204] Example 28: Siglec-9-hIgG1 NSLF shows cooperative binding between sialic acid and Fcγ receptors To determine the effect of Fcγ receptor engagement on the binding of Siglec-9-Fc, S9-hIgG1 NSLF (SEQ ID NO: 48, signal sequence is cleaved during production) and S9.A-hIgG1 LALAPS (SEQ ID NO: 42, signal sequence is cleaved during production) were tested for binding to MDSCs. MDSCs were generated as previously described and incubated with a titrated amount of S9-hIgG1 NSLF for 2 hours on ice in the dark, followed by incubation with fluorescently conjugated anti-mouse IgG (Jackson Immunoresearch) for 30 minutes. Binding was assessed by flow cytometry using a BD FACS Canto and analyzed using FlowJo™ software. As shown in Figure 22, the calculated FACS Kd on MDSCs for S9-hIgG1 NSLF was in the low nM range (Figure 22A) and was approximately 75-fold lower for S9.A-hIgG1 LALAPS (Figure 22B). The FACS Kd calculated for S9.A-hIgG1 LALAPS was more similar to the FACS Kd previously calculated for S9.A-hIgG1 (SEQ ID NO: 40) in a reference cancer cell line, A549, that does not express Fcγ receptors (Figure 22C). These studies show that Siglec-9-Fc binds with higher affinity to human primary bone marrow cells when the Fc-Fcγ receptor binding is intact, and provide further evidence for a cooperative binding mechanism.

[0205] Example 29: Siglec-9-Fc exhibits broad binding of sialic acid moieties To assess the binding of multiple Siglecs to distinct sialic acid glycans, a glycan array consisting of 300 different glycan moieties, including sialic acid-containing and non-sialic acid-containing glycans, was stained with Siglec-3-Fc, Siglec-5-Fc, Siglec-7-Fc, Siglec-10-Fc and Siglec-15-Fc from R&D systems; Siglec-9-hIgG1 (SEQ ID NO: 40, signal sequence is cleaved during production); or an isotype control. Binding was assessed using a fluorescently labeled anti-human antibody. Data were normalized and normalized fluorescence values ​​were calculated. Staining for a subset of sialic acid-containing glycans is shown in FIG. 23. Siglec-9-hIgG1 showed tight binding to many, but not all, types of sialic acid moieties. Binding of Siglec-3-Fc, Siglec-5-Fc, Siglec-7-Fc, Siglec-10-Fc and Siglec-15-Fc was generally more limited. Siglec-9-hIgG1 bound all Siglec10 and 15 ligands, the majority of Siglec3 and 7 ligands, and nearly half of the Siglec5 ligands. These results indicate that Siglec-9-hIgG1 is an efficient blocker of multiple different Siglec ligands compared to other Siglec-Fc constructs and may therefore have significance in a therapeutic setting.

[0206] Example 30: Siglec-9-hIgG1 NSLF exhibits enhanced binding to innate immune cells in human blood compared to Siglec-9-hIgG1 To further demonstrate that cooperative binding of Siglec-9-Fc can occur in whole blood, binding of Siglec-9-Fc was evaluated in blood of healthy donors. 100 μl of whole blood was incubated with serial dilutions of Alexa 647-conjugated S9-hIgG1 (SEQ ID NO: 48, signal sequence is cleaved during production) or S9-hIgG1 NSLF (SEQ ID NO: 45, signal sequence is cleaved during production). Red blood cells (RBCs) were lysed and all samples were acquired on a BD Fortessa™. Mean fluorescence intensity (MFI) and % binding relative to IgG were calculated. S9-hIgG1 NSLF showed enhanced binding to blood monocytes compared to S9-hIgG1 (FIG. 24A). This is consistent with the desired increase in affinity of S9-hIgG1 NSLF for FcγRIIa compared to wild-type hIgG1. The highest binding of S9-hIgG1 NSLF was observed to monocytes, with lower degrees of binding to granulocytes, NK cells, and B cells, and slight binding to T cells and platelets (Figure 24B). These data demonstrate that Siglec-9-Fc binds to immune cells, particularly monocytes, granulocytes, and NK cells, in the presence of serum immunoglobulins.

