WO2023201051A2

WO2023201051A2 - Anti-inflammatory siglec-6 proteins and methods of making and using same

Info

Publication number: WO2023201051A2
Application number: PCT/US2023/018674
Authority: WO
Inventors: Li Peng; Sujata B. NERLE; Lizhi CAO
Original assignee: Palleon Pharmaceuticals Inc.
Priority date: 2022-04-15
Filing date: 2023-04-14
Publication date: 2023-10-19
Also published as: WO2023201051A3

Abstract

The invention relates generally to proteins comprising a recombinant Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, optionally containing a mutation that reduces sialic acid binding activity, and conjugated to multiple serum half-life enhancers, e.g., an immunoglobulin Fc domain and a carboxy-terminal peptide (CTP) from human chorionic gonadotropin (HCG) β-chain. The invention further relates to methods of using the proteins for treating an inflammatory disorder and/or an autoimmune disorder.

Description

ANTI-INFLAMMATORY SIGLEC-6 PROTEINS AND METHODS OF MAKING AND USING SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The invention claims the benefit of and priority to U.S. Provisional Patent Application No. 63/331,609, filed April 15, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The invention relates generally to Siglec-6 proteins, including fusion proteins comprising Siglec-6 extracellular domains (ECDs), and their use in treating inflammatory and/or autoimmune disorders.

BACKGROUND

[0003] Siglecs (Sialic acid-binding immunoglobulin-type lectins) belong to a lectin-based family of cell surface protein receptors that bind to sialic acid, e.g., sial ogly cans, and are predominantly expressed on cells of the hematopoietic system in a manner dependent on cell type and differentiation. Siglecs are Type I transmembrane proteins where the amino terminus is located in the extracellular space and the carboxy terminus is located in the cytosol. Each Siglec protein contains an N-terminal V-set immunoglobulin-like domain (Ig domain) that acts as the binding receptor for sialic acid. Siglecs are lectins, and are categorized into the group of I-type lectins because the lectin domain has a three-dimensional structure similar to an immunoglobulin fold. All Siglecs extend from the cell surface by means of intervening C2-set domains which have no binding activity. Siglecs differ in the number of these C2-set domains. As these proteins contain Ig-like domains, they are members of the Immunoglobulin superfamily (IgSF).

[0004] There are at least 14 different mammalian Siglecs, which together provide an array of different functions based on cell surface receptor-ligand interactions. Most Siglecs, including Siglec-6, are classified as “inhibitory” as they contain an intracellular immunoreceptor tyrosinebased inhibitory motif (ITIM) and ITIM-like motifs in their cytoplasmic tail. On engagement by sialoglycans, ITIMs may become phosphorylated and recruit SH2 domain-containing protein tyrosine phosphatases, SHP1 and SHP2. These phosphatases can inhibit signaling pathways triggered in close proximity and thereby modulate diverse physiological responses depending on the cell type and Siglecs. A few Siglecs, including Siglecs-14, -15, and -16, are known as “activating” and signal through adaptor proteins such as DNAX-activating protein- 10 (DAP- 10) and DNAX-activating protein-12 (DAP-12) upon binding to sialoglycans. These Siglec-glycan interactions can mediate, among other things, cell adhesion and modulate immune cell functions.

[0005] Activation of T cells, for example, occurs through the simultaneous engagement of the T-cell receptor and a co-stimulatory molecule (e.g., CD28 or ICOS) on CD4+ T cells by the major histocompatibility complex (MHCII) peptide and co-stimulatory molecules on the antigen-presenting cell (APC). Both are required for production of an effective immune response. In the absence of co-stimulation, T-cell receptor signaling alone results in anergy. It is believed that the signaling pathways downstream from co-stimulatory molecules usually engage the PI3K pathway generating PIP3 at the plasma membrane and recruiting PH domaincontaining signaling molecules like PDK1 that are essential for the activation of PKC-0, and eventual IL-2 production. In addition, CD4+ cells are useful in the initial antigenic activation of naive CD8 T cells, and sustaining memory CD8+ T cells in the aftermath of an acute infection. Therefore, activation of CD4+ T cells can be beneficial to the action of CD8+ T cells. However, an undesirable T-cell activation can result in the development of an inflammatory and/or autoimmune disorder in a subject.

[0006] Accordingly, there is a need in the art for compositions and methods for treating inflammatory and/or autoimmune disorders, for example, by reducing the number or activity of activated T cells in a subject with an inflammatory and/or autoimmune disorder.

SUMMARY OF THE INVENTION

[0007] The invention is based, in part, upon the discovery that a Siglec-6 extracellular domain (ECD), irrespective of its “inhibitory” cytoplasmic domain, including a Siglec-6 ECD with reduced or no sialic acid binding activity, can act as a ligand and suppress immune responses (such as T cell activation) independent of sialoglycan interaction, by interacting with certain receptors on T cells through a potential protein-protein interaction. The invention is also based, in part, upon the discovery that linking a Siglec-6 ECD protein to both an immunoglobulin Fc domain and a carboxy-terminal peptide results in an unexpectedly beneficial improvement to the half-life of the construct. Accordingly, provided is a protein comprising a Siglec-6 ECD, or a functional fragment or variant thereof, an immunoglobulin Fc domain, and a CTP that can be administered to a subject for the treatment of an inflammatory and/or autoimmune disorder, such as fibrosis and arthritis.

[0008] Accordingly, in one aspect, the invention provides a fusion protein comprising: (a) a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof; (b) a first carboxy-terminal peptide (CTP) derived from human chorionic gonadotropin (HCG); and (c) an immunoglobulin Fc domain.

[0009] In certain embodiments, the Siglec-6 ECD comprises at least one mutation (e.g., 1, 2, 3 or 4 mutations) that reduces sialic acid binding activity. In certain embodiments, the at least one mutation results in the Siglec-6 ECD having less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5%) of the sialic acid binding activity of a corresponding Siglec-6 ECD without the mutation. In certain embodiments, the Siglec-6 ECD is a human Siglec-6 ECD, and the at least one mutation is present in the region from amino acid 112 to 140, or from amino acid 119 to 122, of SEQ ID NO: 21 (wild-type human Siglec-6). In certain embodiments, the mutation is a substitution of an arginine residue at a position corresponding to position 122 of wild-type human Siglec-6 (e.g., R122). In certain embodiments, the Siglec-6 ECD comprises the amino acid sequence of SEQ ID NO: 25.

[0010] In certain embodiments, the immunoglobulin Fc domain is derived from a human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM Fc domain. In certain embodiments, the immunoglobulin Fc domain is derived from a human IgGl, IgG2, IgG3, or IgG4 Fc domain. In certain embodiments, the immunoglobulin Fc domain is derived from a human IgGl Fc domain (e.g., having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 9). The Fc domain can include at least one substitution that alters binding to an Fc receptor, for example, a mutation at position 297 (e.g., an A or a G), according to EU numbering. In certain embodiments, the immunoglobulin Fc domain can comprise the amino acid sequence of SEQ ID NO: 9.

[0011] In certain embodiments, the first CTP comprises the amino acid sequence of SEQ ID NO: 11. The first CTP can be interposed between the Siglec-6 ECD and the immunoglobulin Fc domain. For example, the fusion protein can comprise, from N-terminus to C-terminus: the Siglec-6 ECD, the first CTP, and the immunoglobulin Fc domain.

[0012] In certain embodiments, the fusion protein comprises a second CTP derived from HCG. It is contemplated that the first CTP and the second CTP can comprise same amino acid sequence. In certain embodiments, the second CTP comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments the second CTP is interposed between the Siglec-6 ECD and the immunoglobulin Fc domain, and depending upon the circumstances, the N-terminus of the second CTP can be linked to the C-terminus of the first CTP. For example, an exemplary fusion protein can comprise, from N-terminus to C-terminus: the Siglec-6 ECD, the first CTP, the second CTP, and the immunoglobulin Fc domain.

[0013] In each of the foregoing fusion proteins, the first CTP and/or the second CTP increase the half-life and/or the alpha half-life of the fusion protein by 1.1-fold to 5-fold (e.g., 1.1-fold to 2-fold, 2-fold to 3-fold, 3-fold to 4-fold, or 4-fold to 5-fold) when administered to a subject.

[0014] In certain embodiments, the fusion protein further comprises a first linker. In certain embodiments, the first linker is interposed between any two of the following: the Siglec-ECD, the first CTP, the optional second CTP, and the immunoglobulin Fc. In certain embodiments, the fusion protein further comprises a second linker. In certain embodiments, the first linker and/or the second linker comprise an amino acid sequence selected from the group consisting of: (GGGGS)₂ (SEQ ID NO: 15), GGGGS (SEQ ID NO: 16), EPKSS (SEQ ID NO: 18), (GGP)_n (SEQ ID NO: 19), and (GGGGS)_n (SEQ ID NO: 20). In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus: the Siglec-ECD, a first linker, the first CTP, an optional second CTP, a second linker, and the immunoglobulin Fc.

[0015] In certain embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 33.

[0016] In a related aspect, the disclosure relates to a dimeric protein comprising two fusion proteins according to any of the foregoing embodiments. In certain embodiments, the dimeric protein comprises a first fusion protein and a second fusion protein, wherein the fusion proteins are covalently linked together by one or more disulfide bonds that link the Fc domain of the first fusion protein and the Fc domain of the second fusion protein.

[0017] In a related aspect, the disclosure relates to a pharmaceutical composition comprising a fusion protein of any of the foregoing embodiments and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising a dimeric protein of any of the foregoing embodiments and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is disposed in a sterile container e.g., a bottle or vial). In certain embodiments, the pharmaceutical composition is lyophilized in the sterile container. In certain embodiments, the pharmaceutical composition is present as a solution in the sterile container. In certain embodiments, the sterile container has a label disposed thereon identifying the pharmaceutical composition contained in the container.

[0018] In a related aspect, the disclosure relates to a method of treating an inflammatory and/or autoimmune disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a fusion protein according to any one of the foregoing embodiments, a dimeric protein according to any one of the foregoing embodiments, or a pharmaceutical composition according to any one of the foregoing embodiments.

[0019] These and other aspects and features of the invention are described in the following detailed description and claims.

DESCRIPTION OF THE DRAWINGS

[0020] The invention can be more completely understood with reference to the following drawings, in which:

[0021] FIGURE 1 A depicts a schematic representation of ITIM-containing Siglec molecules that function like a “PD-l-like receptor” and wherein the ITIM-containing Siglecs function as receptors and suppress immune function by binding to sialoglycan on a target cell, e.g., a cancer cell. FIGURE IB depicts a schematic representation of Siglec molecules, e.g., Siglec 6, that function like a “PD-Ll-like ligand” regardless of their intracellular domains being “inhibitory” (ITIM) or “activating” (DAP- 10/12) and wherein such Siglecs function as ligands and suppress immune responses by binding to currently unidentified receptor(s) on T cells in a sialoglycan- independent manner.

[0022] FIGURE 2 depicts a schematic representation of certain exemplary Siglec-6 Extracellular Domain (ECD) fusion proteins. The Siglec-6 ECD fusion protein schematic on the left depicts a dimer, wherein each monomer comprises (1) an ECD having a V-set Ig domain and two C2-set Ig domains, and (2) an IgGl Fc domain. The Siglec-6 ECD fusion protein dimerizes at the Fc domain. The Siglec-6 ECD fusion protein schematic on the right depicts a dimer, wherein each monomer comprises (1) an ECD having a V-set Ig domain and two C2-set Ig domains, (2) a CTP domain, and (3) an IgGl Fc domain. The Siglec-6 ECD fusion protein dimerizes at the Fc domain. For either Siglec-6 ECD fusion protein, the V-set domain can bind sialic acid; however, in certain embodiments, the V-set domain comprises a mutation (e.g., an R122K substitution) that reduces or abolishes sialic acid binding. In addition, the IgGl Fc domain can comprise substitutions that alter binding to an Fc receptor, for example, an N297G substitution.

[0023] FIGURE 3 depicts the in vivo pharmacokinetic clearance curves of exemplary Siglec-6 ECD fusion proteins Sig-6-SAX-lG and Sig-6-SAX-CTP-lG following intraperitoneal administration to mice. [0024] FIGURE 4A is a graph showing secretion of IFN-y following treatment of human T cells from a first donor with the indicated exemplary Siglec-6 ECD fusion protein or with an IgGl N297G isotype control (“IgGl-lG”). The IC50 (pM) of each construct is shown below the figure. FIGURE 4B shows results for a second donor.

[0025] FIGURE 5 depicts a flow chart summarizing a human macrophage polarization assay protocol used to assess activity of Siglec-6 ECD fusion proteins.

[0026] FIGURES 6A and 6B are bar graphs showing secretion of IL-12p40 from either Ml human macrophages (FIGURE 6A) or M2 human macrophages (FIGURE 6B) following treatment with the indicated concentration of Sig-6-SAX-lG, Sig-6-SAX-CTP-lG, or an IgGl N297G isotype control (“IgG-lG”).

[0027] FIGURES 7A and 7B are bar graphs showing secretion of IL-10 from either Ml human macrophages (FIGURE 7A) or M2 human macrophages (FIGURE 7B) following treatment with the indicated concentration of Sig-6-SAX-lG, Sig-6-SAX-CTP-lG, or an IgGl N297G isotype control (“IgG-lG).

[0028] FIGURES 8A and 8B are bar graphs showing secretion of IL-23 from either Ml human macrophages (FIGURE 8A) or M2 human macrophages (FIGURE 8B) following treatment with the indicated concentration of Sig-6-SAX-lG, Sig-6-SAX-CTP-lG, or an IgGl N297G isotype control (“IgG-lG”).

