Cis-binding Siglec agonists and related compositions and methods

Abstract

Cis-binding Siglec agonists are provided. In certain embodiments, the cis-binding Siglec agonist comprises a scaffold having a Siglec ligand and a membrane-tethering domain. Compositions, e.g., pharmaceutical compositions, comprising any of the cis-binding Siglec agonists of the present disclosure are also provided. Methods for agonizing Siglec activity, for example, in an individual in need thereof, are also provided. Kits comprising the cis-binding Siglec agonists and methods for producing the cis-binding Siglec agonists are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 046,140, ​​filed June 30, 2020, which is incorporated herein by reference in its entirety.

[0002] Statement of government support This invention was made with government support under contract CA227942 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] preface Sialic acid-binding IgG-like lectins (Siglecs) are a family of immune checkpoint receptors expressed on all classes of immune cells. They bind to various sialoglycans on target cells and transmit signals to immune cells reporting whether the target is healthy or damaged, "self" or "non-self." Of the 14 human Siglecs, nine contain cytoplasmic inhibitory signaling domains. Therefore, binding of these inhibitory Siglecs by sialoglycans suppresses immune cell activity, resulting in an anti-inflammatory effect. In this respect, inhibitory Siglecs are functionally similar to the T cell checkpoint receptors CTLA-4 and PD-1. Similar to these clinically established targets for cancer immunotherapy, there has been recent interest in antagonizing Siglecs to enhance immune cell reactivity against cancer. Conversely, in the context of anti-inflammatory therapy, binding of Siglecs with agonistic antibodies can suppress immune cell reactivity. This approach has been utilized to achieve B cell suppression in lupus patients by agonism of CD22 (Siglec-2) and to deplete eosinophils for the treatment of eosinophilic gastroenteritis by agonism of Siglec-8. Summary of the Invention

[0004] Cis-binding Siglec agonists are provided. In certain embodiments, the cis-binding Siglec agonist comprises a scaffold having a Siglec ligand and a membrane-tethering domain. Compositions, e.g., pharmaceutical compositions, comprising any of the cis-binding Siglec agonists of the present disclosure are also provided. Methods for agonizing Siglec activity, e.g., in an individual in need thereof, are also provided. Kits comprising the cis-binding Siglec agonists and methods for producing the cis-binding Siglec agonists are also provided. [Brief explanation of the drawings]

[0005] [Figure 1] Glycopolypeptides cluster and agonize Siglecs in cis on effector cells. (A) Phagocytes express activating receptors that mediate "eat-me" signaling of target cells, stimulating phagocytosis and inflammation. (B) Clustering of Siglec-9 by cis ligands stimulates inhibitory signaling that halts phagocyte activation. [Figure 2] Representative synthesis of pS9L-lipid. (a) THF, 3, 6 h at 22 °C, glovebox. (b) Hydrazine monohydrate, MeOH / THF / HO, 24 h at 22 °C, 85% over two steps. (c) 4, sodium pyruvate, Pd26ST, NmCSS, NanA, 20 mM MgCl2 in 200 mM Tris (pH 8.5), 48 h, 50%. (d) Benzhydryl azide, CuSO4, BTTAA, tBuOH / HO, 12 h at 22 °C, 75–100%. [Figure 3]Engineered glycopolypeptides bind to Siglec-9 with high affinity. (A) Glycopolypeptides are based on the same lactosylserine scaffold. pLac contains only lactose moieties. pSia contains terminal Neu5Ac. pS9L contains a Siglec-9 ligand. pS7L contains a Siglec-7 ligand. The N-terminus of the polypeptide was functionalized with either a fluorophore or a biotin moiety. (B) Soluble glycopolypeptides containing N-terminal biotin were bound to a streptavidin-coated tip. Association / dissociation curves were measured by immersing the tip in a recombinant Siglec-Fc fusion protein solution followed by buffer alone. Data are representative of two independent experiments. (C) THP-1 monocytes were coated with lipid-tethered glycopolypeptides and stained with Siglec-9-Fc followed by an anti-human AlexaFluor 647-conjugated secondary antibody. Data are representative of three independent experiments. [Figure 4] pS9L-lipid associates in cis with Siglec-9 but not with Siglec-7. (A) FRET experiments to assess the cis association of pS9L-lipid or pS7L-lipid with Siglec-9 or Siglec-7. The N-terminus of the lipid-conjugated glycopolypeptide (pS9L-lipid or pS7L-lipid) was functionalized with AlexaFluor 555 and loaded into JURKAT cells stably overexpressing either Siglec-9 or Siglec-7. An anti-Siglec antibody bearing AlexaFluor 647 was bound to the Siglec, and the FRET signal was quantified by fluorescence microscopy. (B, D) The relative FRET efficiency was calculated when pS9L-lipid or pS7L-lipid was loaded into Siglec-9-expressing cells. Statistical analysis was performed by one-way t-test, p<0.001, Glass's delta=6.70. (C, E) Relative FRET efficiencies were calculated when pS9L-lipids were loaded into Siglec-9 or Siglec-7 expressing cells. Statistical analysis was performed by one-way t-test, p<0.001, Glass's delta=2.42. All data are representative of at least two independent experiments. Data points in (C) and (E) represent individual cells from a single experiment. Error bars represent standard deviations. [Figure 5] In NF-κB transcription reporter assays, pS9L-lipid agonizes Siglec-9 and inhibits TLR4 signaling. (A) HEKBlue cells coexpress the NF-κB-dependent secreted alkaline phosphatase (SEAP) and TLR4 signaling complex. Upon stimulation with LPS, SEAP can be quantified in the supernatant using a colorimetric assay as a surrogate for NF-κB activity. For these assays, HEKBlue cells were also transfected with a pCMV-Siglec expression vector. (B) Siglec-9-expressing HEKBlue cells were grown on plates coated with antibody (anti-Siglec-9, isotype, or vehicle), and relative NF-κB transcription in response to LPS (10 ng / mL) was measured. (C) Siglec-9-expressing HEKBlue cells were pretreated with pS9L-sol (1 μM), pS9L-lipid (1 μM), or vehicle before LPS stimulation (10 ng / mL). (D) HEKBlue cells were transfected with Siglec-9, Siglec-7, or mock expression vectors, coated with pS9L-lipid (1 μM) or vehicle, and then stimulated with LPS (10 ng / mL). (E) HEKBlue cells were transfected with wild-type, R120A, or Y433 / 456F Siglec-9 expression vectors, coated with pS9L-lipid (1 μM) or vehicle, and then stimulated with LPS (10 ng / mL). Statistics were determined by one-way ANOVA (B, C) or two-way ANOVA (D, E). **=p<0.01, ***=p<0.001, ****=p<0.0001. Error bars represent standard deviation. All data are representative of at least three independent experiments. [Figure 6]Macrophage activation is inhibited by cis-linked pS9L-lipid but not by soluble trans-linked pS9L-sol. (A) Hyperinflammatory macrophages were pretreated with glycopolypeptide (500 nM) and then subjected to LPS stimulation (100 pg / mL). Activation was assayed by cytokine quantification from the supernatant (B), quantitative phosphoproteomics (C-E), or Western blot (F). (B) Macrophages were pretreated with glycopolypeptide (500 nM) and then stimulated with LPS (100 pg / mL) for 18 h. Aliquots of the supernatant were analyzed by multiplex inflammatory cytokine assay. Data are presented as the mean fold-change of technical replicates from three independent experiments compared to vehicle-pretreated cells. Statistics were determined by multiple t-test. *=p<0.05. Error bars represent standard deviation. (C-E) Macrophages were pretreated with glycopolypeptide (500 nM) and then stimulated with LPS (100 pg / mL) for 5 minutes and lysed. Lysates were collected from three independent macrophage differentiations. Lysates were normalized, enriched for phosphoproteins, labeled, and analyzed by quantitative phosphoproteomics. (C) Heat map of fold changes from glycopolypeptide-pretreated macrophages stimulated with or without LPS. (D) Volcano plot of significance relative to fold change from vehicle for macrophages pretreated with pS9L-lipid and stimulated with LPS. Identified significantly altered phosphopeptides are shown in red. Selected unique hits are highlighted in dark blue. (E) Same as D, but for pS9L-sol. (F) Macrophages were treated with glycopolypeptide (500 nM) and stimulated with LPS (100 pg / mL) for 1 h, then lysed and analyzed for total IkB and pIkB (S32 / 36) levels by Western blot. Lane 1 shows control macrophages not treated with glycopolypeptide or LPS. FC is fold change. [Figure 7]pS9L-lipids inhibit macrophage phagocytosis in a Siglec-9-dependent manner. (A) Macrophage phagocytosis can be determined by fluorescence microscopy using beads that fluoresce in acidic (i.e., late endosomal / lysosomal) compartments. (B) Representative merged phase and red fluorescence images at 0 h (top) and 15 h (bottom). (C) THP-1 macrophages were pretreated with polymer (200 nM) and a 1 μm pHrodo red-labeled bead suspension was added at a given effector:target (E:T) ratio. The initial rate of phagocytosis was determined by measuring the increase in red fluorescent area over the first hour. Data are representative of three independent experiments. (D-F) CMAS KO (D), Siglec-9 KO (E), or wild-type (F) THP-1 macrophages were pretreated with glycopolypeptide (200 nM) and assayed for phagocytosis every hour for 10 hours using 1 μm pHrodo red-labeled beads at an E:T ratio of 1:20. (G, H) BV2 mouse microglia carrying either a CRISPR-safe target guide (G) or Siglec-E KO (H) were pretreated with neuraminidase (2 μM) and then loaded with Siglec-E cross-reactive pS7L-lipid (500 nM) and assayed for phagocytosis as in D-F. For (D-H), statistical analysis by two-way ANOVA: # = p < 0.15, * = p < 0.05, ** = p < 0.01. Error bars represent standard error. Data are representative of three independent experiments. [Figure 8]Responses to pS9L-lipids by primary monocyte-derived macrophages are stratified by Siglec-9 expression. (A-D) Monocytes were isolated from PBMCs and differentiated into M1 macrophages by treatment with GM-CSF (50 ng / mL) for 6 days. (A-C) M1 macrophages differentiated from PBMCs isolated from three different donors were treated with glycopolypeptide (500 nM) and then assayed for phagocytosis of pHrodo-labeled beads at an E:T ratio of approximately 1:20. Statistical analysis was performed by two-way ANOVA; *=p<0.05. Error bars represent standard error. (D) M1 macrophages were stained with a fluorescently labeled anti-Siglec-9 antibody by microscopy, and fluorescence was quantified by microscopy. Donors A-C correspond to panels A-C. Normalized expression was determined by calculating the ratio of integrated fluorescence intensity per image to confluency per image. Error bars represent standard deviation. Statistical analysis by two-way ANOVA, ****=p<0.001. Data are from three different donors. [Figure 9] Synthetic glycopolypeptides bearing high-affinity Siglec-9 ligands engage Siglec-9, inducing clustering and signaling. (a) Membrane-tethered cis-binding glycopolypeptide 1 (pS9L) induces Siglec-9 signaling, whereas soluble control polypeptide 2 (pS9L-sol) or nonbinding but membrane-tethered control polypeptide 3 (pLac) do not. (b) Structures of polypeptides pS9L, pS9L-sol, and pLac. All polypeptides are based on an O-lactosylpolyserine-co-alanine scaffold and, in some cases, possess terminal Siglec-9-binding sialic acid analogs and / or C-terminal membrane-tethering lipids. [Figure 10]The cis-binding Siglec-9 agonist (pS9L) inhibits R848-induced NETosis via Siglec-9 and SHP-1. (a-c) Primary neutrophils were co-treated with R848 (10 μM) and glycopolypeptide (500 nM) in IMDM supplemented with 0.5% hiFBS and the membrane-impermeable DNA intercalator Cytotox Green or Red (250 nM). Images were acquired every 15 min for 12 h by fluorescence microscopy. The area of ​​all green fluorescent objects greater than 300 μm was quantified, and the total area of ​​three images per well was averaged. Relative NETosis was determined by normalizing to the maximum NET area from R848 treatment alone (t = 8 h). (a) Representative phase-contrast and fluorescence images at t = 8 h. The scale bar indicates 40 μm. (b) Quantification of NETosis over time as the area under the curve in (c). Error bars represent standard deviation. (c) NET formation and degradation as a function of time. Error bands represent standard error. (d) Treatment of R848-stimulated neutrophils with various glycopolypeptides. Error bars represent standard deviation. (e) pS9L is a mucin-like glycopolypeptide that has a high affinity and specificity for Siglec-9 and is functionalized with a membrane-tethering lipid tail. (f) HL-60 cells were transfected with siRNA against SIGLEC9 (encoding Siglec-9), PTPN6 (encoding SHP-1), or a scrambled control and then grown for 2 days. Cells were then co-treated with R848 (10 μM) and vehicle or pS9L (500 nM). Relative NETosis was determined as in (b), except that all objects larger than 200 μm were quantified; the R848 maximum in dHL-60 cells was observed at 2.5 h postinduction. Error bars represent standard deviation. Statistics were determined by two-way ANOVA (b) or one-way ANOVA (c, d, f). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 11]The Siglec-9 agonist pS9L inhibits neutrophil NETosis induced by COVID-19 plasma. (a, b) Analysis of publicly available single-cell transcriptomics data8 on SIGLEC9 expression (a) and PADI4 expression (b) in peripheral blood neutrophils from healthy donors or COVID-19 patients. Error bars represent standard deviation. Statistics were determined using a mixed-effects model. **=p<0.01, ***=p<0.001. (c, d) Primary neutrophils were cultured for 4 hours in undiluted citrate-anticoagulated plasma from healthy donors or COVID-19 patients. Cells were fixed, stained for extracellular myeloperoxidase, and imaged in DAPI imaging media by fluorescence microscopy. Cells were processed in technical triplicate and imaged across multiple fields of view. (c) Percentage of NET-positive cells across all fields of view. Each dot represents an individual plasma sample. (d) Representative images of plasma samples from COVID-19 patients treated or not with pS9L. Error bars represent standard deviation. Statistics were determined using a mixed-effects model to account for samples with repeated neutrophil donors. ****=p<0.0001. [Figure 12] For example, in COVID-19, local and peripheral inflammatory stimuli induce NETosis and the subsequent hyperinflammatory cascade. Both local inflammatory stimuli at the site of SARS-CoV-2 infection (e.g., virions) and peripheral inflammatory stimuli associated with COVID-19 (e.g., the proinflammatory cytokines IL-8 and G-CSF) have been shown to induce NETosis in vitro. These factors are also suspected to be causative agents of NETosis in vivo, initiating the harmful hyperinflammatory cascade that leads to moderate and severe COVID-19 symptoms. Based on this disclosure, it is predicted that agonists of the neutrophil-associated checkpoint receptor Siglec-9 will inhibit NETosis in general, and in COVID-19 specifically. DETAILED DESCRIPTION OF THE INVENTION

