Multivalent glycan scaffolds and methods of use thereof

Abstract

Multivalent glycan scaffolds and methods for making same, as well as compositions and methods for using same are provided. Multivalent glycan scaffolds typically include a base branched scaffold, such as a dendrimer, having two or more glycans attached to its termini and, optionally, one or more functional elements. The glycans may be synthetic glycans or may be isolated from natural sources. Methods for identifying binding partners, such as cells and / or glycan-binding proteins, using glycan scaffolds are also provided. The method for identifying glycan-based binding interactions may include contacting a target substance with a known multivalent glycan scaffold and identifying an element from the target substance that binds to the scaffold. In other embodiments, the method for identifying glycan-based binding interactions includes contacting a known target substance element with a library of different species of multivalent glycan scaffolds, each having two or more glycans, and identifying a glycan scaffold that binds to the target substance element.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 237,068, filed August 25, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference The sequence listing submitted as an xml file named "NOVAB100PCTST25.xml", created on August 22, 2022, and having a size of 6,353 bytes, is incorporated herein by reference in accordance with 37 CFR § 1.834(c)(1).

[0003] FIELD OF THEINVENTION The present invention is generally in the field of glycan binding, and compositions and methods of use related to glycan binding. [Background technology]

[0004] 2. Background of the Invention Mainly attached to lipids or proteins, glycans are abundantly expressed on the surface of all cells. Due to their enormous structural and compositional diversity, glycans play a major role in numerous biological processes among all organisms, from viruses and bacteria to plants and animals. Proteins that bind to glycans are known as glycan-binding proteins (GBPs) and are typically expressed on the surface of cells to mediate various cell signaling. Thus, the CIS or TRANS binding interaction of a cell surface glycan ligand with its corresponding cell surface GBP is a multivalent interaction, because many copies of each distinct glycan structure and each distinct GBP are present on the cell surface, i.e. hundreds to tens of thousands of identical copies. Such multivalent interactions result in a substantial increase in apparent binding affinity (avidity) due to "forced proximity" (Vauquelin, et al., Br J Pharmacol., 168(8):1771-85, doi:10.1111 / bph.12106(2013)) (the high local "target" concentration experienced by unbound glycan ligands or GBPs when one (or more) glycan ligands are bound to a complementary GBP). Forced proximity significantly increases the binding / rebinding of glycan:GBP complexes, substantially slowing dissociation of the interaction, because all glycan:GBP complexes must dissociate simultaneously. This multivalent avidity enhancement increases apparent affinity by several thousand-fold compared to monovalent binding. This explains the difficulty of inhibiting glycan:GBP-mediated cell-cell interactions despite the poor monovalent affinity typical of glycan:GBP binding.

[0005] To discover and characterize glycan-binding molecules, including lectins, antibodies, and other carbohydrate-binding biomolecules, appropriate glycan epitope bait counterparts are required. Monovalent interactions between glycans and glycan-binding molecules are usually weak, with low affinity and low K dSince the binding affinity of GBPs is in the millimolar to micromolar range, multivalent display of glycans is important to increase the avidity and apparent affinity to enable capture of GBP-glycan complexes or to effectively compete with multivalent GBP-glycan interactions.

[0006] The commercial availability of glycans displayed in multivalent formats is very limited: they are usually coupled to polymers (e.g., polyacrylic acid (PAA)), which display only common glycan epitopes (blood groups, Lewis structures) that are relatively easy to synthesize or purify from natural sources, and are mostly restricted to structures consisting of 1-4 monosaccharide units.

[0007] Multivalent display of glycans has been explored using a wide range of scaffolding constructs, including gold nanoparticles, fullerene multivalent scaffolds, carbon nanotubes, glycodendritic nanoparticles using self-assembling peptides, glycan oligonucleotides, peptide / protein scaffolds, and glycan scaffolds (Spinelli, et al., Chem Soc Rev., 42(11):4557-73, doi: 10.1039 / c2cs35406c (2013);Reichardt, et al., Chem Soc Rev., 42(10):4358-76, doi: 10.1039 / c2cs35427f (2013);Bernardi, et al., Chem Soc Rev., 42(11):4709-27, doi: 10.1039 / c2cs35408j (2013);Muller, et al., Chem Soc Rev., 45(11):3275-302, doi: 10.1039 / c6cs00165c (2016). Despite employing different synthetic strategies, these materials only partially address the problem.Most multivalent glycan constructs reported in the literature display only mono- or disaccharides (Al-Mughaid,et al., J Org Chem., 71(4):1390-8, doi: 10.1021 / jo052045u (2006);Chabre,et al., J Org Chem., 73(14):5602-5, doi: 10.1021 / jo8008935 (2008);Martinez-Avila,et al., Chemistry, 15(38):9874-88, doi: 10.1002 / chem.200900923 (2009);Ribeiro-Viana,et al., Nat Commun., 3:1303, doi: 10.1038 / ncomms2302 (2012);Muñoz, et al., Nat Chem., 8(1):50-7, doi: 10.1038 / nchem.2387 (2016);Budhadev et al., J Am Chem Soc., 142(42):18022-18034, doi: 10.1021 / jacs.0c06793 (2020);Ren, et al., Talanta., 223(Pt 1):121706, doi: 10.1016 / j.talanta.2020.121706 (2021)). Several other "glycoclusters" constructs use derivatives of biantennary N-glycans found in sialoglycopeptides (SGPs) from chicken egg yolk (Tanaka, et al., Angew Chem Int Ed Engl., 49(44): 8195-8200, doi:10.1002 / anie.201000892 (2010); Smirnov, et al., Small.,16(46):e2004831, doi: 10.1002 / smll.202004831(2020)). However, none of these strategies provide a simple and universal method suitable for most glycans and designed to construct multivalent glycan polymers, and larger structures, including complete O-linked and N-linked glycans, are either not available as multivalent polymers or simply not available in any format. It is therefore an object of the present invention to provide alternative and improved means of presenting glycans in a multivalent format. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Vauquelin,et al.,Br J Pharmacol.,168(8):1771-85,doi:10.1111 / bph.12106(2013) [Non-Patent Document 2] Spinelli, et al., Chem Soc Rev., 42(11):4557-73, doi: 10.1039 / c2cs35406c (2013) [Non-Patent Document 3] Reichardt, et al., Chem Soc Rev., 42(10):4358-76, doi: 10.1039 / c2cs35427f (2013) [Non-Patent Document 4] Bernardi, et al., Chem Soc Rev., 42(11):4709-27, doi: 10.1039 / c2cs35408j (2013) [Non-Patent Document 5] Muller, et al., Chem Soc Rev., 45(11):3275-302, doi: 10.1039 / c6cs00165c (2016) [Non-Patent Document 6] Al-Mughaid, et al., J Org Chem., 71(4):1390-8, doi: 10.1021 / jo052045u (2006) [Non-Patent Document 7] Chabre, et al., J Org Chem., 73(14):5602-5, doi: 10.1021 / jo8008935 (2008) [Non-Patent Document 8] Martinez-Avila, et al., Chemistry, 15(38):9874-88, doi: 10.1002 / chem.200900923 (2009) [Non-Patent Document 9] Ribeiro-Viana, et al., Nat Commun., 3:1303, doi: 10.1038 / ncomms2302 (2012) [Non-Patent Document 10] Munoz, et al., Nat Chem., 8(1):50-7, doi: 10.1038 / nchem.2387 (2016) [Non-Patent Document 11] Budhadev et al., J Am Chem Soc., 142(42):18022-18034, doi: 10.1021 / jacs.0c06793 (2020) [Non-Patent Document 12] Ren, et al., Talanta., 223(Pt 1):121706, doi: 10.1016 / j.talanta.2020.121706 (2021) [Non-Patent Document 13] Tanaka, et al., Angew Chem Int Ed Engl., 49(44): 8195-8200, doi:10.1002 / anie.201000892 (2010) [Non-Patent Document 14] Smirnov, et al., Small.,16(46):e2004831, doi: 10.1002 / smll.202004831(2020) Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention Provided are multivalent glycan scaffolds and methods for making them, as well as compositions and methods for using them. Multivalent glycan scaffolds typically include an underlying branched scaffold with two or more glycans linked to its termini and optionally one or more functional elements. The underlying branched scaffold can be, for example, a dendrimer or another branched polymer. The glycans can be synthetic glycans, but are more preferably isolated from natural sources. Each scaffold is typically functionalized with 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycans, which can be a single glycan type (i.e. homogeneous) or a mixture of two or more different glycan types (i.e. heterogeneous). The functional element can be, for example, an agent that facilitates the formation, imaging, localization, identification, isolation, or characterization of the glycan scaffold or its binding partner. Exemplary functional elements include, but are not limited to, nucleic acids with unique barcode sequences that specifically identify the glycans attached to the scaffold, e.g., fluorophores for facilitating scaffold formation, detection, imaging, localization, or sorting, and affinity tags that can be used to isolate or immobilize the scaffold. In an exemplary non-limiting embodiment, the glycan is linked to 2-amino-N-(2-aminoethyl)-benzamide (AEAB), which is directly or indirectly linked to the scaffold. In some embodiments, the glycan is linked to the scaffold directly or indirectly through an amino acid, which may be alone or form part of a polypeptide chain. Thus, in some embodiments, the glycan scaffold is a glycopeptide scaffold. In some embodiments, a single polypeptide chain links and / or displays two or more glycans, which may be the same or different. Exemplary amino acids include, but are not limited to, serine and threonine.

[0010] Also provided are methods of identifying binding partners, such as cells and / or glycan-binding proteins (GBPs), using glycan scaffolds. In some embodiments, the method of identifying glycan-based binding interactions includes contacting a target material with a known multivalent glycan scaffold and identifying an entity from the target material that binds to the scaffold. The target material can be, for example, a population of cells having one, two or more different cell types with one, two or more GBPs, or a cell lysate or other proteinaceous mixture. The method can also be used to determine whether a particular cell type expresses a GBP that binds to a particular glycan. When the target material is a population of cells, the target material entity is typically one or more cell types in the population. Preferably, the one or more cell types preferentially bind to the multivalent glycan scaffold compared to other cell types in the population. Further steps can include isolating the multivalent scaffold to which the cells are bound and / or characterizing the bound cells. When the target material is a cell lysate or other proteinaceous mixture, the target material component is typically one or more GBPs therein.Preferably, the one or more GBPs preferentially bind to the multivalent glycan scaffold compared to other GBPs in the cell lysate or other proteinaceous mixture.Further steps may include isolating and / or characterizing the bound GBP.

[0011] In another embodiment, a method for identifying glycan-based binding interactions includes contacting a known target material element with a library of different species of multivalent glycan scaffolds, each having two or more glycans, and identifying glycan scaffolds that bind to the target material element. The library may include, for example, one or more copies of about 2 to about 10,000 or more, or any subrange or specific integer therein, e.g., 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000 different species of glycan scaffolds. The known target material element may be a cell. Preferably, one or more multivalent glycan scaffolds preferentially bind to the cell compared to other multivalent glycan scaffolds in the library. In this manner, the compositions and methods can be used to identify the glycan specificity of cellular GBPs. In another embodiment, the known target material element is a GBP. Preferably, one or more multivalent glycan scaffolds preferentially bind to the GBP compared to other multivalent glycan scaffolds in the library. Further steps may include isolating and / or characterizing the multivalent glycan scaffold.

[0012] Glycan binding interactions identified according to the provided methods can be used to rationally design multivalent glycan scaffolds for therapeutic and diagnostic applications. For example, a method of treating a subject in need of treatment can include identifying glycan-based binding interactions according to any of the disclosed methods and administering to the subject a composition comprising an effective amount of a therapeutic multivalent glycan scaffold having the identified interacting glycans. Preferably, the scaffold preferentially binds to target cells and / or GBPs in the subject. In some embodiments, the therapeutic glycan scaffold induces or inhibits cellular activity upon binding to an interacting target cell or GBP. In some embodiments, the therapeutic glycan scaffold includes an active agent that functionalizes it. The active agent can be a nucleic acid, a small molecule, a polypeptide, etc. For example, in some embodiments, the target cell is a cancer cell and the active agent is an anti-cancer drug. In other embodiments, the target cell is an immune cell and the active agent is an antigen or encodes an antigen. Similarly, glycan scaffolds functionalized with imaging agents can be used to identify, track, or monitor target cells or GBPs and can be utilized in diagnostic applications.

[0013] Also provided is a composition comprising a glycan scaffold. In some embodiments, the composition comprises a glycan scaffold bound to a cell or GBP. The glycan scaffold, with or without its binding partner, can be immobilized on a surface or substrate, such as a bead, slide, plate, or dish, as required, or can be free in a solution, such as a medium or pharmaceutical composition. [Brief description of the drawings]

[0014] [Figure 1A] FIG. 1A is a flow diagram illustrating how an exemplary glycan containing a reducing end can be obtained commercially (e.g., H-type 2-trisaccharide (Fucα1-3Galβ1-4GlcNAc)), released and purified from natural sources (e.g., by PNGaseF) or by non-reducing alkaline β-elimination. [Figure 1B]FIG. 1B is an illustration of the components of an exemplary multimeric ligand of the disclosed technology. The scaffold determines the ligand valency and size of the structure. The detection tag facilitates detection and capture of the structure. An optional barcode can provide a unique DNA or RNA sequence associated with each distinct ligand present in the library of multimeric ligand structures. [Figure 1C] FIG. 1C is a flow diagram of a library of multimeric ligand particles (MLPs), each displaying a distinct ligand and a DNA or RNA sequence identifier that identifies the ligand to target specific cells and / or tissues.

[0015] [Figure 2A] Figure 2A is a flow diagram illustrating the production of the H3 glycan dendrimer of Example 1. Figure 2A illustrates the azidation of the H3 reducing glycan. [Figure 2B] Figure 2B is a flow diagram illustrating the production of the H3 glycan dendrimer of Example 1. Figure 2B illustrates the click chemistry reaction coupling H3-N3 to an 8-mer PEG biotin / alkyne dendrimer. [Figure 2C] Figure 2C is a bar graph showing the results of a flow cytometry binding experiment with O13 YSD cells (yeast cells expressing lamprey O13 VLRB anti-H3 antibody on their surface) and multivalent H3-PAA biotin, monovalent H3-sp biotin, multivalent H3 glycan dendrimer biotin, and unmodified dendrimer biotin. Only multivalent H3-PAA and H3 glycan dendrimer were bound effectively by O13 YSD cells. [Figure 2D] Figure 2D is a bar graph showing the results of an ELISA binding experiment with O13-human IgG1 Fc fusion protein (coated anti-H3 antibody) and multivalent H3-PAA biotin, multivalent H3 glycan dendrimer biotin, unmodified dendrimer biotin, and multivalent Lewis A-PAA. Multivalent H3-PAA and H3 glycan dendrimer are relatively bound by the O13-hIgG antibody, whereas unmodified dendrimer and Lewis A-PAA compounds are not bound. [Figure 2E] Figure 2E relates to the production and testing of a second batch of H3-dendrimer.Figure 2E is a bar graph showing the PD10 fraction assayed by O13-hIgG anti-H3 antibody. [Figure 2F] Figure 2F relates to the production and testing of a second batch of H3-dendrimer.Figure 2F is a plot showing H3-N3 magnetic beads detected by O13-hIgG antibody. [Figure 2G] Figure 2G relates to the production and testing of a second batch of H3-dendrimer. Figure 2G is a bar graph showing H3-dendrimer-magnetic beads detected by ConA and AAL lectins.

[0016] [Figure 3A] Figure 3A illustrates the generation and testing of Man5 and Man9 glycan dendrimers. Figure 3A illustrates Man5- and Man9-AEAB compounds (NatGlycans). [Figure 3B] Figure 3B illustrates the production and testing of Man5 and Man9 glycan dendrimers. Figure 3B is a flow diagram showing the conjugation reaction to PEG-NHS to produce Man5 and Man9 dendrimers. [Figure 3C] Figure 3C illustrates the production and testing of Man5 and Man9 glycan dendrimers. Figure 3C is a flow diagram showing the conjugation reaction to PEG-NHS to produce Man5 and Man9 dendrimers. [Figure 3D] Figure 3D illustrates the generation and testing of Man5 and Man9 glycan dendrimers. ConA and PNA lectin reactivity to unmodified PEG-NHS, Man5, and Man9 dendrimers.

[0017] [Figure 4A]Figure 4A illustrates the generation and testing of Core1-T, Core2-S, Core3-S, and Core4-S glycan dendrimers (T=threonine, S=serine). Figure 4A illustrates the structures of Core1-tT, Core2, 3, 4-S (Chemily Glycoscience). [Figure 4B] Figure 4B illustrates the production and testing of Core1-T, Core2-S, Core3-S, and Core4-S glycan dendrimers (T = threonine, S = serine). Figure 4B is a flow diagram of the conjugation reaction with PEG-NHS to produce Core1-T, Core2, 3, 4-S dendrimers (Core2-S as an example). [Figure 4C] Figure 4C illustrates the generation and testing of Core1-T, Core2-S, Core3-S, and Core4-S glycan dendrimers (T=threonine, S=serine). Figure 4C is a bar graph showing ConA, PNA, and GSLII lectin reactivity to unmodified PEG-NHS, Core1-T, and Core2-S dendrimers. [Figure 4D] Figure 4D illustrates the generation and testing of Core1-T, Core2-S, Core3-S, and Core4-S glycan dendrimers (T=threonine, S=serine). Figure 4D is a bar graph showing PNA and GSLII lectin reactivity to unmodified PEG-NHS, Core3-S, and Core4-S dendrimers.

