EA044172B1 - THERAPEUTICS WITH CARBOHYDRATE-MEDIATED TARGETED DELIVERY - Google Patents

Description

Cross reference to related application

This application claims priority to co-pending U.S. Provisional Patent Application Serial No. 61/942942, filed February 21, 2014, entitled CARBOHYDRATE-MEDIATED TARGETED THERAPEUTICS

DELIVERY, which is hereby incorporated herein by reference.

Field of technology

The technical scope of the present disclosure relates to pharmaceutically acceptable compositions that are useful in the treatment of transplant rejection, autoimmune disease, food allergy and immune response against a therapeutic agent.

State of the art

US 2012/0039989, US 2012/0178139 and WO 2013/121296 describe targeted delivery of antigens to red blood cells to take advantage of the role of red blood cells in antigen presentation for tolerization. Despite the positive results obtained to date using this approach, the possibility of alternative approaches is still of interest.

Brief description of the invention

In one aspect of the present disclosure there is provided a compound of Formula 1: x+y-4,

Formula 1 where m is equal to an integer from 1 to 100;

X represents an antigen that induces an unwanted immune response; wherein X represents a food antigen or self-antigen;

Y is a linker component, where Y contains a polymeric linker conjugated to the antigen via a disulfide bond or disulfanyl ethyl ether;

wherein each of the disulfide bond or disulfanyl ethyl ether is capable of being cleaved upon administration of the compound to a subject and releasing the antigen from the polymer linker;

where the polymer linker contains a 1-cyano-1-methylpropyl group and methacrylic units containing an ethyl acetamide functional group and

Z is a liver targeting component, wherein Z contains galactose, galactosamine or N-acetylgalactosamine, and wherein the liver targeting component is coupled to a polymer linker via an ethyl acetamide functionality.

The present disclosure also relates to a method of treating an unwanted immune response against an antigen by administering to a mammal in need of such treatment an effective amount of a composition comprising a compound of Formula 1, as noted above. In such a method, the composition can be administered to clear a circulating protein or peptide or antibody that specifically binds to antigenic component X, wherein the circulating protein or peptide or antibody causes graft rejection, immune response against a therapeutic agent, autoimmune disease, hypersensitivity and/or allergies. The composition can be administered in an amount effective to reduce the concentration of antibodies that cause graft rejection, immune response against a therapeutic agent, autoimmune disease, hypersensitivity and/or allergy in the patient's blood by at least 50% w/w, as measured in a time point from about 12 to about 48 hours after administration. The composition can be administered to tolerize the patient against antigenic component X.

In yet another aspect of the present disclosure, there is provided a composition for inducing immune tolerance comprising a compound of formula 1.

In yet another aspect of the present disclosure, the use of a compound of formula 1 for inducing immune tolerance to X is claimed.

The present disclosure also relates to the use of a compound of formula x+y-4 for the treatment of celiac disease, where m is an integer from 1 to 100; X contains an antigen that induces an unwanted immune response and is selected from one or more of high molecular weight glutenin, low molecular weight glutenin, alpha, gamma and omega gliadin, hordein, secalin, avenin; Y contains a polymer linker conjugated to the antigen via a disulfide bond or disulfanylethyl ether; wherein each of the disulfide bond or disulfanylethyl ether is capable of being cleaved upon administration of the compound to a subject and releasing antigen from the polymer linker; wherein the polymer linker contains a 1-cyano-1-methylpropyl group and methacrylic units,

- 1 044172 containing an ethyl acetamide functional group; and Z contains a liver targeting component, wherein Z contains N-acetylgalactosamine, galactose, galactosamine and wherein the liver targeting component is coupled to a polymer linker via an ethyl acetamide functionality.

The present invention also discloses the use of a compound with formula x+y-4 for the treatment of multiple sclerosis and the use of a compound with formula x+y-zL for the treatment of type 1 diabetes.

Brief description of graphic materials

Fig. 1 is a series of graphs showing that galactose conjugate [F1aAPE-m ₄ -n ₈₀ (Gal-PE)] preferentially targets OVA to liver cells including the following: (A.) sinusoidal endothelial cells (LSEC), ( B.) Kupffer cells (KC), (C.) hepatocytes, and (D.) other antigen presenting cells (APCs).

Fig. 2 is a graph showing the proliferation of OTI CD8+ T cells in mice treated with F1aA-OVA-m4-n ₈₀ (Gal-OVA), OVA, or saline (ie, no exposure).

Fig. 3 is a series of graphs showing the percentage of OT-I CD8+ T cells presenting the cell surface markers (A.) PD-1+ and (B.) Annexin-V+ in saline-treated generations of proliferating T cells. OVA or F1aAOVA-m4-n80 (GAL-OVA).

Fig. 4 is a graph showing that galactose conjugate [F1aA-OVA-m ₄ -n ₈₀ (Gal-OVA)] reduces the immunogenicity of OVA as determined by OVA-specific antibody titers (shown in log ^-1 Ab titer) .

In fig. Figure 5 shows that F1aA-OVA-m4-n ₈₀ (Gal-OVA) is able to deplete OVA-specific antibodies in serum.

In fig. Figure 6 shows that F1aA-OVA-m4-n ₈₀ (mGal-OVA), F1b-OVA-m ₁ -n ₄₄ -p ₃₄ (pGal-OVA) and N'-DOMGly ₃ Ser-OVA-Gly ₃ Ser-6xHis -C' (Dom-OVA) are able to attenuate the OVA-specific immune response in draining lymph nodes after intradermal stimulation with OVA and LPS adjuvant.

In fig. Figure 7 shows the characteristics of F1aA-OVA-m ₄ -n ₈₀ and F1b-OVA-m ₁ -n ₄₄ -p ₃₄ . (A). Oscillograms of exclusion HPCL F1aA-OVA-m ₄ -n ₈₀ (magenta), F1b-OVA-m ₁ -n ₄₄ -p ₃₄ (blue) and unconjugated OVA (black). A shift to the left reflects an increase in molecular weight. (B.) Polyacrylamide gel showing the increase in molecular weight after OVA conjugation: (1.) unconjugated OVA, (2.) F1aA-OVA-m ₄ -n ₈₀ and (3.) F1b-OVA-m ₁ -n ₄₄ -p ₃₄ .

Fig. 8 is a graph showing the normalized amount of N-OVAGly3Ser-6xHis-C (OVA), N-DOM-Gly3Ser-OVA-Gly3Ser-6xHis-C (DOM-OVA), or N-OVA-Gly3Ser-DOMGly ₃ Ser- 6xHis-C (OVA-DOM) in the circulation of mice after injection as an iv bolus dose.

Fig. 9 is a series of graphs depicting the titer of anti-OVA IgG antibodies in the circulation of individual mice following treatment with saline, DOM and OVA, DOM-OVA or OVA-DOM. Anti-OVA IgG production was induced by iv injections of OVA alone (a) or OVA and CpG-B (b). Processing times are indicated by vertical dotted lines. The titer was calculated as log ₁₀ of the maximum fold dilution of plasma with detectable anti-OVA IgG.

Detailed description

Two common asialoglycoprotein receptors (ASGPR) are expressed on hepatocytes and liver sinusoidal endothelial cells (or LSECs). Other galactose/galactosamine/N-acetylgalactosamine receptors can be found in various forms on a variety of cell types [eg, dendritic cells, hepatocytes, LSECs and Kupffer cells]. Dendritic cells are considered professional antigen-presenting cells because their primary function is to present antigens to the immune system to generate immune responses. It is known that some cells within the liver are capable of presenting antigens, but it is better known that the liver is involved in tolerogenesis. The implication is that the liver is a tolerogenic organ. For example, in cases of multiple organ transplantation, if the liver is one of the organs transplanted, a reduced incidence of rejection has been reported. References to LSEC appear in the literature relatively recently; consequently, their role in tolerogenesis and/or moderation of inflammatory immune responses is not yet widely confirmed or well understood. However, it is becoming clear that they may also play a significant role in the induction of antigen-specific tolerance.

One of the distinguishing features of the erythrocyte surface is its glycosylation, i.e. the presence of a significant amount of glycosylated proteins. In this regard, glycophorins (eg glycophorin A) have been used as targets for binding erythrocytes. Glycophorins are proteins with many covalently attached sugar chains terminated by sialic acid. As the red blood cell ages and becomes ready for clearance, the terminal sialic acid of its glycophorins is typically lost, leaving N-acetylgalactosamine at the free end. N-acetylgalactosamine is a ligand that is selectively accepted by ASGPR associated with liver cells, resulting in the binding of Nacetylgalactosamine-containing substances to liver cells and their subsequent uptake and processing in the liver.

Until now, it has been understood by those skilled in the art that glycosylation of a therapeutic agent in a manner that results in liver targeting should be avoided because first pass clearance through the liver results in a reduced half-life of the therapeutic agent in the bloodstream. In addition, some monoclonal antibodies need to be specifically glycosylated at ASN297 for optimal binding to their Fc receptors. It has now surprisingly been discovered that galactosylation can be used in such a way as to induce tolerogenesis.

The present disclosure provides certain therapeutic compositions that serve to target (and be taken up by) the liver, particularly hepatocytes, LSECs, Kupffer cells and/or stellate cells, more particularly hepatocytes and/or LSECs, and even more particularly for specific binding of ASGPR. Targeted delivery to the liver promotes two treatment mechanisms: tolerization and clearance. Tolerization uses the role of the liver in eliminating apoptotic cells and processing their proteins for recognition by the immune system as their own, as well as the role of the liver in selecting peripheral proteins for immunological tolerance. Clearance takes advantage of the liver's role in purifying the blood by quickly removing and destroying toxins, polypeptides, etc. These compositions are targeted to the liver using a galactosylated moiety (e.g. galactose, galactosamine and N-acetylgalactosamine, particularly conjugated at C1, C2 or C6), another hepatic targeting moiety (e.g. a monoclonal antibody, or its fragment, or scFv), or by desialylation of the polypeptide that requires such targeted delivery to the liver. The galactosylated or other liver-targeted component can be chemically conjugated or recombinantly fused to the antigen, whereby desialylation exposes the galactose-like component on the antigen polypeptide. The antigen may be endogenous (self antigen) or exogenous (foreign antigen), including, but not limited to: a foreign graft antigen against which transplant recipients develop an unwanted immune response (eg, graft rejection), a foreign food antigen, an animal antigen, an antigen plant origin or antigen from the environment against which patients develop an unwanted immune response (for example, allergy or hypersensitivity), a therapeutic agent against which patients develop an unwanted immune response (for example, hypersensitivity and/or reduced therapeutic activity), self-antigen, against which patients develop an unwanted immune response (eg, an autoimmune disease), or a tolerogenic portion (eg, fragment or epitope); wherein these compositions are useful for inducing tolerization towards an antigen. Alternatively, the galactosylated or other liver-targeted component may be conjugated to an antibody, antibody fragment, or ligand that specifically binds a circulating protein or peptide or antibody, wherein the circulating protein or peptide or antibody causes graft rejection. immune response against a therapeutic agent, autoimmune disease and/or allergy (as discussed above); wherein these compositions are useful for eliminating circulating protein, peptide or antibody. Accordingly, the compositions of the present disclosure can be used to treat an unwanted immune response, for example, transplant rejection, immune response against a therapeutic agent, autoimmune disease and/or allergy. Also provided are pharmaceutical compositions containing a therapeutically effective amount of a composition of the present disclosure mixed with at least one pharmaceutically acceptable excipient. In another aspect, the present disclosure provides methods for treating an unwanted immune response, such as transplant rejection, response to a therapeutic agent, autoimmune disease, or allergy.

Definitions.

As used in the description of the present invention, the following words and phrases are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

Singular forms include reference to plural forms unless the context clearly indicates otherwise.

The term approximately, when used in conjunction with a numerical value, is intended to cover numerical values within a range, typically with a lower limit that is, for example, 5-10% less than the stated numerical value, and an upper limit that, for example , 5-10% more than the specified numerical value.

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An antigen is any substance that serves as a target for receptors in the adaptive immune response, such as a T cell receptor, a B cell receptor, or an antibody. The antigen can be formed within the body (its own, auto or endogenous). The antigen can be produced outside the body (non-self, foreign or exogenous) and can be obtained, for example, by inhalation, ingestion, injection or transplantation. Foreign antigens include, but are not limited to, food antigens, animal antigens, plant antigens, environmental antigens, therapeutic agents, and antigens that are present on an allogeneic graft.

Antigen-binding molecule, as used herein, refers to molecules, in particular proteins, such as immunoglobulin molecules, that contain antibody variable regions that mediate specific binding to an epitope. An antibody variable region may be present, for example, in a complete antibody, an antibody fragment, and a recombinant derivative of the antibody or antibody fragment. The term antigen-binding antibody fragment (or binding moiety) as used herein refers to one or more antibody fragments that retain the ability to specifically bind a target sequence. Antigen binding fragments containing antibody variable regions include, but are not limited to, Fv, Fab and F(ab') ₂ regions, single domain antibodies (sdAbs), nanobodies, single chain Fv (scFv) fragments, tandem scFvs (VHA-V _L A-VHB -V _L B), diabodies, tribodies or tribodies, single chain diabodies (scDb) and bispecific T cell binding fragments (BiTE).

Chemical modification refers to a change in the chemical structure of one or more naturally occurring amino acids of a polypeptide. Such modifications can be carried out at the side chain or end, for example, changing the amino terminus or carboxy terminus. In some embodiments, modifications are useful to provide chemical moieties that can conveniently be used to link polypeptides to other materials or to attach a therapeutic agent.

The term comprising, which is synonymous with the terms including, comprising, or characterized, is inclusive or non-limiting and does not exclude additional, not set forth, elements or process steps. The phrase consisting of excludes any element, step or ingredient that has not been specified. The phrase consisting of actually limits the scope of the object described to specified materials or stages and those that do not materially affect its basic and novel characteristics.

Conservative changes can generally be made to the amino acid sequence without changing activity. These changes are called conservative substitutions or mutations; that is, an amino acid belonging to a group of amino acids with a certain size or characteristics can be replaced by another amino acid. Substituents for an amino acid sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are not expected to significantly affect the average molecular weight determined by polyacrylamide gel electrophoresis or the isoelectric point. Conservative substitutions also include replacing optical isomers in sequences with other optical isomers, in particular D amino acids with L amino acids instead of one or more sequence residues. In addition, all amino acids in the sequence may be subject to substitution of the D-isomer for the L-isomer. Typical conservative substitutions include without limitation Lys to Arg and vice versa, maintaining positive charge; Glu on Asp and vice versa, maintaining a negative charge; Ser to Thr to preserve the free -OH group; and Gln to Asn, retaining the free -NH2 group. Another type of conservative substitution is when amino acids with the required chemical activity are introduced to provide reactive sites for chemical conjugation reactions if there is a need to obtain chemical derivatives. Such amino acids include, but are not limited to, Cys (for insertion of a sulfhydryl group), Lys (for insertion of a primary amine), Asp and Glu (for insertion of a carboxylic acid group), or specialized non-canonical amino acids containing ketone, azide, alkyne, alkene and tetrazine side chains. Conservative substitutions or additions of amino acids bearing free -NH2 or -SH groups may be particularly effective for chemical conjugation with linkers and galactosylated components of Formula 1. In addition, point mutations, deletions and insertions in polypeptide sequences or corresponding nucleic acid sequences in some cases can be produced without loss of function of the polypeptide or nucleic acid fragment. Substitutions may include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more residues. In the embodiment that can be used according to the present invention, a total of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,

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45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200) changes in amino acid sequence (t i.e. substitutions, insertions, deletions, N-terminal truncations and/or Terminal truncations). For the amino acid residues described herein, either a one-letter amino acid designation or a three-letter abbreviation is used in accordance with the standard polypeptide nomenclature, J. Biol. Chem., (1969), 243, 3552-3559. All sequences of amino acid residues are represented herein by formulas in a right-to-left orientation, which corresponds to the usual direction from the amino terminus to the carboxy terminus.

The terms effective amount or therapeutically effective amount refer to that amount of a composition of the present disclosure that is sufficient to effect a treatment as defined below when administered to a mammal in need of such treatment. This amount will vary depending on the subject and the disease state being treated, the weight and age of the subject, the severity of the disease condition, the choice of the particular composition of the present disclosure, the dosage regimen to be followed, the duration of administration, the route of administration, etc., all from which a person skilled in the art can easily determine.

An epitope, also known as an antigenic determinant, is a segment of a macromolecule, such as a protein, that is recognized by the adaptive immune system, such as antibodies, B cells or T cells. An epitope is precisely that part or segment of a macromolecule that is capable of binding to an antibody or its antigen-binding fragment. As used herein, the term binding specifically refers to specific binding. In the context of the present invention, it is preferred that the term epitope refers to a segment of a protein or polyprotein that is recognized by the immune system.

The term galactose is well known in the art and refers to a monosaccharide sugar that exists in both open chain and cyclic form with D- and L-isomers. In the cyclic form, there are two anomers, namely alpha and beta. In the alpha form, the C1 alcohol group is in an axial position, while in the beta form, the C1 alcohol group is in an equatorial position. In particular, galactose refers to the cyclic six-membered pyranose, more specifically the D-isomer and, even more specifically, the α-D form (a-D-galactopyranose). The structure and numbering of galactose is illustrated below.

HE

HE

The term galactosylated component refers to a specific type of component for targeted delivery to the liver. Galactosylated components include, but are not limited to, a galactose, galactosamine and/or N-acetylgalactosamine residue.

The term liver targeting component refers to components having the ability to target, for example, a polypeptide to the liver. The liver contains various types of cells, including, but not limited to, hepatocytes, sinusoidal endothelial cells, Kupffer cells, stellate cells and/or dendritic cells. Typically, the liver targeting component directs the polypeptide to one or more of these cells. On the surface of the corresponding liver cells there are receptors that recognize and specifically bind the component for targeted delivery to the liver. Targeted delivery to the liver can be achieved by chemical conjugation of an antigen or ligand to a galactosylated moiety, desialylation of an antigen or ligand to expose underlying galactosyl moieties, recombinant fusion or chemical conjugation of an antigen or ligand to an ASGPR-binding moiety, or specific binding of an endogenous antibody to an antigen or ligand, where wherein the antigen or ligand is: desialylated to expose galactosyl moieties, conjugated to a galactosylated moiety, or recombinantly fused or chemically conjugated to an ASGPR-binding moiety. Naturally occurring desialylated proteins are not included within the scope of the present disclosure.

The numerical values and ranges presented for the various substituents are intended to cover all integers within the range stated. For example, when defining n as an integer describing a population comprising from about 1 to 100, in particular from about 8 to 90, and more particularly from about 40 to 80 ethylene glycol groups, where the population generally covers an integer, specified as n ± approximately 10% (or in the case of smaller integers from 1 to approximately 25, ±3), it should be understood that n may be an integer

- 5 044172 in numbers from approximately 1 to 100 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110) and that the set described covers ranges such as 1-4, 2-4, 2-6, 3-8, 7-13, 6-14, 18-23, 26-30, 42 -50, 46-57, 60-78, 85-90, 90-110 and 107-113 groups of ethylene glycol. It should be understood that the combined terms approximately and ±10% or ±3 disclose and provide specific support for equivalent ranges in all cases of their application.

The term optional or optional means that the event or condition described thereafter may or may not occur and thus the description includes cases in which the specified event or condition occurs and cases in which it does not occur.

A peptide that specifically binds a particular target is called a ligand for that target.

Polypeptide is a term that refers to a chain of amino acid residues, regardless of post-translational modification (eg, phosphorylation or glycosylation), and/or complexation with additional polypeptides, and/or synthesis into multi-subunit complexes with nucleic acids, and /or carbohydrates, or other molecules. Proteoglycans are therefore also referred to herein as polypeptides. A long polypeptide (having more than about 50 amino acids) is called a protein. A short polypeptide (having less than about 50 amino acids) is called a peptide. Depending on their size, amino acid composition, and three-dimensional structure, certain polypeptides may be referred to as an antigen-binding molecule, antibody, antibody fragment, or ligand. Polypeptides can be produced using a variety of methods, many of which are well known in the art. For example, polypeptides can be obtained by extraction (eg, from isolated cells), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemical synthesis. Polypeptides can be produced, for example, by recombinant technology and expression vectors encoding the polypeptide that are introduced into host cells (eg, by transformation or transfection) to express the encoded polypeptide.

As used herein, a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes any possible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and means for pharmaceutically active substances is well known in the art. Unless any conventional vehicle or agent is incompatible with the active ingredient, it is intended for use in therapeutic compositions. Additional active ingredients may also be included in the compositions.

The term purified, as used herein with reference to a polypeptide, refers to a polypeptide that has been chemically synthesized and thus substantially free of other polypeptides, or that has been separated or isolated from most of the other cellular components that normally accompany it (eg , other cellular proteins, polynucleotides or cellular components). An example of a purified polypeptide is a polypeptide in which at least 70% of the substance, by dry weight, is free of proteins and naturally occurring organic molecules with which it is normally associated. Thus, a purified polypeptide preparation may contain, for example, at least 80%, at least 90%, or at least 99% polypeptide substance, by dry weight. Polypeptides can also be designed to contain a tag sequence (eg, a polyhistidine tag, a myc tag, a FLAG® tag, or other affinity tag) that facilitates purification or staining (eg, capture on an affinity array, microscopic imaging). Thus, a purified composition that contains a polypeptide refers to a purified polypeptide unless otherwise specified. The term isolated means that the polypeptides or nucleic acids of the present disclosure are not in their natural environment. The isolated products of the present disclosure may thus be contained in a culture supernatant, partially enriched, obtained from heterologous sources, cloned into a vector or formulated with a carrier, etc.

The term sequence identity is used in relation to comparisons of polypeptide sequences. This expression specifically refers to the percentage of sequence identity, for example at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% relative to the corresponding reference polypeptide or relative to the corresponding reference polynucleotide. Specifically, the subject polypeptide and the reference polypeptide exhibit said sequence identity over a contiguous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids or over the entire length of the reference polypeptide.

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Specific binding, as a term widely used in biological fields, refers to a molecule that binds to a target with relatively high affinity compared to non-target tissues, and usually involves a variety of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonds, etc. Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and certain protein-receptor interactions; Although such molecules may occasionally bind tissues other than their specific targets, the extent of such off-target binding is not significant and high affinity binding pairs may still meet the definition of specific binding.

The term treating or providing treatment means any treatment of a disease or disorder in a mammal, including preventing or protecting against the disease or disorder, which means that clinical symptoms do not develop;

slowing down a disease or disorder, which means stopping or suppressing the development of clinical symptoms; and/or mitigation of the disease or disorder, meaning that there is a reversal of clinical symptoms.

The term unwanted immune response refers to a response by a subject's immune system that is not desired in a given situation. An immune system response is undesirable if the response does not prevent, reduce, or cure a disease or disorder, but instead causes, intensifies, or worsens the disorder or disease. Typically, the immune system response causes, enhances, or worsens disease if it is directed against the wrong target. For example, an adverse immune response includes, but is not limited to, transplant rejection, an immune response against a therapeutic agent, an autoimmune disease, and an allergy or hypersensitivity.

The term variant should be understood as a protein that, when compared to the protein from which it is derived, differs in one or more changes in its length, sequence or structure. The polypeptide from which the protein variant is derived is also known as the parent polypeptide or polynucleotide. The term variant includes fragments or derivatives of the parent molecule. Typically, fragments are smaller in length or size than the parent molecule, while derivatives have one or more differences in their sequence or structure compared to the parent molecule. Also included are modified molecules, such as, but not limited to, post-translationally modified proteins (eg, glycosylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids, such as methylated DNA. Also, mixtures of different molecules, such as, but not limited to, RNA-DNA hybrids, are encompassed by the term variant. It should be understood that naturally occurring and man-made variants are encompassed by the term variant as used herein. In addition, variants that can be used according to the present invention can be obtained from homologs, orthologues or paralogues of the parent molecule or from an artificially engineered variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active. A variant may have a certain degree of sequence identity to the parent polypeptide from which it is derived. More specifically, in the context of the present disclosure, a protein variant may exhibit at least 80% sequence identity with its parent polypeptide. Preferably, the sequence identity of the protein variants is defined over a contiguous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids.

Compositions.

One aspect of the present disclosure relates to compositions, pharmaceutical compositions and methods of treatment using such compositions as represented by formula 1 x+y-zL

Formula I where m is an integer from about 1 to 100, in particular from about 1 to 20 and, most preferably, from 1 to about 10;

X represents an antigenic component, particularly a foreign antigen or self-antigen, against which the patient develops an unwanted immune response, or a tolerogenic portion (eg, fragment or epitope) of such an antigenic component;

Y is a linker component or direct link, or an antibody, antibody fragment, peptide or other ligand that specifically binds X; And

Z is a liver targeting component, in particular a galactosylated component.

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The value of m in Formula 1 will depend on the nature of X, in the sense that each antigen, antibody, antibody fragment or ligand will have an individual number and density of sites (primarily N-terminal amines, lysine residues and cysteine residues) with which it can be linked by a linker or galactosylated moiety. Antigens with a limited number of such sites can be derivatized, for example at the N- or C-terminus, by adding lysine or cysteine residues (optionally via a cleavable linker, in particular an immunoproteasome cleavage site linker). In general, it is preferred to provide an adequate degree of galactosylation in the compositions of Formula 1 so as to facilitate uptake into liver cells. The pharmaceutical compositions and methods of the present disclosure may use mixtures of compositions of Formula 1, suitably bearing different components X (eg, multiple epitopes associated with a particular unwanted immune response).