[0029] FIGURES 9A and 9B are bar graphs showing CD64 surface expression from either Ml human macrophages (FIGURE 9 A) or from M2 human macrophages (FIGURE 9B) following treatment with the indicated concentration of Sig-6-SAX-lG, Sig-6-SAX-CTP-lG, or an IgGl N297G isotype control (“IgG-lG”). Unstimulated (“Unstim”) and stimulated (“LPS+Inf-G”) macrophages that were not treated with Sig-6-SAX-lG, Sig-6- SAX-CTP-1G, or IgG-lG were used as additional controls.

[0030] FIGURES 10A and 10B depict bar graphs showing secretion of TNF-a (FIGURE 10A) and IL-8 (FIGURE 10B) from cynomolgus pan-T primary T cells following treatment with the indicated concentration of Sig-6-SAX-lG, Sig-6-SAX-CTP-lG, or an IgGl N297G isotype control (“IgG-lG”).

DETAILED DESCRIPTION

[0031] The invention is based, in part, upon the discovery that a Siglec-6 extracellular domain (ECD), irrespective of its “inhibitory” cytoplasmic domain, including a Siglec-6 ECD with reduced or no sialic acid binding activity, can reduce immune system activation (such as T cell activation) and/or dysregulation. The invention provides, among other things, compositions, e.g., pharmaceutical compositions, comprising a Siglec-6 extracellular domain (ECD) that can mediate an anti-inflammatory effect. The invention is also based, in part, upon the discovery that linking a Siglec-6 ECD protein to both an immunoglobulin Fc domain and a carboxyterminal peptide (CTP) results in an unexpectedly beneficial improvement to the half-life of the protein. Furthermore, a protein comprising a Siglec-6 ECD, or a functional fragment or variant thereof, an immunoglobulin Fc domain, and a CTP can be administered to a subject to treat an inflammatory and/or autoimmune disorder, e.g., fibrosis or arthritis.

I. Siglecs And Siglec Biology

[0032] Siglecs (Sialic acid-binding immunoglobulin-type lectins) are cell surface proteins that bind sialic acid. Siglecs comprise a lectin family of surface receptors that bind to sialoglycans and are predominantly expressed on cells of the hematopoietic system in a manner dependent on cell type and differentiation. There are at least 15 different mammalian Siglecs, which together provide an array of different functions based on cell surface receptor-ligand interactions. These receptor-glycan interactions can mediate, among other things, cell adhesion and cell signaling.

[0033] Siglecs are Type I transmembrane proteins where the amino terminus is located in the extracellular space and the carboxy terminus is located in the cytosol. Each Siglec contains an N-terminal V-set immunoglobulin-like domain (Ig domain) that acts as the binding receptor for sialic acid. Siglecs are classified as I-type lectins because the lectin domain is in the form of an immunoglobulin fold. Siglecs extend from the cell surface by means of intervening C2-set domains which do not bind to sialic acid, and various Siglecs differ in the number of these C2- set domains. Given that these proteins contain Ig domains, they are members of the Immunoglobulin superfamily (IgSF).

[0034] As shown in FIGURE 1A, most CD33-like Siglecs, including Siglec-6, are believed to interfere with cellular signaling via their ITIM-containing cytoplasmic domains, thereby inhibiting immune cell activation. In this context, these Siglecs function like “PD-l-like receptors”. For example, and without wishing to be bound by theory, these Siglecs function like receptors on an immune cell which, when bound to their ligands on a cancer cell (e.g., hypersialylated tumor glycans) and on the immune cell itself e.g., sialylated immune cis- ligands), recruit inhibitory proteins, such as SHP phosphatases, via their ITIM domains. It is believed that the tyrosine amino acids contained within the ITIM domain become phosphorylated upon ligand binding and act as docking sites for SH2 domain-containing proteins like SHP phosphatases. This leads to de-phosphorylation of cellular proteins, and downregulation of activating signaling pathways in the immune cell, thereby suppressing immune cell activity and allowing cancer cells to evade the immune system. The alleviation of this suppression can be used to treat a variety of disorders such as cancer.

[0035] Although immune suppression by Siglecs is believed to be mediated by their ITIM- containing cytoplasmic domain upon the extracellular domain’s recognition of sialic acid binding, it has been discovered that a Siglec-6 extracellular domains (ECD) with reduced or no sialic acid binding activity can also reduce immune system activation and/or dysregulation regardless of the nature of its cytoplasmic domain. Without wishing to be bound by theory, it is believed that, in addition to sialic acid-mediated immune suppression of certain immune cells, Siglec-6, can also act through protein-protein interactions in a sialic acid-independent pathway as a ligand for a currently unidentified receptor, on other immune cells, e.g., T-cells, to reduce immune activation. As shown in FIGURE IB, in this capacity, a Siglec such as Siglec-6 can function like a “PD-Ll-like ligand” to suppress the function of certain immune cells, e.g., T- cells, via a receptor disposed on the immune cells, e.g., T-cells, in a sialic acid-independent manner to down regulate the inflammatory activity of the immune cells. The down regulation of this anti-inflammatory activity can be used to treat a variety of disorders such as inflammatory and autoimmune disorders.

[0036] Accordingly, in certain embodiments, the disclosure relates to the use of a Siglec-6 ECD, including a Siglec-6 ECD with reduced or no sialic acid binding activity, to suppress the function of certain immune cells, e.g., the activity of T-cells. Suppression of T-cell activity can be useful to treat diseases or disorders characterized by hyperactivation or dysregulation of the immune system, such as inflammatory and/or autoimmune disorders.

[0037] Further, the disclosure relates to a protein comprising a Siglec-6 ECD, or a functional fragment or variant thereof, conjugated to two serum half-life enhancers. In certain embodiments, the Siglec-6 ECD comprises a mutation that reduces sialic acid binding activity. Exemplary Siglec-6 ECD fusion proteins are shown schematically in FIGURE 2. The Siglec-6 ECD fusion protein schematic on the left depicts a dimer, wherein each monomer comprises (i) an ECD having a V-set Ig domain and two C2-set Ig domains, and (ii) an IgGl Fc domain. The Siglec-6 ECD fusion protein dimerizes at the Fc domain. The Siglec-6 ECD fusion protein schematic on the right depicts a dimer, wherein each monomer comprises (i) an ECD having a V-set Ig domain and two C2-set Ig domains, (ii) a CTP domain, and (iii) an IgGl Fc domain. The Siglec-6 ECD fusion protein dimerizes at the Fc domain. For either Siglec-6 ECD fusion protein, the V-set domain can bind sialic acid; however, in certain embodiments, the V-set domain comprises a mutation (e.g., an R122K substitution) that reduces or abolishes sialic acid binding. In addition, the IgGl Fc domain can comprise substitutions that alter binding to an Fc receptor, for example, a substitution at N297 such as an N297G substitution. In certain embodiments, the disclosure provides Siglec-6 fusion proteins that include one or more mutations to reduce or eliminate sialic acid binding activity and two or more serum half-life enhancers.

[0038] The disclosure further relates to pharmaceutical compositions comprising such fusion proteins and methods of administering such fusion proteins to a subject to treat an inflammatory and/or autoimmune disorder. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier.

[0039] An amino acid sequence of an exemplary human Siglec-6 protein is provided in SEQ ID NO: 21 (NCBI Reference Sequence: NP_001236.4) and a DNA sequence encoding an exemplary human Siglec-6 protein is provided in SEQ ID NO: 22 (NCBI Reference Sequence: NM_198845.5). a. Siglec-6 Extracellular Domains (ECD)

[0040] In certain embodiments described herein, the disclosure relates to a Siglec-6 extracellular domain (ECD). An amino acid sequence of an exemplary human Siglec-6 ECD is provided in amino acid residues 27-347 of SEQ ID NO: 21 (i.e., SEQ ID NO: 23). In certain embodiments, the Siglec-6 ECD comprises an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acid residues 27-347 of SEQ ID NO: 21 (i.e., SEQ ID NO: 23). b. Siglec-6 Extracellular Domain (ECD) Fragments

[0041] As used herein, the term “functional fragment” of a Siglec-6 extracellular domain (ECD) refers to fragment of a Siglec-6 ECD that retains, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the Siglec-6 ECD activity of the corresponding full-length, naturally occurring Siglec-6 ECD. Siglec-6 ECD activity may be assayed by any method known in the art, including, for example, by measuring one or more criteria indicative of the inhibition of immune cell activity, such as (1) the inhibition of NF AT activation in a Jurkat Luciferase cell assay; (2) the inhibition of IFN-y release from activated T cells; (3) reduced hydroxyproline in a mouse idiopathic pulmonary fibrosis (IPF) model; and (4) reduced clinical score in a mouse CAIA (Collagen Antibody Induced Arthritis) model. In certain embodiments, Siglec-6 ECD activity does not include sialic acid binding activity. In certain embodiments, the functional fragment comprises at least 50, at least 75, at least 100, at least 125, or at least 150 consecutive amino acids present in a full-length, naturally occurring Siglec-6 ECD.

[0042] In certain embodiments, the functional fragment of a Siglec-6 ECD comprises a Siglec- 6 V-set immunoglobulin-like domain, e.g., amino acid residues 31-141 of SEQ ID NO: 21 or amino acid residues 27-347 of SEQ ID NO: 21. In certain embodiments, the functional fragment of a Siglec-6 ECD comprises an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acid residues 31-141 of SEQ ID NO: 21 or amino acid residues 27-347 of SEQ ID NO: 21. c. Siglec-6 Extracellular Domain (ECD) Variants

[0043] As used herein, the term “variant” of a Siglec-6 extracellular domain (ECD) refers to variant of a Siglec ECD or a functional fragment thereof that retains, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the Siglec-6 ECD activity of the corresponding full-length, naturally occurring Siglec-6 ECD. Siglec-6 ECD activity may be assayed by any method known in the art, including, for example, by measuring one or more criteria indicative of the inhibition of immune cell activity, such as (1) the inhibition of NF AT activation a Jurkat Luciferase cell assay; (2) reduced hydroxyproline in a mouse idiopathic pulmonary fibrosis (IPF) model; (3) reduced clinical score in a mouse CAIA (Collagen Antibody Induced Arthritis) model; and (4) reduced IFN-y and/or TNF-a secretion from activated T cells. In certain embodiments, Siglec-6 ECD activity does not include sialic acid binding.

[0044] In certain embodiments, the variant of a Siglec-6 ECD comprises a substitution of at least one wild-type cysteine residue.

[0045] In certain embodiments, the variant of a Siglec-6 ECD comprises a conservative substitution relative to a Siglec-6 ECD sequence disclosed herein. As used herein, the term “conservative substitution” refers to a substitution with a structurally similar amino acid. For example, conservative substitutions may include those within the following groups: Ser and Cys; Leu, He, and Vai; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gin, Asn, Glu, Asp, and His. Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution^ matrix (e.g., the PAM 250 matrix). In certain embodiments, the Siglec-6 ECD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions.

[0046] Sequence identity may be determined in various ways that are within the skill of a person skilled in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36:290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402, incorporated by reference herein) are tailored for sequence similarity searching. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6: 119-129, which is fully incorporated by reference herein. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89: 10915-10919, fully incorporated by reference herein). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=l (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent blastp parameter settings may be Q=9; R=2; wink=l; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter (e.g. : -G, Cost to open gap [Integer]: default = 5 for nucleotides/ 11 for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default = -3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits: default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped alignment (in bits): default = 15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty). The equivalent settings in Bestfit protein comparisons are GAP=8 and LEN=2.

[0047] In certain embodiments, the Siglec-6 ECD, or a functional fragment or variant thereof, comprises at least one mutation (e.g., 1, 2, 3, or 4 mutations) that reduces sialic acid binding activity. The mutation can include a deletion, substitution, insertion, or any combination thereof. In certain embodiments, the mutation is a substitution of an alanine (A) for an amino acid present in a wild-type Siglec-6 sequence. In certain embodiments, the mutation is a substitution of a glycine (G) for an amino acid present in a wild-type Siglec-6 sequence. In certain embodiments, the mutation is a substitution of a lysine (K) for an amino acid present in a wildtype Siglec-6 sequence. In certain embodiments, the mutation that reduces sialic acid binding is present in the sialic acid binding region of the Siglec-6 ECD. In certain embodiments, a mutation that reduces sialic acid binding is in at least one conserved residue in the sialic acid binding region of the Siglec-6 ECD. In certain embodiments, the mutation is present in the region from amino acid 112 to 140 of SEQ ID NO: 21 (wild-type human Siglec-6). In certain embodiments, the mutation is present in the region from amino acid 120 to 131 of SEQ ID NO: 21 (wild-type human Siglec-6). In certain embodiments, the mutation is present in the region from amino acid 119-122 of SEQ ID NO: 21 (wild-type human Siglec-6). In certain embodiments, the mutation is present at DI 15, Y119, F120, F121, R122, K129, Y130, Y132, L137, or V139 of SEQ ID NO: 21 (wild-type human Siglec-6). In certain embodiments, the mutation is a substitution or deletion of an arginine residue at a position corresponding to position 122 of wild-type human Siglec-6 (R122), e.g., R122A, R122G, or R122K. In certain embodiments, the Siglec-6 ECD comprises or consists of SEQ ID NO: 24 or SEQ ID NO: 25.

