Polysialic acid conjugate and methods of making and using thereof

Abstract

The present invention, in embodiments, relates to polysialic acid conjugates comprising polysaccharides with repeating units of α-2,8-linked N-acetylneuraminic acid residues that are covalently linked to one or more polypeptide antigens. These polysialic acid-polypeptide conjugates are synthesized chemically to induce antigen-specific immune tolerance via the tolerogenic polysialic acid polysaccharide portion of the conjugate. The conjugates of the present invention can be used to alleviate symptoms arising from food allergies and other gut inflammatory and autoimmune diseases and conditions.

Description

Attorney Docket No. 54880-0002W01POLYSIALIC ACID CONJUGATE AND METHODS OF MAKING AND USING THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is an International Application which claims priority to U.S.Provisional Patent Application No. 63 / 755,596, filed February 7, 2025. The disclosure of the foregoing application is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to polysialic acid conjugates synthesized by chemically linking the polysialic acid to one or more proteins that are known to possess antigenic or allergic properties in mammals. Polysialic acid is a self-antigen with tolerogenic properties. When conjugated to an antigenic protein, polysialic acid can induce antigen-specific immune tolerance responses towards said protein, thereby reversing the effects of inflammatory and allergic responses towards the antigen and can result in alleviation of symptoms arising from food allergies and other gut inflammatory and autoimmune diseases and conditions.BACKGROUND OF THE INVENTION

[0003] The critical role T regulatory (Treg) cells play in inducing immune tolerance in mammals has been increasingly recognized and gaining attention in recent scientific literature. Treg cells are a specialized subset of T cells that play a key role in maintaining immune tolerance and preventing autoimmune responses. Treg cells are characterized by the expression of the transcription factor and surface markers including FoxP3, CD25, and CD4. Treg cells help modulate the immune system by suppressing the activation and proliferation of other immune cells, such as effector T cells (e.g. Thl and Th2 cells) and B cells via various mechanisms and pathways, for example, by promoting the production of antiinflammatory cytokines (e.g. IL-10, TGF-b), blockage of co-stimulatory signals that are necessary for immune activation via direct cell contact with T effector cells or antigen presenting cells (APCs), or consumption of key immune activation growth factor (IL-2).

[0004] In the context of food allergies, Treg cells can induce and maintain immune tolerance to innocuous substances, such as food proteins and polypeptides by dampening the activation of Th2 and B cells, via direct interaction, production of anti-inflammatory cytokines (IL- 10, TGF-b), or modulation of dendritic cells. Inhibition of Th2 and B cells in turn can reduce allergic inflammation by reducing IgE production in food allergies. By physically linkingAttorney Docket No. 54880-0002W01(conjugating) substances that can induce or up-regulate the expression of Treg cells to food allergens, the immunological responses towards these food allergens can be re-programmed towards immune tolerance. Tolerogenic substances and constructs thought to induce or up-regulate Treg cell expression are known in literature, many of which are carbohydrates. For example, oligomannose, sialyllactose, and N-acetylgalactosamine, have been shown to possess such tolerogenic properties (see, for example, US9901633). Conjugation of these tolerogenic carbohydrates to various antigenic polypeptides has demonstrated induction of immune tolerance towards various antigens in vitro and in animal models.

[0005] The instant application describes conjugates and compositions comprising the naturally occurring polysaccharide polysialic acid (also known as PSA, PolySia, or colominic acid), a linear polymer consisting of repeating units of a-2,8-linked N-acetylneuraminic acid residues. The terms “PolySia”, “PSA”, and “colominic acid” are used interchangeably throughout the present disclosure to refer to “polysialic acid.” PolySia is found in both mammalian and bacterial cells. In humans, polysialic acid is a rare post-translational modification found on neural cell adhesion molecules (NCAM) where its putative function is to modulate cell-cell interactions, migration, and plasticity. PolySia is highly expressed during embryonic development, but its expression is limited in most adult tissues. However, aberrant re-expression of PolySia has been observed in various cancers, making it a potential biomarker for cancer progression, prognosis, and diagnosis. By modulating cell surface properties, PolySia enhances cell motility and evasion of cancer cells from immune surveillance. PolySia is also a major structural component of the bacterial capsule in the human pathogens Neisseria meningitidis serotype B and E coli KI; PolySia is a major virulence factor that can help the bacteria to avoid recognition and evade the host immune system via molecular mimicry. As such, unlike other Neisseria meningitidis serotypes (A, C, Y, and W), previous efforts in developing a capsular polysaccharide-conjugate vaccine for Neisseria meningitidis serotype B have been largely unsuccessful. Vaccines targeting serogroup B meningococci use recombinant proteins or outer membrane vesicles rather than the PolySia capsule (e.g. Bexero® and Trumenba®). These observations support the hypothesis that PolySia structure possesses tolerogenic properties, which can be harnessed to induce antigen-specific tolerance as potential treatment for allergies and autoimmune diseases and conditions.Attorney Docket No. 54880-0002W01SUMMARY OF THE INVENTION

[0006] One embodiment of this invention pertains to a poly sialic acid conjugate for inducing antigen-specific immune tolerance responses, wherein the polysialic acid conjugate comprises (a) one or more polysialic acid polysaccharides, comprising repeating units of a-2,8-linked N-acetylneuraminic acid residues; (b) one or more polypeptide antigens; (c) one or more covalent linkages between the polysialic acid polysaccharides and the polypeptide antigens.

[0007] In one embodiment, the polysialic acid polysaccharides are naturally occurring carbohydrates isolated from a micro-organism. In another embodiment, the polysialic acid polysaccharides are made via chemical synthesis.

[0008] In one embodiment, the polysialic acid polysaccharides are a-2, 8 -linked N-acetylneuraminic acid residue repeating units with the following structure:defined herein.

[0009] In one embodiment, the polysialic acid polysaccharides conjugated to one or more polypeptide antigens comprise the following structure:wherein m is defined herein.

[0010] In another embodiment, the polysialic acid polysaccharides conjugated to one or more polypeptide antigens comprise the following structure:Attorney Docket No. 54880-0002W01, wherein the linker is defined herein.

[0011] In another embodiment, the polysialic acid polysaccharides conjugated to one or more polypeptide antigens comprise the following structure:, wherein m is defined herein.

[0012] In one embodiment, the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens by activating one or more of the polysialic acid polysaccharide hydroxyl groups via CDAP chemistry. In another embodiment, the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens byAttorney Docket No. 54880-0002W01activating one or more of the polysialic acid polysaccharide carboxyl groups via EDAC chemistry. In one embodiment, the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens by first de-N-acetylating one or more of the polysialic acid polysaccharide N-Ac groups to provide free primary amino groups at C5.

[0013] In one embodiment, the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the lysine residues of the polypeptide. In another embodiment, the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the cysteine residues of the polypeptide. In one embodiment, the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the glutamine residues of the polypeptide. In one embodiment, the poly sialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the glutamic acid or aspartic acid residues of the polypeptide.

[0014] In one embodiment, the weight ratio of polysialic acid polysaccharides to polypeptide antigens is between about 1% and 75%.

[0015] In one embodiment, the weight ratio of polysialic acid polysaccharides to polypeptide antigens is between about 0.1% and 10%.

[0016] In one embodiment, the weight ratio of polysialic acid polysaccharides to polypeptide antigens is about 50%.

[0017] In one embodiment, the weight ratio of polysialic acid polysaccharides to polypeptide antigens is about 0.9%.

[0018] In one embodiment, wherein the one or more polypeptide antigens is a food allergen.

[0019] In one embodiment, the composition further comprises one or more shortchain fatty acids (SCFA).

[0020] In one embodiment, the one or more SCFA is conjugated to polysialic acid polysaccharides via ester linkages.

[0021] Another aspect of this invention relates to a method for inducing tolerance to a food allergen in a subject, comprising administering a composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to a food allergen. In one embodiment, the subject is a human. In one embodiment, the subject is a dog. In one embodiment, the subject is a cat.

[0022] Another aspect of this invention relates to a method for inducing tolerance to an autoantigen in a subject, comprising administering a composition comprising polysialicAttorney Docket No. 54880-0002W01acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to an autoantigen.BRIEF DESCRIPTION OF THE FIGURES

[0023] For a more complete understanding of the invention and the advantages thereof, reference is made to the following descriptions, taken in conjunction with the accompanying figures, in which:

[0024] FIG 1 shows a representative size-exclusion chromatograph (SEC) of purified polysialic acid-beta casein conjugate overlaid with polysialic acid and beta-casein separately. The difference in elution times on the SEC for peaks corresponding to polysialic acid, betacasein, and polysialic acid-casein conjugate provides experimental evidence that the physicochemical properties of the conjugate are different from the components that give rise to the conjugate.

