Glycotargeting therapeutics

Abstract

To provide pharmaceutically acceptable compositions that are useful in the treatment of transplant rejection, autoimmune disease, food allergy, and immune response against therapeutic agents.SOLUTION: The present invention provides a composition comprising a compound of Formula 1, where m is an integer from about 1 to 100; X comprises an antigen against which a patient develops an unwanted immune response, or a tolerogenic portion thereof; or X comprises an antibody, antibody fragment or ligand that specifically binds a circulating protein or peptide or antibody; Y comprises a linker moiety; and Z comprises a liver-targeting moiety.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to co-pending U.S. Provisional Application No. 61 / 942,942, filed February 21, 2014, entitled “GLYCOTARGETING THRAPEUTICS,” which is incorporated herein by reference.

[0002] Field of the Disclosure The field of the disclosure relates to pharma- ceutically acceptable compositions that are useful in the treatment of transplant rejection, autoimmune diseases, food allergies, and immune responses to therapeutic agents. [Background technology]

[0003] Background of the Disclosure US Application No. 2012 / 0039989, US Application No. 2012 / 0178139 and WO2013 / 121296 describe targeting of antigens to red blood cells, exploiting the role of red blood cells in antigen presentation for tolerization. Despite the positive results that have occurred so far with this approach, there remains interest in possible alternative approaches. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Disclosure An embodiment of the present disclosure provides a composition comprising a compound of formula 1: [ka] where: m is an integer from about 1 to 100; X comprises an antigen, or a tolerogenic portion thereof, to which the patient develops an unwanted immune response; or X comprises an antibody, antibody fragment or ligand that specifically binds to a circulating protein or peptide or antibody that is causally involved in transplant rejection, immune response to a therapeutic agent, autoimmune disease, hypersensitivity and / or allergy; Y comprises a linker moiety; and Z comprises a liver targeting moiety.

[0005] Z can also include, for example, a galactose, galactosamine, or N-acetylgalactosamine conjugated to Y at its C1, C2, or C6.

[0006] Y may be selected from an N-hydroxysuccinimidyl linker, a maleimide linker, a vinylsulfone linker, a pyridyldithiol-poly(ethylene glycol) linker, a pyridyldithiol linker, an n-nitrophenyl carbonate linker, an NHS-ester linker, and a nitrophenoxypoly(ethylene glycol) ester linker.

[0007] Y can further include an antibody, antibody fragment, peptide or other ligand that specifically binds to X; a disulfanylethyl ester; a structure represented by one of formulas Ya through Yp: [ka] [ka] [ka] and [ka] or having a moiety represented by the formula Y'-CMP: [ka] where: the left parenthesis "(" indicates a bond between X and Y; the right or bottom parenthesis and ")" indicates a bond between Y and Z; n is an integer from about 1 to 100; p is an integer from about 2 to 150; q is an integer from about 1 to 44; 8 is -CH 2 -or-CH 2 -CH 2 -C(CH 3 )(CN)-;R 9 is a direct bond or -CH 2 -CH 2 -NH-C(O)-; and Y' represents the remaining portion of Y.

[0008] In another embodiment of the above, n is about 40 to 80, p is about 10 to 100, q is about 3 to 20, and R 8 is -CH 2 -CH 2 -C(CH 3 )(CN)-; and R 9 Ga-CH 2 -CH 2 When Z is -NH-C(O)-, it is a galactose or N-acetylgalactosamine conjugated at its C1.

[0009] In yet another embodiment of the above, Y comprises formula Ya, formula Yb, formula Yh, formula Yi, formula Yk, formula Ym or formula Yn, in particular formula Ya, formula Yb, formula Ym or formula Yn.

[0010] X may further comprise: an exogenous transplant antigen to which the transplant patient develops an unwanted immune response; an exogenous food, animal, plant or environmental antigen to which the patient develops an unwanted immune response; an exogenous therapeutic agent to which the patient develops an unwanted immune response; or a synthetic self-antigen to an endogenous form to which the patient develops an unwanted immune response, or a tolerogenic portion thereof.

[0011] The present disclosure also relates to a method of treatment for an undesired immune response to 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 described above. In such a method, the composition can be administered for clearance of circulating proteins or peptides or antibodies that specifically bind to antigen moiety X, the circulating proteins or peptides or antibodies causally involved in transplant rejection, immune response to therapeutic agents, autoimmune disease, hypersensitivity and / or allergy. The composition can be administered in an amount effective to reduce the concentration of antibodies causally involved in transplant rejection, immune response to therapeutic agents, autoimmune disease, hypersensitivity and / or allergy by at least 50% w / w in the blood of the patient, measured at a time between about 12 and about 48 hours after administration. The composition can be administered for tolerization of the patient to antigen moiety X.