[0018] [Figure 5A] Figure 5A shows the generation and testing of GP100121 (SEQ ID NO: 5) glycopeptide dendrimer. Figure 5A illustrates a synthetic glycopeptide (SEQ ID NO: 5) containing two GalNac linked to one serine and one threonine (GP100120, Sussex Research). [Figure 5B]Figure 5B shows the production and testing of GP100121 (SEQ ID NO: 5) glycopeptide dendrimer. Figure 5B is a flow diagram of the conjugation reaction to PEG-NHS to produce the GP100121 (SEQ ID NO: 5) glycopeptide dendrimer. [Figure 5C] Figure 5C shows the production and testing of GP100121 (SEQ ID NO: 5) glycopeptide dendrimer. Figure 5C is a flow diagram of the conjugation reaction to PEG-NHS to produce the GP100121 (SEQ ID NO: 5) glycopeptide dendrimer. [Figure 5D] Figure 5D shows the generation and testing of GP100121 (SEQ ID NO:5) glycopeptide dendrimer. Figure 5D is a bar graph showing the reactivity of PNA and VVL lectin to unmodified PEG-NHS and GP100121 dendrimers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention I. Definition As used herein, a "dendrimer" is a material composed of identical dendrimer molecules.

[0020] As used herein, a "dendrimer molecule" is a molecule consisting of one or more dendrons arising from a single building block.

[0021] As used herein, a "dendron" is a portion of a molecule with only one free valence, which has exclusively dendritic, terminal constitutional repeating units (CRUs), and where each path from the free valence to any terminal group contains the same number of constitutional repeating units. For purposes of determining the nature of the constitutional repeating units, the free valence is treated as a connection to the CRU. Dendrimer molecules with only one dendron may be referred to as dendrons, monodendrons, or functionalized dendrons. In dendrons, there is no macrocyclic structure of the constitutional units.

[0022] As used herein, a "glycan" is the carbohydrate moiety of a glycoconjugate such as a glycoprotein, glycolipid, or proteoglycan. Also, "glycan" refers to free unbound carbohydrates in a biological medium. Glycans can be single monosaccharides and / or two or more monosaccharides (e.g., polysaccharides) linked glycosidically.

[0023] As used herein, "active agent" refers to a physiologically or pharmacologically active substance that acts locally and / or systemically in the body. An active agent is a substance that is administered to a patient for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), or diagnosis (e.g., diagnostic agent) of a disease or disorder.

[0024] As used herein, "effective amount" and "adequate amount" with respect to a therapeutic agent are at least the minimum concentration required to produce a measurable improvement or prevention of any sign or a particular condition or disorder, to produce a measurable increase in life expectancy, or generally to improve the quality of life of the patient. An effective amount may vary depending on factors such as the disease or condition being treated, the active agent being administered (e.g., a particular targeting construct, etc.), the size of the subject, or the severity of the disease or condition. With respect to cancer, an effective amount may refer to the amount of active agent that reduces or inhibits tumor growth or tumor burden. An effective amount may be associated with a delivery system disclosed herein. For example, in some embodiments, a "therapeutically effective amount" refers to the amount of a therapeutic agent that, when incorporated in and / or on a particle described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without necessitating undue experimentation.

[0025] As used herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0026] As used herein, "antibody" refers to a natural or synthetic antibody that binds to a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that bind to a target antigen. As used herein, "single-chain Fv" and "scFv" refer to an Fv that utilizes the heavy chain variable region (V) of a single polypeptide chain for antigen binding (i.e., Fvs that utilize the heavy chain variable region (V) of a single polypeptide chain for antigen binding). H ) and the light chain variable region (V L (for associating with each other to form an Fv) in a single polypeptide chain joined by linkers that enable the V L and V H V L Area and V H The region may be derived from a parent antibody, or may be chemically or recombinantly synthesized.

[0027] As used herein, "individual," "host," "subject," and "patient" are used interchangeably to refer to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. The subject may be a human patient or a veterinary patient.

[0028] As used herein, "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed to removing the cause of the associated disease, pathological condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to alleviate the symptoms of a disease, pathological condition, or disorder but not to cure it; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to ameliorating the associated disease, pathological condition, or disorder.

[0029] II. Composition Provided are polymeric multifunctional glycan scaffolds and methods of using the same. The scaffolds are typically spherical, branched, or highly branched polymers that can mimic mammalian cells to generate high density multivalent glycans for natural protein-glycan binding events. In addition to displaying multimeric glycans, the scaffolds can be functionalized with a second or further moiety or element that allows characterization and / or isolation of natural binding events, if desired. In a preferred embodiment, the multimeric glycan scaffolds are attached to the surface of cells or glycan-binding proteins (GBPs) to facilitate characterization and / or isolation of the attached cells or proteins. Thus, mixtures of multifunctional glycan scaffolds and cells or proteins, cells or proteins to which the multifunctional glycan scaffolds are attached are also provided. Each of these elements is described in more detail below.

[0030] A. Multivalent Glycan Scaffolds A multimeric glycan scaffold is provided. The glycan scaffold is typically formed from an underlying branched scaffold that is functionalized at its termini with one or more glycans and, optionally, one or more other functional elements.

[0031] An example of a functionalized multivalent glycan scaffold is shown in Figure 1B. Figure 1B shows a multivalent glycan scaffold of the formula: (Ligand) n :Scaffolding:Detection / Separation Tags:Identifier Tags 1 illustrates an embodiment of the present invention. where "ligand" is one or more glycans with n>1 copies (i.e., multivalency); "Scaffold" refers to the underlying branched scaffold, e.g., a dendrimer or a hyperbranched polymer; "Detection / Separation" and "Identifier" "Tags" are exemplary optional functional elements, such as (a) biotin or (HIS)6-8 peptide tags to facilitate separation / isolation, (b) fluorophores to facilitate detection, and / or (c) unique DNA or RNA sequences that can be PCR amplified and sequenced to facilitate identification ("barcodes").

[0032] Alternatively, the "tag" of the formula may be a functional element such as a drug or other material as described elsewhere herein, e.g., a nucleic acid such as DNA, RNA, iRNA, CRISPR guide RNA, or a vesicle containing a drug or other material that is intended to have a therapeutic or cell-modifying effect, or may include an antigen intended to induce (a) a protective immune response against a pathogen or abnormal cell expressing the antigen, e.g., a tumor, or (b) immune tolerance to the antigen as a treatment for an autoimmune disorder against the antigen.

[0033] Each of these elements of the multivalent scaffold is described in more detail below.

[0034] 1. The foundation Materials are provided for forming the underlying scaffold. The underlying scaffold is typically a branched scaffold formed from dendritic molecules. The field of dendritic molecules can be broadly divided into low molecular and high molecular species. The first category includes dendrimers and dendrons, the latter including dendronized polymers, hyperbranched polymers, and polymer brushes. Dendrimers and dendrons are monodisperse and usually highly symmetrical spherical compounds. The underlying scaffold can be formed from any one of these dendritic molecules.

[0035] The properties of dendrimers are controlled in part by the functional groups on the surface of the molecule. However, there are examples of dendrimers with internal functionality. Controllable properties of dendrimers include solubility, toxicity, crystallinity, tectodendrimer formation, and chirality. See also Cloninger, Curr Opin Chem Biol., 6(6):742-8, doi:10.1016 / s1367-5931(02)00400-3 (2002).

[0036] Dendrimers can be thought of as having three main portions: a core, an inner shell, and an outer shell, and can be synthesized with different functionality in each of these portions to control properties such as solubility, thermal stability, and compound attachment. The synthetic process also allows for precise control of the size and number of branches on the dendrimer.

[0037] The preparation of dendrimers requires a level of synthetic control that is achieved by a series of stepwise reactions involving the building up of the dendrimer by each successive group. Dendrimer synthesis can be of the convergent or divergent type. During diffusive dendrimer synthesis, molecules are assembled from the core to the periphery in a stepwise process that involves attaching one generation to the previous generation and then changing the functional group for the next step of reaction. Functional group conversion is essential to prevent uncontrolled polymerization. Such polymerization results in highly branched molecules, known as hyperbranched polymers, rather than monodisperse. Due to steric effects, continued reaction of dendrimer repeat units results in spherical or globular molecules until steric crowding prevents complete reaction at a particular generation, destroying the monodispersity of the molecule. The branched scaffolds that form the basis of the disclosed multivalent glycan scaffolds can be prepared by convergent or diffusive synthesis. The underlying scaffolds can be monodisperse or polydisperse.

[0038] The underlying scaffold is typically nanometer sized, for example, from 100 nm to 1 nm, or any integer subrange thereof, or any discrete integer therebetween. For example, the molecules can be about 100 nm, 75 nm, 50 nm, 25 nm, 20 nm, 15 nm, 12.5 nm, 10 nm, 7.5 nm, 5 nm, 4 nm, 3 nm, or 2 nm in diameter.

[0039] Dendrimers can be classified by generation, which refers to the number of repeated branching cycles performed during their synthesis. For example, if a dendrimer is made by convergent synthesis and the branching reaction is performed three times on the core molecule, the resulting dendrimer is considered a third generation dendrimer. Each successive generation results in a dendrimer with approximately twice the molecular weight of the previous generation. Also, the higher the generation of the dendrimer, the more functional groups are exposed on the surface, which can be used later to customize the dendrimer for a given application.

[0040] The branched scaffold underlying the disclosed multivalent glycan scaffold may be of any generation. Thus, in some embodiments, the dendrimer or other branched molecule may be, for example, any one of generations G1 to G10. In some embodiments, the branched scaffold underlying has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein. In some embodiments, the branched scaffold underlying used herein is G0, G1, G2, or G3. However, the number of possible generations (e.g., 11, 12, 13, 14, 15, 20, or 25) can be increased by decreasing the spacing units in the branched polymer.

[0041] Non-limiting examples of materials that can be used to form the underlying scaffold include, but are not limited to, polyamidoamine (PAMAM) dendrimers, multi-arm polyethylene glycol (PEG), branched polyethyleneimine (PEI) polymers such as poly(propyleneimine), PEG core dendrimers, multi-arm polyethylene glycol (PEG) polymers, poly(acrylates), polyamines, polyamides, polyethers, polyesters, poly(methylacrylate), polyphenylene, and polystyrene.

[0042] For example, in some embodiments, the dendrons of the underlying scaffold are formed from a polyester-8-hydroxyl-1-acetylene bis-MPA dendron, a polyester-6-hydroxyl-1-acetylene bis-MPA dendron, a polyester-32-hydroxyl-1-acetylene bis-MPA dendron, a polyester-8-hydroxyl-1-carboxyl bis-MPA dendron, a polyester-6-hydroxyl-1-carboxyl bis-MPA dendron, or a polyester-32-hydroxyl-1-carboxyl bis-MPA.

[0043] In some cases, the underlying branched scaffold is formed from hyperbranched polymers that form polydisperse dendritic macromolecules with dendrimer-like properties. In many cases, hyperbranched polymers are prepared in a single synthetic polymerization step. For example, hyperbranched polymers can be based on 2,2-bis(hydroxymethyl)propanoic acid (bis-MPA) monomers. Examples of hyperbranched polymers include, but are not limited to, hyperbranched bis-MPA polyester-16-hydroxyl, hyperbranched bis-MPA polyester-32-hydroxyl, and hyperbranched bis-MPA polyester-64-hydroxyl.

[0044] Peptide "arms" and polysacharide "arms" may also be used to form the underlying scaffold.

[0045] In some embodiments, the branched polymer is a polyamidoamine (PAMAM) dendrimer having a number of surface groups (valence sites) ranging from 4 for generation 0 to 4,096 for generation 10, or any integer subrange or specific integer range therein. In other embodiments, the branched polymer is a multi-arm polyethylene glycol (PEG) polymer having a number of valence sites ranging from 2 for Y-shaped PEG to 8 for 8-arm PEG. In other embodiments, the branched polymer comprises a highly branched polyethyleneimine (PEI) polymer having a number of valence sites ranging from 10 for low molecular weight PEI of 800 Da to 211 for high molecular weight PEI of 25,000 Da, or any integer subrange or specific integer range therein.

[0046] The arm length can be customized to different intervals, for example, in certain embodiments, the scaffold has a PEG length per arm of 1-50 PEG units, more preferably 5-25 PEG units.

[0047] In another example, the branched polymer is a dendroalkyne-PEG-dendroalkyne (PEG hyperbranched dendrimer acetylene) (e.g., G4, G5, or G6; PEG 10K). See Creative PEGWorks, catalog numbers HBP-211, HBP-212, and HBP-213.

[0048] In certain embodiments, the multimeric glycan scaffold is between 10 kDa and 50 kDa, between 20 kDa and 60 kDa, or between 20 kDa and 30 kDa (each inclusive), or any integer range thereof, or any particular integer therebetween, and optionally has 8 to 16 arms, e.g., PEG arms. These scaffolds are believed to provide sufficient / suitable spacing and flexibility for multimeric interactions. In some embodiments, the underlying scaffold is larger and / or wider. A larger / wider glycan scaffold potentially provides for the interaction of multiple GBPs at once, which may or may not be desirable depending on the application in which it is used.

[0049] Preferably, the multimeric glycan scaffold is water-soluble.Preferably, the glycan scaffold is stable and / or non-toxic, especially for in vivo applications.For example, when the base scaffold is composed of PEG, the multimeric scaffold is highly water-soluble.Because both PEG and glycan are highly soluble in water and aqueous buffers.

[0050] 2. Linkers and Linkages The disclosed multimeric glycan scaffolds can have two or more glycans, and can also have one or more functional elements, optionally but preferably, at the end of the base scaffold.Thus, the base scaffold is typically produced with one or more terminal groups suitable for linking glycans and optionally additional functional elements.Such groups can be called linkers or linkages.

[0051] The base scaffold may be attached to the glycan or functional element via a terminal group on another bifunctional linker, or via one or more bifunctional linkers that react with the base scaffold. Thus, in some embodiments, the linker is a bifunctional linker that connects the end of the base scaffold to the glycan or functional element via an independent functional group. The number of bifunctional linkers may be one or more. In some embodiments, there is a single bifunctional linker between the base scaffold and the glycan or functional element. In other embodiments, there may be two or more bifunctional linkers between the base scaffold and the glycan or function, for example, one end of the base scaffold is bonded to one end of a first bifunctional linker, and the other end of the first bifunctional linker is attached to one end of a second bifunctional linker. The second bifunctional linker may be attached to the first bifunctional linker via one functional end, and the other functional end may be attached to the glycan. In this way, there can be three, four, or more bifunctional linkers between the underlying scaffold and the glycan or functional element. The number of bifunctional linkers can be selected, for example, to provide better steric properties, for more efficient linking, or to take advantage of commercially available linkers.

[0052] The base scaffold may include at least one end group, which may include, but is not limited to, sodium carboxylate end group, primary amine end group, hydroxyl end group, amidoethanol end group, succinamic acid end group, succinamidyl end group, and a mixture of primary and secondary amine end groups. When the base scaffold includes a terminal amine group, an amine-reactive crosslinker group that binds to primary amines may be used. Groups that can bind to primary amines include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these groups are conjugated to amines by either acylation or alkylation.

[0053] Glycans can generally be attached, for example, via amine groups if the glycan contains amine groups, or via aldehydes or ketones if the glycan contains reducing sugars. For crosslinking to glycans containing reducing sugars, a functional group reactive with an aldehyde or ketone, as appropriate, acts to crosslink the glycan. Aldehyde-reactive crosslinking functional groups include hydrazides that form hydrazone bonds. Aniline can be used to catalyze the hydrazide-aldehyde reaction. Another functional group is an alkoxyamine group, which reacts with an aldehyde to form a stable oxime. If the glycan contains amine groups, an amine-reactive crosslinker group that binds to a primary amine can be used, as described above.

[0054] Thus, the linkage between the underlying scaffold and the glycan or functional element can occur through crosslinking with a moiety that is a bifunctional linker having a first functional end for attachment to the glycan or functional moiety and a second functional end for attachment to the underlying scaffold. In one embodiment, the linker or bifunctional linker comprises one or more functional groups that are succinimidyl, hydrazidyl, aminooxyl, aldehyde, amino, azidyl, biotinyl, carboxyl, epoxy, hydrazinyi, N-hydroxysuccinimidyl, and ketonyl functional groups, with a spacer between the functional groups.

[0055] For example, in other more specific embodiments, the bifunctional linker is a first succinimidyl linker which may be one or more of N,N'-disuccinimidyl carbonate (DSC), N,N'-disuccinimidyl tartrate (DST), N,N'-disuccinimidyl oxalate (DSO), suberic acid bis(N-hydroxysuccinimide ester), N,N'-disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl suberate (DSS), N,N'-disuccinimidyl homobifunctional poly(ethylene glycol) (PEG), and combinations thereof. dihydrazide homobifunctional linker; and a second hydrazidyl homobifunctional linker selected from the group consisting of adipic acid dihydrazide, succinic acid dihydrazide, oxalyl dihydrazide, 9,10-dihydro-9,10-ethanoanthracene-11,12-dicarboxylic acid dihydrazide, ethylmalonic acid dihydrazide, isophthalic acid dihydrazide, pimelic acid dihydrazide, carbohydrazide, thiocarbohydrazide, suberic acid dihydrazide, 6-hydrazinonicotinic acid hydrazide, 4-aminobenzoic acid hydrazide, 2-amino-isonicotinic acid hydrazide, and combinations thereof.

[0056] In some embodiments, the bifunctional linker comprises a homobifunctional material containing succinimidyl, hydrazide, or aminooxyl functional groups; or a heterobifunctional material containing succinimidyl, hydrazide, or aminooxyl functional groups. In some embodiments, the homobifunctional linker comprises N,N'-disuccinimidyl carbonate, adipic acid dihydrazide, or any combination thereof.

[0057] In other embodiments, there are first and second bifunctional linkers between the base scaffold and the glycan or functional element. In one embodiment, the first bifunctional linker is a disuccinimidyl linker, and the second bifunctional linker is a dihydrazide linker or a hydrazide-amine heterobifunctional linker. In this way, the succinimidyl group of the first linker can be attached to the second bifunctional linker via the hydrazide group (of the dihydrazide linker) or the amine group (of the hydrazide amine linker). From there, the hydrazide functional group of the second linker is available to react with the glycan bearing a reducing sugar via its aldehyde or ketone group. Thus, the base scaffold is attached to the succinimidyl group of the first linker via its amine terminal group. The second succinimidyl group of the first linker is then attached to the glycan, alternating through the amine group of the glycan and / or the second succinimidyl group of the first linker is attached to either the amine group or the hydrazide group of the second linker. The second functional group (hydrazide) of the second linker can then be used to attach to the aldehyde or ketone group of the glycan having a reducing end.