[0025] FIG 2 illustrates the experimental design of the mouse allergy model to evaluate effects of polysialic acid-casein conjugate on serum IgG titers in C3H / HeJ mice sensitized towards casein.

[0026] FIG 3A illustrates casein-specific IgG titers in serum samples from Group 1 of the mouse study, where the three mice in this group were sensitized, then treated with polysialic acid-casein conjugate (5 pg per mouse). Mouse 1-1 showed sharp decrease of IgG titers after two treatments with the polysialic acid-casein conjugate to baseline level through the end of the study.

[0027] FIG 3B illustrates casein-specific IgG titers in serum samples from Group 2 of the mouse study, where the three mice in this group were sensitized, then treated with betacasein (100 pg per mouse). Mouse 2-1 was the only mouse that was successfully sensitized towards casein, however, treatment with casein alone did not induce oral tolerance towards casein or reversal of the immunological memory (IgG) against casein.

[0028] FIG 3C illustrates casein-specific IgG titers in serum samples from Group 3 of the mouse study, where the three mice in this group were sensitized, then treated with PBS. Mouse 3-1 was the only mouse that was somewhat sensitized towards casein, however, the IgG titer at the start of the treatment phase was still much lower than the starting IgG titers of mouse 1-1 and 2-1. The slight decrease in IgG titer upon treatment with PBS may have been due to ELISA assay variability.Attorney Docket No. 54880-0002W01

[0029] FIG 3D illustrates the absence of casein-specific IgG titers in serum samples from Group 4 where the three mice in this group were not sensitized towards casein, nor treated with either polysialic acid-casein conjugate or beta-casein, which are potential tolerogenic substances (i.e. negative control). This negative control was included to confirm that any IgG titer observed in the sensitized groups (Groups 1-3) is due to sensitization with casein + cholera toxin adjuvant.DETAILED DESCRIPTION OF THE INVENTION

[0030] Among those benefits and improvements that have been disclosed, other objects and advantages of this disclosure will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the disclosure that may be embodied in various forms. In addition, each of the examples given regarding the various embodiments of the disclosure are intended to be illustrative, and not restrictive.

[0031] Throughout the specifications and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases "in one embodiment," “in an embodiment,” and "in some embodiments" as used herein do not necessarily refer to the same embodiment s), though they may. Furthermore, the phrases "in another embodiment" and "in some other embodiments" as used herein do not necessarily refer to a different embodiment, although they may. All embodiments of the disclosure are intended to be combinable without departing from the scope or spirit of the disclosure.

[0032] As used herein, the term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on”.

[0033] As used herein, terms such as “comprising”, “including” and “having” do not limit the scope of a specific claim to the materials or steps recited by the claim.

[0034] As used herein, the term “about” is used to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%.Attorney Docket No. 54880-0002W01

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skills in the art to which this invention pertains.

[0036] The term “antigen” broadly defines a molecule, moiety, or particulate matter that can be recognized by the immune system. Examples of an antigen include, but are not limited to, proteins, polypeptides, oligosaccharides, polysaccharides, small molecules, lipids, nucleic acids, or conjugates and complexes formed between protein and small molecules, polypeptide and small molecules, protein and oligosaccharides, protein and polysaccharides, polypeptide and polysaccharides, protein and nucleic acids, polypeptide and nucleic acids, lipid-linked polysaccharides, or lipid-linked proteins, etc.

[0037] Antigens can originate either from within the subject’s body (“self-antigen”, “endogenous”, or “autoantigen”) or from the external environment, such as from an infectious micro-organism including bacteria, viruses, protozoans, (e.g. Plasmodium, helminths, fungi), pets and animals (cats, dogs, small rodents, horses, sheep, etc.), or plants. Antigens derived from the external environment are known as “foreign antigen”, “exogenous”, or “non-self antigen.”

[0038] Antigens can be further classified as “T-cell dependent” (T dependent) or “T-cell independent” (T independent) antigens. T dependent antigens require T cell assistance in eliciting antibody production by B cells, whereby T independent antigens can stimulate B cells directly to induce antibody production. Typical examples of T dependent antigens include proteins and polypeptides, which possess antigenic determinants (epitopes) for both B and T cells that are recognized to the corresponding receptors on each cell type. Upon internalization, processing and presentation by B cells, fragments of the T dependent antigens are displayed on the surface of the B cells via major histocompatibility complexes (MHC) class II molecules. T helper cells recognize these MHC Il-presented antigen fragments through T cell receptors (TCR). Furthermore, interaction between B cells and T helper cells via the CD40-CD40L receptor-ligand complex provides the co-stimulatory signals for full activation of both the B and T helper cells, which is characterized by the release of various cytokines by the T helper cells and proliferation and differentiation of the activated B cells into plasma cells and memory B cells, leading to antibody production.

[0039] In contrast, T independent antigens can directly stimulate B cells to produce antibodies without the involvement of T helper cells, since they do not possess epitopes that can be recognized by T helper cells. T independent antigens typically feature repetitive structures and can crosslink multiple BCRs on the surface of a B cell, leading to B cellAttorney Docket No. 54880-0002W01activation. T independent antigens can induce a rapid immune response which is a crucial defense mechanism against pathogens, however, the resulting immune response is usually not as durable and long-lasting compared to those generated by T dependent antigens. Some examples of T independent antigens include polysaccharides that are commonly found in the cell wall (capsule) of bacteria, lipopolysaccharides (LPS) which are found on the outer membrane of Gram-negative bacteria, bacterial flagellin, repetitive antigenic structures (e.g. viral capsid proteins, ferritin nanoparticles, small molecule haptens displayed on viral capsids or ferritin, etc.), and nucleic acids (DNA and RNA).

[0040] The term “immune tolerance,” as used herein, refers to the process by which the immune system becomes unresponsive or less responsive to certain specific antigens, thereby preventing it from attacking the antigen. There are two main types of immune tolerance: central tolerance and peripheral tolerance. Central tolerance occurs in the thymus (for T cells) and bone marrow (for B cells) during the development of the immune cells, where immune cells that have a strong reaction towards self-antigens are eliminated or deactivated to prevent autoimmune responses. Peripheral tolerance occurs outside of the thymus and bone marrow, essentially anywhere in the body but is particularly important in mucosal surfaces along the GI tract (mouth, esophagus, stomach, the intestines), the respiratory tract (nasal cavity, trachea, bronchi, and lungs), the urogenital tract (urethra, bladder, vagina, and cervix), and the conjunctiva of the eye, where frequent exposures to external antigens occur. Peripheral tolerance is actively employed on mucosal surfaces to prevent immune responses towards harmless antigens (e.g. food components and commensal bacteria). The mucosal surfaces are integral to immune regulation, serving as the first line of defense against harmful antigens and pathogens, while simultaneously preventing unnecessary inflammation and damage to tissues and organs from harmless antigens like food, microbiota, or environmental substances.

[0041] Both central and peripheral tolerance mechanisms contribute to the maintenance of tolerance towards self-antigens throughout life; a loss of such tolerance can lead to various autoimmune diseases and conditions. Tolerance can be “natural” or “induced”, where in the case of natural tolerance, the immune system has evolved to recognize certain structures and motifs as harmless and does not mount an immune response against these; the immune system can also be manipulated to create “induced” tolerance against specific antigens. Strategies to induce peripheral tolerance known in the art include induction of oral tolerance, immune modulatory therapy (e.g. anti-cytokine therapies, promotion of Treg cells via TGF-b or IL-10), subcutaneous or sublingual allergen-specificAttorney Docket No. 54880-0002W01immunotherapy (AIT), the use of prebiotics and probiotics to modulate the gut microbiome, and administration of regulatory antibodies that can block costimulatory molecules like CD28 or CD40 to dampen immune cell activation. In the context of food allergies, oral immunotherapy (OIT) has been employed to reduce the risk of severe allergic reactions (i.e. anaphylaxis) and fatality due to accidental exposure to food allergens. In OIT, patients with food allergies are subjected to very low amount of the allergen, which is gradually increased to induce tolerance to the food allergen. This process must be done under close medical supervision, and the determination of the starting dose and rate of increasing dose amounts must be determined carefully to avoid having too high of a starting dose which could trigger anaphylaxis. The exact mechanism of how OIT works is not well understood, although it has been proposed that Treg cells play a key role in facilitating immune tolerance (see, for example, review by Pinheiro-Rosa et al., Immunotherapy Advances 2021). However, it is not known what the “cutoff’ amount of food allergen should be that would induce Treg cells rather than triggering an immune response. Furthermore, durability of the induced tolerance from OIT is not known, and patients must continue to consume the amount of allergen determined to be sufficient for desensitization on a daily or regular basis. Therefore, OIT, as it is currently designed, is not a cure for food allergies.