[0207] Example 31: Siglec-9-hIgG1 NSLF restores T cell proliferation The effect of Siglec-9-hIgG1 NSLF (SEQ ID NO: 45, signal sequence is cleaved during production) was determined using a method similar to that described in Example 10 and Figure 5. MDSCs were generated from human monocytes by culturing with GM-CSF and IL-6 for 5-6 days. MDSCs were harvested and expressed as autologous CD8 +MDSCs were co-cultured with T cells in the presence of anti-CD3 and anti-CD28 antibodies and either Siglec-9-hIgG1 NSLF or control IgG. After 3-5 days, T cell proliferation was assessed. As shown in FIG. 25A, the presence of MDSCs inhibited T cell proliferation, which was restored by Siglec-9-hIgG1 NSLF. As shown in FIG. 25B, the efficacy of Siglec-9-hIgG1 NSLF was assessed in a dose-response manner. A single-digit nM EC50 (approximately 1-2 nM) was observed in restoring T cell proliferation.

[0208] Example 32: Siglec-9-hIgG1 NSLF shows increased potency compared to Siglec-9-hIgG1 Siglec-9-hIgG1 NSLF (SEQ ID NO: 45, signal sequence is cleaved during production) was directly compared to Siglec-9-hIgG1 (SEQ ID NO: 48, signal sequence is cleaved during production) in the MDSC T cell assay described in Example 10. As shown in Figure 26, Siglec-9-hIgG1 NSLF demonstrated approximately 10-fold increased potency compared to Siglec-9-hIgG1. Collectively, these data demonstrate a potent effect of Siglec-9-hIgG1 NSLF in relieving T cell myelosuppression.

[0209] Example 33: Siglec-9-hIgG1 NSLF induces cytokine expression consistent with repolarization The induction of various cytokines, chemokines, and costimulatory molecules by Siglec-9-hIgG1 NSLF (SEQ ID NO: 45, signal sequence is cleaved during production) was analyzed in MDSCs by RNAseq. As shown in Figure 27, Siglec-9-hIgG1 NSLF induced a robust gene expression profile when incubated with MDSCs, which was consistent with repolarization. Similar profiles were also observed in macrophages and dendritic cells (data not shown).

[0210] Example 34: Siglec-9-hIgG1 NSLF repolarizes suppressed myeloid cells better than other checkpoint pathways Siglec-9-hIgG1 NSLF (SEQ ID NO: 45, signal sequence is cleaved during production) was directly compared with antibodies targeting other immune checkpoint pathways for their ability to repolarize suppressed myeloid cells. As shown in Figure 28, Siglec-9-hIgG1 NSLF is highly effective in repolarizing MDSCs compared to those antibodies. Anti-Siglec-15, anti-LILRB2, and anti-PD-L1 are not able to induce CD86 upregulation or CD206 downregulation on the surface of MDSCs as well as Siglec-9-hIgG1 NSLF. These results demonstrate that Siglec-9-hIgG1 NSLF may be a highly effective treatment, potentially more effective than checkpoint inhibitors.

[0211] Example 35: Siglec-9-Fc in combination with anti-PD-L1 reduces E0771 tumor growth The effect of Siglec-9-Fc in combination with anti-PD-L1 was determined using the method as shown in Example 27. E0771 cells were subcutaneously implanted into S3 / 7 / 9BAC mice. Tumors grew to an average of 100 mm 3 Once the tumor reached a median age of 10 days, mice were treated intraperitoneally with 20 mg / kg S9.B-mIgG2a and 10 mg / kg anti-PD-L1 antibody twice per week for 3 weeks. As shown in Figure 29, the combination of Siglec-9-Fc and anti-PD-L1 antibody reduced E0771 tumor growth to a greater extent than either Siglec-9-Fc or anti-PD-L1 antibody treatment alone. At 25 days post-implantation, tumor growth inhibition of 58% was achieved compared to Siglec-9-Fc monotherapy. Mean ± SEM is shown. These studies indicate that the combination of Siglec-9-Fc with PD-1 or PD-L1 inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies, can improve anti-tumor responses.