Compositions of Formula 1 include subclasses wherein X is a foreign graft antigen against which transplant recipients develop an unwanted immune response (eg, graft rejection), a foreign food antigen, an animal antigen, a plant antigen, or an environmental antigen against which patients develop an unwanted immune response (eg, allergy or hypersensitivity), a foreign therapeutic agent against which patients develop an unwanted immune response (eg, hypersensitivity and/or reduced therapeutic activity), or a self antigen against which patients develop an unwanted immune response (eg, autoimmune disease); where Y is a linker of the formula Ya-Yp; and/or where Z represents galactose, galactosamine or N-acetylgalactosamine, as illustrated by formulas 1a-1p, as described below with reference to the reaction schemes.

The present disclosure further provides a pharmaceutically acceptable composition represented by Formula 2

Formula 2 where m' equals zero or an integer from about 1 to 10, m'' equals zero or an integer from about 1 to 10, and the sum of m' + m'' equals an integer from about 1 to 10, but is equal to at least 1;

X represents an antigenic component, particularly a foreign antigen or self-antigen, against which the patient develops an unwanted immune response, or a tolerogenic portion (eg, fragment or epitope) of such an antigenic component;

Y is a linker component or direct link, or an antibody, antibody fragment, peptide or other ligand that specifically binds X; And

Z is a liver targeting component, in particular an ASGPR-targeting antibody, an ASGPR-targeting antibody fragment, an AS GPR-targeting peptide, an AS GPR-targeting scFv or other ligand for ASGPR, wherein such composition optionally includes amino acid sequences to facilitate isolation and purification [eg, His tag or 6xHis with the sequence HHHHHH (SEQ ID NO:1) and additional linkers [eg, Gly3Ser with the sequence GGGS (SEQ ID NO:2)]. In compositions of Formula 2 where m' + m'' is greater than one, the X components may be the same or different, and the Y components may be the same or different. The value of m' + m'' will be less (eg, 3 or less) when X is a complete protein, or greater (up to about 10) when X is a peptide. The Y linker(s) may advantageously contain a cleavage site, in particular a cleavage site for the immunoproteasome.

Compositions of Formula 2 include subclasses wherein X is a foreign graft antigen against which transplant recipients develop an unwanted immune response (eg, graft rejection), a foreign food antigen, an animal antigen, a plant antigen, or an environmental antigen against which patients develop an unwanted immune response (eg, allergy or hypersensitivity), a foreign therapeutic agent against which patients develop an unwanted immune response (eg, hypersensitivity and/or reduced therapeutic activity), or a self antigen against which patients develop an unwanted immune response (eg, autoimmune disease).

Alternatively, in the compositions of Formula 1 and/or Formula 2, X may be an antibody, antibody fragment or ligand that specifically binds a circulating protein or peptide or antibody, wherein the circulating protein or peptide or antibody causes graft rejection, immune response against a therapeutic agent, autoimmune disease, hypersensitivity and/or allergy.

The compositions of Formula 2 can be prepared as fusion proteins and may include several

- 8 044172 components useful in their preparation and purification, such as a signal or leader sequence that directs the expressed polypeptide along a specific transport pathway and is subsequently cleaved from the polypeptide; thus, the signal sequence may be part of the expressed protein and the DNA encoding it, but is not part of the final composition. One example of a mammalian signal sequence used in expression systems is the Ig κ chain sequence that leads to protein secretion: METDTLLLWVLLLWVPGSTG (SEQ ID NO:3). Likewise, one example of a common bacterial signal sequence used in expression systems is the pelB signal sequence, which directs the expressed protein to the bacterial periplasm: MKYLLPTAAAGLLLLLAAQPAMA (SEQ ID NO:4).

Antigens.

The antigen used as X in the compositions of formula 1 and/or formula 2 may be a protein or peptide, for example an antigen may be a complete or partial therapeutic agent, a full-length graft protein or a peptide thereof, a full-length autoantigen or a peptide thereof, a full-length allergen or a peptide thereof and/or a nucleic acid or mimetic of the above-mentioned antigen.

Antigens that are used in putting the present disclosure into practice may be one or more of the following.

Therapeutics that are proteins, peptides, antibodies and antibody-like molecules, including antibody fragments and fusion proteins with antibodies and antibody fragments. These include human proteins, non-human (eg mouse) and non-natural (ie engineered) proteins, antibodies, chimeric antibodies, humanized antibodies and antibody-related binding scaffolds such as fibronectins, DARPin proteins , knottin peptides, etc.

Antigens in human allogeneic transplants against which transplant recipients develop an unwanted immune response.

Self-antigens that cause an unwanted autoimmune response. Those skilled in the art will appreciate that while self antigens are endogenously derived antigens in a patient with an autoimmune disease, the polypeptides used in the compositions of the present disclosure are typically synthesized exogenously (as opposed to those that are purified and concentrated from a source origin).

Foreign antigens, such as food antigens, animal antigens, plant antigens, and environmental antigens, against which the patient mounts an unwanted immune response. Those skilled in the art will appreciate that while a therapeutic protein may also be considered a foreign antigen due to its exogenous origin, for purposes of clarity, such therapeutic agents are described as a separate group throughout the present disclosure. Likewise, a plant antigen or an animal antigen may be consumed and considered a food antigen, and an environmental antigen may be from a plant. All of them, however, are foreign antigens. For the purpose of simplicity, no attempt will be made to describe the characteristics and define all such potentially overlapping groups, as those skilled in the art can identify antigens that can be used in the compositions of the present disclosure, particularly in light of the detailed description and examples.

The antigen may be a complete protein, a portion of a complete protein, a peptide, etc., and may be derivatized (as discussed above) to attach to a linker and/or galactosylated moiety, may be a variant, and/or may contain conservative substitutions, in particularly identity-supporting sequences, and/or may be desialylated.

In embodiments where the antigen is a therapeutic protein, peptide, antibody, or antibody-like molecule, the specific antigens may be selected from: abatacept, abciximab, adalimumab, adenosine deaminase, ado-trastuzumab-emtansine, agalsidase-alpha, agalsidase-beta, aldesleukin , alglucerase, alglucosidase-alpha, α-1-proteinase inhibitor, anakinra, anistreplase (anisoylated plasminogen-streptokinase activator complex), antithrombin III, antithymocyte globulin, ateplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1- esterase inhibitor, canakinumab, carboxypeptidase G2 (glucarpidase and voraxase), certolizumab-pegol, cetuximab, collagenase, Crotalidae immune Fab, darbepoetin-α, denosumab, digoxin immune Fab, dornase-alpha, eculizumab, etanercept, factor VIIa, factor VIII , factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histrelin acetate, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin [including recombinant human insulin (rHu-insulin) and bovine insulin], interferon-a2a , interferon-α2b, interferon-β1a, interferon-β1b, interferon-γ1b, ipilimumab, L-arginase, L-asparaginase, L-methionase, lactase, laronidase, lepirudin/hirudin, mecasermin,

- 9 044172 mecasermin-rinfabate, methoxynatalizumab, octreotide, ofatumumab, oprelvekin, pancreatic amylase, pancreatic lipase, papain, peg-asparaginase, peg-doxorubicin HCl, PEG-epoetin-β, pegfilgrastim, peg-interferon-a2a, peg -interferon- α2b, pegloticase, pegvisomant, phenylalanine ammonia lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, alpha interferon type 1, ustekinumab, vW factor . The therapeutic protein can be obtained from natural sources (eg, concentrated and purified) or synthesized, eg, recombinantly, and includes therapeutic antibody drugs, which are typically monoclonal IgG or fragments or fusions thereof.

Specific therapeutic protein, peptide, antibody or antibody-like molecules include abciximab, adalimumab, agalsidase alpha, agalsidase beta, aldesleukin, alglucosidase alpha, factor VIII, factor IX, infliximab, insulin (including rHu-insulin), L -asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL) or rasburicase (uricase) and usually IgG monoclonal antibodies in their various formats.

Another specific group includes hemostatic agents (factor VIII and IX), insulin (including rHu-insulin) and the non-human therapeutic uricase, PAL and asparaginase.

Adverse immune responses in hematology and transplantation include autoimmune aplastic anemia, graft rejection (in general), and graft-versus-host disease (bone marrow graft rejection). In embodiments in which the antigen is a human allograft antigen, the specific sequences may be selected from protein subunits of various MHC class I and MHC class II haplotypes (eg, donor/recipient differences identified by tissue crossmatch testing) and single amino acid polymorphisms on antigens of minor blood groups, including RhCE, Kell, Kidd, Duffy and Ss. Such compositions can be prepared individually for a given donor/recipient pair.

In embodiments in which the antigen is a self antigen, the specific antigens (and the autoimmune disease with which they are associated) can be selected from the following.

Several major antigens have been identified in type 1 diabetes: insulin, proinsulin, preproinsulin, glutamate decarboxylase-65 (GAD-65 or glutamate decarboxylase 2), GAD-67, glucose 6-phosphatase 2 (IGRP or island-specific glucose-6 catalytic subunit-related protein -phosphatase), insulinoma-associated protein 2 (IA-2) and insulinoma-associated protein 2β (IA-2e); other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, imogen 38, GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, hepatocarcinoma-intestinal-pancreatic-associated protein/pancreatic protein, glucose transporter 2, S100e, glial fibrillary acidic protein, regenerating gene II, pancreatic duodenal homeobox 1, myotonic dystrophy protein kinase, island-specific glucose-6 phosphatase catalytic subunit-related protein, and SST G protein-coupled receptors 1-5. It should be noted that insulin is an example of an antigen that can be characterized as either a self-antigen or a therapeutic protein antigen. For example, rHu insulin and bovine insulin are therapeutic protein antigens (ie, subject to unwanted immune attack), while endogenous human insulin is a self-antigen (ie, subject to unwanted immune attack). Since endogenous human insulin is not available for use in a pharmaceutical composition, a recombinant form is used in the compositions of the present disclosure.

Human insulin, including the exogenously produced form, useful in the compositions of the present disclosure has the following sequence (UNIPROT P01308):

MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALY

LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL

ALEGSLQKRGIVEQCCTSIC SLYQLENYCN (SEQ ID NO:5).

- 10 044172

GAD-65, including the exogenously derived form useful in the compositions of the present disclosure, has the following sequence (UNIPROT Q05329):

MASPGSGFWS FGSEDGSGDS ENPGTARAWC QVAQKFTGGI GNKLCALLYG DAEKPAESGG SQPPRAAARK AACACDQKPC SCSKVDVNYA FLHATDLLPA CDGERPTLAF LQDVMNILLQ YVVKSFDRST KVIDFHYPNE LLQEYNWELA DQPQNLEEIL MHCQTTLKYA IKTGHPRYFN QLSTGLDMVG LAADWLTSTA NTNMFTYEIA PVFVLLEYVT LKKMREIIGW PGGSGDGIFS PGGAISNMYA MMIARFKMFP EVKEKGMAAL PRLIAFSEH

SHFSLKKGAA ALGIGTDSVI LIKCDERGKM IPSDLERRIL EAKQKGFVPF LVSATAGTTV YGAFDPLLAV ADICKKYKIW MHVDAAWGGG LLMSRKHKWK LSGVERANSV TWNPHKMMGV PLQCSALLVR

EEGLMQNCNQ MHASYLFQQD KHYDLSYDTG DKALQCGRHV

DVFKLWLMWR AKGTTGFEAH VDKCLELAEY LYNIIKNREG YEMVFDGKPQ HTNVCFWYIP PSLRTLEDNE ERMSRLSKVA

PVIKARMMEY GTTMVSYQPL GDKVNFFRMV ISNPAATHQD

IDFLIEEIER LGQDL (SEQ ID NO:6).

The IGRP, including the exogenously derived form, useful in the compositions of the present disclosure has the following sequence (UNIPROT QN9QR9): MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFFIYFPLCFQFN QTVGTKMIWVAVIGDWLNLIFKWILFGHRPYWWVQETQIYPNHSSPCLE QFPTTCETGPGSPSGHAMGASCV WYVMVTAALSHTVCGMDKFSITLHR LTWSFLWSVFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHTPGI QTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPIAKKWCANPDWI HIDTTPFAGLVRNLGVLFGLGFAINSEMFLLSCRGGNNYTLSFRLLCALTS LTILQLYHFLQIPTHEEHLFYVLSFCK SASIPLTVVAFIPYSVHMLMKQSG KKSQ (SEQ ID NO:7).

In autoimmune thyroid diseases, including Hashimoto's thyroiditis and Graves' disease, the major antigens include thyroglobulin (TG), thyroid peroxidase (TPO), and thyrotropin receptor (TSHR); other antigens include sodium iodine symporter (NIS) and megalin. In thyroid-associated ophthalmopathy and dermopathy, insulin-like growth factor receptor 1 is an antigen in addition to thyroid autoantigens, including TSHR. In hypoparathyroidism, the main antigen is the calcium-sensing receptor.

In Addison's disease, the main antigens include 21-hydroxylase, 17a-hydroxylase, and P450 side chain cleavage enzyme (P450scc); other antigens include ACTH receptor, P450c21 and P450c17.

In premature ovarian failure, the main antigens include the FSH receptor and α-enolase.

In autoimmune hypophysitis or autoimmune pituitary disease, the major antigens include pituitary gland-specific protein factor (PGSF) 1a and 2; the other antigen is iodothyronine deiodinase type 2.

In multiple sclerosis, major antigens include myelin basic protein (MBP), oligodendrocyte myelin glycoprotein (MOG), and myelin proteolipid protein (PLP).

The MBP, including the exogenously derived form, useful in the compositions of the present disclosure has the following sequence (UNIPROT P02686):

MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNEVFGEA DANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRD APGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKY LATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHP ARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSR FSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDS RSGSPMARR (SEQ ID NO:8).

- 11 044172

MOG, including the exogenously derived form, useful in the compositions of the present disclosure has the following sequence (UNIPROT Q16653):

MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELP

CRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTEL

LKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFY WVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDP HFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF (SEQ ID NO:9).

The PLP, including the exogenously derived form, useful in the compositions of the present disclosure has the following sequence (UNIPROT P60201):

MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIET

YFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQ IFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCHCLGKWLG HPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIG SLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTF HLFIAAFVGA AATLVSLLTFMIATYNFAVLKLMGRGTKF (SEQ ID NO: 10).

Peptides/epitopes useful in the compositions of the present disclosure for the treatment of multiple sclerosis include some or all of the following sequences, individually in compositions of Formula 1, together in a mixture of compositions of Formula 1, or fused into one or more compositions of Formula 2:

MBP13-32: KYLATASTMDHARHGFLPRH (SEQ ID NO: 11);

MVR83-99: ENPWHFFKNIVTPRTP (SEQ ID NO: 12);

MBP111-129: LSRFSWGAEGQRPGFGYGG (SEQ ID NO: 13);

MBP146-170: AQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 14);

MOG1-20: GQFRVIGPRHPIRALVGDEV (SEQ ID NO: 15);

MOG35-55: MEVGWYRPPFSRWHLYRNGK (SEQ ID NO: 16); And

PLP 139-154: HCLGKWLGHPDKFVGI (SEQ ID NO: 17).

In rheumatoid arthritis, major antigens include collagen II, immunoglobulin binding protein, immunoglobulin G fragment region, double-stranded DNA, and natural and citrullinated forms of proteins implicated in the pathology of rheumatoid arthritis, including fibrin/fibrinogen, vimentin, collagen I and II, and alpha -enolase.

In autoimmune gastritis, the main antigen is H+, KY-ATPase.

In pernicious anemia, the main antigen is an intrinsic factor.

In celiac disease, the main antigens are tissue transglutaminase and natural and deamidated forms of gluten or gluten-like proteins such as alpha, gamma and omegagliadin, glutenin, hordein, secalin and avenin. Those skilled in the art will appreciate that, for example, although the primary antigen of celiac disease is alpha-gliadin, alpha-gliadin becomes more immunogenic in the body due to deamidation by tissue glutaminase, which converts alpha-gliadin glutamines to glutamic acid. Thus, while alpha-gliadin is initially a foreign food antigen, once it has been modified in the body to be more immunogenic, it can be characterized as a self-antigen.

In vitiligo, the main antigen is tyrosinase and tyrosinase-related protein 1 and 2.

MART1, melanoma antigen 1 recognized by T cells, Melan-A, including the exogenously derived form useful in the compositions of the present disclosure, has the following sequence (UNIPROT Q16655):

MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRR

RNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVP

NAPPAYEKLSAEQSPPPYSP (SEQ ID NO: 18).

- 12 044172

Tyrosinase, including the exogenously produced form, useful in the compositions of the present disclosure has the following sequence (UNIPROT P14679):

MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRSPCGQL

SGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFN

CGNCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDY

VIPIGTYGQMKNGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDI DF AHEAP AFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDIC TDEYMGGQHPTNPNLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTPEGPL RRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGF A SPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIF EQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGY DYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGL VSLLCRHKRKQLPEEKQPLLMEKEDYHSLYQSHL (SEQ ID NO: 19).

The melanocyte PMEL protein, gp100, including the exogenously derived form useful in the compositions of the present disclosure, has the following sequence (UNIPROT P40967): MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEA QRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIING SQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGG PVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQL RALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTH TYLEPGPVTAQVVLQAAIPLTSGGSSPVPGTTD GHRPTAEAPNTTAGQVPTTEVVG TTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLA EMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMST ESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGE GDAFELTVSCQGGLPKEACME ISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGS GTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRL MKQDFSVPQLPHSSSHWLRLPRIFGSCPIGENSPLLSGQQV (SEQ ID NO:20).

In myasthenia gravis, the main antigen is the acetylcholine receptor.

In pemphigus vulgaris and variants, the main antigens are desmoglein 3, 1 and 4; other antigens include pemfaxine, desmocollins, plakoglobin, perplakin, desmoplakins and acetylcholine receptor.

In bullous pemphigoid, the main antigens include BP180 and BP230; other antigens include plectin and laminin 5.

In Dühring's dermatitis herpetiformis, the main antigens include endomysium and tissue transglutaminase.

In epidermolysis bullosa acquired, the main antigen is collagen VII.

In systemic scleroderma, the main antigens include matrix metalloproteinase 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrillin-1, and PDGF receptor; other antigens include Scl-70, U1 RNP, Th/To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II and III, PM-Slc, fibrillarin and B23.

In mixed connective tissue disease, the main antigen is U1snRNP.

In Sjögren's syndrome, the main antigens are the nuclear antigens SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose) polymerase and topoisomerase, muscarinic receptors, and Fc-gamma receptor IIIb.

In systemic lupus erythematosus, the major antigens include nuclear proteins, including Smith antigen, SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone proteins, and double-stranded DNA (against which autoantibodies are produced in the disease process).

In Goodpasture syndrome, the main antigens include glomerular basement membrane proteins, including collagen IV.

In rheumatic heart disease, the main antigen is cardiac myosin.

In autoimmune polyendocrine syndrome type 1, antigens include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic cytochromes P450 P4501A2 and 2A6, SOX-9, SOX-10 , calcium-sensitive receptor protein and type 1 interferons interferon alpha, beta and omega.

In neuromyelitis optica, the main antigen is AQP4.

- 13 044172

Aquaporin-4, including the exogenously derived form useful in the compositions of the present disclosure, has the following sequence (UNIPROT P55087):

MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGST

INWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKS

VFYIAAQCLGAIIGAGILYLVTPPSWGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIF

ASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWI

YWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVE

TDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV (SEQ ID NO:21).

In uveitis, the major antigens include retinal S antigen or S-arrestin and interphotoreceptor retinol binding protein (IRBP) or retinol binding protein 3.

S-arrestin, including the exogenously derived form, useful in the compositions of the present disclosure has the following sequence (UNIPROT P10523):

MAASGKTSKS EPNHVIFKKI SRDKSVTIYL GNRDYIDHVS

QVQPVDGVVL VDPDLVKGKK LTFRRDLYFS RVQVYPPVGA PFLLTFPDYL PCSVMLQPAP TDAEEDKIPK KSSVRLLIRK FMSDKPLHLA VSLNKEIYFH IKAFVEQVAN VVLYSSDYYV

VYVTLTCAFR YGQEDIDDVIG

ASTPTKLQES LLKKLGSNTY

QDSGKSCGVD FEVKAFATDS

VQHAPLEMGP QPRAEAAWQF

GEPIPVTVTV TNNTEKTVKK

KPVAMEEAQE KVPPNSTLTK

TLTLLPLLAN NRERRGIALD GKIKHEDTNL ASSTIIKEGI DRTVLGILVS

YQIKVKLTVS GFLGELTSSE VATEVPFRLM HPQPEDPAKE SYQDANLVFE EFARHNLKDA GEAEEGKRDK NDVDE (SEQ ID NO:22).

The IRBP, including the exogenously derived form, useful in the compositions of the present disclosure has the following sequence (UNIPROT P10745):

MMREWVLLMSVLLCGLAGPTHLFQPSLVLDMAKVLLDNYCFPENLLGMQEAIQQAI KSHEILSISDPQTLASVLTAGVQSSLNDPRLVISYEPSTPEPPPQVPALTSLSEEELLA WLQRGLRHEVLEGNVGYLRVDSVPGQEVLSMMGEFLVAHVWGNLMGTSALVLDL RHCTGGQVSGIPYIISYLHPGNTILHVD TIYNRPSNTTTEIWTLPQVLGERYGADKDV VVLTSSQTRGVAEDIAHILKQMRRAIVVGERTGGGALDLRKLRIGESDFFFTVPVSRS LGPLGGGSQTWEGSGVLPCVGTPAEQALEKALAILTLRSALPGVVHCLQEVLKDYY TLVDRVPTLLQHLASMDFSTVVSEEDLVTKLNAGLQAASEDPRLLVRAIGPTETPSW PAPDAAAEDSPGVAPELPEDEAIRQALVDSVFQVSVLPGNVGYLRFDSFADASVLG VLAPYVLRQVWEPLQDTEHLIMDLRHNPGGPSSAVPLLLSYFQGPEAGPVHLFTTY DRRTNITQEHFSHMELPGPRYSTQRGVYLLTSHRTATAAEEFAFLMQSLGWATLVG EITAGNLLHTRTVPLLDTPEGSLALTVPVLTFIDNH GEAWLGGGVVPDAIVLAEEALD KAQEVLEFHQSLGALVEGTGHLLEAHYARPEVVGQTSALLRAKLAQGAYRTAVDLE SLASQLTADLQEVSGDHRLLVFHSPGELVVEEAPPPPPAVPSPEELTYLIEALFKTEV LPGQLGYLRFDAMAELETVKAVGPQLVRLVWQQLVDTAALVIDLRYNPGSYSTAIPL LCSYFFEAEPR QHLYSVFDRATSKVTEVWTLPQVAGQRYGSHKDLYILMSHTSGSA AEAFAHTMQDLQRATVIGEPTAGGALSVGIYQVGSSPLYASMPTQMAMSATTGKA WD LAG VE P DITVPMSEALSIAQ DIVALR AKVPTVLQTAG KLVAD N YASAE LG AKM AT KLSGLQSRYSRVTSEVALAEILGADLQMLSGDPHLKAA HIPENAKDRIPGIVPMQIPS PEVFEELIKFSFHTNVLEDNIGYLRFDMFGDGELLTQVSRLLVEHIWKKIMHTDAMIID MRFNIGGPTTSSIPILCSYFFDEGPPVLLDKIYSRPDDSVSELWTHAQVVGERYGSKK SMVILTSSVTAGTAEEFTYIMKRLGRALVIGEVTSGGCQPPQTYHVDDTNLYLTIPTA RSVGASDGSSWEG VGVTPHVVVPAEEALARAKEMLQHNQLRVKRSPGLQDHL (SEQ ID NO:23).

In embodiments in which the antigen is a foreign antigen against which an unwanted immune response may develop, such as food antigens, the specific antigens may occur:

from peanuts: conarchin (Ara h 1), allergen II (Ara h 2), peanut agglutinin, conglutin (Ara h 6); conarchin, for example, has the sequence designated UNIPROT Q6PSU6 from apple: 31 kDa major allergen/homolog of disease resistance protein (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03 D (Mal d 1 );

from milk: α-lactalbumin (ALA), lactotransferrin; from kiwi: actinidia (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), kivellin (Act d 5);

- 14 044172 from egg whites: ovomucoid, ovalbumin, ovotransferrin and lysozyme;

from egg yolks: livetin, apovitillin and vosvetin;

from mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transport protein (Sin a 3), profilin (Sin a 4);

from celery: profilin (Api g 4), high molecular weight glycoprotein (Api g 5);

from shrimp: allergen Pen a 1 (Pen 1), allergen Pen m 2 (Pen m 2), isoform of fast tropomyosin;

from wheat and/or other cereals: high molecular weight glutenin, low molecular weight glutenin, alpha, gamma and omega gliadin, hordein, secalin and/or avenin;

peptides/epitopes useful in the compositions of the present disclosure for the treatment of celiac disease include some or all of the following sequences, individually in compositions of Formula 1, together in a mixture of compositions of Formula 1, or fused into one or more compositions of Formula 2:

corresponding DQ-2, alpha-gliadin 33-mer native:

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:24) corresponding to DQ-2, alpha-gliadin 33-mer deamidated:

LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:25) corresponding to DQ-2, alpha-gliadin:

QQYPSGQGSFQPSQQNPQ (SEQ ID NO:26) corresponding to DQ-2, omega-gliadin (wheat, U5UA46):

QPFPQPEQPFPW (SEQ ID NO:27) from strawberries: the main allergen of strawberries is Fra 1-E (Fra 1); and from bananas: profilin (Mus xp 1).

