Siglec-9 ECD fusion molecule variants and methods of use thereof

Abstract

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

Description

SIGLEC-9 ECD FUSION MOLECULE VARIANTS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No. 63 / 655,515, filed June 3, 2024, which is incorporated by reference herein in its entirety for any purpose.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 20, 2025, is named “01209-0017_00PCT_SL.xml” and is 112,016 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD

[0003] The present disclosure relates to Siglec-9 ECD fusion molecule variants and therapeutic uses of such fusion proteins.BACKGROUND

[0004] 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 microglia, as well as lymphoid cells, such as natural killer cells and subsets of T cells (Crocker et al. (2007) Nat. Rev. Immunol. 7(4):255- 266; O’Reilly and Paulson (2009) Trends in Pharmacol. Sci. 30(5):240-248; and Macauley et al. (2014) Nat. Rev. Immunol. 14(10):653-666). Siglec-9 is a member of the Siglec family of lectins that bind sialic acid residues of glycoproteins and glycolipids. Potential ligands for Siglec proteins are gangliosides, which are glycolipids comprising a ceramide linked to a sialylated glycan. Diversity in the Siglec ligands is generated by the addition of other neutral sugars and sialic acid in different linkages, either branched or terminal, and modification of sialic acid itself.

[0005] Fourteen Siglec proteins have been identified in humans and nine in mice that are comprised of 2-17 extracellular Ig domains including an amino-terminal V-set Ig-like (IgV)domain that contains the sialic acid binding site. The IgV domain contains two aromatic residues and one arginine in a motif that is highly conserved in all Siglecs (Crocker et al. (2007) Nat. Rev. Immunol. 7(4):255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; Von Gunten and Bochner (2008) Ann N Y Acad Sci. 1143:61-82; May et al. (1998) Mol Cell. l(5):719-728; Crocker et al. (1999) Biochem J. 341(2):355- 361; and Crocker and Varki (2001) Trends Immunol. 22(6):337-342). The ligand binding sites have been mapped by crystal structures with and without ligand bound (Attrill et al., (2006) J. Biol. Chem. 281(43):32774-32783; Alphey et al. (2003) J. Biol. Chem. 278(5):3372-3377; Varki et al., Glycobiology, 16(1): 1R-27R; and May et al. (1998) Mol. Cell 1 (5):719-728). Because cell membranes are rich in sialic acids, ligand binding by Siglecs can occur in cis and in trans, which affects their functional properties.Each Siglec has a distinct preference for binding the diverse types of sialylated glycans that are found on the surface of mammalian cells (Crocker et al. (2007) Nat Rev Immunol. 7(4): 255-266).

[0006] Most Siglec proteins, including Siglec-9, are inhibitory receptors that contain one or more immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in their cytoplasmic domains. The inhibitory Siglecs act as negative regulators of immune function (Crocker et al. (2007) Nat Rev Immunol. 7(4):255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; and Von Gunten and Bochner (2008) Ann N Y Acad Sci. 1143:61-82). Other Siglecs are activating receptors that contain immunoreceptor tyrosine-based activating motif (ITAM) sequences in their cytoplasmic domains. Those Siglecs act as positive regulators of immune function (Macauley SM. et al., (2014) Nature Reviews Immunology 14(10):653-666).

[0007] The Siglec protein family plays a role in tumor pathogenesis. Many human tumors robustly upregulate sialic acid ligands that bind Siglec-9, which may enable immune evasion and cancer progression (Jandus et al. (2014) J. Clinic. Invest. 124(4): 1810-1820). In contrast, tumors lacking sialic acid biosynthesis have reduced growth in mice (Stanczak et al. (2018) J Clin Invest. 128(11):4912-4923). Certain SNPs in Siglec-3, 7, 9 are associated with decreased risk of colorectal and lung cancer (Stanczak et al., 2018).

[0008] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.SUMMARY

[0009] The present disclosure is generally directed to Siglec-9 extracellular domain (ECD) fusion proteins and methods of treating cancer and neurodegenerative diseases using Siglec-9 ECD fusion proteins.

[0010] In some embodiments, an isolated polypeptide comprising a Siglec-9 IgV domain is provided. In some embodiments, the isolated polypeptide is a Siglec-9 extracellular domain (ECD) comprising the Siglec-9 IgV domain. In some embodiments, the isolated polypeptide further comprises a C2 type 1 (C2T1) domain. In some embodiments, the isolated polypeptide further comprises a C2 type 2 (C2T2) domain. In some embodiments, the isolated polypeptide is a Siglec-9 extracellular domain (ECD) comprising a deletion of the C2 type 1 (C2T1) domain. In some embodiments, the isolated polypeptide is a Siglec-9 ECD comprising a deletion of the C2 type 2 (C2T2) domain. In some embodiments, the isolated polypeptide is a Siglec-9 ECD comprising a deletion of both the C2T1 domain and the C2T2 domain. In some embodiments, the isolated polypeptide comprises one or more N-glycosylation site modifications relative to an unmodified Siglec-9 ECD. In some embodiments, the isolated polypeptide comprises one or more cysteine modifications relative to an unmodified Siglec-9 ECD, optionally wherein the modifications comprising substitution of a cysteine with serine. In some embodiments, the isolated polypeptide further comprises an Fc domain linked to the isolated polypeptide by a linker.[OH] In some embodiments, the isolated polypeptide is a Siglec-9 ECD comprising the amino acid sequence of any one of SEQ ID NO: 2-6.

[0012] In some embodiments, the isolated polypeptide comprises modification of one or more cysteine residues selected from C158 and C295 of SEQ ID NO: 31 or residues corresponding to amino acid residues C158 and C295 of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, the modification comprises substitution of cysteine with serine.

[0013] In some embodiments, the isolated polypeptide comprises one or more amino acid substitutions relative to the unmodified Siglec-9 ECD set forth in SEQ ID NO:31 selected from N101Q, N138Q, N161Q, N225Q, N231Q, N238Q, N256Q, N334Q, T103A, T140A, T163A, T227A, S233A, T240A, S258A, S336A, and combinations thereof, or one or more amino acid substitutions relative to the unmodified Siglec-9 ECD set forth in SEQ ID NO:31 corresponding to N101Q, N138Q, N161Q, N225Q, N231Q, N238Q, N256Q, N334Q, T103A, T140A, T163A, T227A, S233A, T240A, S258A, S336A, wherein the numbering of the residues is according to EU numbering. In some embodiments, the amino acid substitutions comprise N138Q, C158S, N161Q, N225Q, N231Q, N238Q, N256Q, C295S, and N334Q. In some embodiments, the amino acid substitutions comprise C158S, T163A, T227A, S233A, T240A, S258A, C295S, and S336A.

[0014] In some embodiments, the isolated polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 10-25 or 29-30. In some embodiments, the linker is selected from one or more of SEQ ID NOs 40-43. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 3, 6, or 8.

[0015] In some embodiments, an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30 is provided. In some embodiments, the isolated polypeptide further comprises an Fc domain. In some embodiments, the Fc domain is located at the C-terminus of the polypeptide. In some embodiments, the Fc domain has a human IgGl or IgG4 isotype. In some embodiments, the isolated polypeptide comprises an Fc domain with a human IgGl isotype that has, relative to the IgGl polypeptide of SEQ ID NO:44: (a) reduced binding to FcyRIII; (b) reduced antibody-dependent cellular cytotoxicity (ADCC) and / or reduced complement binding activity; (c) increased binding to FcyRIIa; or any combination of a), b), and / or c). In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOS: 45 or 46. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the Fc domain has an IgG4 isotype. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-80 and 82-85.

[0016] In any of the embodiments provided herein, the isolated polypeptide binds sialic acid on the surface of cells. In some embodiments, the cells are tumor cells. In some embodiments, the cells express FcR. In some embodiments, the cells are myeloid cells. In some embodiments, the cells are selected from monocytes, macrophages, dendritic cells, microglia, and myeloid-derived suppressor cells (MDSCs).

[0017] In any of the embodiments of an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein, the polypeptide: a) blocks cell 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) relieves MDSC-mediated suppression of T-cells, optionally as determined by measuring an increase in IFNy expression or an increase in T-cell proliferation; c) repolarizes MDSCs to a pro-inflammatory phenotype; d) increases expression of CD86 on MDSCs, increases expression of CD1 lb on MDSCs, and / or decreases expression of CD 163 on MDSCs; e) repolarizes tumor macrophages away from an M2 phenotype; f) reduces CD 163+ and / or CD206+ macrophages; g) induces expression of one or more chemokines selected from CCL3, CCL4, CCL5, CCL17, CXCL1, CXCL9, and IL-8 in MDSCs; h) reduces myeloid cell recruitment into 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; j) has increased expression, yield, and / or % monomer following purification relative to a Siglec-9 ECD comprising SEQ ID NO: 1 purified under identical conditions; k) shows higher or comparable binding to A375 cells compared to a Siglec-9 ECD Fc comprising SEQ ID NO: 86; l) repolarizes MDSCs to a pro-inflammatory phenotype to a degree comparable to a Siglec-9 ECD Fc comprising SEQ ID NO: 86; m) has a hydrophobic interaction chromatography (HIC) retention time comparable to a Siglec-9 ECD Fc comprising SEQ ID NO: 86; n) shows < 20% decrease in monomeric content after storage at 40 °C for 2, 7, and / or 14 days compared to the monomeric content at day 0, as measured by analytical size exclusion chromatography (SEC); o) has a melting temperature onset (Tm onset) comparable to that of a Siglec-9 ECD Fc comprising SEQ ID NO: 86, as measured by differential scanning fluorimetry; and / or p) has reduced degree of nonspecific binding to baculovirus particles (BVP) and / or double stranded DNA compared to a Siglec-9 ECD Fc comprising SEQ ID NO: 86, as measured by ELISA.

[0018] In some such embodiments, the MDSCs are human MDSCs and / or the macrophages are human macrophages.

[0019] In some embodiments, an isolated nucleic acid is provided that comprises a nucleic acid sequence that encodes an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein. In some embodiments, an expression vector is provided that comprises the isolated nucleic acid.

[0020] In some embodiments, a host cell is proved, which comprises an isolated nucleic acid or expression vector proved herein. In some embodiments, a host cell is provided, which expresses an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein. In someembodiments, a method of producing the polypeptide is provided, comprising culturing the host cell. In some such embodiments, the polypeptide is isolated.

[0021] In various embodiments, a pharmaceutical composition is provided, which comprises an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may comprise (i) a polypeptide as described herein with its signal peptide, or (ii) a polypeptide lacking its signal peptide; and a pharmaceutically acceptable carrier.

[0022] In some embodiments, a method of treating cancer is provided, comprising administering to a subject with cancer an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) 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, gastric cancer, cervical cancer, and uterine cancer. In some embodiments, the cancer is metastatic. 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.

[0023] In some embodiments, a method of treating a neurological or neurodegenerative disease is provided, comprising administering to a subject with a neurological or neurodegenerative disease an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide.

[0024] In some embodiments, a method of repolarizing myeloid-derived suppressor cells (MDSCs) to a pro-inflammatory phenotype in a subject is provided, comprising administering to a subject with a neurological or neurodegenerative disease an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide. In some such embodiments, the subject has a neurological or neurodegenerative disease. In some embodiments, the neurological or neurodegenerative disease is characterized by dysfunctional or deficient microglia.

[0025] In some embodiments, a method of repolarizing tumor macrophages away from an M2 phenotype in a subject having cancer is provided, comprising administering to the subject an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide.

[0026] In some embodiments, a method of activating myeloid cells in a subject is provided, comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide. In some such embodiments, the myeloid cells are microglia. In some embodiments, the methods of repolarizing myeloid-derived suppressor cells (MDSCs) to a pro-inflammatory phenotype or of activating myeloid cells, are in a subject having cancer. In some such 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, gastric cancer, cervical cancer, and uterine cancer. In some embodiments, the cancer is metastatic.

[0027] In some embodiments, a method of repolarizing tumor macrophages away from an M2 phenotype in a subject having cancer is provided, the method administering to the subject an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) 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, gastric cancer, cervical cancer, and uterine cancer. In some cases, the cancer is metastatic.

[0028] In some embodiments, a method of activating myeloid cells in a subject is provided, the method comprising administering to the subject an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide. In some cases, the myeloid cells are microglia. 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, gastric cancer, cervical cancer, and uterine cancer. In some cases, the cancer is metastatic.

[0029] In some embodiments, the methods of activating myeloid cells, are in a subject having a neurodegenerative disease. In some embodiments, the neurological or neurodegenerative disease is characterized by dysfunctional or deficient microglia. 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, Taupathy disease, multiple sclerosis, immune-mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, pediatric-onset leukoencephalopathy and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

[0030] In some embodiments, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide, wherein an elevated expression level of CD 163 and / or Siglec-9 has been detected in a tumor sample from the subject.

[0031] In some embodiments, a method of predicting a response to treatment with a Siglec-9 extracellular domain (ECD) fusion polypeptide in a subject having cancer is provided, comprising determining an expression level of CD163 and / or Siglec-9 in a tumor sample from the subject, wherein an elevated expression level of CD 163 and / or Siglec-9 predicts response to treatment with the polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceuticalcomposition comprising the polypeptide. In some such embodiments, the method further comprises administering a Siglec-9 ECD fusion polypeptide to the subject.

[0032] In some embodiments, a method of selecting a subject with cancer for treatment with the polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide is provided, comprising selecting the subject for such treatment if a tumor sample from the subject has been determined to have an elevated expression level of CD 163 and / or Siglec-9. In some such embodiments, the method further comprises administering a Siglec-9 ECD fusion polypeptide to the subject.

[0033] In some embodiments, a method of treating cancer in a subject in need there of is provided, comprising determining that a tumor sample from the subject has an elevated expression level of CD163 and / or Siglec-9, and administering the polypeptide comprising a Siglec-9 extracellular domain (ECD) provided herein or a pharmaceutical composition comprising the polypeptide.

[0034] In some such embodiments, the tumor sample from the subject has elevated expression levels of CD163 and Siglec-9. In some embodiments, the tumor sample from the subject has an elevated expression level of CD68. In some embodiments, the tumor sample from the subject has an elevated level of sialic acid. In some embodiments, the tumor sample from the subject is obtained from a tumor biopsy. In some embodiments, the expression level of CD 163 and / or Siglec-9, and / or optionally CD68, and / or optionally the level of sialic acid, is detected by immunohistochemistry (IHC). In some embodiments, the expression level of CD163, Siglec-9, or CD68 or the level of sialic acid is determined to be elevated if IHC detects 20-100 stained cells per high power field, or greater than 100 stained cells per high power field, or if the IHC score on a scale of 0-3 is IHC 2 or IHC 3, for CD163, Siglec-9, CD68 or sialic acid. In some embodiments, the expression level of CD163, Siglec-9, or CD68 or the level of sialic acid is determined to be elevated if the IHC score is IHC 2 or IHC 3. In some embodiments, the expression levels of CD 163 and Siglec-9 are determined to be elevated, and the IHC scores for CD 163 and Siglec-9 are selected from: CD 163 IHC 3 and Siglec-9 IHC 3; CD 163 IHC 2 and Siglec-9 IHC 3; CD 163 IHC 3 and Siglec-9 IHC 2; or CD163 IHC 2 and Siglec-9 IHC 2. In some embodiments, the expression levels of CD163, Siglec-9 and CD68 are determined to be elevated, and the IHC scores for CD163, Siglec-9, and CD68 are selected from: CD163 IHC 3, Siglec-9 IHC 3, and CD68 IHC 3; CD163 IHC 2, Siglec-9 IHC 3, and CD68 IHC 3; CD163 IHC 3, Siglec-9 IHC 2, and CD68 IHC 3; CD163 IHC 2, Siglec-9 IHC 2, and CD68 IHC 3; CD163 IHC 3, Siglec-9 IHC 3, and CD68 IHC 2; CD163 IHC 2, Siglec-9 IHC 3, and CD68 IHC 2; CD163 IHC 3, Siglec-9 IHC 2, and CD68 IHC 2; or CD163 IHC 2, Siglec-9 IHC 2, and CD68 IHC 2. In some embodiments, the expression levels of CD163 and Siglec-9 and the level of sialic acid are determined to be elevated, and the IHC scores for CD 163, Siglec-9, and sialic acid are selected from: CD163 IHC 3, Siglec-9 IHC 3, and sialic acid IHC 3; CD163 IHC 2, Siglec-9 IHC 3, and sialic acid IHC 3; CD 163 IHC 3, Siglec-9 IHC 2, and sialic acid IHC 3; CD 163 IHC 2, Siglec-9 IHC 2, and sialic acid IHC 3; CD163 IHC 3, Siglec-9 IHC 3, and sialic acid IHC 2; CD163 IHC 2, Siglec-9 IHC 3, and sialic acid IHC 2; CD163 IHC 3, Siglec-9 IHC 2, and sialic acid IHC 2; or CD163 IHC 2, Siglec-9 IHC 2, and sialic acid IHC 2. In some embodiments, the expression levels of CD163, Siglec-9 and CD68and the level of sialic acid are determined to be elevated, and the IHC scores for CD 163, Siglec-9, CD68, and sialic acid are selected from: CD163 IHC 3, Siglec-9 IHC 3, CD68 IHC 3, and sialic acid IHC 3; CD163 IHC 2, Siglec-9 IHC 3, CD68 IHC 3, and sialic acid IHC 3; CD163 IHC 3, Siglec-9 IHC 2, CD68 IHC 3, and sialic acid IHC 3; CD163 IHC 2, Siglec-9 IHC 2, CD68 IHC 3, and sialic acid IHC 3; CD163 IHC 3, Siglec-9 IHC 3, CD68 IHC 2, and sialic acid IHC 3; CD163 IHC 2, Siglec-9 IHC 3, CD68 IHC 2, and sialic acid IHC 3; CD163 IHC 3, Siglec-9 IHC 2, CD68 IHC 2, and sialic acid IHC 3; CD163 IHC 2, Siglec-9 IHC 2, CD68 IHC 2, and sialic acid IHC 3; CD163 IHC 3, Siglec-9 IHC 3, CD68 IHC 3, and sialic acid IHC 2; CD163 IHC 2, Siglec-9 IHC 3, CD68 IHC 3, and sialic acid IHC 2; CD163 IHC 3, Siglec-9 IHC 2, CD68 IHC 3, and sialic acid IHC 2; CD163 IHC 2, Siglec-9 IHC 2, CD68 IHC 3, and sialic acid IHC 2; CD163 IHC 3, Siglec-9 IHC 3, CD68 IHC 2, and sialic acid IHC 2; CD163 IHC 2, Siglec-9 IHC 3, CD68 IHC 2, and sialic acid IHC 2; CD163 IHC 3, Siglec-9 IHC 2, CD68 IHC 2, or sialic acid IHC 2; CD163 IHC 2, Siglec-9 IHC 2, CD68 IHC 2, and sialic acid IHC 2.