[0006] Cis-binding Siglec agonists are provided. In certain embodiments, the cis-binding Siglec agonist comprises a scaffold having a Siglec ligand and a membrane-tethering domain. Compositions, e.g., pharmaceutical compositions, comprising any of the cis-binding Siglec agonists of the present disclosure are also provided. Methods for agonizing Siglec activity, for example, in an individual in need thereof, are also provided. Kits comprising the cis-binding Siglec agonists and methods for producing the cis-binding Siglec agonists are also provided.

[0007] Before the Siglec agonists, compositions, kits, and methods of the present disclosure are described in further detail, it is to be understood that the Siglec agonists, compositions, kits, and methods are not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the Siglec agonists, compositions, kits, and methods will be limited only by the appended claims.

[0008] Where a range of values ​​is provided, it is understood that, to the tenth of the unit of the lower limit, each intervening value between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the Siglec agonists, compositions, kits, and methods, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges can independently be included within the smaller ranges and, subject to any specifically excluded limits in the stated range, are also encompassed within the Siglec agonists, compositions, kits, and methods. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the Siglec agonists, compositions, kits, and methods.

[0009] Certain ranges are presented herein with the term "about" preceding the numerical values. The term "about" is used herein to provide literal support for the exact number to which it is attached, as well as for numbers that are near or approximately the number to which it is attached. In determining whether a number is near or approximately a specifically stated number, the near or approximately unstated number may be a number that provides a substantial equivalent to the specifically stated number in the context in which it is presented.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the Siglec agonists, compositions, kits, and methods belong. Although any Siglec agonists, compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the Siglec agonists, compositions, kits, and methods, representative exemplary Siglec agonists, compositions, kits, and methods are now described.

[0011] All publications and patents cited herein are incorporated by reference to disclose and describe the materials and / or methods for which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present Siglec agonists, compositions, kits, and methods are not entitled to antedate such publication, as the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0012] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as the literal basis for the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0013] It is recognized that certain features of the Siglec agonists, compositions, kits, and methods that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the Siglec agonists, compositions, kits, and methods that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure, and such combinations, to the extent they encompass operable processes and / or compositions, are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations listed in embodiments describing such variations are also specifically encompassed by the present Siglec agonists, compositions, kits, and methods, and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed.

[0014] As will be apparent to one skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. A recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0015] Cis-binding Siglec agonists As summarized above, the present disclosure provides cis-binding Siglec agonists (also referred to herein as "Siglec agonists"). According to some embodiments, the Siglec agonists comprise a scaffold bearing a Siglec ligand and a membrane-tethering domain. As demonstrated herein, the Siglec agonists spontaneously insert into cell membranes and bind in cis to specific Siglecs on the surface of immune cells. Siglec agonists find use in a variety of in vitro and in vivo applications. Unexpectedly, when tethered to cell membranes, rather than as solubilized agents at comparable concentrations, the present Siglec ligands bind to Siglecs and inhibit inflammatory activity in reporter systems, macrophage cell lines, and primary macrophages. Thus, the Siglec agonists of the present disclosure represent, among other things, components of a novel modality of immunosuppression by engineering cis interactions into the glycocalyx. Details regarding embodiments of the Siglec agonists of the present disclosure are now described.

[0016] Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of immunomodulatory receptors whose function is regulated by glycan ligands. In humans, the Siglec family consists of 15 family members, expressed on a limited set of cells in the hematopoietic lineage, with the exception of Siglec-4 (MAG) on oligodendrocytes and Schwann cells and Siglec-6 on placental trophoblasts. Siglecs recognize sialic acid-containing glycan ligands on glycoproteins and glycolipids with unique but overlapping specificity via their outermost N-terminal V-set domains. Recognition of these ligands can affect cell signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails. In the majority of Siglecs, these ITIMs have the ability to recruit phosphatases; therefore, these members are referred to as inhibitory Siglecs. Exceptions include Siglec-1 and MAG, which lack such motifs, and activated Siglecs (Siglec-14 to Siglec-16), which associate with adaptor proteins that contain immunoreceptor tyrosine-based activation motifs (ITAMs) via positively charged amino acids in their transmembrane domains.

[0017] Siglecs can be divided into two groups based on their gene homology among mammalian species. The first group is present in all mammals and consists of Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-4, and Siglec-15. The second group consists of CD33-related Siglecs, including Siglec-3 (CD33), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, and Siglec-16. Monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells share a similar Siglec profile: high expression of Siglec-3, Siglec-7, and Siglec-9, low expression of Siglec-10, and expression of Siglec-1 upon stimulation with IFN-α. In contrast, macrophages express primarily Siglec-1, Siglec-3, Siglec-8, Siglec-9, Siglec-11, Siglec-15, and Siglec-16, depending on their differentiation state. Conventional dendritic cells, like monocyte-derived dendritic cells, express Siglec-3, Siglec-7, and Siglec-9, but also express low levels of Siglec-2 and Siglec-15. Plasmacytoid dendritic cells express Siglec-1 and Siglec-5. Downregulation of Siglec-7 and Siglec-9 expression in monocyte-derived dendritic cells was observed after 48 hours of stimulation with LPS, whereas Siglec expression remained unchanged in monocyte-derived macrophages upon stimulation with LPS. Siglecs are also present in other immune cells, such as B cells, basophils, neutrophils, and NK cells.Further details regarding Siglecs can be found in Angata et al. (2015) Trends Pharmacol Sci. 36(10):645-660, Lubbers et al. (2018) Front. Immunol. 9:2807, Bochner et al. (2016) J Allergy Clin Immunol. 135(3):598-608, and Duan et al. (2020) Annu. Rev. Immunol. 38(1):365-395, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0018] As summarized above, in certain embodiments, the Siglec agonist comprises a Siglec ligand-bearing scaffold. By "scaffold" is meant a structure suitable for presenting a Siglec ligand so that the Siglec ligand can bind in cis to one or more corresponding Siglecs. According to some embodiments, the Siglec ligand-bearing scaffold comprises a polymer scaffold. As used herein, a "polymer" is a linear series of monomers linked together by covalent bonds. In certain embodiments, the polymer is a polypeptide. The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to define a linear series of amino acid residues linked together by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 "standard" genetically encodable amino acids, natural amino acids with side chain biological modifications, unnatural amino acids, or combinations thereof. According to some embodiments, the Siglec ligand-bearing scaffold comprises a glycopolypeptide scaffold. Non-limiting examples of suitable glycopolypeptide scaffolds include those described in the experimental section below.