[0058] In certain embodiments, the linker is formed from one or more functional groups selected from acrylamide, alkyne, amine, azide, biotin, carboxylic acid (COOH), dibenzocyclooctyne (DBCO), hydrazine, maleimide, phosphate, succinimidyl NHS ester, and sulfhydrylmercapto (SH).

[0059] Additionally or alternatively, linker may comprise one or more affinity tag pairs. Affinity tag pairs are well known in the art and include epitope / antibody, biotin / avidin, biotin / streptavidin, biotin / neutravidin, glutathione-S-transferase / glutathione, maltose binding protein / amylase, and maltose binding protein / maltose. Examples of suitable epitopes that can be used for epitope / antibody binding pairs include, but are not limited to, HA, FLAG, c-Myc, glutathione-S-transferase, His6, GFP, DIG, biotin, and avidin. Antibodies (both monoclonal and polyclonal, and their antigen-binding fragments) that bind to these epitopes are well known in the art.

[0060] Affinity tags allow highly flexible modular assembly and disassembly of glycans and / or functional elements that are conjugated to affinity tags that form highly specific non-covalent physicochemical interactions with complementary affinity tags that are conjugated to the termini of the underlying scaffold. The underlying scaffold can be conjugated to a single type of affinity tag or to any combination of affinity tag types in any ratio. The ability to vary the number of affinity tag types and their ratios conjugated to the dendrimer arms allows for exquisite control over the number and types of glycans and / or functional elements that can be attached to the scaffold, as well as their ratios.

[0061] In some embodiments, the glycans and / or functional elements are tethered to the underlying scaffold using "click" chemistry. Click chemistry is the 1,3-dipolar cycloaddition of azides and alkynes to form 1,2,3-triazoles. Although many other chemistries can be used to couple glycans to linkers, the choice of click chemistry has several major advantages. Click chemistry functional groups (azides, alkynes, DBCO, etc.) are virtually absent in biological systems and other chemical groups, thus preventing undesired reactions, for example, with glycans and proteins. Azidation of glycans appears to be highly specific, for example only in reducing sugars, and the yields are relatively high, exceeding 70%, for most oligosaccharides tested (Tanaka, et al., Chem Commun (Camb), (23): 3378-9, doi: 10.1039 / b905761g (2009)). Azidation of reducing sugars preserves chemical and structural properties, especially closed ring structures (Tanaka, et al., Chem Commun (Camb), (23): 3378-9, doi: 10.1039 / b905761g (2009)). This is not always the case for alternative tagging chemistries / methods. Click chemistry reactions are one of the most effective reactions available for coupling two molecules. They are fast, reliable, specific, complete, compatible with many reaction conditions, and require very minimal setup. For example, DBCO+N3 requires only two components in solution to catalyze the reaction, and nothing else. In the Examples section below, the reducing end of the glycan was derivatized with an azide (N3) tag and the N3-derivatized glycan was coupled to an alkyne / DBCO-biotin octamer linker (7 arms of alkyne / DBCO and 1 arm of biotin) using click chemistry.

[0062] In some embodiments, the glycans and / or functional elements are tethered to the underlying scaffold using the bifunctional fluorescent linker 2-amino-N-(2-aminoethyl)-benzamide (AEAB) (Song, et al., "Novel fluorescent glycan microarray strategy reveals ligands for galectins," Chem Biol. 2009 Jan 30;16(1):36-47. doi: 10.1016 / j.chembiol.2008.11.004. PMID: 19171304; PMCID: PMC2662446, specifically incorporated herein by reference in its entirety). AEAB can be conjugated to a free glycan via its arylamine group by reductive amination to form a glycan-AEAB (GAEAB), which can then be incorporated into the scaffold, for example, via conjugation to one or more of its arms. See, e.g., the experiments described in Example 2 below, and the associated figures, e.g., Figures 3A-3C. The glycan-AEAB conjugate (GAEAB) retains similar fluorescence to the glycan-2,6-diaminopyridine (DAP) conjugate. However, the arylamine is replaced with a primary alkylamine functional group for more efficient immobilization onto N-hydroxysuccinimide (NHS)-derivatized surfaces.

[0063] In some embodiments, the amino acid serves as a linker, for example, alone or as part of a glycoamino acid or glycopeptide, linking the glycan to the arms of the scaffold. The glycopeptide may contain one or more of the same or different glycans. Examples of dendrimers formed by glycans linked to single amino acids (e.g., serine and threonine) and peptides (e.g., serine and theronine residues) are described in the experiments in Example 2 below. See, for example, Figures 4A-4B and 5A-5C, respectively.

[0064] In some embodiments, the scaffold comprises two or more different linkers (or bifunctional linkers) on one, two or more different ends of the underlying scaffold. Thus, the scaffold can present two or more different terminal linkers for attachment of two or more different moieties (e.g., glycans and another moiety). Using this approach, the ratio of glycans to other functional molecules can be more precisely controlled. The scaffold is typically a multimeric glycan scaffold, and therefore typically presents two or more glycans. In some embodiments, the scaffold presents 0, 1, 2 or more functional elements.

[0065] The possibilities for linker / dendrimer configurations are numerous, and many are commercially available. As a non-limiting example, commercial vendors offer the possibility of virtually any custom synthesis of an 8-arm PEG-X / Y, with one arm bearing a "Y" functional group and seven arms bearing "X" functional groups. Such scaffolds can be used to prepare multivalent glycan scaffolds displaying seven glycans and one functional element.

[0066] The above are exemplary and non-limiting, and other configurations of linkers can be envisioned by one of skill in the art.

[0067] 3. Glycans The glycans presented by each scaffold can be any one or more different glycan species. In some embodiments, the scaffold presents two or more copies of a single type of glycan (i.e., homogeneous for glycans). In other embodiments, the scaffold presents one, two, three or more copies of two or more different glycan species (i.e., heterogeneous for glycans).

[0068] The glycans may be natural or synthetic glycans. Glycans may be obtained from commercial sources or may be isolated from biological sources such as cells, tissues, and glycoproteins.

[0069] Exemplary glycans include, but are not limited to, high mannose N-glycans, complex N-glycans, hybrid N-glycans, O-GalNAc O-glycans, O-mannose O-glycans, O-GlcNAc O-glycans, blood group antigen glycans and Lewis antigen glycans, human milk oligosaccharides, sphingoglycolipid glycans, glycosaminoglycans, bacterial glycans, and glycan-containing natural products.Also, examples of glycans that can be used include all glycans specifically mentioned herein.

[0070] Exemplary commercially available glycans include, but are not limited to, all blood group glycans, such as blood group H2 trisaccharide, Lewis X, sialyl Lewis A, 6-sulfo-sialyl Lewis X, 3'- and 6'-sialylLacNAc, Globo-N-pentaose (Gb5), and Globo-N-tetraose (Gb4).

[0071] Natural sources from which N-glycans can be enzymatically released include, but are not limited to, bovine fetuin, ribonuclease B, chicken ovalbumin, chicken egg yolk sialylglycopeptide, human serum IgG, and cultured cells, including primary and immortalized cells (e.g., Jurkat, THP, Raji, Daudi), or mixtures of populations of any one or more of the above, including combinations of two or more different cell types. For each glycoprotein, the heterogeneity of N-glycans from bovine fetuin, ribonuclease B, chicken egg yolk sialylglycopeptide, and chicken ovalbumin is fairly limited. N-glycans from bovine fetuin are primarily biantennary and triantennary N-glycans modified with 2, 3, or even 4 sialic acids (Jiang, et al., Anal Bioanal Chem., 409(2):421-9, doi:10.1007 / s00216-016-9690-x(2017)). N-glycans from ribonuclease B are restricted to the high mannose type Man5-9 (Jiang, et al., Anal Bioanal Chem., 409(2):421-9, doi:10.1007 / s00216-016-9690-x(2017)). N-glycans from chicken egg yolk sialylglycopeptides are composed exclusively of sialylated biantennary N-glycans (Seko et al., Biochim Biophys Acta., 1335(1-2):23-32, doi:10.1016 / s0304-4165(96)00118-3(1997)). N-glycans from chicken ovalbumin are composed almost exclusively of high mannose and diverse N-acetylglucosamine-terminated N-glycans with little or no modification with galactose, fucose, and sialic acid (Jiang, et al., Anal Bioanal Chem., 409(2):421-9, doi:10.1007 / s00216-016-9690-x(2017)).

[0072] Mucin-derived O-glycans can be released with ammonium hydroxide and reduced with boric acid to make them suitable for azide modification according to the protocol described in the Examples below, which can be prepared using commercially available mucins, such as porcine gastric mucin (PSM) glycan dendrimers, which represent PSM O-glycans.

[0073] Natural glycans can vary widely in monosaccharide composition, size, and structure. Due to limitations in chemical / enzymatic synthesis and costs, commercially available glycans are often restricted to structures consisting of one to four monosaccharide units and therefore represent only limited glycan epitopes. Also, "intact" glycans offer the possibility to present specific glycan epitopes, e.g. Lewis X on the antennae of N-glycans or on O-glycans, in different contexts. Implementations with naturally occurring glycans are representative of glycans that actually occur in nature. Furthermore, natural glycans can be collected in large numbers. While the preparation of synthetic glycans can be practically limited by costs and other resources, naturally occurring glycans can be prepared in relatively large quantities. The disclosed compositions, devices, and methods encompass the isolation and optional utilization of up to hundreds of thousands of glycans.

[0074] Generally, glycans isolated from natural sources will be heterogeneous, especially if isolated from cells, tissues, or organisms. To prepare homogeneous, i.e. monospecific but polyvalent, glycan dendrimers, heterogeneous mixtures of glycans can be separated, for example, by HPLC. It is also possible to generate glycan dendrimers with closely related glycans that differ by only one monosaccharide or one linkage, thus creating libraries that can be used for precision screening of potential glycan ligands.

[0075] As previously introduced, in some embodiments, a single scaffold presents two or more glycans in combination. Glycans can be presented in any possible ratio based on the number of available arms and the number of glycan species linked. As an illustrative non-limiting example, two glycans G1 and G2 are coupled to an 8-arm dendrimer (e.g., 8-arm PEG) alone or in combination with a functional element FE in a ratio of G1:G2:FE of 1:6:1, 2:5:1, 3:4:1, 4:3:1, 5:2:1, 6:1:1, 1:7:0, 2:6:0, 3:5:0, 4:4:0, 5:3:0, 6:2:0, 7:1:0. Such dendrimers can be used experimentally to assay / compare the agonist / antagonist binding effect of two different glycans, e.g., α2-3 vs. α2-6 linkages of sialic acid.

[0076] Methods for isolating and preparing glycans for linkage to the underlying scaffold are described in the Examples below, in the references cited herein, or otherwise known in the art based on the reactive groups used to control the linkage. See, for example, Song, et al., "Oxidative release of natural glycans for functional glycomics," Nat Methods 13, 528-534 (2016), doi.org / 10.1038 / nmeth.3861. For example, hydrazinolysis has been used to both analyze and prepare N- and O-glycans, but is limited due to the toxicity of the reagents and the structural integrity of the glycans. Ammonium salt treatment was originally developed for O-glycans and has also been found to be effective for N-glycans. Pronase digestion of glycoproteins can release sugar amino acids, which can be directly functionalized or further trimmed with N-bromosuccinimide (NBS) for further functionalization. One-pot β-elimination and tagging with 1-phenyl-3-methyl-5-pyrazolone (PMP) have been shown to be effective analytical methods for O-glycan release. Endoceramidase is available to release lipid moieties from glycosphingolipids (GSLs) for glycan analysis, and ozone-initiated glycan release from glycosphingolipids under basic or neutral conditions could potentially be used for large-scale preparations.

[0077] Tanaka et al. first described and established the glycan azido process utilized in the following examples (Tanaka, et al., Chem Commun (Camb), (23): 3378-9, doi: 10.1039 / b905761g (2009)). And also see Streichert et al. (Streichert, et al., Chembiochem., 20 (15): 1914-8, doi: 10.1002 / cbic.201900023 (2019)) for both glycan azidation and click chemistry coupling, each of which is specifically incorporated herein by reference in its entirety. Tanaka demonstrated a reaction in which the terminal reducing end of any glycan can be derivatized with an azido (N3) tag by selective activation of the anomeric hydroxy group while preserving other functional groups of the modified monosaccharide residue, including the N-acetyl group of the non-reducing GlcNAc of the N-glycan. The copper(I)-catalyzed azide-alkyne cycloaddition reaction, or "click chemistry," was introduced 20 years ago by Rostovtsev et al. (Rostovtsev, et al., Angew Chem Int Ed Engl., 41(14):2596-9, doi:10.1002 / 1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4(2002)) and has been adapted to a whole range of biomolecules, including glycans (Baskin, et al., Proc Natl Acad Sci USA, 104(43):16793-7, doi:10.1073 / pnas.0707090104(2007); Zhang, et al.,Molecules,18(6):7145-59.doi:10.3390 / molecules18067145 2013;Streichert,et al.,Chembiochem.,20(15):1914-8,doi:10.1002 / cbic.201900023(2019)).

[0078] N3 tagged glycans can also be clicked onto alkyne or DBCO magnetic beads. Such magnetic beads can be used in pull-down assays and / or flow cytometry type experiments. Specific examples include blood group H2 trisaccharide, human serum IgG-derived N-glycans, bovine fetuin-derived N-glycans, and chicken ovalbumin-derived N-glycans.

[0079] Song, et al. described a method to release glycans from various types of biological samples. Oxidative strategies include the use of bleach and are effective in releasing all types of free reducing N-glycans and O-glycanic acids from glycoproteins, as well as glycan nitriles from glycosphingolipids. The released glycans are directly useful for glycan analysis and / or can be derivatized, e.g., fluorescently. Briefly, NaClO, which is known to degrade proteins, effectively and selectively degrades the aglycone moiety of natural glycoconjugates to release intact glycans. Brief treatment of glycoproteins with NaClO releases free N-glycans that can be specifically derivatized via the reducing end. NaClO degrades glycoproteins within minutes, and the released glycans are easily visualized by their high mobility in thin-layer chromatography (TLC) compared to untreated glycoproteins. The process can be used for all types of common glycoproteins and has been demonstrated, for example, with ovalbumin, bovine IgG, and horseradish peroxidase (HRP), the last of which is resistant to PNGase F digestion due to core α3-fucose modification of the N-glycan. Typically labile sialic acid residues are also well preserved, as demonstrated with bovine fetuin. N-glycans derivatized in this manner can be reacted with amine-based tags, such as the bifunctional fluorescent linker 2-amino-N-(2-aminoethyl)benzamide (AEAB), as well as other fluorescent tags and linkers (e.g., as described above), to facilitate chromatographic separation and purification, and / or incorporation into scaffolds.See, e.g., Song, et al., "Oxidative release of natural glycans for functional glycomics," Nat Methods 13, 528-534 (2016), / / doi.org / 10.1038 / nmeth.3861; Song, et al., "Novel fluorescent glycan microarray strategy reveals ligands for galectins," Chem Biol. 2009 Jan 30;16(1):36-47. doi: 10.1016 / j.chembiol.2008.11.004. PMID: 19171304; PMCID: PMC2662446; U.S. Patent Application No. 2013 / 0331280; and U.S. Patent Application No. 2020 / 0262940, each of which is specifically incorporated by reference herein in its entirety.

[0080] Previous attempts to generate "sugar structures" have focused on building "more multivalent" structures with one to three monosaccharides attached (Bernardi et al., Chem Soc Rev., 42(11):4709-27, doi: 10.1039 / c2cs35408j (2013); Mousavifar, et al., Molecules., 26(9), doi: 10.3390 / molecules26092428 (2021); Reichardt, et al., Chem Soc Rev., 42(10):4358-76, doi: 10.1039 / c2cs35427f (2013); Spinelli, et al., Chem Soc Rev., 42(11):4557-73, doi: 10.1039 / c2cs35406c (2013)). Only a very limited number of studies have attempted to develop methodologies for more complex glycan sets (commercial or naturally released) (Smirnov, et al., Small., 16(46):e2004831, doi:10.1002 / smll.202004831 (2020)). In some embodiments, the disclosed scaffolds are characterized by presenting not only simple multimerization of glycan presentation but also larger glycan sets (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different glycan species on the same scaffold and / or multiple scaffolds with different glycan species linked), including among others naturally released glycans.

[0081] In other embodiments, azide or hydroxy groups are present on or incorporated onto amino acids, e.g., alone or as part of a glycoamino acid or glycopeptide, e.g., a synthetic glycoamino acid or glycopeptide, to generate a glycoamino acid or glycopeptide dendrimer (i.e., not a glycan-only dendrimer). A glycopeptide may contain one or more of the same or different glycans. Examples of dendrimers formed by glycans linked to single amino acids (e.g., serine and threonine) and peptides (e.g., serine and threonine residues) are described in the experiments in Example 2 below. See, e.g., Figures 4A-4B and 5A-5C, respectively. In such cases, the amino acid and / or peptide can be viewed as a linker between the glycan and the arms of the dendrimer.

[0082] Such embodiments can be used to study glycopeptide interactions. For example, glycopeptides known to be required for interactions (meaning that both glycan and peptide moieties are required) can be studied. Rare glycosylation types such as O-Fuc, O-Man, O-GlcNAc (extracellular) are specifically found on a few defined peptide sequences and are usually only a few monosaccharides long (which can be more easily generated synthetically). These are ideal glycopeptides to be used in glycopeptide dendrimers. For example, core proteins modified with GlcNAc can be useful to tease out interacting proteins.