[0042] In subjects with food allergies, a loss of peripheral tolerance in the guts leads to an inappropriate immune response towards harmless food proteins, which the immune system misidentifies as harmful. Impairment or insufficient T regulatory (Treg) cells have been implicated as a significant contributing factor to the development and severity of food allergies. First identified in the late 1980s and early 1990s, Treg cells are a distinct subset of T cells that can suppress the activity of other immune cells to prevent overactive immune responses. Treg cells are characterized by several key markers, including the transcription factor FoxP3, surface markers CD4 and CD25, and CTLA-4, that help identify them among other immune cells. Treg cells exert their immunosuppressive and tolerogenic effects through various mechanisms, including the secretion of anti-inflammatory cytokines IL- 10 and TGF-P, direct cell-cell contact, and competition for resources with effector T cells. Treg cells can also influence antibody production by B cells via several different mechanisms, including induction of apoptosis of B cells, suppression of IgG antibody production, inactivation of autoreactive B cells in the periphery, and selective suppression of B cells in an antigenspecific manner.

[0043] Several strategies are known to induce or activate Treg cells, for example, via cytokine signaling where IL-2, TGF-P, and IL-10, are important in the differentiation,Attorney Docket No. 54880-0002W01development, and functioning of Treg cells; presence of specific small molecules, supplements, and microbiota metabolites, such as retinoic acid (vitamin A), vitamin D, and short-chain fatty acids (SCFAs) such as acetic acid / acetate, propionic acid / propi onate, and butyric acid / butyrate; via influence of the gut microbiota; targeting of checkpoint molecules such as CTLA-4 and PD-1; of particular importance in the present disclosure is the activation of Treg’ s T-cell receptor (TCR) to recognize specific self-antigens in the context of MHC class II molecules, which can result in Treg cell population expansion and activation in vivo. In peripheral tissues, Treg cells can recognize self-antigens that are presented by antigen-presenting cells (APCs), which is crucial in the maintenance of immune tolerance.Therapeutic strategies that use self-antigens to expand or activate Treg cells to induce tolerance are an emerging area of research; some examples include, among others, vaccines based on insulin (for Type 1 diabetes, T1D), Myelin Basic Protein (MBP) (for multiple sclerosis, MS), or collagen (for rheumatoid arthritis, RA).

[0044] While many of the therapeutic strategies utilize protein or polypeptide based self-antigens to induce immune tolerance, carbohydrates have also been shown to have similar tolerogenic effects via Treg induction. For example, oligomannose glycans that bind the mannose receptors such as DC-SIGN (dendritic cell-specific ICAN-3 -grabbing non-integrin) on dendritic cells and MGL (macrophage galactose-type lectin) on macrophages; sialyllactose, which is a glycan consisting of a sialic acid moiety attached to the disaccharide lactose (P-D-galactopyranosyl-(l->4)-D-glucose), binds to Siglecs (sialic acid-binding immunoglobulin-type lectins) that are expressed on the surface of immune cells including dendritic cells, macrophages, and Treg cells, among others. Delivery of these carbohydrates to target the corresponding immune cell surface receptors have been shown to induce Treg cells and suppresses effector T cells (Zhang, W., et al. Front. Immunol. 2021; WO 2022 / 076435; US 9,180,182).

[0045] Sialic acids, which are a class of alpha-keto acid sugars with a nine-carbon backbone, are commonly found as the terminal sugar on glycopeptides and glycolipids in human and other animals, and prokaryotes. The most common mammalian sialic acids are N-acetylneuraminic acid (Neu5Ac), and N-glycolylneuraminic acid (Neu5Gc). However, humans do not naturally synthesize Neu5Gc due to a mutation in the gene responsible for conversion of Neu5Gc from Neu5Ac. Sialic acids can be linked to the underlying glycan via glycosidic linkages, wherein the sialic acids are often found as the terminal sugar residues of the glycan chains that are linked to proteins and lipids where they serve as ligands for lectinsAttorney Docket No. 54880-0002W01(carbohydrate-binding proteins) such as Siglecs, and can play essential roles in cell-cell communication, immune modulation, and other biological processes. There are numerous examples of sialylated glycans in humans, including sialylated N-glycans of transferrin, certain IgG and IgA antibodies, sialylated O-glycans of mucins, sialylated gangliosides (GM1, GDla, GDlb, GTlb), sialylated glycosphingolipids, sialylated proteoglycans, sialylated lectins (selectins), and sialylated neural cell adhesion molecules (NCAMs) in the nervous system. Aside from existing as the terminal residue of N- or O-linked glycans, sialic acids can also be found as a homopolymer. Polysialic acids (PSA, PolySia) are long chain polysaccharides made of Neu5Ac or Neu5Gc repeating units linked together, typically via a2,8-, or a2,9- glycosidic linkages, although in humans, only polysialic acid polysaccharides consisting of Neu5Ac are present. The length of PolySia can vary greatly depending on the source and the method of production, but typically in the range of 5 to 100 repeating units. Due to the presence of the carboxyl groups at Cl of PolySia, it is a negatively-charged (anionic) polymer at physiological pH of around 6.5-7.5.

[0046] Polysialic acids are found on the surface of cells and proteins, most prominently, on the surface of neural cells being a major modification of NCAM in human and other mammals including mice, rats, primates, dogs, birds, reptiles, amphibians, and fish. In humans, NCAMs are cell surface proteins that can affect cell recognition, adhesion, and signaling, especially within the nervous system. NCAMs play a particularly important role in neuronal development and synaptic plasticity. Modification of NCAM with PolySia reduces its adhesive interactions between cells, which can impact neural development and proper functioning of the nervous system. Therefore, PolySia modification of NCAMs is tightly regulated. PolySia is also produced by certain bacteria, such as Neisseria meningitis serotype B (MenB) and E. coli, where PolySia is a major structural component of the outer membrane or capsule of the bacteria that can also help the bacteria evade immune surveillance in the host through “molecular mimicry”, since PolySia is also found in host cell proteins and recognized as a “self’ antigen.

[0047] Polysialic acid can be isolated from natural sources, for example, from E. coli KI bacterial culture (see, for example, Vann et al. 1981 Eur. J. Biochem.). It can also be produced via chemical or enzymatic synthesis (see, for example, Rojas et al. 2001 J. Biol. Chem.; Sutherland et al., 2010, Nat. Chem.; Moremen et al, 2003, Biochemistry; McKee et al., 2006, Glycobiology; Moremen et al, 2011, J. Mol. Biol.). Commercial sources of polysialic acid as sodium salt, include, for example, Millipore Sigma (catalog # C5762 or 27698), Biosynth (catalog # YC11298), or Alfa Chemistry (catalog # ACM70431344), etc.Attorney Docket No. 54880-0002W01Depending on how the polysialic acid is produced by the different manufacturers, quality attributes and characteristics of commercial polysialic acid including identity, purity, average molecular weight, sodium content, etc., are controlled and verified by the manufacturer.

[0048] In some embodiments, compositions for inducing antigen-specific immune tolerance responses comprise polysialic acid polysaccharides with a-2,8-linkedN-acetylneuraminic acid (Neu5Ac) residue repeating units having the following structure:, wherein n is defined herein.

[0049] In some embodiments, the number of repeating units (n) of the PolySia polysaccharide chains can be from 5 to 5000 repeating units, from 5 to 1000 repeating units, from 5 to 500 repeating units, from 5 to 200 repeating units, and from 5 to 50 repeating units. In some embodiments, the number of repeating units (n) of the PolySia polysaccharide chains is at least 5. In some embodiments, the number of repeating units (n) of the PolySia polysaccharide chains is at least 20. In some embodiments, the number of repeating units (n) of the PolySia polysaccharide chains is at least 50. In some embodiments, the number of repeating units (n) of the PolySia polysaccharide chains is at least 100.