II. Serum Half-life Enhancers

[0048] As used herein, the terms “serum half-life enhancer” and “serum half-life extender” are used interchangeably and refer to a moiety that can be associated with (e.g., conjugated to) a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, to enhance its circulating half-life in the serum of a subject. As used herein and unless otherwise indicated or inferred, the terms “serum half-life” and “circulating half-life” refer to the time that it takes following administration of a substance (e.g., a fusion protein as described herein) for the serum concentration of the substance to be reduced by 50%. Two additional half-life measurements may be utilized: the alpha half-life, (T1/201), which corresponds to the decline in serum concentration due to the process of drug redistribution from the central compartment (e.g., the blood) to a peripheral compartment (e.g., a tissue or organ), and the beta half-life (T1/2P) which corresponds to the decline in serum concentration due to the processes of excretion or metabolism. a. Fc Domains and CTPs

[0049] It has been discovered that linking a Siglec-6 ECD protein to both an immunoglobulin Fc domain and a carboxy-terminal peptide (CTP) results in an unexpectedly beneficial improvement to the half-life of the protein. Accordingly, in certain embodiments, a Siglec-6 ECD or functional fragment or variant thereof is conjugated to an Fc domain and a CTP. In certain embodiments, a Siglec-6 ECD or functional fragment or variant thereof is conjugated to an Fc domain, a first CTP, and a second CTP. In certain embodiments, a Siglec-6 ECD or functional fragment or variant thereof is conjugated to an Fc domain and two or more CTPs (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 CTPs).

[0050] The extended half-life resulting from linking (e.g., conjugating) a Siglec-6 ECD, or a functional fragment or variant thereof, to multiple serum half-life enhancers enables a longer pharmacodynamic coverage, leading to increased potency and/or allowing for the Siglec-6 ECD construct to be administered at reduced dosing intervals. In certain embodiments, the serum half-life of a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, conjugated to multiple serum half-life enhancers (e.g., conjugated to an Fc domain and 2 CTPs) is at least 24, 36, 48, or 60 hours. In certain embodiments, the serum half-life of a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, conjugated to multiple serum half-life enhancers (e.g., conjugated to an Fc domain and 2 CTPs) is 12 to 96, 12 to 84, 12 to 72, 12 to 60, 12 to 48, 12 to 36, 12 to 30, 12 to 24, 12 to 18, 18 to 96, 18 to 84, 18 to 72, 18 to

60, 18 to 48, 18 to 36, 18 to 30, 18 to 24, 24 to 96, 24 to 84, 24 to 72, 24 to 60, 24 to 48, 24 to

36, 24 to 30, 30 to 96, 30 to 84, 30 to 72, 30 to 60, 30 to 48, 30 to 36, 36 to 96, 36 to 84, 36 to

72, 36 to 60, 36 to 48, 48 to 96, 48 to 84, 48 to 72, 48 to 60, 60 to 96, 60 to 84, 60 to 72, 72 to

96, 72 to 84, or 84 to 96 hours.

[0051] In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is covalently attached (either directly or indirectly) to the Fc domain (Beck et al., supra) and/or the CTP to form a fusion protein, either by genetic fusion (i.e., production of recombinant fusion protein) or by chemical conjugation. i. Fc domains

[0052] In certain embodiments, a Siglec-6 ECD or a fragment or variant thereof is linked, e.g., directly or indirectly covalently attached, to an immunoglobulin Fc domain. As used herein, unless otherwise indicated, the term “immunoglobulin Fc domain” or “Fc domain” or “Fc” refers to a fragment of an immunoglobulin heavy chain constant region which, either alone or in combination with a second immunoglobulin Fc domain, or unconjugated or conjugated to a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is capable of binding to an Fc receptor. An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain may include, e.g., immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. For example, as used herein, an “Fc” or “Fc domain” can refer to a polypeptide comprising a CH2 domain, a CH3 domain, and optionally a hinge or a portion thereof. This polypeptide can bind (e.g., dimerize) to another polypeptide comprising a CH2 domain, a CH3 domain, and optionally a hinge or a portion thereof, wherein the dimer is capable of binding to an Fc receptor. Boundaries between immunoglobulin hinge regions, CH2, and CH3 domains are well known in the art, and can be found, e.g., in the PROSITE database (available on the world wide web at prosite.expasy.org).

[0053] In certain embodiments, the immunoglobulin Fc domain is derived from a human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM Fc domain. A single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) may be introduced to abolish the heterogeneity observed in recombinant IgG4 antibody. See Angal, S. et al. (1993) MOL. IMMUNOL. 30: 105-108.

[0054] Depending upon the circumstances, the immunoglobulin Fc domain can be derived from a human IgGl isotype or another isotype that elicits antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement mediated cytotoxicity (CDC). In certain embodiments, the immunoglobulin Fc domain is derived from a human IgGl isotype (e.g., SEQ ID NO: 1 and/or SEQ ID NO: 5). Alternatively, the immunoglobulin Fc domain can be derived from a human IgG4 isotype or another isotype that elicits little or no antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement mediated cytotoxicity (CDC). In certain embodiments, the immunoglobulin Fc domain is derived from a human IgG4 isotype.

[0055] In certain embodiments where a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is conjugated to an Fc domain, the Fc domain can have a “knob- into-hole” type format. The “knob” part is engineered by replacing a small amino acid with a larger one, which fits into a “hole”, which is engineered by replacing a large amino acid with a smaller one. The “knob-into-hole” format enhances heterodimer formation but does not suppress homodimer formation. Several approaches to promote heterodimerization have been described, for example in International (PCT) Publication Nos. WO96/27011, W098/050431, W02007/110205, W02007/147901, W02009/089004, W02010/129304, WO2011/90754, WO201 1/143545, WO2012/058768, WO2013/157954, and WO2013/096291, and European Patent Publication No. EP 1870459. Typically, in the approaches known in the art, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both engineered in a complementary manner so that the heavy chain comprising one engineered CH3 domain can no longer homodimerize with another heavy chain of the same structure (e.g. a CH3- engineered first heavy chain can no longer homodimerize with another CH3 -engineered first heavy chain; and a CH3 -engineered second heavy chain can no longer homodimerize with another CH3 -engineered second heavy chain). Thereby the heavy chain comprising one engineered CH3 domain is forced to heterodimerize with another heavy chain comprising the CH3 domain, which is engineered in a complementary manner. As a result, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitutions, such that the first heavy chain and the second heavy chain are forced to heterodimerize, whereas the first heavy chain and the second heavy chain can no longer homodimerize (e.g., for steric reasons).

[0056] Depending upon the certain circumstances, e.g., when the molecule is a heterodimer, the immunoglobulin Fc domain comprises either a “knob” mutation, e.g., T366Y, or a “hole” mutation, e.g., Y407T, for heterodimerization with a second polypeptide (residue numbers according to EU numbering, Kabat, E. A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For example, in certain constructs, the immunoglobulin Fc domain is derived from a human IgGl Fc domain and comprises a Y407T mutation (e.g., the Fc domain comprises SEQ ID NO: 2 and/or SEQ ID NO: 7). In certain other constructs, the immunoglobulin Fc domain is derived from a human IgGl Fc domain and comprises a T366Y mutation (e.g., the Fc domain comprises SEQ ID NO: 3 and/or SEQ ID NO: 6).

[0057] In addition, it is understood that the immunoglobulin Fc domain can be modified to prevent to glycosylation of the Fc domain. For example, in certain constructs, the immunoglobulin Fc domain is derived from a human IgGl Fc domain and comprises a mutation to prevent glycosylation, for example, a mutation at position N297, for example, an N297A mutation or an N297G mutation (residue numbers according to EU numbering, Kabat, E.A., el al., supra}. For example, in certain constructs, the Fc domain comprises SEQ ID NO: 4 and/or SEQ ID NO: 8.

[0058] In certain embodiments, an immunoglobulin Fc domain comprises a modified hinge. For example, in certain embodiments, the Fc domain is derived from a human IgGl Fc domain and comprises a mutation at, e.g., C220. In certain embodiments, the Fc domain comprises a C220S mutation (residue numbers according to EU numbering, Kabat, E.A., et al., supra . In certain embodiments, the Fc domain comprises SEQ ID NO: 18 (EPKSS). In certain embodiments, the Fc domain comprises SEQ ID NO: 18 and SEQ ID NO: 1 (an Fc domain derived from human IgGl), wherein SEQ ID NO: 18 is fused to the N-terminus of SEQ ID NO: 1. In certain embodiments, the Fc domain comprises SEQ ID NO: 18 and SEQ ID NO: 2 (an Fc domain derived from human IgGl with a Y407T mutation), wherein SEQ ID NO: 18 is fused to the N-terminus of SEQ ID NO: 2. In certain embodiments, the Fc domain comprises SEQ ID NO: 18 and SEQ ID NO: 3 (an Fc domain derived from human IgGl with a T366Y mutation), wherein SEQ ID NO: 18 is fused to the N-terminus of SEQ ID NO: 3. In certain embodiments, the Fc domain comprises SEQ ID NO: 18 and SEQ ID NO: 4 (an Fc domain derived from human IgGl with an N297G mutation), wherein SEQ ID NO: 18 is fused to the N-terminus of SEQ ID NO: 4. In certain embodiments, the Fc domain comprises SEQ ID NO: 9 (an Fc domain derived from human IgGl with an N297G mutation and a modified hinge). ii. Carboxy-terminal peptide derived from human chorionic gonadotropin (CTP)

[0059] As used herein, unless otherwise indicated or inferred, the terms “carboxy-terminal peptide”, “CTP”, and “CTP derived from human chorionic gonadotropin” are used interchangeably, and refer to a peptide derived from the hydrophilic, C-terminal peptide from human chorionic gonadotropin P chain, or functional fragment or variant thereof. The native CTP of human chorionic gonadotropin has four O-glycosylation sites, which contributes to the long half-life of wild-type human chorionic gonadotropin. Without wishing to be bound by theory, the presence of O-linked oligosaccharides, ended with negatively charged sialic acids, increases the size and the extent of the negative of the protein, which is thought to slow the rate of renal clearance of the protein and thereby increase the protein’s half-life.

[0060] In certain embodiments, a Siglec-6 ECD or a fragment or variant thereof is linked, e.g., directly or indirectly covalently attached, to a CTP. In certain embodiments, the CTP is a full- length CTP. In certain embodiments, the CTP comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the CTP is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12.

[0061] In certain embodiments, the CTP is a truncated CTP. For example, in certain embodiments, the CTP comprises or consists of the first 5, 6, 7, 8, 9, 10, 11, or the first 12 amino acids of SEQ ID NO: 11.

[0062] In certain embodiments, the CTP is a variant of the native CTP of human chorionic gonadotropin P-chain. For example, in certain embodiments, the variant CTP comprises an amino acid sequence that differs from the wild-type CTP amino acid sequence by 1, 2, 3, 4, or 5 conservative amino acid substitutions, e.g., as described in U.S. Patent No. 5,712,122. In certain embodiments, the CTP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the CTP is encoded by a nucleic acid comprising a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the nucleotide sequence of SEQ ID NO: 12.

[0063] In certain embodiments, a Siglec-6 ECD, or a fragment or variant thereof is linked, e.g., directly or indirectly covalently attached, to more than one CTP. For example, in certain embodiments, a Siglec-6 ECD, or a functional fragment or variant thereof, is covalently attached to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CTPs. In certain embodiments, a Siglec-6 ECD, or a functional fragment or variant thereof, is linked, e.g., directly or indirectly covalently attached, to a first CTP and a second CTP. In certain embodiments, the first CTP comprises the amino acid of SEQ ID NO: 11. Alternatively, in certain embodiments, the first CTP comprises a functional fragment or variant of the wild-type CTP, as describe hereinabove. In certain embodiments, the second CTP comprises the amino acid sequence of SEQ ID NO: 11. Alternatively, in certain embodiments, the second CTP comprises a functional fragment or variant of the wild-type CTP, as described hereinabove. In certain embodiments, the first CTP and the second CTP comprise or consist of the same amino acid sequence. For example, in certain embodiments, the first and second CTP each comprise the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the first CTP and the second CTP are part of the same polypeptide chain. In certain embodiments, the first CTP and the second CTP are directly linked to each other via a peptide bond. b. Other Serum Half Life Enhancers

[0064] As described herein, the disclosure relates, in part, to a Siglec-6 ECD fusion protein comprising a Siglec-6 ECD, or a functional fragment thereof, and an Fc domain and a CTP. In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof is associated with, e.g., conjugated to, additional serum half-life enhancers. For example, in certain embodiments, a Siglec-6 ECD fusion protein comprising a Siglec-6 ECD, or a functional fragment thereof, and an Fc domain and a CTP is further associated with, e.g., conjugated to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 serum half-life enhancers. In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, and multiple serum half-life enhancers are part of the same polypeptide chain.

Exemplary suitable half-life extenders include, e.g., albumin (e.g., human serum albumin (HSA), see, Weimer et al. (2013) Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013, p. 297-323), albumin binding domain (e.g., an HSA binder, see Walker et al. (2013) Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013, p. 325-43), transferrin (see Kim et al. (2010) J. PHARMACOL. EXP. THER. 334:682-92), XTEN (also called recombinant PEG or “rPEG”, see Schellenberger et al. (2009) NAT. BIOTECHNOL. 27: 1186-90), a homo-amino acid polymer (HAP, see Schlapschy et al. (2007) PROTEIN ENG. DES. SEL. 20:273-84)), a proline-alanine-serine polymer (PAS, see Schlapschy et al. (2013) PROTEIN ENG. DES. SEL. 26:489-501), an elastin-like peptide (ELP, see Floss et al. (2013) Fusion protein technologies for biopharmaceuticals: applications and challenges, p. 372-98), gelatin-like protein (GLK, Huang et al. (2010) EUR. J. PHARM.

BIOPHARM. 72:435-41), and a polyethylene glycol (PEG).