In embodiments in which the antigen is a foreign antigen against which an unwanted immune response develops, such as antigens of animal origin, antigens of plant origin and antigens from the environment, the specific antigens may be, for example: antigens originating from cats, mice, dogs, horses, bees, from dust, from trees and goldenrod, including the following proteins or peptides that originate from weeds (including ragweed allergens amb 1, 2, 3, 5 and 6 and Amb 15; amaranth Che 2 and 5; and allergens of other weeds Par j 1, 2 and 3 and Par o 1);

herbs (including the main allergens Cyn d 1, 7 and 12; Dac g 1, 2 and 5; Hol I 1.01203; Lol p 1, 2, 3, 5 and 11; Mer 1; Pha 1; Poa p 1 and 5 );

pollen from ragweed and other weeds (including curly sorrel, white goosefoot, amaranth, plantain, small sorrel and wormwood), grasses (including Bermuda grass, Johnson grass, meadow grass, hedgehog grass, sweet grass and timothy grass) and trees (including catalpa, elm, hickory, olive, pecan, sycamore and walnut);

dust (including the main allergens from Dermatophagoides pteronyssinus species, such as Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21 and 23; from Dermatophagoides farina species, such as Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18, 22 and 24; from Blomia tropicalis species, such as Blo t 1, 2, 3 , 4, 5, 6, 10, 11, 12, 13, 19 and 21; as well as allergens Eur m 2 from Euroglyphus maynei, Tyr p 13 from Tyrophagus putrescentiae and allergens Bla g 1, 2 and 4; Per 1, 3 and 7 from a cockroach);

pets (including cats, dogs, rodents and farm animals; major cat allergens include Fel d 1-8, feline IgA, BLa g 2 and feline albumin; major dog allergens include Can f 1-6 and canine albumin);

bee venom, including the main allergens Api m 1-12; and fungi, including allergens that originate from Aspergillus and Penicillium species, as well as Alternaria alternata, Davidiella tassiana and Trichophyton rubrum species.

As will be appreciated by those skilled in the art, a patient can be tested to identify the antigen against which an unwanted immune response has developed, and a protein, peptide, or the like can be developed from that antigen. and included as X in the composition of the present disclosure.

Sialylated antigens, antibodies, antibody fragments.

The following are examples of antigens, antibodies, antibody fragments with sialylation that can be removed to leave glycosylation for specific targeting to the ASGPR: follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), luteinizing hormone (LH), osteopontin, thyroid stimulating hormone ( TSH), agalsidase-alpha, agalsidase-beta (FABRAZYME®; Genzyme), epoetin-alpha and epoetin-beta, follitropin-alpha (GONAL-F®; Merck/Serono) and follitropin-beta (FOLLISTIM®; Schering-Plough) , insulin-like growth factor binding protein 6 (IGFBP-6), lutropin-alpha (LUVERIS®; Merck/Serono), transforming growth factor β1, antithrombin (ATryn®/TROMBATE-III®; Genzyme/Talecris Biotherapeutics), thyrotropin-alpha ( THYROGEN®; Genzyme), lenograstim, sargramostim (LEUKINE®; Genzyme), interleukin-3, prourokinase, lymphotoxin, C1-esterase inhibitor (Berinert®; CSL), IgG-like antibodies, interferon-beta, coagulation factor VIIa (NOVOSEVEN ®; Novo Nordisk), blood clotting factor VIII (morocto- 15 044172 cog-alpha), blood clotting factor IX (nonacog-alpha) (BENEFIX®; Wyeth) and tumor necrosis receptor p55 fusion protein; (See: Byrne et al., Drug Discovery Today, Vol 12, No. 7/8, pages 319-326, Apr. 2007 and Sola et al., BioDrugs. 2010; 24(1): 9-21). Pharmaceutically relevant proteins that have previously been hyperglycosylated and can be desialylated for hepatocyte targeting-ASGPR include: interferon alpha and gamma, luteinizing hormone, Fv antibody fragments, asparaginase, cholinesterase, darbepoetin alpha (AraNESP®; Amgen), thrombopoietin, leptin, FSH, IFN-a2, serum albumin and corifollitropin-alpha.

Proteins with glycans that do not typically contain sialic acids at their ends, including proteins produced in bacteria or yeast (such as arginase, some insulins, and uricase), will not undergo desialylation.

Those skilled in the art will appreciate that publicly available references such as UNIPROT disclose the presence and location of desialylation sites on most, if not all antigens, antibodies, antibody fragments and ligands of interest.

Antibodies and peptide ligands.

In embodiments that utilize an antibody, antibody fragment, or ligand, such components are selected to specifically bind a target circulating protein or peptide or antibody, resulting in liver uptake of the circulating target component, possibly as an adduct with a targeting component. delivery, which ultimately leads to clearance and inactivation of the circulating target component. For example, liver-targeted factor VIII will bind to and clear circulating factor VIII antibodies. Techniques for identifying such components will be familiar to those skilled in the art.

Linkers.

Linkers used in the compositions of the present disclosure (Y in Formula 1) may include N-hydroxysuccinamidyl linkers, malic acid amide linkers, vinyl sulfone linkers, pyridyl dithiol poly(ethylene glycol) linkers, pyridyl dithiol linkers, nitrophenyl carbonate linkers , NHS ester linkers, nitrophenoxy-poly(ethylene glycol) ester linkers, etc.

One particular group of linkers of the formula Y'-CMP is given below (where Y' represents the remainder of the linker and R ⁹ and Z are as defined). More specifically, in the group of linkers comprising the formula Y'-CMP, the substituent R ⁹ is an ethyl acetamide group, and even more particularly, ethyl acetamide is conjugated to C1 N-acetylgalactosamine, p N ^Z .

Formula Y'-CMP

Dithiol-containing linkers, in particular disulfanylethyl carbamate-containing linkers (referred to as free amine X-incorporating, otherwise referred to as disulfanylethyl ester without free amine X inclusion), are particularly preferred in the compositions of the present disclosure as they have the ability to cleave and release antigen in its original form after entering the cell, such as illustrated below (where Y' represents the remainder of the linker and X' and Z are as defined).

- 16 044172

Particularly with respect to the linkers shown below as Formula Ya - Formula Yp:

the left parenthesis ( denotes the relationship between X and Y;

the right or lower parenthesis and ) denotes the relationship between Y and Z;

n equals an integer representing a population including from about 1 to 100, in particular from about 8 to 90 (for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90 or 95), more specifically from about 40 to 80 (for example, 39, 40, 43, 45 , 46, 48, 50, 52, 53, 55, 57, 60, 62, 65, 66, 68, 70, 73, 75, 78, 80 or 81) groups of ethylene glycol, where the collection usually covers an integer defined as n ±10%;

p equals an integer representing a population comprising from about 2 to 150, in particular from about 20 to 100 (for example, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 105) and more particularly from about 30 to 40 (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43 or 44), where the population usually covers an integer defined as p ±10%;

q is equal to an integer representing a population including from about 1 to 44, in particular from about 3 to 20 (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, or 22) and more particularly from about 4 to 12 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13), where the plurality is typically covers an integer defined as q ±10%; And

R ⁸ is -CH ₂ - (methyl) or -CH ₂ -CH2-C(CH ₃ )(CN)- (1-cyano-1-methylpropyl or CMP).

R ⁹ is a direct linkage or -CH ₂ -CH ₂ --NH-C(O)-(ethyl acetamide group or EtAcN), as illustrated in the following structures of Formula 1 (where the EtAcN group is shown and the remainder of the linker is designated Y') :

he he he and Z represents galactose, galactosamine or N-acetylgalactosamine conjugated at C1.

SG

NN^

Formula Ya

- 17 044172

Formula Yb

Formula Yc

Formula Yg

- 18 044172

Formula Yn (Formula Yn linkers can be synthesized via certain precursors that make Yn particularly suitable for conjugation to hydrophobic antigens)

- 19 044172 o

Formula Yp.

The linkers given above in the form of formulas Yh-Yn are synthesized as isomers, which are used without separation. For example, linkers of formulas Yh, Yi, Yj and Yn will be a mixture of 8,9dihydro-1H-dibenzo[b,f][1,2,3]triαzolo[4,5-d]αzocin-8-yl and 8 ,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl structures illustrated below:

Linkers of formulas Yk, Yl and Ym will be a mixture of 8,9-dihydro-Shdibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8-yl and 8 ,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-9-yl structures illustrated below

The presence of such isomeric mixtures does not impair the functionality of compositions in which such linkers are used.

Galactosylated components.

The galactosylated components used in the compositions of the present disclosure serve to target the compositions to liver cells (eg, specifically binding hepatocytes) and can be selected from: galactose, galactosamine or N-acetylgalactosamine. It has been reported that the affinity for ASGPR can be maintained by modifying one of the two sides of the C3/C4 diol anchor of galactose (Mamidyala, Sreeman K., et al., J. Am. Chem. Soc. 2012, 134, 1978-1981), therefore The conjugation points are located, in particular, at C1, C2 and C6.

Specific galactosylated components include C1- or C6-conjugated galactose, C2-conjugated galactosamine, and C6-conjugated N-acetylgalactosamine. Other specific galactosylated components include N-acetylgalactosamine conjugated at C2, more particularly conjugated to a linker bearing an ^R9 substituent which is _CH2 . Third galactosylated moieties include galactose, galactosamine or N-acetylgalactosamine conjugated at C1, more particularly conjugated to a linker bearing an R ⁹ substituent that is an ethyl acetamide group.

ASGPR-targeting antibodies.

The ASGPR-specific antibodies used in the compositions of the present disclosure also serve to target the compositions of the present disclosure to liver cells and can be selected from commercially available products such as anti-asialoglycoprotein receptor antibody

- 20 044172 (ab42488) from Abcam plc, Cambridge, UK, and ASGPR1/2 (FL-291) (sc28977) from Santa Cruz Biotechnology, Inc., Dallas, TX. Alternatively, such antibodies can be expressed using any of a variety of published sequences, such as Dom26h-196-61, a single domain anti-ASGPR antibody:

EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRIS

ARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEH

TRFDSWGQGTLVTVSS (SEQ.ID.No:6)

[Coulstock E., et al., (2013) Liver-targeting of interferon-alpha with tissue-specific domain antibodies. PLoS One. 8(2):e57263 and US 2013/0078216], or such a sequence with conservative substitutions. The aforementioned US patent application discloses liver-targeted molecules such as DOM26h-196-61 for the delivery of certain therapeutic agents [including interferon (interferon-alpha 2, interferon-alpha 5, interferon-alpha 6 or consensus interferon) , ribavirin, nexavar/sorafenib, erbitus/cetuximab, avastatin/bevacizumab and Herceptin/trastuzumab] for the treatment of liver diseases. Formula 2 compositions using DOM26h-196-61 or another liver-targeted molecule described in US 2013/0078216 do not include interferon (interferon-alpha 2, interferon-alpha 5, interferon-alpha 6, or consensus interferon), ribavirin, nexavar/sorafenib, erbitus/cetuximab, avastatin/bevacizumab and Herceptin/trastuzumab, within their scope.

New sequences for an antibody, antibody fragment or peptide that specifically target ASGPR can be discovered using various methods known to those skilled in the art. These methods may include, but are not limited to, vaccination technology, hybridoma technology, display library technologies (including bacterial and phage platforms), endogenous antigen repertoire screening technologies, directed evolution, and rational design.

Nomenclature.

Compositions of Formula 1 may be named using a combination of IUPAC and trivial names. For example, a compound corresponding to formula 1, where X is the cyclobutyl moiety (shown in place of the antigen by way of illustration), Y is the formula Ya, m is 1, n is 4, and Z is N-acetylgalactosamin-2-yl:

may be called (Z)-(21-cyclobutyl-1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7, 10,13-tetraoxa-16,17-dithiagenicosan-21-ylidene)triaz-1-in2-ium chloride, so the corresponding composition of the present disclosure, where X is tissue transglutaminase may be called (Z)-(21-(tissue transglutaminase)-1-oxo-1-((2,4,5trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiagenicosan -21-ylidene)triaz-1-yne-2-ium chloride. A corresponding composition of the present disclosure, where X' is tissue transglutaminase, m is 2, n is 4, and Z' is N-acetylgalactosamin-2-yl, may be referred to as (3Z)-((tissue transglutaminase)-1. 3-diylbis(1-oxo1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17- dithiagenicosan-21-yl-21-ylidene))-bis-(triaz-1-in-2-ium) chloride.

To simplify the compositions of formulas 1 may be named using an alternative naming system by referring to X and matching one of formulas 1a-1p (as illustrated in the reaction schemes) followed by repetition of integers for the variables m, n, p and/or q , R ⁸ , R ⁹ and identification of the galactosylated moiety and the position at which it is conjugated. According to this system, the composition of formula 1a, where X is ovalbumin, m is 2, n is 4, and Z is X-acetylgalactosamin-2-yl, can be called F1a-OVA-m ₂ -n ₄ 2NAcGAL.

- 21 044172

Similarly, the following composition of formula 1,

may be called 2-((2-(((3-(3-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro2H-pyran-2-yl)oxy)-16- cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbonothioyl)thio)-3,6,9,12-tetraoxa-20-azadocosyl)-3,9-dihydro-8H-dibenzo[b, f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)carbamoyl)oxy)ethyl)disulfanyl)ethyl insulin carboxylate.

Isomer

may be called 2-((2-(((3-(1-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro2H-pyran-2-yl)oxy)-16- cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbonothioyl)thio)-3,6,9,12-tetraoxa-20-azadocosyl)-1,9-dihydro-8H-dibenzo[b, f][1,2,3]triαzolo[4,5-d]αzocin-8-yl)-3-oxopropyl)carbamoyl)-oxy)ethyl)disulfanyl)ethyl insulin carboxylate (bold text added for ease of identification of the difference between formal names). By using the naming system adopted for the present disclosure, both isomers may be referred to as F1n-insulinm ₁ -n ₁ -p ₁ -q ₄ -CMP-EtAcN-1NAcGAL, where CMP indicates that R ⁸ is 1-cyano-1- methylpropyl, EtAcN indicates that R ⁹ is ethyl acetamide, and 1NAcGAL indicates that Z'' is N-acetylgalactosamine conjugated at C1. The absence of the abbreviation EtAcN before the Z designation will indicate that R ⁹ represents a direct connection.

In formula 2 compositions, left-to-right orientation should not be taken to indicate the order from N to C unless specifically stated to the contrary. For example, a compound of Formula 2 where m' is 1, m'' is 0, X is ovalbumin, Y is Gly ₃ Ser, and Z is an anti-ASGPR antibody, Dom26h-196-61, may be referred to as OVA-Gly ₃ Ser-DOM and be construed to cover both of the following:

N-OVA-Gly ₃ Ser-DOM-C and

N-DOM-Gly ₃ Ser-OVA-C

Compositions of Formula 2, for example, where m' is 1, m'' is 0, X is ovalbumin, Y is Gly ₃ Ser, and Z is anti-ASGPR Dom26h-196-61 (with a purification tag attached through additional linker) can be named as follows:

N-OVA-Gly ₃ Ser-DOM-Gly ₃ Ser-6xHis-C or

N- DOM- Gly ₃ Ser- OVA- Gly ₃ Ser-6xHis -C where the C'-terminal group of Gly ₃ Ser-6xHis is an amino acid sequence that facilitates isolation and purification.

Composition of formula 2, where m' is 1, m'' is 1, each X is factor VIII, each Y is Gly ₃ Ser, and Z is an anti-ASGPR antibody, Dom26h-196-61 (purification labeled, attached via an additional linker) may be called FVIIIGly ₃ Ser-DOM-Gly ₃ Ser-FVIII-Gly ₃ Ser-6xHis and covers both of the following:

- 22 044172

N-FVIII-Gly ₃ Ser-DOM-Gly ₃ Ser-FVIII-Gly ₃ Ser-6xHis-C

N-6xHis-Gly ₃ Ser-FVIII-Gly ₃ Ser-DOM-Gly ₃ Ser-FVIII-C.

Composition of formula 2, where m' is 3, m' is 0, one, three X antigens are, respectively, alpha-gliadin 33-mer deamidated (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26 ) and omega-gliadin (SEQ ID NO:27), each Y is a linker with an immunoproteasome cleavage site (IPC), and Z is an anti-ASGPR antibody, Dom26h-196-61, may be called alpha-gliadin 33-mer deamidated-IPC-alpha-gliadin-IPC-omega-gliadin-IPCDOM.

Preparation of compositions according to the invention

Compositions of Formula 1 can be prepared, for example, by amending the procedures described in Zhu, L., et al., Bioconjugate Chem. 2010, 21, 2119-2127. The synthesis of certain compositions of formula 1 is also described below with reference to reaction schemes 1-14. Other approaches for synthesis will be apparent to those skilled in the art.

Formula 101 (below) is an alternative representation of X

X ¹ --R ¹

101 where R ¹ represents a free surface amino (-NH2) or thiol (-SH) moiety located in the three-dimensional structure of X' so as to be accessible for conjugation to the linker, and X' represents the remainder of X excluding the designated free amino group (amino groups) [(X'' is used in reaction schemes to denote the rest of X excluding the free thiol group(s)]. Depending on the individual composition of X, it will have at least one (N-terminal amine) and may have several R ¹ -groups (primarily lysine residues or cysteine residues that are not involved in the formation of disulfide bonds), as represented by m, which is equal to an integer from about 1 to 100, more typically 1 or from about 4 to 20, and most typically from 1 to about 10.

The variables used in the reaction schemes are as defined above, and additionally include the following, which are to be understood as having their given meanings unless specifically stated to the contrary with respect to a particular reaction scheme or step.

R ² represents OH or a protecting group;

R ³ represents OH, NH2, NHAc, a protecting group or an NH-protecting group;

R ⁴ represents OH or a protecting group;

R ⁵ represents OH or a protecting group;

R ⁶ represents OH or a protecting group;

Z' is C1 or C6 conjugated galactose, C2 conjugated galactosamine, or C6 conjugated N-acetylgalactosamine;

R ⁸ is -CH ₂ - or -CH2-CH2-C(CH ₃ )(CN)- and

R ⁹ represents a direct bond and Z'' represents N-acetylgalactosamine conjugated at C2;

R ⁹ represents an ethyl acetamide group, and Z'' represents galactose, galactosamine or N-acetylgalactosamine conjugated at C1.

Synthesis reaction parameters.

The terms solvent, inert organic solvent, or inert solvent mean a solvent that is inert under the reaction conditions described in connection therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine, etc.]. Unless otherwise indicated, the solvents used in the reactions of the present disclosure are inert organic solvents.

The term q.s. means adding an amount sufficient to achieve the stated purpose, for example, bringing the solution to the required volume (i.e. 100%).

Isolation and purification of the compounds and intermediates described herein, if necessary, can be accomplished using any suitable separation or purification technique, such as, for example, filtration, extraction, crystallization, column chromatography, thin layer chromatography or thick layer chromatography, centrifugal size exclusion chromatography, high performance liquid chromatography, recrystallization, sublimation, fast protein liquid chromatography, gel electrophoresis, dialysis or a combination of these techniques. Specific illustrations of suitable separation and isolation techniques may be available by reference to the following examples. However, other equivalent separation or isolation techniques can of course also be used.

Unless otherwise indicated (including in the Examples), all reactions are carried out at standard atmospheric pressure (approximately 1 atmosphere) and ambient (or room) temperature (approximately 20°C), with a pH of approximately 7.0-8.0.

Characterization of the reaction products can be carried out using conventional means, for example, proton and carbon NMR, mass spectrometry, size exclusion chromatography, infrared spectroscopy, gel electrophoresis.

Reaction Scheme 1 illustrates the preparation of compositions of formula 1, where Z can be galactose, galactosamine or N-acetylgalactosamine. In this regard, and as defined above, Z' used in reaction scheme 1 covers galactose conjugated at C1 and C6 and corresponding to the following structures according to formula 1:

galactosamine conjugated at C2 and corresponding to the following structure according to formula 1:

and N-acetylgalactosamine conjugated at C6 and corresponding to the following structure according to formula 1:

Reaction scheme 1

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As illustrated above in Reaction Scheme 1, Step 1, surface thiol group(s) can be produced on an antigen, antibody, antibody fragment or ligand with free surface amine group(s) (Formula 101') by contacting Trout's reagent (Formula 102 ) at a pH of approximately 8.0 for approximately 1 hour to obtain Formula 103', wherein unreacted Trout's reagent is removed, for example, using centrifugal size exclusion chromatography. The two structures shown below illustrate the product of reaction scheme 1, step 1, respectively, showing the free surface amino group(s) initially found on X (i.e., formula 103', where X' represents the remainder of X excluding the free ones indicated

- 25 044172 surface amino groups), and excluding free surface amino group(s) (i.e. formula

103). This corresponds to the illustrated difference between X and formula 101. This convention was followed in subsequent reaction schemes.

In Reaction Scheme 1, Step 2, pyridyl dithiol poly(ethylene glycol) ester and NHS (Formula 104) are contacted with galactosamine (Formula 105, where R ³ is NH ₂ and R ² , R ⁴ , R ⁵ and R ⁶ is OH) under stirring conditions at pH approximately 8 for 1 hour to obtain the corresponding pyridyl-dithiol-poly(ethylene glycol) sugar of formula 106A, which can be used without further purification.

In Reaction Scheme 1, Step 3, 4,4'-dithiodipyridine (Formula 107) is contacted with thiol-poly(ethylene glycol)propanoic acid (Formula 108) to give the corresponding pyridyldithiol-poly(ethylene glycol)propanoic acid (Formula 109).

In Reaction Scheme 1, Step 4, an acid of formula 109 is contacted with a protected galactose or N-acetylgalactosamine of formula 105, where R ² is OH and R ³ , R ⁴ , R ⁵ and R ⁶ are protecting groups (PG) where R ⁶ represents OH and R ² , R ³ , R ⁴ and R ⁵ represent PG, or where R ⁶ represents N-acetyl and R ² , R ³ , R ⁴ and R ⁵ represent PG, to give the corresponding pyridyl-dithiol-poly(ethylene glycol) sugars of formulas 106B, 106C and 106D, which can be used after deprotection.

In Reaction Scheme 1, Step 5, an excess (corresponding to the m value) of the product from Step 2 or Step 4 (Formula 106, i.e. 106A, 106B, 106C or 106D) is added to a stirred solution of the product from Step 1 (Formula 103') for approximately 1 hour, followed by centrifugal size exclusion chromatography to remove any remaining free reactants to obtain the corresponding product according to Formula 1a, respectively, Formula 1aA, Formula 1aB, Formula 1aC and Formula 1aD.

Compositions corresponding to formula 1a may be named accordingly, for example as follows:

"FlaA-X'-m _m -n _n " or "Fla-X'-m _m -n _n -2NGAL", "FlaB-X'-m _m -n _n " or "Fla-X'-m _m - n _n -lGAL", "FlaC-X'-m _m -n _n " or "Fla-X'-m _m -n _n -6GAL", "FlaD-X'-m _m -n _n " or "Fla- X'-m _m -n _n -6NAcGAL", respectively, for products obtained using the intermediate according to formulas 106A-D.

Referring to reaction schemes 2-14 and for purposes of nomenclature used in connection therewith, except where expressly stated otherwise, Z'' refers to N-acetylgalactosamine conjugated at C2:

or to galactose, galactosamine or N-acetylgalactosamine conjugated at C1. It should be noted that C1-conjugated compositions must be protected during synthesis, for example by cyclization of the amine with a C3-hydroxyl, and subjected to deprotection followed by incorporation of the protected galactosamine into an adjacent portion of the linker.

Poly(galactose-methacrylate) reagents of formulas 201, 401, 501, 601, 701, 803 and 1401 can be prepared by adding a methacrylate group to galactose, for example by contacting galactosamine and methacrylic anhydride, followed by polymerization by reversible addition and fragmentation (RAFT) using the appropriate RAFT tool in

- 26 044172 the presence of azobiisobutyronitrile (AIBN) in a suitable solvent, starting with freeze-thaw cycles followed by heating to about 60-80°C, preferably 70°C for 5-8, preferably about 6 hours. The polymer can be precipitated in a lower alkanol , preferably methanol.

Reaction scheme 2

As illustrated in Reaction Scheme 2, the resulting antigen, antibody, antibody fragment or ligand with free surface thiol group(s), for example, as described with reference to Reaction Scheme 1, Step 1 (Formula 103'), is contacted with excess ( corresponding to the value of m) pyridyl-dithiol-poly(ethylene glycol) of formula 201 for 1 hour to obtain the corresponding product according to formula 1b.