[0212] Example 36: Potential pharmacodynamic markers of Siglec-9-Fc To clarify the mechanism of action and to identify potential pharmacodynamic (PD) markers of response, immune monitoring studies were performed. Mice were inoculated with E0771 tumor cells at 100 mm 3 Mice were randomized into two groups with an average volume of 1000 mg / kg / day and administered S9.B-mIgG2a (SEQ ID NO: 44, signal sequence is cleaved during production) or isotype control three times every 3-4 days. 24 hours after the last dose, mice were euthanized and spleens and tumors were harvested for flow cytometry analysis. CD11b is a pleiotropic regulator of myeloid cell function, including regulation of adhesion, migration, phagocytosis, and cell activation. S9.B-mIgG2 induced a significant increase in CD11b and CD86 expression on splenic myeloid cells (Figure 30). These changes in splenic myeloid cells are consistent with those observed in human MDSCs and indicate potential pharmacodynamic markers of Siglec-9-Fc.

[0213] Example 37: Additional Siglec-9 variants within and outside the IgV domain Additional Siglec-9-Fc variants were generated that may improve properties such as stability and / or PK. The specific variants generated are shown in FIG. 31, and all of the contemplated variants are included in the sequence listing below. In the variants designated S9.32-S9.38, a single tryptophan (W38) in an undesired hydrophobic patch in the IgV domain was replaced with a less hydrophobic residue. The variants designated S9.39 and S9.41-S9.45 contain additional substitutions in the IgV domain that may further reduce the effect of the undesired hydrophobic patch. The variants designated S9.47-S9.53 contain substitutions outside the IgV domain that may confer stability. As shown in FIG. 31, specific variants were tested for specific properties. In addition, specific variants were tested in assays similar to those described in Example 13 to examine the effect on markers of repolarization in MDSCs. As shown in Figure 32, variants S9.36, S9.37 and S9.38 behaved comparable to Siglec-9-Fc-hIgG1, exhibiting a decrease in CD163 (Figure 32A) and a decrease in CD206 (Figure 32B) and an increase in CD86 (Figure 32C).

[0214] Example 38: Fc variants to improve FcRn binding and half-life Further substitutions and modifications were made in the Fc region of Siglec-9-hIgG1 NSLF (SEQ ID NO: 45) so that its half-life could be improved. The Fc region of Siglec-9-hIgG1 NSLF is predicted to be bound by the neonatal Fc receptor (FcRn) in the acidic environment of the endosome when Siglec-9-hIgG1 NSLF is taken up into cells in vivo. As a result of this binding, Siglec-9-hIgG1 NSLF will be directed back to the cell surface and released into the extracellular environment under physiological pH conditions, instead of being degraded in the acidic endosome. By "recycling" Siglec-9-hIgG1 NSLF back to the extracellular environment after internalization, this process may increase the amount of Siglec-9-hIgG1 NSLF in circulation, thereby improving its half-life. This may then allow for smaller dosages or less frequent dosing.

[0215] Therefore, substitutions and modifications were made in the Fc region of Siglec-9-hIgG1 NSLF (SEQ ID NO: 45) to improve its binding to FcRn in vitro and potentially improve its ability to be recycled in vivo. These substitutions and modifications include "YTE" and "LS" substitutions, as well as cysteine-containing loop insertions, as described in Dall' Acqua et al. (2002) J. Immunol. 169:5171-5180; Zalevsky et al. (2010) Nat. Biotechnol. 28:157-159; and U.S. Patent No. 9,688,756, each of which is incorporated herein by reference in its entirety. The sequences of the resulting modified constructs are shown in SEQ ID NOs: 228-230 (substitutions and modifications are indicated by the double-underlined residues in the sequence listing below). The modified constructs are tested for improved binding to FcRn in vitro, for example by surface plasmon resonance, and then examined for improved PK and PD in vivo. Modified constructs that contain "YTE" or "LS" substitutions or cysteine-containing loop insertions in the Fc but not NSLF substitutions are also contemplated. These constructs are shown in SEQ ID NOs: 231-233.

[0216] Example 39: Siglec-9-hIgG1 NSLF has improved serum PK compared to Siglec-9-hIgG1 The pharmacokinetic properties of Siglec-9-hIgG1 NSLF (SEQ ID NO: 45, signal sequence is cleaved during production) and Siglec-9-hIgG1 (SEQ ID NO: 48, signal sequence is cleaved during production) were compared. Cynomolgus monkeys were treated with a single dose of 80 mg / kg intravenous injection of Siglec-9-hIgG1 or Siglec-9-hIgG1 NSLF. The mean concentration-time profiles of Siglec-9-hIgG1 and Siglec-9-hIgG1 NSLF in the serum of Cynomolgus monkeys were determined. Figure 33 shows that Siglec-9-hIgG1 NSLF (squares) has improved PK over Siglec-9-hIgG1 (circles).