[0065] Suitable serum half-life enhancers also include a variety of polymers, such as those described in U.S. Patent No. 7,842,789. For example, block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides which comprise the saccharide monomers such as D-mannose, D- and L- galactose, fucose, fructose, D-xylose, L-arabinose, and D-glucuronic acid can be used. In other embodiments, the serum half-life enhancer can be a hydrophilic polyvinyl polymer such as polyvinyl alcohol and polyvinylpyrrolidone (PVP)-type polymers. The serum half-life enhancer can be a functionalized polyvinylpyrrolidone, for example, carboxy or amine functionalized on one (or both) ends of the polymer (as available from Polymer Source). Alternatively, the serum half-life enhancer can include Poly N-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), Poly(N-isopropylacrylamide) or functionalized poly (N-i sopropy 1 aery 1 ami de) .

[0066] In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is covalently attached (either directly or indirectly) to a naturally long-half-life polypeptide or protein such transferrin (Kim et al., supra), or albumin (Weimer el al., supra) to form a fusion protein, either by genetic fusion (i.e., production of recombinant fusion protein) or by chemical conjugation.

[0067] In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is covalently attached to an inert polypeptide such as an XTEN (also called recombinant PEG or “rPEG”, see Schellenberger, supra), a homo amino acid polymer (HAP, see Schlapschy et al. (2007), supra), a proline-alanine-serine-polymer (PAS, see Schlapschy et al., (2013), supra), an elastin-like peptide (ELP, see Floss et al., supra), or gelatinlike protein (GLK, Huang et al., supra) to form a fusion protein, either by genetic fusion i.e., production of recombinant fusion protein) or by chemical conjugation. Inert polypeptides function, among other things, to increase the size and hydrodynamic radius of a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, thereby to extend halflife. In certain embodiments, an XTEN polypeptide has a length from about 25 amino acids to about 1500 amino acids (e.g., from about 25 amino acids to about 100 amino acids, from about 25 amino acids to about 250 amino acids, from about 25 amino acids to about 500 amino acids, from about 25 amino acids to about 750 amino acids, from about 25 amino acids to about 1,000 amino acids, from about 25 amino acids to about 1250 amino acids, from about 100 amino acids to about 250 amino acids, from about 100 amino acids to about 250 amino acids, from about 100 amino acids to about 500 amino acids, from about 100 amino acids to about 750 amino acids, from about 100 amino acids to about 1,000 amino acids, from about 100 amino acids to about 1250 amino acids, from about 100 amino acids to about 1,500 amino acids, from about 250 amino acids to about 1250 amino acids, from about 250 amino acids to about 1,000 amino acids, from about 250 amino acids to about 750 amino acids, from about 250 amino acids to about 500 amino acids, from about 500 amino acids to about 750 amino acids, from about 500 amino acids to about 1000 amino acids, from about 500 amino acids to about 1,250 amino acids, from about 500 amino acids to about 1,500 amino acids, from about 750 amino acids to about 1000 amino acids, from about 750 amino acids to about 1250 amino acids, from about 750 amino acids to about 1500 amino acids, from about 1,000 amino acids to about 1,250 amino acids, from about 1000 amino acids to about 1,500 amino acids, or from about 1,250 amino acids to about 1,500 amino acids.

[0068] In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is chemically conjugated to a repeat chemical moiety such as PEG or hyaluronic acid (see, Mero et al. (2013) CARB. POLYMERS 92:2163-70), which increases the hydrodynamic radius of the Siglec-6 extracellular domain (ECD), or the functional fragment or variant thereof, thereby to extend half-life.

[0069] In certain embodiments, a Siglec-6 ECD or a functional fragment or variant thereof, is conjugated to polyethylene glycol (PEG) or derivative thereof (e.g., alkoxy polyethylene glycol, for example, methoxypolyethylene glycol, ethoxypolyethylene glycol and the like). In one embodiment, the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, as described herein, can be covalently attached to at least one PEG having an actual MW of at least about 20,000 D. In another embodiment, the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is covalently attached to at least one PEG having an actual MW of at least about 30,000 D. In another embodiment, the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is covalently attached to at least one PEG having an actual MW of at least about 40,000 D. In certain embodiments, the PEG is methoxyPEG(5000)-succinimidylpropionate (mPEG-SPA), methoxy PEG(5000)- succinimidylsuccinate (mPEG-SS). Such PEGS are commercially available from Nektar Therapeutics or SunBiowest or LaysanBio or NOF. It is contemplated that the PEG may be branched, or Y-shaped, as available from JenKem USA or NOF, or comb-shaped, or synthesized by coupling two or more PEGs to a small molecule such as glutamic acid. The omega position of PEG may include a hydroxyl group or a methoxy group and the PEG may also contain an amino group in the omega position. Such an amino group can in turn be coupled to a variety of agents. In certain embodiments, the serum half-life enhancer can be a pegylated poly-L-lysine or a pegylated poly-D-lysine.

[0070] Attachment sites on a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, for a PEG or a derivative thereof include the N-terminal amino group and epsilon amino groups found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. PEG may be covalently bonded directly to the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, with or without the known use of a multifunctional (ordinarily bifunctional) crosslinking agent using chemistries and used in the art. For example, the PEG modifier can be conjugated to the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, by using a thiol reactive cross linker and then reacting with a thiol group on the PEG. In certain embodiments, sulfhydryl groups can be derivatized by coupling to maleimido-substituted PEG (e.g. alkoxy-PEG amine plus sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate), or PEG-mal eimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.).

[0071] In certain embodiments, a Siglec-6 ECD or a fragment or variant thereof can be linked, e.g., directly or indirectly covalently attached, to human serum albumin (HSA) or to an HSA- binding peptide (see, e.g., PCT Publication Nos. WO2013128027A1 and WO2014140358A1). Human serum albumin (HSA) (molecular mass ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/mL (600 pM), and has a half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. The neonatal Fc receptor (FcRn) appears to be involved in prolonging the life-span of albumin in circulation (see, Chaudhury et al. (2003) J. EXP. MED., 3: 315-22). Albumin and IgG bind noncooperatively to distinct sites of FcRn and form a tri-molecular (see id). Binding of human FcRn to HSA and to human IgG is pH dependent, stronger at acidic pH and weaker at neutral or physiological pH (see id.). This observation suggests that proteins and protein complexes containing albumin, similar to those containing IgG (particularly Fc), are protected from degradation through pH-sensitive interaction with FcRn (see id.). Using surface plasmon resonance (SPR) to measure the capacity of individual HSA domains to bind immobilized soluble human FcRn, it has been shown that FcRn and albumin interact via the D-III domain of albumin in a pH-dependent manner, on a site distinct from the IgG binding site (see, Chaudhury et al. (2006) BlOCHEM. 45:4983-90 and PCT Publication No. W02008068280A1).

[0072] Exemplary HSA-binding proteins are known in the art. For example, U.S. Patent Application Publication No. US20130316952A1 discloses a polypeptide that binds serum albumin having the amino acid sequence of LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA (SEQ ID NO: 38). In certain embodiments, the HSA-binding protein is an HSA-specific antibody, derivative, or HSA- binding fragment thereof. Additional exemplary polypeptides that bind HSA are described in U.S. Patent Nos. 8,188,223, and 9,284,361, PCT Publication Nos. WO2017085172, and W02018050833, and Dennis et al. (2002) J. BIOL. CHEM., 277: 35035-43; Jacobs et al. (2015) PROTEIN ENG. DES. SEL., 28: 385-93; and Zorzi et al. (2017) NAT. COMMUN., 8: 16092. [0073] In certain embodiments, a Siglec-6 ECD or a fragment or variant thereof can be linked, e.g, directly or indirectly covalently attached, to an HSA-binding moiety. HSA has multiple fatty acid binding sites and, in certain embodiments, the HSA-binding moiety may be a fatty acid moiety that is conjugated or linked to the Siglec-6 extracellular domain or fragment thereof (e.g., the Siglec-6 extracellular domain or fragment thereof may be acylated or lipidated). Without wishing to be bound by theory, a linked fatty acid or lipid moiety is thought to enhance protein half-life by facilitating reversible binding to HSA. Methods of generating acylated proteins are known in the art including, e.g., as described in PCT Publication No. W02000055119 and in U.S. Patent No. 8,791,236.

[0074] In certain embodiments, a Siglec-6 ECD or a fragment or variant thereof can be linked, e.g., directly or indirectly covalently attached, to transferrin or a fragment thereof. Transferrin is a high molecular weight protein (molecular mass ~76 kDa) that is normally present at a high concentration in human serum (approximately 3-4 mg/mL). Transferrin has a half-life of approximately 14 to 17 days in humans in its glycosylated form, or approximately 7-10 days in its non-glycosylated form. Transferrin binds circulating iron ions in a pH-dependent manner and mediates their transport throughout the body and into cells via interaction with its receptor. When iron-loaded transferrin is bound to its cell surface receptor, the receptortransferrin complex is endocytosed, and the transition to the low-pH of the endosome triggers the release of iron ions. The naturally long half-life of transferrin is thought to be a function both of a recycling mechanism that returns endocytosed transferrin to circulation, in addition to the protein’s large size. Transferrin fusion proteins are known in the art, e.g., those described in U.S. Patent Nos. 5,672,683, 5977,307, and 7,176,278. In certain embodiments, a Siglec-6 ECD or a fragment or variant thereof can be linked, e.g., directly or indirectly covalently attached, to a protein having affinity for transferrin, such as an anti-transferrin antibody or derivative thereof, or transferrin-binding fragment thereof. Exemplary transferrin-binding proteins are known in the art, such as those described in U.S. Patent Application No. 16/755,268.

[0075] In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, is itself polysialylated or covalently attached to a negatively charged, highly sialylated protein. For example, in certain embodiments, a Siglec-6 ECD is linked e.g., directly or indirectly covalently attached, to a carboxy -terminal peptide (CTP) derived from chorionic gonadotropin (CG) P-chain (see Duijkers el al. (2002) HUM REPROD 17: 1987-93). In certain embodiments, a Siglec-6 ECD is linked, e.g., directly or indirectly covalently attached, to multiple CTPs. In certain embodiments, a Siglec-6 ECD is linked, e.g., directly or indirectly covalently attached, to two CTPs. In certain embodiments, a Siglec-6 ECD is linked, e.g., directly or indirectly covalently attached, to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CTPs.

[0076] In certain embodiments, a serum half-life enhancer may have a molecular weight from about 2 kDa to about 5 kDa, from about 2 kDa to about 10 kDa, from about 2 kDa to about 20 kDa, from about 2 kDa to about 30 kDa, from about 2 kDa to about 40 kDa, from about 2 kDa to about 50 kDa, from about 2 kDa to about 60 kDa, from about 2 kDa to about 70 kDa, from about 2 kDa to about 80 kDa, from about 2 kDa to about 90 kDa, from about 2 kDa to about 100 kDa, from about 2 kDa to about 150 kDa, from about 5 kDa to about 10 kDa, from about 5 kDa to about 20 kDa, from about 5 kDa to about 30 kDa, from about 5 kDa to about 40 kDa, from about 5 kDa to about 50 kDa, from about 5 kDa to about 60 kDa, from about 5 kDa to about 70 kDa, from about 5 kDa to about 80 kDa, from about 5 kDa to about 90 kDa, from about 5 kDa to about 100 kDa, from about 5 kDa to about 150 kDa, from about 10 kDa to about 20 kDa, from about 10 kDa to about 30 kDa, from about 10 kDa to about 40 kDa, from about 10 kDa to about 50 kDa, from about 10 kDa to about 60 kDa, from about 10 kDa to about 70 kDa, from about 10 kDa to about 80 kDa, from about 10 kDa to about 90 kDa, from about 10 kDa to about 100 kDa, from about 10 kDa to about 150 kDa, from about 20 kDa to about 30 kDa, from about 20 kDa to about 40 kDa, from about 20 kDa to about 50 kDa, from about 20 kDa to about 60 kDa, from about 20 kDa to about 70 kDa, from about 20 kDa to about 80 kDa, from about 20 kDa to about 90 kDa, from about 20 kDa to about 100 kDa, from about 20 kDa to about 150 kDa, from about 30 kDa to about 40 kDa, from about 30 kDa to about 50 kDa, from about 30 kDa to about 60 kDa, from about 30 kDa to about 70 kDa, from about 30 kDa to about 80 kDa, from about 30 kDa to about 90 kDa, from about 30 kDa to about 100 kDa, from about 30 kDa to about 150 kDa, from about 40 kDa to about 50 kDa, from about 40 kDa to about 60 kDa, from about 40 kDa to about 70 kDa, from about 40 kDa to about 80 kDa, from about 40 kDa to about 90 kDa, from about 40 kDa to about 100 kDa, from about 40 kDa to about 150 kDa, from about 50 kDa to about 60 kDa, from about 50 kDa to about 70 kDa, from about 50 kDa to about 80 kDa, from about 50 kDa to about 90 kDa, from about 50 kDa to about 100 kDa, from about 50 kDa to about 150 kDa, from about 60 kDa to about 70 kDa, from about 60 kDa to about 80 kDa, from about 60 kDa to about 90 kDa, from about 60 kDa to about 100 kDa, from about 60 kDa to about 150 kDa, from about 70 kDa to about 80 kDa, from about 70 kDa to about 90 kDa, from about 70 kDa to about 100 kDa, from about 70 kDa to about 150 kDa, from about 80 kDa to about 90 kDa, from about 80 kDa to about 100 kDa, from about 80 kDa to about 150 kDa, from about 90 kDa to about 100 kDa, from about 90 kDa to about 150 kDa, or from about 100 kDa to about 150 kDa.

[0077] Methods for making and using the foregoing serum half-life enhancers are known in the art. See also, e.g., Strohl (2015) BIODRUGS 29:215-239.