Compositions of Formula 1b may be named as follows:

"Flb-X'-rn _m -n _n -p _p -2NAcGAL" or "Flb-X'-rn _m -n _n -p _p -EtAcN-Z".

For example, a composition of formula 1b where X' is uricase, m is 1, n is 4, p is 4, and Z'' is N-acetylgalactosamine conjugated at C2 may be referred to as F1 b-uricase-m ₁ -n ₄ -p ₄ -2NAcGAL or 30-(uricase)-3,5,7,9-tetramethyl-12-oxo-1-phenyl-1-thioxo3,5,7,9-tetrakis((2,4,5- trihydroxy-6-(hydroxymethyl)tetrahydro-2H-πpyran-3-yl)carbamoyl)13,16,19,22-tetraoxa-2,25,26-tritiatriacontane-30-iminium.

Reaction scheme 3

As illustrated in Reaction Scheme 3, an antigen, antibody, antibody fragment or ligand with native free surface thiol group(s) (cysteines) [formula 101 corresponds to formula 101 and illustrates X, as the term will be used hereinafter, representing X for excluding the designated free surface thiol group(s)] are contacted with an excess (corresponding to the m value) of pyridyl-dithiol-poly(ethylene glycol) of formula 201 to obtain the corresponding product according to formula 1c.

Compositions corresponding to Formula 1c may be named as follows: "Flc-X'-rn _m -n _n -p _p -2NAcGAL" or "Flc-X'-m _m -n _n -p _p -EtAcN-Z".

- 27 044172

Reaction scheme 4

As illustrated in Reaction Scheme 4, an antigen, antibody, antibody fragment or ligand with native free surface thiol group(s) of Formula 101 is contacted with an excess (corresponding to the m value) of pyridyl dithiol of Formula 401 to obtain the corresponding product of Formula 1 d.

Compositions corresponding to Formula 1d may be named as follows: "Fld-X'-m _m -p _p -2NAcGAL" or "Fld-X'-m _m -p _p -EtAcN-Z".

Reaction scheme 5

As illustrated in Reaction Scheme 5, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of Formula 101' is contacted with an excess (corresponding to the m value) of n-nitrophenyl carbonate of Formula 501 to obtain the corresponding product of Formula 1e.

Compositions corresponding to Formula 1e may be named as follows: "Fle-X'-m _m -p _p -2NAcGAL" or "Fle-X'-m _m -p _p -EtAcN-Z".

Reaction scheme 6 g

If formula

As illustrated in Reaction Scheme 6, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of Formula 101' is contacted with an excess (corresponding to the m value) of an n-nitrophenyl carbonate poly(ethylene glycol) ester. formulas

601 to obtain the corresponding product according to formula 1f.

Compositions corresponding to formula 1f may be named as follows:

"Flf-X'-m _m -n _n -p _p -2NAcGAL" or "Flf-X'-m _m -n _n -p _p -EtAcN-Z".

Reaction scheme 7

As illustrated in Reaction Scheme 7, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of Formula 101' is contacted with an excess (corresponding to the m value) of an NHS ester and poly(ethylene glycol) of Formula 701 to obtain the corresponding product according to formula 1g.

Compositions corresponding to Formula 1g may be named as follows:

- 29 044172

Reaction scheme 8

As illustrated in Reaction Scheme 8, Step 1, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of Formula 101' is contacted with an excess (corresponding to the m value) of the amine-reactive linker for a click chemistry reaction of Formula 801 to obtain the corresponding product according to formula 802.

In Reaction Scheme 8, Step 2, the product of Formula 802 is then contacted with an equivalent amount (again corresponding to the m value) of galactose(amine) polymer of Formula 803 to yield the corresponding isomeric product of Formula 1h. The two isomers illustrated above are obtained by nonspecific cyclization of an azide of formula 803 with a triple bond of formula 802. Such nonspecific cyclization occurs in the synthesis of other compositions where Y is selected from the formulas Yh - Yn, but will not be shown in every case.

Compositions corresponding to formula 1h may be named as follows:

"F 111-Х' -rn _m -n _n -p _p -q _q -2NAcGAL" or "F Ih-X'-m _m -n _n -p _p -q _q -EtAcN-Ζ".

- 30 044172

Reaction scheme 9

As illustrated in Reaction Scheme 9, Step 1, an antigen, antibody, antibody fragment or ligand with native free surface thiol group(s) of formula 101'' is contacted with an excess (corresponding to the m value) of a thiol-reactive linker for a click chemistry reaction formula 901 to obtain the corresponding product according to formula 902''.

In Reaction Scheme 9, Step 2, the product of Formula 902'' is then contacted with an equivalent amount (again corresponding to the m value) of galactose(amine) polymer of Formula 803 to produce the corresponding isomeric product of Formula 1i.

Compositions corresponding to Formula 1i may be named as follows:

Proceeding according to the procedures described for reaction scheme 9, but replacing starting material 101 with compound of formula 103' (derivatized with Trout's reagent), the corresponding isomeric product of formula 1j is obtained, as shown below.

Compositions corresponding to formula 1j may be named as follows:

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Reaction scheme 10

As illustrated in Reaction Scheme 10, Step 1, an antigen, antibody, antibody fragment or ligand with native free surface thiol group(s) of formula 101'' is contacted with an excess (corresponding to the m value) of a thiol-reactive linker for a click chemistry reaction formula 1001 to obtain the corresponding product according to formula 1002.

In Reaction Scheme 10, Step 2, the product of Formula 1002 is then contacted with an equivalent amount (again corresponding to the m value) of galactose(amine) polymer of Formula 803 to produce the corresponding isomeric product of Formula 1k.

Compositions corresponding to formula 1k can be named as follows:

"Flk-X'-m _m -n _n -p _p -q _q -2NAcGAL" or "Flk-X'-m _m -n _n -p _p -q _q -EtAcN-Z".

Proceeding according to the procedures described with respect to reaction scheme 10, but replacing starting material 101'' with the compound of formula 103' derivatized with Trout's reagent), the corresponding isomeric product of formula 1L is obtained, as shown below.

Compositions corresponding to Formula 1L may be named as follows: “FlL-X'-m _m -n _n -p _p -q _q -2NAcGAL” or “FlL-X'-m _m -n _n -p _p -q _q - EtAcN-Z".

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Reaction scheme 11

1103

- 33 044172

- 34 044172

1139 + 1107

As illustrated in Reaction Scheme 11, Step 1, galactose, protected galactosamine, or N-acetyl-D-galactosamine (formula 1101, where R ³ and R ⁴ are OH, R ³ is an NH protecting group (e.g., cyclized with R ⁴ ) or R ³ is NHAc and R ⁴ is OH, respectively) are brought into contact with 2-chloroethan-1-ol, followed by cooling and adding acetyl chloride dropwise. The solution was warmed to room temperature and then heated to 70°C for several hours. Ethanol was added to the crude product and the resulting solution was stirred in the presence of carbon and then filtered followed by removal of the solvent to obtain the corresponding product of formula 1102.

As illustrated in Reaction Scheme 11, Step 2, the product of formula 1102 was added to excess sodium azide and heated to 90°C for several hours, then filtered followed by removal of the solvent to obtain the corresponding product of formula 1103.

As illustrated in Reaction Scheme 11, Step 3, the product of formula 1103 was added to a solution of carbon-supported palladium catalyst and ethanol, and stirred under hydrogen gas (3 atm) for several hours, then filtered, followed by removal of the solvent to obtain the corresponding product formulas 1104.

As illustrated in Reaction Scheme 11, Step 4, the product of formula 1104 was added to a solution of methacrylic acid anhydride. Triethylamine was added and the reaction mixture was stirred for 2 hours, followed by removal of the solvent and isolation to give the corresponding product of formula 1105.

As illustrated in Reaction Scheme 11, Step 5, the azide-modified uRAFT agent (Formula 1106) was added to a solution of the product of Formula 1105 with azobiisobutyronitrile, subjected to 4 freeze-pump-thaw cycles and then stirred at 70°C. After several hours, the corresponding polymer product of formula 1107 was precipitated by adding a lower alkanol followed by removal of the solvent. If R ³ is an NH protecting group (eg, cyclized with R ⁴ ) the protecting group(s) are removed at this point.

As illustrated in Reaction Scheme 11, Step 6, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of formula 101' is added to a pH 8.0 buffer and contacted with an excess (corresponding to the m value) of dioxopyrrolidine formula 1108 with stirring. After 1 hour, the unreacted compound of Formula 1108 is removed and the resulting product of Formula 1109 is used without further purification.

- 35 044172

As illustrated in Reaction Scheme 11, Step 7, the product of Formula 1107 is added to a pH 8.0 buffer to which the product of Formula 1109 has been added. After stirring for 2 hours, the excess of the compound of Formula 1107 is removed to yield the corresponding isomeric product of Formula 1m.

By replacing N-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)methacrylamide with the product of formula 1105 in step 5 and continuing with steps 6 and 7, the corresponding isomeric product of formula 1m is obtained, where Z '' is N-acetylgalactosamine conjugated at C2.

Compositions corresponding to formula 1m may be named as follows: “Flm-X'-m _m -n _n -pp-q _q .EtAcN-Z”, where Z” represents 1GAL, 1NGAL or

INAcGAL, or "F lm-X'-m _m -n _n -pp-q _q .2NAcGAL"

Reaction scheme 12

The synthesis method of reaction scheme 12 is particularly suitable for hydrophobic antigens, antibodies, antibody fragments and ligands (eg insulin) due to the use of organic solvents.

As illustrated in Reaction Scheme 12, Step 1, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of formula 101' is dissolved in an organic solvent (eg, DMF) containing triethylamine. To this is added a certain amount (corresponding to the m value) of the compound of formula 1201, followed by stirring and addition of tert-butyl methyl ether. The corresponding product of formula 1202 is isolated as a precipitate

The product of formula 1202 is resuspended in an organic solvent and an amount (corresponding to the m value) of the compound of formula 1107 (prepared, for example, as described with reference to reaction scheme 11) is added, followed by stirring. The reaction product was precipitated by adding dichloromethane, followed by filtration and removal of the solvent. Purification (eg resuspension in PBS followed by centrifugal size exclusion chromatography) gives the corresponding isomeric product of formula 1n.

- 36 044172

Compositions corresponding to formula 1n may be named as follows: “Fln-X'-m _m -n _n -p _p -q _q .EtAcN-Z”, where Z” represents 1GAL, 1NGAL or

INAcGAL, or "F lm-X'-n _m -n _n -p _p -q _q .2NAcGAL."

Reaction scheme 13

In Reaction Scheme 13, Step 1, nitrophenoxycarbonyl-oxyalkyl-dithiol-poly(ethylene glycol) ester and NHS (Formula 1301) are contacted with galactose, galactosamine or N-acetylgalactosamine (Formula 105) to give the corresponding product of Formula 1302 along with the other two products illustrated, from which, before proceeding to the next step, the necessary nitrophenoxycarbonyl-dithiol-poly(ethylene glycol)carboxyethylgalactose, galactosamine or N-acetylgalactosamine of formula 1302 was isolated.

As illustrated in Reaction Scheme 13, Step 2, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) of formula 101' is contacted with an excess (corresponding to the m value) of the product of formula 1302 to obtain the corresponding product of formula 1o.

Compositions corresponding to formula 1o may be named as follows:

"F1 o-X' -m _m -n _n -Z '"

Reaction scheme 14

As illustrated in Reaction Scheme 14, an antigen, antibody, antibody fragment or ligand with native free surface amine group(s) (Formula 101') is brought into contact with excess

- 37 044172 (corresponding to the m value) of pyridyl-dithiol-poly(ethylene glycol) ester and NHS formula

1401 to obtain the corresponding product according to formula 1p.

Compositions corresponding to formula 1p can be named as follows:

"Flp-X'-rn _m -n _n -p _p -2NAcGAL" or "Flp-X'-rn _m -n _n -p _p -EtAcN-Z".

Preparation of fusion proteins.

The fusion protein of the compositions of formula 2 can be expressed using art-standard techniques using commercially available expression vectors for mammalian, bacterial, yeast or insect cells and published, discovered or engineered gene sequences. The X, Y and Z coding sequences along with the tag sequences can be cloned into an expression vector, for example the mammalian expression vector pSecTag A, where the fusion protein is inserted C-terminally relative to the Ig κ chain secretion leader sequence. Various other cloning techniques can be used, including site-directed mutagenesis and variations of the QuikChange protocol (Geiser, et al.), and those known to those skilled in the art. The fusion proteins can be transiently expressed in mammalian cells [eg, embryonic kidney (HEK293) cells or Chinese hamster ovary (CHO) cells] by transient transfection with the polyethylenimine vectors described above. Transfected cells are cultured in a suitable medium (eg, FreeStyle 293 medium, Life Technologies), supplemented with, for example, valproic acid or DMSO, for approximately 7 days, after which the cells are removed by centrifugation and the culture supernatants are collected and sterilized by filtration.

Alternatively, the fusion proteins can be stably expressed by generating stably transfected mammalian cell lines. In addition, expression vectors can be used to produce fusion proteins in bacteria such as Escherichia coli, Corynebacterium or Pseudomonas fluorescens using compatible media (e.g. LB, 2XYT, SOB, SOC, TB and other broths), additives (e.g. glycerol, glucose and other additives) and appropriate conditions for growth and expression. When using yeast expression systems, Saccharomyces cerevisiae and Pichia pastoris, or other organisms of the genera Saccharomyces, Pichia, Kluyveromyces and Yarrowia, can usually be used, and organisms such as the filamentous fungi Aspergillus, Trichoderma or Myceliophthora thermophila C1 less commonly used. When using insect expression systems, baculovirus-infected insect cells or non-lytic expression in insect cells can be used to achieve high protein expression levels; the most common insect cells include, but are not limited to, Sf9 and Sf21 (from Spodoptera frugiperda), Hi-5 (from Trichoplusia ni) and Schneider 2 and 3 (from Drosophila melanogaster).

Expressed fusion protein products can be purified from culture supernatants using affinity chromatography (eg, using a HisTrap Ni ²⁺ Sepharose column, GE Healthcare, for His-tagged fusion protein), followed by other chromatographic purification steps such as size exclusion chromatography (for example, using a Superdex 75 column, GE Healthcare) or ion exchange chromatography. The purity of the protein can be checked, for example, by staining SDS-PAGE gels with Coomassie Brilliant Blue and Western blotting (eg, Western blotting for the 6xHis tag in the case of a His-tagged fusion protein). Protein concentration can be determined using the Lambert-Beer law by measuring absorbance at 280 nm, for example using a NanoDrop 2000 (Thermo Scientific). Molecular weight and extinction coefficient can be estimated based on the amino acid sequence of the protein, for example using the ExPASy ProtParam tool. Endotoxin levels can be measured, for example, using the HEK-Blue TLR4 reporter cell line (Invivogen) according to the manufacturer's instructions.

Preparation of desialylated antigens, antibodies, antibody fragments and ligands.

Desialylated proteins can be prepared by art-standard techniques using the commercially available enzyme neuraminidase (also known as acetyl-neuraminyl hydrolase or sialidase) or sulfuric acid. To enzymatically desialylate a protein of interest, the protein can be incubated with neuraminidase at 37°C for 1 hour or longer if necessary. For chemical desialylation by acid hydrolysis, the protein of interest can be treated with 0.025N. sulfuric acid at 80°C for 1 hour or longer if necessary. The desialylated protein can then be purified from the reaction mixture using metal ion affinity chromatography (e.g. using a HisTrap Ni ²⁺ Sepharose column, GE Healthcare), followed by size exclusion chromatography (e.g. using a Superdex 75 column, GE Healthcare) . Protein purity can be checked, for example, by staining SDS-PAGE gels with Coomassie brilliant blue and Western blotting for a 6xHis tag. Desialation can be tested, for example, by lectin detection of the presence of sialic acid in the protein in Western blots or colorimetric quantification of sialic acid using commercially available kits (e.g.

- 38 044172 measures Abcam, ProZyme or Sigma). The concentration of desialylated protein can be determined using the Lambert-Beer law by measuring absorbance at 280 nm, for example using a NanoDrop 2000 (Thermo Scientific). Molecular weight and extinction coefficient can be estimated based on the amino acid sequence of the protein, for example using the ExPASy ProtParam tool. Endotoxin levels can be measured, for example, using the HEK-Blue TLR4 reporter cell line (Invivogen) according to the manufacturer's instructions.

Specific methods and final stages.

The compound of formula 103' is contacted with an excess (corresponding to the m value) of the compound of formula 106 to obtain the corresponding product of formula 1a.

The compound of formula 103' is contacted with an excess (corresponding to the m value) of the compound of formula 201 to obtain the corresponding product of formula 1b.

A compound of formula 802, 902 or 1002 is contacted with an excess (corresponding to the m value) of a compound of formula 803 to obtain the corresponding product of formula 1h, formula 1i or formula 1k, respectively.

The compound of formula 1109 is contacted with an excess (corresponding to the m value) of the compound of formula 1107 to obtain the corresponding product of formula 1m, in particular, where n is about 80, p is about 30, q is about 4 and m, determined by the antigen, is from about 2 to 10.

The compound of formula 1202 is contacted with an excess (corresponding to the m value) of the compound of formula 1107 to obtain the corresponding product of formula 1n, in particular, where n is about 1, p is about 30, q is about 4, and m, as determined by the antigen, is from about from 2 to 10.

Specific compositions.

By way of non-limiting example, a particular group preferred for the compositions, pharmaceutical formulations, methods of preparation and use of the present disclosure are the following combinations and combinations of substituent groups of Formula 1 (divided into subgroups, respectively, in order of increasing preference):

X represents a foreign graft antigen against which transplant recipients develop an unwanted immune response, a foreign antigen against which patients develop an unwanted immune response, a therapeutic protein against which patients develop an unwanted immune response, a self antigen against which patients develop an unwanted immune response, or its tolerogenic part.

X is a therapeutic protein to which patients develop an unwanted immune response selected from: abatacept, abciximab, adalimumab, adenosine deaminase, ado-trastuzumab-emtansine, agalsidase-alpha, agalsidase-beta, aldesleukin, alglucerase, alglucosidase-alpha, inhibitor α-1-proteinase, anakinra, anistreplase (anisoylated plasminogen and streptokinase activator complex), antithrombin III, antithymocyte globulin, ateplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitor, canakinumab, carboxypeptidase G2 (glucarpidase and voraxase), certolizumabpegol, cetuximab, collagenase, Crotalidae immune Fab, darbepoetin-a, denosumab, digoxin immune Fab, dornase-alpha, eculizumab, etanercept, factor VIIa, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histrelin acetate, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin (including rHu-insulin and bovine insulin), interferon-a2a, interferon-α2b, interferon-β1a, interferon-β1b, interferon-γ1b, ipilimumab, L-arginase, L-asparaginase, L-methionase, lactase, laronidase, lepirudin/hirudin, mecasermin, mecasermin-rinfabate, methoxyofatumumab, natalizumab, octreotide, oprelvequin, pancreatic amylase, pancreatic lipase, papain, peg-asparaginase, peg- doxorubicin HCl, PEG-epoetin-β, pegfilgrastim, peg-interferon-a2a, peg-interferon-α2b, pegloticase, pegvisomant, phenylalanine ammonia lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostima , streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, interferon alpha type 1, ustekinumab and vW factor.

In particular, where X is abciximab, adalimumab, agalsidase-alpha, agalsidazubeta, aldesleukin, alglucosidase-alpha, factor VIII, factor IX, infliximab, L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL) or rasburicase (uricazu).

In particular, where X is factor VIII, factor IX, uricase, PAL or asparaginase.

X is a self-antigen polypeptide selected for the treatment of type 1 diabetes mellitus, pediatric multiple sclerosis, juvenile rheumatoid arthritis, celiac disease or general alopecia.

In particular, where X is a self-antigen polypeptide selected for the treatment of new-onset type 1 diabetes mellitus, pediatric multiple sclerosis or celiac disease

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X is a foreign antigen against which patients develop an unwanted immune response

From peanuts, including conarchin (Ara h 1).

From wheat, including alpha-gliadin 33-mer native (SEQ ID NO:24), alpha-gliadin 33-mer deamidated (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26) and omega-gliadin (SEQ ID NO:27).

From cats, including Fel d 1A (UNIPROT P30438) and feline albumin (UNIPROT P49064).

From dogs, including Can f 1 (UNIPROT O18873) and canine albumin (UNIPROT P49822).

X is a foreign graft antigen against which transplant recipients develop an unwanted immune response, such as a human leukocyte antigen protein.

X is an antibody, antibody fragment or ligand that specifically binds a circulating protein or peptide or antibody, wherein the circulating protein or peptide or antibody leads to graft rejection, immune response against a therapeutic agent, autoimmune disease and/or allergy.

Specifically, where X binds an endogenous circulating protein, or peptide, or antibody.

Y is a linker selected from: formula Ya, formula Yb, formula Yh, formula Yi, formula Yk, formula Ym, formula Yn, formula Yo and formula Yp.

In particular, where n is from 8 to 90 ±10%, p is from 20 to 100 ±10% and q is from 3 to 20 ±3.

Specifically, where n is from 40 to 80 ±10%, p is from 30 to 40 ±10%, and q is from 4 to 12 ±3.

In particular, where Y is a formula Ya, a formula Yb, a formula Ym or a formula Yn.

Specifically, where n is from 8 to 90 ±10%, p is from 20 to 100 ±10%, and q is from 3 to 20 ±3.

More specifically, where n is from 40 to 80 ±10%, p is from 30 to 40 ±10%, and q is from 4 to 12 ±3.

Specifically, where Z is conjugated to Y via an ethyl acetamide group.

More specifically, where Z is conjugated to Y at its C1.

More specifically, where R ⁸ represents CMP.

More specifically, where R ⁸ represents CMP.

In particular, where R ⁸ represents CMP.

Z represents galactose, galactosamine or N-acetylgalactosamine.

In particular, where Z represents galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

In particular, where Z represents galactose or N-acetylgalactosamine conjugated at C1 or C2.

More specifically, where Z is N-acetylgalactosamine conjugated at C1.

Each of the groups and subgroups described above are preferred individually and may be combined to describe additional preferred aspects of the present disclosure, for example, but not by way of limitation, as follows:

X is a self-antigen polypeptide selected for the treatment of type 1 diabetes mellitus, pediatric multiple sclerosis, juvenile rheumatoid arthritis, celiac disease or general alopecia.

In particular, where X is a self-antigen polypeptide selected for the treatment of new-onset type 1 diabetes mellitus, pediatric multiple sclerosis or celiac disease

In particular, where Y is a linker selected from: formula Ya, formula Yb, formula Yh, formula Yi, formula Yk, formula Ym, formula Yn, formula Yo and formula Yp.

In particular, where n is from 8 to 90 ±10%, p is from 20 to 100 ±10% and q is from 3 to 20 ±3.

Specifically, where n is from 40 to 80 ±10%, p is from 30 to 40 ±10%, and q is from 4 to 12 ±3.

In particular, where Y is a formula Ya, a formula Yb, a formula Ym or a formula Yn.

Specifically, where n is from 8 to 90 ±10%, p is from 20 to 100 ±10%, and q is from 3 to 20 ±3.

More specifically, where n is from 40 to 80 ±10%, p is from 30 to 40 ±10%, and q is from 4 to 12 ±3.

Even more specifically, where Z is conjugated to Y via an ethyl acetamide group.

More specifically, where Z is conjugated to Y via an ethyl acetamide group.

Specifically, where Z is conjugated to Y via an ethyl acetamide group.

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In particular, where Z represents galactose, galactosamine or N-acetylgalactosamine.

In particular, where Z represents galactose or N-acetylgalactosamine conjugated at

C1, C2 or C6.

More specifically, where Z represents galactose or N-acetylgalactosamine conjugated at C1 or C2.

Even more specifically, where Z is N-acetylgalactosamine conjugated at C1.

In particular, where Z represents galactose, galactosamine or N-acetylgalactosamine.

In particular, where Z represents galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

In particular, where Z represents galactose or N-acetylgalactosamine conjugated at C1 or C2.

More specifically, where Z is N-acetylgalactosamine conjugated at C1.

In particular, where Y is a linker selected from: formula Ya, formula Yb, formula Yh, formula Yi, formula Yk, formula Ym, formula Yn, formula Yo and formula Yp.

Specifically, where n is from 8 to 90 ±10%, p is from 20 to 100 ±10%, and q is from 3 to 20 ±3.

More specifically, where n is from 40 to 80 ±10%, p is from 30 to 40 ±10%, and q is from 4 to 12 ±3.

Specifically, where Y is a formula Ya, a formula Yb, a formula Ym, or a formula Yn.

More specifically, where n is from 8 to 90 ±10%, p is from 20 to 100 ±10%, and q is from 3 to 20 ±3.

More preferably, where n is from 40 to 80 ±10%, p is from 30 to 40 ±10% and q is from 4 to 12 ±3.

More specifically, where Z is conjugated to Y via an ethyl acetamide group.

In particular, where Z represents galactose, galactosamine or N-acetylgalactosamine.