[0083] 4.Functional group One or more arms of the scaffold can be functionalized with a functional element. The functional element can be linked to the scaffold using a linker as described above. The moiety is typically selected based on the planned use of the scaffold. The functional element can be, for example, a small molecule, a protein or polypeptide, a carbohydrate, a nucleic acid, or a combination thereof. The functional element can perform a variety of different functions, such as tracking or otherwise monitoring or identifying the scaffold, characterizing the binding activity of the scaffold, and isolating the scaffold. The scaffold can include a combination of two or more moieties of the same or different types. The moieties can be in series or in parallel. In other words, two or more functional elements can be tethered to the same arm of the underlying scaffold (i.e., in series) or to separate arms of the underlying scaffold (i.e., in parallel), as shown in FIG. 1B. Exemplary functional elements are described in more detail below and include oligomeric bioconjugates (Manimala, et al., Glycobiology.,17(8):17C-23C, doi: 10.1093 / glycob / cwm047 (2007)), polymers (Becer, Macromol Rapid Commun., 33(9):742-52, doi: 10.1002 / marc.201200055 (2012)), quantum dots (Hild, et al., Eur J Pharm Biopharm., 2008;68(2):153-68, doi: 10.1016 / j.ejpb.2007.06.009 (2008), and nanoparticles (Song, et al., J Am Chem Soc., 132(33):11428-30, doi: 10.1021 / ja103351m (2010), Wang, et al., Adv Sci (Weinh), 7(16):2001264. Epub 2020 / 08 / 25. doi: 10.1002 / advs.202001264 (2020)).

[0084] Imaging and Contrast Agents In some embodiments, the scaffold is functionalized with an agent to identify the presence, determine the location, and / or quantify the amount of the scaffold. Agents useful for this purpose include fluorescent tags, radionuclides, and imaging agents.

[0085] Suitable imaging agents include fluorescent molecules such as those described by Molecular Probes (Handbook of fluorescent probes and research products), e.g., Rhodamine, Fluorescein, Texas Red, Acridine Orange, Alexa Fluor (various), Allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various), Calcein, Calcium Crimson, Calcium Green, Calcium Orange, 6-Carboxyrhodamine 6G, Cascade Blue, Cascade Yellow, DAPI, DiA, DiD, Dil, DiO, DiR, ELF 97, Eosin, ER Tracker Blue-White, EthD-1, Indium Bromide, Fluo-3, Fluo4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342, 7-Hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissamine Rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensor Green, LysoSensor Yellow / Blu, LysoSensor Green FM, Magnesium Green, Marina Blue, MitoTracker Green FM, MitoTracker Orange CMTMRos, MitoTracker Red CMXRos, Monobromobimane, NBD-amine, NeruoTrace 500 / 525 Green, Nile Red, Oregon Green, Pacific Blue, POP-1, Propidium Iodide, Rhodamine 110, Rhodamine Red, R-Phycoerythrin, Resorfin, RH 414, Rhod-2, Rhodamine Green, Rhodamine 123, ROX dye, SodiumGreen, SYTO Blue (various), SYTO Green (various), SYTO Orange (various), SYTOX Blue, SYTOX Green, SYTOX Orange, Tetramethylrhodamine B, TOT-1, TOT-3, X-rhod-1, YOYO-1, YOYO-3, and 2-amino-N-(2-aminoethyl)benzamide (AEAB).

[0086] A NanoLuc tag can be added to the peptide sequence and used for luciferase imaging and / or to label dendrimers, for example in place of biotin.

[0087] In addition, radionuclides can be used as imaging agents. Suitable radionuclides include, but are not limited to, Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(I1), indium, gallium, and technetium radioactive species. Other suitable imaging agents include metal ions that are commonly used for chelation in paramagnetic T1-type MIR contrast agents, including divalent and trivalent cations such as copper, chromium, iron, gadolinium, manganese, erbium, europium, dysprosium, and holmium. Metal ions that can be chelated and used for radionuclides imaging include, but are not limited to, metals such as gallium, germanium, cobalt, calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium, technetium, rhenium, platinum, thallium, and samarium. In addition, metal ions known to be useful in neutron capture radiotherapy include boron and other metals with large nuclear cross sections. Also suitable are metal ions useful in ultrasound contrast and x-ray contrast compositions.

[0088] b. Antibodies and Ligands In some embodiments, the scaffold is functionalized with a nucleic acid, or a peptide or protein, such as an antibody or a receptor ligand. The ligand can be, for example, a growth factor, a cytokine, and a chemokine, including a chemokine. The antibody can be polyclonal, monoclonal, fragment, recombinant, or single chain, many of which are commercially available or readily obtained using standard techniques. The antibody can be a fragment of a full-length antibody, or a fusion protein formed for a segment of an antibody. Exemplary antibody fragments and fusions include, but are not limited to, single chain antibodies, single chain variable fragments (scFv), di-scFv, tri-scFv, diabodies, triabodies, teratbodies, disulfide-linked Fv (sdFv), Fab', F(ab')2, Fv, and single domain antibody fragments (sdAb).

[0089] Ligands and antibodies can be used to enhance targeting or attachment of the scaffold to a surface or cell, isolation of the scaffold (ie, affinity chromatography), and the like.

[0090] c. Isolation tag In some embodiments, the scaffold is functionalized with a tag to enhance the isolation or purification of the scaffold. As introduced above, this can be an antibody or a ligand, or a polypeptide purification tag. Polypeptide purification tags are known in the art and include, but are not limited to, His-tags, which typically contain six or more consecutive histidine residues, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E-binding protein, protein A, and small ubiquitin-related modifiers (SUMO). More specific examples include FLAG-tags, which contain the sequence DYKDDDDK (SEQ ID NO: 1); haemagglutinin (HA)-tags, which contain the sequence YPYDVP (SEQ ID NO: 2); or MYC-tags, which contain the sequence ILKKTAYIL (SEQ ID NO: 3) or EQKLISEEDL (SEQ ID NO: 4). Methods of using purification tags to facilitate protein purification are known in the art and include, for example, a chromatography step in which the tag reversibly binds to a chromatography resin. Similar methods can be used to isolate scaffolds.

[0091] d. Barcode In some embodiments, the underlying scaffold and / or glycan are functionalized with a nucleic acid barcode, such that the glycan scaffold is ultimately functionalized with one or more nucleic acid barcodes. For example, in some embodiments, the barcode is attached to a glycan ligand, and then a glycan-barcode compound is attached to the underlying scaffold to form a multimeric structure. In other embodiments, the barcode and the glycan are each separately attached to the underlying scaffold. A nucleic acid barcode, also referred to as a barcode, unique molecular identifier, or UMI, is a short sequence of nucleotides (e.g., DNA, RNA, or a combination thereof) that is used as an identifier for a target molecule and / or an associated molecule such as a target nucleic acid, e.g., a cell type or phenotype, a specific genomic perturbagen, or, as used herein, more typically, a multivalent glycan scaffold.

[0092] The nucleic acid barcode or UMI may have a length of at least, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides, and may be in single-stranded or double-stranded form. One or more nucleic acid barcodes and / or UMIs may be attached or "tagged" onto one or more arms of the glycan scaffold. This attachment may be direct (e.g., covalent or non-covalent attachment of the barcode to the target molecule) or indirect (e.g., via an additional molecule, such as a specific binding agent, such as an antibody (or other protein) or a barcode-receptor adaptor (or other nucleic acid molecule)). In some embodiments, the barcode is in tandem with another functional element, such as a fluorescent tag, a purification tag, etc., as shown in Figure IB. The UMI can be flanked by a common amplification sequence that allows two or more different UMI sequences to be amplified using a common primer. Thus, a barcode can consist of a UMI sequence, such as a UMI sequence and one or more additional sequences, such as a common primer binding sequence.

[0093] Glycan scaffolds can be labeled with multiple nucleic acid barcodes in a combinatorial manner, such as a nucleic acid barcode concatemer. Typically, the nucleic acid barcodes are used to identify the glycan scaffold as having come from a particular compartment or library, having particular physical properties (e.g., glycans, other functional elements, etc.), in contact with a particular cell type, etc., or having been subjected to particular treatment conditions. Glycan scaffolds can be associated with multiple nucleic acid barcodes to provide information about all of these characteristics (and beyond). Meanwhile, each member of a given population of UMIs is typically associated with an individual member of a particular set of the same specific (e.g., glycans, other functional elements, etc.) nucleic acid barcodes (e.g., covalently attached to or components of the same molecule).

[0094] To identify a UMI, the nucleic acid is amplified, if necessary (e.g., by PCR), and the UMI is "read," for example, by sequencing, hybridization to a known complementary sequence (e.g., microarrays, fluorescently tagged nucleic acids, etc.), or any other suitable means.

[0095] e. Active agent In some embodiments, the multivalent glycan scaffold is functionalized with one or more active agents that can be delivered, for example, to a subject or cell, using the disclosed scaffolds.

[0096] The active agents to be delivered include therapeutic agents, nutritional agents, diagnostic agents, and prophylactic agents. The active agents can be small molecule active agents or biopolymers, such as proteins, polypeptides, or nucleic acids. Suitable small molecule active agents include organic compounds and organometallic compounds. Small molecule active agents can be hydrophilic, hydrophobic, or amphiphilic compounds.

[0097] Exemplary therapeutic agents that can be incorporated onto the scaffold include antigens, e.g., tumor antigens, CD4+ T cell epitopes, cytokines, chemotherapeutic agents, radionuclides, small molecule signaling inhibitors, photothermal antennae, monoclonal antibodies, immunological danger signaling molecules, other immunotherapeutics, enzymes, antibiotics, antivirals (protease inhibitors, especially, alone or in combination with nucleosides for the treatment of HIV or Hepatitis B or C), antiparasitic agents (helminths, protozoans), growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and biologically active fragments thereof, including humanized antibodies, single chain antibodies, and chimeric antibodies, antigen and vaccine formulations, including adjuvants, peptide drugs, anti-inflammatory agents, immunomodulatory agents (agents that bind to Toll-like receptors to activate the innate immune system, and agents that inhibit the innate immune system, such as erythrocytes, cerebrospinal fluids, and endothelial cells). These include activating ligands (including, but not limited to, CpG oligonucleotides), molecules that mobilize and optimize the adaptive immune system, molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells, and helper T cells, and molecules that inactivate or downregulate suppressor or regulatory T cells), agents that promote the incorporation of multivalent glycan scaffolds into cells (including dendritic cells and other antigen presenting cells), dietary supplements such as vitamins, and oligonucleotide drugs (including DNA, RNA, antisense, aptamers, small interfering RNA, ribozymes, external guide sequences for ribonuclease P, triplex formers, crispr guide RNAs (e.g., gRNA, sgRNA), etc.).

[0098] In certain embodiments, the therapeutic agent is a bivalent structure containing an E3 ligase ligand and a binder or ligand for the protein to be destroyed (e.g., PROTACS®). Such agents do not function as traditional enzyme inhibitors, but rather act by inducing selective intracellular protein degradation (i.e., ubiquitination and proteosomal destruction (proteolysis) of the target). PROTACS® requires cellular uptake, which likely occurs when multimeric glycan ligands crosslink cellular GBPs, and cell type specificity that can be achieved by glycan ligand:GBP interactions. For a review, see Scheepstra, et al., Comput Struct Biotechnol J, 17:160-176. doi:10.1016 / j.csbj.2019.01.006 (2019).

[0099] Exemplary diagnostic agents have been introduced above and include, but are not limited to, paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents.

[0100] Toxins and "lethal agents" can be applied to glycan dendrimers to target and kill cancer cells / tumors (Cousin, et al., Int J Mol Sci., 17(9), doi: 10.3390 / ijms17091566 (2016); Torres-Perez, et al., Front Oncol., 10:605037, doi: 10.3389 / fonc.2020.605037 (2020); Veprek, et al., J Med Chem., 49(21):6400-7, doi: 10.1021 / jm050741g (2006); Smirnov, et al. Small.,16(46):e2004831, doi: 10.1002 / smll.202004831(2020)). Thus, in some embodiments, the active agent is an anticancer drug. In certain embodiments, the multivalent glycan scaffold comprises one or more anticancer drugs.Representative anticancer drugs include alkylating agents (e.g., cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (e.g., fluorouracil (5-FU), gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), antimitotic agents (taxa such as paclitaxel and dextaxel), and anticancer drugs (e.g., cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide). and vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, and actinomycins such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), topoisomerase inhibitors (camptothecins such as camptothecin, irinotecan, topotecan, etc. and derivatives of epipodophyllotoxin such as amsacrine, etoposide, etoposide phosphate, and teniposide; antibodies against vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®), other anti-VEGF compounds; thalidomide (THALOMID®) and its derivatives such as lenalidomide (REVLIMID®); endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors such as sunitinib ( SUTENT®; tyrosine kinase inhibitors, such as sorafenib (Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib; transforming growth factor-α or transforming growth factor-β inhibitors, and antibodies against the epidermal growth factor receptor, such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

[0101] In certain embodiments, the polyvalent glycan scaffold comprises one or more immunomodulatory agents. Exemplary immunomodulatory agents include cytokines, xanthines, interleukins, interferons, oligodeoxynucleotides, glucans, growth factors (e.g., TNF, CSF, GM-CSF, and G-CSF), hormones such as estrogens (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN® (fluoxymesterone)), progestins (MEGACE® (megestrol acetate), PROVERA® (medroxyprogesterone acetate)), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

[0102] Examples of immunological adjuvants that can be associated with particles include, but are not limited to, TLR ligands, C-type lectin receptor ligands, NOD-like receptor ligands, RLR ligands, and RAGE ligands. TLR ligands can include lipopolysaccharide (LPS) and its derivatives, and lipid A and its derivatives, including, but not limited to, monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4'-monophosphoryl lipid A. In certain embodiments, the immunological adjuvant is MPL. In another embodiment, the immunological adjuvant is LPS. TLR ligands can also include, but are not limited to, TLR3 ligands (e.g., polyinosinic-polycytidylic acid (poly(I:C)), TLR7 ligands (e.g., imiquimod and resiquimod), and TLR9 ligands.

[0103] The multivalent glycan scaffold may also include antigens and / or adjuvants (i.e., molecules that enhance immune responses). Antigens may be peptides, proteins, polysaccharides, sugars, lipids, glycolipids, nucleic acids, or combinations thereof. Antigens may be derived from any source, including, but not limited to, viruses, bacteria, parasites, plants, protozoa, fungi, tissues, or transformed cells, such as cancer or leukemia cells, and may be whole cells or immunogenic components thereof, such as cell wall components or molecular components thereof.

[0104] Suitable antigens are known in the art and are available from commercial government and scientific sources. Antigens can be purified or partially purified polypeptides derived from tumors, or viral or bacterial sources. Criteria for identifying and selecting effective antigenic peptides (e.g., minimal peptide sequences capable of eliciting an immune response) can be found in the art. For example, Apostolopoulos, et al. (Curr. Opin. Mol. Ther., 2:29-36 (2000)) describe a strategy for identifying minimal antigenic peptide sequences based on understanding the three-dimensional structure of antigen-presenting molecules and their interactions with both antigenic peptides and T cell receptors. Shastri (Curr. Opin. Immunol., 8:271-7 (1996)) discloses a method for identifying rare peptides that are useful for activating T cells from the thousands of peptides that normally bind to MHC molecules. Antigens can be recombinant polypeptides produced by expressing DNA encoding polypeptide antigens in a heterologous expression system. The antigen may be DNA encoding all or part of an antigenic protein. The DNA may be in the form of vector DNA, such as plasmid DNA.

[0105] Antigens may be provided as single antigens or in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids. Peptide, protein, and DNA-based vaccines can be used to induce immunity against various diseases or conditions. Cell-mediated immunity is necessary to detect and destroy virus-infected cells. Most traditional vaccines (e.g., protein-based vaccines) can only induce humoral immunity. Nucleic acid vaccines, such as RNA and DNA-based vaccines, represent a unique means to vaccinate against viruses or parasites because they can induce both humoral and cell-mediated immunity. In addition, nucleic acid-based vaccines are potentially safer than traditional vaccines. And they can be more stable and more cost-effective for production and storage. Nucleic acid vaccines include two major components, a carrier (or delivery vehicle) and a nucleic acid encoding the antigen. The carrier protects the nucleic acid from degradation and can facilitate its entry into specific tissues or cells and expression at efficient levels.

[0106] In some embodiments, the antigen is a tolerogenic antigen. As used herein, "tolerogenic" refers to a condition or ability to stimulate or increase tolerance. Appropriate tolerogenic antigens are selected based on the desired therapeutic outcome and the disease, disorder, or condition to be treated. Exemplary antigens are known in the art. See, for example, US Patent Application Publication No. 2014 / 0356384, specifically incorporated herein by reference in its entirety. This explains that tolerogenic antigens can be derived from therapeutic proteins to which tolerance is desired, from proteins administered to humans lacking therapeutic antibodies and antibody-like molecules, proteins that are non-human, human allograft antigens, autoantigens to which the patient has exerted or may exert an autoimmune response, foreign antigens (e.g., food antigens or drug antigens) to which the patient has exerted an unwanted immune response. Tolerance can also be induced by administering the antigen in conjunction with a tolerance-inducing signal. CD22 is an "inhibitory" receptor on B lymphocytes that recognizes glycan ligands (e.g., Neu5Acα1-6Galβ1-4GlcNAc and / or Neu5Acα2-6Galβ1-4(6S)GlcNAc). When an antigen is bound by the B cell antigen receptor (BCR) and CD22 is also bound by its ligand, the B cell becomes unresponsive or anergic. Qadura, et al., Haemophilia, 17:288e95 (2017); Orgel, et al., J Allergy Clin Immunol 2017;139:366e9.e2; and Bednar, et al., ACS Chem Biol, 14:644e54 (2019).

[0107] In certain embodiments, the multivalent glycan scaffold core contains two or more active agents.

[0108] f. Particle In some embodiments, the scaffold functionalizes the delivery vehicle, such as nanoparticles, microparticles, micelles, synthetic lipoprotein particles, or carbon nanotubes. Such delivery vehicles can be loaded with other materials, such as active agents, including those specifically mentioned above, and can be used to facilitate their delivery. In some embodiments, the delivery vehicle provides controlled release of the compound. In some embodiments, the release of the active agent is controlled by the diffusion of the compound from the particle and / or the degradation of the polymer particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers that slowly dissolve in an aqueous environment to form a gel, such as hydroxypropylmethylcellulose or polyethylene oxide, can also be suitable as materials for the drug-containing particles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and its copolymers, poly-4-hydroxybutyrate (P4HB) and its copolymers, polycaprolactone and its copolymers, and combinations thereof.