III. Linkers

[0078] It is understood that, depending upon the circumstances, the Siglec-6 ECD, or a functional fragment or variant thereof, can be linked or fused directly to a serum half-life enhancer (e.g., an immunoglobulin Fc domain and/or a CTP). Alternatively, the Siglec-6 ECD, or a functional fragment or variant thereof, can be covalently bound to a serum half-life enhancer by a linker and/or two serum half-life enhancers (e.g., an Fc domain and a CTP and/or two CTPs) can be covalently bound together via a linker.

[0079] The linker may couple, with one or more amino acids (natural, unnatural or a combination thereof), the Siglec-6 ECD, or a functional fragment or variant thereof, to a serum half-life enhancer where the amino acid (e.g., a cysteine amino acid) may be introduced by site- directed mutagenesis. In certain embodiments, the linker couples, with one or more amino acids (natural, unnatural or a combination thereof), two serum half-life enhancers (e.g., an Fc domain and a CTP and/or two CTPs), where the amino acid (e.g., a cysteine amino acid) may be introduced by site-directed mutagenesis. The linker may include one or more unnatural amino acids. It is contemplated that, in certain circumstances, a linker containing for example, one or more sulfhydryl reactive groups (e.g., a mal eimide) may covalently link a cysteine in the Siglec- 6 ECD, or a functional fragment or variant thereof, or in a serum half-life enhancer that is a naturally occurring cysteine residue or is the product of site-specific mutagenesis. In certain embodiments, a linker containing for example, one or more sulfhydryl reactive groups (e.g., a maleimide) may covalently link a cysteine in a serum half-life enhancer (e.g., an Fc or a CTP) to a cysteine in another half-life enhancer (e.g., a CTP), wherein the cysteine may be naturally occurring or be introduced by site-directed mutagenesis.

[0080] The linker may be a cleavable linker or a non-cleavable linker. Optionally or in addition, the linker may be a flexible linker or an inflexible linker.

[0081] The linker should be a length sufficiently long to allow the Siglec-6 ECD, or a functional fragment or variant thereof, and a serum half-life enhancer to be linked without steric hindrance from each other (or to allow two half-life enhancers to be linked without steric hindrance from each other) and sufficiently short to retain the intended activity of the fusion protein. The linker preferably is sufficiently hydrophilic to avoid or minimize instability of the fusion protein. The linker preferably is sufficiently hydrophilic to avoid or minimize insolubility of the fusion protein. The linker should be sufficiently stable in vivo (e.g., it is not cleaved by serum, enzymes, etc.) to permit the fusion protein to be operative in vivo.

[0082] The linker may be from about 1 angstroms (A) to about 150 A in length, or from about 1 A to about 120 A in length, or from about 5 A to about 110 A in length, or from about 10 A to about 100 A in length. The linker may be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or greater angstroms in length and/or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or fewer A in length. Furthermore, the linker may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and 120 A in length.

[0083] In certain embodiments, the linker comprises a polypeptide linker that connects or fuses a Siglec-6 ECD to a serum half-life enhancer (e.g., immunoglobulin Fc domain or CTP). In certain embodiments, the linker comprises a polypeptide linker that connects or fuses two half-life enhancers to each other (e.g., an Fc domain to a CTP or two CTPs to each other). For example, it is contemplated that a gene encoding a Siglec-6 ECD linked directly or indirectly (for example, via an amino acid containing linker) to a serum half-life enhancer can be created and expressed using conventional recombinant DNA technologies. When a linker is employed, the linker may comprise hydrophilic amino acid residues, such as Gin, Ser, Gly, Glu, Pro, His and Arg. In certain embodiments, the linker is a peptide containing 1-25 amino acid residues, 1- 20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, e.g., (GlyGlyPro)n (SEQ ID NO: 19) or (GlyGlyGlyGlySer)_n (SEQ ID NO: 20), where n is 1-5. In certain embodiments, the linker comprises, consists, or consists essentially of GGGGS (SEQ ID NO: 16). In certain embodiments, the linker comprises, consists, or consists essentially of GGGGSGGGGS (SEQ ID NO: 15). In certain embodiments, the linker comprises, consists, or consists essentially of GGGGS GGGGS GGGGS (SEQ ID NO: 17). In certain embodiments, the linker comprises, consists, or consists essentially of EPKSS (SEQ ID NO: 18). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Patent Nos. 5,482,858 and 5,525,491. IV. Proteins Comprising a Siglec Extracellular Domain (ECD) Conjugated to Multiple Serum Half-Life Enhancers

[0084] In certain embodiments, the disclosure provides a fusion protein comprising a Siglec-6 ECD, or a functional fragment or variant thereof, conjugated to multiple serum half-life enhancers. The fusion protein can comprise a Siglec-6 ECD, or a functional fragment or variant thereof, disclosed herein and any combination of serum half-life enhancers disclosed herein.

The Siglec-6 ECD, or a functional fragment or variant thereof, and the serum half-life enhancers can be fused directly, or can comprise any linker as disclosed herein. All combinations of Siglec-6 ECDs, or functional fragments or variants thereof, linkers, and serum half-life enhancers are contemplated herein.

[0085] In certain embodiments, the fusion protein comprises a Siglec-6 ECD or a functional fragment or variant thereof, an Fc domain, and a CTP. In further embodiments, the fusion protein comprises one or more linkers, e.g., a first and second linker.

[0086] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Siglec-6 ECD or a functional fragment or variant thereof, a CTP, and an Fc domain. In certain embodiments, the fusion protein further comprises a first linker and/or a second linker, optionally wherein each of the linkers is independently (i) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain, or (ii) interposed between the CTP and the Fc domain.

[0087] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a CTP, a Siglec-6 ECD or a functional fragment or variant thereof, and an Fc domain. In certain embodiments, the fusion protein further comprises a first linker and/or a second linker, optionally wherein each of the linkers is independently (i) interposed between the CTP and the Siglec-6 ECD or functional fragment or variant thereof, or (ii) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain.

[0088] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Siglec-6 ECD or functional fragment or variant thereof, an Fc domain, and a CTP. In certain embodiments, the fusion protein further comprises a first linker and/or a second linker, optionally wherein each of the linkers is independently i) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain, or ii) interposed between the Fc domain and the CTP. [0089] In certain embodiments, the fusion protein comprises a Siglec-6 ECD or a functional fragment or variant thereof, an Fc domain, a first CTP, and a second CTP. In certain embodiments, the first CTP and the second CTP are independently selected from a wild-type CTP, a functional fragment thereof, and a functional variant thereof. In certain embodiments, the first CTP and the second CTP comprise the same amino acid sequence, e.g., SEQ ID NO: 11. In certain embodiments, the C-terminus of the first CTP is covalently attached to the N-terminus of the second CTP, e.g., by a peptide bond. In certain embodiments, the C-terminus of the second CTP is covalently attached to the N-terminus of the first CTP, e.g., by a peptide bond.

[0090] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Siglec-6 ECD or a functional fragment or variant thereof, a first CTP, a second CTP, and an Fc domain. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each of the linkers is independently i) interposed between the Siglec-6 ECD or functional fragment or variant thereof, ii) interposed between the first CTP and the second CTP, or iii) interposed between the second CTP and the Fc domain.

[0091] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first CTP, a second CTP, a Siglec-6 ECD or a functional fragment or variant thereof, and an Fc domain. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each of the linkers is independently (i) interposed between the first CTP and the second CTP, (ii) interposed between the second CTP and the Siglec-6 ECD or functional fragment or variant thereof, or (iii) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain.

[0092] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Siglec-6 ECD or a functional fragment or variant thereof, an Fc domain, a first CTP, and a second CTP. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each of the linkers is independently i) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain, ii) interposed between the Fc domain and the first CTP, or iii) interposed between the first CTP and the second CTP.

[0093] In certain embodiments, the fusion protein comprises a “CTP-repeat domain,” wherein the CTP-repeat domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more CTPs. In certain embodiments, each CTP in the CTP-repeat domain comprises the same amino acid sequence, e.g., SEQ ID NO: 11. In certain embodiments, each of the CTPs in the CTP-repeat domain are covalently attached end-to-end in a single polypeptide chain. In certain embodiments, the CTP- repeat domain comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the fusion protein comprises a Siglec-6 ECD or a functional fragment or variant thereof, an Fc domain, and a CTP -repeat domain.

[0094] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Siglec-6 ECD or a functional fragment or variant thereof, a CTP-repeat domain, and an Fc domain. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each of the linkers is independently i) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the CTP-repeat domain, or ii) interposed between the CTP-repeat domain and the Fc domain.

[0095] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a CTP repeat domain, a Siglec-6 ECD or a functional fragment or variant thereof, and an Fc domain. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each of the linkers is independently i) interposed between the CTP-repeat domain and the Siglec-6 ECD or functional fragment or variant thereof, or ii) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain.

[0096] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a Siglec-6 ECD or a functional fragment or variant thereof, an Fc domain, and a CTP-repeat domain. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each of the linkers is independently i) interposed between the Siglec-6 ECD or functional fragment or variant thereof and the Fc domain, or ii) interposed between the Fc domain and the CTP-repeat domain.

[0097] In certain embodiments, the fusion protein comprises a Siglec-6 ECD, or a functional fragment or variant thereof, a first CTP, a second CTP, and an Fc domain. In certain embodiments, the fusion protein further comprises one or more linkers, optionally wherein each linker is interposed between any two of the preceding components. In certain embodiments, the Siglec-6 ECD comprises or consists of amino acids 27-347 of SEQ ID NO: 21 (SEQ ID NO: 23). In certain embodiments, the Siglec-6 ECD comprises or consists of amino acids 27-347 of SEQ ID NO: 21 with an R122A substitution (SEQ ID NO: 24). In certain embodiments, the Siglec-6 ECD comprises or consists of amino acids 27-347 of SEQ ID NO: 21 with an R122K mutation (SEQ ID NO: 25). In certain embodiments, the one or more linkers comprise a GGGGSGGGGS (SEQ ID NO: 15) linker or an EPKSS (SEQ ID NO: 18) linker. In certain embodiments, the Fc domain is a human IgGl Fc domain, optionally with an N297A or an N297G mutation. In certain embodiments, the Fc domain comprises SEQ ID NO: 4. In certain embodiments, the Fc domain comprises SEQ ID NO: 9. In certain embodiments, the first CTP and/or the second CTP comprise SEQ ID NO: 11. In certain embodiments, the first CTP and the second CTP comprise SEQ ID NO: 11, and the C-terminus of the first CTP is fused directly to the N-terminus of the second CTP (SEQ ID NO: 13) by a peptide bond. In certain embodiments, the fusion protein comprises SEQ ID NO: 33, or a functional fragment thereof. In certain embodiments, the protein is encoded by SEQ ID NO: 34. In certain embodiments, the protein comprises an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In certain embodiments, the protein is encoded by a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.

[0098] In certain embodiments, the inclusion of one or more (e.g., 2) CTPs into a fusion protein comprising an Fc domain and a Siglec-6 ECD, or functional fragment or variant thereof, increases the serum half-life of the fusion protein as compared to an appropriate control protein lacking the one or more CTPs. Depending upon the circumstances, the increase in the serum half-life comprises an increase in the alpha half-life, an increase in the beta half-life, or an increase in both the alpha half-life and beta half-life. Furthermore, the inclusion of the one or more CTPs can result in a 1.1- to 5-fold, 1.1- to 4.5-fold, 1.1- to 4-fold, 1.1- to 3.5-fold, 1.1- to 3-fold, 1.1- to 2.75-fold, 1.1- to 2.5-fold, 1.1- to 2.25-fold, 1.1- to 2-fold, 1.1- to 1.9-fold, 1.1- to 1.8 fold, 1.1- to 1.7-fold, 1.1- to 1.6-fold, 1.1- to 1.5-fold, 1.1- to 1.4-fold, 1.1- to 1.3-fold, 1.1 to 1.2-fold, 1.2- to 5-fold, 1.2- to 4.5-fold, 1.2- to 4-fold, 1.2- to 3.5-fold, 1.2- to 3 -fold, 1.2- to 2.75-fold, 1.2- to 2.5-fold, 1.2- to 2.25-fold, 1.2- to 2-fold, 1.2- to 1.9-fold, 1.2- to 1.8 fold, 1.2- to 1.7-fold, 1.2- to 1.6-fold, 1.2- to 1.5-fold, 1.2- to 1.4-fold, 1.2- to 1.3-fold, 1.3- to 5-fold, 1.3- to 4.5-fold, 1.3- to 4-fold, 1.3- to 3.5-fold, 1.3- to 3-fold, 1.3- to 2.75-fold, 1.3- to 2.5-fold, 1.3- to 2.25-fold, 1.3- to 2-fold, 1.3- to 1.9-fold, 1.3- to 1.8 fold, 1.3- to 1.7-fold, 1.3- to 1.6-fold, 1.3- to 1.5-fold, 1.3- to 1.4-fold, 1.4- to 5-fold, 1.4- to 4.5-fold, 1.4- to 4-fold, 1.4- to 3.5-fold, 1.4- to

3-fold, 1.4- to 2.75-fold, 1.4- to 2.5-fold, 1.4- to 2.25-fold, 1.4- to 2-fold, 1.4- to 1.9-fold, 1.4- to 1.8 fold, 1.4- to 1.7-fold, 1.4- to 1.6-fold, 1.4- to 1.5-fold, 1.5- to 5-fold, 1.5- to 4.5-fold, 1.5- to