In particular, where Z represents galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

More specifically, where Z represents galactose or N-acetylgalactosamine conjugated at C1 or C2.

More preferably, where Z is N-acetylgalactosamine conjugated at C1.

m is equal to an integer from approximately 1 to 100.

m equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 or 110.

In particular, m is from about 1 to 20.

m equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.

More specifically, m is equal to approximately 10.

m equals 9, 10 or 11.

n equals an integer representing a population ranging from approximately 1 to 100.

n equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110.

In particular, n is from about 8 to 90.

Specifically, n equals 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50 , 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95 or 99.

More specifically, n is from about 40 to 80.

More specifically, n equals 37, 4θ, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, or 88.

n represents the population covering the ranges 1-4, 2-4, 2-6, 3-8, 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82, 85-90, 90-110 and 107-113.

Specifically, n represents a population spanning the ranges 7-13, 6-14, 15-25, 26-30, 4250, 46-57, 60-82, 85-90, and 82-99.

More specifically, n represents the population spanning the ranges 36-44, 42-50, 46-57, 6082 and 75-85.

p equals an integer representing a population ranging from approximately 2 to 150.

p equals 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 or 165.

Specifically, where n equals an integer representing a population ranging from approximately 1 to 100.

Specifically, n equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30 , 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110 .

More specifically, where n is from about 8 to 90.

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More specifically, n equals 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41.

45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95 or 99.

Even more specifically, where n is from about 40 to 80.

Even more specifically, n equals 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, or 88.

More specifically, p equals 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 110.

Specifically, where n equals an integer representing a population ranging from approximately 1 to 100.

Specifically, n equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30 , 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110 .

More specifically, where n is from about 8 to 90.

More specifically, n equals 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50 , 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95 or 99.

Even more specifically, where n is from about 40 to 80.

Even more specifically, n equals 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, or 88.

More specifically, p equals 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

Specifically, where n equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105 or 110.

More specifically, where n is from about 8 to 90.

More specifically, n equals 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 35, 37, 40, 41, 45, 50 , 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95 or 99.

Even more specifically, where n is from about 40 to 80.

Even more specifically, n equals 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, or 88.

q is equal to an integer representing a population ranging from approximately 1 to 44.

q equals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 44 or 48.

By way of non-limiting example, a particular group preferred for the compositions, pharmaceutical formulations, methods of preparation and use of the present disclosure are the following combinations and combinations of substituent groups of Formula 2 (divided into subgroups, respectively, in order of increasing preference):

X is a therapeutic protein to which patients develop an unwanted immune response selected from: abatacept, abciximab, adalimumab, adenosine deaminase, adotrastuzumab-emtansine, agalsidase-alpha, agalsidase-beta, aldesleukin, alglucerase, alglucosidase-alpha, α-inhibitor 1-proteinase, anakinra, anistreplase (anisoylated plasminogen and streptokinase activator complex), antithrombin III, antithymocyte globulin, ateplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitor, canakinumab, carboxypeptidase G2 (glucarpidase and voraxase), certolizumab-pegol, cetuximab, collagenase, Crotalidae immune Fab, darbepoetin-α, denosumab, digoxin immune Fab, dornase-alpha, eculizumab, etanercept, factor VIIa, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histrelin acetate, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin, interferon-a2a, interferon-α2b, interferon-β1a, interferon-β1b, interferon-γ1b, ipilimumab, L-arginase, L-asparaginase , L-metionase, lactase, laronidase, lepirudin/girudin, Mekazrasmin, Mekhazrasmin-Rinfabata, methoxiophaumuba, nalizumab, octreotide, ripe, pancreatic amylase, pancreatic lipase, papain, PAG-asparaginase, PEG-codeshopin HCl, PEG-EPOE. Tina-β, pegfilgrastim, peg-interferon-a2a, peg-interferon-α2b, pegloticase, pegvisomant, phenylalanine ammonia lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab ( atlizumab), trastuzumab, alpha interferon type 1, ustekinumab and vW factor; provided that interferon (interferon-alpha 2, interferon-alpha 5, interferon-alpha 6 or consensus interferon), ribavirin, nexavar/sorafenib, erbitus/cetuximab, avastatin/bevacizumab or Herceptin/trastuzumab are excluded from the scope of claim 2 when m '+ m' is 1 and Z represents DOM 26h-196-61 or other liver-targeted molecules described in US Patent 2013/0078216.

In particular, where X is abciximab, adalimumab, agalsidase-alpha, agalsidazubeta, aldesleukin, alglucosidase-alpha, factor VIII, factor IX, infliximab, L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL) or rasburicase (uricazu).

In particular, where X is factor VIII, factor IX, uricase, PAL or asparaginase.

X is a self-antigen polypeptide selected for the treatment of type 1 diabetes mellitus, pediatric multiple sclerosis, juvenile rheumatoid arthritis, celiac disease or general alopecia.

In particular, where X is a self-antigen polypeptide selected for the treatment of new-onset type 1 diabetes mellitus, pediatric multiple sclerosis or celiac disease.

- 42 044172

X is a foreign antigen against which patients develop an unwanted immune response.

From peanuts, including conarchin (Ara h 1).

From wheat, including alpha-gliadin 33-mer native (SEQ ID NO:24), alpha-gliadin 33-mer deamidated (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26) and omega-gliadin (SEQ ID NO:27).

From cats, including Fel d 1A (UNIPROT P30438) and feline albumin (UNIPROT P49064).

From dogs, including Can f 1 (UNIPROT O18873) and canine albumin (UNIPROT P49822).

X is a foreign graft antigen against which transplant recipients develop an unwanted immune response, such as a human leukocyte antigen protein.

X is an antibody, antibody fragment or ligand that specifically binds a circulating protein or peptide or antibody, wherein the circulating protein or peptide or antibody leads to graft rejection, immune response against a therapeutic agent, autoimmune disease and/or allergy.

Specifically, where X binds an endogenous circulating protein, or peptide, or antibody.

Y represents Gly ₃ Ser.

Z is the sequence of the anti-ASGPR antibody, Dom26h-196-61, or a sequence containing a conservative substitution.

m' + m'' equals 1 or 2, and X represents full-length protein, including protein therapeutics.

m' + m'' equals 1 or 2, where m' equals 1 and m'' equals 0 or 1.

m' + m'' is from 2 to about 10, where X represents a group of self-antigen peptides (epitopes) that are known to be associated with a particular autoimmune disease, or that serve to treat genetically distinct target populations.

m' + m'' is from about 4 to 7, wherein the autoimmune disease is multiple sclerosis, and X is independently selected from: MBP13-32 (SEQ ID NO:11), MBP83-99 (SEQ ID NO:12), MBP111 -129 (SEQ ID NO:13), MBP146-170 (SEQ ID NO: 14), MOG1-20 (SEQ ID NO: 15), MOG35-55 (SEQ ID NO: 16) and PLP139-154 (SEQ ID NO : 17).

m' + m'' is 7 and X represents, respectively, MBP13-32 (SEQ ID NO: 11), MBP83-99 (SEQ ID NO:12), MBP111-129 (SEQ ID NO:13), MBP146- 170 (SEQ ID NO:14), MOG1-20 (SEQ ID NO:15), MOG35-55 (SEQ ID NO:16) and PLP139-154 (SEQ ID NO: 17).

As with the above discussion regarding Formula 1, each of the groups and subgroups described above for Formula 2 are individually preferred and may be combined to describe additional preferred aspects of the present disclosure.

Applicability, testing and introduction.

General applicability.

The compositions of the present disclosure are useful in a variety of applications, including, as will be appreciated by those skilled in the art, in the treatment of transplant rejection, immune response against a therapeutic agent, autoimmune disease, and food allergy.

In a preferred embodiment, the compositions of the present disclosure are used to modulate, in particular down-regulate, an antigen-specific unwanted immune response.

The compositions of the present disclosure are useful for binding and clearing from the bloodstream specific unwanted proteins, including antibodies produced endogenously in a patient (i.e., not related to exogenous antibodies administered to the patient), peptides, etc., that cause autoimmune reaction and associated pathologies, allergies, inflammatory immune responses and anaphylaxis.

In the present disclosure, antigens are targeted to the liver for presentation by antigen presenting cells to specifically down regulate the immune system or to clear unwanted circulating proteins. This differs from previous applications of liver targeting, such as those described in US Pat. No. 2013/0078216, where the purpose of liver targeting molecules such as DOM26h-196-61 is to deliver therapeutics for the treatment of liver diseases such as fibrosis, hepatitis , cirrhosis and liver cancer.

The present disclosure provides compositions and methods for treating an unwanted immune response to self antigens and foreign antigens, including, without limitation: a foreign graft antigen against which transplant recipients develop an unwanted immune response (e.g., graft rejection), a foreign antigen against which patients develop an unwanted immune response (eg, allergy or hypersensitivity), a therapeutic agent to which patients develop an unwanted immune response (eg, hypersensitivity and/or reduced therapeutic activity), self-antigen to which patients develop an unwanted immune response (eg, autoimmune disease).

- 43 044172

Autoimmune disease conditions that can be treated using the methods and compositions presented herein include, without limitation: acute disseminated encephalomyelitis (ADEM); acute interstitial allergic nephritis (drug allergy); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; alopecia areata; generalized alopecia; ankylosing spondylitis; juvenile arthritis; psoriatic arthritis; rheumatoid arthritis; atopic dermatitis; autoimmune aplastic anemia; autoimmune gastritis; autoimmune hepatitis; autoimmune hypophysitis; autoimmune oophoritis; autoimmune orchitis; autoimmune polyendocrine syndrome type 1; autoimmune polyendocrine syndrome type 2; autoimmune thyroiditis; Behçet's disease; bronchiolitis obliterans; bullous pemphigoid; celiac disease; Churg-Strauss syndrome; chronic inflammatory demyelinating polyneuropathy; cicatricial pemphigoid; Crohn's disease; Coxsackie myocarditis; Dühring's dermatitis herpetiformis; diabetes mellitus (type 1); erythema nodosum; acquired epidermolysis bullosa, giant cell arteritis (arteritis of the temporal arteries); giant cell myocarditis; Goodpasture's syndrome; Graves' disease; Guillain-Barre syndrome; Hashimoto's encephalitis; Hashimoto's thyroiditis; IgG4-related sclerosing disease; Lambert-Eaton syndrome; mixed connective tissue disease; Mucha-Habermann disease; multiple sclerosis; myasthenia gravis; optic neuritis; neuromyelitis of the optic nerve; pemphigus vulgaris and variants; pernicious anemia; autoimmune disease of the pituitary gland; polymyositis; postcardiotomy syndrome; premature loss of ovarian function; primary biliary cirrhosis; primary sclerosing cholangitis; psoriasis; rheumatic heart disease; Sjögren's syndrome; systemic lupus erythematosus; systemic scleroderma; nonspecific ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vitiligo and Wegener's granulomatosis.

A specific group of autoimmune disease conditions that can be treated using the methods and compositions presented herein include, but are not limited to: acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; psoriatic arthritis; rheumatoid arthritis; autoimmune aplastic anemia; autoimmune hypophysitis; autoimmune gastritis; autoimmune polyendocrine syndrome type 1; bullous pemphigoid; celiac disease; Coxsackie myocarditis; Dühring's dermatitis herpetiformis; diabetes mellitus (type 1); acquired epidermolysis bullosa; giant cell myocarditis; Goodpasture's syndrome; Graves' disease; Hashimoto's thyroiditis; mixed connective tissue disease; multiple sclerosis; myasthenia gravis; neuromyelitis of the optic nerve; pernicious anemia; pemphigus vulgaris and variants; autoimmune disease of the pituitary gland; premature loss of ovarian function; rheumatic heart disease; systemic scleroderma; Sjögren's syndrome; systemic lupus erythematosus and vitiligo.

In embodiments using an antigen against which an unwanted immune response develops, such as food antigens, treatment of reactions against, for example: peanut, apple, milk, egg white, egg yolk, mustard, celery, shrimp, wheat (and other grains) can be provided. cereals), strawberries and bananas.

As will be appreciated by those skilled in the art, a patient can be tested to identify a foreign antigen against which an unwanted immune response has developed, and a composition of the present disclosure can be formulated based on that antigen.

Testing.

In determining the usefulness of the compositions and methods of the present disclosure, the specificity of binding to antigen presenting cells in the liver (specifically, binding to hepatocytes and specifically to ASGPR) should first be determined. This can be achieved, for example, by using a marker (such as a fluorescent marker phycoerythrin (PE)) in the composition of the present disclosure. The composition is administered to suitable experimental subjects. Controls, eg, unconjugated PE or vehicle (saline), are administered to another group(s) of subjects. The composition and controls are allowed to circulate in the blood for a period of 1 to 5 hours, after which the spleen and liver of the subjects are collected and fluorescence is measured. The specific cells in which fluorescence is detected can then be identified. When tested in this manner, the compositions of the present disclosure show higher concentration levels in antigen presenting cells of the liver compared to unconjugated PE or vehicle.

The effectiveness of immunomodulation can be tested by measuring the proliferation of OT-I CD8+ cells (transplanted into host mice) in response to administration of a composition of the present disclosure comprising a known antigen, such as ovalbumin (OVA), compared to administration of the antigen alone or vehicle alone. When tested in a similar manner, the compositions of the present disclosure show an increase in OT1 cell proliferation compared to antigen alone or vehicle, demonstrating an increase in cross-priming of CD8+ T cells. To distinguish T cells that undergo expansion and development into a functional effector phenotype from those that undergo expansion and deletion, proliferating OT-I CD8+ T cells can be analyzed phenotypically for molecular signatures of exhaustion [such as programmed death protein-1 (PD-1). 1), FasL and others], as well as annexin-V as a hallmark of apoptosis

- 44 044172 and thus deletions. OT-I CD8+ T cells can also be assessed for their ability to respond to antigen stimulation with an adjuvant to demonstrate lack of a functional response and thus immune tolerance to the antigen. To this end, cells are analyzed for inflammatory signatures after administration of the compositions of the present disclosure into host mice followed by antigenic stimulation. When tested in this manner, the compositions of the present disclosure show very low (eg, background) levels of inflammatory responses with OT-I CD8+ T cells to OVA, thereby demonstrating immune tolerance.

The humoral immune response can be tested by administering a composition of the present disclosure comprising a known antigen, such as OVA, versus administering the antigen alone or the vehicle alone and measuring the levels of antibodies produced. When tested in this manner, the compositions of the present disclosure show very low (eg, background) levels of antibody formation in response to their administration and vehicle administration, with significantly higher levels of antibody formation in response to antigen administration.

The effectiveness of tolerization against an antigen can be tested as described above with reference to the humoral immune response, where several weeks after treatment(s) with the composition of the present disclosure, a group of subjects were stimulated by administering the antigen alone, followed by measurement of antibody levels to the antigen. When tested in a similar manner, the compositions of the present disclosure show low levels of antibody formation in response to antigen stimulation in groups pretreated with such compositions compared to groups that were not pretreated.

Disease-specific experimental models are well known to those skilled in the art and include the NOD (or non-obese diabetic) mouse model of autoimmunity and tolerance and the EAE (experimental autoimmune encephalomyelitis) model for the human inflammatory demyelinating disease multiple sclerosis. In particular, NOD mice develop spontaneous autoimmune diabetes (similar to type 1a diabetes in humans). Groups of NOD mice are treated with a test compound or a negative control compound, followed by measurement of blood glucose levels. Successful treatment is consistent with treating diabetes in humans or providing proof of mechanism approaches to treating other autoimmune diseases; (See, for example, Anderson and Bluestone, Annu. Rev. Immunol. 2005;23:447-85.)

Introduction.

The compositions of the present disclosure are administered in a therapeutically effective dosage, for example, in a dosage sufficient to provide treatment for the disease states previously described. Administration of the compounds of the present disclosure or their pharmaceutically acceptable salts can be accomplished using any conventional means of administration that serve such applications.

Although human dosage levels have yet to be optimized for the compounds of the present disclosure, they can initially be extrapolated based on approximately 10 μg to 100 μg doses administered to mice. Typically, an individual human dose is from about 0.01 to 2.0 mg/kg body weight, preferably from about 0.1 to 1.5 mg/kg body weight, and most preferably from about 0.3 to 1.0 mg/kg body weight. The drug may be administered over one day or over several days and may be repeated at intervals of several days, one or more weeks, or one or more months. Administration may be as a single dose (eg, as a bolus dose) or as an initial bolus dose followed by a continuous infusion of the remainder of the full dose over an extended period of time, eg, 1 to 7 days. The amount of active compound administered will, of course, depend on any one or all of the following: the subject and disease state being treated, the severity of the lesion, the route and schedule of administration, and the judgment of the attending physician. It should also be understood that the amounts administered will depend on the molecular weight of the antigen, antibody, antibody fragment or ligand, as well as the size of the linker.

The compositions of the present disclosure can be administered either alone or in combination with other pharmaceutically acceptable excipients. Although all typical routes of administration are contemplated, it is currently preferred to provide liquid dosage forms suitable for injection. The compositions will generally include a conventional pharmaceutical carrier or excipient, as well as a composition of the present disclosure or a pharmaceutically acceptable salt thereof. In addition, these compositions may include other therapeutic agents, pharmaceuticals, carriers, and the like, including, without limitation, a therapeutic protein, peptide, antibody, or antibody-like molecule corresponding to the antigen (X) that is used in the composition of the present disclosure , and other active agents that can act as immunomodulatory agents and, more specifically, can have inhibitory effects on B cells, including antifolates, immunosuppressives, cytostatics, mitotic inhibitors and anti-metabolites or combinations thereof.

- 45 044172

Typically, depending on the intended route of administration, the pharmaceutically acceptable composition will contain from about 0.1 to 95%, preferably from about 0.5 to 50%, by weight of the composition of the present disclosure, the balance being suitable pharmaceutical excipients, carriers, etc. .P. Dosage forms or compositions can be prepared containing the active ingredient in the range of 0.005 to 95% with the balance consisting of a non-toxic carrier.

Liquid pharmaceutical compositions suitable for administration can be prepared, for example, by dissolving, dispersing, etc. the active composition of the present disclosure (for example, a lyophilized powder); and optionally pharmaceutical adjuvants in a carrier such as, for example, water (water for injection), saline, aqueous dextrose, glycerin, glycols, ethanol or the like. (except for galactoses), thus obtaining a solution or suspension. If necessary, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic excipients such as wetting agents, emulsifying agents, stabilizing agents, solubilizing agents, pH buffering agents and the like, for example sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate and triethanolamine oleate, etc., osmolytes, amino acids, sugars and carbohydrates, proteins and polymers, salts, surfactants, chelating agents and antioxidants, preservatives and specific ligands. Current methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd Edition, 2012. The composition or composition to be administered will in any case contain the active substance in an amount effective to treat the symptoms of the subject being treated.

Examples

The following examples serve to more fully describe the manner in which the above disclosure may be applied, as well as to set forth the best modes provided for carrying out the various aspects of the present disclosure. It is understood that these examples do not serve in any way to limit the true scope of the present disclosure, but rather are presented for illustrative purposes. All sources referenced in this document are incorporated by reference in their entirety.

Example 1. F1aA-OVA-m ₄ -n ₈₀ (or F1a-OVA-m ₄ -n ₈₀ -2NGAL)

1A. Formula 103', where X' represents OVA and m equals 4.

In an endotoxin-free tube, OVA (5.0 mg, 0.00012 mmol) was added to 100 μl of PBS, pH 8.0, containing 5 mM EDTA and mixed. Separately, 1 mg of Trout's reagent was dissolved in 100 μl of PBS, pH 7.0, and 16 μl (0.00119 mmol) of the Trout's reagent solution thus obtained was added to the stirred OVA solution with continuous stirring. After 1 hour, excess Trout's reagent was removed using a size exclusion chromatography column to give the corresponding product of formula 103'.

1B. Formula 106A, where n is 80.

In an endotosin-free tube, galactosamine (10.0 mg, 0.04638 mmol) was dissolved with stirring in 100 μl of PBS, pH 8.0, containing 5 mM EDTA. Pyridyl dithiol poly(ethylene glycol) NHS ester (formula 104, where n is 80) (16.23 mg, 0.00464 mmol) dissolved in 100 μl PBS, pH 7.0, was added to the stirred galactosamine solution. After 1 hour, the resulting pyridyl-dithiol-poly(ethylene glycol)-N-acetylgalactosamine (formula 106A) was ready for use without further purification.

1C. Formula 1aA, where X' is OVA, m is 4, n is 80 (and Z' is C2-galactosamine).

The purified OVA-Trout reagent conjugate of formula 103' obtained in Example 1A was added directly to the stirred product of formula 106A obtained in Example 1B. After 1 hour, the resulting product of formula 1a was purified by passing the reaction mixture through a centrifugal size exclusion chromatography column. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product (see Fig. 5.)

1D. Other compounds of formula 103'.

Proceeding according to the procedures described in Example 1A and replacing the OVA with the following: abciximab, adalimumab, agalsidase-alpha, agalsidase-beta, aldesleukin, alglucosidase-alpha, factor VIII, factor IX, L-asparaginase, laronidase,

- 46 044172 octreotide, phenylalanine ammonia lyase, rasburicase, insulin (SEQ ID NO:5),

GAD-65 (SEQ ID NO:6),

IGRP (SEQ ID NO: 7) MBP (SEQ ID NO:8), MOG (SEQ ID NO:9), PLP (SEQ ID NO:10), MBP13-32 (SEQ ID NO:11), MBP83-99 ( SEQ ID NO: 12), MBP111-129 (SEQ ID NO:13), MBP146-170 (SEQ ID NO:14), MOG1-20 (SEQ ID NO:15), MOG35-55 (SEQ ID NO: 16) , PLP139-154 (SEQ ID NO:17), MARTI (SEQ ID NO:18), tyrosinase (SEQ ID NO:19), PMEL (SEQ ID NO:20), aquaporin-4 (SEQ ID NO:21), S-arrestin (SEQ ID NO:22), IRBP (SEQ ID NO:23), conarchin (UNIPrOt Q6PSU6), alpha-gliadin 33-mer native (SEQ ID NO:24), alpha-gliadin 33-mer deamidated (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26), omega-gliadin (SEQ ID NO:27), Fel d 1A (UNIPROT P30438), feline albumin (UNIPROT P49064),

Can f 1 (UNIPROT O18873), canine albumin (UNIPROT P49822), and RhCE example (UNIPROT P18577) prepared the following corresponding compounds of formula 103', where X is abciximab and m is 10.

X represents adalimumab and m equals 11,

X is agalasidase alpha and m is 14,

X represents agalasidase-beta and m is 14,

X represents aldesleukin and m equals 6,

X represents alglucosidase-alpha and m is 13,

X represents factor VIII and m equals 100,

X represents factor IX and m equals 18,

X represents L-asparaginase and m is 5,

X represents laronidase and m is equal to 7.

X represents octreotide and m equals 1,

X is phenylalanine ammonia lyase and m is 12,

X represents rasburicase and m equals 12,

X is insulin (SEQ ID NO:5) and m is 2, X is GAD-65 (SEQ ID NO:6) and m is 8, X is IGRP (SEQ ID NO:7) and m is 7, X is MBP (SEQ ID NO:8) and m is 6, X is MOG (SEQ ID NO:9) and m is 5, X is PLP (SEQ ID NO:10) and m is 8, X is is MBP13-32 (SEQ ID NO:11) and m is 1, X is MBP83-99 (SEQ ID NO: 12) and m is 1, X is MBP111-129 (SEQ ID NO:13) and m is 1, X is MBP146-170 (SEQ ID NO:14) and m is 2, X is MOG1-20 (SEQ ID NO: 15) and m is 1, X is MOG35-55 (SEQ ID NO: 16 ) and m is 2, X is PLP139-154 (SEQ ID NO:17) and m is 3, X is MART1 (SEQ ID NO:18) and m is 4, X is tyrosinase (SEQ ID NO:19 ) and m is 8, X is PMEL (SEQ ID NO:20) and m is 5,

- 47 044172

X is aquaporin-4 (SEQ ID NO:21) and m is 4,

X is S-arrestin (SEQ ID NO:22) and m is 12,

X is IRBP (SEQ ID NO:23) and m is 21,

X represents conarchine and m equals 21,

X is alpha-gliadin 33-mer native (SEQ ID NO:24) and m is 1.

X is alpha-gliadin 33-mer deamidated (SEQ ID NO:25) and m is 1.

X is alpha-gliadin (SEQ ID NO:26) and m is 1.

X is omega-gliadin (SEQ ID NO:27) and m is 1.

X represents Fel d 1 and m equals 4,

X represents feline albumin and m equals 16,

X represents Can f 1 and m equals 6,

X represents canine albumin and m equals 23 and

X is an example of RhCE and m is equal to 10.

1E. Other compounds of formula 1aA.