[0109] The functional elements can be linked to the scaffold using the same or similar means (e.g., linkers and glycans) as above, or using other methods known in the art. In Sago et al., RNA was encapsulated in lipid nanoparticles via phase mixing without any particular chemical action (Sago, et al., Proc Natl Acad Sci US A., 115(42):E9944-E52, doi:10.1073 / pnas.1811276115(2018)). Sojitra et al. used a glycan-phage conjugate strategy in which each phage incorporates a different DNA-encoded barcode. The desired glycan is chemically conjugated (via click chemistry) to a subset of 2,700 copies of the main coat protein pVIII to generate a multivalent display. IDT commercializes, for example, functionalized DNA, modified dithiols, which can be coupled to the glycan scaffold via maleimide thiol reactive groups. DBCO-conjugated DNA from IDT has also been used to couple DNA to carboxylated polystyrene microspheres via an amine-PEG-N3 linker (Oh, et al., Langmuir., 36(13):3583-9, doi:10.1021 / acs.langmuir.9b03386(2020)).

[0110] B. Compositions Comprising Multivalent Glycan Scaffolds Compositions are provided that include the disclosed multivalent glycan scaffolds.The composition can be, for example, a surface or substrate to which the scaffolds are covalently or non-covalently linked, or a solution in which the scaffolds are dispersed.In some embodiments, the surface, substrate, solution, etc., includes two or more scaffolds, which can be the same type of scaffold (e.g., with the same underlying scaffold, glycan, and optionally functional element), different types of scaffold (e.g., with different underlying scaffolds, glycans, and / or functional elements), or a combination thereof.

[0111] For example, in some embodiments, a surface, substrate, solution, etc. comprises at least 1, 5, 10, 25, 50, 75, 100, 150, 250, 500, 1,000, 1,500, 2,500, 5,000, 10,000 multivalent glycan scaffolds of one or more of the same or different species.

[0112] A collection of two or more species of scaffolds can be referred to as a library. In some embodiments, a surface, substrate, solution, etc. comprises a library of 5, 10, 25, 50, 75, 100, 150, 250, 500, 1,000, 1,500, 2,500, 5,000, 10,000, or more different species of multivalent glycan scaffolds.

[0113] 1. Surfaces and Substrates The disclosed multivalent glycan scaffolds can be covalently or non-covalently attached to various surfaces and substrates. Substrates can include, but are not limited to, transparent and refractive solid phase materials such as glass (such as silicate glass), ceramics, indium tin oxide (ITO), metals, plastic polymers, and silica slides. Plastic polymers include, for example, polystyrene (Example 8), polypropylene, polycarbonate, polyethylene, high density polyethylene (HDPE), polyvinyl chloride, polyamide, acrylonitrile butadiene styrene (ABS), and polyurethane. Surfaces and substrates include, but are not limited to, beads, dishes including tissue culture dishes, plates including microtiter plates, microscope slides, point-of-care diagnostic chips, miniature biosensors, microreactors, and microelectrodes. See also U.S. Patent Application Publication No. 2019 / 0128881, the entirety of which is specifically incorporated herein by reference.

[0114] In some embodiments, the multivalent glycan scaffold is attached to a surface or substrate via one or more arms.More specifically, in some embodiments, a linker or bifunctional linker connects the scaffold to a surface or substrate.Any of the linkers or bifunctional linkers described herein or elsewhere can be used.

[0115] In some embodiments, the surface is a chromatography column.In such an embodiment, a multivalent glycan scaffold having one of a pair of affinity tags (e.g., His tag) is bound to the column surface having a corresponding / second pair of affinity tags (e.g., nickel, cobalt, or copper ions in / on the column).

[0116] 2. Cells and Proteins As described in more detail below, the disclosed multivalent glycan scaffolds can be used in various cell and protein binding assays. Such assays can be used to identify glycan-cell and / or glycan-protein / receptor binding events and activities, and isolate cells or proteins based on this. Thus, in some embodiments, the multivalent glycan scaffold binds to cells or tissues or organs and / or proteins to form multivalent glycan scaffold-cell and / or multivalent glycan scaffold-protein complexes.

[0117] In a preferred embodiment, binding is regulated via the glycan, optionally but preferably by binding to a glycan-binding molecule such as a glycan-binding protein. Binding is typically non-covalent and can occur in the context of a cell surface or in another context such as a free or immobilized protein.

[0118] The protein may be recombinant or naturally occurring, and may be purified or unpurified (e.g., in a lysate). The protein may be, for example, a cell surface protein, on the surface of a cell, or a soluble protein.

[0119] The binding may be transient or stable.

[0120] The cells can be, for example, primary cells or established cell lines. Exemplary cells, tissues, and organs include embryonic cells, immune cells, unipotent cells, multipotent cells, and pluripotent cells. cell); stem cells including embryonic stem cells, and adult stem cells such as hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells, neural stem cells, and muscle satellite cells, and differentiated cells including cells derived from the ectoderm, endoderm, and mesoderm including, but not limited to, fibroblasts, parenchymal cells, hematopoietic cells, white blood cells including lymphocytes, platelets, stromal cells, adipocytes, lung cells, ductal cells, bone cells including osteoclasts, epithelial cells, and skin cells such as keratinocytes, muscle tissue including smooth muscle, skeletal muscle, and cardiac muscle, vascular tissue including endothelial cells, liver tissue including hepatocytes, and nervous tissue including neurons, glia, e.g., astroglia or astrocytes, oligoglia or oligodendrocytes, microglia, beta cells, pituitary cells, synovial cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, white blood cells, granulocytes, tumor cells, or any combination thereof. Tumor cells, as used herein, include primary cancer cells and metastatic cells. Tumor cells are sometimes used interchangeably with cancer cells herein, but also include non-malignant (non-cancerous) cells that exhibit increased proliferation compared to normal cells.

[0121] The cell may be isolated from a subject. The subject may be a mammalian subject. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected of having a tumor, e.g., a malignant tumor.

[0122] The cells can be obtained from any part of the subject, for example, from one or more organs or tissues of the subject. For example, but not limited to, the one or more cells can be derived from one or more tissues or organs selected from the group consisting of the heart, liver, lung, muscle, testis, blood, brain, spleen, breast, skin, pancreas, mouth, esophagus, stomach, small intestine, large intestine, gallbladder, and kidney of the subject. The one or more cells can be obtained from cancer tissue, such as a tumor, or from a non-cancerous tissue. The one or more cells can be obtained from one or more cells from one or more organs or tissues of a healthy subject or a subject with a particular disease or disorder. In some embodiments, the one or more cells can be obtained from one or more organs or tissues of a subject with cancer. Optionally, the one or more cells can be obtained from a biopsy of a tissue or organ of the subject.

[0123] In some embodiments, a population of cells, i.e., two or more cells, is obtained from a tissue or organ of a subject. Optionally, one or more cell populations can be obtained from one or more tissues or organs of a subject. Optionally, one or more subpopulations of cells can be isolated from a population of cells obtained from a tissue or organ of a subject, for example, by fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), ELISA, or microfluidic-based sorting to isolate one or more specific cell types from a population of cells. By isolating a specific cell type from a population of cells, the methods provided herein can be used to identify nanoparticles suitable for delivery to one or more specific cell types or subpopulations of cells in a tissue or organ of a subject. For example, tumor cells can be isolated from a sample containing a population of liver cells, including cancer cells and non-cancerous cells, using one or more cancer markers.

[0124] Also provided is a cell composition, which refers to a composition having at least one cell. In some embodiments, the cell composition comprises a single cell. In some embodiments, the cell composition comprises at least 2, 3, 4, 5, 10, 25, 50, 100, 500, 1,000, or more cells. The cell composition can be obtained from a variety of different sources, such as in vivo sources, such as body fluids or tissue samples from animals, or in vitro sources, such as cultured cells or cultured tissues. Body fluids and tissue samples often contain multiple cell types, for example, brain tissue can contain neurons, glial cells, and many other types of cells. In certain embodiments, the cells are peripheral blood mononuclear cells (PBMCs) from the blood of an animal. Thus, in some embodiments, the cell composition comprises at least 2, 3, 4, or 5 cell types. The cell composition can also include a medium, such as a cell culture medium.

[0125] Any one or more of the cells or cell compositions referred to herein may be used in any of the methods described below.

[0126] 3. Media and Pharmaceutical Compositions In some embodiments, the multivalent glycan scaffold, alone or covalently or non-covalently bound to a surface, substrate, cell, protein, etc., is immersed in a solution, preferably a medium or a pharma- ceutically acceptable carrier.

[0127] The solution can be obtained from a variety of different sources, such as in vivo sources, such as body fluids or synthetic sources, such as pharmaceutical compositions or media, such as cell culture media. Media can vary depending on the sample and cell type used. In some embodiments, the cell composition includes one or more amino acids, vitamins, inorganic salts, glucose, serum, growth factors, hormones, and attachment factors. Exemplary media include Ames medium, BGJb medium with or without Fitton-Jackson modification, Crick medium, CMRL-1066 medium, Fisher medium, Glascow's minimum essential medium (GMEM), Iscove's modified Dulbecco's medium (IMDM), L-15 medium (Leibovitz), McCoy's 5A modified medium, NCTC medium, Swim S-77 medium, Waymouth medium, William's medium E, Dulbecco's modified Eagle's medium (DMEM), and Eagle's minimum essential medium (EMEM).

[0128] A pharmaceutical composition may be suitable for, but need not be, administered to a subject by, for example, parenteral (intramuscular (IM), intraperitoneal (IP), intravenous (IV), or subcutaneous injection (SubQ)), transdermal (passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration. The formulation may be in the form of a suspension or emulsion. In general, a pharmaceutical composition may include pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may include diluents, sterile water, buffered saline with various pH and ionic strength and various buffer contents (e.g., Tris-HCl, acetate, phosphate); and, if necessary, additives such as detergents and solubilizers (e.g., TWEEN® 20, TWEEN® 80, also called polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.

[0129] The formulation can be lyophilized and redissolved / resuspended immediately prior to use. The formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0130] In some embodiments, especially in those in which active agents are tethered, the scaffold can be administered to a subject in need of disease or disorder treatment to treat the disease or disorder.In some embodiments, the composition is administered systemically, for example by intravenous or intraperitoneal administration, in an amount effective for delivering the composition to target cells.Other possible routes include, but are not limited to, transdermal and oral.

[0131] In certain embodiments, the composition is administered locally, for example by direct injection into the site to be treated. In some embodiments, such as for the treatment of cancer, the composition is injected or otherwise administered directly into one or more tumors. Typically, local injection causes an increase in local concentration of the composition that is higher than can be achieved by systemic administration. In some embodiments, the composition is delivered locally to the appropriate cells by using a catheter or syringe. Other means of locally delivering such compositions to cells include using an infusion pump (e.g., from Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant (e.g., P. Johnson and JG Lloyd-Jones, eds., Drug Delivery Systems: Fundamentals and Techniques (Chichester, England: Ellis Horwood Ltd., 1988 ISBN-10:0895735806), which can affect a sustained release of drug to the immediate area of ​​the implant.

[0132] The composition can be provided to a subject or cell directly, such as by contacting the subject or cell, or indirectly, such as by the action of any biological process. For example, the scaffold can be formulated in a physiologically acceptable carrier and injected into the tissue or fluid surrounding the cells.

[0133] As further studies are performed, information will emerge regarding dosage levels appropriate for the treatment of various conditions in various patients, and those skilled in the art will be able to ascertain appropriate dosing, taking into account the treatment status, age, and health of the recipient. The dosage selected will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. Generally, dosage levels of 0.001 to 10 mg / kg body weight / day are administered to mammals. In general, dosages may be lower for intravenous injection or infusion. In general, the total amount of active agent administered to an individual using the disclosed vesicles may be less than the amount of unassociated active agent that must be administered for the same desired or intended effect and / or may exhibit reduced toxicity.

[0134] III.How to use Glycans are abundantly expressed on the surface of all cells. Proteins that bind cell surface glycans, glycan-binding proteins (GBPs), are also typically expressed on the surface of cells. Thus, the binding interaction of a cell surface glycan ligand with its corresponding cell surface GBP is a multivalent interaction, because many copies of each distinct glycan structure and each distinct GBP are present on the cell surface, i.e., hundreds to tens of thousands of identical copies. Such multivalent interactions result in a substantial increase in apparent binding affinity (avidity) due to "forced proximity" (Vauquelin, et al., Br J Pharmacol., 168(8):1771-85(2013).doi:10.1111 / bph.12106) (the high local "target" concentration experienced by an unbound glycan ligand or GBP when one (or more) glycan ligands bind to a complementary GBP). Forced proximity significantly increases the binding / rebinding of glycan:GBP complexes, substantially slowing the dissociation of the interaction, because all glycan:GBP complexes must dissociate simultaneously. This multivalent avidity enhancement increases the apparent affinity by several thousand-fold compared to monovalent binding. This explains the difficulty of inhibiting glycan:GBP-mediated cell-cell interactions, despite the poor monovalent affinity typical of glycan:GBP binding.

[0135] The disclosed multivalent glycan scaffolds solve this problem, as shown by the following experimental data: monomeric H3 binding to anti-H3 antibodies is not detected by flow cytometry, and furthermore, multimerization of H3 glycans by the disclosed scaffolds results in multimeric H3:anti-H3 antibody complexes that are readily detected by flow cytometry.

[0136] Thus, methods are provided for using the scaffolds to identify specific glycan:GBP binding interactions, for example, to identify glycan:GBP binding partners, to understand structural / chemical determinants of binding specificity / discrimination, to isolate both GBPs that bind to specific glycan structures and glycans that bind to specific GBPs, and / or to target cells expressing GBPs with specificity for specific glycan structures for diagnostic detection and / or therapeutic elimination / inhibition or activation.

[0137] For example, a scaffold containing a detection tag, e.g., a fluorophore, and / or an identifier tag, e.g., a unique DNA sequence, associated with each distinct glycan can be used to identify cells and tissues to which each distinct glycan will direct the localization of the structure in vivo, facilitating the design and delivery of cell / tissue specific targeting.

[0138] These structures are therefore useful for (a) identifying and isolating receptors for ligands of interest, (b) identifying cells and tissues that express the receptor, (c) targeting cells and tissues that express receptors for the ligand to induce signaling events or deliver therapeutic entities, including genes or gene-modifying materials, and (d) delivering glycan ligands that target antigens, such as CD22 on B lymphocytes, to specific cell types, such as dendritic cells, that can process the antigen to induce optimal immunity or induce antigen-specific tolerance.

[0139] The methods and steps used in the identification and analysis of glycan binding events and their subsequent application or use are described in more detail below.

[0140] A. Contact of the glycan scaffold with the target substance A typical method may include contacting one or more species (e.g., a library) of multivalent glycan scaffolds with a target substance, such as a population of cells or proteins, such as the lysate introduced above. In some embodiments, either the scaffold or the target substance is immobilized upon contact. In other embodiments, both the scaffold and the target substance are free or mobile, for example in solution.

[0141] Either the scaffold glycan or the target substance, or both, can be used as bait. Thus, in some embodiments, known or unknown cell types or GBPs are used as baits to capture known or unknown glycan binding partners from a library of glycan scaffolds. In other embodiments, glycan scaffolds with known or unknown glycans are used to capture known or unknown cells or GBPs from a heterogeneous population of cells or proteins (e.g., lysates). Figure 1C is a flow diagram of a library of multimeric ligand particles (MLPs), each of which displays distinct glycans and DNA or RNA sequence identifiers (barcodes) that identify glycans that target specific cells and / or tissues.

[0142] Thus, embodiments are provided in which either the bait or the target is unknown and the other is unknown, in which both the bait and the target are known, and in which both the bait and the target are unknown.

[0143] In one embodiment, a library of polymeric glycan scaffolds is barcoded such that each scaffold displays a distinct polymeric glycan and a distinct barcode that uniquely identifies the glycan. This library is exposed to cells (e.g., human blood cells) (e.g., a mixture) with a mixture of cell type-specific antibodies with each cell type-specific antibody labeled with a distinct fluorophore so that individual blood cell types can be separated and collected by FACS. PCR amplification and sequencing of the barcodes in each collected cell type "pool" will identify which cell type expresses the GBP for the identified glycan. This strategy can also be used with a mixture of GBPs provided that they are paired with a reagent, e.g., an anti-GBP antibody, or a distinct chemical or recombinant tag for each GBP. The anatomical location of the cell / tissue in vivo will identify the presence of the GBP associated with that cell / tissue that is responsible for the localization of the polymeric glycan scaffold. Additionally, the methods can aid in the identification of glycan structures that confer cellular uptake of multimeric glycan scaffolds, and such identified glycans can be utilized for targeting and intracellular delivery.

[0144] In some embodiments where both the bait and the target are unknown, the method resembles a shotgun glycan microarray approach (e.g., as described by Rick Cummings, specifically with human milk oligosaccharides, see, e.g., Yu, et al., Mol Cell Proteomics, 13(11):2944-60 (2014). doi:10.1074 / mcp.M114.039875). Without knowing what the oligosaccharides are, we fractionated them by HLPC, printed them on a microarray slide, and then probed them with neonatal rotavirus. The process of printing / immobilizing the ligand on a solid phase material results in multivalent presentation of the ligand. Positive spots (signaling interactions) could be further investigated by going back to the fraction of interest and identifying the glycans in the binding fraction using standard glycan analysis methods. This allows the viral lectins to be further investigated, now knowing the appropriate glycan ligands. A similar approach can be utilized with the disclosed glycan scaffolds. The mixture of glycans, (e.g. N3) tags are fractionated to generate glycan scaffolds (e.g. dendrimers) with unique identifiers (e.g. nucleic acid barcodes), cell / protein samples are screened and subjected to PCR to find glycan-dendrimer binders, and then the corresponding fractions are returned to analyze their contents.