[0050] Various chemistries can be employed to produce conjugates of polysialic acid and proteins or polypeptides. Examples of common chemistries used in carbohydrate activation known in the art include, but are not limited to, modification of carbohydrate hydroxy groups with cyanylating agents (e.g., CNBr and CDAP) or carbonyldiimidazole to form active esters; periodate oxidation of vicinal diols in the sugar ring or at glycerol moiety (e.g. in sialic acid) to generate aldehydes ready for conjugation to the amino groups of proteins via reductive amination; activation of carboxyl groups of carbohydrate uronic acids by carbodiimide (e.g. DCC, DIC, EDC (ED AC), or CMCT). Alternatively, amino groups can be introduced with ammonium salts or with di-hydrazide spacers to the carbohydrate, which can react directly with carboxylic acids of proteins through carbodiimide chemistry or coupled to a variety of bifunctional linkers to incorporate squaric ester, mal eimide, thiol, azide, hydrazide, hydrazine, or alkyne moieties for further conjugation. In cases where chemoselective chemistry (e.g., Michael reaction, click chemistry) is used in the preparation of conjugates between carbohydrates and proteins, the protein is modified accordingly to introduce the appropriate orthogonal chemical groups to react with the linker or chemical groups introduced onto the carbohydrate. In many cases, the activation of carbohydratesAttorney Docket No. 54880-0002W01and / or modification of the protein is random where multiple sites of attachment are possible, resulting in a heterogeneous mixture of cross-linked, lattice-type conjugates.

[0051] In some embodiments, the polysialic acid polysaccharides are covalently linked to one or more polypeptide antigens by activating one or more of the poly sialic acid polysaccharide hydroxyl groups via CDAP chemistry. In some embodiments, the polysialic acid polysaccharides are covalently linked to one or more polypeptide antigens by activating one or more of the polysialic acid polysaccharide carboxyl groups via peptide coupling chemistry, e.g., ED AC chemistry. In some embodiments, the N-acetyl groups of the N-acetylneuraminic acid repeating units in the PolySia polysaccharides are partially removed to yield free primary amino groups, which are then covalently linked to the glutamic acid or aspartic acid residues present in the one or more polypeptide antigens using standard peptide coupling chemistry. In some embodiments, the N-acetyl groups of the N-acetylneuraminic acid repeating units in the PolySia polysaccharides are partially removed to yield free primary amino groups, which are then covalently linked to one or more polypeptide antigens using transglutaminase-mediated cross-coupling reaction to the glutamine residues present in the one or more polypeptide antigens.

[0052] As used herein, the term “conjugate” means one or more polysialic acid polysaccharide chains linked (i.e., covalently linked) to a polypeptide or protein. Conjugation of protein molecules to PolySia can occur via the free hydroxyl groups at C4, C7 and / or C9 of each A-acetylneuraminic acid residue (repeating unit), the carboxyl groups at C2, or the amino groups at C5 after removal of the N-acetyl groups.

[0053] In some embodiments, the composition comprises polysialic acids conjugated to one or more polypeptide antigens having the following structure:wherein m is defined herein.Attorney Docket No. 54880-0002W01

[0054] Conjugation to the protein at any of the viable positions (C2, C4, C7, C9, or C5-amino) of any Neu5Ac residue does not imply conjugation at the same positions in all Neu5Ac residues within the same PolySia chain. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the hydroxyl groups at C4 of any Neu5Ac residue, so that Ri is X where X represents the antigen. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the hydroxyl groups at C7 of any Neu5Ac residue, so that R2 is X where X represents the antigen. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the hydroxyl groups at C9 of any Neu5 Ac residue, so that R3 is X where X represents the antigen. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the carboxyl groups at C2 of any Neu5Ac residue, so that R4 is Y where Y represents the antigen. In some embodiments, at least one of Ri, R2, or R3 is H. In some embodiments, Ri, R2, and R3 are H and R4 is Y. In some embodiments, at least one of Ri, R2, or R3 is X and R4 is Y. In some embodiments, at least one of Ri, R2, or R3 is H and R4 is Y. In some embodiments, at least one of Ri, R2, or R3 is X. In some embodiments, Ri or R2, is X. In some embodiments, R2 or R3, is X. In some embodiments, Ri or R3, is X. In some embodiments, both Ri and R2 are X. In some embodiments, both R2 and R3 are X. In some embodiments, both Ri and R3 are X. In some embodiments, Ri, R2 and R3 are X. In some embodiments, R2 or R3, is X and R4 is OH. In some embodiments, Ri or R3, is X and R4 is OH. In some embodiments, both Ri and R2 are X and R4 is OH. In some embodiments, both R2 and R3 are X and R4 is OH. In some embodiments, both Ri and R3 are X and R4 is OH. In some embodiments, Ri, R2 and R3 are X and R4 is OH.

[0055] In some embodiments, the composition comprises polysialic acids conjugated to one or more polypeptide antigens having the following structure:Attorney Docket No. 54880-0002W01

[0056] In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the amino groups at C5 of any Neu5Ac residue, so that Rs is P or Q, where P represents a polypeptide antigen and Q represents a polypeptide antigen with a linker.Primary amino groups at C5 of Neu5Ac can be obtained via partial de-N-acetylation by acidic or basic hydrolysis. Subsequently, the free primary amino groups can be conjugated to either carboxylic acid groups of aspartic acid or glutamic acid residues using suitable coupling reagents such as ED AC, or to the glutamine residues present in the polypeptide antigens using transglutaminase-catalyzed cross-coupling reaction. The transglutaminase used for cross-coupling reaction can be derived from a microbial source (e.g. Millipore Sigma Cat #. SAE0159) or a mammalian source (e.g., guinea pig liver, Millipore Sigma Cat #. T5398), and can be either purified in its native form or modified to have enhanced catalytic activity (e.g. eMTG from Merck KGaA, Millipore Sigma Cat #. SAE0217). In some embodiments, R s Z where Z is a fluorophore. As defined herein, a fluorophore is a molecule that absorbs light at one wavelength (excitation) and re-emits it at a longer, lower-energy wavelength (emission), acting as a fluorescent tag in biological imaging (microscopy) and flow cytometry applications. Commonly used small-molecule fluorophores are known to those skilled in the art, for example, fluorescein, Alexa Fluor™, coumarins, cyanins, XFD488 hydroxlamine, etc. In some embodiments, Rs is P and R4 is Z. In some embodiments, Rs is PAttorney Docket No. 54880-0002W01and R4 is OH. In some embodiments, R5 is Q and R4 is Z. In some embodiments, R5 is Q and R4 is OH.

[0057] As used herein, “polypeptides” or “proteins” are polymers of amino acids having, for example, from 2 to about 1000 or more amino acid residues. The terms “polypeptides” and “proteins” are used interchangeably herein. In some embodiments, “polypeptides” have from 10 to about 130 amino acids, from 10 to about 220 amino acids, from 10 to about 500 amino acids, or from 10 to about 730 amino acids. Any naturally occurring or synthetic amino acid can form the polypeptide. Polypeptides can also include modifications such as glycosylations, phosphorylation, lipidation, carb amyl ati on, deamidation, and other post-translational modifications.

[0058] Conjugation of the protein or polypeptide to the PolySia polysaccharide chain can occur with both natural and non-natural amino acid residues present in the sequence of the polypeptide chains. In some embodiments, the proteins or polypeptides are conjugated to an activated hydroxyl or carboxyl groups of PolySia via lysine residues. In some embodiments, the proteins or polypeptides are conjugated to an activated hydroxyl or carboxyl groups of PolySia via cysteine residues. In some embodiments, the proteins or polypeptides are conjugated to an activated hydroxyl or carboxyl groups of PolySia via tyrosine residues. In some embodiments, the proteins or polypeptides are conjugated to the free amino groups of PolySia. In some embodiments, the proteins or polypeptides are conjugated to the free amino groups of PolySia via glutamine residues in transglutaminase-mediated cross-coupling reaction. In some embodiments, the proteins or polypeptides are conjugated to the free amino groups of PolySia via glutamic acid or aspartic acid residues using peptide coupling chemistry (e.g., ED AC). In some embodiments, the proteins or polypeptides have been modified to include non-natural amino acid residues for click chemistry, such as / ?-azido-phenylalanine, / ?-propynyl-phenylalanine, amino acids with dibenzocyclooctyne (DBCO) groups, amino acids with benzyloctyne (BCO) groups, amino acids with tetrazine groups, or amino acids with furan groups.