[0217] Example 40: Pharmacokinetics of Siglec-9-Fc variants As shown in Example 37, the pharmacokinetic properties of certain Siglec-9-Fc variants were determined. S9.1, S9.36, S9.37, S9.38, and S9.45 were given as a single dose by intravenous bolus injection to Siglec3 / 7 / 9BAC transgenic mice. As shown in Figure 34, S9.37 showed a C max and AUC 0-inf The mean T 1 / 2 is similar to other variants, but the increased AUC may indicate improved exposure (bioavailability) of S9.37 specifically compared to other variants.

[0218] Table of specific sequences In the table below, the bolded and underlined residues in a particular SEQ ID NO: represent residues in which the variant Siglec-9 ECD sequence differs from the native Siglec-9 ECD sequence. The double underlined residues in SEQ ID NO:228-233 represent variant Fc domain residues. In some cases, the residue numbers used in the name (e.g., S35X) for a particular Siglec-9 variant in the "Description" column may not match the numbering of the residues in the SEQ ID NO: in the "Sequence" column (e.g., due to the presence or absence of a signal sequence), as can be seen when comparing the bolded and underlined mutant residues with their position in the SEQ ID NO: below. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 8-22

Table 8-23

Table 8-24

Table 8-25

Table 8-26

Table 8-27

Table 8-28

Table 8-29

Table 8-30

Table 8-31

Table 8-32

Table 8-33

Table 8-34

Table 8-35

Table 8-36

Table 8-37

Table 8-38

Table 8-39

Table 8-40

Table 8-41

Table 8-42

Table 8-43

Table 8-44

Table 8-45

Table 8-46

Table 8-47

Table 8-48

Table 8-49

Table 8-50

Table 8-51

Table 8-52

Table 8-53

Table 8-54

Table 8-55

Table 8-56

Table 8-57

Table 8-58

Table 8-59

Table 8-60

Table 8-61

Table 8-62

Table 8-63

Table 8-64

Table 8-65

Table 8-66

Table 8-67

Table 8-68

Table 8-69

Table 8-70

Table 8-71

Table 8-72

Table 8-73

Table 8-74

Table 8-75

Table 8-76

Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:78 bound to an Fc domain at its C-terminus, wherein the Fc domain has a human IgG1 isotype.

2. wherein the Fc domain is, relative to the IgG1 polypeptide of SEQ ID NO: 142: a) reduced binding to FcγRIII; b) reduced antibody-dependent cellular cytotoxicity (ADCC) and / or reduced complement fixation activity; c) increased binding to FcγRIIa; or d) any combination of a), b), and / or c).

2. The isolated polypeptide of claim 1, having the following structure:

3. 3. The isolated polypeptide of claim 1 or 2, wherein the Fc domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 142-144 and 234-239.

4. 2. The isolated polypeptide of claim 1, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

142.

5. 2. The isolated polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:

10.

6. 3. The isolated polypeptide of claim 1 or 2, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

143.

7. 2. The isolated polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

227.

8. An isolated polypeptide as described in claim 1, comprising an amino acid sequence selected from any one of SEQ ID NOs: 45-48 and 228-233, lacking amino acid residues 1-19.

9. 2. The isolated polypeptide of claim 1, comprising an amino acid sequence selected from any one of SEQ ID NOs: 45-48 and 228-233.

10. The isolated polypeptide of claim 8, comprising the amino acid sequence of SEQ ID NO:45 lacking amino acid residues 1-19.

11. 10. The isolated polypeptide of claim 9 comprising the amino acid sequence of SEQ ID NO:

45.

12. The isolated polypeptide of claim 8, comprising the amino acid sequence of SEQ ID NO:48 lacking amino acid residues 1 to 19.

13. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:78 linked at its C-terminus to SEQ ID NO:

143.

14. 14. The isolated polypeptide of any one of claims 1 to 13, wherein the polypeptide binds to sialic acid on the surface of a cell.