[0019] The Siglec agonist of the present disclosure can comprise any suitable number of Siglec ligands. In certain embodiments, the Siglec agonist of the present disclosure comprises 2 to 200 Siglec ligands, e.g., 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 25, or 2 to 10 Siglec ligands, e.g., 4 to 8 (e.g., 6) Siglec ligands. The Siglec agonist of the present disclosure can comprise a single type of Siglec ligand. In other embodiments, the Siglec agonist comprises two or more different types of Siglec ligands, e.g., multiple different types of Siglec ligands for binding to the same Siglec or two or more different Siglecs.

[0020] According to some embodiments, the Siglec ligand comprises a ligand for a specific Siglec. In certain such embodiments, the Siglec ligand comprises only a ligand for a specific Siglec. "Only" in this context means that the Siglec ligand comprises only a ligand for a specific Siglec, including, but not limited to, selective or specific ligands for a specific Siglec. "Selectively" means that the ligand preferentially binds to a specific Siglec, e.g., in a sample and / or in vivo, compared to binding to one or more other Siglecs (e.g., all other Siglecs). In certain embodiments, the Siglec ligand comprises, for example, about 10 4 M -1 A molecule is "specific" for a particular Siglec if it binds to or associates with the Siglec with an affinity or Ka (i.e., the association rate constant in units of 1 / M of the specific binding interaction) that is equal to or greater than the equilibrium dissociation constant (KD) (e.g., 10 -5 M~10 -13 In certain embodiments, specific binding can be defined as a binding intensity of a Siglec ligand that is greater than or equal to about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8M or less, or about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 This means that the Siglec ligand binds to a specific Siglec with a KD of no more than M. The binding affinity of a Siglec ligand for a Siglec can be readily determined using conventional techniques, for example, by biolayer interferometry (BLI) (e.g., using an Octet RED96 device from ForteBio), by competitive ELISA (enzyme-linked immunosorbent assay), by equilibrium dialysis, using surface plasmon resonance (SPR) technology (e.g., with a BIAcore 2000 or BIAcore T200 instrument using the general procedures outlined by the manufacturer), by radioimmunoassay, or the like.

[0021] The Siglec agonist of the present disclosure may comprise an immunoinhibitory Siglec ligand. As used herein, an "immunosuppressive Siglec ligand" refers to a ligand for a Siglec that contains a cytoplasmic inhibitory signaling domain, and engagement of the Siglec by the ligand suppresses the activity of an immune cell expressing the Siglec (e.g., results in an anti-inflammatory effect). In certain embodiments, the Siglec agonist of the present disclosure may comprise an immunoinhibitory Siglec ligand, including a ligand for a CD33-associated Siglec. Examples of CD33-associated Siglecs include Siglec-3 (CD33), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, and Siglec-16. According to some embodiments, the Siglec ligand comprises a Siglec-3 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-3 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-5 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-5 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-6 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-6 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-7 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-7 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-8 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-8 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-9 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-9 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-10 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-10 ligand.According to some embodiments, the Siglec ligand comprises a Siglec-11 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-11 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-14 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-14 ligand. According to some embodiments, the Siglec ligand comprises a Siglec-16 ligand. In certain such embodiments, the Siglec ligand comprises only a Siglec-16 ligand. In certain embodiments, the Siglec agonists of the present disclosure may comprise immunosuppressive Siglec ligands, including ligands for Siglec-2 (CD22). In certain such embodiments, the Siglec ligand comprises only a Siglec-2 ligand.

[0022] Siglec ligands (e.g., sialosides and / or analogs thereof) for binding to one or more Siglecs of interest that can be used in the Siglec agonists of the present disclosure are known, including, for example, those described in Courtney et al. (2009) Proc. Natl. Acad. Sci. 106(8):2500-2505, Spence et al. (2015) Sci. Transl. Med. 7(303):1-13, Perdicchio et al. (2016) Proc. Natl. Acad. Sci. 113(12):3329-3334, Shahraz et al. (2015) Sci. Rep. 5:1-17, Nycholat et al. (2019) J. Am. Chem. Soc. 141(36):14032-14037, and Rillahan et al. al. (2012) Angew. Chemie-Int. Ed. 51(44):11014-11018, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0023] As summarized above, the Siglec agonist of the present disclosure comprises a membrane-tethering domain. A "membrane-tethering domain" refers to a domain (e.g., a chemical moiety) that can stably associate with the cell membrane of a cell (e.g., an immune cell) that expresses a Siglec on its surface that is agonized in cis by the Siglec agonist. In certain embodiments, "stably associated" refers to a physical association between two entities in which the average half-life of the association is at least one day in PBS at 4°C. In some embodiments, the physical association between the two entities has an average half-life of at least one day, at least one week, at least one month (including at least six months, e.g., at least one year) in PBS at 4°C. According to some embodiments, the stable association results from a covalent bond between the two entities, a non-covalent bond (e.g., an ionic or metallic bond) between the two entities, or other forms of chemical attraction, such as hydrogen bonding, van der Waals forces, etc.

[0024] Suitable membrane-tethering domains include, but are not limited to, chemical moieties adapted to insert into the plasma membrane of a cell. The basic structure of the plasma membrane is a phospholipid bilayer, which forms a stable barrier between two aqueous compartments. The plasma membrane of animal cells contains four major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin), which together account for more than half of the lipids in most membranes. These phospholipids are asymmetrically distributed between the two halves of the membrane bilayer. The outer leaflet of the plasma membrane is primarily composed of phosphatidylcholine and sphingomyelin, while phosphatidylethanolamine and phosphatidylserine are the predominant phospholipids in the inner leaflet. A fifth phospholipid, phosphatidylinositol, is also localized in the inner half of the plasma membrane. Although phosphatidylinositol is a quantitatively minor membrane component, it plays an important role in cell signaling. The head groups of both phosphatidylserine and phosphatidylinositol are negatively charged, and therefore, their predominance in the inner leaflet results in a net negative charge on the cytoplasmic face of the plasma membrane. In certain embodiments, the membrane-tethering domain is a homodimeric coiled-coil protein domain or a multisubunit tethering complex (MTC), including but not limited to, those described in Zhi et al. (2014) F1000 Prime Rep. 6:74. According to some embodiments, the membrane-tethering domain comprises a lipid membrane-tethering domain. Non-limiting examples of lipid membrane-tethering domains include those used in the experimental section below.

[0025] Suitable membrane-tethering domains also include chemical moieties (including their constituents (e.g., membrane-associated proteins such as transmembrane proteins)) adapted to stably bind to cell membranes. In certain embodiments, such chemical moieties include small molecules. By "small molecule" is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. According to some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain embodiments, the small molecule is not made up of repeating molecular units such as those present in a polymer.

[0026] According to some embodiments, the chemical moiety (including any of its components) adapted for stable binding to a cell membrane is an antibody. The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer, which in turn is composed of two dimers of heavy and light chain polypeptides); single-chain antibodies; fragments of antibodies (e.g., whole antibodies or single-chain antibody fragments) that retain specific binding to a cell surface molecule of a target cell, including, but not limited to, single-chain Fvs (scFvs), Fabs, (Fab')2, (scFv')2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments), and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0027] In certain embodiments, the chemical moiety adapted to stably bind to a cell membrane is a ligand for a cell surface molecule (e.g., a cell surface receptor) expressed on the cell surface. The ligand can be a circulating factor, secreted factor, cytokine, growth factor, hormone, peptide, polypeptide, small molecule, nucleic acid, etc. that forms a complex with the cell surface molecule on the surface of the cell. In some embodiments, when the chemical moiety is a ligand, the ligand is modified in such a way that complex formation with the cell surface molecule occurs but the normal biological consequences of such complex formation are not achieved. In certain embodiments, the ligand is a ligand for a cell surface receptor present on a target cell. Cell surface receptors of interest include, but are not limited to, receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (non-RTKs), growth factor receptors, cytokine receptors, etc.

[0028] In some embodiments, the chemical moiety (including its components) adapted for stable binding to cell membranes is an aptamer. "Aptamer" refers to a nucleic acid (e.g., an oligonucleotide) that has specific binding affinity for a cell surface molecule. Aptamers exhibit desirable properties for targeted delivery of Siglec agonists, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and universal synthetic accessibility. Aptamers that find use in the Siglec agonists of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1):39-45, Sun et al. (2014) Mol. Ther. Nucleic Acids 3:e182, and Zhang et al. (2011) Curr. Med. Chem. 18(27):4185-4194.

[0029] According to certain embodiments, the chemical moiety (including any components thereof) adapted to stably bind to a cell membrane is a nanoparticle. As used herein, a "nanoparticle" is a particle having at least one dimension within the range of 1 nm to 1000 nm, 20 nm to 750 nm, 50 nm to 500 nm (including 100 nm to 300 nm, e.g., 120 to 200 nm). The nanoparticle may have any suitable shape, including, but not limited to, a sphere, a spheroid, a rod, a disk, a cone, a cube, a cylinder, a nanohelix, a nanospring, a nanoring, an arrow, a teardrop, a tetrapod, a prism, or any other suitable geometric or non-geometric shape. In certain aspects, the nanoparticle comprises on its surface one or more of the other chemical moieties described herein, such as antibodies, ligands, aptamers, small molecules, etc. Nanoparticles that find use with the Siglec agonists of the present disclosure include those described in Wang et al. (2010) Pharmacol. Res. 62(2):90-99, Rao et al. (2015) ACS Nano 9(6):5725-5740, and Byrne et al. (2008) Adv. Drug Deliv. Rev. 60(15):1615-1626.