[0059] Depending on the conjugation chemistry, the average length of the PolySia polysaccharide chains, the number of available chemical groups on the protein antigen for conjugation, and the input ratios of the PolySia repeating units versus the protein coupling partner, different molar or weight ratios of PolySia:Protein in the resulting conjugates can be obtained. Moreover, since there are multiple hydroxyl and carboxyl groups throughout the PolySia polymer chains, as well as multiple linking sites on the protein coupling partner, theAttorney Docket No. 54880-0002W01PolySia chains can be linked to the protein molecules with a single or multiple points of attachment. Cross-linking between multiple strands of PolySia polysaccharide chains and protein antigen molecules is also possible, leading to a lattice-type structure. The average number of PolySia conjugation sites to a protein antigen molecule is represented by “m”, where m can be from about 1 to about 5, from about 1 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 30, and from about 30 to about 40. In some embodiments, m is at least about 1. In some embodiments, m is at least about 3. In some embodiments m is at least about 7. In some embodiments, m is at least about 10. In some embodiments, m is at least about 12. In some embodiments, m is at least about 15. In some embodiments, m is at least about 17. In some embodiments, m is at least about 20. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 0.1% to about 75%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 1% to about 75%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 1% to about 20%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 1% to about 50%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 0.1% to about 50%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 5% to about 75%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 5% to about 40%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 5% to about 20%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 5% to about 10%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 20% to about 50%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 10% to about 20%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 50%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 0.1% to about 10%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 0.1% to about 5%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is between about 0.5% to about 1%. In some embodiments, the weight ratio of polysialic acid polysaccharides to the polypeptide antigens is about 0.9%.Attorney Docket No. 54880-0002W01

[0060] The polysialic acid polysaccharides can be linked (i.e., conjugated) to the protein antigen with or without a linking molecule, a linker or a spacer. As used herein, the term “linker” or “spacer” does not contain any carbohydrate residues, amino acid residues, or polypeptides; thus, it is neither a carbohydrate residue, an oligosaccharide, a polysaccharide, an amino acid residue, or a polypeptide. A linker, spacer or crosslinking agent, as generally understood by an artisan of ordinary skills in the art, is typically a small molecule or polymer, linear or not, having a molecular weight of approximately <500 daltons and is non-pyrogenic and non-toxic in the final product form, in particular, in the framework of an in vivo use, or when the final product is a composition for use in modulating immune responses. Different linkers or spacers with various lengths can be used, including but are not limited to, adipic acid dihydrazide, aminoalkyl, hydrazine-PEG-hydrazine, etc., where the polyethylene glycol (PEG) moieties in the linker may prolong circulation in the body.

[0061] In some embodiments, the composition comprises polysialic acids conjugated to one or more polypeptide antigens having the following structure:, wherein the linker is defined herein.

[0062] Conjugation to the protein, with or without the use of a linker, at any of the viable positions (C2, C4, C7, or C9) of any Neu5Ac residue does not imply conjugation at the same positions in all Neu5Ac residues within the same PolySia chain. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the hydroxyl groups at C4 of any Neu5 Ac residue, so that Ri is X where X represents the antigen. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the hydroxyl groups at C7 of any Neu5Ac residue, so that R2 is X where X represents the antigen. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the hydroxylAttorney Docket No. 54880-0002W01groups at C9 of any Neu5Ac residue, so that R3 is X where X represents the antigen. In some embodiments, the site of conjugation to the protein or polypeptide antigen is via the carboxyl groups at C2 of any Neu5Ac residue, so that R4 is Y where Y represents the antigen. In some embodiments, at least one of Ri, R2, or R3 is H. In some embodiments, Ri, R2, and R3 are H and R4 is Y. In some embodiments, at least one of Ri, R2, or R3 is X and R4 is Y. In some embodiments, at least one of Ri, R2, or R3 is H and R4 is Y. In some embodiments, at least one of Ri, R2, or R3 is X. In some embodiments, Ri or R2, is X. In some embodiments, R2 or R3, is X. In some embodiments, Ri or R3, is X. In some embodiments, both Ri and R2 are X. In some embodiments, both R2 and R3 are X. In some embodiments, both Ri and R3 are X. In some embodiments, Ri, R2 and R3 are X. In some embodiments, R2 or R3, is X and R4 is OH. In some embodiments, Ri or R3, is X and R4 is OH. In some embodiments, both Ri and R2 are X and R4 is OH. In some embodiments, both R2 and R3 are X and R4 is OH. In some embodiments, both Ri and R3 are X and R4 is OH. In some embodiments, Ri, R2 and R3 are X and R4 is OH.

[0063] The present disclosure describes the physical coupling (conjugation) of one or more polypeptide antigens to PolySia. In some embodiments, the one or more polypeptide antigens are food allergens. Numerous food allergens are known to trigger mild to severe, and sometimes life-threatening, immune responses in humans and some domesticated mammals (e.g., dogs and cats), wherein the most common food allergens include proteins and polypeptides found in cow milk, chicken eggs, gluten proteins from wheat, barley, or rye, tree nuts, peanuts, sesame seed, fish, crustacean shellfish, soy, or berries. In some embodiments, the one or more polypeptide food allergens derive from cow milk. In some embodiments, the one or more cow milk allergens are selected from alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin, or beta-lactoglobulin. In some embodiments, the polySia is covalently linked to cow milk beta-casein. In some embodiments, the polySia is covalently linked to gluten proteins. In some embodiments, the polySia is covalently linked to gliadins or glutenins. In some embodiments, the PolySia is covalently linked to a mixture of gliadins. In some embodiments, the PolySia is covalently linked to a -gliadins. In some embodiments, the PolySia is covalently linked to P-gliadins. In some embodiments, the PolySia is covalently linked to y-gliadins. In some embodiments, the PolySia is covalently linked to co -gliadins. In some embodiments, the one or more polypeptide food allergens derive from peanuts. In some embodiments, the one or more peanut allergens comprises peanut extracts. In some embodiments, the one or more peanut allergens are selected from Ara hl, Ara h2, Ara h3, AraAttorney Docket No. 54880-0002W01h6, or Ara h8. In some embodiments, the polySia is covalently linked to Ara hl. In some embodiments, the PolySia is covalently linked to Ara h2.

[0064] Short-chain fatty acids (SCFAs) are fatty acids with fewer than six carbon atoms and have been shown to play important roles in immune regulation; for example, butyrate has been shown to promote the differentiation of naive CD4+ T cells into Treg cells and maintain Treg phenotypic stability by promoting the expression of HDAC3. SCFAs are produced by gut microbiota from dietary fiber, which helps in regulating gut-associated immune responses and maintaining gut homeostasis. In some embodiments, the compositions for inducing antigen-specific immune tolerance responses further comprise one or more SCFA. In some embodiments, the compositions for inducing antigen-specific immune tolerance responses further comprise one or more SCFA selected from acetic acid, acetate, propionic acid, propionate, butyric acid, or butyrate. In some embodiments, the compositions for inducing antigen-specific immune tolerance responses further comprise butyric acid or butyrate. The SCFA can be included in the composition as free fatty acids or as a conjugate where the SCFA molecules are grafted onto a PolySia polysaccharide molecule, or a different nontoxic polysaccharide such as starch. In some embodiments, the one or more SCFA is conjugated to polysialic acid polysaccharides via ester linkages. In some embodiments, the one or more SCFA is present in the composition as free fatty acids. In some embodiments, the one or more SCFA is present in the composition as conjugate of starch.

[0065] The present disclosure describes a method for inducing tolerance to one or more food allergens in a subject by administering a composition comprising polysialic acid conjugated to food allergens. The composition may comprise polysialic acid conjugated to a single food allergen, or to more than one food allergens simultaneously. Alternatively, the composition may comprise a mixture of polysialic acid conjugates, each of which having the polysialic acid conjugated to a single, different food allergen. The composition may further comprise short-chain fatty acids (SCFAs), which may be conjugated to the polysialic conjugate or admixed with the polysialic acid conjugates to yield the final composition.

[0066] The present disclosure describes a method for inducing tolerance to an autoantigen in a subject by administering a composition comprising polysialic acid covalently linked to an autoantigen. Examples of autoantigens include proteins implicated in Type 1 diabetes (T1D), where loss of tolerance to these autoantigens results in inflammation and damage to the pancreatic beta cells, which produce insulin. The autoantigens implicated in T1D include, for example, insulin, glutamic acid decarboxylase, insulinoma associated protein 2, zinc transporter 8, or fragments thereof. In addition, composition comprisingAttorney Docket No. 54880-0002W01poly sialic acid conjugated to an autoantigen that are human myelin proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), neurofascin, or fragments thereof, can be administered to a subject with multiple sclerosis (MS), where a loss of immune tolerance to one or more of these proteins is believed to be involved in the pathogenesis and disease progression of MS.