[0035] In some embodiments, IHC for Siglec-9 is conducted with an anti-Siglec-9 antibody comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 87, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 89, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 90, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 91, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, IHC for Siglec-9 is conducted with an anti-Siglec-9 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, optionally wherein the antibody is a murine IgG2A antibody. 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, head and neck cancer, breast cancer, cervical cancer, and gastric cancer. In some embodiments, the cancer is metastatic. In some embodiments, the methods further comprise comprising 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 methods further comprise administering a chemotherapeutic agent.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 shows the Siglec-9 ECD model that was obtained from Alphafold(alphafold.ebi.ac.uk / entry / Q9Y336) and is shown with the cartoon of the hlgGl hinge. The N- glycosylation sites, the cysteine pairs, and the domains are annotated.

[0037] FIG. 2 shows the Siglec-9-ECD Fc variants and the number of variants tested.

[0038] FIG. 3 shows SDS-PAGE analysis of the purified proteins of Siglec-9 ECD Fc variants 1 and 2, as described in Example 2.

[0039] FIGS. 4A-4C show illustrations of the Siglec-9 ECD Fc control (FIG. 4A), Siglec-9 ECD Fc variant 2 (FIG. 4B), and Siglec-9 ECD Fc variant 3 (FIG. 4C).

[0040] FIG. 5 shows a comparison of the truncation sites for Siglec-9 ECD Fc variant 1 (left) and Siglec-9 ECD Fc variant 2 (right).

[0041] FIG. 6 shows A375 cell binding of Siglec-9 ECD Fc control, Siglec-9 ECD Fc variant 2 (fraction 1), Siglec-9 ECD Fc variant 2 (fraction 2), Siglec-9 ECD Fc variant 3, as compared to an isotype control, as described in Example 3.

[0042] FIG. 7 shows A375 cell binding of Siglec-9 ECD Fc variants compared to Siglec-9 ECD Fc control and an isotype control, as described in Example 3.

[0043] FIGS. 8A-8F show CD86 (FIG. 8A, FIG. 8B), CD163 (FIG. 8C, FIG. 8D), and CD206 (FIG. 8E, FIG. 8F) expression from two different donor MDSCs contacted with Siglec-9ECD Fc control, Siglec- 9ECD Fc variant 2 (fraction 1), Siglec-9ECD Fc variant 2 (fraction 2), and controls, as described in Example 4.

[0044] FIGS. 9A-9C show CD86 (FIG. 9A), CD163 (FIG. 9B), and CD206 (FIG. 9C) expression from donor MDSCs contacted with Siglec-9ECD Fc control or Siglec-9ECD Fc variants, as described in Example 4.

[0045] FIG. 10 shows hydrophobicity profiles of the Siglec-9 ECD Fc variants using analytical hydrophobic interaction chromatography (HIC).

[0046] FIG. 11 shows stability of Siglec-9-ECD Fc variants under heat stress using an analytical Size Exclusion Chromatography (aSEC) assay.

[0047] FIG. 12 shows Siglec-9 ECD Fc control, Siglec-9 ECD Fc variant 2 (fraction 1), Siglec-9ECD Fc variant 2 (fraction 2), and Siglec-9ECD Fc variant 3 charge heterogeneity, as described in Example 7.

[0048] FIG. 13 shows a comparison of average Tm onsets of Siglec-9 ECD Fc variants as measured by differential scanning fluorimetry (DSF), as described in Example 8.

[0049] FIG. 14 shows the terminal titer of stable pool cells comparing Siglec-9 ECD Fc control, Siglec- 9 ECD Fc variant 2, and Siglec-9 ECD Fc variant 3, as described in Example 9.

[0050] FIGS. 15A-15B show that Siglec-9 ECD Fc truncated variants showed reduced nonspecific binding (BVP and dsDNA), as described in Example 10.

[0051] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.DETAILED DESCRIPTION

[0052] Provided herein are polypeptides comprising the extracellular domain of Siglec-9 and a fusion partner, e.g., an Fc domain. Siglec-9 ECD-Fc fusion molecules unexpectedly show cooperative binding to myeloid cells, resulting in potent activation of these innate immune cells, compared to antibodies against Siglec-9 or other Siglec proteins. Such activation is useful, e.g., 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 are polypeptides comprising variants of the Siglec-9 extracellular domain, and in the IgV domain, the C2 type 1 (C2T1) domain and / or the C2 type 2 (C2T2), which are engineered to improve stability, solubility, ligand binding and / or other properties. Such variants are useful in fusion molecules for activating the immune response as described above. Other embodiments are further described herein.

[0053] Definitions

[0054] The terms “Siglec-9 extracellular domain” and “Siglec-9 ECD” refer to an extracellular domain polypeptide of Siglec-9 or a fragment thereof that binds sialic acid on the surface of cells. The terms include natural and engineered variants thereof. In some embodiments, a Siglec-9 ECD comprises the IgV domain of Siglec-9. In some embodiments, a Siglec-9 ECD comprises the IgV domain and the C2 type 1 (C2T1) domain and / or the C2 type 2 (C2T2) domain of Siglec-9. In some embodiments, a Siglec- 9 ECD comprises one or more N-glycosylation site modifications and / or the Siglec-9 ECD comprises one or more cysteine modifications. In some embodiments, Siglec-9 ECD comprises a linker. Nonlimiting exemplary Siglec-9 ECDs are shown in SEQ ID NOs: 2-25 and 27-30.

[0055] The term “Siglec-9 ECD fusion molecule” refers to a molecule comprising a Siglec-9 ECD and a covalently-attached 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 the Siglec-9 ECD. A Siglec-9 ECD fusion molecule in which the fusion partner is an Fc domain may also be referred to herein as a “Siglec-9 ECD-Fc fusion molecule,” a “Siglec-9 ECD-Fc,” or a “Siglec-9-Fc.” Nonlimiting exemplary Siglec-9 ECD-Fc fusion molecules are shown in the amino acid sequences of SEQ ID NOs: 57-80 and 82-85, including those sequences with or without their associated signal peptides.

[0056] The term “specific binding” or “specifically binds” or is “specific for” a target moiety means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a test molecule for the target moiety compared to binding of the test molecule for a control moiety. The test molecule specifically binds the 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 stronger than the binding affinity for the control moiety. For the avoidance of doubt, specific binding does not require that a test molecule does not bind any other moieties.

[0057] An “amino acid modification” at a specified position, e.g., of a Siglec-9 ECD of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion “adjacent” to a specified residue means insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue. The preferred amino acid modification herein is a substitution.

[0058] The term “Fc region” herein is used to mean a 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 might vary, the human IgG heavy chain Fc region is generally defined as including a polypeptide from an amino acid residue at position Cys226 or from Pro230, to thecarboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of an Fc region-containing polypeptide, or by recombinantly engineering the nucleic acid encoding the Fc region-containing polypeptide. Suitable native-sequence Fc regions for use in the present disclosure include human IgGl, IgG2, IgG3 and IgG4.

[0059] 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 a native sequence human IgGl Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

[0060] A “variant Fc region” comprises an amino acid sequence which 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 compared to a native sequence Fc region, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0061] ‘ ‘Fc receptor” or “FcR” describes a receptor that binds to the Fc region. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG Fc region (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRII A contains an immunoreceptor tyrosine-based activation motif (“ITAM”) in its cytoplasmic domain. Inhibiting receptor FcyRIIB 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 comprise Fc regions.

[0062] Binding to FcR in vivo and serum half-life of human FcR high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcR, or in primates to which the polypeptides having a variant Fc region are administered. WO 2000 / 042072 (Presta) describes Fc region variants with improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001).

[0063] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a reference polypeptide sequence refers to the percentage of amino acid residues in a query sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes ofdetermining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full-length of the sequences being compared.

[0064] As used herein, “substitutions corresponding to “n,” wherein n is any number, refers to an amino acid position of a subject polypeptide that aligns with position n of a reference polypeptide after aligning the amino acid sequences of the subject and reference polypeptides and introducing gaps. Alignment for purposes of whether a position of a subject polypeptide corresponds with position n of a reference polypeptide can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any parameters needed to achieve maximal alignment over the full length of two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.

[0065] The term “N-glycosylation site” denotes the amino acid residue within an N-glycosylation site consensus sequence to which a glycan is or can be attached. Generally N-linked glycans are attached to the amid nitrogen atom of an asparagine amino acid (Asn, N) side chain. The N-glycosylation site consensus sequence is Asn-X-Ser / Thr, wherein X can be any amino acid residue except proline.

[0066] As used herein, the terms “nucleic acid” and “polynucleotide” and “oligonucleotide” are used interchangeably, and refer to a polymer of 3 nucleotides or more. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5 -bromouridine, C5 -fluorouridine, C5 -iodouridine, C5 -propynyl-uridine, C5 - propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modifiedsugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long. When a number of nucleotides is used as an indication of size, e.g., of a fusion polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a fusion polynucleotide. In some embodiments, a nucleic acid is linear, i.e., beginning at a 5’ end and ending at a 3’ end. In some embodiments, a nucleic acid is circular, such as a circular RNA molecule.

[0067] An “isolated” nucleic acid molecule encoding a polypeptide, such as a polypeptide comprising a Siglec-9 ECD of the present disclosure, is a nucleic acid molecule that is identified and separated from at least one contaminant molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with most or substantially all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides herein are distinguished from nucleic acids existing naturally in cells.

[0068] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.

[0069] “Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include 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.

[0070] A “host cell” includes an individual cell or cell culture that can contain or contains a vector(s) or other exogenous nucleic acid, e.g., that incorporates a polynucleotide insert(s). In some embodiments, the vector or other exogenous nucleic acid is incorporated into the genome of the host cell. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells comprising (e.g., transfected with) a polynucleotide (s) of this invention.

[0071] As used herein, “expression level” and “level” refer to a measurement that is made using any analytical method for detecting a nucleic acid, a protein, or other molecule in a biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, ratio of measured levels, or the like, of, for, or corresponding to a protein, such as CD163, CD68, or Siglec-9, or to another molecule or moiety, such as sialic acid, in the biological sample. The exact nature of the “level” depends on the specific design and components of the particular analytical method employed for detection. In some embodiments, an expression level of a protein or level of another molecule or moiety is determined by immunohistochemistry (IHC).

[0072] As used herein, “elevated expression level” or “elevated level” refers to a measurement that shows an increased concentration or amount of a protein, molecule or moiety in a biological sample, such as a tumor sample, relative to a reference biological sample or falling within a specified range predetermined to indicate an increased concentration or amount. In some embodiments, the measurement may be performed by immunohistochemistry (IHC). As described in certain exemplary embodiments herein, an elevated expression level or elevated level as determined by IHC may be characterized by a score of IHC 2 or IHC 3.

[0073] A “tumor sample” herein refers to a sample comprising or expected to comprise tumor cells that is obtained from a patient. A tumor sample may further comprise immune cells, e.g., innate immune cells, such as myeloid-derived suppressor cells (MDSCs) or macrophages, and / or adaptive immune cells, such as T-cells. By way of example, a tumor sample can be a biopsy obtained from a solid tumor, or a tumor sample can be a blood or plasma sample in the case of a blood-based cancer. In some cases, the tumor sample can be fixed onto a slide for analysis of levels of certain proteins or other molecules in cells from the sample.

[0074] ‘ ‘Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered 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) polypeptide; 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as a polysorbate, such as TWEEN™, polyethylene glycol (PEG), and a poloxamer such as PLURONICS™.

[0075] As used herein, the term “preventing” includes providing prophylaxis with respect to occurrence or recurrence of a particular disease, disorder, or condition in an individual. An individual may be predisposed to, susceptible to a particular disease, disorder, or condition, or at risk of developing such a disease, disorder, or condition, but has not yet been diagnosed with the disease, disorder, or condition.

[0076] As used herein, an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.

[0077] As used herein, the terms “treat,” “treatment,” “treating,” and the like refer to clinical intervention designed to alter the natural course of a clinical pathology in the individual being treated. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, remission or improved prognosis, and / or alleviating or lessening the symptoms of a particular disease, disorder, or condition. An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated. In certain embodiments, a patient is successfully “treated” for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infdtration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition of or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or stable disease (SD); a decrease in progressive disease (PD); reduced time to progression (TTP); or any combination thereof.

[0078] The terms “administer,” “administering,” “administration,” and the like refer to methods that may be used to enable delivery of a therapeutic agent such as a Siglec-9 ECD fusion molecule (e.g., a Siglec-9 ECD-Fc fusion molecule). Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0079] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. An effective amount herein may 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 one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “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, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0080] An “individual” or “subject” or “patient” for purposes of treatment, prevention, or reduction of 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, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual is human.

[0081] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. The cancer may be a primary tumor or may be advanced or metastatic cancer. A “refractory” cancer is one that progresses even though an anti-tumor treatment has been administered to the cancer patient. A “recurrent” cancer, or a cancer that has “recurred,” is one that has regrown, either at the initial site or at a distant site, after a response to initial therapy. A “relapsed” patient is one who has signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy.

[0082] As used herein, administration of an agent or composition “in conjunction” or “in combination” with another agent or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration. In some embodiments, administration in conjunction means administration as a part of the same treatment regimen. In some embodiments, administration of an agent in combination with another agent results in “synergy” or a“synergistic effect,” i.e., the effect achieved when the agents are used together is greater than the sum of the effects that result 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 that result from using the agents separately.

[0083] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0084] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.

[0085] It is understood that aspect and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0086] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.Polypeptides comprising Siglec-9 extracellular domains

[0087] In some embodiments, a Siglec-9 ECD or Siglec-9 ECD fusion molecule according to any of the embodiments herein may incorporate any of the features, singly or in combination, as described herein.