[0012] Yet another aspect of the disclosure provides a composition comprising a compound of formula 2: [ka] wherein: if m'+m" is equal to 1 and Z is DOM 26h-196-61, then m' is zero or an integer from about 1 to 10, m" is zero or an integer from about 1 to 10, and the sum of m'+m" is an integer from about 1 to 10, but is at least 1, with the proviso that if X is not an interferon, ribavirin, nexavar / sorafenib, erbitux / cetuximab, avastatin / bevacizumab, or herceptin / trastuzumab; each X is a foreign or autoantigen, or a tolerogenic portion thereof, to which the patient develops an unwanted immune response; each Y is a linker moiety or a direct bond, or an antibody, antibody fragment, peptide, or other ligand that specifically binds to X; and Z is a liver targeting moiety.

[0013] In the above embodiment involving formula 2, Z may comprise an ASGPR-targeting antibody, an ASGPR-targeting antibody fragment, an ASGPR-targeting peptide, an ASGPR-targeting scFv, or another ASGPR ligand. Y may be a linker with an immune proteasome cleavage site. X may comprise a group of relevant antigens to which a patient develops an undesirable immune response, or a group of immune tolerogenic fragments thereof. For example, X may be selected from the group including: two or more of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 GAD-67, glucose-6-phosphatase 2, insulinoma-associated protein 2, insulinoma-associated protein 2β, ICA69, ICA12, carboxypeptidase H, Imogen38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter type 2, hepatocellular carcinoma-enteric-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regenerate gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST G protein-coupled receptor 1-5; two or more of myelin basic protein, myelin oligodendrocyte glycoprotein, myelin proteolipid protein, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and two or more of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0014] The present disclosure also relates to a method of treatment for an undesired immune response to an antigen by administering to a mammal in need of such treatment an effective amount of a composition comprising a compound of formula 2 above. In such a method, the composition can be administered for clearance of circulating proteins or peptides or antibodies that specifically bind to antigen moiety X, the circulating proteins or peptides or antibodies causally involved in transplant rejection, immune response to therapeutic agents, autoimmune disease, hypersensitivity and / or allergy. The composition can be administered in an amount effective to reduce the concentration of antibodies causally involved in transplant rejection, immune response to therapeutic agents, autoimmune disease, hypersensitivity and / or allergy by at least 50% w / w in the blood of the patient, measured at a time between about 12 and about 48 hours after administration. The composition can be administered for tolerization of the patient to antigen moiety X. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 is a series of graphs showing that galactose conjugation [F1aA-PE-m4-n80 (Gal-PE)] preferentially targets OVA to cells in the liver, including: (A.) sinusoidal endothelial cells (LSECs), (B.) Kupffer cells (KCs), (C.) hepatocytes, and (D.) other antigen presenting cells (APCs).

[0016] [Diagram 2] FIG. 2 is a graph showing the proliferation of OTI CD8+ T cells in mice treated with F1aA-OVA-m4-n80 (Gal-OVA), OVA or saline (ie, naive).

[0017] [Diagram 3] FIG. 3 is a series of graphs showing the percentage of OT-I CD8+ T cells presenting surface markers (A.) PD-1+ and (B.) Annexin V+ in the generation of proliferating T cells treated with saline, OVA, or F1aA-OVA-m4-n80 (GAL-OVA).

[0018] [Figure 4] FIG. 4 is a graph showing that galactose conjugation [F1aA-OVA-m4-n80 (Gal-OVA)] reduces the immunogenicity of OVA as determined by OVA-specific antibody titers (shown as Ab titers log-1).

[0019] [Diagram 5] FIG. 5 shows that F1aA-OVA-m4-n80 (Gal-OVA) can deplete OVA-specific antibodies from serum.

[0020] [Figure 6] FIG. 6 shows that F1aA-OVA-m4-n80 (mGal-OVA), F1b-OVA-m1-n44-p34 (pGal-OVA), and N′-DOM-Gly3Ser-OVA-Gly3Ser-6xHis-C′ (Dom-OVA) can reduce OVA-specific immune responses in draining lymph nodes after intradermal challenge with OVA and adjuvant LPS.