4-fold, 1.5- to 3.5-fold, 1.5- to 3-fold, 1.5- to 2.75-fold, 1.5- to 2.5-fold, 1.5- to 2.25-fold, 1.5- to 2-fold, 1.5- to 1.9-fold, 1.5- to 1.8 fold, 1.5- to 1.7-fold, 1.5- to 1.6-fold, 1.6- to 5-fold, 1.6- to 4.5-fold, 1.6- to 4-fold, 1.6- to 3.5-fold, 1.6- to 3-fold, 1.6- to 2.75-fold, 1.6- to 2.5-fold, 1.6- to 2.25-fold, 1.6- to 2-fold, 1.6- to 1.9-fold, 1.6- to 1.8 fold, 1.6- to 1.7-fold, 1.7- to 5-fold, 1.7- to 4.5-fold, 1.7- to 4-fold, 1.7- to 3.5-fold, 1.7- to 3-fold, 1.7- to 2.75-fold, 1.7- to 2.5-fold, 1.7- to

2.25-fold, 1.7- to 2-fold, 1.7- to 1.9-fold, 1.7- to 1.8 fold, 1.8- to 5-fold, 1.8- to 4.5-fold, 1.8- to 4-fold, 1.8- to 3.5-fold, 1.8- to 3-fold, 1.8- to 2.75-fold, 1.8- to 2.5-fold, 1.8- to 2.25-fold, 1.8- to 2-fold, 1.8- to 1.9-fold, 1.9- to 5-fold, 1.9- to 4.5-fold, 1.9- to 4-fold, 1.9- to 3.5-fold, 1.9- to 3- fold, 1.9- to 2.75-fold, 1.9- to 2.5-fold, 1.9- to 2.25-fold, 1.9- to 2-fold, 2- to 5-fold, 2- to 4.5- fold, 2- to 4-fold, 2- to 3.5-fold, 2- to 3-fold, 2- to 2.75-fold, 2- to 2.5-fold, 2- to 2.25-fold, 2.25- to 5-fold, 2.25- to 4.5-fold, 2.25- to 4-fold, 2.25- to 3.5-fold, 2.25- to 3-fold, 2.25- to 2.75-fold,

2.25- to 2.5-fold, 2.5- to 5-fold, 2.5- to 4.5-fold, 2.5- to 4-fold, 2.5- to 3.5-fold, 2.5- to 3-fold,

2.5- to 2.75-fold, 2.75- to 5-fold, 2.75- to 4.5-fold, 2.75- to 4-fold, 2.75- to 3.5-fold, 2.75- to 3- fold, 3- to 5-fold, 3- to 4.5-fold, 3- to 4-fold, 3- to 3.5-fold, 3.5- to 5-fold, 3.5- to 4.5-fold, 3.5- to 4-fold, 4- to 5-fold, 4- to 4.5-fold, or 4.5- to 5-fold increase in the serum half-life, the alpha halflife, and/or the beta half-life as compared to an appropriate control protein lacking the one or more CTPs.

V. Methods of Making Siglec-6 Extracellular Domain (ECD) Conjugated to Multiple Serum Half-Life Enhancers

[0099] Methods for producing a fusion protein comprising Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, are known in the art. For example, DNA molecules encoding a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof and, if appropriate, one or more protein-based serum half-life enhancers (e.g., a CTP or a peptide that can dimerize to produce an Fc domain) either linked directly or via a peptide linker, can be synthesized chemically or by recombinant DNA methodologies. The sequences of interest can be cloned by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using the appropriate synthetic nucleic acid primers. The resulting DNA molecules encoding the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, and optionally, the one or more serum half-life extenders and optional linker(s), can be ligated to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art.

[0100] Nucleic acids encoding a desired Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (EEK 293) cells, HeLa cells, baby hamster kidney (BEK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof.

[0101] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed protein may be secreted. The expressed protein may accumulate in refractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the protein may be refolded and/or cleaved by methods known in the art.

[0102] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers and introns. The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0103] A polypeptide comprising the components described herein can be produced by growing (culturing) a host cell transfected with an expression vector encoding such a variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags. In certain embodiments, in order to express a protein, e.g., a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, as a secreted protein, a native N-terminal signal sequence of the protein is replaced, e.g., with MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 35), MGWSCIILFLVATATGVHS (SEQ ID NO: 36), or MEFGLSWLFLVAILKGVQC (SEQ ID NO: 37). In certain embodiments, to express a protein, e.g., a fusion protein, as a secreted protein, an N-terminal signal sequence, c.g, MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 35), MGWSCIEFLVATATGVHS (SEQ ID NO: 36), or MEFGLSWLFLVAILKGVQC (SEQ ID NO: 37) is added. Additional exemplary N-terminal signal sequences include signal sequences from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

VI. Pharmaceutical Compositions

[0104] For therapeutic use, a protein comprising a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, linked to multiple serum half-life enhancers (e.g., an Fc and 2 CTPs), preferably is combined with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0105] The term “pharmaceutically acceptable carrier” as used herein refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975], Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0106] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants (see, Remington ’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0107] In certain embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (See Anselmo et al. (2016) BIOENG. TRANSL. MED. 1 : 10-29).

[0108] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2- hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3 -hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0109] Pharmaceutical compositions containing a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, conjugated to multiple serum half-life enhancers (e.g., an Fc and 2 CTPs), as disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, it is contemplated that the constructs derived herein can be administered by IV infusion. Alternatively, it is contemplated that the constructs can be administered by intratumoral injection.

[0110] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington ’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[OHl] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0112] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0113] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein are generally administered by parenteral administration. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion.

[0114] Generally, a therapeutically effective amount of active component, for example, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, linked to multiple serum half-life enhancers (e.g., an Fc and 2 CTPs), is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, conjugated to multiple serum half-life enhancers (e.g., an Fc and 2 CTPs), and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. In certain embodiments, a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, linked to multiple serum half-life enhancers (e.g., an Fc and 2 CTPs), is lyophilized, and then reconstituted in buffered saline, at the time of administration.

VII. Therapeutic Uses

[0115] The compositions and methods disclosed herein can be used to treat various forms of immune system disorders in a subject. The present invention provides methods for decreasing an unwanted immune or inflammatory response in a subject, by administering to the subject an effective amount of a Siglec-6 ECD, or a functional fragment or variant thereof, linked to multiple serum half-life enhancers (e.g., an Fc and 2 CTPs), wherein the Siglec-6 ECD, or the functional fragment or variant thereof, reduces the unwanted immune or inflammatory response in the subject. In one embodiment, the method is used to decrease the number of T cells, e.g., CD4 T cells and/or CD8 T cells, in a subject. In another embodiment, the method is used to decrease the activity of T cells, e.g., CD4 T cells and/or CD8 T cells, in a subject. In certain embodiments, the T cells are hyperactive. Siglec-6 ECDs of the present disclosure act primarily on activated T cells with little observed effects on non-activated T cells.

[0116] Activation of T cells occurs through the simultaneous engagement of a T-cell receptor and a co-stimulatory molecule (like CD28, or ICOS) on CD4+ T cells by the major histocompatibility complex (MHCII) peptide and co-stimulatory molecules on the APC. Both are required for production of an effective immune response. In the absence of co-stimulation, T cell receptor signaling alone results in anergy. The signaling pathways downstream from co- stimulatory molecules usually engages the PI3K pathway generating PIP3 at the plasma membrane and recruiting PH domain containing signaling molecules like PDK1 that are essential for the activation of PKC-0, and eventual IL-2 production. Optimal CD8+ T cell response relies on CD4+ signaling. CD4+ cells are useful in the initial antigenic activation of naive CD8 T cells, and sustaining memory CD8+ T cells in the aftermath of an acute infection. Therefore, activation of CD4+ T cells can be beneficial to the action of CD8+ T cells. However, the unwanted immune or inflammatory activated T cell response can result in an inflammatory or autoimmune disorder.

[0117] Thus, the invention provides a method of treating an inflammatory disorder and/or an autoimmune disorder in a subject. The method comprises administering to the subject an effective amount of a protein construct described herein either alone or in a combination with another therapeutic agent to treat the inflammatory disorder and/or the autoimmune disorder in the subject. The term “effective amount” as used herein refers to the amount of an active agent (e.g., Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, linked to multiple serum half-life enhancers, according to the present disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0118] As used herein, “treat”, “treating” and “treatment” mean the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0119] Examples of inflammatory disorders include, but are not limited to: acute respiratory distress syndrome (ARDS), acute lung injury (ALI), alcoholic liver disease, allergic inflammation of the skin, lungs, and gastrointestinal tract, allergic rhinitis, ankylosing spondylitis, asthma (allergic and non-allergic), atopic dermatitis (also known as atopic eczema), atherosclerosis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic respiratory distress syndrome (CRDS), colitis, dermatitis, diabetes, eczema, endocarditis, fatty liver disease, fibrosis (e.g., idiopathic pulmonary fibrosis, scleroderma, kidney fibrosis, and scarring), food allergies (e.g., allergies to peanuts, eggs, dairy, shellfish, tree nuts, etc.), gastritis, gout, hepatic steatosis, hepatitis, inflammation of body organs including joint inflammation including joints in the knees, limbs or hands, inflammatory bowel disease (IBD) (including Crohn's disease or ulcerative colitis), intestinal hyperplasia, irritable bowel syndrome, juvenile rheumatoid arthritis, liver disease, metabolic syndrome, multiple sclerosis, myasthenia gravis, neurogenic lung edema, nephritis (e.g., glomerular nephritis), non-alcoholic fatty liver disease (NAFLD) (including non-alcoholic steatosis and non-alcoholic steatohepatitis (NASH)), obesity, prostatitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis sinusitis, splenitis, seasonal allergies, sepsis, systemic lupus erythematosus, uveitis, and UV-induced skin inflammation.