[0088] Provided herein are polypeptides 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: 37. As previously shown, the IgV domain of Siglec-9 is sufficient for binding to sialic acid on the surface of cells. See Example 2 of WO 2021 / 091885, which is incorporated by reference in its entirety and for any reason. In some embodiments, polypeptides are provided that comprise a Siglec-9 extracellular domain (ECD) comprising the IgV domain, and optionally further comprising the C2 type 1 (C2T1) domain, and / or the C2 type 2 (C2T2) domain. The Siglec-9 C2T1 domain comprises amino acids 146-229 of human Siglec-9 of SEQ ID NO: 37, and the Siglec-9 C2T2 domain comprises amino acids 236-336 of human Siglec-9 of SEQ ID NO: 37. In some embodiments, a Siglec-9 ECD comprises amino acids 20- 336 of SEQ ID NO: 37, optionally with one or more amino acid modifications. In some embodiments, the Siglec-9 ECD may comprise the IgV, C2T1 and C2T2 domains, but may lack, for example, the last one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve C-terminal (membrane proximal) amino acids of the ECD. The twelve C-terminal (membrane proximal) amino acids of the ECD are shown in SEQ ID NO: 39. Examples include SEQ ID NOs: 7-31, for instance, which comprise the IgV, C2T1 and C2T2 domains and which lack the C-terminal membrane proximal region of SEQ ID NO: 39.In some embodiments, a polypeptide comprises a Siglec-9 IgV domain comprising one or more amino acid substitutions that improve stability of the polypeptide, improve the binding affinity for sialic acid, improve the function of the polypeptide, improve the pharmacokinetic properties of the polypeptide (e.g., half-life, Cmax, or AUC), or any combination of the foregoing. In some embodiments, a polypeptide comprises a Siglec-9 IgV domain comprising one or more amino acid substitutions that improve stability of the polypeptide, improve the binding affinity for sialic acid, improve the function of the polypeptide, improve the pharmacokinetic properties of the polypeptide (e.g., half-life, Cmax, or AUC), or any combination of the foregoing, with reference to a wild-type Siglec-9 IgV domain, such as the Siglec-9 IgV domain of the human Siglec-9 protein set forth in SEQ ID NO:38. In some embodiments, a polypeptide comprises a Siglec-9 IgV domain having an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30.

[0089] In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain and a C2T1 domain. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain and C2T1 domain with a C2T1 native linker. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain and a C2T1 domain with a C2T1 native linker and a non-native linker. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain and a native IgV domain linker. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain with an amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain with a C to S amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, an IgV domain native linker, and an amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, an IgV domain native linker, and a C to S amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, an IgV domain native linker, a nonnative linker, and an amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, an IgV domain native linker, a non-native linker, and a C to S amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid sequence of any one of SEQ ID NOS: 2-6.

[0090] In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, a native IgV domain linker, a C2T1 domain, a C2T1 native linker, a C2T2 domain, and a C2T2 domain native linker. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, a native IgV domain linker, a C2T1 domain, a C2T1 native linker, a C2T2 domain, a C2T2 domain native linker, and a non-native linker, wherein the non-native linker is between the Siglec-9 EDC and an Fc. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid sequence of SEQ ID NO: 7 or 8.

[0091] In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising one or more amino acid substitutions that improve stability of the polypeptide, improve the binding affinity for sialic acid, improve the function of the polypeptide, improve the pharmacokinetic properties of the polypeptide (e.g., half-life, Cmax, or AUC), or any combination of the foregoing. In some embodiments, theseimprovements are with reference to a wild-type Siglec-9 ECD, such as the wild-type Siglec-9 ECD of the human Siglec-9 protein set forth in SEQ ID NO:38. In some embodiments, a polypeptide comprises a Siglec-9 ECD having an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30. In some embodiments, a polypeptide comprises a Siglec-9 ECD having an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30, optionally which lack the C-terminal membrane proximal region of SEQ ID NO: 39. In some embodiments, one or more cysteines in the Siglec-9 ECD is modified. In some embodiments, one or more cysteines in the Siglec-9 ECD is modified, wherein the modification is a substitution of cysteine with serine. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of one or more cysteine residues selected from C158 and C295 of SEQ ID NO: 31 or residues corresponding to amino acid residues C158 and C295 of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of one or more cysteine residues selected from C158 and C295 of SEQ ID NO: 31 or residues corresponding to amino acid residues C158 and C295 of SEQ ID NO: 31, wherein the modification comprises substitution of cysteine with serine, and wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of both C158 and C295 of SEQ ID NO: 31 or residues corresponding to amino acid residues C158 and C295 of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of both C158 and C295 of SEQ ID NO: 31 or residues corresponding to amino acid residues C158 and C295 of SEQ ID NO: 31, wherein the modification comprises substitution of cysteine with serine, and wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of C158 of SEQ ID NO: 31 or residues corresponding to amino acid residue C158 of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of C158 of SEQ ID NO: 31 or residues corresponding to amino acid residue C158 of SEQ ID NO: 31, wherein the modification comprises substitution of cysteine with serine, and wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of C295 of SEQ ID NO: 31 or residues corresponding to amino acid residue C295 of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising modification of C295 of SEQ ID NO: 31 or residues corresponding to amino acid residue C295 of SEQ ID NO: 31, wherein the modification comprises substitution of cysteine with serine, and wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain with a C36S amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, an IgV domain native linker, and a C36S amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an IgV domain, an IgV domain native linker, a non-native linker, and a C36S amino acid substitution. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acidsequence of any one of SEQ ID NOs: 4-6, 9, 27, and 28. In any of the preceding embodiments, the amino acid substitutions may be with reference to the Siglec-9 IgV or Siglec-9 ECD of a wild-type Siglec-9 protein, such as the human Siglec-9 protein set forth in SEQ ID NO:38.

[0092] In some embodiments, one or more N-glycosylation sites of the polypeptide may be modified. In some embodiments, the one or more N-glycosylation sites are modified with reference to a wild-type Siglec-9 IgV domain, ECD, or protein, such as an IgV domain, ECD, or protein of the human Siglec-9 protein set forth in SEQ ID NO:38. In some embodiments, the N-glycosylation site modification comprises one or more amino acid substitutions. The modification may be to an Asn, Ser, and / or Thr residue in the N-glycosylation site consensus sequence. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising one or more amino acid substitutions selected from N101Q, N138Q, N161Q, N225Q, N23 IQ, N238Q, N256Q, N334Q, T103A, T140A, T163A, T227A, S233A, T240A, S258A, S336A, and combinations thereof, of SEQ ID NO: 31, or one or more amino acid substitutions corresponding to N10IQ, N138Q, N161Q, N225Q, N23 IQ, N238Q, N256Q, N334Q, T103A, T140A, T163A, T227A, S233A, T240A, S258A, S336A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising amino acid substitutions N138Q, C158S, N161Q, N225Q, N231Q, N238Q, N256Q, C295S, and N334Q of SEQ ID NO: 31 or substitutions corresponding to N138Q, C158S, N161Q, N225Q, N23 IQ, N238Q, N256Q, C295S, and N334Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising amino acid substitutions C158S, T163A, T227A, S233A, T240A, S258A, C295S, and S336A of SEQ ID NO: 31 or substitutions corresponding to C158S, T163A, T227A, S233A, T240A, S258A, C295S, and S336A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering.

[0093] In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution ofNIOlQ of SEQ ID NO: 31 or a substitution corresponding to N101Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution ofN138Q of SEQ ID NO: 31 or a substitution corresponding to N138Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution ofN161Q of SEQ ID NO: 31 or a substitution corresponding to N161Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of N225Q of SEQ ID NO: 31 or a substitution corresponding to N225Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution ofN231Q of SEQ ID NO: 31 or a substitution corresponding to N23 IQ of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of N238Q of SEQ ID NO: 31 or a substitution corresponding to N238Q of SEQ ID NO: 31,wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution ofN256Q of SEQ ID NO: 31 or a substitution corresponding to N256Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of N334Q of SEQ ID NO: 31 or a substitution corresponding to N334Q of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of T103A of SEQ ID NO: 31 or a substitution corresponding to T103A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of T140A of SEQ ID NO: 31 or a substitution corresponding to T140A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of T163A of SEQ ID NO: 31 or a substitution corresponding to T163A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of T227A of SEQ ID NO: 31 or a substitution corresponding to T227A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of S233A of SEQ ID NO: 31 or a substitution corresponding to S233A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of T240A of SEQ ID NO: 31 or a substitution corresponding to T240A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of S258A of SEQ ID NO: 31 or a substitution corresponding to S258A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid substitution of S336A of SEQ ID NO: 31 or a substitution corresponding to S336A of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering. In some embodiments, a polypeptide comprises a Siglec-9 ECD comprising an amino acid sequence of any one of SEQ ID NOS: 10-25 and 29-30.

[0094] In any of the embodiments provided herein, a polypeptide may further comprise a fusion partner. Nonlimiting exemplary fusion partners include Fc domains, albumin, and polyethylene glycol (PEG). In some embodiments, the fusion partner is covalently linked to the C-terminus of a Siglec-9 ECD. In some aspects, the fusion partner comprises an Fc domain. In some embodiments, a polypeptide comprising a Siglec-9 ECD and an Fc domain is provided herein, wherein the Fc domain is optionally fused to the C- terminus of the Siglec-9 ECD with or without an intervening linker sequence. A “linker sequence” as used herein refers to a polypeptide sequence not found in a native Siglec-9 ECD or its fusion partner (e.g., an Fc domain), wherein such polypeptide sequence is disposed between the Siglec-9 ECD and its fusion partner. In some embodiments, a 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 someembodiments, the Fc domain is fused to the C-terminus with a linker sequence. In various embodiments, a polypeptide comprises a Siglec-9 ECD and an IgGl Fc domain, e.g., the IgGl Fc domain of SEQ ID NO: 44. In some embodiments, a polypeptide comprising a Siglec-9 ECD comprises an IgGl Fc domain comprising NSLF substitutions and a C220S substitution, e.g., SEQ ID NO: 45. In some embodiments, a polypeptide comprising a Siglec-9 ECD comprises an IgGl Fc domain comprising NSLF substitutions, e.g., SEQ ID NO: 46. In some embodiments, a polypeptide comprising a Siglec-9 ECD comprises an IgGl Fc domain comprising a K322A substitution, e.g., SEQ ID NO: 47. In some embodiments, a polypeptide comprising a Siglec-9 ECD comprises an IgG4 Fc domain or an IgG4 Fc domain comprising a S228P substitution, e.g., as shown in SEQ ID NOs: 48 or 49, respectively.

[0095] In some embodiments, a Siglec-9 ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-80, and 82-85. In some cases, the Siglec-9 ECD comprises the IgV, C2T1, and C2T2 domains. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region sequence of SEQ ID NO: 39 (MPR). In some embodiments, the Siglec-9 ECD comprises the IgV, C2T1, and C2T2 domains and lacks the MPR. In some embodiments, a Siglec-9 ECD comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30. In some embodiments, a Siglec-9 ECD comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30 and lacks the MPR of SEQ ID NO: 39. In some embodiments, a Siglec-9 ECD consists of an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30. In some aspects, the Siglec-9 ECD is part of a Siglec-9 ECD fusion molecule, comprising the ECD and a fusion partner. In some embodiments, the fusion partner is an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49.

[0096] In some embodiments, a Siglec-9 ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-80 and 82-85, lacking a signal sequence. In some embodiments, a Siglec-9 ECD fusion molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-80 and 82-85, including a signal sequence. In some embodiments, a Siglec-9 ECD fusion molecule consists of an amino acid sequence selected from any one of SEQ ID NOs: 57-80 and 82-85, lacking a signal sequence. In some embodiments, a Siglec-9 ECD fusion molecule consists of anamino acid sequence selected from any one of SEQ ID NOs: 57-80 and 82-85, including a signal sequence.

[0097] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 2. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 2 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 2 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 2 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 57.

[0098] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 3. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 3 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 3 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 3 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In someembodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 58.

[0099] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 4. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 4 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 4 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 4 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 59.

[0100] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the Siglec-9 ECD lacks the membrane proximalregion (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 5. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 5 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 5 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 5 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 60.

[0101] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 6. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 6 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 6 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 6 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c)increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 61.

[0102] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 7. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 7 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 7 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 7 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 62.

[0103] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 8. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 8 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 8 including the signal sequence. In some cases, theSiglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 8 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 63.

[0104] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 9. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 9 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 9 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 9 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50-55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus,in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 64.

[0105] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 10. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 10 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 10 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 10 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 65.

[0106] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 11. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 11 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 11 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 11 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is anFc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 66.

[0107] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 12. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 12 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 12 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 12 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 67.

[0108] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 13. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 13 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 13 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 13 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 68.

[0109] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 14. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 14 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 14 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 14 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In someembodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 69.

[0110] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 15. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 15 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 15 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 15 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 70.[oni] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 16. In some cases, the Siglec-9 ECD comprises or consists of theamino acid sequence of SEQ ID NO: 16 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 16 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 16 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 71.

[0112] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 17. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 17 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 17 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 17 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F(NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 72.

[0113] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 18. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 18 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 18 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 18 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 73.

[0114] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 19. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 19 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 19 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 19 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising theECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 74.

[0115] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 20. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 20 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 20 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 20 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 75.In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 75.

[0116] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 21. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 21 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 21 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 21 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 76.

[0117] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 22. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 22 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 22 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 22 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). Insome embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 77.

[0118] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 23. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 23 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 23 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 23 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 78.

[0119] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Siglec-9 ECD lacks the membraneproximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 24. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 24 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 24 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 24 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 79.

[0120] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 25. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 25 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 25 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 25 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c)increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 80.

[0121] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 27. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 27 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 27 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 27 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 82.

[0122] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 28. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 28 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 28 including the signal sequence. In somecases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 28 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 83.

[0123] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 29. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 29 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 29 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 29 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and 50- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus,in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 84.

[0124] In some embodiments, a Siglec-9 ECD or a Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the Siglec-9 ECD lacks the membrane proximal region (MPR) sequence of SEQ ID NO: 39. In some cases, the Siglec-9 ECD consists of the amino acid sequence of SEQ ID NO: 30. In some cases, the Siglec-9 ECD comprises or consists of the amino acid sequence of SEQ ID NO: 30 lacking the signal sequence, but wherein the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 30 including the signal sequence. In some cases, the Siglec-9 ECD has been expressed from a nucleic acid encoding SEQ ID NO: 30 lacking the signal sequence. In some cases, the Siglec-9 ECD is a Siglec-9 ECD fusion molecule comprising the ECD and a fusion partner. In some such embodiments, the fusion partner may be an Fc, albumin, or PEG. In some embodiments, the fusion partner is an Fc. In some embodiments, the fusion partner is an Fc and it is located at the C-terminus of the molecule (i.e., the Fc is attached to the C-terminus of the Siglec-9 ECD either directly or via a linker). In some embodiments, the Fc is a human IgGl (hlgGl). In some embodiments, the Fc comprises the amino acid sequence of any one of SEQ ID NOs: 44-47 and SO- 55. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the Fc domain has an hlgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 45 or 46. In some embodiments, the Fc is a human IgG4, with or without an S228P substitution. Thus, in some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the Siglec-9 ECD fusion molecule comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the Siglec-9 ECD fusion molecule consists of the amino acid sequence of SEQ ID NO: 85.

[0125] The sequence table below depicts the sequences corresponding to SEQ ID NOs listed herein. Exemplary Fc Domains

[0126] In some embodiments of any of the Siglec-9 ECD fusion molecules provided herein, the fusion molecule may comprise an Fc domain. In some embodiments, the Fc domain is a human IgGl, IgG2, IgG3, and / or IgG4 isotype.

[0127] In certain embodiments of any of the Siglec-9 ECD fusion molecules provided herein, the Fc domain has an IgGl isotype. In some embodiments, the Siglec-9 ECD fusion molecule contains a murine IgGl Fc domain. In some embodiments, the Siglec-9 ECD fusion molecule contains a human IgGl Fc domain (hlgGl), e.g., as provided in SEQ ID NO: 44. In some embodiments, the human IgGl Fc domainof the Siglec-9 ECD fusion molecule binds an activating Fc receptor. In certain embodiments, the activating Fc receptor is selected from any one or more of FcyRI, FcyRIIa and lie, and FcyRIIIa and Illb.