[0021] [Figure 7-A] Figure 7 shows the characterization of F1aA-OVA-m4-n80 and F1b-OVA-m1-n44-p34. (A.) Size-exclusion HPLC traces of F1aA-OVA-m4-n80 (magenta), F1b-OVA-m1-n44-p34 (blue) and unconjugated OVA (black). The shift to the left represents an increase in molecular weight. (B.) Polyacrylamide gel showing the increased molecular weight after OVA conjugation: (1.) unconjugated OVA, (2.) F1aA-OVA-m4-n80 and (3.) F1b-OVA-m1-n44-p34. [Figure 7-B] Same as above

[0022] [Figure 8]FIG. 8 is a graph showing the normalized amounts of N-OVA-Gly3Ser-6xHis-C (OVA), N-DOM-Gly3Ser-OVA-Gly3Ser-6xHis-C (DOM-OVA), or N-OVA-Gly3Ser-DOM-Gly3Ser-6xHis-C (OVA DOM) in the circulation of mice after injection as an intravenous bolus.

[0023] [Figure 9(a)] Figure 9 is a series of graphs showing titers of anti-OVA IgG antibodies in the circulation of individual mice after treatment with saline, DOM and OVA, DOM-OVA, or OVA-DOM. Anti-OVA IgG production was induced by intravenous injection of OVA alone (a) or OVA and CpG-B (b). Treatment times are indicated by vertical dashed lines. Titers are calculated as the log10 of the highest fold dilution of plasma with detectable anti-OVA IgG. [Figure 9(b)] Same as above

[0024] Detailed Description Two known 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 multiple cell types (e.g., 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 for generating an immune response. Although some cells in the liver are known to be capable of presenting antigens, it is more known that the liver is involved in immune tolerance induction. The liver is understood to be a tolerogenic organ. For example, a lower incidence of rejection has been reported in cases of multiple organ transplants when one of the transplanted organs is the liver. The case of LSECs is newer than the literature; consequently, their role in immune tolerance induction and / or attenuation of inflammatory immune responses has not yet been widely recognized or well understood. However, it is becoming clear that they may also play an important role in the induction of antigen-specific tolerance.

[0025] One of the unique features of the erythrocyte surface is its glycosylation, i.e., the presence of a significant number of glycosylated proteins. Indeed, glycophorins (e.g., glycophorin A) have been exploited as targets for erythrocyte binding. Glycophorins are proteins with many covalently attached glycans and a terminal sialic acid. As erythrocytes age and become ripe for clearance, the terminal sialic acid of the glycophorin tends to be lost, leaving N-acetylgalactosamine at the free end. N-acetylgalactosamine is the ligand selectively received by hepatocyte-associated ASGPR, leading to the binding of N-acetylgalactosamine-containing substances by hepatocytes and their subsequent uptake and processing in the liver.

[0026] Up to now, it has been understood by those skilled in the art that the glycosylation of therapeutic agents in a manner that leads to liver targeting should be avoided due to the first-pass clearance by the liver, which leads to a poor circulation half-life of therapeutic agents.Similarly, some monoclonal antibodies need to be specifically glycosylated with ASN297 for optimal binding to their Fc receptors.Surprisingly, it has now been found that galactosylation can be used in a manner that induces immune tolerance induction.