[0120] Examples of autoimmune diseases or disorders include, but are not limited to: arthritis, including rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis; inflammatory hyperproliferative skin diseases; psoriasis, such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails; atopy, including atopic diseases such as hay fever and Job's syndrome; dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis; x-linked hyper IgM syndrome; allergic intraocular inflammatory diseases; urticaria, such as chronic allergic urticaria, chronic idiopathic urticaria, and chronic autoimmune urticaria; myositis; polymyositis/dermatomyositis; juvenile dermatomyositis; toxic epidermal necrolysis; scleroderma, including systemic scleroderma; sclerosis, such as systemic sclerosis, multiple sclerosis (MS), spino-optical MS, primary progressive MS (PPMS), relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, and ataxic sclerosis; neuromyelitis optica (NMO); inflammatory bowel disease (IBD), including Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis, ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, transmural colitis, and autoimmune inflammatory bowel disease; bowel inflammation; pyoderma gangrenosum; erythema nodosum; primary sclerosing cholangitis; respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS); meningitis; inflammation of all or part of the uvea; iritis; choroiditis; an autoimmune hematological disorder; rheumatoid spondylitis; rheumatoid synovitis; hereditary angioedema; cranial nerve damage, as in meningitis; herpes gestationis; pemphigoid gestationis; pruritis scroti; autoimmune premature ovarian failure; sudden hearing loss due to an autoimmune condition; IgE-mediated diseases, such as anaphylaxis and allergic and atopic rhinitis; encephalitis, such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis; uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis; glomerulonephritis (GN) with and without nephrotic syndrome, such as chronic or acute glomerulonephritis, primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN; proliferative nephritis; autoimmune polyglandular endocrine failure; balanitis, including balanitis circumscripta plasmacellularis; balanoposthitis; erythema annulare centrifugum; erythema dyschromicum perstans; eythema multiform; granuloma annulare; lichen nitidus; lichen sclerosus et atrophicus; lichen simplex chronicus; lichen spinulosus; lichen planus; lamellar ichthyosis; epidermolytic hyperkeratosis; premalignant keratosis; pyoderma gangrenosum; allergic conditions and responses; allergic reaction; eczema, including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema; asthma, such as asthma bronchiale, bronchial asthma, and auto-immune asthma; conditions involving infiltration of T cells and chronic inflammatory responses; immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy; chronic pulmonary inflammatory disease; autoimmune myocarditis; leukocyte adhesion deficiency; lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), cutaneous SLE, subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus; juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, and diabetic large-artery disorder; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; tuberculosis; sarcoidosis; granulomatosis, including lymphomatoid granulomatosis; Wegener's granulomatosis; agranulocytosis; vasculitides, including vasculitis, large-vessel vasculitis, polymyalgia rheumatica and giant-cell (Takayasu's) arteritis, medium- vessel vasculitis, Kawasaki's disease, polyarteritis nodosa/periarteritis nodosa, microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, systemic necrotizing vasculitis, ANCA-associated vasculitis, Churg- Strauss vasculitis or syndrome (CSS), and ANCA-associated small-vessel vasculitis; temporal arteritis; aplastic anemia; autoimmune aplastic anemia; Coombs positive anemia; Diamond Blackfan anemia; hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa); Addison's disease; pure red cell anemia or aplasia (PRC A); Factor VIII deficiency; hemophilia A; autoimmune neutropenia; pancytopenia; leukopenia; diseases involving leukocyte diapedesis; CNS inflammatory disorders; multiple organ injury syndrome, such as those secondary to septicemia, trauma or hemorrhage; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; anti-phospholipid antibody syndrome; allergic neuritis; Behcet's disease/syndrome; Castleman's syndrome; Goodpasture's syndrome; Reynaud's syndrome; Sjogren's syndrome; Stevens-Johnson syndrome; pemphigoid, such as pemphigoid bullous and skin pemphigoid, pemphigus, pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus; autoimmune polyendocrinopathies; Reiter's disease or syndrome; thermal injury; preeclampsia; an immune complex disorder, such as immune complex nephritis, and antibody-mediated nephritis; polyneuropathies; chronic neuropathy, such as IgM polyneuropathies and IgM-mediated neuropathy; thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, autoimmune or immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), and chronic or acute ITP; scleritis, such as idiopathic cerato-scleritis, and episcleritis; autoimmune disease of the testis and ovary including, autoimmune orchitis and oophoritis; primary hypothyroidism; hypoparathyroidism; autoimmune endocrine diseases, including thyroiditis, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes, autoimmune polyglandular syndromes, and polyglandular endocrinopathy syndromes; paraneoplastic syndromes, including neurologic paraneoplastic syndromes; Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome; stiff-man or stiff-person syndrome; encephalomyelitis, such as allergic encephalomyelitis, encephalomyelitis allergica, and experimental allergic encephalomyelitis (EAE); myasthenia gravis, such as thymoma-associated myasthenia gravis; cerebellar degeneration; neuromyotonia; opsoclonus or opsoclonus myoclonus syndrome (OMS); sensory neuropathy; multifocal motor neuropathy; Sheehan's syndrome; hepatitis, including autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis, and autoimmune chronic active hepatitis; lymphoid interstitial pneumonitis (LIP); bronchiolitis obliterans (non-transplant) vs NSIP; Guillain-Barre syndrome; Berger's disease (IgA nephropathy); idiopathic IgA nephropathy; linear IgA dermatosis; acute febrile neutrophilic dermatosis; subcorneal pustular dermatosis; transient acantholytic dermatosis; cirrhosis, such as primary biliary cirrhosis and pneumonocirrhosis; autoimmune enteropathy syndrome; Celiac or Coeliac disease; celiac sprue (gluten enteropathy); refractory sprue; idiopathic sprue; cryoglobulinemia; amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease); coronary artery disease; autoimmune ear disease, such as autoimmune inner ear disease (AIED); autoimmune hearing loss; polychondritis, such as refractory or relapsed or relapsing polychondritis; pulmonary alveolar proteinosis; Cogan's syndrome/nonsyphilitic interstitial keratitis; Bell's palsy; Sweet's disease/syndrome; rosacea autoimmune; zoster-associated pain; amyloidosis; a non-cancerous lymphocytosis; a primary lymphocytosis, including monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS); peripheral neuropathy; channelopathies, such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS; autism; inflammatory myopathy; focal or segmental or focal segmental glomerulosclerosis (FSGS); endocrine ophthalmopathy; uveoretinitis; chorioretinitis; autoimmune hepatological disorder; fibromyalgia; multiple endocrine failure; Schmidt's syndrome; adrenalitis; gastric atrophy; presenile dementia; demyelinating diseases, such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy; Dressier's syndrome; alopecia areata; alopecia totalis; CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia); male and female autoimmune infertility (e.g., due to anti-spermatozoan antibodies); mixed connective tissue disease; Chagas' disease; rheumatic fever; recurrent abortion; farmer's lung; erythema multiforme; post-cardiotomy syndrome; Cushing's syndrome; bird-fancier's lung; allergic granulomatous angiitis; benign lymphocytic angiitis; Alport's syndrome; alveolitis, such as allergic alveolitis and fibrosing alveolitis; interstitial lung disease; transfusion reaction; leprosy; malaria; Samter's syndrome; Caplan's syndrome; endocarditis; endomyocardial fibrosis; diffuse interstitial pulmonary fibrosis; interstitial lung fibrosis; pulmonary fibrosis; idiopathic pulmonary fibrosis; cystic fibrosis; endophthalmitis; erythema elevatum et diutinum; erythroblastosis fetalis; eosinophilic fasciitis; Shulman's syndrome; Felty's syndrome; flariasis; cyclitis, such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis; Henoch-Schonlein purpura; sepsis; endotoxemia; pancreatitis; thyroxicosis; Evan's syndrome; autoimmune gonadal failure; Sydenham's chorea; poststreptococcal nephritis; thromboangitis ubiterans; thyrotoxicosis; tabes dorsalis; choroiditis; giant-cell polymyalgia; chronic hypersensitivity pneumonitis; keratoconjunctivitis sicca; epidemic keratoconjunctivitis; idiopathic nephritic syndrome; minimal change nephropathy; benign familial and ischemia-reperfusion injury; transplant organ reperfusion; retinal autoimmunity; joint inflammation; bronchitis; chronic obstructive airway/pulmonary disease; silicosis; aphthae; aphthous stomatitis; arteriosclerotic disorders; aspermiogenese; autoimmune hemolysis; Boeck's disease; cryoglobulinemia; Dupuytren's contracture; endophthalmia phacoanaphylactica; enteritis allergica; erythema nodo sum leprosum; idiopathic facial paralysis; febris rheumatica; Hamman-Rich's disease; sensoneural hearing loss; haemoglobinuria paroxysmatica; hypogonadism; ileitis regionalis; leucopenia; mononucleosis infectiosa; traverse myelitis; primary idiopathic myxedema; nephrosis; ophthalmia symphatica; orchitis granulomatosa; pancreatitis; polyradiculitis acuta; pyoderma gangrenosum; Quervain's thyreoiditis; acquired splenic atrophy; non-malignant thymoma; vitiligo; toxic-shock syndrome; food poisoning; conditions involving infiltration of T cells; leukocyte-adhesion deficiency; immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; diseases involving leukocyte diapedesis; multiple organ injury syndrome; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; allergic neuritis; autoimmune polyendocrinopathies; oophoritis; primary myxedema; autoimmune atrophic gastritis; sympathetic ophthalmia; rheumatic diseases; mixed connective tissue disease; nephrotic syndrome; insulitis; polyendocrine failure; autoimmune polyglandular syndrome type I; adult-onset idiopathic hypoparathyroidism (AOIH); cardiomyopathy such as dilated cardiomyopathy; epidermolysis bullosa acquisita (EBA); hemochromatosis; myocarditis; nephrotic syndrome; primary sclerosing cholangitis; purulent or nonpurulent sinusitis; acute or chronic sinusitis; ethmoid, frontal, maxillary, or sphenoid sinusitis; an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils; anaphylaxis; seronegative spondyloarthritides; polyendocrine autoimmune disease; sclerosing cholangitis; chronic mucocutaneous candidiasis; Bruton's syndrome; transient hypogammaglobulinemia of infancy; Wiskott-Aldrich syndrome; ataxia telangiectasia syndrome; angiectasis; autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization; allergic hypersensitivity disorders; glomerulonephritides; reperfusion injury; ischemic reperfusion disorder; reperfusion injury of myocardial or other tissues; lymphomatous tracheobronchitis; inflammatory dermatoses; dermatoses with acute inflammatory components; multiple organ failure; bullous diseases; renal cortical necrosis; acute purulent meningitis or other central nervous system inflammatory disorders; ocular and orbital inflammatory disorders; granulocyte transfusion-associated syndromes; cytokine-induced toxicity; narcolepsy; acute serious inflammation; chronic intractable inflammation; pyelitis; endarterial hyperplasia; peptic ulcer; valvulitis; and endometriosis.

[0121] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0122] In certain embodiments, a method or composition described herein, is administered in combination with one or more additional therapies, e.g., surgery, radiation therapy, or administration of another therapeutic preparation. In certain embodiments, the additional therapy may include chemotherapy, e.g., a cytotoxic agent. In certain embodiments the additional therapy may include a targeted therapy, e.g. a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In certain embodiments, the additional therapy may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, e.g., a steroid, a biologic immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g. methotrexate), or an NSAID. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes.

[0123] The invention also provides a method of decreasing the expression of HLA-DR, CD86, CD83, CD64, IFNy, IL-lb, IL-12 (e.g., IL-12p40), TNFa, IL-17A, IL-2, IL-23, or IL-6 in a cell, tissue, or subject. The method comprises contacting the cell, tissue, or subject with an effective amount of a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof, linked to multiple serum half-life enhancers, disclosed herein (e.g., an Fc domain and 2 CTPs). In certain embodiments, the cell is selected from a dendritic cell and a peripheral blood mononuclear cell (PBMC). In certain embodiments, expression of HLA-DR, CD86, CD83, CD64, IFNy, IL-lb, IL-12 (e.g., IL-12p40), TNFa, IL-17A, IL-2, IL-23, or IL-6 is decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. In certain embodiments, expression of HLA-DR, CD86, CD83, CD64, IFNy, IL-lb, IL-12 (e g., IL-12p40), TNFa, IL-17A, IL-2, IL-23, or IL-6 is decreased by about 5% to about 100%, by about 5% to about 90%, by about 5% to about 80%, by about 5% to about 70%, by about 5% to about 60%, by about 5% to about 50%, by about 5% to about 40%, by about 5% to about 30%, by about 5% to about 20%, by about 5% to about 15%, by about 5% to about 10%, by about 10% to about 100%, by about 10% to about 90%, by about 10% to about 80%, by about 10% to about 70%, by about 10% to about 60%, by about 10% to about 50%, by about 10% to about 40%, by about 10% to about 30%, by about 10% to about 20%, by about 10% to about 15%, by about 15% to about 100%, by about 15% to about 90%, by about 15% to about 80%, by about 15% to about 70%, by about 15% to about 60%, by about 15% to about 50%, by about 15% to about 40%, by about 15% to about 30%, by about 15% to about 20%, by about 15% to about 10%, by about 20% to about 100%, by about 20% to about 90%, by about 20% to about 80%, by about 20% to about 70%, by about 20% to about 60%, by about 20% to about 50%, by about 20% to about 40%, by about 20% to about 30%, by about 30% to about 100%, by about 30% to about 90%, by about 30% to about 80%, by about 30% to about 70%, by about 30% to about 60%, by about 30% to about 50%, by about 30% to about 40%, by about 40% to about 100%, by about 40% to about 90%, by about 40% to about 80%, by about 40% to about 70%, by about 40% to about 60%, by about 40% to about 50%, by about 50% to about 100%, by about 50% to about 90%, by about 50% to about 80%, by about 50% to about 70%, by about 50% to about 60%, by about 60% to about 100%, by about 60% to about 90%, by about 60% to about 80%, by about 60% to about 70%, by about 70% to about 100%, by about 70% to about 90%, by about 70% to about 80%, by about 80% to about 100%, by about 80% to about 90%, or by about 90% to about 100%. Gene expression may be measured by any suitable method known in the art, for example, by ELISA, or by Luminex multiplex assays.

[0124] In certain embodiments, expression of HLA-DR, CD86, CD83, IFNy, IL-lb, IL-10, TNFa, IL-17A, IL-2, or IL-6 in the cell, tissue, or subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to a similar or otherwise identical cell or tissue that has not been contacted with a construct described herein. Gene expression may be measured by any suitable method known in the art, for example, by ELISA, or by Luminex multiplex assays.

[0125] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0126] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0127] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0128] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0129] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0130] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0131] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0132] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

EXAMPLES

[0133] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way. Example 1

[0134] This Example describes the in vivo characterization of Siglec-6 extracellular domain (ECD) fusion proteins in mice.

[0135] The Siglec-6 ECD fusion proteins described in this and the following Examples are depicted schematically in FIGURE 2 and summarized in TABLE 1. One fusion protein (Sig-6- SAX-CTP-1G) had (i) a mutant Siglec-6 ECD with a sialic acid loss-of-binding domain (SAX) mutation (R122K), (ii) a human IgGl Fc domain having an N297G mutation removing an N- linked glycosylation site, and (iii) two carboxy-terminal peptides (CTPs; each comprising SEQ ID NO: 11) derived from human chorionic gonadotropin (CG) P chain interposed between the Siglec-6 ECD and the Fc domain. A second fusion protein (Sig-6-SAX-lG) was designed that lacked the two CTPs, but which was otherwise identical to Sig-6-SAX-CTP-lG. Placing the CTP domains at alternative locations, e.g., as N-terminal fusions before the Siglec-6 ECD V-Set domain, was found to lower expression.

TABLE 1

[0136] The proteins indicated in TABLE 1 were recombinantly expressed and purified.

Briefly, proteins were expressed in a 200 mL transfection of Expi293 human cells using the pFusion mammalian expression vector. Proteins were purified using Protein A chromatography, followed by dialysis into PBS (pH 7.2). Purified proteins were assayed for endotoxin and characterized for purity by SDS-PAGE and SEC-HPLC.

[0137] Mice were grouped (6 mice/group) and intraperitoneally administered a 10 mg/kg dose of either Sig-6-SAX-lG or Sig-6-SAX-CTP-lG, as summarized in TABLE 2. A control group of 2 mice was not administered a Siglec-6 ECD fusion protein. Plasma was collected from pairs of mice at the timepoints indicated in TABLE 2. [0138] Briefly, Siglec-6 ECD constructs in samples and standards were prepared in neat mouse plasma, and were captured onto an ELISA plate overnight at 4 °C using 1.5 pg/mL of a mouse anti-human Siglec-6 antibody. The following day, plates were blocked with blocking buffer (Superblock), diluted 1 :20 into assay buffer (PBS, 0.1% Tween-20, 1% BSA), and incubated on the plate for 1 hour at room temperature. Samples were incubated with 0.5 pg/mL HRP-conjugated anti -human Fc for 15 minutes at room temperature, and the plates were developed with 3,3',5,5'-Tetramethylbenzidine (TMB). Data were fit to standard curve by a 4- point sigmoidal fit.

TABLE 2

[0139] FIGURE 3 depicts the concentration of either Sig-6-SAX-lG and Sig-6-SAX-CTP-lG (ng/ml) detected in mouse plasma at the indicated time following injection. The Siglec-6 ECD fusion protein comprising two CTP domains in addition to an Fc domain (Sig-6-SAX-CTP-lG) demonstrated an extended half-life in vivo as compared to Sig-6- SAX- 1G. This extended halflife enables a longer pharmacodynamic coverage, leading to increased potency and/or allowing for the Siglec-6 ECD fusion construct to be administered at reduced dosing intervals. Siglec-6- SAX-1G demonstrated an alpha half-life (Ti/2a) of 0.33 hours and a beta half-life (T1/2P) of 33.8 hours, whereas Siglec-6-SAX-CTP-lG demonstrated a T1/201 of 0.64 hours and T1/2P of 28.9 hours.