[0067] An “effective amount” of PSA-polypeptide conjugates or PSA-polypeptide conjugate-containing composition to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the subject. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration required to obtain the optimal therapeutic effect. In some embodiments, the subject receiving the PSA-polypeptide conjugates or PSA-polypeptide conjugate-containing composition is a mammal, for example, human, non-human primates, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, pigs, and horses. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mouse. In some embodiments, the subject is a dog. In some embodiments, the subject is a cat.

[0068] The compositions described herein may be prepared as pharmaceutically acceptable compositions with suitable pharmaceutically acceptable carriers, excipients, tonicity modifiers, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co, Easton Pa. 18042, USA). As described herein, excipients refer to inert substances used as diluent or vehicle for a therapeutic agent. Pharmaceutically acceptable carriers are used, in general, with a therapeutic agent to make it useful for therapy or as a product. In general, for any substance, a pharmaceutically acceptable carrier is a material that is combined with the substance for delivery to an animal. Conventional pharmaceutical carriers that are aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. In some cases, the carrier is essential for delivery, e.g., to solubilize an insoluble compound for liquid delivery; a buffer for control of the pH of the substance to preserve its activity; or a diluent to prevent loss of the substance in the storage vessel or to control the concentration of the active ingredient. In other cases, however, the carrier is for convenience, e.g., a liquid or a capsule for more convenient administration. Pharmaceutically acceptable salts of the conjugates described herein may be prepared according to methods known to those skilled in the arts. Thus, pharmaceutically acceptable compositions are highly purified to be free of contaminants, biocompatible and non-toxic, and are suitable forAttorney Docket No. 54880-0002W01administration to a patient. In the case of water as the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins and microorganisms.

[0069] In some embodiments, the composition is administered via inhalation.

[0070] In some embodiments, the composition is administered subcutaneously.

[0071] In some embodiments, the composition is administered intramuscularly.

[0072] The conjugates described herein are typically to be administered as admixtures with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The deliverable conjugate may be made in a form suitable for oral, enteral (buccal, sublingual), or parenteral administration, which can include, via inhalation, intravenous injection, intramuscular injection, or subcutaneous injection. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. Suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flowinducing agents, and melting agents may be included as carriers, e.g., for pills. For instance, an active component can be combined with an oral, non -toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, di calcium phosphate, calcium sulfate, mannitol, sorbitol and the like. The conjugates can be administered orally in solid dosage forms, such as capsules, tablets, and as powders in a sachet, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active conjugates can also be administered parentally, in sterile liquid dosage forms. Buffers for achieving a physiological pH and osmolarity may also be used.EXAMPLES

[0073] Example 1: Synthesis of polysialic acid-beta casein conjugate using CDAP chemistry

[0074] A 5 mg / mL solution of colominic acid sodium salt (Biosynth, catalog # YC11298) in 0.9% w / v aqueous NaCl, 5 mg / mL beta casein (Millipore Sigma, Cat# C6905) in 0. IM HEPES buffer pH 8.0, and a 200 mg / mL solution of l-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in acetonitrile were prepared by dissolving the appropriate amounts of the reagents in the corresponding diluent to reach the target concentration. A 2.5M dimethylaminopyridine (DMAP) (Millipore Sigma, catalog # 107700) solution was prepared by suspending 3.0 g of DMAP in 5 mL of deionized water in a glass bottle. The suspension was mixed by vortexing, and a 10 N HC1 solution was added toAttorney Docket No. 54880-0002W01the cloudy suspension in 50 pL increments until the solution turned clear. The resulting clear solution was placed in an ice bath and additional aliquots of the ION HC1 solution was added to it until the pH of the DMAP solution reaches pH 9.0. The pH-adjusted DMAP solution was transferred to a 10-mL volumetric flask, diluted to mark with deionized water, and pH checked and re-adjusted with small amounts of 10 N NaOH back to pH 9.0 after the dilution.

[0075] For the CDAP activation of colominic acid, 1.0 mL of the 5 mg / mL colominic acid solution was placed in a 10-mL glass vial and placed on an ice bath. To the cooled colominic acid solution was added 100 pL of the 2.5M DMAP solution dropwise with gentle stirring using a magnetic stirrer to mix. The pH of the resulting solution was adjusted to pH 9.0 by adding small aliquots of a 0.1 N HC1 solution. Subsequently, 100 pL of a 200 mg / mL CDAP solution was added, and the resulting reaction mixture was incubated at 5 °C for 15 min with gentle stirring, during which time the pH of the reaction mixture was monitored closely and maintained at pH 9.0 by adding small aliquots of a 0.1 NNaOH solution as needed. Afterwards, 1.0 mL of a 5 mg / mL beta-casein solution was added to the CDAP-activated colominic acid at 5 °C, and the reaction mixture was incubated at room temperature for 1 hour with gentle stirring. The crude colominic acid-beta casein conjugate was purified by size-exclusion chromatography (SEC) using a BioSEP SEC-S 3000300 x 7.8 mm column. The crude product was eluted isocratically with lx PBS at a flow rate of 1.0 mL / min, with a total run time of 30 min. Peak detection was done with a UV DAD detector at 205 nm. A representative SEC chromatogram of the purified poly sialic acid-casein conjugate, overlaid with polysialic acid and casein as separate traces, is illustrated in FIG 1. The protein concentration of the purified conjugate was measured using the Pierce BCA Protein Assay Kit (ThermoFisher catalog # 23225). The carbohydrate content was measured using a commercial sialic acid quantification kit (e.g. Millipore Sigma Product # SIALICQ), HPLC quantitation of DMB-derivatized sialic acid after acidic hydrolysis, or by quantitative 'H-NMR, using TSP-d6 or DMSO as internal quantitation standard.

[0076] Example 2: Synthesis of polysialic acid-beta casein conjugate using EDAC chemistry

[0077] A 5 mg / mL solution of colominic acid sodium salt (Biosynth, catalog # YC11298) in 100 mM MES pH 6.0 buffer, 5 mg / mL beta casein (Millipore Sigma, catalog # C6905) in 100 mM MES pH 6.0 buffer, and a 10 mg / mL EDAC (Millipore Sigma, catalog # 03450) solution in deionized water were prepared by dissolving the appropriate amounts of the reagents in the corresponding diluent to reach the target concentration.Attorney Docket No. 54880-0002W01

[0078] For the ED AC activation of colominic acid, 10.0 mL of the 5 mg / mL colominic acid solution was placed in a glass round bottom flask and placed on an ice bath. To the cooled colominic acid solution was added 1.25 mL of the 10 mg / mL EDC solution in one portion. The resulting reaction mixture was incubated at 5 °C for 1 hour with stirring. Afterwards, 10 mL of a 5 mg / mL beta-casein solution was added to the EDC-activated colominic acid at room temperature, and the reaction mixture was incubated at room temperature for 1 hour with stirring. The crude reaction mixture was filtered through a 0.22-pm sterile vacuum filter. The crude colominic acid-beta casein conjugate was purified by size-exclusion chromatography (SEC) using a Superdex 200 prep grade XK 26 / 100 column. The crude product was eluted isocratically with lx PBS at a flow rate of 1.0 mL / min, with a total run time of 30 min. Peak detection was done with a UV DAD detector at 205 nm.Fractions were collected and analyzed by analytical SEC to select the fractions containing the conjugate to combine. The protein concentration of the purified conjugate was measured using the micro BCA Protein Assay Kit (ThermoFisher catalog # 23235). The carbohydrate content was measured using a commercial sialic acid quantification kit (e.g. Millipore Sigma Product # SIALICQ), HPLC quantitation of DMB-derivatized sialic acid after acidic hydrolysis, or by quantitative1H-NMR, using TSP-d6 or DMSO as internal quantitation standard.