By following the procedures described in Example 1C and substituting compounds of formula 103', for example, with those obtained in Example 1D, the following corresponding compounds of formula 1aA were obtained:

F1 aA-abciximab-m ₁₀ -n ₈₀ , F1 aA-adalimumab-m ₁ 1 -n ₈₀ , F1 aA-agalasidase-alpha-m ₁₄ -n ₈₀ , F1 aA-agalasidase-beta-m ₁ 4-n ₈₀ , F1 aA-aldesleukin-m ₆ -n ₈₀ ,

F1 aA-alglucosidase-alpha-m ₁₃ -n ₈₀ ,

F1aA factor VIII-m ₁₀₀ -n ₈₀ ,

F1aA factor IX-m ₁₈ -n ₈₀ ,

F1 aA-L-asparaginase-m ₅ -n ₈₀ ,

F1 aA-laronidase-m ₇ -n ₈₀ , F1 aA-octreotide-m ₁ -n ₈₀ , F1aA-phenylalanine-ammonia-lyase-m ₁₂ -n ₈₀ , F1aA-rasburicase-m ₁₂ -n ₈₀ ,

F1 aA-insulin-m ₂ -n ₈₀ , F1aA-GAD-65-m ₈ -n ₈₀ , F1 aA-IGRP-m ₇ -n ₈₀ , F1 aA-MBP-m ₆ -n ₈₀ , F1 aA-MOG- m ₅ -n ₈₀ , F1 aA-PLP-m ₈ -n ₈₀ , F1 aA-MBP13-32-m1 -n ₈₀ , F1 aA-MBP83-99-m1 -n ₈₀ , F1aA-MBP111-129-m1-n ₈₀ , F1aA-MBP146-170-m ₂ -n ₈₀ , F1aA-MOG1-20-m1-n ₈₀ , F1aA-MOG35-55-m ₂ -n ₈₀ , F1aA-PLP139-154-m3-n80, F1 aA- MART 1 -m4-n ₈₀ , F1 aA-tyrosinase-m ₈ -n ₈₀ , F1 aA-PMEL-m ₅ -n ₈₀ , F1 aA-aquaporin-4-m ₄ -n ₈₀ , F1 aA-S-arrestin- m ₁₂ -n ₈₀ , F1aA-IRBP-m ₂₁ -n ₈₀ , F1 aA-conarchin-m ₂ 1 -n ₈₀ , F1aA-alpha-gliadin 33-mer native-m ₁ -m ₈₀ , F1aA-alpha-gliadin 33 -meric deamidated-m ₁ -n ₈₀ , F1 aA-alpha-gliadin-m ₁ -n ₈₀ , F1 aA-omega-gliadin-m ₁ -n ₈₀ , F1aA-Fel d 1-m ₄ -n ₈₀ ,

F1aA-feline albumin-m ₁₆ -n ₈₀ ,

F1aA-Can f 1-m6-n ₈₀ ,

F1aA-canine albumin-m ₂₃ -n ₈₀ and

F1 aA-RhCE-m ₁₀ -n ₈₀ .

- 48 044172

1F. Other compounds of formula 106A.

Proceeding according to the procedures described in Example 1B and replacing the pyridyldithiol-poly(ethylene glycol) ester and NHS (formula 104, where n is 80) with the following:

formula 104 where n is 12, formula 104 where n is 33, formula 104 where n is 40, formula 104 where n is 43, formula 104 where n is 50, formula 104 where n is 60, formula 104 where n is 75 and formula 104 where n is 80, the following corresponding compounds of formula 106A were prepared where n is 12, n is 33, n is 40, n is 43, n is 50, n is 60, n is 75 and n equals 84,

1G. Other compounds of formula 1aA.

By following the procedures described in Example 1E and replacing the compounds of formula 106A with those prepared in Example 1F, the corresponding compounds of formula 1aA were obtained, where n is 12, 33, 40, 43, 50, 60, 75 and 84, such as

F1 aA-insulin-m ₂ -n ₁₂ ,

F1 aA-insulin-m ₂ -n ₃₃ ,

F1 aA-insulin-m ₂ -n4 ₀ ,

F1 aA-insulin-m ₂ -n4 ₃ ,

F1 aA-insulin-m ₂ -n ₅₀ ,

F1 aA-insulin-m ₂ -n ₆₀ ,

F1aA-insulin-m ₂ -n ₇₅ and

F1 aA-insulin-m ₂ -n ₈ 4.

Example 2. F1b-OVA-m ₁ -n4-p34-2NAcGAL.

2A. Formula 103', where X' represents ovalbumin and m equals 1.

In an endotoxin-free tube, OVA (6.5 mg, 0.000155 mmol) was added to 200 μl of PBS, pH 8.0, containing 5 mM EDTA and mixed. Separately, 1 mg of Trout's reagent was dissolved in 100 μl of PBS, pH 7.0, and 43 μl (0.00310 mmol) of the Trout's reagent solution thus obtained was added to the stirred OVA solution with continuous stirring. After 1 hour, unreacted Trout's reagent was removed using a size exclusion chromatography column to give the product of formula 103'.

2B. Formula 1b, where X' is ovalbumin, m is 1, n is 4, p is 34, R ⁹ is a direct bond and Z'' is 2NAcGAL.

In a microcentrifuge tube, poly(galactosamine methacrylate)-(pyridyl disulfide) (formula 201) (20.0 mg, 0.0020 mmol) was solubilized in 50 μl of PBS, pH 8.0, containing 5 mM EDTA. To this was added the purified product OVA Trout's reagent from Example 2A, followed by stirring for 1 hour. The resulting product of formula 1b was purified by passing the reaction mixture through a centrifugal size exclusion chromatography column. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product; (see Fig. 5).

2C. Other compounds of formula 1b

By following the procedures described in Example 2B and substituting compounds of formula 103', for example, with those obtained in Example 1D, the corresponding compounds of formula 1b were prepared

F1b-abciximab-m ₁₀ -n4-p ₃ 4-2NAcGAL,

F 1b-adalimumab-m ₁ 1 -n ₄ -p ₃ 4-2NAcGAL,

F1b-agalasidase-alpha-m ₁ 4-n4-p ₃ 4-2NAcGAL,

F1b-agalasidase-beta-m ₁ 4-n4-p ₃ 4-2NAcGAL,

F1b-aldesleukin-m ₆ -n4-p ₃ 4-2NAcGAL,

F1b-alglucosidase-alpha-m ₁₃ -n4-p ₃ 4-2NAcGAL,

F 1b-factor VIII-m1 ₀₀ -n4-p ₃₄ -2NAcGAL,

F1b factor IX-m ₁₈ -n4-p ₃ 4-2NAcGAL,

F1b-L-asparaginase-m5-n4-p ₃ 4-2NAcGAL,

F1b-laronidase-m ₇ -n4-p ₃ 4-2NAcGAL,

F1b-octreotide-m ₁ -n4-p ₃ 4-2NAcGAL,

- 49 044172

F1b-phenylalanine ammonia lyase-m ₁ 2-n ₄ -p ₃₄ -2NAcGAL,

F1b-rasbyricase-m ₁ 2-n ₄ -p ₃₄ -2NAcGAL,

F1b-insulin-m ₂ -n ₄ -p ₃₄ -2NAcGAL,

F1b-GAD-65-m ₈ -n ₄ -p ₃₄ -2NAcGAL,

F1b-IGRP-m ₇ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MBP-m6-n ₄ -p ₃₄ -2NAcGAL,

F1b-MOG-m ₅ -n ₄ -p ₃₄ -2NAcGAL,

F1b-PLP-m ₈ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MBP 13-32-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MBP83-99-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MBP 111- 129-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MBP 146-170-m ₂ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MOG 1 -20-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MOG35-55-m ₂ -n ₄ -p ₃₄ -2NAcGAL,

F1b-PLP 139-154-m ₃ -n ₄ -p ₃₄ -2NAcGAL,

F1b-MART 1 -m ₄ -n ₄ -p ₃₄ -2NAcGAL,

F1b-Tupo3UHa3a-m ₈ -n ₄ -p ₃₄ -2NAcGAL,

F1b-PMEL-m ₅ -n ₄ -p ₃₄ -2NAcGAL,

F1b-aKeanopuH-4-m ₄ -n ₄ -p ₃₄ -2NAcGAL,

F1b-S-appecTUH-m ₁₂ -n ₄ -p ₃₄ -2NAcGAL,

F1b-IRBP-m ₂₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-KOHapxuH-m ₂₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-alpha-gliadin 33-mer native-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-alpha-gliadin 33-mer deamidated-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-alpha-gliadin-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

F1b-omega-gliadin-m ₁ -n ₃ -p ₃₃ -2NAcGAL.,

F1b-Fel d 1-m ₄ -n ₄ -p ₃₄ -2NAcGAL,

F1b-cat albumin-m ₁₆ -n ₄ -p ₃₄ -2NAcGAL,

F1b-Can f 1-m ₆ -n ₄ -p ₃₄ -2NAcGAL,

F1b-canine albumin-m2 ₃ -n ₄ -p ₃₄ -2NAcGAL and

F1b-RhCE-m ₁₀ -n ₄ -p ₃₄ -2NAcGAL.

Example 3. F1f-OVA-m ₁ -n ₄ -p33-2NAcGAL.

3A. Formula 1f, where X' is ovalbumin and m is 1, n is 4, p is 33, R ⁹ is direct bond and Z'' is 2NAcGAL.

In an endotoxin-free tube, OVA (4.0 mg, 0.0000952381 mmol) was added to 0.1 ml PBS, pH 7.4, and mixed. Separately, poly(N-acetylgalactosamine)-p-nitrophenol carbonate of formula 601, where n is 4 and p is 33 (33.0 mg, 0.002380952 mmol), was added to 100 μl of PBS, pH 7.5, and stirred for vortex until dissolved. Both solutions were mixed and the mixture was stirred vigorously for 1 hour. The mixture was then collected and dialyzed for 3 days against PBS, pH 7.4 (molecular weight cut-off 30 kDa) to obtain the product of formula 1f.

Example 4. F1g-PVA-m ₁ -p ₉₀ -2NAcGAL.

4A. Formula 1g, where X' is ovalbumin and m is 1, p is 90, R ⁹ is a direct bond and Z'' is 2NAcGAL.

In an endotoxin-free tube, OVA (5.0 mg, 0.000119048 mmol) was added to 0.2 ml PBS, pH 7.4, and mixed. To the stirred solution was added 75 mg (0.00297619 mmol) poly(galactosamine methacrylate)-NHS (formula 701) dissolved in 0.4 ml PBS, pH 7.4. The mixture was left to stir for 2 hours. The mixture was then collected and dialyzed for 3 days against PBS, pH 7.4 (molecular weight cut-off 30 kDa) to give the product of formula 1g.

Example 5. F1h-OVA-m2-n ₄₅ -p ₅₅ -q4-2NAcGAL.

5A. Formula 802', where X' is ovalbumin, m is 2 and n is 45.

In an endotoxin-free tube, OVA (3.0 mg, 0.0000714286 mmol) was added to 150 μl of PBS, pH 8.0, containing 5 mM EDTA and mixed. Dibenzocyclooctine-PEG-(p-nitrophenylcarbonate) (Formula 801) (5.265 mg, 0.002142857 mmol) dissolved in DMF was added to the OVA solution and stirred for 1 hour. Excess dibenzocyclooctine-PEG-(p-nitrophenylcarbonate) was removed using a column for centrifugal size exclusion chromatography to obtain the product of formula 802'.

5V. Formula 1h, where X' is ovalbumin, m is 2, n is 45, p is 55, q is 4, R ⁸ is CH2, R ⁹ is direct bond and Z'' is 2NAcGAL.

Poly(galactosamine methacrylate)-N3 (formula 803, where p is 55, q is 4 and Z'' is N-acetylgalactosamine) (33 mg, 0.002142857 mmol) was dissolved in 100 μl PBS, pH 7.4, and added to the product from Example 5A with stirring. After 1 hour, the resulting product of formula 1h was purified by centrifugal size exclusion chromatography.

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Example 6. F1j-OVA-m ₁₀ -n ₄₅ -p ₅₅ -q ₄ -2NAcGAL.

6A. Formula 103', where X' represents ovalbumin and m equals 10.

In an endotoxin-free tube, OVA (5.0 mg, 0.00019 mmol) was added to 150 μl of PBS, pH 8.0, containing 5 mM EDTA and mixed. Separately, 1 mg of Trout's reagent was dissolved in 100 μl of PBS, pH 7.0, and 16 μl (0.0019 mmol) of the Trout's reagent solution thus obtained was added to the stirred OVA solution with continuous stirring. After 1 hour, unreacted Trout's reagent was removed using a size exclusion chromatography column to give the product of formula 103'.

6B. Formula 902'', where X' is ovalbumin, m is 10 and n is 45.

Dibenzocyclooctyne-PEG-(pyridyl disulfide) (formula 901, where n is 45) (6.0 mg, 0.00238 mmol) was dissolved in DMF and the resulting solution was added to the OVA solution prepared in Example 6A and stirred for 1 hour. Excess dibenzocyclooctyne-PEG-(pyridyl disulfide) was removed using centrifugal size exclusion chromatography to give the product of formula 902''.

6C. Formula 1j, where X' is ovalbumin, m is 10, n is 45, p is 55, q is 4, R is CH ₂ , R ⁹ is direct bond and Z'' is 2NAcGAL.

Poly(galactosamine methacrylate)-N3 (formula 803, where p is 55, q is 4, and Z'' is N-acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in 150 μl PBS, pH 7.4, and added to the product from Example 6B with stirring. After 1 hour, the resulting product of formula 1j was purified (excess p(GMA)-N3 was removed) by centrifugal size exclusion chromatography. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

Example 7. F1L-OVA-m ₂ -n ₈₀ -p ₅₅ -q ₄ -2NAcGAL.

7A. Formula 1002, where X' is ovalbumin, m is 2 and n is 80.

Dibenzocyclooctyne-PEG-(pyridyl disulfide) (formula 1001, where n is 80) (9.0 mg, 0.00238 mmol) was dissolved in DMF and the resulting solution was added to purified OVA solution of formula 103' (where X' is ovalbumin and m equals 2), for example, prepared as described in Example 6A, and stirred for 1 hour. Excess dibenzocyclooctine-PEG-(pyridyl disulfide) was removed using centrifugal size exclusion chromatography to obtain the product of formula 1002.

7B. Formula 1L, where X' is ovalbumin, m is 2, n is 80, p is 55, q is 4, R ⁸ is CH ₂ , R ⁹ is direct bond and Z'' is 2NAcGAL.

Poly(galactosamine methacrylate)-No. (formula 803, where p is 55, q is 4, and Z'' is N-acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in 150 μl PBS, pH 7.4, and added to the product from Example 7A with stirring. After 1 hour, the resulting product of formula 1L was purified (excess poly(galactosamine methacrylate)-N was removed) using centrifugal size exclusion chromatography. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

Example 8. Preparation of poly(galactosamine-methacrylate) polymers.

8A. Galactosamine methacrylate.

To stirred galactosamine hydrochloride (2.15 g, 10.0 mmol) was added 0.5 M sodium methoxide (22 ml, 11.0 mmol). After 30 minutes, methacrylic anhydride (14.694 g, 11.0 mmol) was added and stirring was continued for 4 hours. The resulting galactosamine methacrylate was loaded onto silica gel on a rotary evaporator and purified by column chromatography using DCM:MeOH (85:15) .

8B. Formula 201, where n is 4 and p is 30

Galactose methacrylate (600 mg, 2.43 mmol), 2-(2-(2-(2-(pyridin-2-yldisulfanyl)ethoxy)ethoxy)ethoxy)ethyl-2-((phenylcarbonothioyl)thio)acetate (44 .8 mg, 0.081 mmol) and AIBN (3.174089069 mg, 0.016 mmol) were added to 1.5 ml DMF in a Schlenk flask. The reaction mixture was subjected to 4 freeze-thaw cycles and then stirred at 70°C for 6 hours. The desired polymer product of formula 201 was precipitated in 12 ml of methanol and excess solvent was removed under reduced pressure.

Example 9. Preparation of F1aA-PE-m ₃ -n ₈₀ .

9A. Formula 103', where X' represents phycoerythrin.

In an endotoxin-free tube, phycoerythrin (PE) (purchased from Pierce) (200 μL, 0.000004 mmol) was added to 50 μL of PBS, pH 8.0, containing 5 mM EDTA and mixed. Separately, 1 mg of Trout's reagent was dissolved in 100 μl of PBS, pH 7.0, and 2 μl (0.00013 mmol) of the Trout's reagent solution thus obtained was added to the stirred PE solution with continuous stirring. After 1 hour, excess Trout's reagent was removed using a size exclusion chromatography column to give the product of formula 103'.

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9B. Formula 106A, where n is 80.

In an endotoxin-free tube, galactosamine (7.0 mg, 0.03246 mmol) was dissolved with stirring in 100 μl of PBS, pH 8.0, containing 5 mM EDTA. Pyridyl dithiol poly(ethylene glycol) NHS ester (formula 104, where n is 80) (16.23 mg, 0.00464 mmol) dissolved in 50 μl PBS, pH 7.0, was added to the stirred galactosamine solution. After 1 hour, the resulting product of formula 106A was ready for use without further purification.

9C. Formula 1a, where X' is phycoerythrin, m is 3, n is 80 and Z' is galactosamine.

The purified PE-Trout reagent conjugates obtained in Example 9A were added directly to the stirred product of Formula 106A obtained in Example 9B. After 1 hour, the resulting product of formula 1a was purified by passing the reaction mixture through a centrifugal size exclusion chromatography column. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

Example 10. OVA-DOM.

10A. Obtaining the expression vector.

The mammalian cell expression vector pSecTag A was purchased from Life Technologies. The gene encoding the anti-ASGPR antibody domain, Dom26h-196-61, herein referred to as DOM, was purchased from the supplier GenScript as a codon-optimized sequence for expression of the protein in human cells. Sequences encoding OVA, DOM, flexible linker Gly ₃ Ser and 6xHis tag were cloned into the mammalian cell expression vector, pSecTag A, C-terminal to the Ig κ chain secretion leader sequence by site-directed mutagenesis following a variation of the QuikChange protocol (Geiser, et al.).

10V. Expression and purification of a compound of formula 2, where m' is 1, m' is 0, X is ovalbumin, Y is Gly ₃ Ser, Z is anti-ASGPR antibody, Dom26h-196-61.

HEK293 cells were transiently transfected with modified pSecTag A vectors prepared, for example, as described in Example 10A using polyethylenimine. Transfected cells were cultured in FreeStyle 293 medium (Life Technologies) supplemented with valproic acid for 7 days, after which the cells were removed by centrifugation and culture supernatants were collected and sterilized by filtration. OVA/DOM fusion proteins of formula 2 were purified from culture supernatants by metal ion affinity chromatography using a HisTrap Ni ²⁺ -Sepharose column (GE Healthcare), followed by size exclusion chromatography using a Superdex 75 column (GE Healthcare), they had the following general structure:

N-OVA-Gly ₃ Ser-DOM-Gly ₃ Ser-6xHis-C and the following amino acid sequence:

GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQI NKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYA EERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVL QPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMP FRVTEQESKPVQMMY QIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTE WTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAE SLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVCm.FRADHPFLFCIKHIAT NAVLFFGRCVSPGGGSEVQLLESGG GLVQPGGSLRLSCAASGFTFEKYAMAW VRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSHHHHHH (SEQ ID NO:28) Protein purity was checked by staining SDS-PAGE gels with Coomassie Brilliant Blue and Western blotting against 6 xHis tags. Protein concentration was determined using the Lambert-Beer law by measuring absorbance at 280 nm using a NanoDrop 2000 (Thermo Scientific), and molecular weight (57.3 kDa) and extinction coefficient (57090 M ^-1 cm ^-1 ) were estimated based on amino acid sequence of a protein using the ExPASy ProtParam tool. Endotoxin levels were measured using the HEK-BLUE TLR reporter cell line (Invivogen) according to the manufacturer's instructions.

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10C. Other compositions of formula 2.

By following the procedures described in Examples 10A and 10B and making appropriate substitutions in the pSecTag A vectors, the following fusion proteins of Formula 2 were prepared:

N-DOM-Gly ₃ Ser-0 VA-Gly ₃ Ser-6xHis-C with the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSGSIGAAS MEFCFDVFKELKVHHANENIFYCPiAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCG TS VNVHSSLRDILNQiTKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELIN SWVESQTNGiIRNVLQPSSVDSQTAMVLVNAiVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMY QIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVME ERKIKVY LPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGV DAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH (SEQ ID NO:29); And

N-OVA-Gly ₃ Ser-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis -C with the following amino acid sequence:

GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSI EAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAA DQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMP FRVTEQES KPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVM EERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVV GSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSEVQLLESGGGLVQ PGGSLRL SCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSGSIGAASMEFCFDVFKELKVHHANENIF YCPIAIMSALAMVYLGAKDSTRTQiNKVVRFDKLPGFGDSEAQCGTSVNVHSSLRDILNQITKPN DVY SFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGilRNVLQPSSV DSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKiLELP FASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNL TSVLMAMG ITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHI ATNAVLFFGRCVSPGGGSHHHHHH (SEQ ID NO:30).

10D. Other compositions of formula 2.

Proceeding according to the procedures described in Examples 10A and 10B, and replacing Gly ₃ Ser in the vectors with an immunoproteasome cleavage site (IPC) linker, and OVA in the vectors with

MVR13-32 (SEQ ID NO:11),

МВР83-99 (SEQ ID NO:12),

MBP111-129 (SEQ ID NO:13),

MBP146-170 (SEQ ID NO:14),

MOG1-20 (SEQ ID NO:15),

MOG35-55 (SEQ ID NO:16), and

PLP139-154 (SEQ ID NO:17), or in alpha-gliadin 33-mer deamidated (SEQ ID NO:25) alpha-gliadin (SEQ ID NO:26) and omega-gliadin (SEQ ID NO:27) vectors , the following fusion proteins of formula 2 were obtained:

MBP13-32-IPC-MBP83-99-IPC-MBP111-129-IPC-MBP146-170-IPC-MOG1-20-IPC-MOG35-55-IPCPLP139-154-IPC-DOM and alpha-gliadin 33-mer deamidated- IPC-alpha-gliadin-IPC-omega-gliadin-IPC-DOM.

Example 11 Desialylated OVA.

11A. Obtaining the expression vector.

The mammalian cell expression vector pSecTag A was purchased from Life Technologies. Sequences encoding OVA, a Gly ₃ Ser flexible linker, and a 6xHis tag were cloned into pSecTag A C-terminal to the Ig κ chain secretion leader sequence by site-directed mutagenesis following a variation of the QuikChange protocol (Geiser, et al.).

11B. Expression and purification of OVA.

HEK293 cells were transiently transfected with a modified pSecTag A vector prepared, for example, as described in Example 10A using polyethylenimine. Transfected cells were cultured in FreeStyle 293 medium (Life Technologies) supplemented with valproic acid for 7 days, after which the cells were removed by centrifugation and the supernatants

- 53 044172 cultures were collected and sterilized by filtration. The OVA protein was purified from the culture supernatant by metal ion affinity chromatography using a HisTrap Ni ²⁺ -Sepharose column (GE Healthcare), followed by size exclusion chromatography using a Superdex 75 column (GE Healthcare), they had the following structure:

Ν’- OVA Gly3Ser 6xHis -C’, and the following amino acid sequence:

GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQI NKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYA EERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVL QPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMP FRVTEQESKPVQMMY QIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTE WTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAE

SLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIAT

NAVLFFGRCVSPGGGSHHHHHH (SEQ ID NO:31)

Protein purity was checked by staining SDS-PAGE gels with Coomassie brilliant blue and Western blotting for the 6xHis tag. Protein concentration was determined using the Lambert-Beer law by measuring absorbance at 280 nm using a NanoDrop 2000 (Thermo Scientific), and molecular weight (43.8 kDa) and extinction coefficient (31525 M ^-1 cm ^-1 ) were estimated based on amino acid sequence of a protein using the ExPASy ProtParam tool.

11C. Desialization.

OVA was desialylated by incubation with neuraminidase for 1 hour at 37°C (New England Biolabs). Desialated OVA was purified from the reaction mixture by metal ion affinity chromatography (eg, using a HisTrap Ni ²⁺ -Sepharose column, GE Healthcare), followed by size exclusion chromatography (eg, using a Superdex 75 column, GE Healthcare).

Protein purity was checked by staining SDS-PAGE gels with Coomassie brilliant blue and Western blotting for the 6xHis tag. Desialation was tested using Sambucus nigra lectin-based detection of the presence of sialic acid within the protein in Western blots (Vector Biolabs) or using a sialic acid-mediated fluorescence assay (ProZyme). The concentration of desialylated protein was determined using the Lambert-Beer law as described above for the protein before desialylation. Endotoxin levels were measured using the HEK-Blue TLR4 reporter cell line (Invivogen) according to the manufacturer's instructions.

Example 12. Distribution in the liver.

12A. F1aA-PE-m ₃ -n ₈₀ was prepared, for example, as described in example 9. For injection, a 30 μg/100 μl solution in sterile saline was prepared.

A solution of F1aA-PE-m ₃ -n ₈₀ (30 μg) was administered to one of three groups of C57 black 6 mice (3 in each group) by injection into the tail vein. Two other groups of mice received an equivalent volume of phycoerythrin in 100 μl of saline or vehicle saline. Three hours after administration, the livers and spleens of these animals were collected and the level of cellular fluorescence in these organs was determined using flow cytometry as an indicator of cellular PE content.