Example 2 [0140] This Example describes the effects of Siglec-6 extracellular domain (ECD) fusion proteins on cytokine expression in activated T cells.

[0141] On day 1, human PBMCs were thawed and enriched for T cells using the STEMCELL T isolation kit (Catalog: 19051). T cells were stimulated with anti-CD3 (clone OKT3) and anti- CD28 (clone CD28.2) antibodies at a final concentration of 1 pg/ml in complete RPMI media (supplemented with 10% heat-inactivated FBS, non-essential amino acids, and sodium pyruvate). On day 2, floating cells were collected and re-plated in fresh complete RPMI media and anti-CD3 and anti-CD28 antibodies were replenished at 1 pg/mL to stimulate cells continuously. On day 4 or 5, cells were spun down and resuspended in fresh RPMI media (10% complete) and supplied with low levels of IL-2 (5 ng/mL or ~50 IU). On day 7, U-bottom well plates were coated with anti-CD3 antibody (clone OKT3) at 1 pg/mL and incubated on a shaker for 2 hours. Activated T cells were harvested, centrifuged, resuspended in fresh media, and counted. Sig-6-SAX-lG or Sig-6-SAX-CTP-lG was diluted and added to each well at the desired concentration. An IgGl antibody with an N297G mutation (IgGl-lG) was used as an isotype control. Then, approximately 200,000 activated T cells were added to each well to a final volume of 200 pL. Cultures were incubated overnight, and the supernatants were harvested for analysis the next day. Cytokine concentration was measured using a Biolegend LEGENDplex kit (Catalog No. 741035).

[0142] FIGURE 4 depicts secretion of IFN-y from human T cells treated with Sig-6-SAX-lG or Sig-6-SAX-CTP-lG. FIGURE 4A and FIGURE 4B depict the results from two individual donors. The IC50 (pM) for each test article is shown below each figure. Sig-6-SAX-CTP-lG exhibited a comparable ability to inhibit IFN-y secretion as Sig-6-SAX-lG, demonstrating that a Siglec-6 ECD-Fc fusion protein and a Siglec-6 ECD-CTP-Fc fusion protein are each capable of suppressing immune cell activity, and that the inclusion of the CTP domain in the fusion protein does not adversely affect the immunosuppressive activity of the Siglec-6 ECD.

Example 3

[0143] This Example describes the effects of Siglec-6 extracellular domain (ECD) fusion proteins on cytokine expression in Ml and M2 polarized human macrophages.

[0144] FIGURE 5 summarizes a human macrophage polarization assay described in this Example. Briefly, Ml or M2 macrophages were generated using CD 14+ monocytes isolated from fresh or frozen human PBMCs. Ml macrophages were generated by culturing monocytes in 50 ng/ml GM-CSF, whereas M2 macrophages were generated by culturing monocytes in presence of 50 ng/mL M-CSF. The media and cytokines were replenished every 2 or 3 days until day 6. On day 6, macrophages were harvested using Accutase (Innovative Cell Technologies) and gentle scraping. Harvested macrophages were re-seeded in a flat 96-well plate at approximately 50,000 cells/well in RPMI 1640 media (supplemented with 10% FBS, non-essential amino acids, and sodium pyruvate). On the next day, media was decanted and replaced with fresh media, and the test article (Sig-6-SAX-lG or Sig-6-SAX-CTP-lG) was diluted and added to the culture at the desired concentration. IgG-lG was used as an control. Cells were stimulated via overnight incubation with 50 ng/mL LPS and 10 ng/mL IFN-y. The next day, supernatants were harvested for cytokine analysis and cells were processed for the identification of cell-surface markers.

[0145] FIGURES 6A-B depict the effect of Siglec-6 ECD fusion proteins on IL-12p40 secretion from either Ml human macrophages (FIGURE 6A) or M2 human macrophages (FIGURE 6B)

[0146] FIGURES 7A-B depict the effect of Siglec-6 ECD fusion proteins on IL-10 secretion from either Ml human macrophages (FIGURE 7A) or M2 human macrophages (FIGURE 7B).

[0147] FIGURES 8A-B depict the effect of Siglec-6 ECD fusion proteins on IL-23 secretion from either Ml human macrophages (FIGURE 8A) or M2 human macrophages (FIGURE 8B).

[0148] For both Ml and M2 macrophages, Sig-6-SAX-CTP-lG treatment resulted in very similar changes in cytokine secretion as Sig-6-SAX-lG treatment, and especially for M2 macrophages. This again demonstrates that a Siglec-6 ECD-Fc fusion protein and a Siglec-6 ECD-CTP-Fc fusion protein are each capable of suppressing immune cell activity, and that inclusion of the CTP domain in the fusion protein does not impact the immunosuppressive activity of the Siglec-6 ECD.

[0149] A second human macrophage polarization assay was used to assess activity of Siglec-6 ECD fusion proteins on cell surface markers. This polarization protocol was identical to the one described hereinabove and summarized in FIGURE 5, except that the CD 14+ monocytes were started on plasma-treated plates on Day 1. TABLE 3 summarizes the cell staining panel used for flow cytometry analysis of Ml and M2 human macrophages following treatment with test articles. Untreated macrophages (stimulated or unstimulated) and IgG-lG-treated macrophages were used as controls.

[0150] TABLE 3

[0151] FIGURES 9A-B depict the effect of Siglec-6 ECD fusion proteins on CD64 surface expression for either Ml human macrophages (FIGURE 9A) or M2 human macrophages (FIGURE 9B). For both Ml and M2 macrophages, Sig-6-SAX-CTP-lG treatment had a very similar effect on macrophage CD64 surface expression as Sig-6- SAX- 1G treatment. This again demonstrates that a Siglec-6 ECD-Fc fusion protein and a Siglec-6 ECD-CTP-Fc fusion protein are each capable of suppressing immune cell activity, and that the inclusion of the CTP domain did not affect the immunosuppressive activity of the Siglec-6 ECD.

Example 4

[0152] This Example describes the effects of Siglec extracellular domains (ECDs) on cytokine expression in Cynomolgus T cells.

[0153] Siglec-6-SAX-lG and Sig-6-SAX-CTP-lG are tested using pre-screened commercially available (IQ Biosciences) enriched cyno Pan-T cells (primary; not cultured activated) to evaluate if at high concentrations of Siglec-6 ECD fusion proteins can modulate secretion of cytokines. Cyno Pan-T cells were seeded (approximately 100,000/well) onto anti-CD3 coated (2 pg/ml, clone FN18) 96 well plate and treated with 5 serial 3-fold dilutions of test articles (from 3 pM to 0.03 pM), or with no test article. Treatment with IgG-lG was used as a control.

Following an overnight incubation, supernatants were analyzed for cytokines using Biolegend Legendplex kit for Non-Human Primate (NHP) samples.

[0154] FIGURE 10 depicts the effect of Siglec-6 ECD fusion proteins on TNF-alpha expression (FIGURE 10A) and on IL-8 expression (FIGURE 10B) from Cynomolgus T cells. Treatment with either Sig-6-SAX-lG or Sig-6-SAX-CTP-lG resulted in strong down regulation of TNF-alpha secretion from cynomolgus T cells, again demonstrating that a Siglec-6 ECD-Fc fusion protein and a Siglec-6 ECD-CTP-Fc fusion protein are each capable of suppressing immune cell activity, and that the inclusion of the CTP domain in the fusion protein does not impact the immunosuppressive activity of the Siglec-6 ECD. This result also demonstrates the cross reactivity of human Sig-6 ECD activity on cynomolgus cells. Sig-6-SAX-lG and Sig-6- SAX-CTP-1G demonstrated very little down regulation of IL-8 secretion (FIGURE 10B). Other analytes/ cytokines that were also analyzed include IL-6, IL-10, IP-10, IL-lbeta, IL-17A,

IL-12p40, Inf-G, GM-CSF and MCP-1, but these analytes/cytokines were only detectable at low levels following anti-CD-3 stimulation.

INCORPORATION BY REFERENCE

[0155] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0156] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

SEQUENCE LISTING

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising:

(a) a Siglec-6 extracellular domain (ECD), or a functional fragment or variant thereof;

(b) a first carboxy-terminal peptide (CTP) derived from human chorionic gonadotropin (HCG); and

(c) an immunoglobulin Fc domain.

2. The fusion protein of claim 1, wherein the Siglec-6 ECD comprises a mutation that reduces sialic acid binding activity.

3. The fusion protein of claim 2, wherein the mutation results in the Siglec-6 ECD having less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5%) of the sialic acid binding activity of a corresponding Siglec-6 ECD without the mutation.

4. The fusion protein of claim 2 or claim 3, wherein the Siglec-6 ECD is a human Siglec-6 ECD, and the mutation is present in the region from amino acid 112 to 140 or from amino acid 119 to 122 of SEQ ID NO: 21 (wild-type human Siglec-6).

5. The fusion protein of any one of claims 2-4, wherein the mutation is a substitution of an arginine residue at a position corresponding to position 122 of wild-type human Siglec-6 (e.g., R122).

6. The fusion protein of claim 5, wherein the Siglec-6 ECD comprises the amino acid sequence of SEQ ID NO: 25.

7. The fusion protein of any one of claims 1-6, wherein the immunoglobulin Fc domain is derived from a human IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM Fc domain.

8. The fusion protein of claim 7, wherein the immunoglobulin Fc domain is derived from a human IgGl, IgG2, IgG3, or IgG4 Fc domain.

9. The fusion protein of claim 8, wherein the immunoglobulin Fc domain is derived from a human IgGl Fc domain.

10. The fusion protein of claim 9, wherein the immunoglobulin Fc domain comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 9.

11. The fusion protein of claim 9 or claim 10, wherein the immunoglobulin Fc domain comprises a mutation at position 297 (e.g., an A or a G), according to EU numbering.

12. The fusion protein of any one of claims 1-11, wherein the first CTP comprises the amino acid sequence of SEQ ID NO: 11.

13. The fusion protein of any one of claims 1-12, wherein the first CTP is interposed between the Siglec-6 ECD and the immunoglobulin Fc domain.

14. The fusion protein of any one of claims 1-13, wherein the fusion protein comprises, from N-terminus to C-terminus: the Siglec-6 ECD, the CTP, and the immunoglobulin Fc domain.

15. The fusion protein of any one of claims 1-14, wherein the fusion protein comprises a second CTP derived from HCG.

16. The fusion protein of claim 15, wherein the first CTP and the second CTP comprise the same amino acid sequence.

17. The fusion protein of claim 15 or 16, wherein the second CTP comprises the amino acid sequence of SEQ ID NO: 11.

18. The fusion protein of any one of claims 15-17, wherein the second CTP is interposed between the Siglec-6 ECD and the immunoglobulin Fc domain.

19. The fusion protein of any one of claims 15-18, wherein the N-terminus of the second CTP is linked to the C-terminus of the first CTP.

20. The fusion protein of any one of claims 15-19, wherein the fusion protein comprises, from N-terminus to C-terminus: the Siglec-6 ECD, the first CTP, the second CTP, and the immunoglobulin Fc domain.

21. The fusion protein of any one of claims 1-20, wherein the first CTP and/or the second CTP increase the half-life of the fusion protein by 1.1-fold to 5-fold e.g., 1.1-fold to 2-fold, 2- fold to 3-fold, 3-fold to 4-fold, or 4-fold to 5-fold) when administered to a subject.

22. The fusion protein of any one of claims 1-21, wherein the first CTP and/or the second CTP increase the alpha half-life of the fusion protein by 1.1-fold to 5-fold (e.g., 1.1-fold to 2- fold, 2-fold to 3 -fold, 3 -fold to 4-fold, or 4-fold to 5-fold) when administered to a subject.

23. The fusion protein of any one of claims 1-22, further comprising a first linker.

24. The fusion protein of any one of claim 1-23, wherein the first linker is interposed between any two of the following: the Siglec-ECD, the first CTP, the second CTP, and the immunoglobulin Fc.

25. The fusion protein of any one of claims 1-24, further comprising a second linker.

26. The fusion protein of any one of claim 1-25, wherein the first linker and/or the second linker comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

27. The fusion protein of any one of claims 1-26, wherein the fusion protein comprises, from N-terminus to C-terminus: the Siglec-ECD, the first linker, the first CTP, the second CTP, the second linker, and the immunoglobulin Fc.

28. The fusion protein of any one of claims 1-22, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 33.

29. A dimeric protein, the dimeric protein comprising two fusion proteins of any one of claims 1-28.

30. The dimeric protein of claim 29, wherein the dimeric protein comprises a first fusion protein and a second fusion protein, wherein the first and second fusion proteins are covalently linked together by one or more disulfide bonds that link the Fc domain of the first fusion protein and the Fc domain of the second fusion protein.

31. A pharmaceutical composition comprising the fusion protein of any one of claims 1-28 or the dimeric protein of claim 29 or 30, and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is disposed in a sterile container (e.g., a bottle or vial).

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is lyophilized in the sterile container.

34. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is present as a solution in the sterile container.

35. The pharmaceutical composition of any one of claims 32-34, wherein the sterile container has a label disposed thereon identifying the pharmaceutical composition contained in the container.

36. A method of treating an inflammatory and/or autoimmune disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the fusion protein of any of claims 1-28, the dimeric protein of claim 29 or 30, or the pharmaceutical composition of any one of claims 31-35.