[0027] The present disclosure provides certain therapeutic compositions targeted for delivery to (and uptake by) the liver, particularly hepatocytes, LSECs, Kupffer cells and / or stellate cells, more particularly hepatocytes and / or LSECs, and even more particularly, certain therapeutic compositions that specifically bind to ASGPR. Liver targeting facilitates two mechanisms of treatment: tolerization and clearance. Tolerization exploits the liver's role in clearing apoptotic cells and processing those proteins recognized by the immune system as "self", as well as in sampling peripheral proteins for immune tolerance. Clearance exploits the liver's role in blood purification by rapidly removing and degrading toxins, polypeptides, etc. Targeting of these compositions to the liver is accomplished by a galactosylation moiety (e.g., conjugated with galactose, galactosamine and N-acetylgalactosamine, particularly C1, C2 or C6), by another liver targeting moiety (e.g., a monoclonal antibody, or fragment or scFv thereof), or by de-sialylation of the polypeptide for which such liver targeting is desired. Galactosylation or other liver targeting moieties can be chemically conjugated or recombinantly fused to the antigen, while desialylation exposes galactose-like moieties on the antigen polypeptide. Such antigens can be endogenous (self-antigens) or exogenous (foreign antigens) and include, but are not limited to: foreign transplant antigens to which the transplant patient develops an unwanted immune response (e.g., transplant rejection), foreign food, animal, plant or environmental antigens to which the patient develops an unwanted immune (e.g., allergic or hypersensitivity) response, therapeutic agents to which the patient develops an unwanted immune response (e.g., hypersensitivity and / or reduced therapeutic activity), self-antigens to which the patient develops an unwanted immune response (e.g., autoimmune disease), or tolerogenic portions thereof (e.g., fragments or epitopes); these compositions are useful for inducing tolerization to antigens.Alternatively, galactosylation or other liver targeting moieties can be conjugated to antibodies, antibody fragments or ligands that specifically bind to circulating proteins or peptides or antibodies that are causally involved in transplant rejection, immune responses to therapeutic agents, autoimmune diseases, and / or allergies (as described above); these compositions are useful for clearing circulating proteins, peptides or antibodies. Thus, the compositions of the present disclosure can be used to treat undesired immune responses, such as transplant rejection, immune responses to therapeutic agents, autoimmune diseases, and / or allergies. Also provided is a pharmaceutical composition that contains a therapeutically effective amount of the compositions of the present disclosure mixed with at least one pharma- ceutically acceptable excipient. In another aspect, the present disclosure provides a method for treating undesired immune responses, such as transplant rejection, responses to therapeutic agents, autoimmune diseases, or allergies.

[0028] definition

[0029] As used herein, 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.

[0030] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] The term "about" when used in connection with a numerical value is meant to encompass numerical values ​​within a range typically having a lower limit, i.e., a value that is, for example, 5-10% less than the indicated numerical value, and an upper limit, i.e., a value that is, for example, 5-10% greater than the indicated numerical value.

[0032] An "antigen" is any substance that serves as a target for a receptor of the adaptive immune response, such as a T cell receptor, a B cell receptor, or an antibody. An antigen may originate from within the body ("self", "auto" or "endogenous"). An antigen may also originate from outside the body ("non-self", "foreign" or "endogenous"), for example, introduced 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 present in allogeneic transplants.

[0033] As used herein, an "antigen-binding molecule" refers to a molecule, particularly a protein such as an immunoglobulin molecule, that contains an antibody variable region that provides specific binding to an epitope. The antibody variable region can be present, for example, in an intact antibody, an antibody fragment, and a recombinant derivative of an antibody or antibody fragment. As used herein, the term "antigen-binding fragment" (or "binding portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to a target sequence. Antigen-binding fragments containing antibody variable regions include, without limitation, "Fv," "Fab," and "F(ab')2" regions, "single domain antibodies (sdAb)," "nanobodies," "single-chain Fv (scFv)" fragments, "tandem scFvs" (VHA-VLA-VHB-VLB), "diabodies," "triabodies" or "tribodies," "single-chain diabodies (scDb)," and "bi-specific T-cell engagers (BiTEs)."

[0034] "Chemical modification" refers to a change in the naturally occurring chemical structure of one or more amino acids of a polypeptide. Such modifications can be at the side chain or at the terminus, e.g., altering the amino or carboxyl terminus. In some embodiments, modifications are useful to create chemical groups that can be conveniently used to link the polypeptide to other materials or to attach to therapeutic agents.

[0035] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The phrase "consisting of" excludes any element, step, or ingredient not specified. The phrase "consisting essentially of" limits the scope to which the subject matter is described of specified materials or steps that do not materially affect its basic and novel characteristics.