15. The isolated polypeptide of claim 14, wherein the cell is a tumor cell.

16. The isolated polypeptide of claim 14, wherein the cell expresses an FcR.

17. The isolated polypeptide of claim 14, wherein the cell is a bone marrow cell.

18. The isolated polypeptide of claim 17, wherein the bone marrow cells are selected from monocytes, macrophages, dendritic cells, microglial cells, and myeloid derived suppressor cells (MDSCs).

19. a) blocking cellular binding of any one or more Siglec family members selected from Siglec-3, Siglec-5, Siglec-7, Siglec-9, Siglec-10, and Siglec-15; b) relieving MDSC-mediated suppression of T cells; c) repolarizing MDSCs towards a pro-inflammatory phenotype; d) increasing the expression of CD86 on MDSCs, increasing the expression of CD11b on MDSCs, and / or decreasing the expression of CD163 on MDSCs; e) repolarizing tumor macrophages away from the M2 phenotype; f) reducing CD163+ and / or CD206+ macrophages; g) inducing expression of one or more chemokines selected from CCL3, CCL4, CCL5, CCLI7, CXCL1, CXCL9, and IL-8 in MDSCs; h) reducing the recruitment of myeloid cells to the tumor microenvironment; i) binds to MDSCs with an affinity of less than 100 nM, less than 50 nM, less than 25 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 2 nM, 1-50 nM, 1-25 nM, 1-20 nM, 1-10 nM, 1-5 nM, or 1-2 nM; or j) The isolated polypeptide of any one of claims 1 to 18, which performs any one or more of (a) to (i).

20. The isolated polypeptide described in claim 19, wherein in b), relieving MDSC-mediated suppression of T cells is determined by measuring an increase in IFNγ expression or an increase in T cell proliferation.

21. 20. The isolated polypeptide of claim 19, wherein the MDSC is a human MDSC and / or the macrophage is a human macrophage.

22. 22. An isolated nucleic acid comprising a nucleic acid sequence encoding the isolated polypeptide of any one of claims 1 to 21.

23. 23. The isolated nucleic acid of claim 22, encoding an amino acid sequence selected from any one of SEQ ID NOs: 45-48 and 228-233.

24. 23. The isolated nucleic acid of claim 22, encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 78 or 227.

25. An expression vector comprising the isolated nucleic acid of any one of claims 22 to 24.

26. 26. A host cell comprising the isolated nucleic acid of claim 22 or 23 or the expression vector of claim 25.

27. A host cell expressing the isolated polypeptide of any one of claims 1 to 21.

28. 28. A method for producing a polypeptide comprising culturing a host cell according to claim 26 or claim 27 and optionally comprising isolating the polypeptide.

29. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 21 and a pharma- ceutically acceptable carrier.

30. A medicament for treating cancer, comprising a polypeptide according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 29.

31. The pharmaceutical composition of claim 30, wherein the cancer is a solid tumor associated with a tumor microenvironment that includes myeloid cells.

32. The pharmaceutical agent of claim 30 or 31, wherein the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer and uterine cancer.

33. The pharmaceutical composition according to any one of claims 30 to 32, wherein the cancer is metastatic.

34. The pharmaceutical agent according to any one of claims 30 to 33, further comprising administering an antagonist of PD-1 or PD-L1, said antagonist of PD-1 or PD-L1 being an antibody that binds to PD-1 or PD-L1, respectively.

35. The pharmaceutical composition according to any one of claims 30 to 34, further comprising a chemotherapeutic agent.

36. A medicament for repolarizing myeloid-derived suppressor cells (MDSCs) in a subject to a pro-inflammatory phenotype, comprising a polypeptide according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 29.

37. A medicament for repolarizing tumor macrophages from the M2 phenotype in a subject with cancer, comprising a polypeptide according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 29.

38. A medicament for activating bone marrow cells in a subject, comprising a polypeptide according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 29.

39. The method of claim 38, wherein the bone marrow cells are microglial cells.

40. The pharmaceutical of any one of claims 36, 38 or 39, wherein the subject has cancer.

41. The pharmaceutical of claim 37 or 40, wherein the cancer is a solid tumor associated with a tumor microenvironment that includes myeloid cells.

42. 42. The pharmaceutical of claim 40 or 41, wherein the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck cancer, breast cancer and uterine cancer.

43. The method according to any one of claims 40 to 42, wherein the cancer is metastatic.

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