[0030] According to some embodiments, the Siglec agonist of the present disclosure comprises a polymer scaffold, a Siglec ligand, a membrane-tethering domain, or any combination thereof, independently selected from any of the cis-binding Siglec agonists described in the Experimental section below.

[0031] The Siglec agonists of the present disclosure may be detectably labeled, for example, with an in vivo imaging agent, a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. The Siglec agonist may be further conjugated to other chemical moieties, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair).

[0032] Also provided are methods for producing cis-binding Siglec agonists (e.g., any of the cis-binding Siglec agonists of the present disclosure). In certain embodiments, such methods include synthesizing a polymer scaffold comprising a membrane-tethering domain at its terminus and attaching a Siglec ligand to a subunit of the polymer scaffold. According to some embodiments, the attaching includes sialylating the subunit of the polymer scaffold. A variety of approaches suitable for synthesizing polymer scaffolds and attaching a Siglec ligand to a subunit of such a polymer scaffold are available. Non-limiting examples of such approaches include those used in the experimental section below.

[0033] composition The present disclosure also provides compositions comprising one or any combination of the cis-binding Siglec agonists of the present disclosure.

[0034] In certain embodiments, the compositions of the present disclosure comprise a cis-binding Siglec agonist of the present disclosure present in a liquid medium, which may be an aqueous liquid medium such as water, a buffer solution, or the like. One or more additives may be present in such compositions, such as salts (e.g., NaCl, MgCl, KCl, MgSO), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)sulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc.

[0035] Aspects of the present disclosure further include pharmaceutical compositions, hi some embodiments, a pharmaceutical composition of the present disclosure comprises a cis-binding Siglec agonist of the present disclosure and a pharmaceutically acceptable carrier.

[0036] The Siglec agonists of the present disclosure can be incorporated into various formulations for therapeutic administration. More specifically, the Siglec agonists can be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, and aerosols.

[0037] Formulations of Siglec agonists for administration to an individual (e.g., suitable for human administration) are generally sterile and may be free of detectable pyrogens or other contaminants that would contraindicate administration to a patient via the selected route of administration.

[0038] In pharmaceutical dosage forms, Siglec agonists may be administered in the form of their pharmaceutically acceptable salts, and may be used alone or in appropriate association with or combination with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0039] For oral preparations, the Siglec agonist may be used alone or in combination with suitable additives for producing tablets, powders, granules, or capsules, for example, conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; grinding agents such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0040] The Siglec agonist can be formulated for parenteral administration (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.). In certain embodiments, the Siglec agonist is formulated for injection by dissolving, suspending, or emulsifying the Siglec agonist in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, a synthetic fatty acid glyceride, a higher fatty acid ester, or a propylene glycol ester, and, if desired, with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0041] Pharmaceutical compositions containing Siglec agonists can be prepared by mixing a Siglec agonist having the desired purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers (such as phosphate, citric acid, and other organic acids), antioxidants (including ascorbic acid, glutathione, cysteine, methionine, and citric acid), preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof), amino acids (arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, hydroxybenzoates ... , leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof), monosaccharides, disaccharides, and other carbohydrates, low molecular weight (less than about 10 residues) polypeptides, proteins (such as gelatin or serum albumin), chelating agents (such as EDTA), sugars (such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid), and / or non-ionic surfactants (such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG)).

[0042] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, with the lyophilized preparation being reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of purified water (typically equal to the volume removed during lyophilization), although solutions containing antimicrobial agents may be used to produce pharmaceutical compositions for parenteral administration.

[0043] Aqueous formulations of Siglec agonists can be prepared in pH buffer solutions, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively at about 5.5. Examples of buffers suitable for pHs within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The concentration of the buffer can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0044] To adjust the tonicity of the formulation, an isotonicity agent may be included in the formulation. Exemplary isotonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may also be suitable. The term "isotonic" refers to a solution that has the same tonicity as another solution, such as saline or serum, to which it is being compared. The isotonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, 100 mM to 350 mM.

[0045] Surfactants can also be added to the formulation to reduce aggregation and / or minimize the formation of fine particles in the formulation and / or reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trade name Tween 20™) and polysorbate 80 (sold under the trade name Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trade name Brij™. An example of the concentration of the surfactant can be in the range of about 0.001% to about 1% (w / v).

[0046] A lyoprotectant can also be added to protect the Siglec agonist from unstable conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant can be present in an amount of about 10 mM to 500 nM.

[0047] In some embodiments, the pharmaceutical compositions comprise a Siglec agonist and one or more of the above-identified ingredients (e.g., surfactants, buffers, stabilizers, tonicity agents), and are essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at a concentration of about 0.001 to about 2% (w / v).

[0048] method The present disclosure also provides a method for using the cis-binding Siglec agonist of the present disclosure. In certain embodiments, a method for agonizing Siglec activity is provided, comprising contacting a cell expressing Siglec with any of the Siglec agonists of the present disclosure under conditions in which the membrane-tethering domain is inserted into the cell membrane and the Siglec ligand binds to one or more Siglecs expressed by the cell in cis. As an example, the method may be a method for agonizing Siglec-9 activity, in which the Siglec agonist comprises a Siglec-9 ligand. According to some embodiments, the method of the present disclosure is carried out in vitro.

[0049] In certain embodiments, the method is performed in vivo. For example, provided is a method of agonizing Siglec activity in an individual in need thereof, comprising administering to the individual an effective amount of any of the Siglec agonists disclosed herein. An "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic result (including a prophylactic result), such as a reduction in symptoms caused by immune cell (e.g., macrophage) activity compared to a control. An effective amount can be administered in one or more administrations.

[0050] In some embodiments, the individual is an individual in need of suppression of immune cell reactivity, and the Siglec ligand comprises an immunoinhibitory Siglec ligand, e.g., one or more of any of the immunoinhibitory Siglec ligands described elsewhere herein, e.g., a ligand for one or more CD33-associated Siglecs (e.g., Siglec-9), a ligand for Siglec-2, or any combination thereof.

[0051] In certain embodiments, the individual has an inflammatory disease, and the Siglec agonist is administered to the individual in an amount effective to treat the inflammatory disease. "Treating" or "treatment" refers to at least ameliorating one or more symptoms associated with the inflammatory disease in the individual, where amelioration is used broadly to refer to at least a decrease in the magnitude of a parameter (e.g., a symptom) associated with the inflammatory disease being treated. Thus, treatment also includes situations in which the inflammatory disease or at least one or more symptoms associated therewith are completely suppressed, e.g., prevented from developing, or halted, e.g., terminated, so that the individual no longer suffers from the inflammatory disease or at least the symptoms that characterize it.

[0052] Non-limiting examples of inflammatory diseases that can be treated according to the present methods include age-related macular degeneration, neutrophilic acute respiratory distress syndrome, systemic lupus erythematosus (SLE), eosinophilic gastroenteritis, allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, transplant rejection, and any combination thereof.

[0053] The pharmaceutical compositions can be administered to any of a variety of individuals. In certain aspects, the individual is a "mammal" or "mammalian," where these terms are used broadly to describe organisms within the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is a human. In certain aspects, the individual is an animal model (e.g., a mouse model, a primate model, etc.) of a condition characterized by immune cell reactivity (e.g., an inflammatory disease).

[0054] In some embodiments, an effective amount of a cis-binding Siglec agonist (e.g., present in a pharmaceutical composition comprising same) is an amount that, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is effective to reduce symptoms of a condition characterized by immune cell reactivity (e.g., an inflammatory disease) in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, 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 more, compared to the individual's symptoms in the absence of treatment with the Siglec agonist.

[0055] Dosing depends on the severity and responsiveness of the condition characterized by immune cell reactivity (e.g., inflammatory disease) being treated. Optimal dosing schedules can be calculated from measurements of Siglec agonist accumulation in the individual's body. The administering physician can determine the optimal dosage, administration method, and repetition rate. Optimal dosages may vary depending on the relative potency of individual Siglec agonists and are generally determined based on EC2A1-C1 ... 50 The dosage can be estimated based on the following: Generally, dosage is 0.01 μg to 100 g per kg of body weight and can be administered one or more times daily, one or more times weekly, one or more times monthly, or one or more times yearly. The treating physician can estimate the dosing repetition rate based on the measured residence time and concentration of the Siglec agonist in bodily fluids or tissues. After successful treatment, it may be desirable to have the individual undergo maintenance therapy to prevent recurrence of the disease state, in which case the Siglec agonist is administered at a maintenance dose ranging from 0.01 μg to 100 g per kg of body weight once or more times daily to once every several months, once every six months, once a year, or any other suitable frequency.

[0056] The therapeutic methods of the present disclosure may involve administering to an individual one Siglec agonist, or may involve administering two or more Siglec agonists, either separately or as a cocktail of different Siglec agonists.

[0057] The Siglec agonists of the present disclosure can be administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes. Conventional pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intraocular, intravenous, intraarterial, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined as desired or tailored depending on the particular Siglec agonist and / or the desired effect. The Siglec agonist may be administered in a single dose or multiple doses. In some embodiments, the Siglec agonist is administered parenterally, e.g., intravenously, intraarterially, or similarly. In some embodiments, the Siglec agonist is administered by injection, e.g., for systemic delivery (e.g., intravenous infusion), or to a local site, e.g., a site of local inflammation.

[0058] kit As summarized above, the present disclosure also provides kits. The kits find use, for example, in practicing the methods of the present disclosure. According to some embodiments, the kits include any of the pharmaceutical compositions of the present disclosure and instructions for administering an effective amount of the pharmaceutical composition to an individual in need thereof. According to some embodiments, the kits of the present disclosure include a pharmaceutical composition comprising a cis-binding Siglec agonist, including an immunosuppressive Siglec ligand. Such kits may include instructions for administering an effective amount of the pharmaceutical composition to an individual in need of suppressed immune cell reactivity. Such kits may include instructions for administering an effective amount of the pharmaceutical composition to an individual with an inflammatory disease, non-limiting examples of which include age-related macular degeneration, neutrophilic acute respiratory distress syndrome, systemic lupus erythematosus (SLE), eosinophilic gastroenteritis, allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, transplant rejection, and any combination thereof.