[0128] In some embodiments, the human IgGl Fc domain of the Siglec-9 ECD fusion molecule does not bind or has reduced binding to FcyRIII (CD16) and / or Clq. In some embodiments, the human IgGl Fc domain of the Siglec-9 ECD fusion molecule has reduced antibody-dependent cellular cytotoxicity (ADCC) and / or complement binding activity, respectively, which in each case may reduce undesired killing of cells, e.g., myeloid cells, to which the Siglec-9 ECD fusion molecule binds. The above effects may be achieved by certain amino acid modifications, e.g., the “NSLF” mutations, in which an IgGl Fc domain contains the mutations N325S and L328F (by EU numbering of the IgGl Fc domain), as shown, e.g., in SEQ ID NO: 46. In another embodiment, the human IgGl Fc domain comprises the “NSLF” mutations and the third cysteine in the hinge is removed by a C220S modification (EU numbering), e.g. as provided in SEQ ID NO: 45. In another embodiment, the human IgGl Fc domain comprises a mutation corresponding to K322A (EU numbering), e.g., as provided in SEQ ID NO: 47.

[0129] Exemplary modifications to the IgGl Fc domain are listed below in Table 1.Table 1: Exemplary modifications to the IgGl Fc domain_

[0130] For example, in some embodiments, the Fc domain has a human IgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID NO: 44. In some cases, the Fc domain comprises SEQ ID NO: 45 or 46. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions.

[0131] In some embodiments, substitutions and variations can also be made in the Fc region of a Siglec- 9-hIgGl NSLF (see, e.g., SEQ ID NO:45 or 46), for example, to improve its binding to FcRn in vitro, and therefore potentially improve its ability to be recycled in vivo. Exemplary substitutions and variations include the “YTE” and “LS” substitutions, and 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 US Patent No. 9,688,756, which are each incorporated herein by reference in their entirety. In some embodiments, an Fc domain may have a sequence as shown in SEQ ID NOs: 50-55. Modified constructs can be tested for improved binding to FcRn in vitro, e.g., via surface plasmon resonance, and then examined for pharmacokinetics (PK) and pharmacodynamics (PD) in vivo.Modified Fc constructs may also contain the “YTE” or “LS” substitution or cysteine-containing loop insertion, but not the NSLF substitution, in the Fc. Such constructs are shown in SEQ ID Nos: 53-55.

[0132] In certain embodiments of any of the Siglec-9 ECD fusion molecules provided herein, the Fc domain has an IgG2 isotype. In some embodiments, the Siglec-9 ECD fusion molecule contains a murine IgG2 Fc domain, e.g., murine IgG2a (mIgG2a). In some embodiments, the Siglec-9 ECD fusion molecule contains a human IgG2 Fc domain (hIgG2). In some embodiments, the human IgG2 Fc domain of the Siglec-9 ECD fusion molecule binds an activating Fc receptor. In certain embodiments, the activating Fc receptor is selected from any one or more of FcyRI, FcyRIIa and lie, and FcyRIIIa and Illb.

[0133] In certain embodiments of any of the Siglec-9 ECD fusion molecules provided herein, the Fc domain has an IgG4 isotype. In some embodiments, the Siglec-9 ECD fusion molecule contains a human IgG4 Fc domain (hIgG4), e.g., as provided in SEQ ID NO: 48. In some embodiments, the human IgG4 Fc region of the Siglec-9 ECD fusion molecule binds an activating Fc receptor. In certain embodiments, the activating Fc receptor is selected from any one or more of FcyRI, FcyRIIa and lie, and FcyRIIIa and Illb. In certain embodiments, the human IgG4 Fc region comprises a mutation corresponding to S228P (by EU numbering), e.g., as provided in SEQ ID NO: 49.

[0134] Polypeptide Variants

[0135] 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.Substitution, Insertion, and Deletion Variants

[0136] In some embodiments of any of the polypeptides provided herein, polypeptide variants having one or more amino acid substitutions are provided. Amino acid sequence variants of polypeptide may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the polypeptide, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the polypeptide. TABLE 2: Amino Acid Substitutions

[0137] Modifications in the biological properties of a polypeptide may be accomplished by selecting substitutions that differ in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, 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 divided into groups based on common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro; and(6) aromatic: Trp, Tyr, Phe.

[0138] For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class. Such substituted residues can be introduced, for example, into regions of a human polypeptide that are homologous with non-human polypeptides, or into the non- homologous regions of the molecule.In making changes to the polypeptide described herein, according to certain embodiments, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They 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).

[0139] The importance of the hydropathic amino acid index in conferring interactive biological function on 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 having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. Incertain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included.

[0140] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0141] The following hydrophilicity values have been assigned to these amino acid residues: 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). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those which are within ±1 are included, and in certain embodiments, those within ±0.5 are included.

[0142] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides comprising a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.

[0143] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to a polypeptide to improve its stability.Other polypeptide modifications

[0144] In some embodiments of any of the polypeptides, the polypeptides is a derivative. The term “derivative” refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain embodiments, a chemically modified polypeptide can have a greater circulating half-life than polypeptide that is not chemically modified. In certain embodiments, a chemically modified polypeptide can have improved targeting capacity for desired cells, tissues, and / or organs. In some embodiments, a derivative polypeptide is covalently modified to include 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. 4640835, 4496689, 4301144, 4670417, 4791192 and 4179337. In certain embodiments, a derivative polypeptide comprises one or more polymer, including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, , copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers orrandom copolymers), poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide / ethylene oxide co-polymer, poly oxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers.

[0145] In certain embodiments, a derivative is covalently modified with polyethylene glycol (PEG) subunits. In certain embodiments, one or more water-soluble polymer is bonded at one or more specific position, for example at the amino terminus, of a derivative. In certain embodiments, one or more water- soluble polymer is randomly attached to one or more side chains of a derivative. In certain embodiments, PEG is used to improve the therapeutic capacity of a polypeptide. Certain such methods are discussed, for example, in U.S. Pat. No. 6133426, which is hereby incorporated by reference for any purpose. Nucleic acids, vectors, and host cells

[0146] Siglec-9 ECD fusion molecules of the present disclosure may be produced using recombinant methods and compositions. In some embodiments, isolated nucleic acids (polynucleotides) having a nucleotide sequence encoding any of the Siglec-9 ECD fusion molecules of the present disclosure are provided. For example, nucleic acids herein may encode a polypeptide of any one of SEQ ID Nos: 57-80 and 82-85.

[0147] In some embodiments, a nucleic acid encodes a Siglec-9 ECD fusion molecule that includes a signal sequence. In some embodiments, the signal sequence is a native signal sequence. A native human Siglec-9 signal sequence is shown in SEQ ID NO: 35. In some embodiments, the signal sequence is a non-native signal sequence. One skilled in the art would understand that any signal sequence may be used that appropriately effects intracellular trafficking of the encoded polypeptide, cleavage of the signal sequence, and secretion of the encoded polypeptide from a cell. In some such embodiments, the nucleic acid encodes a Siglec-9 ECD fusion molecule comprising a signal sequence that improves intracellular trafficking of the encoded polypeptide, signal sequence cleavage and / or secretion of the encoded polypeptide (efficiency and / or yield) relative to the native human Siglec-9 signal sequence. In some such embodiments, the nucleic acid encodes a Siglec-9 ECD fusion molecule comprising a signal sequence, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, a signal sequence of SEQ ID NO: 36 improves production of the Siglec-9 ECD fusion molecule.

[0148] In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 57. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 58. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 59. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 60. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 61. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 62. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 63. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 64. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ IDNO: 65. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 66. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 67. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 68. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 69. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 70. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 71. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 72. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 73. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 74. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 75. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 76. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 77. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 78. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 79. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 80. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 82. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 83. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 84. In some embodiments, one or more nucleic acids herein may encode the amino acid sequence of SEQ ID NO: 85.

[0149] In some embodiments, one or more vectors (e.g., expression vectors) comprising any of the above nucleic acids are provided. In some embodiments, the nucleic acids or polynucleotides herein may be composed of RNA or DNA, and may be included within various types of vectors, for example, to improve expression of the encoded fusion polypeptides. For example, vectors comprising nucleic acids encoding polypeptides herein include viral vectors, nonlimiting examples of which include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector or a lentivirus vector or an RNA vector, among others. In some embodiments, a nucleic acid or polynucleotide is or comprises RNA. In some embodiments, a nucleic acid or polynucleotide is or comprises messenger RNA (mRNA). It is understood that an RNA polynucleotide comprising a specified nucleotide sequence herein comprises the version of that sequence in which T is replaced by U. The RNA may be single stranded in some cases, or it may be double stranded. In some embodiments, the RNA may be chemically modified, such as modified at the 5’ and / or 3’ end. In some embodiments, the RNA may be circular RNA rather than linear RNA. In some embodiments, a fusion polynucleotide is or comprises DNA. In some embodiments, the DNA may be chemically modified, such as modified at the 5’ and / or 3’ end. In some embodiments, an RNA polynucleotide may also comprise one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof, such as a modified Uand / or C. In cases where a nucleic acid comprising a particular sequence herein also comprises a modified nucleotide, such as, for instance, a modified C or U, it is understood that when referring to the sequence by its SEQ ID NO, the wild type nucleotide, such as C or U, in the SEQ ID NO will be replaced by its modified version.

[0150] In some embodiments, the polynucleotide comprises a 5 ’ untranslated region and / or a 3 ’ untranslated region. In some cases, the polynucleotide comprises a poly-A tail following the 3’ untranslated region, which may be, for example, from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues in length.

[0151] In some cases herein, the polynucleotide is RNA, and comprises a 5’ cap. In some cases, the 5’ cap may comprise the structure of a m7G(5’)ppp(5’)(2’OMeA)pG, i.e. a 7-methyl guanosine triphosphate linked to a 2’O-methyl adenosine - guanine dinucleotide. A commercially available 5’ capping reagent, for example, includes CleanCap® Reagent AG (N-7113; TriLink Biotechnologies, a division of Maravai Life Sciences).

[0152] In cases where the polynucleotide is RNA, the RNA may be single or double stranded, and may be encoded by one or more of the DNA sequences. For example, in some embodiments, a DNA polynucleotide may be prepared and used to transcribe an RNA polypeptide, which may then be administered to a subject in order to cause expression of the fusion polypeptide in the subject.

[0153] In some embodiments, a host cell comprising such nucleic acid is also provided. In some embodiments, the host cell comprises (e.g., has been transduced with) a vector comprising a nucleic acid that encodes the Siglec-9 ECD fusion molecule. In some embodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

[0154] Methods of making a Siglec-9 ECD fusion molecule of the present disclosure are provided. In some embodiments, the method includes culturing a host cell of the present disclosure comprising a nucleic acid encoding the Siglec-9 ECD fusion molecule, under conditions suitable for expression of the Siglec-9 ECD fusion molecule. In some embodiments, the Siglec-9 ECD fusion molecule is subsequently recovered from the host cell (or host cell culture medium).

[0155] For recombinant production of a Siglec-9 ECD fusion molecule of the present disclosure, a nucleic acid encoding the Siglec-9 ECD fusion molecule is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures.

[0156] Suitable vectors comprising a nucleic acid sequence encoding any of the Siglec-9 ECD fusion molecules of the present disclosure include, without limitation, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in selecting clones comprising the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0157] Suitable host cells for cloning or expression of Siglec-9 ECD fusion molecule-encoding vectors include prokaryotic or eukaryotic cells. For example, Siglec-9 ECD fusion molecules of the present disclosure may be produced in eukaryotes, in particular when glycosylation and Fc effector function contribute to the activity of the molecule.

[0158] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are also suitable cloning or expression hosts for Siglec-9 ECD fusion molecule-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of a Siglec-9 ECD fusion molecule with a partially or fully human glycosylation pattern (e.g., Gemgross Nat. Biotech. 22: 1409-1414 (2004); and Li et al. Nat. Biotech. 24:210-215 (2006)).

[0159] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al. J. Gen Virol. 36:59 (1977)), which were used to recombinantly produce the Siglec-9 ECD fusion molecules of the Examples herein; baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., 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 (MMT 060562); TRI cells, as described, e.g., in Mather et al. Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 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.

[0160] In other cases, DNA nucleic acids may be used directly in pharmaceutical compositions, or may be used for transcription to RNA polynucleotides, such as in vitro transcription. In some cases, transcribed polyribonucleotides may contain modifications comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof, as described above. For example, to incorporate such modifications, ribonucleotides comprising a modified ribose, modified backbone, or modified nucleobase may be used in place of one of the four natural ribonucleotides, either fully or partially, in order to obtain an RNA polynucleotide including such modifications. In some embodiments, the modifications may be as described above. For example, in order to obtain a modified C or modified U at a desired frequency in an RNA, for instance, one could use a particular percentage of unmodified and modified C or U ribonucleotide triphosphate starting materials so as to give the desired percentage ofmodified C or U in the final RNA transcribed in vitro. The same process may be used with other modified bases or backbones.Exemplary activities of Siglec-9 ECD fusion molecules

[0161] Provided herein are polypeptides comprising a Siglec-9 ECD, wherein the polypeptide binds sialic acid on the surface of cells. The polypeptide comprising a Siglec-9 ECD may be a Siglec-9 ECD fusion molecule such as a Siglec-9 ECD-Fc fusion molecule. A polypeptide comprising a Siglec-9 ECD may bind cells comprising sialic acid on the surface with an affinity (Kd) of 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. In some embodiments, the polypeptide binds cells comprising sialic acid on the 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-9 ECD or Siglec-9 ECD fusion molecule may bind to MDSCs with a Kd of, for example, less than 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 cells are myeloid-derived suppressor cells (MDSCs). In some cases, the MDSCs are human MDSCs.

[0162] A nonlimiting exemplary assay for determining affinity is as follows. MDSCs, such as human MDSCs, are isolated and incubated with titrating amounts of a polypeptide comprising a Siglec-9 ECD- Fc fusion molecule. A fluorescently-tagged anti-Fc domain antibody (e.g., an antibody that binds IgGl Fc domain) is used for detection, and binding is evaluated by flow cytometry. In some embodiments, a non-human Fc domain (e.g., a mouse IgGl Fc domain) is used in the fusion molecule, in order to reduce background binding of the fluorescently-tagged anti-Fc domain antibody to the MDSCs. An exemplary assay is provided in Example 7 of WO 2021 / 091885, which is incorporated by reference in its entirety and for any reason. Example 7 demonstrates that Siglec-9 ECD fusion molecules bind cells comprising sialic acid on the surface.

[0163] In some embodiments, a polypeptide comprising a Siglec-9 ECD repolarizes myeloid-derived suppressor cells (MDSCs). The polypeptide comprising a Siglec-9 ECD may be a Siglec-9 ECD fusion molecule such as a Siglec-9 ECD-Fc fusion molecule. Repolarization of MDSCs may be determined, for example, by measuring increased chemokine expression from MDSCs incubated with the polypeptides. Nonlimiting exemplary chemokines whose expression may be increased, indicating repolarization of MDSCs, include CCL3, CCL4, CCL5, CCL17, CXCL1, CXCL9, and IL-8. An assay to determine repolarization may measure expression of one, two, three, four, five or more chemokines. Repolarization of MDSCs may also be determined by measuring expression of CD86 and / or CD 163 expression on the MDSCs cultured in the presence of a polypeptide comprising a Siglec-9 ECD. CD86 is a pro- inflammatory marker, and an increase in CD86 expression is consistent with repolarization of MDSCs. CD 163 is an M2 macrophage marker, and a decrease in CD 163 expression is consistent with repolarization of MDSCs toward a pro-inflammatory phenotype. An exemplary assay is provided inExample 8 of WO 2021 / 091885. Example 8 demonstrates that Siglec-9 ECD fusion molecules potently repolarize MDSCs. See also Example 4.

[0164] In some embodiments, a polypeptide comprising a Siglec-9 ECD relieve MDSC-mediated suppression of T cells. The polypeptide comprising a Siglec-9 ECD may be a Siglec-9 ECD fusion molecule such as a Siglec-9 ECD-Fc fusion molecule. A nonlimiting exemplary assay for determining relief of MDSC-mediated suppression of T cells is as follows. MDSCs are isolated and cultured, e.g., for 48 hours, with the polypeptide. The MDSCs are then co-cultured with isolated T cells (e.g., CD8+ T cells) and T-cell activator, such as Dynabeads® Human T-Activator CD3 / CD28. T cell activation may be determined by measuring IFNy expression. In some embodiments, IFNy expression is increased, indicating T cell activation, when MDSCs are incubated with a polypeptide comprising the Siglec-9 ECD, compared to control polypeptide. An exemplary assay is provided in Example 9 of WO 2021 / 091885.