As shown in FIG. 1, sinusoidal endothelial cells (LSEC), hepatocytes, Kupffer cells (KC) and other antigen presenting cells (APC) from the liver of mice that were treated with F1aA-PE-m ₃ -n ₈₀ exhibited at least a threefold increase in fluorescence compared to animals that received the PE solution. There was no detectable difference in the fluorescence of spleen cells collected from these three groups. These results confirm that F1 aAPE-m3-n80 had sufficient specificity to bind to antigen-presenting cells in the liver.

12V. Proceeding according to the procedures described in Example 12A and replacing F1aA-PE-m ₃ -n ₈₀ with compounds F1b-PE-m ₃ -n ₄ -p ₃₄ -2NAcGAL, F1f-PE-m ₃ -n ₄ -p ₃₃ -2NAcGAL , F1g-PE-m ₃ -p ₉₀ -2NAcGAL, F1h-PE-m ₃ n ₄₅ -p ₅₅ -q ₄ -2NAcGAL, F 1j -PE-m ₃ -n ₄₅ -p ₅₅ -q ₄ -2NAcGAL, F1 L-PE-m ₃ -n ₈₀ -p ₅₅ -q ₄ -2NAcGAL, F1 m-PE-m ₃ -n ₈₀ -p ₃₀ q ₄ -CMP-2NHAc, F1m-PE-m ₃ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH, F1n-PE-m ₃ -n1-p ₃₀ -q ₄ -CMP-2NHAc and F1n-PE-m ₃ -n ₃₃ -p ₃₀ q ₈ -CMP-2OH, obtained, for example, as described with reference to Example 9, by replacing X in Examples 2B, 3, 4, 5B, 6B, 7B, 19G, 19L, 20B and 20F, respectively, it was confirmed that compounds in which F1aA-PE-m ₃ -n ₈₀ was replaced by compounds F1b-PE-m ₃ -n ₄ -p ₃₄ -2NAcGAL, F1f-PE-m ₃ -n 4 -p ₃₃ -2NAcGAL, F1gPE _- m3-p90-2NAcGAL, F1h-PE-m3-n45-p55 -q4-2NAcGAL, F1j-PE-m3-n45-p55-q4-2NAcGAL, F1L-PE-m3-n80-p55q ₄ -2NAcGAL, F1m-PE-m ₃ -n ₈₀ -p ₃₀ -q ₄ -CMP- 2NHAc, F1m-PE-m ₃ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH, F1n-PE-m ₃ -n ₁ -p ₃₀ -q ₄ CMP-2NHAc and F1n-PE-m3-n33-p30 -q8-CMP-2OH had sufficient specificity to bind to antigen-presenting cells in the liver.

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Example 13. Proliferation of antigen-specific OT1 CD8+ T cells.

13A. F1aA-OVA-m ₄ -n ₈₀ , synthesized, for example, as described in example 1, was prepared for injection at a concentration of 10 μg/100 μl in physiological solution. On day 0, 10 ⁶ RT-1 T cells were fluorescently labeled and adoptively transferred into 3 groups of CD 45.2 mice (5 per group) by tail vein injection. The next day (ie, day 1), each of the 3 groups of mice was administered, respectively, 10 μg of F1aA-OVA-m ₄ -n ₈₀ , OVA or saline by tail vein injection. On day 6, animals were sacrificed and the % of proliferating OT-1 cells in the spleen was determined by fluorescence-activated cell sorting.

The results of this study (see Fig. 2) show that the percentage of proliferating OTI T cells in mice treated with F1aA-OVA-m ₄ -n ₈₀ (Gal-OVA in Fig. 2) was significantly higher than percentage of proliferating OTI cells in the spleen of mice treated with OVA or saline (untreated in Fig. 2). Increased OTI cell proliferation demonstrates increased cross-priming of CD8+ T cells in animals treated with F1aA-OVA-m ₄ -n ₈₀ compared to other treatments. Taken together with the results from Example 12, these results indicate that the ability of F1aA-OVA-m ₄ -n ₈₀ to target antigens to the liver enhances the presentation of OVA by antigen presenting cells in the liver to OVA-specific OTI T cells.

13V. To distinguish T cells undergoing expansion and development into a functional effector phenotype from those undergoing expansion and deletion, proliferating OTI CD8+ T cells were analyzed for annexin-V, as a hallmark of apoptosis and thus deletion, as well as exhaustion marker, programmed death protein-1 (PD-1). As shown in FIG. 3, F1aA-OVA-m ₄ -n ₈₀ (Gal-OVA in Fig. 3) induced much higher numbers of annexed-Y+ and PD-1+ proliferating OTI CD8+ T cells than soluble OVA.

13C. By following the procedures described in Examples 13A and 13B and replacing F1aA-OVA-m ₄ -n ₈ with compounds of formula 1 prepared, for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it was shown that the compounds of Examples 3A, 4A, 5B, 6C, 7B and 19G induce much higher numbers of annexin-V and PD-1+ proliferating OTI CD8+ T cells than soluble OVA.

13D. Proceeding according to the procedures described in Examples 13A and 13B, and by replacing F1aAOVA-m ₄ -n ₈ with compounds of formulas 1 and 2 prepared, for example, as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and replacing OVA with antigens corresponding to X (or X', or X''), respectively, the compounds of Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F were shown to induce much more higher numbers of annexed-V ⁺ and PD-1+ proliferating OTI CD8+ T cells than soluble X antigen.

Example 14 F1aA-OVA-m ₄ -n ₈ does not induce a response with OVA-specific antibodies.

14A. To assess the humoral immune response to F1aA-OVA-m4-n8, mice were treated with weekly iv injections of either F1aA-OVA-m ₄ -n ₈ or OVA, and then blood levels of OVA-specific antibodies were measured. On days 0, 7, and 14 of the experiment, mice received an IV injection of 100 μl of saline containing one of the following: 1) 6 μg of OVA; 2) 6 μg F1aA-OVA-m ₄ -n ₈ ; 3) 30 µg OVA; 4) 30 μg F1aA-OVA-m ₄ -n ₈ , or 5) saline solution only. Each group consisted of 5 mice. On day 19, mice were bled by cheek puncture and the titer of OVA-specific antibodies in the blood of each mouse was determined by ELISA. The results of this study showed that while OVA-specific antibody titers increased in mice treated with 6 and 30 μg of OVA, mice treated with 6 and 30 μg of F1aA-OVA-m ₄ -n ₈ (GalOVA in Fig. 4), blood titers were similar to those in saline-treated mice (ie, vehicle-treated animals) (Fig. 4). For example, mice treated with 6 and 30 μg of OVA had mean antibody titers of 3.5 and 2.5, respectively; whereas, mice treated with 6 and 30 μg OVA had mean antibody titers of 0.75 and 0.25, respectively.

14V. By following the procedures described in Example 14A and replacing F1aA-OVA-m ₄ -n ₈ with compounds of formula 1 prepared, for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it was shown that for mice treated with the compounds of Examples 3A, 4A, 5B, 6C, 7B, and 19G exhibited OVA-specific antibody titers similar to those in saline-treated mice.

14C. Proceeding according to the procedures described in Example 14A and replacing F1aA-OVA-m ₄ -n ₈ with compounds of formulas 1 and 2 prepared, for example, as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and replacing OVA with antigens corresponding to X (or X', or X''), respectively, it was shown that for mice treated with the compounds of Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F , titers of antigen X-specific antibodies are similar to those in mice treated with saline.

Example 15 F1aA-OVA-m ₄ -n ₈ depletes OVA-specific antibodies.

15A. Mice that had varying titers of OVA antibodies in the blood (each mouse had a titer ranging from 0 to 4.5) were treated with an iv injection of 20 μg F1aA-OVA-m ₄ -n ₈ dissolved in 100 μl physio

- 55 044172 logical solution. Mice received iv injections of F1aA-OVA-m ₄ -n ₈ on days 0, 5, 7, 12 and 14 (F1aA-OVA-m ₄ -n ₈ injections are designated Gal-OVA and are shown as green arrows on the x-axis in Fig. 5). To determine the ability of F1aA-OVA-m ₄ -n ₈ to deplete OVA-specific serum antibodies, mice were bled on day -1 to establish an initial antibody titer, and then subsequent blood draws were performed after each injection of F1aA-OVA-m ₄ -n ₈ on days 2, 6, 9, 13 and 16. The antibody titer of each mouse was determined using ELISA. The results of this study indicate that F1aA-OVA-m ₄ -n ₈ is able to deplete serum antibody levels in mice. For example, one day after the first injection of F1aA-OVA-m ₄ -n ₈ (ie, day 2), mice with positive anti-OVA antibody titers exhibited a 5- to 100-fold decrease in serum antibody levels (Fig. 5). The results show that although antibody titers increased in some mice over the course of the 19-day experiment, titer levels never reached the initial antibody titre measured on day -1, and subsequent doses of F1aA-OVA-m4-n8 were effective in reducing these short-term increases antibody titers. These results demonstrate that F1aA-OVA-m ₄ -n ₈ has the binding specificity of OVA-specific serum antibodies and the kinetics required to deplete OVA-specific serum antibodies.

15V. By following the procedures described in Example 15A and replacing F1aA-OVA-m ₄ -n ₈ with compounds of formula 1 prepared, for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it was shown that the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G are characterized by the binding specificity of OVA-specific serum antibodies and the kinetics required to deplete OVA-specific serum antibodies.

15C. Proceeding according to the procedures described in Example 15A and replacing F1aA-OVA-m ₄ -n ₈ with compounds of formulas 1 and 2 prepared, for example, as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and replacing OVA with antigens corresponding to X (or X', or X''), respectively, the compounds of Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F were shown to have antigen binding specificity serum X-specific antibodies and the kinetics required for antigen depletion of serum X-specific antibodies.

Example 16. Model from OT-1 stimulation to tolerance development.

16A. By applying the standard model from OTI stimulation to tolerance development (Liu, Iyoda, et al., 2002), the ability of F1aA-OVA-m ₄ -n ₈ (mGal-OVA), F1b-OVA-m ₁ -n ₄ -p ₃₄ was demonstrated (pGal-OVA) and N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C (Dom-OVA) prevent subsequent immune responses to vaccine-mediated antigen stimulation, even when stimulation includes a very strong bacterial adjuvant (i.e. lipopolysaccharide). To develop tolerance, 233 nmol of one of F1aA-OVA-m ₄ -n ₈ , F1b-OVA-m ₁ -n ₄ -p ₃₄ , N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser6xHis-C or soluble OVA was administered intravenously in 100 μl saline on days 1 and 6, followed by adoptive transfer of OTI CD8 ⁺ (CD45.2+) T cells into CD45.1+ mice (n=5 mice per group). After 9 additional days to allow for potential deletion of the transferred T cells, recipient mice were then challenged with OVA (10 μg) adjuvanted lipopolysaccharide (LPS) (50 ng) by intradermal injection. Characterization of the draining lymph nodes 4 days after stimulation allowed us to determine whether a deletion had actually occurred or not.

16V. Intravenous administration of F1aA-OVA-m ₄ -n ₈ , Flb-OVA-m ₁ -n ₄ -p ₃₄ and N-DOM-Gl·y ₃ Ser-OVA-Gl·y ₃ Ser6xHis-C resulted in a significant reduction in OTI populations CD8+ T cells in draining lymph nodes compared with mice treated with unmodified OVA before antigen stimulation with LPS, demonstrating deletion tolerance. For example, in FIG. Figure 6 shows that draining lymph nodes from mice treated with one of F1aA-OVA-m4-n8 (mGal-OVA), F1b-OVA-m ₁ -n ₄ -p ₃₄ (pGal-OVA) and N-DOM- Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C (Dom-OVA) contained more than 9-fold fewer OTI CD8+ T cells compared to OVA-treated mice and more than 43-fold fewer compared to with control stimulation mice that did not receive intravenous injections of antigen; responses in spleen cells were similar. These results demonstrate that F1aA-OVA-m ₄ -n ₈ , F1b-OVA-m ₁ -n ₄ -p ₃₄ and N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C attenuated OVA-specific immune response after stimulation with adjuvanted OVA.

16C. Following the procedures described in Examples 16A and B and substituting F1aA-OVA-m ₄ -n ₈ , F1b-OVA-m ₁ -n ₄ -p ₃₄ , and N-DOM-Gl·y ₃ Ser-OVA-Gl y ₃ Ser-6xHis-C compounds of formula 1 prepared, for example, as described in examples 3A, 4A, 5B, 6C, 7B and 19G, it was shown that the compounds of examples 3A, 4A, 5B, 6C, 7B and 19G attenuate the OVA-specific immune response following stimulation with adjuvanted OVA.

16D. Proceeding according to the procedures described in Examples 16A and B and substituting F1aA-OVA-m4-n8, F1b-OVA-m ₁ -n ₄ -p ₃₄ , and N-DOM-Gl·y ₃ Ser-OVA-Gl·y ₃ Ser-6xHis-C compounds of formulas 1 and 2, prepared, for example, as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and replacing the OVA with antigens corresponding to X (or X', or X''), respectively, the compounds of Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F have been shown to attenuate the antigen X-specific immune response following stimulation with an adjuvanted antigen X.

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Example 17. Pharmacokinetics.

17A. OVA and N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C and N-OVA-Gly ₃ Ser-DOM-Gly ₃ Ser6xHis-C fusion proteins (prepared, for example, as described in Example 10) were labeled with using IRDye 800CW (LI-COR Biosciences) according to the manufacturer's instructions. Unreacted dye was removed using Zeba desalting columns (Thermo Scientific). Tagged proteins, 50 μg in 100 μl PBS per dose, were administered iv or sc to C57BL/6 mice (5 mice per group). At time points = 0, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 96 h postinjection, blood samples were collected from the tail tip into heparin-coated capillary tubes. Samples were stored at 4°C, protected from light, until analysis.

On the day of analysis, blood samples were centrifuged to remove cellular components. Plasma was transferred to fresh capillary tubes and scanned using an Odyssey infrared imaging system (LI-COR Biosciences). The signal was detected at the 800 nm channel. Using the image processing program ImageJ (US National Institutes of Health), each sample was fitted as a line of width 2, and the average intensity along the line was determined as a relative measure of the amount of circulating protein at that time point.

Fluorescence signals (normalized to fluorescence at time = 0) as a function of time of administration of OVA and DOM-OVA and OVA-DOM fusion proteins, along with bi-exponential decay curves plotted in FIG. 8 indicate that clearance of ASGPR-targeted OVA fusion proteins from the circulation was faster than with unmodified OVA.

17V. By following the procedures described in Example 17A and replacing the N-DOMGly3Ser-OVA-Gly3Ser-6xHis-C and OVA fusion protein with aliased Factor VIII and Factor VIII, respectively, clearance of aliased Factor VIII from the circulation was shown to be faster than in in the case of unmodified factor VIII.

Example 18: Biodistribution.

18A. OVA and N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C fusion protein (prepared, for example, as described in Example 10) were labeled using Alexa Fluor 647 (Life Technologies) according to the manufacturer's instructions. Unreacted dye was removed using Zeba desalting columns (Thermo Scientific). Tagged proteins, 50 μg in 100 μl PBS per dose, were administered iv or sc to C57BL/6 mice (5 mice per group). Two hours after injection, mice were sacrificed by CO ₂ asphyxiation. Blood, heart, intestines, kidneys, liver, lungs, stomach, spleen, and the rest of the body were imaged using the IVIS Spectrum imaging system (Caliper Life Sciences). Data were acquired and analyzed using Living Image software (Caliper Life Sciences). Suspensions of individual liver and spleen cells were obtained and stained with fluorescently labeled antibodies to MHC class II, CD1d, CD3, CD4, CD8a, CD11b, CD11c, CD14, CD19, CD45, CD123 and/or Lin in PBS with 0.1% (weight/volume) BSA. Samples were analyzed using an LSRII flow pitometer and FACS Diva software (BD Biosciences).

A histogram of fluorescence signal densities (photons/weight) for each organ and each protein shows that animals injected with the N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C fusion protein had the strongest fluorescence signal in the liver compared to those treated with unmodified OVA.

The histogram with the percentages of N-DOM-Gly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C- and OVA-positive cells shows that animals injected with N-DOM-Gly3Ser-OVAGly3Ser-6xHis-C fusion proteins are characterized by significantly more high percentage of positive hepatocytes compared to those treated with unmodified OVA.

18V. By following the procedures described in Example 18A and replacing the N-DOMGly3Ser-OVA-Gly3Ser-6xHis-C and OVA fusion protein with asialylated factor VIII and factor VIII, respectively, it was shown that animals treated with asialylated factor VIII were significantly more high percentage of positive hepatocytes compared to those treated with unmodified factor VIII.

18C. Proceeding according to the procedures described in Example 18A and replacing the N-DOMGly ₃ Ser-OVA-Gly ₃ Ser-6xHis-C fusion protein with compounds of formula 1 prepared, for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it was shown that animals administered the compounds of Examples 3A, 4A, 5B, 6C, 7B and 19G had a significantly higher percentage of positive hepatocytes compared to those treated with OVA.

18D. Following the procedures described in Examples 18A and B and substituting F1aA-OVA-m4-n ₈ , F1b-OVA-m1-n4-p ₃ 4 , and N-DOM-Gl y ₃ Ser-OVA-Gl y ₃ Ser-6xHis-C compounds of formulas 1 and 2, prepared, for example, as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and replacing the OVA with antigens corresponding to X (or X', or X''), respectively, it was shown that animals administered compounds of Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F had a significantly higher percentage of positive hepatocytes compared to those treated with using antigen X.

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Example 19. F1m-OVA-m2-n ₈₀ -p ₃₀ -q4-CMP-2NHAc.

19A. Formula 1102, where R ³ represents NHAc and R ⁴ represents OH.

N-Acetyl-D-galactosamine (formula 1101, where R ³ is NHAc and R ⁴ is OH) (5 g, 22.6 mmol) was added to a stirred solution of chloroethanol (200 ml) at room temperature. The solution was cooled to 4°C and acetyl chloride was added dropwise to the solution. The solution temperature was brought to room temperature and then heated to 70°C. After 4 h, unreacted chloroethanol was removed under reduced pressure. 100 ml of ethanol was added to the crude product and the resulting solution was stirred in the presence of carbon for 2 hours. The solution was filtered and the solvent was removed under reduced pressure. The corresponding product of formula 1102, N-(2-(2chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide, was used without further purification.

19V. Formula 1103, where R ³ represents NHAc and R ⁴ represents OH.

N-(2-(2-Chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide prepared in Example 19A (2 g, 7.4 mmol) was added to stirred solution of DMF (100 ml) and sodium azide (4 g, 61.5 mmol). The solution was heated at 90°C for 12 hours and then filtered. The residual solvent was removed under reduced pressure and the crude product was purified by flash chromatography (10% MeOH in dichloromethane) to give the corresponding product of formula 1103, N-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl )tetrahydro-2H-pyran-3yl)acetamide.

19C. Formula 1104, where R ³ represents NHAc and R ⁴ represents OH.

N-(2-(2-Azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide prepared in Example 19B (2 g, 6.9 mmol) was added to a solution of a carbon-supported palladium catalyst and ethanol (50 ml). The solution was stirred under hydrogen gas (3 atm) for 4 hours. The resulting solution was filtered and the residual solvent was removed under reduced pressure to give the corresponding product of formula 1104, N-(2-(2-aminoethoxy)-4,5dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide, which was used without further purification.

19D. Formula 1105, where R ³ represents NHAc and R ⁴ represents OH.

N-(2-(2-Aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide prepared in Example 19C (1.0 g, 3.78 mmol), was added to a solution of methacrylic anhydride (0.583 g, 3.78 mmol) in DMF (50 ml). Triethylamine was then added to the solution and the reaction mixture was stirred for 2 hours at room temperature. After 2 hours, the excess solvent was removed under reduced pressure and the corresponding product of formula 1105, N-(2-((3-acetamido-4,5-dihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl) methacrylamide was isolated using flash chromatography.

19E. Formula 1107, where p is 30, q is 4, R ³ is NHAc, R ⁴ is OH and R ⁸ is CMP.

Azide-modified uRAFT of formula 1106, where q is 4 (28 mg), was added to a solution of N-(2-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)ethyl)methacrylamide prepared in Example 19D (579 mg, 1.74 mmol) and azobisisobutyronitrile (2.2 mg, 0.0116 mmol) in DMF. The reaction mixture was subjected to 4 freeze-pump-thaw cycles and then stirred at 70°C. After 12 hours, the polymer product of formula 1107, where p is 30 and q is 4, was precipitated from the reaction mixture by adding methanol. The solvent was decanted from the solid and the solid was collected, and the residual solvent was removed under reduced pressure.

19F. Formula 1109, where X' is OVA, m is 2, and n is 80.

Ovalbumin (5 mg, 0.00012 mmol) was added to 100 μl sodium phosphate buffer (pH 8.0) and mixed. To this solution was added 5 mg of the compound of formula 1108, where n is 80. After 1 hour, the unreacted compound of formula 1108 was removed from the solution by centrifugal size exclusion chromatography. The resulting buffered solution containing the corresponding product of formula 1109 was used in the next reaction without further purification.

19G. Formula 1m, where X' is OVA, m is 2, n is 80, p is 30, q is 4, R ³ is NHAc and R ⁸ is CMP.

The solution obtained in Example 19F was added to 100 μl of sodium phosphate buffer (pH 8.0) which contained 10 mg of the product of formula 1107 obtained in Example 19E. The reaction mixture was allowed to stir for 2 hours and then excess compound of formula 1107 was removed by centrifugal size exclusion chromatography to obtain the corresponding isomeric product of formula 1m in solution, which was used in biological studies without further purification. The R ³ substituent is designated in the name of the title compound as 2NHAc.

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19N. Other compounds of formula 1109.

Proceeding according to the procedures described in Example 19F and replacing the OVA with the following: abciximab, adalimumab, agalasidase-alpha, agalasidase-beta, aldesleukin, alglucosidase-alpha, factor VIII, factor IX, L-asparaginase, laronidase, octreotide, phenylalanine ammonia- lyase, rasburicase, insulin (SEQ ID NO:5),

GAD-65 (SEQ ID NO:6),

IGRP (SEQ ID NO: 7) MBP (SEQ ID NO:8), MOG (SEQ ID NO:9), PLP (SEQ ID NO:10), MBP13-32 (SEQ ID NO:11), MBP83-99 ( SEQ ID NO: 12), MBP111-129 (SEQ ID NO:13), MBP146-170 (SEQ ID NO:14), MOG1-20 (SEQ ID NO:15), MOG35-55 (SEQ ID NO:16) , PLP139-154 (SEQ ID NO:17), MARTI (SEQ ID NO:18), tyrosinase (SEQ ID NO:19), PMEL (SEQ ID NO:20), aquaporin-4 (SEQ ID NO:21), S-arrestin (SEQ ID NO:22), IRBP (SEQ ID NO:23), conarchin (UNIPrOt Q6PSU6), alpha-gliadin 33-mer native (SEQ ID NO:24), alpha-gliadin 33-mer deamidated (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26), omega-gliadin (SEQ ID NO:27), Fel d 1A (UNIPROT P30438), feline albumin (UNIPROT P49064), Can f 1 (UNIPROT O18873) , canine albumin (UNIPROT P49822) and an example of RhCE (UNIPROT P18577), the following corresponding compounds of formula 1109 were prepared, where n is 80: X is abciximab and m is 10, X is adalimumab and m is 11, X is agalsidase- alpha and m is 14, X is agalsidase-beta and m is 14, X is aldesleukin and m is 6, X is alglucosidase-alpha and m is 13, X is factor VIII and m is 100, X is factor IX and m is 18, X is L-asparaginase and m is 5, X is laronidase and m is 7, X is octreotide and m is 1, X is phenylalanine ammonia lyase and m is 12, X represents rasburicase and m is equal to 12,

X is insulin (SEQ ID NO:5) and m is 2,

X is GAD-65 (SEQ ID NO:6) and m is 8,

X is IGRP (SEQ ID NO:7) and m is 7,

X is MBP (SEQ ID NO:8) and m is 6,

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X is MOG (SEQ ID NO:9) and m is 5,

X is PLP (SEQ ID NO:10) and m is 8,

X is MBP13-32 (SEQ ID NO:11) and m is 1.

X is MBP83-99 (SEQ ID NO:12) and m is 1.

X is MBP111-129 (SEQ ID NO:13) and m is 1.

X is MBP146-170 (SEQ ID NO:14) and m is 2.

X is MOG1-20 (SEQ ID NO: 15) and m is 1.

X is MOG35-55 (SEQ ID NO: 16) and m is 2.

X is PLP139-154 (SEQ ID NO:17) and m is 3.

X represents MARTI (SEQ ID NO:18) and m equals 4,

X is tyrosinase (SEQ ID NO:19) and m is 8,

X is PMEL (SEQ ID NO:20) and m is 5,

X is aquaporin-4 (SEQ ID NO:21) and m is 4.

X is S-arrestin (SEQ ID NO:22) and m is 12,

X is IRBP (SEQ ID NO:23) and m is 21,

X represents conarchine and m equals 21,

X is alpha-gliadin 33-mer native (SEQ ID NO:24) and m is 1.

X is alpha-gliadin 33-mer deamidated (SEQ ID NO:25) and m is 1.