[0145] The contacting can be performed in vitro or in vivo. For in vitro applications, the method typically includes contacting a glycan scaffold and a cell or protein, or other target substance, and allowing a sufficient time for the scaffolds to bind to one or more elements of the target substance. For in vivo applications, the method typically includes administering a glycan scaffold to a subject, allowing a sufficient time for the scaffolds to bind to one or more target GBPs on one or more cells in the subject, and optionally obtaining one or more cells from the subject. Exemplary in vivo embodiments, including imaging, diagnostic, and therapeutic applications, are described below.

[0146] B. Identification and Analysis of Binding Events After a suitable period of time to allow binding between the scaffold glycan and one or more elements of the target material, and one or more optional washing steps, the binding event can be analyzed. For example, the separation tag can be used to isolate the scaffold, and the labeling tag can be used to detect or isolate the scaffold, for example, by staining, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), ELISA, immunohistochemistry, etc., and optionally, but preferably, the captured target element is isolated or analyzed.

[0147] 1. Identification and Analysis of Glycan Scaffolds Either method may include sequencing the scaffold barcode to determine specific information about the (e.g., isolated or collected) scaffold, such as the nature of the underlying scaffold, glycans, and functional elements. This may facilitate, for example, the identification of unknown glycans that bind to known cells or proteins. Optionally, the barcode sequence is amplified. Those skilled in the art know how to design amplification primers that specifically amplify the barcode identifier sequence and / or the unique molecular identifier sequence. Optionally, the amplification primers are designed such that detection of the amplified barcode identifier sequence is sufficient to identify the barcode identifier sequence, thereby identifying the scaffold. In certain embodiments, the amplification is performed using polymerase chain reaction (PCR), such as quantitative PCR.

[0148] Optionally, one or more amplified barcode sequences and / or unique molecular identifier sequences are quantified. By determining the number of one or more barcode identifier sequences, it can be determined whether the scaffold can bind to a particular tissue or organ, and / or the effectiveness or specificity or frequency of binding to one or more cell types, tissues, or organs can be evaluated.

[0149] Barcode sequence and / or unique molecular identifier sequence are sequenced as required.Sequencing methods include, but are not limited to, shotgun sequencing, bridge PCR, Sanger sequencing (including microfluidic Sanger sequencing), pyrosequencing, massively parallel signature sequencing, nanopore DNA sequencing, single molecule real-time sequencing (SMRT) (Pacific Biosciences, Menlo Park, Calif.), ion semiconductor sequencing, ligation sequencing, sequencing by synthesis (Illumina, San Diego, Ca), polony sequencing, 454 sequencing, solid-phase sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, mass spectrometry sequencing, pyrosequencing, Supported Oligo Ligation Detection (SOLiD) sequencing, DNA microarray sequencing, RNAP sequencing, tunneling current DNA sequencing, and any other DNA sequencing method that will be identified in the future. One or more of the sequencing methods described herein can be used for high-throughput sequencing.The term high-throughput sequencing used herein refers to any method related to the sequencing of nucleic acid, in which multiple nucleic acid sequences are sequenced at a given time.Amplified products can be pooled before sequencing.

[0150] In a preferred embodiment, the barcode can be used to code or determine the specific composition of the associated glycan scaffold. (Sago, et al., Proc Natl Acad Sci US A.,115(42):E9944-E52. Epub 2018 / 10 / 03. doi: 10.1073 / pnas.1811276115. PubMed PMID: 30275336; PMCID: PMC6196543 (2018)) and see also U.S. Published Application No. 2020 / 0110083.

[0151] 2. Identification and analysis of target substances The cells and / or the bound target substances, such as GBPs, can be identified and / or analyzed. For example, the target proteins can be subjected to protein analysis and / or identification, such as Western blotting (e.g., immunoblotting), Edman degradation, mass spectrometry, such as peptide mass fingerprinting or de novo peptide sequencing, light scattering, such as batch dynamic light scattering (DLS), static light scattering (SLS), charge and zeta potential, multi-detection GPC / SEC, circular dichroism (CD), or isothermal titration calorimetry (ITC). The target cells can be identified or characterized by one or more protein or gene expression analysis, phenotypic and / or histological analysis, for example, by microscopy, including optical, fluorescent, and / or electron microscopy. In some embodiments, the target substances are released from the scaffold before or during the identification and / or analysis. In other embodiments, the target substances are not released from the scaffold. In such embodiments, the scaffold and / or bait, etc. can be subtracted from the analysis as necessary. For example, in the case of mass spectrometry, the signal generated by the scaffold and / or bait can be subtracted from the spectrograph.

[0152] Analysis of the target substance can also include assaying for induction and / or inhibition of cellular pathways (eg, signaling pathways) that are modulated by the glycans of the attached scaffold.

[0153] C. Application and Treatment Methods Once a binding relationship between a glycan and a target substance, such as a cell or protein, is established, scaffolds featuring the relevant glycans can be rationally designed for targeted delivery of one or more active agents to cells, tissues, organs, and / or proteins.

[0154] 1. Targeted Delivery of Active Agents Using Glycan Scaffolds In a preferred embodiment, the analysis reveals that one or more glycan scaffolds preferentially bind to a particular target protein or cell. Preferential binding means that more of a particular glycan scaffold species binds to one protein or cell type compared to other proteins or cell types. For example, such preferential binding can be across organs or tissues (e.g., liver instead of pancreas), across diseased cells / normal cells (e.g., cancer vs. non-cancerous), or across cells within the same tissue or organ (e.g., lymphocytes as opposed to monocytes). By identifying preferentially binding scaffolds, one skilled in the art can identify targeted scaffolds that, when administered to a subject, preferentially deliver their functional elements, such as tags, labels, or active agents, to a particular cell type or a particular tissue or organ of the subject. Once a scaffold-binding event has been identified for a desired cell type, depending on the tissue or organ, one skilled in the art can select or prepare targeted glycan scaffolds that are functionalized with one or more active agents for delivery to cells, tissues, or organs in the subject. The active agent is selected based on the desired treatment, which may include delivery of small molecules, proteins, nucleic acid therapeutics, and vaccine components such as antigens and / or adjuvants, as introduced above. Thus, any method may further include in vitro or in vivo delivery of one or more active agents.

[0155] In other embodiments, the analysis identifies that the glycan itself induces or inhibits a cellular action or activity (e.g., cell signaling). In such embodiments, similar principles of preferential binding are used to identify the desired binding relationship and therapeutic outcome, but the glycan scaffold may optionally lack functionalization with additional functional elements (e.g., active agents, such as drugs, or tags, etc.) because the glycan itself is the active agent that modulates (e.g., activates or inhibits) the therapeutic outcome.

[0156] Thus, the in vivo method can include, for example, administering to a subject in need thereof a pharmaceutical composition containing an effective amount of a glycan scaffold, optionally together with an active agent that functionalizes the glycan scaffold.

[0157] In other embodiments, the glycan scaffold in suspension or immobilized on a substrate is used instead of or as an adjunct to plasma exchange. For example, once a GBP (i.e., IgG, etc.) has been identified from blood or another fluid, the corresponding glycan scaffold can be used to remove the GBP from the blood or other fluid by contacting the blood or other fluid with the glycan scaffold and then separating the glycan scaffold from the blood or other fluid. The GBP binds to the glycan scaffold and remains bound to the glycan scaffold when the blood or other fluid is removed. Such methods can be performed ex vivo, as in conventional plasmapheresis, or typically in vivo, where the GBP-bound glycan scaffold is subsequently collected so that the GBP can be removed.

[0158] Similarly, the imaging method may include administering a pharmaceutical composition containing a glycan scaffold with an imaging agent that functionalizes the glycan scaffold (e.g., a luciferase substrate tag or a fluorophore on a dendrimer) to a subject in need of the pharmaceutical composition. A method for detecting a target substance in a subject may include imaging the subject and determining or locating the target substance in the subject when and / or where the glycan scaffold is detected. If the target substance indicates a disease or disorder, the subject may then be diagnosed with the disease or disorder.

[0159] 2. Exemplary Usage Strategies Exemplary, non-limiting usage strategies are also provided.

[0160] For example, glycans (self or pathogen-derived) can be used to stimulate the innate and adaptive immune system, e.g., via Siglecs (Murugesan, et al., Curr Opin Chem Biol., 62:34-42, doi:10.1016 / j.cbpa.2021.01.001(2021)) and C-type lectins (Richardson, et al., Front Immunol., 5:288, doi:10.3389 / fimmu.2014.00288(2014)), such as DC-SIGN and MGL.

[0161] Pathogen interactions with / recognition by host cells are often glycan-dependent, and glycan scaffolds can be used to explore, define, and neutralize such interactions.Several studies have explored these possibilities using di- / trisaccharides (Cordoba, et al., Nanomedicine, 9(7):972-84, doi: 10.1016 / j.nano.2013.03.004 (2013); Clayton, et al., Bioconjug Chem., 22(10):2186-97, doi: 10.1021 / bc200331v (2011); Garcia-Oliva, et al., Chemistry, 26(7):1588-96, doi: 10.1002 / chem.201903788 (2020); Bernardi et al., Chem Soc Rev., 42(11):4709-27, doi: 10.1039 / c2cs35408j (2013), Kensinger, et al., Antimicrob Agents Chemother, 48(5):1614-23, doi: 10.1128 / AAC.48.5.1614-1623.2004 (2004), Imberty, et al., Chemistry, 14(25):7490-9, doi: 10.1002 / chem.200800700 (2008), Hoyos, et al., Chemistry, 27(28):7593-624, doi: 10.1002 / chem.202005065 (2021), Gingras, et al., Chem Soc Rev., 42(11):4823-41, doi: 10.1039 / c3cs60090d (2013), Wells, et al., Adv Ther (Weinh), 2021:2000210, doi: 10.1002 / adtp.202000210 (2021), Varga, et al., Biomaterials, 35(13):4175-84, doi: 10.1016 / j.biomaterials.2014.01.014 (2014), Mintzer, et al., Mol Pharm., 9(3):342-54, doi: 10.1021 / mp2005033 (2012)).The possibility of using full length complex N-glycans on the disclosed glycan dendrimers allows for example new studies with host cell N-glycans / O-glycans to be carried out.

[0162] Cell surface glycan-binding protein receptors can be targets for intracellular drug delivery / targeting. For example, Cattiaux, et al., Bioorg Med Chem., 26(5):1006-15, doi: 10.1016 / j.bmc.2017.12.036 (2018), Krist, et al., Biochem Biophys Res Commun., 287(1):11-20, doi: 10.1006 / bbrc.2001.5537 (2001), Bernardes, et al., Org Biomol Chem., 8(21):4987-96. Epub 2010 / 09 / 08. doi: 10.1039 / c0ob00372g (2010), Bezouska, et al., FEBS Lett., 426(2):243-7, doi: 10.1016 / s0014-5793(98)00340-8 (1998), Thoma, et al., Chemistry, 12(1):99-117. Epub 2005 / 10 / 19. doi: 10.1002 / chem.200500901 (2005), Lepenies, et al., Adv Drug Deliv Rev., 65(9):1271-81, doi: 10.1016 / j.addr.2013.05.007 (2013), Doores, et al., Chemistry., 12(3):656-65, doi: 10.1002 / chem.200500557 (2006), Zaal, et al. al., Front Immunol. 11:305, doi: 10.3389 / fimmu.2020.00305 (2020). In certain embodiments, the compositions and methods are used to target cancer cells that express specific ligands for the glycans of the glycan scaffold. In other embodiments, the compositions and methods are used to reduce inflammation.