[0165] In some embodiments, a polypeptide comprising a Siglec-9 ECD, blocks binding of other Siglecs to MDSCs. In some such embodiments, the polypeptide blocks binding of Siglec-3, Siglec-5, Siglec-7, Siglec-9, and / or Siglec-10 to MDSCs. Binding may be measured, for example, using the flow cytometry assay described herein for measuring Kd. An exemplary assay is provided in Example 19 of WO 2021 / 091885.

[0166] In some embodiments, a Siglec-9 ECD fusion molecule may comprise the amino acid sequence of any one of SEQ ID NOs: 2-25 and 27-30 joined at its C-terminus to an Fc domain, either directly or via a linker molecule, such as the amino acid sequence of any one of SEQ ID NOs: 57-80 and 82-85, with or without the signal sequence. In some such cases, the molecule may bind to MDSCs, such as human MDSCs, with a Kd of, for example, less than 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.

[0167] For example, in some embodiments, the Fc domain has a human IgGl isotype that has: a) reduced binding to FcyRIII; b) reduced antibody-dependent cellular cytotoxicity (ATCC) and / or reduced complement binding activity; c) increased binding to FcyRIIa; or any combination of a), b), and / or c), relative to the IgGl polypeptide of SEQ ID No: 44. In some cases, the Fc domain comprises SEQ ID NO: 45 or 46. In some cases, the Fc domain comprises a human IgGl isotype with N325S and L328F (NSLF) substitutions. In some such cases, such a molecule may also have increased potency in inducing IFNy production in the presence of MDSCs compared to a Siglec-9 ECD with the same amino acid sequence, but joined at its C-terminus to an hlgGl wild-type Fc molecule. In some embodiments, the molecule may relieve MDSC-mediated suppression of T-cells, for example, as determined by measuring an increase in IFNy expression or an increase in T-cell proliferation. In some cases, such a molecule may increase expression of CD86 on MDSCs and / or may decrease expression of CD206 on MDSCs. In some cases, such a molecule may also bind to MDSCs, such as human MDSCs, with a Kd that is lower thanthat of a molecule comprising a Siglec-9 ECD of the same amino acid sequence but joined at its C- terminus to an hlgGl wild-type Fc.Pharmaceutical compositions / formulations

[0168] Provided herein are pharmaceutical compositions that comprise a polypeptide disclosed herein (e.g., a fusion polypeptide), a polynucleotide disclosed herein, or an expression vector comprising a polynucleotide. In some embodiments, provided herein are pharmaceutical compositions comprising a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, such as a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, provided herein are pharmaceutical compositions comprising the Siglec-9 ECD fusion molecules, polynucleotides, or expression vectors of the present disclosure having the 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 nontoxic to recipients at the dosages and concentrations employed.

[0169] In various embodiments, pharmaceutical compositions comprising a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector are provided in formulations with a pharmaceutically 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, which can comprise antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0170] In some embodiments, a polynucleotide herein may be formulated in a lipid nanoparticle (LNP) formulation, or in another medium intended for use with pharmaceutical polynucleotide molecules. In some such embodiments, the polynucleotide is RNA. In other cases, the polynucleotide is DNA. In some cases, the polynucleotide is RNA comprising one or more modified nucleotides, as described above. The RNA may be single stranded, or it may be double stranded.

[0171] Such LNP formulations may be provided in a variety of pharmaceutically suitable media, such as aqueous buffers, and may optionally comprise further excipients or carriers.

[0172] For example, in some cases, the polynucleotide comprises an RNA, e.g., an mRNA, that is formulated in an LNP formulation. Accordingly, in some embodiments, the disclosure provides an LNP formulation comprising a polynucleotide comprising an RNA, e.g., mRNA.Therapeutic uses

[0173] As disclosed herein, Siglec-9 ECD fusion molecules, polynucleotides, or expression vectors, e.g., Siglec-9 ECD-Fc fusion molecules, polynucleotides, or expression vectors, of the present disclosure may be used for preventing, reducing risk, or treating diseases and disorders. In addition, Siglec-9 ECD fusion molecules, polynucleotides, or expression vectors, e.g. Siglec-9 ECD-Fc fusion molecules,polynucleotides, or expression vectors, of the present disclosure may be used in methods of repolarizing myeloid-deprived suppressor cells (MDSCs) to a pro-inflammatory phenotype, e.g., wherein the subject has cancer or a neurological or neurodegenerative disease, as described below. Siglec-9 ECD fusion molecules, polynucleotides, or expression vectors, e.g. Siglec-9 ECD-Fc fusion molecules, polynucleotides, or expression vectors, of the present disclosure may also be used in methods of activating myeloid cells, e.g., wherein the subject has cancer or a neurological or neurodegenerative disease, as described below. Siglec-9 ECD fusion molecules, polynucleotides, or expression vectors, e.g. Siglec-9 ECD-Fc fusion molecules, polynucleotides, or expression vectors, of the present disclosure may be further used in methods of repolarizing tumor macrophages away from an M2 phenotype in a subject with cancer as described herein.

[0174] In one aspect of the invention, a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, e.g., a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, is used as a therapeutic agent. A 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-9 ECD fusion molecule.

[0175] As further detailed below, a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, e.g., a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, can be used in combination with an additional therapeutic agent that is used to treat the disease or pathology provided herein. The terms “in combination” and “in conjunction” are used interchangeably in the present disclosure. The additional therapeutic agent being administered in combination with the Siglec-9 ECD fusion molecule may be administered before, after, or concurrently with the Siglec-9 ECD fusion molecule, polynucleotide, or expression vector.

[0176] In some embodiments, the disease or disorder to be treated is cancer. In certain embodiments, the cancer is a solid tumor. The solid tumor may be associated with a tumor microenvironment comprising myeloid cells, e.g., macrophages, monocytes, microglia (in the CNS), dendritic cells, neutrophils, and / or granulocytes. In certain embodiments, the tumor microenvironment comprises macrophages and monocytes. In certain embodiments, myeloid cells create an immunosuppressive tumor microenvironment in which a tumor can evade the immune system. Treatment with a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector herein may alleviate this suppression by activating myeloid cells and promoting an anti -tumor immune response.

[0177] In certain embodiments, a cancer to be prevented or treated by the methods of the present disclosure includes, without limitation, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, small -cell lung cancer, non-small cell lung cancer (NSCLC), squamous nonsmall cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, non-squamous NSCLC, glioma, cancer of the peritoneum, hepatocellular cancer, gastric cancer or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, colorectal cancer, endometrial cancer, hepatic carcinoma, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractoryprostate adenocarcinoma), thyroid cancer, neuroblastoma, sarcoma, pancreatic cancer, brain cancer (e.g., astrocytoma such as glioblastoma (glioblastoma multiforme)), cervical cancer, bladder cancer, hepatoma, 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 malignant melanoma, such as cutaneous or intraocular malignant melanoma), thyroid cancer, bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, carcinoma of the fallopian tubes, vulval cancer, cholangiocarcinoma, 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, gastric cancer, cervical cancer, and uterine cancer.

[0178] In certain embodiments, a cancer to be prevented or treated by the methods of the present disclosure includes, without limitation, a hematopoietic cancer, such as a leukemia, lymphoma, or myeloma.

[0179] 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 second site derived from any of the above types of cancer.

[0180] In some embodiments, the present disclosure provides methods of treating an individual having cancer, wherein the individual has a cancer that is refractory to checkpoint inhibitor therapy, by administering to the individual an effective amount of a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, e.g., a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, of the present disclosure. In certain embodiments, the individual has a cancer that is refractory to therapy with a PD-1 or PD-L1 antagonist, e.g., a PD-1 or PD-L1 antibody, such as those provided below.

[0181] In some embodiments, the present disclosure provides methods of treating an individual having cancer, wherein the individual has a cancer that has recurred after checkpoint inhibitor therapy, by administering to the individual a therapeutically effective amount of a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, e.g., a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, of the present disclosure. In certain embodiments, the individual has a cancer that has recurred after therapy with a PD-1 or PD-L1 antagonist, e.g., a PD-1 or PD-L1 antibody, such as those provided below

[0182] In some embodiments, a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, e.g., a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, of the present disclosure may 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, an antagonist of PD-1 is an antibody to PD-1. PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), MEDI-0680 (AMP-514; WO2012 / 145493), camrelizumab (SHR-I2I0), tislelizumab (BGB-A317), or spartalizumab (NPVPDR001, NVS240118, PDR001). A recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) lused to the Fc portion of IgGl, called AMP-224, can also be used to antagonize the PD-1 receptor. In some embodiments, an antagonist of PD-L1 is an antibody toPD-L1. PD-L1 antibodies include, for example, TECENTRIQ (atezolizumab), durvalumab (MEDI4736), BMS-936559 (W02007 / 005874), MSB0010718C (WO2013 / 79174) or rHigM12B7. In some embodiments, a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present invention is administered in combination with radiation therapy and / or a chemotherapeutic agent.

[0183] In some embodiments, methods are provided for treating a neurological or neurodegenerative disorder by administering to a patient in need thereof a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, such as a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector. In some embodiments, the neurological or neurodegenerative disorder is characterized by dysfunctional (e.g., hypoactive) or deficient microglia. Microglia are innate immune cells that reside specifically in the brain and that function as macrophages, clearing debris and dead neurons through the process of phagocytosis and providing other supportive functions for maintaining brain health. Without being limited by theory, the activation of microglia by a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector would treat the neurological or neurodegenerative disorder. In some embodiments, the patient has symptoms of a neurological or neurodegenerative disorder, and the Siglec-9 ECD fusion molecule, polynucleotide, or expression vector is administered to treat the neurological or neurodegenerative disorder. In some embodiments, the patient is at risk of a neurological or neurodegenerative disorder, and the Siglec-9 ECD fusion molecule, polynucleotide, or expression vector is administered to reduce risk, slow onset, or prevent the neurological or neurodegenerative disorder. In some embodiments, the neurological or neurodegenerative disorder is selected from dementia, including 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, pediatric -onset leukoencephalopathy and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).Dementia

[0184] Dementia is a non-specific syndrome (i.e., a set of signs and symptoms) that presents as a serious loss of global cognitive ability in a previously unimpaired person, beyond what might be expected from normal ageing. Dementia may be static as the result of a unique global brain injury. Alternatively, dementia may be progressive, resulting in long-term decline due to damage or disease in the body. While dementia is much more common in the geriatric population, it can also occur before the age of 65. Cognitive areas affected by dementia include, without limitation, memory, attention span, language, and problem solving. Generally, symptoms must be present for at least six months to before an individual is diagnosed with dementia.

[0185] Exemplary forms of dementia include, without limitation, frontotemporal dementia, Alzheimer's disease, vascular dementia, semantic dementia, and dementia with Lewy bodies.

[0186] In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure can prevent, reduce the risk, and / or treat dementia. In someembodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, may modulate one or more Siglec-9 activities in an individual having dementia.Frontotemporal dementia

[0187] Frontotemporal dementia (FTD) is a condition resulting from the progressive deterioration of the frontal lobe of the brain. Over time, the degeneration may advance to the temporal lobe. Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for 20% of pre-senile dementia cases. The clinical features of FTD include memory deficits, behavioral abnormalities, personality changes, and language impairments (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, J. R., Neurology 58: 1615-1621 (2002)).

[0188] A substantial portion of FTD cases are inherited in an autosomal dominant fashion, but even in one family, symptoms can span a spectrum from FTD with behavioral disturbances, to Primary Progressive Aphasia, to Cortico-Basal Ganglionic Degeneration. FTD, like most neurodegenerative diseases, can be characterized by the pathological presence of specific protein aggregates in the diseased brain. Historically, the first descriptions of FTD recognized the presence of intraneuronal accumulations of hyperphosphorylated Tau protein in neurofibrillary tangles or Pick bodies. A causal role for 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 show no accumulation of hyperphosphorylated Tau but do exhibit immunoreactivity to ubiquitin (Ub) and TAR DNA binding protein (TDP43) (Neumann, M., et al., Arch. Neurol. 64: 1388- 1394 (2007)). A majority of those FTD cases with Ub inclusions (FTD-U) were shown to carry mutations in the Progranulin gene.

[0189] In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure, can prevent, reduce the risk, and / or treat FTD. In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, may modulate one or more Siglec-9 activities in an individual having FTD.Alzheimer’s disease

[0190] Alzheimer’s disease (AD) is the most common form of dementia. There is no cure for the disease, which worsens as it progresses, and eventually leads to death. Most often, AD is diagnosed in people over 65 years of age. However, the less-prevalent early-onset Alzheimer's can occur much earlier. Common symptoms of Alzheimer’s disease include, behavioral symptoms, such as difficulty in remembering recent events; cognitive symptoms, confusion, irritability and aggression, mood swings, trouble with language, and long-term memory loss. As the disease progresses bodily functions are lost, ultimately leading to death. Alzheimer’s disease develops for an unknown and variable amount of time before becoming fully apparent, and it can progress undiagnosed for years.

[0191] Reported herein is also the observation that the minor allele of rs2075803, a SNP at the Siglec-9 locus on chromosome 19, is associated with an increase in both Siglec-9 levels in plasma and Alzheimer’s Disease risk. Additionally, reported herein is the observation that the minor allele ofrsl2983058, a SNP at the Siglec-7 locus on chromosome 19, is associated with an increase in both Siglec-7 levels in plasma and Alzheimer’s Disease risk.

[0192] Accordingly, in some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure can prevent, reduce the risk, and / or treat Alzheimer’s disease. In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector may modulate one or more Siglec-9 activities in an individual having Alzheimer’s disease.Parkinson ’s disease

[0193] Parkinson’s disease, which may be referred to as idiopathic or primary parkinsonism, hypokinetic rigid 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 leads to the major symptoms of Parkinson’s. Most often, Parkinson’s disease is diagnosed in people over 50 years of age. Parkinson’s disease is idiopathic (having no known cause) in most people. However, genetic factors also play a role in the disease.

[0194] Symptoms of Parkinson’s disease include, without limitation, tremors of the hands, arms, legs, jaw, and face, muscle rigidity in the limbs and trunk, slowness of movement (bradykinesia), postural instability, difficulty walking, neuropsychiatric problems, changes in speech or behavior, depression, anxiety, pain, psychosis, dementia, hallucinations, and sleep problems.

[0195] In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure can prevent, reduce the risk, and / or treat Parkinson’s disease. In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector may modulate one or more Siglec-9 activities in an individual having Parkinson’s disease.Amyotrophic lateral sclerosis (ALS)

[0196] As used herein, amyotrophic lateral sclerosis (ALS), motor neuron disease, or Lou Gehrig's disease are used interchangeably and refer to a debilitating disease with varied etiology characterized by rapidly progressive weakness, muscle atrophy and fasciculations, muscle spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (dyspnea).

[0197] 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 protects again the damage caused by ALS causing proteins such as TDP-43 (Laird, AS et al., (2010). PLoS ONE 5: e 13368). It was also demonstrated that pro-NGL induces p75 mediated death of oligodendrocytes and corticospinal neurons following 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).

[0198] In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure can prevent, reduce the risk, and / or treat ALS. In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector may modulate one or more Siglec-9 activities in an individual having amyotrophic lateral sclerosis.Huntington ’s disease

[0199] Huntington’s disease (HD) is an inherited neurodegenerative disease caused by an autosomal dominant mutation in the Huntingtin gene (HTT). Expansion of a cytokine-adenine-guanine (CAG) triplet repeat within the Huntingtin gene results in production of a mutant form of the Huntingtin protein (Htt) encoded by the gene. This mutant Huntingtin protein (mHtt) is toxic and contributes to neuronal death. Symptoms of Huntington’s disease most commonly appear between the ages of 35 and 44, although they can appear at any age.

[0200] Symptoms of Huntington’s disease, include, without limitation, motor control problems, jerky, random movements (chorea), abnormal eye movements, impaired balance, seizures, difficulty chewing, difficulty swallowing, cognitive problems, altered speech, memory deficits, thinking difficulties, insomnia, fatigue, dementia, changes in personality, depression, anxiety, and compulsive behavior.

[0201] In some embodiments, administering as a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure can prevent, reduce the risk, and / or treat Huntington’s disease (HD). In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector may modulate one or more Siglec-9 activities in an individual having Huntington’s disease.Tauopathy disease

[0202] Tauopathy diseases, or Tauopathies, are a class of neurodegenerative disease caused by aggregation of the microtubule-associated protein tau within the brain. Alzheimer’s disease (AD) is the most well-known tauopathy disease and involves an accumulation of tau protein within neurons in the form of insoluble neurofibrillary tangles (NFTs). Other tauopathy diseases and disorders include progressive supranuclear palsy, dementia pugilistica (chromic traumatic encephalopathy), frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease (Parkinson-dementia complex of Guam), Tangle-predominant 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.