X is alpha-gliadin (SEQ ID NO:26) and m is 1.

X is omega-gliadin (SEQ ID NO:27) and m is 1.

X represents Fel d 1 and m equals 4,

X represents feline albumin and m equals 16,

X represents Can f 1 and m equals 6,

X represents canine albumin and m equals 23 and

X is an example of RhCE and m is equal to 10.

19I. Other compounds of formula 1m.

By following the procedures described in Example 19G and replacing compounds of formula 1109 with, for example, those obtained in Example 19H, the following corresponding compounds of formula 1m were prepared:

F1m-abciximab-m ₁₀ -n ₈₀ -p ₃ 0-q4-CMP-2NHAc,

F1m-adalimumab-m ₁ 1 -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-agalsidase-alpha-m ₁ 4-n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-agalsidase-beta-m ₁ 4-n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-aldesleukin-m ₆ -n ₈₀ -p ₃₀ -q4-SMP-2NHAc,

F1m-aglucosidase-alpha-m ₁₃ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-factor VIII-m ₁₀₀ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m factor IX-m ₁₈ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-L-asparaginase-m ₅ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-laronidase-m ₇ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-octreotide-m ₁ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-phenylalanine ammonia lyase-m ₁₂ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-rasburicase-m ₁₂ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-insulin-m ₂ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-GAD-65-m ₈ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-IGRP-m ₇ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-MBP-m ₆ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-MOG-m ₅ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-PLP-m ₈ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-MBP13-32-mi -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-MBP83-99-m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-MBP111-129-m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-MBP146-170-m ₂ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-MOG1-20 -m1 -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1 m-MOG3 5-55 -m ₂ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-PLP 139-154-m ₃ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-MART 1 -m4-n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-tyrosinase-m ₈ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-PMEL-m ₅ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-aquaporin-4-m4-n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-S-arrestin-m ₁₂ -n ₈₀ -p ₃₀ -q4-CMP-2NHAc,

F1m-IRBP-m ₂₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

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F1m-conarchin-m ₂₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

Flm-alpha-gliadin 33-mer native-t1 -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

Flm-alpha-gliadin 33-mer deamidated-t1 -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-alpha-gliadin-m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-omega-gliadin-m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-Fel d 1-m ₄ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

Flm-feline albumin-m ₁₆ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

F1m-Can f 1-m ₆ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

Flm-canine albumin-m ₂₃ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc and

F1m-RhCE-m ₁₀ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc.

19J. Formula 1107, where p is 30, q is 8, R ³ is OH, R ⁴ is OH and R ⁸ is CMP.

Proceeding according to the procedures described in Example 19A, and replacing N-acetyl-D-galactosamine with galactose, and following the procedures described in Example 19E, except using the azide-modified uRAFT of formula 1106, where q is 8, gave compound of formula 1107, where where p is 30, q is 8, R ³ is OH, R ⁴ is OH and R ⁸ is CMP.

19K. Formula 1109, where n is 62 and where X' and m are the same as in example 19H.

By following the procedures described in Example 19F, replacing OVA with the compounds described in Example 19H, and using the compound of formula 1108, where n is 62, the corresponding compounds of formula 1109, where n is 62, were prepared.

19L. Other compounds of formula 1m.

By following the procedures described in Example 19G and replacing the compound of formula 1107 with the compounds obtained in Example 19J, and replacing the compound of formula 1109 with the compounds obtained in Example 19K, the corresponding compounds of formula 1m were obtained:

F1m-abciximab-m ₁₀ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-adalimumab-m ₁ 1 -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1 m-agalsidase-alpha-m ₁₄ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-agalsidase-beta-m ₁₄ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-aldesleukin-m ₆ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-aglucosidase-alpha-m ₁₃ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-factor VIII-m ₁₀₀ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m factor IX-m ₁₈ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-L-asparaginase-m ₅ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-laronidase-m ₇ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-octreotide-m ₁ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-phenylalanine ammonia lyase-m ₁₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-rasburicase-m ₁₂ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-insulin-m ₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-GAD-65-m ₈ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-IGRP-m ₇ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MBP-m ₆ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MOG-m ₅ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-PLP-m ₈ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MBP13-32-m ₁ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MBP83-99-m ₁ -n ₆₀ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MBP111-129-m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MBP146-170-m ₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MOG1-20-m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MOG35-55-m ₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-PLP 139-154-m ₃ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-MART 1 -m ₄ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-tyrosinase-m ₈ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-PMEL-m ₅ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-aquaporin-4-m ₄ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-S-arrestin-m ₁₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-IRBP-m ₂₁ -n6 ₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-conarchin-m ₂₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-alpha-gliadin 33-mer native-m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-alpha-gliadin 33-mer deamidated-m ₁ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-alpha-gliadin-m ₁ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH,

F1m-omega-gliadin-m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

- 61 044172

F1m-Fel d 1-m ₄ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

Flm-feline albumin-m ₁₆ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

F1m-Can f 1-m ₆ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

Flm-canine albumin-m ₂₃ -n _¢2 -p ₃₀ -q ₈ -CMP-2OH, and

F1m-RhCE-m ₁₀ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH.

Example 20. F1n-insulin-m ₂ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc.

20A. Formula 1202, where X' represents insulin, m equals 2, and n equals 1.

Recombinant human insulin (5 mg) was added to 100 μl of DMF containing 10 μl of triethylamine and mixed until the insulin became soluble. To this solution was added 10 mg (0.0161 mmol) of the linker precursor of formula 1201, where n is 1, and the reaction mixture was allowed to stir. After 1 hour, 1.3 ml of tert-butyl methyl ether was added to isolate the corresponding product of formula 1202, which precipitated. Residual DMF and tert-butyl methyl ether were removed under reduced pressure. Characterization by liquid chromatography-mass spectroscopy and polyacrylamide gel electrophoresis confirmed the identity of the product. The modified insulin product of formula 1202 was used without further purification.

20V. Formula 1n, where X' is insulin, m is 2, n is 1, p is 30, q is 4 and R ⁸ is CMP.

The product of formula 1202 obtained in example 20A was resuspended in 100 μl of DMF. The polymer product of Formula 1107 obtained in Example 19E (10 mg) was added and the reaction mixture was left stirring for 1 hour. After 1 hour, the reaction products were precipitated by adding dichloromethane (1.3 ml). The product was filtered and the residual solvent was removed under reduced pressure. The crude product was then resuspended in 500 μl of PBS and low molecular weight components were removed by centrifugal size exclusion chromatography to obtain the corresponding isomeric product of formula 1n. Characterization by liquid chromatography, mass spectroscopy, and polyacrylamide gel electrophoresis confirmed the identity of the product.

The modified insulin product of formula 1202 was used without further purification. 20C. Other compounds of formula 1202.

Proceeding according to the procedures described in Example 19F and replacing OVA and insulin with the following: abciximab, adalimumab, agalsidase-alpha, agalsidase-beta, aldesleukin, alglucosidase-alpha, factor VIII, factor IX, L-asparaginase, laronidase, octreotide, phenylalanine- ammonia lyase, rasburicase,

GAD-65 (SEQ ID NO:6),

IGRP (SEQ ID NO: 7)

MBP (SEQ ID NO:8),

MOG (SEQ ID NO:9),

PLP (SEQ ID NO:10),

MBP13-32 (SEQ ID NO:11),

MBP83-99 (SEQ ID NO:12), MBP111-129 (SEQ ID NO:13), MBP146-170 (SEQ ID NO:14), MOG1-20 (SEQ ID NO:15), MOG35-55 (SEQ ID NO:16), PLP139-154 (SEQ ID NO:17), MARTI (SEQ ID NO:18), Tyrosinase (SEQ ID NO:19), PMEL (SEQ ID NO:20), Aquaporin-4 (SEQ ID NO :21), S-arrestin (SEQ ID NO:22), IRBP (SEQ ID NO:23), conarchin (UNIPrOt Q6PSU6), alpha-gliadin 33-mer native (SEQ ID NO:24),

- 62 044172 alpha-gliadin 33-mer deamidated (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26), omega-gliadin (SEQ ID NO:27),

Fel d 1A (UNIPROT P30438), feline albumin (UNIPROT P49064),

Can f 1 (UNIPROT O18873), canine albumin (UNIPROT P49822) and RhCE example (UNIPROT P18577) prepared the following corresponding compounds of formula 1202, where n is 1:

X represents abciximab and m equals 10,

X represents adalimumab and m equals 11,

X is agalsidase-alpha and m is 14,

X is agalsidase beta and m is 14,

X represents aldesleukin and m equals 6,

X represents alglucosidase-alpha and m is 13,

X represents factor VIII and m equals 100,

X represents factor IX and m equals 18,

X represents L-asparaginase and m is 5,

X represents laronidase and m is equal to 7.

X represents octreotide and m equals 1,

X is phenylalanine ammonia lyase and m is 12,

X represents rasburicase and m equals 12,

X is GAD-65 (SEQ ID NO:6) and m is 8,

X is IGRP (SEQ ID NO:7) and m is 7,

X is MBP (SEQ ID NO:8) and m is 6,

X is MOG (SEQ ID NO:9) and m is 5,

X is PLP (SEQ ID NO:10) and m is 8,

X is MBP13-32 (SEQ ID NO:11) and m is 1.

X is MBP83-99 (SEQ ID NO: 12) and m is 1.

X is MBP111-129 (SEQ ID NO: 13) and m is 1.

X is MBP146-170 (SEQ ID NO: 14) and m is 2.

X is MOG1-20 (SEQ ID NO: 15) and m is 1.

X is MOG35-55 (SEQ ID NO: 16) and m is 2.

X is PLP139-154 (SEQ ID NO:17) and m is 3.

X is MART1 (SEQ ID NO:18) and m is 4.

X is tyrosinase (SEQ ID NO:19) and m is 8,

X is PMEL (SEQ ID NO:20) and m is 5,

X is aquaporin-4 (SEQ ID NO:21) and m is 4.

X is S-arrestin (SEQ ID NO:22) and m is 12,

X is IRBP (SEQ ID NO:23) and m is 21,

X represents conarchine and m equals 21,

X is alpha-gliadin 33-mer native (SEQ ID NO:24) and m is 1.

X is alpha-gliadin 33-mer deamidated (SEQ ID NO:25) and m is 1.

X is alpha-gliadin (SEQ ID NO:26) and m is 1.

X is omega-gliadin (SEQ ID NO:27) and m is 1.

X represents Fel d 1 and m equals 4,

X represents feline albumin and m equals 16,

X represents Can f 1 and m equals 6,

X represents canine albumin and m equals 23 and

X is an example of RhCE and m is equal to 10.

20D. Other compounds of formula 1n.

By following the procedures described in Example B and replacing compounds of formula 1202 with, for example, those obtained in Example 20C, the following corresponding compounds of formula 1m were prepared:

F1n-abciximab-m ₁₀ -n ₁ -p ₃₀ -q4-CMP-2NHAc,

F1n-adalimumab-m ₁ 1 -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-agalsidase-alpha-m ₁ 4-n ₁ -p ₃₀ -q4-CMP-2NHAc,

F1n-agalsidase-beta-m ₁ 4-n ₁ -p ₃₀ -q4-CMP-2NHAc,

F1n-aldesleukin-m ₆ -n ₁ -p ₃₀ -q4-CMP-2NHAc,

F1n-aglucosidase-alpha-m ₁₃ -n ₁ -p ₃₀ -q4-CMP-2NHAc,

F1n factor VIII-m ₁₀₀ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

- 63 044172

Fln factor IX-m ₁₈ -n1-p ₃₀ -q ₄ -CMP-2NHAc,

F1n-L-asparaginase-m ₅ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-laronidase-m ₇ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-OKTpeoTug-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-phenylalanine ammonia lyase-m ₁₂ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-pac6ypuKa3a-m ₁₂ -n1-p ₃₀ -q ₄ -CMP-2NHAc,

F1n-GAD-65-m ₈ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-IGRP-m ₇ -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MBP-m ₆ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MOG-m ₅ -n1-p ₃₀ -q ₄ -CMP-2NHAc,

F1n-PLP-m8-n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MBP 13-32-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MBP83-99-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MBP 111-129-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MBP 146-170-m ₂ -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MOG 1 -20-m1 -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MOG35-55-m ₂ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-PLP 139-154-m ₃ -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-MART 1 -m ₄ -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-Tupo3UHa3a-m ₈ -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-PMEL-m ₅ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-aKeanopuH-4-m ₄ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-S-appecTUH-m ₁₂ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-IRBP-m ₂₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1 p-conarchin-t ₂ 1 -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-alpha-gliadin 33-mer anti-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-alpha-gliadin 33-mer deamidated-m ₁ -n1 -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-alpha-gliadin-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-omega-gliadin-m ₁ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-Fel d 1-m ₄ -n1-p ₃₀ -q ₄ -CMP-2NHAc,

F1n-cat albumin-m ₁₆ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-Can f 1-m ₆ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

F1n-canine albumin-m ₂₃ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc, and

F1 n-RhCE-m1 ₀ -n1 -p ₃₀ -q ₄ -CMP-2NHAc.

20E. Formula 1202, where n is 33 and where X' and m are the same as in Example 20C.

By following the procedures described in Example 19F, replacing insulin with the compounds described in Example 20C, and using the compound of formula 1201, where n is 33, the corresponding compounds of formula 1202, where n is 33, were prepared.

20F. Other compounds of formula 1n.

By following the procedures described in Example 20B and replacing the compound of formula 1107 with the compounds obtained in Example 19J, and replacing the compound of formula 1202 with the compounds obtained in Example 20E, the following corresponding compounds of formula 1n were obtained:

F1n-abciximab-m ₁₀ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1 n-adalimymab-m ₁ 1 -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-agalsidase-alpha-m ₁₄ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-agalsidase-beta-m ₁₄ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-aldesleukin-m ₆ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-aglucosidase-alpha-m ₁₃ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n factor VIII-m ₁₀₀ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n factor IX-m ₁₈ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-L-asparaginase-m ₅ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-laronidase-m ₇ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-octreotide-m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-phenylalanine ammonia lyase-m ₁₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1 n-rasbyricase-m ₁₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-GAD-65-m8-n33-P30-q8-CMP-2OH,

F1n-IGRP-m7-n33-p30-q8-CMP-2OH,

F1n-MBP-m6-n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-MOG-m5-n33-p30-q8-CMP-2OH,

F1n-PLP-m8-n33-p30-q8-CMP-2OH,

F1n-MBP 13-32-m1-n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-MBP83-99-m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

- 64 044172

F1n-MBP 111- 129-m1 -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-MBP146- 170-m ₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-MOG 1 -20-m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-MOG35-55-m ₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-PLP 139-154-m ₃ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-MART 1 -m ₄ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-Tupo3UHa3a-m ₈ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1 n-PMEL-m ₅ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1 p-aquaporin-4 -t4-p ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-S-appecTUH-m ₁₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-IRBP-m ₂₁ -n ₃ 3-p3o-q ₈ -CMP-2OH,

F1 p-cocharin-t ₂ 1 -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-alpha-gliadin 33-mer native-m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-alpha-gliadin 33-mer deamidated -m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-alpha-gliadin-m ₁ -n ₃₃ -p30-q ₈ -CMP-2OH,

F1n-omega-gliadin-m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-Fel d 1-m ₄ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-feline albumin-m ₁₆ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-Can f 1-m ₆ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

F1n-canine albumin-m ₂₃ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH and

F1n-RhCE-m ₁₀ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH.

Example 21. Clearance of OVA-specific antibodies from the bloodstream.

To induce the production of OVA-specific antibodies for subsequent depletion, C57BL/6 mice were administered iv up to five doses of 10 μg of OVA until the plasma anti-OVA IgG titer, determined by ELISA, reached 3 (Fig. 9A). Plasma samples were obtained by centrifuging blood collected in EDTA-coated tubes at 2000 x g for 10 min at room temperature and stored at -20°C until analysis. The titer was determined as log ₁₀ of the maximum fold dilution of plasma with detectable anti-OVA IgG. In this case, plasma samples were analyzed at 10-, 50-, 100-, 500-, 1000-, 5000-, 10000- and 50000-fold dilutions to obtain potential titer values of 0, 1, 1.7, 2, 2.7 , 3, 3.7, 4 and 4.7. Mice with titers below 3 after five doses of 10 μg OVA were given two additional doses of 10 μg OVA adjuvanted with 10 μg CpG-B to produce anti-OVA IgG and therefore the amount of circulating anti-OVA IgG available for depletion ( Fig. 9B).

To evaluate the effectiveness of OVA targeting hepatocyte ASGPR on the clearance of anti-OVA antibodies, mice with circulating anti-OVA IgG were administered i.v. saline solution or saline solution containing molar equivalents of DOM and OVA, DOM-OVA or OVA-DOM. Specifically, mice were treated with molar equivalents of 10, 50, 100, or 200 μg of OVA as indicated in FIG. 9. At time points t = -1 day, +3 hours, +1 day and +7 days relative to the time of injection treatment, blood samples were collected to assess the amount of anti-OVA IgG in the bloodstream by analyzing plasma by ELISA. Clearance of anti-OVA IgG from the bloodstream is expected to reduce the amount of anti-OVA IgG in plasma and therefore reduce the plasma anti-OVA IgG titer determined by ELISA. In mice immunized with OVA alone and treated with DOM-OVA or OVA-DOM, anti-OVA IgG titers were reduced by 3 or 2, respectively, representing a 1000- or 100-fold reduction in circulating anti-OVA IgG. up to 50-fold more effective than control treatments using saline or DOM and OVA (Fig. 9A). In mice immunized with CpG-B-adjuvanted OVA, clearance of circulating anti-OVA IgG was not observed (Fig. 9B).

Example 22. NOD mice.

Nonobese diabetic (NOD) mice are prone to spontaneous onset of autoimmune diabetes mellitus, which results from an autoimmune response to various pancreatic autoantigens. Diabetes develops in NOD mice as a result of insulitis, characterized by the infiltration of various leukocytes into the pancreatic islets.

To evaluate the effectiveness of diabetes treatment, NOD mice were divided into control or test groups starting at 6 weeks of age and treated, respectively, with weekly intravenous injections of the test formulation (10 μg) or an inactive control such as saline. Injections were continued for 18 consecutive weeks.

Blood glucose concentrations in mice were measured weekly. Mice that maintained blood glucose concentrations below 300 mg/mL during the experiment were considered not to have diabetes. Additionally, mouse pancreases were collected at the end of the study and T cell infiltration into the pancreas was determined using immunohistochemistry as an assessment of insulitis. Tolerance induction was assessed by the depletion of autoantigen-specific CD8 T cells compared with saline-treated mice that developed

Claims (22)

Diabetes occurred. The presence of autoantigen-specific CD8 T cells was determined using the assay ELISpot. When tested as described above, compositions of formulas 1 and 2 wherein X is insulin, such as F1m-insulin-m ₂ -n ₁ -p ₃ oq ₄ -2NAcGAL, demonstrated effectiveness in the treatment of diabetes mellitus. While the present disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications can be made to adapt a particular situation, material, mixture of substances, method, process step or steps to the purpose, spirit and scope of the present disclosure. All such modifications are provided within the scope of the appended claims. All patents and publications cited above are hereby incorporated by reference. CLAIM 1. Compound of formula 1 for inducing immune tolerance x+yz] _m Formula 1 where m is equal to an integer from 1 to 100; X represents an antigen that induces an unwanted immune response; wherein X represents a food antigen or self-antigen; Y is a linker component, where Y contains a polymeric linker coupled to the antigen via a disulfide bond or disulfanyl ethyl ether; wherein each of the disulfide bond or disulfanyl ethyl ether is capable of being cleaved upon administration of the compound to a subject and releasing the antigen from the polymer linker; wherein the polymer linker contains a 1-cyano-1-methylpropyl group and methacrylic units containing an ethyl acetamide functional group; And Z is a liver targeting component, wherein Z contains galactose, galactosamine or N-acetylgalactosamine, and wherein the liver targeting component is coupled to a polymer linker via an ethyl acetamide functionality. 2. A compound according to claim 1, containing Z conjugated to Y at its C1, C2 or C6. 3. A compound according to claim 1 or 2, where Z represents a beta anomer. 4. A compound according to any one of claims 1 to 3, wherein the antigen is a food antigen associated with celiac disease and contains one or more of SEQ ID NO:25, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO :27, or wherein the antigen is a multiple sclerosis associated self antigen and contains one or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:12 or SEQ ID NO:13. 5. The compound of claim 4, wherein the antigen is a food antigen associated with celiac disease and comprises SEQ ID NO:25, wherein the liver targeting component comprises N-acetylgalactosamine. 6. The compound of claim 4, wherein the antigen is a multiple sclerosis associated self antigen and comprises one or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:12 or SEQ ID NO:13 and where the component for targeted delivery to the liver contains N-acetylgalactosamine. 7. A compound according to any one of claims 1 to 3, where X represents one or more of conarchin (Ara h 1), allergen II (Ara h 2), peanut agglutinin, conglutin (Ara h 6), 31 kDa main allergen/ homolog of disease resistance protein (Mal d 2), lipid transfer protein precursor (Mal d 3), main allergen Mal d 1.03D (Mal d 1), α-lactalbumin (ALA), lactotransferrin, actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), quivellin (Act d 5), ovomucoid, ovalbumin, ovotransferrin and lysozyme, livetin, apovitillin, vosvetin, 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transport protein (Sin a 3), profilin (Sin a 4), profilin (Api g 4), high molecular weight glycoprotein (Api g 5), allergen Pen a 1 (Pen a 1), allergen Pen m 2 (Pen m 2), isoforms of fast tropomyosin, high molecular weight glutenin, low molecular weight glutenin, alpha, gamma and omega gliadin, hordein, secalin, avenin, the main strawberry allergen Fra a 1-E (Fra a 1) or profilin (Mus xp 1) . 8. A compound according to any one of claims 1 to 3, wherein X is one or more of high molecular weight glutenin, low molecular weight glutenin, alpha, gamma and omega gliadin, hordein, secalin or avenin. - 66 044172 9. A compound according to any one of claims 1 to 3, where X represents gliadin. 10. A compound according to any one of claims 1 to 3, where X represents one or more of insulin, proinsulin, preproinsulin, glutamate decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), Insulinoma-associated protein 2β (IA-2e), ICA69, ICA12 (SOX-13), carboxypeptidase H, imogen 38, GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E, peripherin, hepatocarcinoma-associated glucose transporter 2- intestinal-pancreatic/pancreatic protein, S100e, glial fibrillary acidic protein, regenerating gene II, pancreatic duodenal homeobox 1, myotonic dystrophy protein kinase, islet-specific glucose 6-phosphatase catalytic subunit-related protein or SST receptors 1-5 coupled G protein. 11. A compound according to any one of claims 1 to 3, wherein X is one or more of myelin basic protein, oligodendrocyte myelin glycoprotein or proteolipid protein. 12. A compound according to any one of claims 1 to 3, wherein X represents one or more of insulin, proinsulin or preproinsulin. 13. A compound according to any one of claims 1 to 3, wherein X is a polypeptide containing the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:25. 14. A compound according to any one of claims 1 to 3, wherein X is a polypeptide corresponding to SEQ ID NO:5. 15. The compound of claim 14, wherein X is a polypeptide containing the amino acid sequence corresponding to SEQ ID NO:5 and IA-2. 16. A compound according to any one of claims 1-3, wherein X is a polypeptide corresponding to at least one of SEQ ID NO:8-17. 17. A compound according to any one of claims 1 to 3, where X is a self-antigen associated with multiple sclerosis, type 1 diabetes mellitus, pemphigus vulgaris or myasthenia gravis. 18. Composition for inducing immune tolerance, containing a compound according to any one of claims 1 to 17. 19. Use of a compound according to any one of claims 1 to 17 for inducing immune tolerance to X. 20. A method of treating an unwanted immune response against an antigen, comprising administering to a mammal in need of such treatment an effective amount of a compound according to any one of claims 1 to 17. 21. Application of compound with formula for the treatment of celiac disease, where m is an integer from 1 to 100; X contains an antigen that induces an unwanted immune response and is selected from one or more of high molecular weight glutenin, low molecular weight glutenin, alpha, gamma and omegagliadin, hordein, secalin, avenin; Y contains a polymer linker conjugated to the antigen via a disulfide bond or disulfanylethyl ether; wherein each of the disulfide bond or disulfanyl ethyl ether is capable of being cleaved upon administration of the compound to a subject and releasing the antigen from the polymer linker; wherein the polymer linker contains a 1-cyano-1-methylpropyl group and methacrylic units containing an ethyl acetamide functional group; And Z contains a component for targeted delivery to the liver, where Z contains N-acetylgalactosamine, galactose, galactosamine; and wherein the liver targeting component is coupled to a polymer linker via an ethyl acetamide functionality. 22. Application of a compound with formula for the treatment of multiple sclerosis, where m is an integer from 1 to 100; X contains an antigen that induces an unwanted immune response and is selected from one or more of myelin basic protein, oligodendrocyte myelin glycoprotein and proteolipid protein; Y contains a polymer linker conjugated to the antigen via a disulfide bond or disulfanylethyl ether; wherein each of the disulfide bond or disulfanyl ethyl ether is capable of being cleaved upon administration of the compound to a subject and releasing the antigen from the polymer linker; wherein the polymer linker contains a 1-cyano-1-methylpropyl group and methacrylic units containing an ethyl acetamide functional group; And -