[0163] The present invention can be further understood by the following numbered paragraphs: Paragraph 1. A method for identifying glycan-based binding interactions, comprising contacting a target material with a known multivalent glycan scaffold comprising two or more glycans, and identifying an entity from the target material that binds to the scaffold. Paragraph 2. The method of paragraph 1, wherein the target material is a population of cells comprising one or more different cell types, or a cell lysate or other proteinaceous mixture comprising one or more glycan binding proteins (GBPs). Paragraph 3. The method of paragraph 2, wherein the target material is a population of cells comprising two or more different cell types, or a cell lysate or other proteinaceous mixture comprising two or more glycan binding proteins (GBPs). Paragraph 4. The method of paragraph 3, wherein the target material is a population of cells and the elements of the target material are one or more cell types within the population. Paragraph 5. The method of Paragraph 4, wherein the one or more cell types preferentially bind to the multivalent glycan scaffold compared to other cell types in the population. Paragraph 6. The method of any one of paragraphs 2 to 5, further comprising isolating the multivalent scaffold to which the cells are attached. Paragraph 7. The method of any one of paragraphs 2 to 6, further comprising characterizing the bound cells. Paragraph 8. A composition comprising a glycan-scaffold-bound cell as defined in any one of paragraphs 2 to 7. Paragraph 9. The method of paragraph 2, wherein the target material is a cell lysate or other proteinaceous mixture, and the components of the target material are one or more GBPs within the cell lysate or other proteinaceous mixture. Paragraph 10. The method of Paragraph 9, wherein the one or more GBPs preferentially bind to the multivalent glycan scaffold compared to other GBPs in the cell lysate or other proteinaceous mixture. Paragraph 11. The method of any one of paragraphs 9 or 10, further comprising characterizing the bound GBP. Paragraph 12. The method of any one of Paragraphs 9 to 10, further comprising isolating the bound GBP. Paragraph 13. A composition comprising a glycan scaffold-binding GBP as defined in any one of Paragraphs 9 to 12. Paragraph 14. A method for identifying glycan-based binding interactions, comprising contacting one or more known target substance entities with one or more species of multivalent glycan scaffolds each comprising two or more glycans, and identifying a glycan scaffold that binds to the target substance entity. Paragraph 15. The method of Paragraph 14, comprising contacting the one or more known target material elements with a library of different species of multivalent glycan scaffolds each comprising two or more glycans, and identifying glycan scaffolds that bind to the target material elements, optionally wherein the library comprises from about 2 to about 10,000, or any subrange or specific integer therein, e.g., 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000 different species of glycan scaffolds. Paragraph 16. The method of Paragraphs 14 or 15, wherein the known target material components are one or more cell types. Paragraph 17. The method of Paragraph 16, wherein the one or more multivalent glycan scaffolds preferentially bind to the one or more cell types compared to other multivalent glycan scaffolds in the library. Paragraph 18. The method of any one of paragraphs 16 or 17, further comprising characterizing the attached multivalent glycan scaffold. Paragraph 19. The method of any one of Paragraphs 16 to 18, further comprising isolating the multivalent glycan scaffold. Paragraph 20. A composition comprising a glycan-scaffold-bound cell as defined in any one of Paragraphs 14 to 19. Paragraph 21. The method of Paragraphs 14 or 15, wherein the known target substance elements are one or more GBPs. Paragraph 22. The method of Paragraph 21, wherein the one or more multivalent glycan scaffolds preferentially bind to the one or more GBPs compared to other multivalent glycan scaffolds in the library. Paragraph 23. The method of any one of Paragraphs 21 or 22, further comprising characterizing the attached multivalent glycan scaffold. Paragraph 24. The method of any one of Paragraphs 21 to 23, further comprising isolating the multivalent glycan scaffold. Paragraph 25. A composition comprising a glycan scaffold-binding GBP as defined in any one of Paragraphs 21 to 24. Paragraph 26. A method for identifying glycan-based binding interactions, comprising contacting a target substance with one or more unknown multivalent glycan scaffolds comprising two or more glycans, and identifying entities from said target substance that bind to the multivalent glycan scaffold, optionally wherein said one or more unknown multivalent glycan scaffolds form a library comprising about 2 to about 10,000, or any subrange or specific integer therein, e.g., 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000 different species of glycan scaffolds. Paragraph 27. The method of Paragraph 26, wherein the target material is a population of cells comprising one or more different cell types, or a cell lysate or other proteinaceous mixture comprising one or more glycan binding proteins (GBPs). Paragraph 28. The method of Paragraph 27, wherein the target material is a population of cells comprising two or more different cell types, or a cell lysate or other proteinaceous mixture comprising two or more glycan binding proteins (GBPs). Paragraph 29. The method of Paragraph 28, wherein the target material is a population of cells and the elements of the target material are one or more cell types within the population. Paragraph 30. The method of Paragraph 29, wherein the one or more cell types preferentially bind to the multivalent glycan scaffold compared to other cell types in the population. Paragraph 31. The method of any one of Paragraphs 26 to 30, further comprising isolating the multivalent scaffold to which the cells are attached. Paragraph 32. The method of any one of Paragraphs 26 to 31, further comprising characterizing the bound cells and / or the bound glycans to cells. Paragraph 33. A composition comprising a glycan-scaffold-bound cell as defined in any one of Paragraphs 27 to 32. Paragraph 34. The method of Paragraph 27, wherein the target material is a cell lysate or other proteinaceous mixture, and the components of the target material are one or more GBPs within the cell lysate or other proteinaceous mixture. Paragraph 35. The method of Paragraph 34, wherein the one or more GBPs preferentially bind to the multivalent glycan scaffold compared to other GBPs in the cell lysate or other proteinaceous mixture. Paragraph 36. The method of any one of Paragraphs 34 or 35, further comprising characterizing the bound GBP and / or the glycan bound to a GBP. Paragraph 37. The method of any one of Paragraphs 34 to 36, further comprising isolating the bound GBP. Paragraph 38. A composition comprising a glycan scaffold-binding GBP as defined in any one of Paragraphs 34 to 37. Paragraph 39. The method or composition of any one of Paragraphs 1 to 38, wherein the multivalent glycan scaffold comprises an underlying branched scaffold having two or more glycans linked to its termini and optionally one or more functional elements. Paragraph 40. The method or composition of Paragraph 39, wherein the underlying branched scaffold is a dendrimer. Paragraph 41. The method or composition of Paragraph 39 or 40, wherein the two or more glycans are all the same glycan species. Paragraph 42. The method or composition of Paragraph 39 or 40, wherein the two or more glycans comprise two or more different glycan species. Paragraph 43. The method or composition of any one of Paragraphs 39 to 42, wherein the one or more functional elements are agents that facilitate the formation, imaging, localization, identification, isolation, and / or characterization of the glycan scaffold and / or elements of the target substance bound to the glycan scaffold. Paragraph 44. The method or composition of any one of Paragraphs 39 to 43, wherein at least one of the functional elements is a nucleic acid comprising a barcode that specifically identifies the glycan scaffold. Paragraph 45. The method or composition of any one of Paragraphs 39 to 44, wherein at least one of the functional elements is a fluorophore. Paragraph 46. The method or composition of any one of Paragraphs 39 to 45, wherein the glycan is linked to a 2-amino-N-(2-aminoethyl)-benzamide (AEAB) that is linked directly or indirectly to a terminus of the underlying branched scaffold. Paragraph 47. A method or composition according to any one of Paragraphs 39 to 45, wherein at least one of the functional elements is an affinity tag. Paragraph 48. The method or composition of any one of Paragraphs 39 to 47, wherein the glycan is linked to an amino acid that is directly or indirectly linked to a terminus of the underlying branched scaffold. Paragraph 49. The method of Paragraph 48, wherein the amino acid is not part of a polypeptide chain. Paragraph 50. The method of Paragraph 48, wherein the amino acid is part of a polypeptide chain. Paragraph 51. The method of Paragraph 50, wherein the polypeptide chain comprises two or more amino acids having linked glycans. Paragraph 52. The method of any one of Paragraphs 48 to 51, wherein the amino acid is serine or threonine. Paragraph 53. A method or composition according to any one of Paragraphs 1 to 52, wherein the glycan scaffold or targeting material element is optionally immobilised on a surface or substrate, such as a bead, slide, plate or dish. Paragraph 54. The method or composition of any one of Paragraphs 1 to 52, wherein neither the glycan scaffold nor the target substance is immobilized. Paragraph 55. The method or composition of Paragraph 54, wherein the glycan scaffold and targeting agent are in solution. Paragraph 56. A method of treating a subject in need of treatment, comprising identifying a glycan-based binding interaction according to any of the preceding methods, and administering to the subject a composition comprising an effective amount of a therapeutic multivalent glycan scaffold comprising the glycan of the identified interaction. Paragraph 57. The method of Paragraph 56, wherein the therapeutic glycan induces or inhibits cellular activity upon binding to a cell or GBP of said interaction. Paragraph 58. The method of Paragraph 56, wherein the therapeutic glycan comprises an active agent that functionalizes it. Paragraph 59. The method of Paragraph 58, wherein the active agent is a nucleic acid, a small molecule, or a polypeptide. Paragraph 60. The method of Paragraph 59, wherein said cells are cancer cells and said active agent is an anti-cancer drug. Paragraph 61. The method of Paragraph 59, wherein the cell is an immune cell and the active agent is an antigen or encodes an antigen. Paragraph 62. A composition comprising a therapeutic glycan according to any one of Paragraphs 56 to 62. Paragraph 63. A multivalent glycan scaffold comprising an underlying branched scaffold having two or more glycans and one or more functional elements linked to its termini. Paragraph 64. The multivalent glycan scaffold of Paragraph 63, wherein at least one of the functional elements is an agent that facilitates the formation, imaging, localization, identification, isolation, and / or characterization of the glycan scaffold and / or a targeting element bound to the glycan scaffold. Paragraph 65. A multivalent glycan scaffold according to any one of Paragraphs 63 or 64, wherein at least one of the functional elements is a fluorophore. Paragraph 66. The multivalent glycan scaffold of any one of Paragraphs 63 to 65, wherein the glycans are linked to 2-amino-N-(2-aminoethyl)-benzamide (AEAB) that is linked directly or indirectly to the termini of the underlying branched scaffold. Paragraph 67. A multivalent glycan scaffold according to any one of Paragraphs 63 to 66, wherein at least one of the functional elements is a nucleic acid comprising a barcode that specifically identifies the glycan scaffold. Paragraph 68. The multivalent glycan scaffold of any one of Paragraphs 63 to 67, wherein at least one of the functional elements is an affinity tag. Paragraph 69. The method or composition of any one of Paragraphs 63 to 68, wherein the glycan is linked to an amino acid that is directly or indirectly linked to a terminus of the underlying branched scaffold. Paragraph 70. The method of Paragraph 69, wherein the amino acid is not part of a polypeptide chain. Paragraph 71. The method of Paragraph 69, wherein the amino acid is part of a polypeptide chain. Paragraph 72. The method of Paragraph 71, wherein the polypeptide chain comprises two or more amino acids having linked glycans. Paragraph 73. The method of any one of Paragraphs 69 to 72, wherein the amino acid is serine or threonine. Paragraph 74. The multivalent glycan scaffold of any one of Paragraphs 63 to 73, wherein the underlying branched scaffold is a dendrimer. Paragraph 75. The multivalent glycan scaffold of any one of Paragraphs 63 to 74, wherein the underlying scaffold is formed from 2 to 16 arms (inclusive), optionally 8 to 16 arms. Paragraph 76. The multivalent glycan scaffold of any one of Paragraphs 63 to 75, wherein the underlying scaffold and / or its arms are formed from polyethylene glycol (PEG) polymers. Paragraph 77. The multivalent glycan scaffold of any one of Paragraphs 63 to 76, wherein the underlying scaffold is formed from 8-arm PEG-biotin / alkyne, biotin(1) / alkyne(7), MW 20k; dendroalkyne-PEG-dendroalkyne, PEG 10k, G4 32 alkyne; dendroalkyne-PEG-dendroalkyne, PEG 10k, G5 64 alkyne; or dendroalkyne-PEG-dendroalkyne, PEG 10k, G6 128 alkyne. Paragraph 78. The multivalent glycan scaffold of any one of Paragraphs 63 to 77, wherein the multimeric glycan scaffold is between 10 kDa and 50 kDa, between 20 kDa and 60 kDa, or between 20 kDa and 30 kDa (each inclusive), or any integer range thereof, or any specific integer therebetween. Paragraph 79. The multivalent glycan scaffold of any one of Paragraphs 63 to 78, wherein the two or more glycans are all the same glycan type. Paragraph 80. The multivalent glycan scaffold of any one of Paragraphs 63 to 79, wherein the two or more glycans comprise two or more different glycan species. Paragraph 81. Formula: (Ligand) n :Scaffolding:Detection / Separation Tags:Identifier Tags where "ligand" is one or more glycan species that are present and have n>1 copies (i.e., multivalent); "Scaffold" refers to an underlying branched scaffold, such as a dendrimer or hyperbranched polymer; A functional element, in which one or both of a "detection / separation tag" and an "identifier tag" are present; For example, a vesicle containing (a) a biotin or (HIS) 6-8 peptide tag to facilitate separation / isolation, (b) a fluorophore to facilitate detection, (c) a unique DNA or RNA sequence that can be PCR amplified and sequenced to facilitate identification (a "barcode"); and / or (d) a drug or other material as described elsewhere herein, which may include a nucleic acid such as DNA, RNA, iRNA, CRISPR guide RNA, or an antigen that is intended to have a therapeutic or cell-modifying effect or to induce (a) a protective immune response against a pathogen or abnormal cell expressing the antigen, e.g., a tumor, or (b) immune tolerance to the antigen as a treatment for an autoimmune disorder against the antigen. 81. The multivalent glycan scaffold of any one of paragraphs 63 to 80. Paragraph 82. The multivalent glycan scaffold of any one of Paragraphs 63 to 81, wherein the glycan scaffold is not immobilized. Paragraph 83. A multivalent glycan scaffold as described herein, including but not limited to the text and / or figures. Paragraph 84. A composition comprising a plurality of multivalent glycan scaffolds according to any one of Paragraphs 63 to 83 in solution. Paragraph 85. The composition of Paragraph 84, wherein said multiple multivalent glycan scaffolds are not immobilized. EXAMPLES

[0164] Working Example Example 1: Generation of multivalent, multifunctional glycan dendrimers material and method reagent 2-Chloro-1,3-dimethylimidazolinium chloride (DMC), Sigma Aldrich (#529249-5G) Sodium azide (NaN3), Fisher Scientific (#BP922I-500) 2,6-Lutidine, Sigma Aldrich (#336106-100ML) Acetonitrile (ACN), Fisher Scientific (#AA42311K2) Trifluoroacetic acid (TFA), Fisher Scientific (#AAL06374AC) Carbograph Column 150mg, BGB Analytes (#SPU-5122423) ·PNGaseF, New England Biolabs (#P0704) ·Blood type H 2-trisaccharide, Chemilly Glycoscience(#OS03006) Lewis X tetrassacharide, Carbosynth (#OL09569) ·3'-sialylLacNAc, Carbosynth (#OS00732) ·6'-sialylLacNAc, Dextrauk (#SLN306) Globo-N-pentaose (Gb5), Chemily Glycoscience (#OS03012) Globo-N-tetraose (Gb4); Elicityl (#GLY121-90%) 8-mer PEG dendrimer (7 arms alkyne functionalized; 1 arm biotin functionalized), Creative PEGWorks (#CPW-8841) Multivalent H3-PAA-biotin, Glycotech (#01-034) Monovalent H3-sp-biotin, Glycotech (#02-034) Polyvalent Lewis A-PAA-biotin, Glycotech (#01-035) ·Jurkat cell, Ipracell (#CC-05-10-50) Bovine fetuin, MyBioSource (#MBS634403) Chicken egg albumin, Fisher Scientific (#50-136-8468) Ribonuclease B (RNAseB), Worthington (#LS005710) Human IgG, Athens Research & Technology (#16-16-090707) Porcine gastric mucin (PSM), Sigma Aldrich (#M2378-100G) ·CuSO4, Sigma Aldrich(#451657-10G) Tris-hydroxypropyltriazolylmethylamine (THPTA), Fisher Scientific (#NC1486137) Sodium Ascorbate, Sigma Aldrich (#11140-50G) Sodium phosphate buffer (PBS), 100 mM monosodium phosphate, RPI (#S23185-500.0), 100 mM disodium phosphate, RPI (#S23175-500.0), pH 7.4 PD-10 Sephadex G-25 desalting column, Cytiva (#17085101) - Tris, RPI (#T60040-1000.0) NaCl, RPI (#S23025-3000.0) CaCl2, RPI (#C36200-500.0) MgCl2, RPI (#M24000-100.0) · Sodium Carbonate Anhydrous, RPI (#25025-1000.0) Sodium Bicarbonate, RPI (#S22060-1000.0) Bovine serum albumin (BSA), RPI (#A30075-100.0) Streptavidin-PE, Invitrogen (#SNN1007) Propidium iodide (PI), Miltenyi Biotec (#130-093-233) ·Neutralite Avidin-HRP, SouthernBiotech(#7200-05) ·TMB ELISA substrate, AbCam (#ab171524)

[0165] Azide derivatization of glycans Adapted from Tanaka, et al., Chem Commun (Camb), (23): 3378-9, doi: 10.1039 / b905761g (2009) and Streichert, et al., Chembiochem., 20(15): 1914-8, doi: 10.1002 / cbic.201900023 (2019), which are specifically incorporated by reference in their entirety. Commercially available glycans, including blood group H2 trisaccharide, Lewis X tetrasaccharide, 3'- and 6'-sialylLacNAc, Globo-N-pentaose (Gb5), and Globo-N-tetraose (Gb4), were derivatized at a concentration of 50 mM in a final volume of 45 μL to 60 μL. Glycans were first solubilized in a minimal volume (10-15 μL) of MilliQ water to which solutions of NaN3 and 2,6-lutidine were added to final concentrations of 3.5 M and 1.5 M, respectively. DMC was added to a final concentration of 750 mM and the final solution was incubated at 4° C. for 72 h with occasional mixing to homogenize the samples. See Table 1.

[0166] [Table 1]

[0167] N3 derivatized glycans were purified on a carbograph column (150 mg volume) conditioned with 1 column volume (CV) of ACN+0.1% TFA, 1 CV of MilliQ water+0.1% TFA, 1 CV of 10% ACN+0.1% TFA, and 1 CV of MilliQ water+0.1% TFA. 1 mL of MilliQ water+0.1% TFA was added to the glycan azide reaction solution before loading onto the carbograph column. The reaction tube was rinsed with 1 mL of 10% ACN+0.1% TFA and loaded onto the carbograph column. The carbograph column was then washed with 3 CV of 10% ACN+0.1% TFA. Glycans were eluted with 2 CV of 25% ACN+0.1% TFA followed by 1 CV of 50% ACN+0.1% TFA. Fractions were pooled and lyophilized.

[0168] For larger N-glycan type glycans (MW approximately 1,500-3,000 Da), conditions were adjusted. Final concentrations / volume / mol are shown in Table 2.

[0169] [Table 2]

[0170] Glycan coupling to 8-arm PEG biotin / alkyne linker Glycan-N3 and 8-arm PEG-biotin / alkyne linker were resuspended in PBS at 20 mg / mL and 15 mg / mL, respectively. Fresh solutions of 20 mM CuSO4, 20 mM THPTA, and 50 mM sodium ascorbate were prepared in PBS. The coupling reaction was carried out with a 21 molar excess of glycan-N3 compared to 8-arm PEG-biotin / alkyne under the following conditions: glycan-N3 0.45 mM, 8-arm PEG-biotin / alkyne 9.45 mM, CuSO4 0.2 mM, THPTA 1 mM, and sodium ascorbate 5 mM in a final volume of 200 μL sodium phosphate buffer. The reaction was incubated overnight at room temperature with gentle rotation.

[0171] The conjugated glycan-dendrimers were purified on a PD-10 G-25 column equilibrated with 10 mL of sodium phosphate buffer. The glycan-dendrimer solution was loaded onto the column and the column was washed with 10 mL of sodium phosphate buffer. Fractions of 1 mL were collected and fractions containing glycan-dendrimers were pooled and lyophilized. The dried octamer-H3 was reconstituted in 1 mg / mL sodium phosphate buffer, aliquoted, and frozen at -20°C.

[0172] Flow cytometry 1-2.10 7O13 YSD cells were washed once in TSM-BSA buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 1% BSA) and then incubated with 100 mM H3-PAA-biotin or H3-sp-biotin in 250 μL of TSM-BSA and 200 mM H3 glycan dendrimer or unmodified dendrimer with gentle shaking for 1 h at 4° C. O13 YSD cells were then washed three times with TSM-BSA and then incubated with 250 μL of streptavidin-PE (1:400 dilution) in TSM-BSA for 30 min at 4° C. in the dark with gentle shaking. O13 YSD cells were then washed three times with ice-cold TMS-BSA and resuspended in 250 μL TMS-BSA, to which 1 μL PI was added to stain live / dead cells. 125 μL of each sample was used for flow cytometry measurements.

[0173] ELISA Wells of a 96-well plate were coated with 200 μL of 0.05 M carbonate-bicarbonate buffer pH 9.6 containing 2 μg / ml O13-hIgG1 and incubated for 30 min at 37° C. Wells were blocked with TSM+3% BSA for 1 h at room temperature and then washed 3 times with TSM+0.05% Tween® 20 (TMS-T). Wells were incubated with either 100 nM H3-PAA-biotin, H3 glycan dendrimer, Lewis A-PAA-biotin, or unmodified dendrimer in 200 μL of TSM buffer for 1 h at room temperature. After washing 3 times with TSM-T, wells were incubated with 200 μL of TSM containing a 1:2000 dilution of avidin-HRP for 1 h at room temperature. Signal per well was revealed by adding 150 μL of TMB and reading using a plate reader equipped with a 630 nm filter.

[0174] result Blood H2-type trisaccharides (H3) were derivatized with an azide (N3) functional group on the C1 of the reducing monosaccharide (N-acetylglucosamine in the case of H3) (Figure 2A). The N3-tagged H3 glycans were "clicked" onto the alkyne arms of an 8-mer PEG-biotin / alkyne dendrimer (Figure 2B).

[0175] The H3 glycan dendrimers were tested by flow cytometry with a yeast clone expressing VLR O13, a variable lymphocyte receptor (VLR) highly specific for H3 (Collins, et al., Structure, 25(11):1667-78e4, doi:10.1016 / j.str.2017.09.003(2017)). Commercially available multivalent H3-PAA-biotin was used as a positive control, which has previously shown robust binding by O13 YSD cells. Commercially available monovalent H3-sp-biotin and unmodified 8mer PEG-biotin / alkyne dendrimer were used as negative controls.

[0176] The results showed that H3-PAA-biotin and H3 glycan dendrimer were bound by O13 YSD cells to a similar extent, if not greater extent, than for the H3 glycan dendrimer. Both unmodified 8-mer PEG-biotin / alkyne dendrimer and monovalent H3-sp-biotin showed no binding by O13 yeast (Figure 2C).

[0177] The generation of H-trisaccharide (H3) dendrimers described initially was repeated in full (azidation of H3, coupling to alkyne dendrimer, and functional assays). All data were similar and reproducible. Figure 2E shows the reactivity of PD10 fractions with O13-hIgG anti-H3 antibody after conjugation of PEG-alkyne dendrimer with H3-N3. Figure 2F shows the reactivity of O13-hIgG anti-H3 antibody to DBCO magnetic beads conjugated or not with H3-N3 (conjugated only with H3-N3, not with H3-dendrimer, this was done to demonstrate N3 modification of H3). FIG. 2G shows that H3-dendrimer coupled to magnetic beads is reactive to Aleuria Aurantia lectin (AAL, specific for α-linked fucose) and non-reactive to Concanavalin A (ConA, specific for mannose).