[0203] In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure, can prevent, reduce the risk, and / or treat tauopathy disease. In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector may modulate one or more Siglec-9 activities in an individual having a tauopathy disease.Multiple sclerosis

[0204] Multiple sclerosis (MS) can also be referred to as disseminated sclerosis or encephalomyelitis disseminata. MS is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. MS affects the ability of nerve cells in the brain and spinal cord to communicate with each other effectively. Nerve cells communicate by sending electrical signals called action potentials down long fibers called axons, which are contained within an insulating substance calledmyelin. In MS, the body’s own immune system attacks and damages the myelin. When myelin is lost, the axons can no longer effectively conduct signals. MS onset usually occurs in young adults, and is more common in women.

[0205] Symptoms of MS include, without limitation, changes in sensation, such as loss of sensitivity or tingling; pricking or numbness, such as hypoesthesia and paresthesia; muscle weakness; clonus; muscle spasms; difficulty in moving; difficulties with coordination and balance, such as ataxia; problems in speech, such as dysarthria, or in swallowing, such as dysphagia; visual problems, such as nystagmus, optic neuritis including phosphenes, and diplopia; fatigue; acute or chronic pain; and bladder and bowel difficulties; cognitive impairment of varying degrees; emotional symptoms of depression or unstable mood; Uhthoff s phenomenon, which is an exacerbation of extant symptoms due to an exposure to higher than usual ambient temperatures; and Lhermitte's sign, which is an electrical sensation that runs down the back when bending the neck.

[0206] In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure, can prevent, reduce the risk, and / or treat MS. In some embodiments, administering a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector may modulate one or more Siglec-9 activities in an individual having MS.Administration

[0207] A Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, such as a Siglec-9 ECD- Fc fusion molecule, polynucleotide, or expression vector, provided herein (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intratumoral, intralesional administration, intracerobrospinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusions include intramuscular, intravenous administration as a bolus or by continuous infusion over a period of time, intraarterial, intra-articular, intraperitoneal, or subcutaneous administration. In some embodiments, the administration is intravenous administration. In some embodiments, the administration is subcutaneous. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0208] For the prevention or treatment of disease, the appropriate dosage of a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the invention, such as a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of fusion molecule, the severity and course of the disease, whether the fusion molecule is administered for preventive or therapeutic purposes, previous therapy, 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.Diagnostic uses

[0209] In some embodiments, the Siglec-9 ECD fusion molecules provided herein is useful for detecting the presence of a Siglec ligand, e.g., sialic acid, in a sample or an individual. The term “detecting” as used herein encompasses quantitative or qualitative detection. Provided herein are methods of using the Siglec-9 ECD fusion molecules of this disclosure for diagnostic purposes, such as the detection of sialic acid in an individual or in tissue samples derived from an individual. In some embodiments, the individual is a human.

[0210] The detection method may involve quantification of the sialic acid-bound Siglec-9 ECD fusion molecule. Such detection in biological samples may occur with 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-9 ECD fusion molecule is radiolabeled, for example with 18F and subsequently detected utilizing micro-positron emission tomography analysis. Sialic acid binding may also be quantified in a patient 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 axial tomography (CAT).Methods of Determining CD 163, Siglec-9, CD68, and Sialic Acid Levels

[0211] In some embodiments, the detection of expression levels may be quantitative or qualitative detection, e.g., determining CD163, Siglec-9, CD68, and / or sialic acid levels. Such detection in tumor samples may occur with any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micropositron emission tomography. In certain embodiments, the detection is by immunohistochemistry (IHC).

[0212] In some embodiments, an expression level determined by IHC may be categorized by an IHC score. In some embodiments, the IHC score comprises a 4-point scale. In some such cases, the score is either 0, 1, 2, or 3, where IHC 0 comprises less than 10 stained cells per high power field, IHC 1 comprises 10-20 stained cells per high power field, IHC 2 comprises 20-100 stained cells per high power field, and IHC 3 comprises greater than 100 stained cells per high power field. In some embodiments, an elevated expression level comprises an IHC score of either 3 or 2 for each molecule or protein measured. In some embodiments, a “high power filed” is a tumor sample section, such as plated on a slide, that is measured at x 400 magnification with conventional light microscopy.

[0213] In some embodiments, CD 163 and / or Siglec-9, and optionally further CD68 may be detected individually, and an elevated expression level determined. Sialic acid may be detected individually, and an elevated level determined. In some embodiments, CD 163 and Siglec-9 are detected and an elevated expression level determined. In some embodiments, elevated expression levels of CD163, Siglec-9, CD68, and / or sialic acid levels may be detected according to the IHC scores shown in Table 3.Table 3: Elevated IHC Scores

[0214] In some embodiments, detection of CD163, Siglec-9, CD68, and / or sialic acid levels may be conducted with a commercially available antibody. In some embodiments, detection of Siglec-9 may be conducted with an anti-Siglec-9 antibody such as antibody 2D4, described in WO 2017 / 075432. In some embodiments, the anti-Siglec-9 antibody comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 87, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 89, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 90, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 91, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, the anti- Siglec-9 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the anti-Siglec-9 antibody is a murine IgGl, IgG2A, or IgG2B antibody. In some embodiments, the anti-Siglec-9 antibody is a murine IgG2A antibody, and thus, comprises a murine IgG2A constant region. In some cases, sialic acid is detected using a Siglec-9 ECD-murine IgGl Fc fusion polypeptide as IHC detection reagent. In some cases, CD 163 is detected by IHC using a rabbit anti-human monoclonal antibody D6U1J (Cell Signaling Cat. No. 93498). In some cases, CD68 isdetected by IHC using a rabbit anti-human CD68 monoclonal antibody (Cell Signaling, Cat#76437, Lot# 1).

[0215] In some cases, using IHC, contrasting staining may be used to distinguish and co-localize the different proteins or markers such as CD163, Siglec-9, CD68, and sialic acid in a tumor sample. For example, different color fluorescent stains may be used for detection of different proteins within the sample. In some cases, and IHC score may then be determined by counting the apparent number of cells stained by the stain for the particular protein or molecule to be detected.

[0216] In certain embodiments, the detection of one or more of CD 163, Siglec-9, CD68, or sialic acid may occur within the subject, and subsequently detected utilizing micro-positron emission tomography analysis. CD163, Siglec-9, CD68, expression levels or sialic acid levels may also be quantified in a subject, such as in a tumor in the subject, 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 axial tomography (CAT).Articles of Manufacture

[0217] Provided herein are articles of manufacture (e.g., kits) comprising a Siglec-9 ECD fusion molecule, polynucleotide, or expression vector, e.g., a Siglec-9 ECD-Fc fusion molecule, polynucleotide, or expression vector, as described herein. Article of manufacture may include one or more containers comprising a Siglec-9 ECD fusion molecule described herein. 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. The containers may be unit doses, bulk packages (e.g., multi -dose packages) or sub-unit doses.

[0218] In some embodiments, the kits may further include a second agent. In some embodiments, the second agent is a pharmaceutically acceptable buffer or diluting agent including, but not limited to, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer's solution and dextrose solution. In some embodiments, the second agent is a pharmaceutically active agent as described above.

[0219] In some embodiments of any of the articles of manufacture, the article of manufactures further includes instructions for use in accordance with the methods of this disclosure. The instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. In some embodiments, these instructions comprise a description of administration of the Siglec-9 ECD fusion molecule, polynucleotide, or expression vector of the present disclosure to prevent, reduce risk, or treat an individual having a disease, disorder, or injury selected from 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, adenocarcinoma of the lung, squamous carcinoma of the lung, non-squamous NSCLC, glioma, cancer of the peritoneum, hepatocellular cancer, gastric cancer or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, colorectal cancer, endometrial cancer, hepatic carcinoma, 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, hepatoma, 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 malignant melanoma, such as cutaneous or intraocular malignant melanoma), thyroid cancer, bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, carcinoma of the fallopian tubes, vulval cancer, cholangiocarcinoma, esophageal cancer, dementia, including 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, pediatric-onset leukoencephalopathy and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), according to any methods of this disclosure. In some embodiments, the instructions include instructions for use of the Siglec-9 ECD fusion molecule, polynucleotide, or expression vector and the second agent (e.g., second pharmaceutically active agent).

[0220] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.EXAMPLES

[0221] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of parameters that could be changed or modified to yield essentially similar results.Example 1: Generation of Siglec-9 ECD variants

[0222] A Siglec-9 ECD Fc fusion protein (hlgGl NSLF Fc) utilizing the wildtype sequence of human Siglec-9 was described in WO 2021 / 091885. The inventors herein desired to improve production yield and formulation stability of this fusion protein while maintaining its biological properties.

[0223] Previous efforts were made to engineer a Siglec-9-Fc fusion protein with improved biophysical properties. See WO 2021 / 091885. WO 2021 / 091885 described methods to engineer a Siglec-9-Fc fusion protein, such as domain truncation and removal of hydrophobic patches. The ECD of Siglec-9 includes 3 domains: a V-set immunoglobulin (IGV) domain, and two C2-set immunoglobulin domains (C2T1 and C2T2) (Figure 1) with the IGV domain being the predicted site of sialic acid binding (Crocker et al, Nat Rev Immunol 7, 255-266, 2007). For domain truncation, four constructs were previously tested including IGV-Fc, IGV-C2T1-Fc, C2T1-C2T2-Fc, and the full length ECD as a control (IGV-C2T1-C2T2-Fc). The expression yield and in vitro function (A375 cell binding) were compared between those constructs as shown in Example 2 of WO 2021 / 091885. All three truncated variants showed significant decrease in yield compared to the control. Truncated variants were also screened in an A375 cell binding assay to assess retention of function. For A375 cell binding, only the variants that contain the IGV domain retained cell binding; while the variant that did not contain IGV domain (C2T1-C2T2-Fc) did not bind to A375 cells. These results showed that previous efforts to truncate the domains within the ECD led to a decrease in expression yield and confirmed that the IGV is the main domain required for target binding.

[0224] In addition to domain truncation, another engineering effort was described in WO 2021 / 091885 that focused on removing hydrophobic patches in the IGV binding domain. A homology model of Siglec- 9 IGV was created using the Molecular Operating Environment software (MOE, Chemical Computing Group) based on a high-resolution structure of the homologous Siglec-7 protein. Electrostatic and hydrophobic surface patches were calculated using MOE. Several engineered variants were generated with an aim to reduce hydrophobic patches or to improve stability. The variants were expressed, purified, and assessed for yield and protein stability, binding to A375 cells, and functional activity on myeloid- derived suppressor cells (MDSCs) such as induction of CD86. Results showed that some of the variants had a neutral or positive impact on the production yield and stability. However, those variants showed decreased binding to A375 cells and decreased functional activity on MDSCs. In conclusion, previous engineering efforts were unable to identify a variant with both an increase in yield and stability, and maintenance of in vitro functional properties.

[0225] The present disclosure describes a novel engineering effort that aimed to reduce the cost of manufacturing a Siglec-9-ECD Fc (such as SEQ ID NO: 86), while maintaining biological and biophysical properties of the molecule. The Siglec-9 ECD sequence (SEQ ID NO: 26) was analyzed for molecular engineering strategies, such as domain truncation, amino acid mutation, or linker modification, that could potentially lead to engineered variants with improved properties, such as increased expression titer, improved purification yield, or reduced protein A resin cost during purification.

[0226] To evaluate the sequence of Siglec-9-ECD Fc, a Siglec-9 model was obtained from Alphafold (alphafold.ebi.ac.uk / entry / Q9Y336). As shown in FIG. 1, Siglec-9 ECD is shown with a depiction of the hlgGl hinge region. Potential liability sites such as N-glycosylation sites and cysteines were investigated using the model. For N-glycosylation, Siglec-9 ECD Fc (SEQ ID NO: 86) contains 18 N-glycosylation sites in total, 8 N-glycosylation sites in each Siglec-9 ECD, and 1 N-glycosylation site in each Fc. For cysteine pairs, the hinge region in the Fc of Siglec-9 ECD Fc (SEQ ID NO: 86) contains 3 cysteine pairs. In addition to the cysteine pairs in the Fc hinge region, each of the Siglec-9 ECD contains two cysteines that can form intradomain cysteine pairs between each ECD (interdomain cysteine pairs are not shown).

[0227] From the sequence analysis described above, several engineering strategies were investigated. As outlined in FIG. 2, these engineering strategies included domain truncations, removal of N-glycosylation sites, removal of potential free cysteines, and linker engineering between the Siglec-9 ECD and Fc domains.1. Domain Truncation Variants

[0228] For the domain truncation strategy, the focus was on removal of the C2-set domains. The C2T1 and C2T2 domains are thought to function by protruding the IGV domain away from the membrane, in order to facilitate binding of the IGV domain to sialylated membrane proteins, but not to be directly involved with binding. Therefore, it was of interest to investigate whether one or both of these domains could be deleted to increase manufacturability while retaining sialic acid binding. Domain truncations included a selection of linkers and additionally removal of a potential free cysteine site (C36), which forms an intra-domain disulfide bond in the native structure between the IGV and C2T1 domains.Crucially, the AlphaFold structure of Siglec-9 ECD allowed for better prediction of the domain boundaries as compared to the truncations described in WO 2021 / 091885. Six truncated variants were tested, and their sequences are shown in the Table of Certain Sequences as SEQ ID NOs: 1-6.2. Full-Length Variants:Insertion of Linkers

[0229] Protein A purification is one of the major costs in manufacture of therapeutic antibodies due to the high cost of the resin. Protein A resin binding capacity of the Siglec-9 ECD Fc (SEQ ID NO: 86) molecule is low (~20 mg protein per mL resin), in comparison to a typical antibody binding capacity (~60 mg protein per mL resin), which results in a higher manufacturing cost due to the larger amount of resin needed. Due to the size and highly glycosylated nature of the Siglec-9 ECD, it was hypothesized, without being bound by theory, that the ECD might be blocking the protein A binding site in the Fc domain of the fusion protein through steric hindrance. Therefore, a variety of linkers were added between the Siglec-9 ECD and the Fc domain to test whether the linkers would increase the protein A binding capacity. Two Siglec-9 ECD variants with varying linkers were constructed, and their sequences are shown in the Table of Certain Sequences as SEQ ID NOs: 7 and 8.Removal of Free Cysteines

[0230] The hinge region in the Fc of Siglec-9 ECD Fc (SEQ ID NO: 86) contains three cysteine pairs. In addition to the cysteine pairs in the Fc hinge region, each of the Siglec-9 ECD contains two cysteines that can form intra-ECD cysteine pairs (other cysteine pairs within each of the ECD are not shown). Because the cysteine pairs in the hinge region should be sufficient for dimer formation of the ECD-Fc, those two cysteines in the Siglec-9 ECD were mutated to serine (C158S, C295S, or both). Three Siglec-9 ECD variants were constructed (C158S, C295S, or both), and their sequences are shown in the Table of Certain Sequences as SEQ ID NOs: 9, 27, and 28.Removal of N-glycosylation Sites

[0231] Since Siglec-9-ECD contains large numbers of N-glycosylation sites, a strategy was used to remove N-glycosylation sites by mutating the amino acids in the N-glycosylation consensus site N-X- S / T. The strategy was to mutate Asparagine (N) to Glutamine (Q), or mutate Serine(S) or Threonine (T) to Alanine (A). Siglec-9 ECD contains multiple N-glycosylation sites, and a large amount of sialylated glycan can lead to charge heterogeneity of the protein. The isoelectric point of Siglec-9 ECD Fc (SEQ ID NO: 86) includes many peaks ranging from pH 5-8 (FIG. 12). Therefore, removal of N-glycosylation sites could potentially reduce charge heterogeneity and potentially lead to improved purification yield, especially during ion exchange steps that separate proteins based on charge. Using this strategy, a total of 16 Siglec-9 ECD variants were engineered, and the sequences of the Siglec-9 ECD variants are shown in the Table of Certain Sequences as SEQ ID NOs: 10-25.