[0059] The kit may include a quantity of the composition in a unit dosage, e.g., an ampoule, or a multi-dose format. Thus, in certain embodiments, the kit may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of a composition comprising a Siglec agonist of the present disclosure. The term "unit dosage," as used herein, refers to a physically discrete unit suitable for a unit dosage in human or animal subjects, each containing a predetermined amount of the composition calculated to be sufficient to produce a desired effect. The amount of a unit dosage depends on various factors, such as the specific Siglec agonist used in the subject, the effect to be achieved, and the pharmacodynamics associated with the Siglec agonist. In yet other embodiments, the kit may include a single, multi-dose of the composition.

[0060] The components of the kit may be in separate containers, or multiple components may be in a single container. Suitable containers include single tubes (e.g., vials), ampoules, plates of one or more wells (e.g., 96-well plates, 384-well plates, etc.), etc.

[0061] The instructions included in the kit (e.g., instructions for use (IFU)) can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present in the kit as a package insert, on the label of the kit's container or a component thereof (i.e., associated with the packaging or sub-packaging), or the like. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g., a portable flash drive, DVD, CD-ROM, diskette, or the like. In still other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the Internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. Like the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0062] Regardless of the scope of the appended claims, the present disclosure is also defined by the following embodiments. 1. A scaffold bearing a Siglec ligand; a membrane-tethering domain; cis-binding Siglec agonists, including 2. The Siglec agonist of embodiment 1, wherein the scaffold bearing the Siglec ligand comprises a polymer scaffold. 3. The Siglec agonist of embodiment 2, wherein the scaffold bearing the Siglec ligand comprises a glycopolypeptide scaffold. 4. The Siglec agonist of any one of embodiments 1 to 3, wherein the scaffold comprises 2 to 50 Siglec ligands. 5. The Siglec agonist of embodiment 4, wherein the scaffold comprises 2 to 10 Siglec ligands. 6. The Siglec agonist of any one of embodiments 1-5, wherein the Siglec ligand comprises an immunosuppressive Siglec ligand. 7. The Siglec agonist of embodiment 6, wherein said Siglec ligand comprises a ligand for one or more CD33-associated Siglecs. 8. The Siglec agonist of embodiment 7, wherein said Siglec ligand comprises a Siglec-9 ligand. 9. The Siglec agonist of embodiment 8, wherein said Siglec ligand comprises only Siglec-9 ligands. 10. The Siglec agonist of embodiment 7, wherein said Siglec ligand comprises a Siglec-7 ligand. 11. The Siglec agonist of embodiment 10, wherein said Siglec ligand comprises only Siglec-7 ligands. 12. The Siglec agonist of any one of embodiments 1 to 5, wherein the membrane-tethering domain comprises a lipid membrane-tethering domain. 13. A composition comprising the Siglec agonist of any one of embodiments 1-12 present in a liquid medium. 14. A composition comprising the Siglec agonist of any one of embodiments 1 to 12, which is present in lyophilized form. 15. A Siglec agonist according to any one of embodiments 1 to 12, a pharmaceutically acceptable carrier; and A pharmaceutical composition comprising: 16. The pharmaceutical composition of embodiment 15, wherein the composition is formulated for parenteral administration. 17. The pharmaceutical composition of embodiment 16, wherein the composition is formulated for intravenous administration. 18. A method for agonizing Siglec activity, comprising contacting a cell expressing a Siglec with a Siglec agonist described in any one of embodiments 1 to 12 under conditions in which the membrane tethering domain is inserted into the cell membrane and the Siglec ligand binds in cis to one or more Siglecs expressed by the cell. 19. The method of embodiment 18, wherein the method is performed in vitro. 20. The method of embodiment 18, wherein the method is performed in vivo. 21. The method of any one of embodiments 18-20, wherein the method is a method of agonizing Siglec-9 activity and the Siglec agonist comprises a Siglec-9 ligand. 22. A method for agonizing Siglec activity in an individual in need thereof, comprising administering to the individual an effective amount of a Siglec agonist described in any one of embodiments 1 to 12. 23. The method of embodiment 22, wherein the individual is an individual in need of suppression of immune cell reactivity and the Siglec ligand comprises an immunoinhibitory Siglec ligand. 24. The method of embodiment 22 or 23, wherein the individual has an inflammatory disease, and the Siglec agonist is administered to the individual in an amount effective to treat the inflammatory disease. 25. The method of embodiment 24, wherein the individual has an inflammatory disease selected from the group consisting of age-related macular degeneration, neutrophilic acute respiratory distress syndrome, systemic lupus erythematosus (SLE), eosinophilic gastroenteritis, allergy, asthma, autoimmune disease, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, transplant rejection, and any combination thereof. 26. The method of embodiment 22 or 23, wherein the individual is an individual with a viral infection. 27. The method of embodiment 26, wherein the viral infection is a coronavirus infection. 28. The method of embodiment 27, wherein the coronavirus infection is a SARS-CoV-2 infection. 29. The method of any one of embodiments 22 to 28, wherein the Siglec agonist inhibits neutrophil activation in the individual. 30. The method of any one of embodiments 22-29, wherein the Siglec agonist inhibits NETosis in the individual. 31. The method of any one of embodiments 22 to 30, wherein the Siglec ligand comprises a ligand for one or more CD33-associated Siglecs. 32. The method of embodiment 31, wherein the Siglec ligand comprises a Siglec-9 ligand. 33. The method of embodiment 32, wherein the Siglec ligand comprises only Siglec-9 ligands. 34. The method of embodiment 31, wherein the Siglec ligand comprises a Siglec-7 ligand. 35. The method of embodiment 34, wherein the Siglec ligands comprise only Siglec-7 ligands. 36. A pharmaceutical composition according to any one of embodiments 15 to 17; instructions for administering an effective amount of said pharmaceutical composition to an individual in need thereof; Includes a kit. 37. The kit of embodiment 36, wherein the Siglec ligand comprises an immunosuppressive Siglec ligand. 38. The kit of embodiment 37, wherein the instructions are for administering an effective amount of the pharmaceutical composition to an individual in need of suppression of immune cell reactivity. 39. A method for producing a cis-binding Siglec agonist, comprising: synthesizing a polymer scaffold comprising a membrane-tethering domain at its terminus; attaching a Siglec ligand to a subunit of said polymer scaffold; A method comprising: 40. The method of embodiment 39, wherein said conjugating comprises sialylating a subunit of the polymer scaffold. The following examples are offered by way of illustration and not by way of limitation. [Example]

[0063] experiment Example 1 - Glycopolypeptide synthesis by N-carboxyanhydride polymerization The design of biomimetic cis-ligands for Siglecs was inspired by mucins, highly glycosylated polypeptides that are natural Siglec ligands. To construct the glycopolypeptide backbone, we used an N-carboxyanhydride (NCA) polymerization platform. NCA monomers were polymerized using a lipid-tethered initiator to yield lipid-tethered polypeptides that spontaneously insert into cell membranes. To elaborate glycopolypeptide scaffolds, we combined a previously used enzymatic method by Chen and coworkers (Angew. Chemie-Int. Ed. 2006, 45(24), 3938-3944) (Membrane-Tethered Mucin-Like Polypeptides Sterically Inhibit Binding and Slow Fusion Kinetics of Influenza A Virus. ChemRxiv 2019) with sialic acid analogs previously reported by Paulson and coworkers (Angew. Chemie-Int. Ed. 2012, 51(44), 11014-11018) to bind individual Siglec receptors with high affinity and selectivity. Here, we evaluated whether such lipid-linked sialoglycopolypeptides could insert into the plasma membrane and cluster adjacent Siglec receptors via cis-binding.

[0064] Glycopolypeptide scaffolds were synthesized by polymerization of an equimolar mixture of alanine NCA 1 and O-β-peracetyllactoselenate NCA 2 (Figure 2). Polymerization was initiated either with a Ni(0) complex to yield a soluble glycopolypeptide or by precomplexing Ni(0) with lipid-conjugated N-allylcarboxyleucine amide to form an activated Ni(II) initiator complex 3. The lipid-conjugated initiator results in a C-terminal conjugated lipid on the polypeptide. After polymerization, the carbohydrate was deprotected with hydrazine to yield the O-lactosyl glycopolypeptide pLac-sol or pLac-lipid, respectively.

[0065] Using a one-pot multienzyme system, the common pLac precursor was α-2,6-sialylated with N-acetylneuraminic acid (pSia), 9-N-propargylcarboxy-N-acetylneuraminic acid (pS7L), or N-propargylcarboxymannosamine 4 (pS9L, Figure 2) in conjunction with sodium pyruvate and neuraminic acid aldolase. After enzymatic elaboration, high-affinity Siglec ligands were synthesized by Huisgen cycloaddition using either adamantyl azide (pS7L) or benzhydryl azide (pS9L). This yielded glycopolypeptides bearing either a C-terminal lipid or solubilizing group, a free N-terminus, and a glycan bearing the terminal high-affinity Siglec ligand. Finally, the polypeptides were N-terminally labeled with commercially available biotin or AlexaFluor NHS esters (Methods).

[0066] Example 2 - pS9L-lipid inserts into the plasma membrane and binds to Siglec-9 in cis A series of N-terminally labeled sialylated glycopolypeptides were constructed from a common precursor, pLac-lipid or pLac-sol (Figure 3, panel A). The binding of the constructs to recombinant soluble Siglec-Fc fusion proteins was tested in vitro and on cell surfaces. For in vitro binding, N-terminally biotinylated lipid-free glycopolypeptides were immobilized on a streptavidin-coated chip and immersed in a Siglec-Fc fusion protein solution. Each glycopolypeptide specifically bound to its cognate Siglec receptor (Figure 3, panel B). For example, pS9L bound Siglec-9-Fc with high affinity, whereas none of the other glycopolypeptides tested bound to it (Figure 3, panel B). Similar specificity was observed when the lipid-linked versions were inserted into the cell membrane and cells were stained with recombinant Siglec-9-Fc (Figure 3, panel C). There was no substantial difference between the insertions of the various glycopolypeptides. The Siglec-9 mutation R120A, a mutant that abolishes sialic acid binding, abolished the effects observed in both in vitro and flow experiments, and staining with SNA showed no increase in binding to either structure.