[0178] The results show that monovalent glycans (2H-type trisaccharide antigens or H3) to the H3-specific GBP (anti-H3 antibody) cannot be detected by flow cytometry, but binding of the anti-H3 antibody GBP to multimeric H3 generated by the disclosed scaffold is readily detected by flow cytometry. The dissociation constant (K d ) is 2 × 10 -6 M / L, which indicates excellent K for the glycan:GBP interaction. d It is thought that many glycans: GBP K d is > 10 -5 M / L, possibly 10 -3 M / L may be very poor. d =k off / k on and k on >>10 6 M -1 s -1Therefore, (see Fundamental Immunology 5th Edition, 2003 (William Paul) Chapter 4 Antigen-Antibody Interactions and Monoclonal Antibodies; Thermodynamics and Kinetics, Jay A. Berzofsky, Ira J. Berkower, Suzanne L. Epstein) off is K d =2×10 -6 About M / L <<2s -1 and the half-life of the monomeric H3:anti-H3 antibody complex is < <t 1 / 2 =ln2 / k off = 0.35s. Such transient complexes are unlikely to survive the washes / incubations required to remove unbound ligands (H3 glycans) and non-specifically bound materials prior to detection of specifically bound ligands. This is shown by experimental data: monomeric H3 bound to anti-H3 antibodies is not detected by flow cytometry, and furthermore, multimerization of H3 glycans by the disclosed scaffolds results in multimeric H3:anti-H3 antibody complexes that are easily detected by flow cytometry.

[0179] In addition to H3, several other glycans, including Lewis X tetrasaccharide, 3'- and 6'-sialylLacNAc, Globo-N-pentaose (Gb5), and Globo-N-tetraose (Gb4), have been azide-derivatized and used to generate the corresponding glycan dendrimers.

[0180] N-glycans from bovine fetuin, RNAse B, chicken ovalbumin, and human T lymphocyte Jurkat cells have been enzymatically released and will be used to confirm that this approach can be used with naturally released glycans.

[0181] In summary, these experiments illustrate the derivatization and incorporation of an azide tag on the H2-type trisaccharide (H3) using click chemistry, and the subsequent generation of H3 glycan dendrimers. Flow cytometry binding experiments showed that the H3 glycan dendrimer, but not the unmodified 8-mer PEG linker, was effectively bound by O13 YSD cells to a level similar to that of the commercially available H3-PAA. These results support the use of this strategy as a platform for the derivatization and display of any glycan (e.g., with a reducing end), including commercially available, synthetic, or naturally released glycans, in a multivalent manner. Monovalent glycans have very weak interactions with their glycan-binding partners, and as in nature, multivalent glycan display is required to support robust binding. The provided multivalent glycan dendrimers can be used, for example, to screen new glycan-binding molecules in any biological system to inhibit or activate glycan-dependent cell surface receptors and block glycan-dependent pathogen infection.

[0182] These experiments illustrate glycan dendrimers that support the multimeric display of glycans with biotin-functionalized linkers. Glycans with reducing ends from any source can be functionalized and coupled to octameric biotin linkers. These strategies provide an easy and adaptable alternative to currently commercially available multivalent glycan polymers.

[0183] The biotin handle provides experimental flexibility. For example, fluorescent tags can be added for flow cytometry / FACS and imaging experiments. Glycan dendrimers can also be captured by avidin / streptavidin handles for pull-down experiments. By using avidin / streptavidin functionalized beads, glycan dendrimers can be used to probe glycan-bound molecules in a flow cytometry setting, and by using bifunctional amine-avidin linkers, glycan dendrimers can be coupled to MagPlex® or MultiPlex® beads to be compatible with Multiplex Luminex technology.

[0184] Currently available commercial multivalent glycan polymers are rather limited and restricted to "small" glycans (1-4 monosaccharides), which in turn restricts experimental applications. The provided glycan dendrimer technology significantly expands the number of multivalent glycan polymers potentially available for a variety of applications, including probing carbohydrate-binding molecules, inhibition assays, and glycan-induced cell signaling experiments. Homogeneous purified glycans, as well as heterogeneous pools of glycans released from glycoproteins or complex tissues, can be derivatized and coupled to bifunctional multivalent linkers to create diverse multivalent glycan dendrimers.

[0185] In addition, the methodology provided offers the possibility to reconstitute the heterogeneity of cell or tissue glycones on multivalent bifunctional dendrimers.

[0186] Example 2: Generation of Glycan Dendrimers Using NHS / NH2 Coupling Chemistry material and method Conjugation of Man5- / Man9-AEAB to 8-arm-PEG-NHS compounds 8-arm-PEG-NHS (7 arms containing NHS groups, 1 arm containing a biotin group) (average molecular weight approx. 21 kDa) was purchased as a custom compound from CreativePEGWorks. Man5- and Man9-AEAB (1 mg) were purchased from NatGlycan LLC.

[0187] 500 μg of Man5-AEAB (approximately 360 nmol) or Man9-AEAB (approximately 240 nmol) was lyophilized in a 1.5 mL tube. 8-arm-PEG-NHS was resuspended at 21 mg / mL in 100 mM sodium phosphate, pH 8.5. 17.2 μL of 8-arm-PEG-NHS solution (approximately 17.2 nmol) was added to the tube containing the lyophilized Man5-AEAB, and 11.5 μL of 8-arm-PEG-NHS solution (approximately 11.5 nmol) was added to the tube containing the lyophilized Man9-AEAB. The mixture was incubated at room temperature for 3 hours with gentle rotation. The reaction was stopped and any unreacted NHS groups were quenched by adding 200 μL of 1 M Tris, pH 7.5. The dendrimer solution was buffer exchanged with PBS using a 5,000 MWCO Vivaspin 500 filter unit (Sartorius). The glycan-dendrimers were finally resuspended in PBS at 1 mg / mL.

[0188] Conjugation of Core 1-Thr, Core 2, 3, 4-Ser to 8-arm-PEG-NHS compounds Core 1-Thr, Core 2-Ser, Core 3-Ser, and Core 4-Ser were purchased as custom products from Chemily Glycoscience.

[0189] 350 nmol of Core1-T (~550 μg), Core2-S (~750 μg), Core3-S (~580 μg), or Core4-S (~780 μg) were lyophilized in 1.5 mL tubes. 8-arm-PEG-NHS was resuspended at 21 mg / mL in 100 mM sodium phosphate, pH 8.5. 50 μL of 8-arm-PEG-NHS solution (~50 nmol) was added to the tubes containing lyophilized Core1-T and Core2, 3, 4-S and incubated for 3 h at room temperature with gentle rotation. The reaction was then stopped and any unreacted NHS groups were quenched by adding 200 μL of 1 M Tris, pH 7.5. Finally, the dendrimer solution was buffer exchanged with PBS using 5,000 MWCO Vivaspin 500 tubes (Sartorius). The glycan-dendrimers were finally resuspended in PBS at 1 mg / mL.

[0190] Conjugation of GP100120 to 8-arm-PEG-NHS compounds Synthetic fibroin glycopeptide GP100120 (GAT*ES*RGAGAGA-NH2, where S*=Ser*=GalNAcα-O-Ser, T*=Thr*=GalNAcα-O-Thr (SEQ ID NO:5), containing a C-terminal amide) was purchased from Sussex Research.

[0191] 312 nmol of GP100120 (approximately 440 μg) was lyophilized in a 1.5 mL tube. 8-arm-PEG-NHS was resuspended at 21 mg / mL in 100 mM sodium phosphate, pH 8.5. 15 μL of the 8-arm-PEG-NHS solution (approximately 15 nmol) was added to the tube containing the lyophilized GP100120 and incubated at room temperature for 3 hours with gentle rotation. The reaction was then stopped and any unreacted NHS groups were quenched by adding 200 μL of 1 M Tris, pH 7.5. Finally, the dendrimer solution was buffer exchanged with PBS using a 5,000 MWCO Vivaspin 500 tube (Sartorius). The glycan-dendrimers were finally resuspended in PBS at 1 mg / mL.

[0192] Lectin assay of dendrimers To facilitate detection of the glycan-dendrimer, it was coupled to streptavidin magnetic beads (MagnaBind™, Fisher Scientific) via its biotin handle. Separation and washing of the magnetic beads was performed with a DynaMag™-Spin Magnet (Fisher Scientific). 30 μL of beads were washed once with 200 μL of PBS and then resuspended in 44 μL of PBS before adding 6.3 μL of glycan-dendrimer at 1 mg / mL. The bead-dendrimer mixture was incubated for 30-45 min at room temperature with gentle rotation. After removal of the solution, the beads were incubated with 3 drops of biotin solution (Streptavidin / Biotin Blocking Kit, Vector Laboratories) for 30 min at room temperature with gentle rotation. After removal of the biotin solution, the beads were further blocked by incubating with 200 μL of Tris Buffer Saline (TBS) + 5% BSA (30-45 min) at room temperature with gentle rotation. After removal of the TBS-5% BSA, the beads were washed once with PBS and resuspended in 50 μL of PBS.

[0193] Lectins ConA, PNA, GSLII, VVL, and AAL were purchased from Vector Laboratories as biotinylated proteins. Avidin-HRP was purchased from SouthernBiotech. Lectins and avidin-HRP were pre-complexed by incubating lectins (0.5 μg / mL ConA and 2 μg / mL all other lectins) and avidin-HRP (1:500 dilution) in TBS-1% BSA for 30-45 min at room temperature with gentle rotation. 3 μL of dendrimer beads were used per assay. Beads were incubated with 100 μL of pre-complexed lectin-avidin-HRP solution for 30-45 min at room temperature with gentle rotation. Beads were then washed 3 times with 250 μL of TBS+0.1% Tween® 20. 100 μL of TMB (Abcam) was added to the beads and transferred to a 96-well plate for signal detection.

[0194] result Additional glycan dendrimers were generated using an 8-arm-PEG compound (Creative PEGWorks) that contains an N-hydroxysuccinimide (NHS) reactive group (instead of the alkyne group used previously), which reacts with primary amines to form covalent bonds under very simple reaction conditions.

[0195] Man5 and Man9 N-glycans tagged with AEAB (NatGlycan) generate Man5- and Man9-dendrimers. The AEAB tag has a primary amine and reacts with NHS in phosphate buffer, pH 8.5 (Figure 3A-C). Incorporation of Man5 or Man9 N-glycans was confirmed by selective reactivity with concanavalin A lectin (ConA) but not with peanut agglutinin lectin (PNA). Unmodified PEG-NHS dendrimers showed no signal with either lectin (Figure 3D).

[0196] The same 8-arm-PEG-NHS compound was also used to couple the O-glycans Core1-Thr, Core2,3,4-Ser glycan-amino acid (Chemily Glycoscience) to generate Core1,2,3,4-dendrimers (Figure 4A-B). Core1-T- and Core2-S-dendrimers were found to be positive by Peanut Agglutinin (PNA, specific for galactose as found in T antigen) detection and negative for both ConA and Griffonia Simplicifolia Lectin II (GSLII, specific for terminal α- or β-linked N-acetylglucosamine) (Figure 4C). Similarly, Core3-S- and Core4-S-dendrimers showed moderate and high reactivity, respectively, for GSLII while being negative for PNA (Figure 4D).

[0197] A synthetic glycopeptide (GP100120, Sussex Research) containing two GalNacs linked to one serine and one threonine was conjugated to the same 8-arm-PEG-NHS compound (Figure 5A-C). The GP100120-dendrimer was found to be positive by Vicia Villosa lectin (VVL, specific for terminal N-acetylgalactosamine) detection and negative for PNA staining (Figure 5D).

[0198] 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 disclosed invention belongs. Publications cited herein and the materials cited therein are specifically incorporated by reference.

[0199] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. a multivalent glycan scaffold comprising an underlying branched scaffold having two or more glycans; the multivalent glycan scaffold is not immobilized; the underlying branched scaffold is a synthetic dendrimer or a synthetic hyperbranched polymer; and A multivalent glycan scaffold, wherein the multivalent glycan scaffold provides sufficient / appropriate spacing and flexibility for multimeric interactions between the multivalent glycan scaffold and a target substance that binds to the multivalent glycan scaffold.

2. 2. The multivalent glycan scaffold of claim 1, wherein the multivalent glycan scaffold is between 10 kDa and 50 kDa, or between 20 kDa and 60 kDa, or between 20 kDa and 30 kDa (inclusive), or any integer range thereof, or any specific integer therebetween.

3. The multivalent glycan scaffold of claim 1, comprising one or more functional elements linked to its termini, optionally wherein: (a) at least one of the functional elements is an agent that facilitates the formation, imaging, localization, identification, isolation, and / or characterization of the glycan scaffold and / or targeting element(s) bound to the glycan scaffold; and / or (b) a multivalent glycan scaffold, wherein at least one of the functional elements is a fluorophore, a nucleic acid comprising a barcode that specifically identifies the glycan scaffold, or an affinity tag. (a) the glycan is linked to 2-amino-N-(2-aminoethyl)-benzamide (AEAB), which is linked directly or indirectly to the terminus of the underlying branched scaffold; or (b) the glycan is linked to an amino acid that is linked directly or indirectly to a terminus of the underlying branched scaffold, and optionally wherein: (i) the amino acid is not part of a polypeptide chain; or (ii) the amino acid is part of a polypeptide chain; And further optionally, the polypeptide chain comprises two or more amino acids having linked glycans.

5. (a) the underlying scaffolding is formed of 2 to 16 arms (inclusive), optionally 8 to 16 arms; (b) the underlying scaffold and / or its arms are formed from polyethylene glycol (PEG) polymers; and / or (c) The multivalent glycan scaffold of claim 1, wherein the underlying scaffold is formed from 8-arm PEG-biotin / alkyne, biotin(1) / alkyne(7), MW 20k; dendroalkyne-PEG-dendroalkyne, PEG 10k, G4 32 alkyne; dendroalkyne-PEG-dendroalkyne, PEG 10k, G5 64 alkyne; or dendroalkyne-PEG-dendroalkyne, PEG 10k, G6 128 alkyne.

6. The multivalent glycan scaffold of claim 1 , wherein the two or more glycans are all the same type of glycan or are composed of two or more different types of glycans.

7. formula: (Ligand) ｎ : Scaffold: Detection / Separation Tag: Identifier Tag where "ligand" is one or more types of glycan that are present and have n>1 copies (i.e., multivalent); "Scaffold" is the underlying branched scaffold that is present and is selected from synthetic dendrimers or synthetic hyperbranched polymers; "detection / separation tag" refers to a vesicle in which one or both of a "detection / separation tag" and an "identifier tag" are present and contain a functional element, for example, (a) biotin or a (HIS) 6-8 peptide tag to facilitate separation / isolation, (b) a fluorophore to facilitate detection, (c) a unique DNA or RNA sequence ("barcode") that can be PCR amplified and sequenced to facilitate identification; and / or (d) a drug or other material as described elsewhere herein, e.g., a nucleic acid such as DNA, RNA, iRNA, crispr guide RNA, etc., or an antigen that is intended to have a therapeutic or cell-modifying effect or that may include (a) a protective immune response against pathogens or abnormal cells expressing the antigen, e.g., tumors, or (b) immune tolerance to the antigen as a treatment for autoimmune disorders against the antigen. The multivalent glycan scaffold of claim 1 .

8. A composition comprising a plurality of multivalent glycan scaffolds according to claim 1 in solution.

9. 10. A composition comprising a multivalent glycan scaffold according to any one of claims 1 to 8 for use in a method for identifying glycan-based binding interactions, the method comprising contacting a target substance with the multivalent glycan scaffold, the method comprising: (i) contacting a target substance with a known multivalent glycan scaffold according to any one of claims 1 to 8, comprising two or more glycans, and identifying an entity from the target substance that binds to the scaffold; or (ii) contacting one or more known target substance elements with one or more species of multivalent glycan scaffolds according to any one of claims 1 to 8, each comprising two or more glycans, and identifying glycan scaffolds that bind to the target substance elements; and optionally, the known target material component is one or more cell types.

10. the target material is a population of cells comprising one or more different cell types, or a cell lysate or other proteinaceous mixture comprising one or more glycan binding proteins (GBPs); Optionally, the target material is a population of cells and the target material element is one or more of the cell types within the population.

11. 10. The composition of claim 9, wherein the one or more cell types preferentially bind to the multivalent glycan scaffold compared to other cell types in the population.

12. The composition described in claim 9, wherein the method further comprises isolating the multivalent scaffold to which the cells are bound, and wherein the method further comprises, if necessary, characterizing the bound cells.

13. The method of claim 12, wherein the target substance is a cell lysate or other proteinaceous mixture, and the target substance component is one or more of the GBPs therein; Optionally, the one or more GBPs preferentially bind to the multivalent glycan scaffold relative to other GBPs in the cell lysate or other proteinaceous mixture; 10. The composition of claim 9, wherein the method optionally further comprises characterizing the bound GBP and / or the glycan bound to the GBP.

14. The method comprising (i), and further comprising contacting the one or more known target material elements with a library of different species of the multivalent glycan scaffolds, each comprising two or more glycans, and identifying glycan scaffolds that bind to the target material elements, optionally wherein the library comprises from about 2 to about 10,000, or any subrange or specified integer therein, e.g., 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000, or 10. The composition of claim 9, wherein the method comprises (ii), and the method further comprises contacting a target substance with one or more unknown multivalent glycan scaffolds comprising two or more glycans, and identifying elements from the target substance that bind to the multivalent glycan scaffolds, wherein optionally the one or more unknown multivalent glycan scaffolds form a library comprising about 2 to about 10,000, or any subrange or specified integer therein, e.g., 2, 5, 10, 25, 50, 75, 100, 250, 500, 1,000 different species of glycan scaffolds.

15. 10. A composition comprising the multivalent glycan scaffold of any one of claims 1 to 8 for use in a method of treating a subject in need thereof, said method comprising identifying glycan-based binding interactions according to any of the preceding methods, and administering to said subject an effective amount of a composition comprising said therapeutic multivalent glycan scaffold comprising said glycans of said identified interactions, and optionally (a) the therapeutic glycan induces or inhibits cellular activity upon binding to the cell or GBP of said interaction; or (b) the therapeutic glycan comprises an active agent functionalized thereon, e.g., an active agent selected from the group consisting of nucleic acids, small molecules, and polypeptides, or the cell is a cancer cell and the active agent is an anti-cancer drug; or The composition, wherein the cell is an immune cell and the active agent is an antigen or encodes an antigen.

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