[0232] The sequence of the Siglec-9-ECD wild-type is shown in the Table of Certain Sequences as SEQ ID NO: 26.Combination Mutations

[0233] In addition, combinations of the above mutations were made to determine any additive effects. Combination mutations were made that remove N-glycosylation sites in C2T1 and C2T2 domains while maintaining the N-glycosylation sites in the IGV domain so as to not interfere with the binding domain. The N-glycosylation site removal was achieved through N to Q mutations or S / T to A mutations. This was tested in combination with C158S and C295S mutations, as those cysteine pairs between the Siglec- 9-ECD may not be necessary for ECD-Fc dimer formation. The sequences of the combination Siglec-9 ECD variants are shown in the Table of Certain Sequences as SEQ ID NOs: 29 and 30.

[0234] Moreover, the Siglec-9 ECD Fc molecule (SEQ ID NO: 86) contains three cysteine pairs in the hinge region of the hlgGl NSLF Fc. Two of these cysteine pairs are in the hinge region, which is crucial for dimer formation of the ECD-Fc. The third cysteine pair (C220 in Eu numbering scheme) would normally pair with the light chain in a conventional monoclonal antibody and is thus not necessary for dimer formation and may cause improper disulfide bond formation in the fusion protein format utilized due to lack of a light chain. Therefore, the ECD variants in this investigation were fused to an hlgGl NSLF Fc that contains two cysteine pairs in the hinge region with C220 mutated to serine (SEQ ID NO: 45).Example 2: Siglec-9 ECD Fc variants yield and percent monomer

[0235] To examine the yield and percent (%) monomer of the Siglec-9-ECD Fc variants, expression plasmids for the variants were generated, and transiently transfected using ExpiCHO™ cells in 30 mL scale according to the manufacturer’s protocol (Invitrogen). The Siglec-9-ECD Fc variants were harvested at day 5 post transfection, and expression titer was quantitated from the supernatant using protein A tips (Gator BLI). The proteins were purified using a standard protein A purification protocol and analyzed for yield, percent monomer, percent HMW and percent LMW species using analytical size exclusion chromatography.1. Yield and Percent Monomer of Domain Truncation Variants

[0236] For the domain truncation strategy, variants 1-6 (SEQ ID NOs: 56-61, respectively) were designed to either include IGV-C2T1 domains (variants 1-3), or IGV domain only (variants 4-6), with variation in the truncation site or linker between the ECD and Fc. Variant 1 had very low yield and a high level of aggregation (Table 4 below). Variants 2 and 3 had higher yield and comparable percent monomer to Siglec-9-ECD baseline control. In addition, SDS-PAGE analysis of the purified protein was performed. FIG. 3 shows that variant 1 had low yield and multiple higher MW bands representing protein aggregates, while variant 2 had higher expression yield with a main band at the expected molecular weight. In FIG. 3, “reduced" and “non-reduced refer to whether the disulfide bonds were broken with a reducing agent such as beta-mercaptoethanol prior to running the SDS Page gel.

[0237] Table 4 shows the yield and percent monomer results of truncated variants 4-6, in which the IGV domain was fused to hlgGl NSLF Fc with a variation in the linker between ECD and Fc. Since there is one intradomain cysteine pair between the IGV and C2T1 domains, the cysteine in the IGV domain at amino acid position 36 was mutated to serine (C36S) to avoid a potential unpaired cysteine. All three IGV-Fc variants had low yield and higher aggregation than Siglec-9-ECD Fc baseline control, suggestingthat it is difficult to delete both C2T1 and C2T2 domains and still maintain the desired yield and high percent monomer.

[0238] As a comparison, the truncation site used in WO 2021 / 091885 was based on Uniprot domain annotation (uniprot.org / uniprotkb / Q9Y336 / entry), which is the truncation site used in variant 1 that had low yield and high percent of aggregation. FIG. 5 shows that, based on the AlphaFold structure, the truncation site in variant 1 may cut into the hydrophobic core of the C2T1 domain and result in exposed hydrophobic regions that could lead to aggregation and reduced yield. Variant 2 used a novel truncation site based on the AlphaFold structure that does not cut into the C2T1 protein fold, leading to a variant with relatively high yield and percent monomer. Variant 3 had the same truncation site as variant 2 with an additional GGSGG linker to test for potential increase in protein A binding capacity. The results suggested that the additional linker did not interfere with yield and percent monomer (Table 4). Overall, this result demonstrated that with an improved design of the truncation site, truncated variants containing the IGV-C2T1 domains (variant 2 and 3) can be produced with high yield and comparable percent monomer to the Siglec-9 ECD Fc baseline control. Therefore, out of 6 truncated variants, variants 2 and 3 were selected for further investigation. Depictions of variants 2 and 3 are shown in FIGS. 4B and 4C, respectively, in comparison to the Siglec-9 ECD Fc shown in FIG. 4A.2. Yield and Percent Monomer of Full-Length Variants

[0239] Full-length Siglec-9-ECD Fc with point mutations or linker additions were also tested for yield and percent monomer, as shown in Table 4. The addition of a linker between Siglec-9-ECD and Fc (variants 7 and 8, SEQ ID NOs: 62 and 63, respectively) did not impact the yield or percent monomer compared to the baseline control (variant 26, SEQ ID NO: 86). Removal of cysteine pairs in the Siglec-9 ECD variants were made by C158S, C295S, or both C158S and C295S substitutions. These substitutions did not impact the yield and the percent monomer (variants 9, 27, 28, SEQ ID NOs: 64, 82, 83). This result demonstrates that a linker can be added between the Siglec-9-ECD and Fc, and the cysteine pairs between the Siglec-9 ECD-Fc can be removed without any impact on yield and percent monomer.

[0240] Point mutations in the full-length variants to remove individual N-glycosylation sites showed varied range of yield and percent monomer depending on the mutation sites and whether the mutation was an N to Q mutation or an S / T to A mutation (variants 10-25 in Table 4, SEQ ID NOs: 65-80, respectively). In addition to single point mutations, two variants were examined that had combinations of mutations to remove six N-glycosylation sites in C2T1 and C2T2 domains and remove the two cysteine pairs (variants 29 and 30, SEQ ID NOs: 84 and 85). Interestingly, the expression titer of those two variants was very low (lOmg / L and 18 mg / L).

[0241] To remove multiple N-glycosylation sites and maintain the yield and percent monomer, a new mutation combination design can be tested based on the results of those single point mutation variants. For example, a combination of N161Q, N238Q, N256Q, N334Q, C158S and C295S amino acid mutations can be tested.

[0242] Overall, the yield and percent monomer of Siglec-9 ECD-Fc engineered variants were investigated. The domain truncation strategy using a novel truncation site resulted in truncated variants(IGV-C2T1-Fc), for example, that showed high yield and percent monomer. Some of the full-length variants maintained similar yield and percent monomer as the Siglec-9 ECD-Fc control. Therefore, the truncated and full-length variants that had high yield and percent monomer were selected for further investigation of the biological and biophysical properties as shown in the following examples.Table 4:Example 3: Siglec-9-ECD Fc variant A375 cell binding

[0243] To determine whether the truncated variants maintain the biological properties of the Siglec-9 ECD Fc control molecule, binding of those variants to A375 cells, a melanoma cancer cell line, was assessed in a first experiment. The truncated variants were polished to remove aggregates and tested for A375 cell binding in comparison to the Siglec-9-ECD Fc control. For variant 2, the protein fractions from the polishing step were pooled into two groups. For variant 3, the fractions from the polishing step were pooled into one group. The analytical size exclusion chromatography (aSEC) analysis of the polished protein showed >90% monomer. To examine cell binding, A375 cells were incubated with multiple concentrations of the variants 2 and 3, or the Siglec-9-ECD Fc control for 30 mins, washed and stained with Alexa Fluor 647 conjugated goat anti-human IgG antibody Fc gamma fragment specific,washed and analyzed by flow cytometry. As shown in FIG. 6, the cell binding of the truncated variant (variant 2) from both groups, and the truncated variant with GGSGG linker (variant 3) was comparable or higher than the Siglec-9-ECD Fc control molecule at multiple concentrations. This result demonstrates that the truncated variants retain comparable or even enhanced functional binding activity.

[0244] In addition, full length variants were selected that showed high yield and percent monomer were polished to remove aggregates. The fractions were pooled into 1 or 2 groups and subjected to A375 cell binding analysis as described above. The protein concentration used for the binding assay shown in FIG. 7 was 0.23 nM (28 pg / ml for full-length variants and 23 pg / ml for truncated variants). Group 1 and Group 2 refer to different groups of fractions that showed slightly different aSEC elution times (different hydrodynamic radius). The Siglec-9 ECD Fc variant 2 samples were polished by prep-SEC. The other variants were polished by cation exchange (CEX). FIG. 7 shows that the A375 cell binding of the full- length variants was comparable or higher than the Siglec-9-ECD Fc control molecule at a single concentration of 0.23 nM, suggesting that the full-length variants retain comparable or higher binding activity.Example 4: Siglec-9-ECD Fc variants reprogram myeloid-derived suppressor cells

[0245] To determine the ability of the truncated variants to reprogram suppressive myeloid cells, myeloid derived suppressor cells (MDSCs) were generated from monocytes of healthy donors by culturing with 100 ng / ml GM-CSF and 100 ng / ml IL-6 for 7 days. Cells were then harvested and treated with increasing doses of truncated variants, IgG controls, or Siglec-9 ECD Fc control as a positive control. After 48 hours of treatment, cells were harvested and MDSC reprogramming was examined by staining for surface CD86, CD163, and CD206, followed by analysis by flow cytometry. CD86 is upregulated, and CD 163 and CD206 are downregulated, upon suppressive myeloid cell reprogramming to a proinflammatory phenotype.

[0246] FIGS. 8A-8F demonstrate that the truncated variants upregulated CD86 and downregulated CD 163 and CD206 in a dose dependent manner similarly to the Siglec-9 ECD Fc control. The two groups of truncated variant 2 were tested, along with two negative controls (hlgGl with or without the NSLF mutation) and the Siglec-9 ECD Fc control. Of note, donor 1854 appeared to be more sensitive to the treatment, although both donors revealed similar trends. Overall, according to cell surface markers, the truncated variants appeared to reprogram MDSCs in a manner that is equivalent to the Siglec-9 ECD Fc control.

[0247] Next, full length variants with single point mutations were tested for the ability to reprogram MDSCs compared to the Siglec-9 ECD Fc control. MDSCs were generated from healthy donors as described above, followed by treatment for 48 hours with 10 ug / ml of the full length variants, IgG controls, or the Siglec-9 ECD Fc control as a positive control. The cell surface markers CD86, CD163, and CD206 were analyzed by flow cytometry.

[0248] FIGS. 9A-9C show that the single point mutation, full length Siglec-9 ECD Fc variants reprogrammed MDSCs (donor 1846) similar to the Siglec-9 ECD Fc control at a fixed concentration of10 ug / ml. Taken together, as with the truncated variants, the single point mutation full length variants appeared to reprogram MDSCs in a manner that is equivalent to the Siglec-9 ECD Fc control.Example 5: Siglec-9-ECD Fc Truncated and full-length variants showed comparable hydrophobic characteristics assessed by hydrophobic interaction chromatography (HIC)

[0249] The truncated and full-length Siglec-9 ECD Fc variants were assessed for hydrophobic characteristics using analytical hydrophobic interaction chromatography (HIC). Hydrophobic characteristics can predict the propensity of the molecule to self-associate or aggregate, which may lead to undesirable non-specific binding. For the HIC experimental method, using a high salt buffer, a 30- minute gradient increased the percentage of IX PBS to elute sample off the column matrix. At time zero (T=0), a 100 mg sample was loaded into a HIC column (Propac HIC-10 2.1 x 100 mm, Thermo #063653) and retention time was assessed on an HPLC instrument (Agilent 1100 HPLC). Slower elution indicates higher hydrophobicity. NIST mAb (Millipore Sigma NIST8671), a commercial reference antibody, was used as a positive control for the analytical technique. Samples in formulation buffer (300mM Arginine, pH 6.5) were tested, and HPLC column elution retention times were observed. As shown in Table 5, the HPLC column elution retention times ranged from 16.6 to 17.97 minutes. These retention times are all consistent with the passing criteria (<25 minutes). FIG. 10 also shows the hydrophobicity profiles of the variants and that there was no impact from removal of the glycosylation site. Based on the HIC data, the truncated variants did not show any changes in the HIC retention time compared to the Siglec-9 ECD Fc control, suggesting similar hydrophobic characteristics of the molecule.Table 5Example 6: Siglec-9-ECD Fc variants response to heat stress

[0250] To assess the stability of Siglec-9-ECD Fc variants, a 2-week accelerated stability study was conducted. Stability was evaluated using an analytical Size Exclusion Chromatography (aSEC) assay with a SEC column (BEH 200A 300 mm SEC column, Waters Technologies #186005226) through an Ultra Performance Liquid Chromatography system (Thermo Vanquish UPLC). Tests utilized a 25 pg sample load using 2X PBS + 10% EtOH mobile phase. The purity of the monomer was determined by percent (%) of aSEC chromatogram main peak area. Samples were assessed at time zero (T=0) by aSEC test to determine initial purity. Aliquots were prepared and stored at 40°C. Sample time points were collected after T=2 Days, T=7 Days and T=14 days (T=2wk) and assessed by purity (%monomer by aSEC). The passing criteria is <20% change in %monomer of the starting material at the 2-week timepoint.

[0251] FIG. 11 shows the %change in monomeric content after 0, 2, 7, and 14 days of incubation at 40C. All engineered variants showed acceptable thermostability <20% decrease in monomeric content compared to day 0.Example 7: Siglec-9-ECD Fc charge heterogeneity

[0252] The variants were tested for charge heterogeneity. Charge heterogeneity can be measured using imaged iso-electric focusing (iCIEF). As the Siglec-9 ECD Fc control is focused across a pH gradient by capillary electrophoresis, the charge variant peaks will separate and eventually focus where their net charge equals zero. The range of charge variants is imaged and calibrated between the lower pl marker of 5.00 and upper marker 9.50. Peak height differences indicate the difference in abundance of the charge variants starting at the baseline noise. When the Siglec-9 ECD Fc control is run on iCIEF, the resulting charge variants are shown as a broad isoform profile with a pl range from approximately 5.24 to 8.54. The charge isoforms are heterogenous, likely due to the 18 glycosylation sites, and high amounts of glycosylated variants occupying the extracellular domain (ECD) region.

[0253] The results of charge heterogeneity measurements that were performed on the variants are shown in FIG. 12. In the truncated Siglec-9 ECD Fc variant 2, fraction 1, two main peaks were present at 6.585 and 8.941, flanked by smaller charge variants. This indicates there are two discrete populations in the pl range 6.585 to 9.156. Due to the modification, the charge variant peaks are reduced as there are less glycosylation sites, less glycosylated variants, and overall, less charge heterogeneity. In the truncated Siglec-9 ECD Fc variant 2, fraction 2, two main peaks were present at 6.928 and 8.879, bordered by smaller charge variants. This indicates there are two discrete populations in the pl range 6.928 to 8.988. Post modification, the charge variant peaks are reduced due to less glycosylation sites, less glycosylated variants, and in general, less charge heterogeneity.

[0254] In the truncated Siglec-9 ECD Fc variant 3 with the GGSGG linker, two main peak regions were present at 6.534 and 7.116-8.780. Due to the modifications including a shorter GGSGG linker, a broader array was observed for the higher pl charge variant population compared to the lower pl charge variant population, indicating more heterogeneity contributed by the glycosylation sites and the amounts of glycosylated variants.Example 8: Siglec-9-ECD Fc variant Tm Onset

[0255] To determine thermal stability of Siglec-9 ECD Fc control and its variants, the apparent melting temperature onset (Tm onset) of the domains was measured by differential scanning fluorimetry (DSF). The Tm analysis of Siglec-9 ECD Fc control and its variants (at a concentration of 0.5mg / mL or less) was conducted with a temperature ramping performance from 25 to 99°C at a rate of 0.05° C per second. The rate of change of Rox fluorescence over temperature is measured using a Protein Thermal Shift Dye Kit and a QuantStudio 3 real time PCR instrument (Applied Biosystems / ThermoFisherScientific). As the temperature increases, a protein begins to unfold exposing hydrophobic patches, and the passive fluorescent Rox dye then binds to these patches and the normalized fluorescent emission can be quantified by the qPCR instrument. The initial rate of change in fluorescence between the beginning and ending baseline (observed as first derivative) is called out as the differential melting temperature onset (ATm onset). A higher Tm onset value suggests a more stable protein.