[0036] "Conservative changes" can generally be made to an amino acid sequence without altering activity. These changes are referred to as "conservative substitutions" or "mutations"; that is, an amino acid belonging to a group of amino acids having a particular size or characteristics can be substituted for another amino acid. Substitutes for an amino acid sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (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 substantially affect the apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Conservative substitutions also include replacing an optical isomer of a sequence with another optical isomer, specifically replacing a D amino acid with an L amino acid for one or more residues of a sequence. Additionally, amino acids in a sequence may undergo D to L isomeric substitutions. Exemplary conservative substitutions are Arg to Lys and vice versa, which maintain a positive charge; Asp to Glu and vice versa, which maintain a negative charge; Thr to Ser, which maintains a free OH; and free NH 2Conservative substitutions include, but are not limited to, substitutions of Asn with Gln that maintain the NH group. If a requirement for chemical derivatization arises, yet another type of conservative substitution constitutes where an amino acid with the desired chemical reactivity is introduced to provide a reactive site for a chemical conjugation reaction. Such amino acids include, but are not limited to, Cys (inserting a sulfhydryl group), Lys (inserting a primary amine), Asp and Glu (inserting a carboxylic acid group), or unusual non-standard amino acids containing ketone, azide, alkyne, alkene, and tetrazine side chains. Free NH 2 Conservative substitutions or additions of amino acids with or without SH may be particularly advantageous for chemical conjugation with the linker and galactosylation moieties of formula 1. Furthermore, point mutations, deletions, and insertions of the polypeptide sequence or the corresponding nucleic acid sequence may be made, in some cases, 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. The variants that can be used in the present invention may exhibit a total of up to 200 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or up to 200) changes in amino acid sequence (i.e., exchanges, insertions, deletions, N-terminal truncations, and / or C-terminal truncations). The amino acid residues described herein utilize either the single-letter amino acid code or the three-letter abbreviations in accordance with standard polypeptide nomenclature, J. Biol. Chem., (1969), 243, 3552-3559. All amino acid residue sequences are represented herein by formulas with left-right orientation in the conventional direction from amino terminus to carboxy terminus.

[0037] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition of the present disclosure sufficient to effect treatment as defined below when administered to a mammal in need of such treatment. This amount will vary depending on the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the dosing regimen according to the particular composition of the present disclosure selected, the timing of administration, the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

[0038] 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, e.g., by an antibody, a B cell, or a T cell. An epitope is a part or segment of a macromolecule that is capable of binding to an antibody or an antigen-binding fragment thereof. In this context, the term "binding" particularly relates to specific binding. In the context of the present invention, the term "epitope" preferably refers to a segment of a protein or polyprotein that is recognized by the immune system.

[0039] The term galactose is well known in the art and refers to a monosaccharide that exists both in open-chain and cyclic forms, 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 the axial position, while in the beta form, the C1 alcohol group is in the equatorial position. In particular, "galactose" refers to a cyclic six-membered pyranose, more particularly the D-isomer, and even more particularly the alpha-D-form (α-D-galactopyranose). The structure and numbering of galactose are shown below:

[0040] [ka]

[0041] The term "galactosyl moiety" refers to a specific type of liver targeting moiety. Galactosyl moieties include, but are not limited to, galactose, galactosamine and / or N-acetylgalactosamine residues.

[0042] The term "liver targeting moiety" refers to a moiety that has the ability to direct, for example, a polypeptide to the liver. The liver contains different cell types, including, but not limited to, hepatocytes, sinusoidal epithelial cells, Kupffer cells, stellate cells, and / or dendritic cells. Typically, a liver targeting moiety directs a polypeptide to one or more of these cells. On the surface of each hepatocyte, a receptor is present that recognizes and specifically binds to the liver targeting moiety. Liver targeting can be achieved by chemical conjugation of an antigen or ligand to a galactosyl moiety, desialylation of an antigen or ligand to expose potential 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 the antigen or ligand is: desialylated to expose potential galactosyl moieties, conjugated to a galactosyl moiety, or recombinantly fused or chemically conjugated to an ASGPR-binding moiety. Naturally occurring desialylated proteins are not encompassed within the scope of this disclosure.

[0043] The "numeric values" and "ranges" provided for the various substituents are intended to encompass all integers within the stated ranges. For example, when defining n as an integer representing a mixture containing about 1 to 100, particularly about 8 to 90, and more particularly about 40 to 80 ethylene glycol groups, typically including integers specified as n ± about 10% (or 1 to about 25, ±3 for integers smaller), n can be any integer from about 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, The range of the ethylene glycol moiety may be 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 ethylene glycol groups, and the disclosed mixtures should be understood to encompass 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 ethylene glycol groups. The terms "about" and "±10%" or "±3" in combination should be understood to disclose and provide specific support for the equivalent range wherever used.

[0044] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0045] A peptide that specifically binds to a particular target is called a "ligand" for that target.

[0046] "Polypeptide" is a term that refers to a chain of amino acid residues, regardless of post-translational modification (e.g., phosphorylation or glycosylation) and / or complex formation with additional polypeptides and / or synthesis into multi-subunit complexes with nucleic acids and / or carbohydrates, or other molecules. Thus, proteoglycans are also referred to herein as polypeptides. Long polypeptides (having more than about 50 amino acids) are referred to as "proteins". Short polypeptides (having about 50 amino acids or less) are referred to as "peptides". Depending on the size, amino acid composition, and three-dimensional structure, certain polypeptides may be referred to as "antigen-binding molecules", "antibodies", "antibody fragments", or "ligands". Polypeptides can be produced by a number of methods, many of which are well known in the art. For example, a polypeptide can be obtained by extraction (e.g., from isolated cells), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemical synthesis. A polypeptide can be produced, for example, by recombinant techniques and an expression vector encoding the polypeptide that is introduced (e.g., by transformation or transfection) into a host cell for expression of the encoded polypeptide.