[0067] To determine whether lipid-tethered glycopolypeptides associate in cis with Siglecs, Förster resonance energy transfer (FRET) was measured between the fluorophore of a lipid-linked glycopolypeptide N-terminally labeled with an AlexaFluor 555 donor fluorophore and the fluorophore of an anti-Siglec antibody conjugated to an AlexaFluor 647 acceptor (Figure 4, panel A). The donor fluorophore was excited with a 535 nm laser, and fluorescence was detected at both 555 nm and 647 nm emission wavelengths. The FRET signal was calculated as the relative efficiency (E), which is the ratio of the acceptor fluorescence intensity to the sum of the acceptor and donor fluorescence intensities. rel ) was used to quantify FRET efficiency.

[0068] Glycopolypeptide specificity was analyzed using pS9L-lipid and pS7L-lipid in Siglec-9-expressing cells (Figure 4, panels B and D), and Siglec specificity was analyzed using pS9L-lipid in Siglec-9- or Siglec-7-expressing cells (Figure 4, panels C and E). Strikingly, a dramatic increase in relative FRET efficiency was observed only when pS9L-lipid was combined with Siglec-9, but not in the mismatched pair. Furthermore, intense puncta were observed in the Siglec-9 / pS9L-lipid pair. To account for differences in antibody binding affinity or antibody / fluorophore ratio, the intensity of acceptor fluorophore emission in the FRET case compared with the single-color control was also determined. A substantial increase in acceptor emission intensity was observed between the FRET case of Siglec-9 / pS9L-lipid and the acceptor-only control, but not in the case of Siglec-7 / pS9L-lipid.

[0069] Example 3 - Cis-linked glycopolypeptides inhibit TLR4-induced NF-κB activity in Siglec-9-expressing cells To investigate the effects of membrane-tethered glycopolypeptides on inflammatory signaling, we developed a reporter system for Siglec activity based on the HEKBlue hTLR4 reporter assay. It has previously been shown that CD33-associated Siglecs regulate hTLR4 in transgenic HEK cells. In this reporter line, LPS-induced TLR4 signaling initiates NF-κB transcription of alkaline phosphatase (SEAP) secreted into the supernatant. NF-κB activity correlates with SEAP activity in a colorimetric assay. We modified this assay by transfecting these cells with a Siglec expression vector (Figure 5, panel A). We validated this assay by plating Siglec-9-expressing HEKBlue cells on anti-Siglec-9-coated plates to engage Siglec-9 signaling. A substantial decrease in activity was observed compared to vehicle-treated or isotype-coated plates (Figure 5, panel B).

[0070] HEKBlue cells were transfected with Siglec-9, coated with glycopolypeptides (1 μM), and stimulated with LPS. A decrease in relative NF-κB activity was observed with cis-linked pS9L-lipid, but not with soluble trans-linked pS9L-sol (Figure 5, panel C) or other lipid-tethered glycopolypeptides.

[0071] To test the Siglec specificity of pS9L-lipid, HEKBlue cells were transfected with Siglec-9, Siglec-7, or a mock vector, coated with pS9L-lipid (1 μM), and then stimulated with LPS. Only when cells expressed Siglec-9, pS9L-lipid inhibited NF-κB activity compared with vehicle-treated cells (Figure 5, panel D). Transfection of Siglec-9 constructs with the R120A mutation or the Y433 / 456F double mutant, which prevent tyrosine phosphorylation of the ITIM / ISIM domain, rescued NF-κB activity in response to pS9L-lipid (Figure 5, panel E).

[0072] Example 4 - Cis-binding but not trans-binding pS9L inhibits MAPK signaling in macrophages Pathologically relevant cell types that express Siglec-9 in inflammatory diseases are primarily those of the macrophage lineage. THP-1 cells are an immortalized monocyte lineage that is plastic and has been used to study macrophage biology. THP-1 monocytes were transfected with phorbol-12-myristate-13-acetate to express Siglec-9. + We differentiated them into macrophages and used them to investigate the effects and mechanism of action of pS9L-lipid. Hyposialylated THP-1 macrophages were used as a model of hyperinflammatory macrophages, comparable to hyposialylated THP-1 macrophages previously used to validate their activity, recapitulating the hyposialylated state of hyperinflammatory phagocytes. We assessed whether this model would allow us to deconvolute the confounding effects of natural cis-ligands and isolate the pS9L-lipid signaling pathway. By coating cells with glycopolypeptides and analyzing them with or without stimulation with LPS, we compared the effects of soluble trans-linked pS9L-sol and membrane-tethered cis-linked pS9L-lipid with those of vehicle-treated cells. The early signaling cascade was analyzed using quantitative phosphoproteomics, complementing this technique with cytokine quantification at later time points (Figure 6, Panel A).

[0073] Cellular cytokine production was analyzed using a multiplex cytometric bead assay of six inflammatory human cytokines. Macrophages were pretreated with glycopolypeptide (200 nM) and then stimulated with either vehicle or LPS for 18 hours. Supernatant samples were collected and assayed for cytokine content. A significant decrease in IL-1β, IL-8, and TNFα was observed when treated with cis-linked pS9L-lipid, but not with the trans-linked soluble analog pS9L-sol (Figure 6, panel B). IL-10 and IL-12p70 were below the detection limit of this assay (<20 pg / mL).

[0074] Using a similar protocol, phosphoproteome changes were analyzed from lysates after either glycopolypeptide loading or 5 min of stimulation with LPS (Figure 6, panels C–E). Minimal changes were observed in unstimulated macrophages. Dramatic changes in phosphorylation were observed when LPS-stimulated cells were pretreated with cis-linked pS9L-lipid, but not with pS9L-sol or pLac-lipid. Notably, phosphorylation correlated with decreased MAPK signaling activity. Differential phosphorylation of SH2 domain-containing proteins (e.g., SHIP2 and PTN7) was also observed. Downstream MAPK signaling was verified by analyzing total IκB protein phosphorylation by Western blot (Figure 6, panel F). pS9L-lipid was found to have both higher total IκB and lower IκB phosphorylation at the site signaling IκB degradation (S32 / 36) compared to pS9L-sol. No differential phosphorylation of phosphotyrosine on Siglec-9 was observed at any of the time points assayed.

[0075] Example 5 - Cis-ligands of Siglec-9 and Siglec-E inhibit phagocytosis by macrophages and microglia Engagement of Siglec receptors has been shown to inhibit phagocytosis. In this example, we evaluated whether pS9L-lipids could inhibit phagocytosis via Siglec-9. This was investigated by microscopy monitoring the phagocytosis of beads that turn on fluorescently at low pH (pHrodo red) (Figure 7, panels A and B).

[0076] The initial rate of phagocytosis was analyzed at multiple effector:target (E:T) ratios (Figure 7, panel C). pS9L-lipid was compared with its soluble analog (pS9L-sol), a sugar variant containing only inactive lactose (pLac-lipid), and untreated cells to analyze potential interactions of pS9L glycan binding or nonspecific effects caused by lipid insertion. After loading wild-type THP-1 macrophages with glycopolypeptides (200 nM), various amounts of target pHrodo-labeled beads were added. Phagocytosis was then monitored microscopically immediately and 1 h after target addition to determine the initial rate of phagocytosis. Phagocytosis was quantified as the area of ​​fluorescence above a background threshold, observed on five images per well with three wells per sample. In the case of pS9L-lipid, we observed a dramatic decrease in the phagocytosis rate and apparent maximum phagocytosis rate at all given E:T ratios, while both control glycopolypeptides produced results comparable to vehicle-treated cells.

[0077] To determine whether the observed effects were mediated by Siglec-9 agonism, we generated two CRISPR knockouts: one for Siglec-9 and one for CMAS, a gene required for sialic acid biosynthesis. Knocking out CMAS resulted in sialic acid-deficient macrophages, which we assessed to determine whether it mediated the above-mentioned effects. pS9L-lipids potently inhibited CMAS-knockout macrophages (Figure 7, panel D), and knocking out Siglec-9 abolished the effects of pS9L-lipids (Figure 7, panel E).

[0078] Next, we tested a small number of glycovariants based on the same scaffold as pS9L-lipid (Figure 7, panel F). We observed that only pS9L-lipid was able to significantly inhibit phagocytosis. A trend toward inhibition by Siglec-7-conjugated pS7L lipid was observed, but this was not statistically significant. THP-1 macrophages express low levels of Siglec-7. We also assayed a panel of soluble trans-linked glycopolypeptides with the same glycan and similar molecular weight, but no effect on phagocytosis was observed. We determined that inhibition by pS9L-lipid was dose-dependent on glycopolypeptide pretreatment and could also be observed with alternative targets, including zymosan fungal particles.

[0079] To assess the generality of the inhibition of Siglec clustering by cis-ligands, we further investigated the mild inhibition by pS7L-lipid. While S7L sialoside has some affinity for Siglec-7, it is the most potent ligand for Siglec-E, the mouse ortholog of Siglec-7 / 9, as discovered by Paulson and colleagues. Therefore, we assessed the potential inhibitory effect on Siglec-E-expressing cells. Indeed, we observed a tendency toward inhibition in mouse microglia pretreated with pS7L-lipid (Figure 7, Panel G), which was abolished by CRISPR knockout of Siglec-E (Figure 7, Panel H). Compared to the control polymer, a more potent and statistically significant effect was observed.

[0080] To demonstrate the clinical relevance of these findings, we performed a similar assay using human primary macrophages. Monocytes were isolated from PBMCs from healthy donors and differentiated into resting (M0), M1, or M2 macrophages. When pretreated with glycopeptides, we observed that the phagocytic activity of M0 and M1 macrophages, but not M2 macrophages, from five of six donors was inhibited by treatment with pS9L-lipid, but not by treatment with the control polymers pS9L-sol or pLac-lipid (Figure 8, panels A–C). Follow-up of one non-responder donor determined that macrophages from this donor had dramatically lower levels of Siglec-9 expression (Figure 8, panel D).

[0081] Methods for Examples 1-5 statistical analysis All statistical analyses were performed using GraphPad Prism 6.