[0079] Example 3: Establishment of cow milk -casein allergy model in C3H / HeJ mice and evaluation of efficacy of polysialic acid-beta casein conjugates

[0080] 6-week-old female C3H / HeJ mice were procured from Jackson Laboratories (strain #: 000659). Upon arrival at the facility, the mice were housed in Innovive disposable caging under standard conditions (temperature: 20-26°C, humidity: 30-70%, 12-hour light / dark cycle) on a filtered rack, with ad libitum access to Teklad irradiated standard rodent chow (Inotiv, product # 8904) and irradiated water (Innovive). The mice underwent an acclimation period of at least three days before study procedures began. The study was conducted under Institutional Animal Care and Use Committee (IACUC) protocol #001, approved on 10 / 31 / 2023. Statistical analysis and data plotting were performed using Microsoft Excel or GraphPad Prism v.10.2.3 (GraphPad Software, San Diego, CA, USA).

[0081] Mice were randomized into four experimental groups (n = 3 mice / group) and weighed twice weekly throughout the study, with daily monitoring during the challenge phases to assess health status of the animals. FIG 2 illustrates the experimental design of the mouse study. Sensitization, treatments, and challenge doses were administered via oralAttorney Docket No. 54880-0002W01gavage using disposable plastic feeding tubes (20G x 38mm) while the mice were conscious. For the sensitization phase, each mouse was administered with 200 pL of a suspension containing beta-casein (Sigma-Aldrich, Cat# C6905) at 50-75 mg / mL (10-15 mg per mouse), Cholera toxin (CT) (Sigma- Aldrich, Cat# C8052) at 10-20 pg / mL (2-4 pg per mouse) once weekly for a total of 10 weeks. Blood samples were collected each week prior to the sensitization procedures via cheek bleed. All blood samples were processed immediately upon collection. Serum was collected in specialized collection tubes (Sarstedt), centrifuged at 10,000g for 10 min at 4°C, and transferred into Eppendorf 1.5 mL tubes (ThermoFisher Scientific) for long-term storage at -80°C prior to ELISA assay to measure IgG against betacasein (Example 4).

[0082] Ten days after the final sensitization, mice were fasted overnight, and prechallenge blood samples were collected via cheek bleed. The challenge phase consisted of two consecutive oral gavage doses of beta-casein (20 mg / mouse), administered 30 minutes apart. Post-challenge plasma collection was conducted after the second dose, and mice were observed for 45 minutes post-challenge by two independent investigators for clinical symptom assessment. There was no observable treatment related clinical symptoms for all groups, nor were any statistically significant differences in the trending of the average body weights for all treatment versus control groups.

[0083] As illustrated in FIG 3A-3D, inducing serum IgG response (as surrogate biomarker for casein-specific allergic response) in C3H / HeJ mice was not straightforward. Out of 9 mice (Groups 1-3) that were subjected to the beta-casein + CT sensitization procedures, only one mouse showed casein-specific IgG titers above 3 absorbance units after 6 weekly sensitizations with beta-casein (Day 43 samples). Furthermore, it was observed that a rather high concentration of casein was required for sensitization; in the first six sensitizations, for each animal 10 mg of casein with 10 pg of Cholera toxin as adjuvant were used where only one mouse had IgG titers above 3 absorbance units. The other eight mice had IgG titers below 0.4 absorbance units, or very close to baseline level. Subsequently, both the amounts of the casein and Cholera toxin adjuvant were increased, which appeared to be more effective in inducing IgG responses. After four additional sensitizations using the higher amounts of casein and CT (Day 72), two mice had IgG titers higher than 3 absorbance units (“high responders”); one mouse had IgG titers between 0.3-0.8 absorbance units (“medium responders”), three mice had IgG titers between 0.1-0.3 absorbance units (“low responders”), and the remaining four mice still had IgG titers close to baseline level (“nonAttorney Docket No. 54880-0002W01responders”). The two “high responders” were re-assigned to Groups 1 and 2 (one mouse per group); the one “medium responders” was re-assigned to Group 3 (treatment with PBS negative control); the three “low responders” were re-assigned to Groups 1, 2, 3 (one mouse per group), and the four non-responders were re-assigned to Groups 1 and 2 (one mouse per group), and Group 3 (two mice per group). There was no change to the assignment of Group 4, which was the non-sensitized negative control to demonstrate that the serum IgG titer was a result of sensitization. As expected, all three mice in Group 4 did not appear to have IgG titers significantly above baseline at the end of the sensitization phase (Day 72).

[0084] After the 10thsensitization (Day 72), the animals were rested for 10 days, fasted one day before the challenge, and high dose casein (twice with 20 mg each, 30 min apart) was administered to the mice via oral gavage on Day 82 to see if any clinical symptoms were present. There were no observable clinical symptoms before or after the casein challenge; the average body weights of all groups dropped slightly but there is no statistically significant difference among the groups (Groups 1-3, sensitized vs. Group 4, not sensitized; average = 20.93 g, %CV = 3.31%). We concluded that the decrease in the average body weight is likely due to the fasting prior to challenge rather than an immunological response to the high dose casein challenge. The ELISA data showed no significant change in the IgG titers in all the animals pre- and post- casein challenge, indicating that serum IgG titers, once induced, remained at a certain level regardless of whether casein is present or not.

[0085] Following the challenge phase, the study continued with the treatment phase where a once-weekly oral gavage regimen was performed for six weeks, during which groups received either polysialic acid-beta casein conjugate (Group 1), beta-casein alone (Group 2), or phosphate buffered saline (PBS) (Groups 3 & 4). Body weight measurements were recorded twice per week, and health checks were conducted twice weekly. Blood samples were collected weekly during the treatment period. There was a 3 -week pause on the treatment between the 4thand the 5thdose to assess the effects of pausing treatment on the serum IgG titers. After the 6thtreatment dose (Day 154), the mice were rested for 6 days, followed by fasting for one day and then a second beta-casein challenge (2 doses of 20 mg beta-casein / mouse, administered 30 minutes apart) on Day 161. Blood samples were collected immediately before and 2 days after the challenge dose. All blood samples were processed immediately upon collection. Serum was collected in specialized collection tubes (Sarstedt), centrifuged at 10,000g for 10 min at 4°C, and transferred into Eppendorf 1.5 mL tubes (Thermo Scientific) for long-term storage at -80°C prior to ELISA assay.Attorney Docket No. 54880-0002W01

[0086] As illustrated in FIG 3, the “high responder” (Mouse 1-1 in FIG 3A) in Group 1 showed significant decrease of serum IgG titers upon treatment with polysialic acid-casein conjugates; in contrast, the “high responder” in Group 2 (Mouse 2-1 in FIG 3B), which received beta-casein at approximately 20 times higher than the amount of casein in the poly sialic acid-casein conjugate Group 1 mice received, did not show any reduction in the serum casein-specific IgG titers. The “low responder” (Mouse 1-2) in Group 1 showed a delayed increase in IgG titer on Day 105 (after 1stdose of polysialic acid-casein conjugate) to approximately 2.6 absorbance units, but it began to decrease after the 2ndtreatment with the conjugate, and increased again during the casein challenge that occurred after 6 doses of treatment. Taken together, the data suggest that the polysialic acid-conjugate may reduce the serum IgG titers, presumably via Treg-mediated inhibition of IgG production by B cells. On the other hand, casein on its own did not reduce the IgG titers at the dose used in the current experimental design, indicating that casein conjugated to polysialic acid is recognized differently by the immune system compared to casein alone. Further studies and repetition of the mouse study are under way to confirm these results, and to investigate the potential mechanism of action of the reversal of the IgG responses effected by poly sialic acid-casein conjugate.

[0087] Example 4: ELISA method for measuring p-casein specific IgG antibody responses in mouse blood serum samples

[0088] One day prior to running the ELISA assay, Costar high-binding 96-well flat bottom microtiter plates were coated with 52 pL of a 10 pg / mL solution of cow milk beta casein in 50 mM carbonate buffer at pH 9.4 using multichannel automatic pipet. The coated plates were incubated overnight at 2-8 °C. On the following day, residual liquid from coating was removed by blotting with a paper towel. All wells were blocked with a 3% BSA solution in PBS (pH 7.3) at 300 pL per well. Plate blocking was performed at room temperature for 3 hours.Mouse serum samples were diluted 1 : 100 with sample diluent (1% BSA in PBS, pH 7.3). The diluted samples were added to the coated / b locked microtiter plates at 100 pL per well, and incubated at room temperature for 30 minutes. After the incubation period, plates were washed on a Bioteck Washer five times with PBST. After washing, 100 pL of a 1 :2000 dilution of the detection antibody (goat anti-mouse IgG HRP, Thermo Fisher, catalog # 31437) in HRP diluent (ScyTek Laboratories, catalog # HSB500) was added to each well. The plates were incubated for 30 minutes at room temperature upon addition of the detectionAttorney Docket No. 54880-0002W01antibody. After the incubation, the plates were washed 5 times on a Biotek Washer with PBST. Subsequently, 100 pL of TMB (ScyTek Laboratories, catalog # TM4125) was added to each well, followed by incubation at room temperature for 15 minutes. At the end of the incubation period, 100 pL of the Stop Solution (ScyTek Laboratories, catalog # TSB125) was added to each well. Absorbance at 450 nm was measured using a Perkin Elmer UV-Vis plate reader.