[0047] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0048] The term "purified" as used herein with respect to a polypeptide refers to a polypeptide that has been chemically synthesized and thus is substantially uncontaminated by other polypeptides, or that has been separated or isolated from most of the other cellular components with which it is naturally associated (e.g., other cellular proteins, polynucleotides, or cellular components). An example of a purified polypeptide is a polypeptide that is at least 70% by dry weight free from naturally associated proteins and naturally occurring organic molecules. Thus, a preparation of purified polypeptide can be, for example, at least 80%, at least 90%, or at least 99% by dry weight of the polypeptide. The polypeptide can also be engineered to contain a tag sequence (e.g., polyhistidine tag, myc tag, FLAG® tag, or other affinity tag) that facilitates purification or labeling (e.g., incorporation into an affinity matrix, visualization by microscopy). Thus, unless otherwise indicated, a purified composition that includes a polypeptide refers to a purified polypeptide. The term "isolated" indicates that the polypeptides or nucleic acids of the disclosure are not in their natural environment. Thus, an isolated product of the present disclosure may be contained in a culture supernatant, partially concentrated, produced from a heterologous source, cloned into a vector, formulated with a vehicle, or the like.

[0049] The term "sequence identity" is used in relation to polypeptide sequence comparisons. This expression particularly refers to a percentage of sequence identity, e.g., a percentage of sequence identity of 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 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the respective reference polypeptide or respective reference polynucleotide. The subject and reference polypeptides in particular exhibit sequence identity over a 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.

[0050] As commonly used in the biological arts, the term "specific binding" refers to molecules that bind to a target with relatively high affinity compared to non-target tissues and generally involves multiple non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, etc. Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and certain protein-receptor interactions; while high-affinity binding pairs may still fall within the definition of specific binding, such molecules may bind to tissues in addition to their specific targets, occasionally to the extent that such non-target binding is insignificant.

[0051] The term "treatment" or "treating" refers to any treatment of a disease or disorder in a mammal: Preventing or protecting against disease or disability, i.e. preventing clinical symptoms from developing; Inhibiting a disease or disorder, i.e., arresting or suppressing the onset of clinical symptoms; and / or This includes alleviating the disease or disorder, ie, causing regression of clinical symptoms.

[0052] The term "undesirable immune response" refers to a reaction by a subject's immune system that is undesirable in a given situation. A reaction of the immune system is undesirable if such a reaction does not lead to the prevention, reduction, or cure of a disease or disorder, but instead causes, enhances, or exacerbates the disorder or disease. Typically, an immune system reaction causes, enhances, or exacerbates a disease if it is directed against an inappropriate target. Exemplary undesirable immune responses include, but are not limited to, transplant rejection, immune responses to therapeutic agents, autoimmune diseases, and allergic or hypersensitivity disorders.

[0053] The term "variant" is understood as a protein that differs from the protein from which it is derived by one or more changes in its length, sequence, or structure. The polypeptide from which a 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" exhibit one or more differences in their sequence or structure compared to the parent molecule. Modified molecules also include, but are not limited to, modified nucleic acids, such as, for example, post-translationally modified proteins (e.g., glycosylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and methylated DNA. Mixtures of different molecules, such as RNA-DNA hybrids, are also encompassed by the term "variant", but are not limited thereto. Naturally occurring and artificially constructed variants are understood to be encompassed by the term "variant" as used herein. Moreover, variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule, or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent molecule, i.e., is functionally active. A variant may be characterized by a certain degree of sequence identity to the parent polypeptide from which it is derived. More precisely, a protein variant in the context of the present disclosure may exhibit at least 80% sequence identity to its parent polypeptide. Preferably, the sequence identity of a protein variant spans a stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] composition