[0082] Glycopolypeptide synthesis Glycopolypeptides were synthesized as previously described (Delaveris et al. (2019) Membrane-Tethered Mucin-Like Polypeptides Sterically Inhibit Binding and Slow Fusion Kinetics of Influenza A Virus. ChemRxiv). Briefly, alanine and the N-carboxyanhydride of O-lactosylserine were polymerized using a pre-complexed initiator to yield lipid-linked or soluble protected glycopolypeptides. Glycans were deacetylated using hydrazine and purified by dialysis. A one-pot multienzyme system was then used to elaborate polylactosyl scaffolds to yield various sialosides on the glycopolypeptide scaffold. Unnatural sialosides bearing alkyne handles were then reacted with azides to provide polymeric display of the aforementioned high-affinity Siglec ligands.

[0083] human cell culture Cell lines were cultured in either DMEM (HEKBlue hTLR4, BV2) or RPMI (JURKAT, THP-1) supplemented with 10% heat-inactivated FBS. THP-1 cells were further supplemented with 50 μM beta-mercaptoethanol. THP-1 monocytes were differentiated into macrophages by activation with PMA for 24 hours followed by recovery in standard medium for 24 hours. PBMCs were isolated from buffy coats or LRS chambers from whole blood using Ficoll-Paque gradient centrifugation. Monocytes were isolated by adherence to tissue culture plastic and differentiated into macrophages for 7 days in RPMI-1640 medium containing 20% ​​heat-inactivated FBS either without exogenous cytokines (M0), with GM-CSF (immature M1), with GM-CSF for 5 days followed by LPS and IFN-γ in 10% heat-inactivated FBS for 2 days (activated M1), or with M-CSF for 5 days followed by IL-4 and IL-13 in 10% heat-inactivated FBS for 2 days (M2).

[0084] In vitro protein binding Biotinylated ligand (200 nM) in PBS with 0.1% BSA was loaded onto a streptavidin-coated chip, and protein binding was recorded for 60 seconds with OctetRed96 (response of approximately 0.4 nM). The chip was then immersed in serial dilutions of Siglec-Fc for 30 seconds to allow association, followed by 30 seconds of dissociation in buffer. The chip was regenerated between washes with glycine buffer (pH 1.5).

[0085] Flow cytometry Cells were harvested and incubated for 10 min in serum-free medium with gentle agitation every 15 min. 7 Fluorophore-conjugated glycopolypeptides were loaded at a density of 1000 cells / mL for 1 hour. Cells were then washed and stained with either a fluorophore-conjugated primary antibody or an unconjugated primary antibody with a fluorophore-conjugated anti-IgG secondary antibody at 4°C, followed by three post-staining washes. All flow analyses were performed on unfixed cells.

[0086] Fluorescence microscopy FRET data were collected by confocal microscopy. JURKAT cells expressing Siglec-7 or Siglec-9 were cultured in serum-free RPMI for 10 min. 7 Cells were suspended at 1000 cells / mL and labeled with AlexaFluor 555-labeled glycopolypeptides (2 μM) for 1 hour with gentle agitation every 15 minutes. Cells were washed and then labeled with AlexaFluor 647-labeled anti-Siglec-7 or anti-Siglec-9 antibodies in complete medium for 30 minutes at room temperature. Cells were washed with PBS and then plated onto 8-well borosilicate glass #1.5 coverslips pre-coated with fibronectin for live cell imaging. Cells were then imaged. For Siglec-9 immunocytochemistry, cells were fixed with 10% formalin, washed, and stained with AlexaFluor 488-conjugated anti-Siglec-9 on ice for 1 hour. Cells were then washed and imaged using an Incucyte microscope at 488 nm fluorescence, with five images collected per well in triplicate wells per condition.

[0087] Cloning The PmNanA expression plasmid was constructed by InFusion cloning using IDT gBlock ligation into PCR-linearized pET22b vector. CRISPR plasmids were constructed using the optimization guide, cloned into the LentiCRISPR v2 plasmid using the Gecko protocol, and purified by MiraPrep. pCMV Siglec-9 mutants were generated using the Q5 mutagenesis kit.

[0088] Protein expression and purification PmNanA, Pd26ST, and NmCSS were expressed in BL21(DE3) E. coli and isolated.

[0089] HEKBlue hTLR4 reporter assay The HEKBlue hTLR4 assay was generally performed according to the manufacturer's instructions. Cells were transfected 24 hours prior to the assay using Lipofectamine LTX. For antibody-coated plate assays, 96-well plates were prepared by incubating with antibody solution (10 ng / mL) in PBS for 2 hours at 37°C, followed by three washes with PBS before plating the transfected cells. For glycopolypeptide assays, cells were harvested from the transfection plate, pelleted by centrifugation (300 rcf, 5 minutes), and resuspended in a solution of glycopolypeptide (1 μM) in serum-free DMEM. Cells were mixed every 15 minutes for 1 hour, at which point they were washed with complete medium (1 mL), counted, and plated.

[0090] Cytokine bead assay CMAS KO THP-1 macrophages were cultured and labeled with glycopolypeptide (200 nM) for 3 hours. At this time, medium and either vehicle or LPS (100 pg / mL) were added, and the cells were cultured for 18 hours. Aliquots of medium were then taken and flash-frozen at -80°C. Quantification with BD human inflammatory cytokine beads was then performed on thawed samples from three biological replicates in one batch, according to the manufacturer's instructions.

[0091] Phosphoproteomics CMAS KO THP-1 macrophages were cultured and labeled with glycopolypeptides (500 nM) in serum-free medium for 3 hours. At this time, medium and either vehicle or LPS (100 pg / mL) were added to stimulate the cells for 5 minutes. Cells were then lysed in cold RIPA buffer containing benzonase, pelleted by centrifugation (18,000 rcf, 15 minutes, 4°C), and supernatant protein concentrations were quantified using a Rapid Gold BCA.

[0092] Proteins were digested into tryptic peptides using the S-trap protocol (Protifi) and then labeled with 10-plex TMT (Tandem Mass Tags, Thermo Fisher Scientific). Phosphopeptides were enriched with Ti(IV)-IMAC beads (ReSyn Biosciences). Phosphopeptide and protein abundance samples were analyzed by LC-MS / MS using a Dionex Ultimate 3000 RPLC nanosystem connected to an Orbitrap Fusion (Thermo Fisher Scientific). Peptides were loaded onto a trap column (Acclaim PepMap 100 C18, 5 μm particles, 20 mm length, Thermo Fisher Scientific) and separated on a 25 cm EasySpray reversed-phase LC column (75 μm i.d., 2 μm, 100 Å, packed with PepMap C18 particles, Thermo Fisher Scientific) using water with 0.2% formic acid (mobile phase A) and acetonitrile with 0.2% formic acid (mobile phase B). Total acquisition time for all methods was 180 min per run. Raw files were searched using the Andromeda algorithm and processed with MaxQuant. Results were then processed with Perseus to calculate statistically significant changes in the phosphoproteome. Data were deposited with the ProteomeXchange Consortium via the PRIDE partner repository under dataset identifier PXD018774.

[0093] Western blot CMAS KO THP-1 macrophages were cultured and labeled with glycopolypeptide (500 nM) in serum-free medium for 3 hours. At this time, cells were stimulated for 60 minutes by adding medium and either vehicle or LPS (100 pg / mL). Cells were then lysed in cold RIPA buffer containing benzonase and pelleted by centrifugation (18,000 rcf, 15 minutes, 4°C). Supernatant protein concentrations were quantified by BCA. Lysates were then run on SDS-PAGE using 4-12% bis-acrylamide gels in XT-MES at 200V for 1 hour. Gels were transferred to nitrocellulose using a TransBlot Turbo using standard TurboBlot conditions. Blots were blocked with 5% BSA in TBS and stained with primary antibodies overnight at 4°C, followed by incubation with IR dye-conjugated secondary antibodies for 1 hour at room temperature. Blots were imaged using LiCOR.

[0094] Phagocytosis assay Phagocytes were treated with glycopolypeptides in serum-free medium for 3 hours. Cells were washed and coated with 100 μL of serum-free medium. Targets were then added as a suspension in 100 μL of serum-free medium. Plates were briefly centrifuged (300 rcf, 1 minute) to sediment the targets, after which phagocytosis was monitored by fluorescence microscopy in an Incucyte. Five images per well were collected in three wells per condition. For BV2 phagocytosis, BV2 cells were pretreated with recombinant endotoxin-free V. cholera sialidase at 2 μM for 1 hour before treatment with glycopolypeptides.

[0095] Example 6 - Cis-binding Siglec agonists inhibit neutrophil activation The TLR-7 / 8 agonist R848 induces NETosis in primary neutrophils in vitro Neutrophils are myeloid-lineage immune cells involved in numerous innate immune functions. Neutrophils have been suggested to induce the hyperinflammatory response in COVID-19 through a death process called NETosis, in which neutrophils rapidly decondense chromatin and expel neutrophil extracellular traps (NETs), an amalgam of genomic DNA, intracellular proteins (e.g., histones), and tissue-damaging enzymes (e.g., neutrophil elastase, myeloperoxidase). It has been proposed that tissue damage from extracellular DNA and NET-associated enzymes acts as a proinflammatory signal to other immune cells, initiating a hyperinflammatory cascade in COVID-19 that leads to ARDS and potentially death. Consistent with this hypothesis, NETs have been widely observed both at the site of infection (i.e., lung tissue) and in the periphery (i.e., serum and plasma).

[0096] In COVID-19, evidence of widespread NETosis can be observed in infected lungs, and SARS-CoV-2 virions have been shown to infect and induce NETosis in healthy neutrophils in vitro. These reports implicate TLR-7 and / or TLR-8 in the induction of NETosis in neutrophils at the site of infection. Notably, TLR-7 and TLR-8 are single-stranded RNA receptors for which numerous substrates have been identified in the SARS-CoV-2 genome. Consistent with the hypothesis that SARS-CoV-2 induces TLR-7 / 8-mediated immunity, human genetic variation in TLR7 has been associated with severe COVID-19. Therefore, TLR-7 / 8 agonists may provide a convenient means to model local inflammation induced by viral infection in vitro without using live virus.