[0256] FIG. 13 illustrates that, as was measured by DSF, Tml onset of Siglec-9 ECD Fc control was 58°C, which was comparable to the variants Tml onset range of 56-60°C. Tm2 and Tm3 onsets for the variants measured were in the range of 69-71°C and 83-86°C, respectively, and comparable with the Siglec-9 ECD Fc control Tm2 onset of 70°C and Tm3 onset of 83°C. The Tm onset values are within assay variability.Example 9: Siglec-9 ECD Fc variant stable pool titer

[0257] To assess the improvements in the expression of the truncated variants, stable pool cells were generated by integrating DNA plasmid with the gene of interest and the glutamine synthease gene into a CHO K-l GS null host cell line by electroporation. The integration was facilitated by transposase and cells expressing the gene of interest were selected by removing glutamine from the cell culture media. Once viability of cell culture exceeds 95%, the cells are set up in a fed batch process to assess the final expression. The fed batch process is a 14 day process with additions of nutrient feeds on days 3, 4, 7, 10 and 12. The cell culture fluid is harvested on either day 14 or when cell viability drops below 70%.

[0258] The stable pool expression of the Siglec-9 ECD Fc control cell culture harvest on day 14 was 800 mg / L. Truncated variants 2 and 3 expressed greater than 2 g / L on day 13 harvest as shown in FIG. 14. Example 10: Nonspecific binding

[0259] To assess nonspecific binding of the truncated variants and Siglec-9 ECD Fc control, an ELISA assay was performed to assess binding to baculovirus particles (BVP) and double stranded DNA (dsDNA). BVP particles derived from insect cells contain phospholipid, carbohydrate, glycoproteins, extracellular matrix, nucleic acids, viral capsid, allowing for detection of electrostatic and hydrophobic interactions (Hotzel et al., mAbs, 2012). dsDNA is one of the agents used in ELISA assay to studypolyreactivity in natural antibody repertoires during B-cell maturation. Majority of the antibodies expressed from early immature B cells showed ELISA binding to dsDNA, ssDNA, insulin, and LPS (Wardemann et al., 2003).

[0260] An ELISA plate was coated with either BVP particles or dsDNA, washed and incubated with blocking buffer, washed and incubated with the samples, followed by detection with anti-human IgG HRP antibody. A BVP score was calculated from the OD450 value of the sample wells to the background (no protein) wells. As shown in FIG. 15A, the truncated variant (group 1 and group 2), and the truncated variant with linker, showed reduced BVP score in comparison to Siglec-9 ECD Fc control.

[0261] For dsDNA ELISA, as shown in FIG. 15B, the truncated variant (group 1 and group 2), and the truncated variant with linker, showed reduced binding in comparison to Siglec-9 ECD Fc control. The reduced binding was also observed in ELISA assay using non-coated plate (Nunc MaxiSorp) containing highly charged polystyrene surface. These results suggested that the reduced nonspecific binding is likely due to reduced charge-charge interaction of the truncated variants in comparison to Siglec-9 ECD Fc control. An earlier study showed antibodies that had increased risk for fast clearance to BVP ELISA binding (Hotzel et al., mAbs, 2012). Accordingly, the reduced BVP score of the truncated variant may show improved pharmacokinetic (PK) properties and reduced serum clearance in vivo.Table of Certain SequencesIn the table below, bold and underlined residues in certain SEQ ID NOs show variant Siglec-9 ECD sequences represent residues that differ from the native Siglec-9 ECD sequence. Underlined residues represent linkers. In some cases, residue numbers used in the name for a particular Siglec-9 variant in the “Description” column (e.g. C36 to S) may not match the numbering of the residues in the SEQ ID Nos of the “Sequence” column, (for example, due to the absence or presence of a signal sequence).

Claims

What is claimed is:

1. An isolated polypeptide comprising a Siglec-9 IgV domain.

2. The isolated polypeptide of claim 1 further comprising a C2 type 1 (C2T1) domain.

3. The isolated polypeptide of claim 1 or claim 2 further comprising a C2 type 2 (C2T2) domain.

4. The isolated polypeptide of claim 1, wherein the isolated polypeptide is a Siglec-9 extracellular domain (ECD) comprising a deletion of the C2 type 1 (C2T1) domain.

5. The isolated polypeptide of claim 1 or claim 4, wherein the isolated polypeptide is a Siglec-9 ECD comprising a deletion of the C2 type 2 (C2T2) domain.

6. The isolated polypeptide of any one of claims 1, 4, or 5, wherein the isolated polypeptide is a Siglec-9 ECD comprising a deletion of both the C2T1 domain and the C2T2 domain.

7. The isolated polypeptide of any one of claims 1-6, wherein the isolated polypeptide comprises one or more N-glycosylation site modifications relative to an unmodified Siglec-9 ECD.

8. The isolated polypeptide of any one of claims 1-7, wherein the isolated polypeptide comprises one or more cysteine modifications relative to an unmodified Siglec-9 ECD, optionally wherein the modifications comprise substitution of a cysteine with serine.

9. The isolated polypeptide of any one of claims 1-8, further comprising an Fc domain linked to the isolated polypeptide by a linker.

10. The isolated polypeptide according to any one of claims 1-3, 5, or 7-9, wherein the isolated polypeptide is a Siglec-9 ECD comprising the amino acid sequence of any one of SEQ ID NO: 2-6.

11. The isolated polypeptide of any one of claims 8-10, wherein the isolated polypeptide comprises modification of one or more cysteine residues selected from C158 and C295 of SEQ ID NO: 31 or residues corresponding to amino acid residues C158 and C295 of SEQ ID NO: 31, wherein the numbering of the residues is according to EU numbering.

12. The isolated polypeptide of claim 11, wherein the modification or modifications comprise substitution of cysteine with serine.

13. The isolated polypeptide according to any one of claims 7-9 or 11-12, comprising one or more amino acid substitutions relative to the unmodified Siglec-9 ECD set forth in SEQ ID NO:31 selected from N10IQ, N138Q, N161Q, N225Q, N231Q, N238Q, N256Q, N334Q, T103A, T140A, T163A, T227A, S233A, T240A, S258A, S336A, and combinations thereof, or one or more amino acid substitutions relative to the unmodified Siglec-9 ECD set forth in SEQ ID NO:31 corresponding to N101Q, N138Q, N161Q, N225Q, N231Q, N238Q, N256Q, N334Q, T103A, T140A, T163A, T227A, S233A, T240A, S258A, S336A, wherein the numbering of the residues is according to EU numbering.

14. The isolated polypeptide of claim 13, wherein the amino acid substitutions comprise N138Q, C158S, N161Q, N225Q, N231Q, N238Q, N256Q, C295S, and N334Q.

15. The isolated polypeptide of claim 13, wherein the amino acid substitutions comprise C158S, T163A, T227A, S233A, T240A, S258A, C295S, and S336A.

16. The isolated polypeptide of any one of claims 7-9 or 11-15, wherein the isolated polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 10-25 or 29-30.

17. The isolated polypeptide according to any one of claims 9-16, wherein the linker is selected from one or more of SEQ ID NOs 40-43.

18. The isolated polypeptide of claim 17, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 3, 6, or 8.

19. An isolated polypeptide comprising a Siglec-9 extracellular domain (ECD) comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-25 and 27-30.

20. The isolated polypeptide of claim 19, wherein the polypeptide further comprises an Fc domain.

21. The isolated polypeptide of any one of claims 9- 18 or 20, wherein the Fc domain is located at the C-terminus of the polypeptide.

22. The isolated polypeptide of any one of claims 9-18 or 20-21, wherein the Fc domain has a human IgGl or IgG4 isotype.

23. The isolated polypeptide of claim 22, wherein the Fc domain has a human IgGl isotype that has, relative to the IgGl polypeptide of SEQ ID NO:44: a) reduced binding to FcyRIII, b) reduced antibody-dependent cellular cytotoxicity (ADCC) and / or reduced complement binding activity, c) increased binding to FcyRIIa, or d) any combination of a), b), and / or c).

24. The isolated polypeptide of any one of claims 9-18 or 20-23, wherein the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 45 and 46.

25. The isolated polypeptide of claim 24, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 45.

26. The isolated polypeptide of any one of claims 19-25, wherein the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-80 and 82-85.

27. The isolated polypeptide of any one of claims 1-26, wherein the polypeptide binds sialic acid on the surface of cells.

28. The isolated polypeptide of claim 27, wherein the cells are tumor cells.

29. The isolated polypeptide of claim 27, wherein the cells express FcR.

30. The isolated polypeptide of claim 27, wherein the cells are myeloid cells.

31. The isolated polypeptide of claim 30, wherein the myeloid cells are selected from monocytes, macrophages, dendritic cells, microglia, and myeloid-derived suppressor cells (MDSCs).

32. The isolated polypeptide of any one of claims 1-31, wherein the polypeptide: a) blocks cell 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) relieves MDSC-mediated suppression of T-cells, optionally as determined by measuring an increase in IFNy expression or an increase in T-cell proliferation; c) repolarizes MDSCs to a pro-inflammatory phenotype;d) increases expression of CD86 on MDSCs, increases expression of CD1 lb on MDSCs, and / or decreases expression of CD 163 on MDSCs; e) repolarizes tumor macrophages away from an M2 phenotype; f) reduces CD 163+ and / or CD206+ macrophages; g) induces expression of one or more chemokines selected from CCL3, CCL4, CCL5, CCL17, CXCL1, CXCL9, and IL-8 in MDSCs; h) reduces myeloid cell recruitment into 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; j) has increased expression, yield, and / or % monomer following purification relative to a Siglec-9 ECD comprising SEQ ID NO: 1 purified under identical conditions; k) shows higher or comparable binding to A375 cells compared to a Siglec-9 ECD Fc comprising SEQ ID NO: 86; l) repolarizes MDSCs to a pro-inflammatory phenotype to a degree comparable to a Siglec- 9 ECD Fc comprising SEQ ID NO: 86; m) has a hydrophobic interaction chromatography (HIC) retention time comparable to a Siglec-9 ECD Fc comprising SEQ ID NO: 86; n) shows < 20% decrease in monomeric content after storage at 40 °C for 2, 7, and / or 14 days compared to the monomeric content at day 0, as measured by analytical size exclusion chromatography (SEC); o) has a melting temperature onset (Tm onset) comparable to that of a Siglec-9 ECD Fc comprising SEQ ID NO: 86, as measured by differential scanning fluorimetry; and / or p) has reduced degree of nonspecific binding to baculovirus particles (BVP) and / or double stranded DNA compared to a Siglec-9 ECD Fc comprising SEQ ID NO: 86, as measured by ELISA.

33. The isolated polypeptide of claim 32, wherein the MDSCs are human MDSCs and / or the macrophages are human macrophages.

34. An isolated nucleic acid molecule comprising a nucleic acid sequence that encodes the isolated polypeptide of any one of claims 1-33.

35. An expression vector comprising the isolated nucleic acid of claim 34.

36. A host cell comprising the isolated nucleic acid of claim 34 or the expression vector of claim 35.

37. A host cell that expresses the isolated polypeptide of any one of claims 1-33.

38. A method of producing a polypeptide comprising culturing the host cell of claim 36 or claim 37.

39. The method of claim 38, comprising isolating the polypeptide.

40. A pharmaceutical composition comprising the polypeptide of any one of claims 1-33, the isolated nucleic acid of claim 34, or the expression vector of claim 35, and a pharmaceutically acceptable carrier.

41. A method of treating cancer comprising administering to a subject with cancer the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40.

42. The method of claim 41, wherein the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells.

43. The method of claim 41 or 42, 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, gastric cancer, cervical cancer, and uterine cancer.

44. The method of any one of claims 41-43, wherein the cancer is metastatic.

45. The method of any one of claims 41-44, further comprising 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- Ll, respectively.

46. The method of any one of claims 41-45, further comprising administering a chemotherapeutic agent.

47. A method of treating a neurological or neurodegenerative disease, comprising administering to a subject with a neurological or neurodegenerative disease the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40.

48. A method of repolarizing myeloid-derived suppressor cells (MDSCs) to a pro-inflammatory phenotype in a subject, comprising administering to the subject the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40.

49. The method of claim 48, wherein the subject has a neurological or neurodegenerative disease.

50. The method of claim 49, wherein the neurological or neurodegenerative disease is characterized by dysfunctional or deficient microglia.

51. A method of repolarizing tumor macrophages away from an M2 phenotype in a subject having cancer, the method comprising administering to the subject the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40.

52. A method of activating myeloid cells in a subject, the method comprising administering to the subject the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40.

53. The method of claim 52, wherein the myeloid cells are microglia.

54. The method of any one of claims 48, 52, or 53, wherein the subject has cancer.

55. The method of claim 51 or 54, wherein the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells.

56. The method of claim 54 or 55, 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, gastric cancer, cervical cancer, and uterine cancer.

57. The method of any one of claims 54-56, wherein the cancer is metastatic.

58. The method of claim 50, 52, or 53, wherein the subject has a neurodegenerative disease.

59. The method of claim 47-50, or 58, wherein the neurodegenerative disease is selected from dementia, frontotemporal dementia, Alzheimer’s disease, vascular dementia, and mild cognitiveimpairment, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s disease, Taupathy disease, multiple sclerosis, immune -mediated neuropathies (such as neuropathic pain), Nasu-Hakola disease, pediatric -onset leukoencephalopathy and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

60. A method of treating cancer in a subject in need thereof, comprising administering to the subject the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40, wherein an elevated expression level of CD 163 and / or Siglec-9 has been detected in a tumor sample from the subject.

61. A method of predicting a response to treatment with a Siglec-9 extracellular domain (ECD) fusion polypeptide in a subject having cancer, comprising determining an expression level of CD163 and / or Siglec-9 in atumor sample from the subject, wherein an elevated expression level of CD163 and / or Siglec-9 predicts response to treatment with the polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40.

62. A method of selecting a subject with cancer for treatment with a Siglec-9 extracellular domain (ECD) fusion polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40, comprising selecting the subject for such treatment if a tumor sample from the subject has been determined to have an elevated expression level of CD 163 and / or Siglec-9.

63. The method of claim 61 or 62, further comprising administering a Siglec-9 ECD fusion polypeptide to the subject.

64. A method of treating cancer in a subject in need thereof, comprising determining that a tumor sample from the subject has an elevated expression level of CD 163 and / or Siglec-9, and administering a Siglec-9 extracellular domain (ECD) fusion polypeptide of any one of claims 1-33 or the pharmaceutical composition of claim 40 to the subject.

65. The method of any one of claims 60-64, wherein the tumor sample from the subject has elevated expression levels of CD163 and Siglec-9.

66. The method of any one of claims 60-65, wherein the tumor sample from the subject has an elevated expression level of CD68.

67. The method of any one of claims 60-66, wherein the tumor sample from the subject has an elevated level of sialic acid.

68. The method of any one of claims 60-67, wherein the tumor sample from the subject is obtained from atumor biopsy.

69. The method of any one of claims 60-68, wherein the expression level of CD163 and / or Siglec-9, and / or optionally CD68, and / or optionally the level of sialic acid, is detected by immunohistochemistry (IHC).

70. The method of claim 69, wherein the expression level of CD 163, Siglec-9, or CD68 or the level of sialic acid is determined to be elevated if IHC detects 20-100 stained cells per high power field, or greater than 100 stained cells per high power field, or if the IHC score on a scale of 0-3 is IHC 2 or IHC 3, for CD 163, Siglec-9, CD68 or sialic acid.

71. The method of claim 69 or 70, wherein the expression level of CD163, Siglec-9, or CD68 or the level of sialic acid is determined to be elevated if the IHC score is IHC 2 or IHC 3.

72. The method of any one of claims 69-71, wherein IHC for Siglec-9 is conducted with an anti- Siglec-9 antibody comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 87, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 89, an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 90, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 91, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 92.

73. The method of any one of claims 69-72, wherein IHC for Siglec-9 is conducted with an anti- Siglec-9 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, optionally wherein the antibody is a murine IgG2A antibody.

74. The method of any one of claims 60-73, wherein the cancer is a solid tumor associated with a tumor microenvironment comprising myeloid cells.

75. The method of any one of claims 60-74, wherein the cancer is selected from renal cell carcinoma, sarcoma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, lung cancer, melanoma, head and neck cancer, breast cancer, cervical cancer, and gastric cancer.

76. The method of any one of claims 60-75, wherein the cancer is metastatic.

77. The method of any one of claims 60 or 63-76, further comprising 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.

78. The method of any one of claims 60 or 63-77, further comprising administering a chemotherapeutic agent.

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