[0055] One aspect of the disclosure relates to compositions, pharmaceutical formulations, and methods of treatment utilizing such compositions represented by Formula 1: [ka] where: m is an integer from about 1 to 100, particularly from about 1 to 20, and most particularly from 1 to about 10; X is an antigenic moiety, particularly a foreign antigen, or a self-antigen to which the patient develops an unwanted immune response, or a tolerogenic portion of such an antigenic moiety (e.g., a fragment or epitope); Y is a linker moiety or a direct bond, or an antibody, antibody fragment, peptide or other ligand that specifically binds to X; and Z is a liver targeting moiety, particularly a galactosylating moiety. The value for m in formula 1 will depend on the nature of X, with each antigen, antibody, antibody fragment or ligand having a particular number and density of sites (mainly N-terminal amines, lysine residues and cysteine ​​residues) to which a linker or galactosylation moiety can be attached. 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, particularly a linker with an immunoproteasome cleavage site). In general, providing a sufficient degree of galactosylation in the composition of formula 1 is preferred to facilitate uptake by hepatocytes. The pharmaceutical formulations and methods of the present disclosure can utilize a mixture of compositions of formula 1, each bearing a different X moiety (e.g., several epitopes associated with a particular undesirable immune response).

[0056] The compositions of Formula 1 include subgenera where X is an exogenous transplant antigen to which the transplant patient develops an unwanted immune response (e.g., transplant rejection), an exogenous food, animal, plant or environmental antigen to which the patient develops an unwanted immune response (e.g., allergic or hypersensitivity), an exogenous therapeutic agent to which the patient develops an unwanted immune response (e.g., hypersensitivity and / or reduced therapeutic activity), or an autoantigen to which the patient develops an unwanted immune response (e.g., autoimmune disease); Y is a linker of formulas Ya to Yp; and / or Z is galactose, galactosamine, or N-acetylgalactosamine, as illustrated by Formulas 1a through 1p, described below with respect to reaction schemes.

[0057] The present disclosure further provides a pharma- ceutically acceptable composition represented by Formula 2: [ka] where: m' is zero or an integer from about 1 to 10, m" is zero or an integer from about 1 to 10, and the sum of m'+m" is an integer from about 1 to 10, but is at least 1; X is an antigenic moiety, particularly a foreign antigen, or a self-antigen to which the patient develops an unwanted immune response, or a tolerogenic portion of such an antigenic moiety (e.g., a fragment or epitope); Y is a linker moiety or a direct bond, or an antibody, antibody fragment, peptide or other ligand that specifically binds to X; and Z is a liver targeting moiety, in particular an ASGPR-targeting antibody, an ASGPR-targeting antibody fragment, an ASGPR-targeting peptide, an ASGPR-targeting scFv, or another ASGPR ligand; Such compositions may optionally contain an amino acid sequence that facilitates isolation and purification [e.g., a "His tag" or "6xHis" having the sequence: HHHHHH (SEQ ID NO:1)] as well as an additional linker [e.g., a "Gly" tag having the sequence: GGGS (SEQ ID NO:2)]. 3 In compositions of formula 2 where m'+m" is greater than 1, the X moieties can be the same or different, and the Y moieties can be the same or different. The value for m'+m" is smaller (e.g., 3 or less) when X is a whole protein, or larger (up to about 10) when X is a peptide. The linker "Y" may advantageously include a cleavage site, particularly an immunoproteasome cleavage site.

[0058] The compositions of Formula 2 include subgenera where X is an exogenous transplant antigen to which the transplant patient develops an unwanted immune response (e.g., transplant rejection); an exogenous food, animal, plant or environmental antigen to which the patient develops an unwanted immune response (e.g., allergic or hypersensitivity); an exogenous therapeutic agent to which the patient develops an unwanted immune response (e.g., hypersensitivity and / or reduced therapeutic activity); or an autoantigen to which the patient develops an unwanted immune response (e.g., autoimmune disease).

[0059] Alternatively, in the compositions of Formula 1 and / or Formula 2, X can be an antibody, antibody fragment or ligand that specifically binds to a circulating protein or peptide or antibody that is causally involved in transplant rejection, immune responses to therapeutic agents, autoimmune disease, hypersensitivity and / or allergy.

[0060] The compositions of formula 2 can be prepared as fusion proteins and can contain several components useful in their preparation and purification, such as a signal sequence or leader sequence that directs the expressed polypeptide to a specific transport pathway and is subsequently cleaved from the polypeptide; thus, the signal sequence can be a 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 kappa chain sequence, which directs protein secretion: METDTLLLWVLLLWVPGSTG (SEQ ID NO:3). Similarly, one example of a common bacterial signal sequence used in expression systems is the pelB signal sequence, which directs expressed proteins to the bacterial periplasm: MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:4).