[0097] TLR agonists were assayed using a live-cell imaging technique in which freshly isolated neutrophils were cultured in low-serum medium in the presence of a fluorogenic, membrane-impermeable, DNA-intercalating dye (Cytotox Green). Externalization of genomic DNA by NETosis causes the dye to intercalate, resulting in increased fluorescence. As previously demonstrated, NETs are much larger than the nuclei of apoptotic cells, and therefore, when observed microscopically, NETotic cells produce a much larger fluorescent area than apoptotic cells. Therefore, the larger (i.e., 100 μm) 2 Apoptotic cells can be excluded by counting only fluorescent objects (super-fluorescent).

[0098] In this study, we found that the TLR-7 / 8 agonist R848 was sufficient to induce NETosis in healthy neutrophils in vitro (Figure 10a-c). The citrullination status of the PADI4 substrate H3 was also assayed by Western blot, and we observed that R848 rapidly induced citrullination at R2, R8, and R17. While citrullination is an important aspect of NETosis, the degree of citrullination does not necessarily indicate the degree of NETosis, as, for example, PMA-induced NETosis only results in modest citrullination (data not shown). In addition, we also performed quantitative phosphoproteomics using lysates from neutrophils treated with medium, phorbol-12-myristate-13-acetate (PMA), or R848. Similar results to previously published datasets were observed using neutrophils stimulated with either R848 or PMA. Furthermore, several phosphosites, including those involved in neutrophil degranulation and calcium flux, were found to be differentially regulated in both datasets, consistent with the described mechanism of NETotic cell death. These results demonstrate that the TLR-7 / 8 agonist R848 induces NETosis in primary neutrophils. Therefore, this compound can be used to model local inflammation associated with viral infections, including COVID-19.

[0099] Siglec-9 agonists inhibit TLR-7 / 8-induced NETosis via SHP-1 Previous studies have shown that engagement of Siglec-9 leads to apoptotic and non-apoptotic death pathways and immunosuppression of neutrophils. Therefore, we hypothesized that Siglec-9-mediated immunosuppression and cell death could override the NETotic effects of antiviral TLR signaling. To test this concept, we used the Siglec-9 agonist pS9L and two control glycopolypeptides, pLac and pS9L-sol (Figure 9). We assayed anti-NETotic activity by co-treatment of glycopolypeptides (500 nM) and R848 (10 μM) in primary neutrophils in the live cell assay described above (Figure 10). We observed that pS9L was sufficient to inhibit NETosis induced by R848 treatment (Figure 10a-c). Furthermore, neither control polymer inhibited R848-induced NETosis (Figure 10d). We also confirmed that pS9L inhibited NETosis equivalently to a high concentration of cross-linked anti-Siglec-9 antibody (clone 191240). Mitochondrial-derived reactive oxygen species (ROS) generation has previously been described as a critical signaling step in Siglec-9-induced apoptotic signaling. In the absence of any TLR ligands to avoid NADPH-derived ROS in inflammatory signaling, we found that treatment with pS9L induced oxidative burst similar to treatment with cross-linked anti-Siglec-9 antibody. Furthermore, this oxidative burst was inhibited by the addition of the SHP-1 / 2 inhibitor NSC-87877, suggesting that SHP-1 and / or SHP-2 mediate pS9L-induced oxidative burst in neutrophils, consistent with Siglec-9 engagement.

[0100] Quantitative phosphoproteomics was performed using lysates from R848-stimulated primary neutrophils co-treated with vehicle, pS9L, or pLac. Notably, increased phosphorylation of hycin (HYCCI / FAM126A), a key component in the phosphorylation of phosphoinositides, a class of signaling molecules involved in mediating NETosis, was observed. Additionally, increased phosphorylation of RASAL3 (RASL3), a negative regulator of the MAPK signaling pathway, was observed. These data suggest that pS9L inhibits calcium flux and NADPH activity required for NETosis, as well as the MAPK inhibitory effect previously described for pS9L in macrophages.

[0101] To determine whether the anti-NETotic effect of pS9L is specifically mediated by Siglec-9 signaling, we replicated these results in the promyelocytic leukemia cell line HL-60. These cells can be differentiated into neutrophil-like cells (dHL-60) using all-trans retinoic acid (ATRA, 100 nM) and dimethyl sulfoxide (DMSO, 1.25% (v / v)). Notably, dHL-60 cells have previously been used to study NETosis in vitro. Consistent with previous reports, R848 induced NETosis in dHL-60 cells. We further observed that pS9L inhibited NETosis, and siRNA knockdown of Siglec-9 (encoded by SIGLEC9) or SHP-1 (encoded by PTPN6) abolished the effect of pS9L (Figure 10e). Thus, the Siglec-9 agonist pS9L inhibits TLR7 / 8-induced NETosis via Siglec-9 and SHP-1.

[0102] Siglec-9 is upregulated in severe COVID-19 and can suppress NETosis induced by COVID-19 plasma Serum and plasma from COVID-19 patients have been shown to induce NETosis in vitro in neutrophils isolated from healthy donors. While the causative components are unknown, potential factors include viral TLR ligands, damage-associated molecular patterns that bind to TLRs, activated platelets, and pro-inflammatory cytokines. Recent reports have documented increased levels of neutrophil-activating cytokines, primarily IL-8 and G-CSF, in COVID-19 plasma. This study also observed that the combination of IL-8 and G-CSF was sufficient to induce NETosis in vitro. Additionally, transcriptome analysis of peripheral bone marrow cells and neutrophils from COVID-19 patients revealed increased SIGLEC9 (Figure 11a) and PADI4 (Figure 11b) expression. This led to the hypothesis that Siglec-9 expression on over-expressed NETotic neutrophils represents an exhaustion-like phenotype, similar to that observed for Siglec-9 on exhausted tumor-infiltrating T cells. These observations further support Siglec-9 as an attractive target for therapeutic blockade of hyperinflammatory NETosis in general and in COVID-19 in particular.

[0103] To test the hypothesis that pS9L can inhibit NETosis induced by COVID-19 plasma, neutrophils isolated from whole blood of healthy donors were treated with citrate-anticoagulated heterologous plasma from healthy donors or COVID-19 patients. Neutrophils in undiluted plasma were co-treated with pS9L (500 nM), the non-binding analog pLac (500 nM), or vehicle. To comply with biosafety regulations, cells were incubated in the presence of COVID-19 plasma for 4 hours and, after fixation, assayed for extracellular complexes of myeloperoxidase (MPO) and DNA (DAPI) (Figures 11c and 11d). These staining combinations, which indicate NETosis when observed extracellularly, suggest the presence of NETs in the context of COVID-19. +It has previously been used to identify cells. In this study, we observed that COVID-19 plasma induced NETosis in neutrophils from healthy donors, as indicated by the formation of web-like NET structures (Figure 11d). Similar to previous experiments using R848, NETosis stimulated with COVID-19 plasma was inhibited by pS9L treatment (Figures 11c and 11d). Furthermore, pLac did not inhibit NETosis induced by COVID-19 plasma, and neither pS9L nor pLac affected basal NETosis in in vitro-cultured neutrophils (Figure 11c). Similar experiments were performed by staining neutrophils treated with 10% plasma or undiluted plasma in IMDM for extracellular H1 / DNA complexes, another marker of NETs, ​​and comparable results were observed.

[0104] Collectively, these data suggest that Siglec-9 agonism can inhibit NETosis induced by COVID-19 patient plasma and thus suppress peripheral inflammation in patients with COVID-19. Additionally, Siglec-9 agonists may resolve NET-associated pathologies in general, including those observed in COVID-19 and other conditions such as immune thrombosis and sepsis.

[0105] Accordingly, the foregoing merely illustrates the principles of the present disclosure. It should be understood that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the art, and should not be construed as limiting to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. a glycopolypeptide scaffold bearing a Siglec ligand; A cis-binding Siglec agonist comprising a membrane-tethering domain.

2. The Siglec agonist of claim 1, wherein the scaffold comprises 2 to 50 Siglec ligands.

3. The Siglec agonist of claim 2, wherein the scaffold comprises 2 to 10 Siglec ligands.

4. The Siglec agonist according to any one of claims 1 to 3, wherein the Siglec ligand comprises an immunosuppressive Siglec ligand.

5. The Siglec agonist of claim 4 , wherein the Siglec ligand comprises a ligand for one or more CD33-associated Siglecs.

6. The Siglec agonist of claim 5, wherein the Siglec ligand comprises a Siglec-9 ligand.

7. The Siglec agonist of claim 5, wherein the Siglec ligand comprises a Siglec-7 ligand.

8. The Siglec agonist of any one of claims 1 to 7, wherein the membrane-tethering domain comprises a lipid membrane-tethering domain.

9. A Siglec agonist according to any one of claims 1 to 8; and a pharmaceutically acceptable carrier.

10. A composition for use in a method for agonizing Siglec activity in an individual in need thereof, the composition comprising a Siglec agonist described in any one of claims 1 to 8, the method comprising administering to the individual an effective amount of the Siglec agonist.

11. The composition of claim 10 , wherein the individual is an individual in need of suppression of immune cell reactivity, and the Siglec ligand comprises an immunosuppressive Siglec ligand.

12. The composition of claim 10 or 11, wherein the individual has an inflammatory disease, and the Siglec agonist is administered to the individual in an amount effective to treat the inflammatory disease.

13. 13. The composition of claim 12, wherein the individual has an inflammatory disease selected from the group consisting of age-related macular degeneration, neutrophilic acute respiratory distress syndrome, systemic lupus erythematosus (SLE), eosinophilic gastroenteritis, allergy, asthma, autoimmune disease, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury, transplant rejection, and any combination thereof.

14. The composition of claim 10 or 11, wherein the individual has a viral infection, and the viral infection may be a coronavirus infection.

15. The composition of any one of claims 10 to 14, wherein the Siglec agonist inhibits neutrophil activation in the individual.

16. The composition of any one of claims 10 to 15, wherein the Siglec agonist inhibits NETosis in the individual.

17. The pharmaceutical composition of claim 9 ; and instructions for administering an effective amount of the pharmaceutical composition to an individual in need thereof.

18. 1. A method for making a cis-binding Siglec agonist, comprising: synthesizing a glycopolypeptide scaffold comprising a membrane-tethering domain at its terminus; and attaching a Siglec ligand to a subunit of said glycopolypeptide scaffold.

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