Claims

1. Attorney Docket No. 54880-0002W01What is claimed is:

1. A polysialic acid conjugate for inducing antigen-specific immune tolerance responses, wherein the polysialic acid conjugate comprises:a) one or more polysialic acid polysaccharides, comprising repeating units of oc-2,8-linked N-acetylneuraminic acid residues;b) one or more polypeptide antigens;c) one or more covalent linkages between the polysialic acid polysaccharides and the polypeptide antigens.

2. The conjugate of claim 1, wherein polysialic acid polysaccharides are naturally occurring carbohydrates isolated from a micro-organism.

3. The conjugate of claim 1, wherein the polysialic acid polysaccharides are made via chemical synthesis.

4. The conjugate of claim 1, wherein the polysialic acid polysaccharides comprise oc-2,8-linked N-acetylneuraminic acid residue repeating units with the following structure:

5. The conjugate of claim 1, wherein the polysialic acid polysaccharides conjugated to one or more polypeptide antigens comprise the following structure:Attorney Docket No. 54880-0002W016. The conjugate of claim 1, wherein the polysialic acid polysaccharides conjugated to one or more polypeptide antigens comprises the following structure:the linker is polyethyleneglycol (PEG).

7. The conjugate of claims 5 and 6, wherein at least one of Ri, R2, or R3 is X.

8. The conjugate of claims 5 and 6, wherein Ri, R2, or R3 is X.

9. The conjugate of claims 5 and 6, wherein at least one of Ri, R2, or R3 is H.Attorney Docket No. 54880-0002W0110. The conjugate of claims 5 and 6, wherein Ri, R2, and R3 are H and R4 is Y.

11. The conjugate of claims 5 and 6, wherein at least one of Ri, R2, or R3 is X and R4 is Y.

12. The conjugate of claim 4, wherein n is at least 5.

13. The conjugate of claims 5 and 6, wherein m is at least 1.

14. The conjugate of claim 1, wherein the polysialic acid polysaccharides conjugated to one or more polypeptide antigens comprises the following structure:

15. The conjugate of claims 14, wherein m is at least 1.

16. The conjugate of claims 14, wherein R5 is P and R4is OH.

17. The conjugate of claims 14, wherein R5 is P and R4 is Z.

18. The conjugate of claims 14, wherein R5 is Q and R4is OH.Attorney Docket No. 54880-0002W0119. The conjugate of claims 14, wherein R5 is Q and R4 is Z.

20. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens by activating one or more of the poly sialic acid polysaccharide hydroxyl groups via CDAP chemistry.

21. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens by activating one or more of the poly sialic acid polysaccharide carboxyl groups via ED AC chemistry.

22. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens by first de-N-acetylating one or more of the polysialic acid polysaccharide N-Ac groups to provide free primary amino groups at C5.

23. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the lysine residues of the polypeptide.

24. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the cysteine residues of the polypeptide.

25. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the glutamine residues of the polypeptide.

26. The conjugate of claim 1, wherein the polysialic acid polysaccharides are covalently linked to the one or more polypeptide antigens via the glutamic acid or aspartic acid residues of the polypeptide.

27. The conjugate of claim 1, wherein the weight ratio of polysialic acid polysaccharides to polypeptide antigens is between about 0.1% and 10%.

28. The conjugate of claim 1, wherein the weight ratio of polysialic acid polysaccharides to polypeptide antigens is about 0.9%.Attorney Docket No. 54880-0002W0129. The conjugate of claim 1, wherein the one or more polypeptide antigens is a food allergen.

30. The conjugate of claim 29, wherein the one or more polypeptide food allergens derive from cow milk, chicken egg, wheat, tree nuts, peanuts, sesame seed, fish, crustacean shellfish, soy, or berries.

31. The conjugate of claim 29, wherein the one or more polypeptide food allergens derive from cow milk.

32. The conjugate of claim 31, wherein the one or more cow milk allergens are selected from alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin, or beta-lactoglobulin.

33. The conjugate of claim 31, comprising one or more polysialic acid polysaccharides covalently linked to cow milk beta-casein.

34. The conjugate of claim 29, wherein the one or more polypeptide food allergens derive from wheat.

35. The conjugate of claim 34, wherein the one or more wheat allergens are selected from gliadins or glutenins.

36. The conjugate of claim 34, wherein the one or more wheat allergens are gliadins.

37. The conjugate of claim 29, wherein the one or more polypeptide food allergens derive from peanuts.

38. The conjugate of claim 37, wherein the one or more peanut allergens are selected from Ara hl, Ara h2, Ara h3, Ara h6, or Ara h8.

39. The conjugate of claim 37, comprising one or more polysialic acid polysaccharides covalently linked to peanut Ara h2.Attorney Docket No. 54880-0002W0140. The conjugate of claim 1, further comprising one or more short-chain fatty acids (SCFA).

41. The conjugate of claim 40, wherein the one or more SCFA is selected from acetic acid, acetate, propionic acid, propionate, butyric acid, or butyrate.

42. The conjugate of claim 41, wherein the one or more SCFA comprises butyric acid or butyrate.

43. The conjugate of claim 40, wherein the one or more SCFA is conjugated to polysialic acid polysaccharides via ester linkages.

44. A composition comprising the polysialic acid conjugate of any one of claims 1-43 and one or more carriers.

45. A method for inducing tolerance to one or more food allergens in a subject, comprising administering a composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to the food allergens.

46. The method of claim 45, wherein the composition is administered orally.

47. The method of claim 45, wherein the composition is administered subcutaneously.

48. The method of claim 45, wherein the composition is administered intramuscularly.

49. The method of claim 45, wherein the subject is a human.

50. The method of claim 45, wherein the subject is a dog.

51. The method of claim 45, wherein the subj ect is a cat.

52. A method for inducing tolerance to an autoantigen in a subject, comprising administering a composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to an autoantigen.Attorney Docket No. 54880-0002W0153. The method of claim 52, wherein the autoantigen comprises insulin, glutamic acid decarboxylase, insulinoma associated protein 2, zinc transporter 8, or fragments thereof.

54. The method of claim 52, wherein the autoantigen comprises one or more human myelin proteins selected from myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), or neurofascin, or fragments thereof.

55. The method of claim 52, wherein the composition is administered orally.

56. The method of claim 52, wherein the composition is administered subcutaneously.

57. The method of claim 52, wherein the composition is administered intramuscularly.

58. A method for inducing tolerance to an environmental allergen in a subject, comprising administering a composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to an environmental allergen.

59. The method of claim 58, wherein the environmental allergen comprises one or more polypeptides derived from cats, dogs, dust mites, grass pollen, or wheat pollen.

60. The method of claim 58, wherein the composition is administered orally.

61. The method of claim 58, wherein the composition is administered via inhalation.

62. The method of claim 58, wherein the composition is administered subcutaneously.

63. The method of claim 58, wherein the composition is administered intramuscularly.

64. A composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to one or more food allergens for use in inducing tolerance to the food allergens in a subject, comprising administering the composition to the subj ect.Attorney Docket No. 54880-0002W0165. A composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to an autoantigen for use in inducing tolerance to the autoantigen in a subject, comprising administering the composition to the subject.

66. The composition for use of claim 65, wherein the autoantigen comprises insulin, glutamic acid decarboxylase, insulinoma associated protein 2, zinc transporter 8, or fragments thereof.

67. The composition for use of claim 65, wherein the autoantigen comprises one or more human myelin proteins selected from myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), or neurofascin, or fragments thereof.

68. A composition comprising polysialic acid (poly-alpha-2, 8-linked N-acetyl neuraminic acid) polysaccharide covalently linked to an environmental allergen for use in inducing tolerance to the environmental allergen in a subject, comprising administering the composition to the subject.

69. The composition for use of claim 68, wherein the environmental allergen comprises one or more polypeptides derived from cats, dogs, dust mites, grass pollen, or wheat pollen.

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