[0061] antigen

[0062] The antigen utilized as X in the composition of Formula 1 and / or Formula 2 can be a protein or peptide, e.g., the antigen may be a full or partial therapeutic agent, a full-length transplant protein or peptide thereof, a full-length autoantigen or peptide thereof, a full-length allergen or peptide thereof, and / or a nucleic acid, or a mimetic of the above-mentioned antigens.

[0063] Antigens utilized in the practice of this disclosure may be one or more of the following: Therapeutic agents that are proteins, peptides, antibodies and antibody-like molecules, including antibody fragments, and fusion proteins with antibodies and antibody fragments. These include human, non-human (such as mouse) and non-natural (i.e., engineered) proteins, antibodies, chimeric antibodies, humanized antibodies, and non-antibody binding scaffolds, such as fibronectin, DARPins, knottins, etc. · Human allograft antigens to which transplant patients develop undesirable immune responses. Autoantigens that trigger unwanted autoimmune responses. One of skill in the art will recognize that autoantigens are of endogenous origin in an autoimmune disease patient, while polypeptides utilized in the compositions of the present disclosure are typically exogenously synthesized (as opposed to purified and enriched from a source). Foreign antigens, such as food, animal, plant and environmental antigens to which the patient experiences an undesirable immune response. While therapeutic proteins may also be considered foreign antigens due to their exogenous origin, those skilled in the art will recognize that for clarity of description in this disclosure, such therapeutic agents are described as separate groups. Similarly, plant or animal antigens may be edible and are considered food antigens, and environmental antigens may be derived from plants. They are all foreign antigens, however. In the interest of brevity, no attempt will be made to distinguish and define all such potentially overlapping groups that a skilled artisan may recognize antigens that may be utilized in the compositions of the present disclosure, especially in light of the detailed description and examples. The antigen can be a complete protein, a portion of a complete protein, a peptide, etc., and can be derivatized for attachment to a linker and / or galactosylation moiety (as described above), can be mutant and / or contain conservative substitutions, particularly while maintaining sequence identity, and / or can be desialylated.

[0064] In embodiments in which the antigen is a therapeutic protein, peptide, antibody or antibody-like molecule, the specific antigen may be selected from the following: abatacept, abciximab, adalimumab, adenosine deaminase, ado-trastuzumab emtansine, agalsidase alfa, agalsidase beta, aldesleukin, alglucerase, alglucosidase alfa, alpha-1-protease inhibitor, anakinra, anistreplase (anisoylated plasminogen streptokinase activating complex), antithrombin III, antithymocyte globulin, alteplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitor, canakinumab, carboxypeptidase G2 (glucarpidase and voraxaze), certolizumab. Pegol, cetuximab, collagenase, Crotalidae immune Fab, darbepoetin-α, denosumab, digoxin immune Fab, dornase alfa, eculizumab, etanercept, activated factor VII, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histrelin acetate, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin [recombinant human insulin ("rHu insulin") and bovine insulin], interferon-alpha 2a, interferon-alpha 2b, interferon-beta 1a, interferon-beta 1b, interferon-gamma 1b, ipilimumab, L-arginase, L-asparaginase, L-methioninase, lactase, laronidase, lepirudin / hirudin, mecasermin, mecasermin rinfavert, methoxynatalizumab, octreotide, ofatumumab, oprelvekin, pancreatic amylase, pancreatic lipase, papain, PEG-asparaginase, PEG-doxorubicinHCl, PEG-epoetin-β, pegfilgrastim, Peg-interferon-α2a, Peg-interferon-α2b, pegloticase, pegvisomant, phenylalanine ammonia lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, type 1 alpha-interferon, ustekinumab, vW factor. Therapeutic proteins can be obtained (e.g., enriched and purified) from natural sources or synthesized, e.g., recombinantly, and include antibody therapeutics that are typically IgG monoclonal or fragments or fusions.

[0065] Specific therapeutic proteins, peptides, antibodies or antibody-like molecules include various forms of abciximab, adalimumab, agalsidase alfa, agalsidase beta, aldesleukin, alglucosidase alfa, factor VIII, factor IX, infliximab, insulin (including rHu insulin), L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL), or rasburicase (uricase), and generally IgG monoclonal antibodies.

[0066] Another particular group includes hemostatic agents (Factors VIII and IX), insulin (including rHu insulin), and the non-human therapeutics uricase, PAL and asparaginase.

[0067] Undesirable immune responses in hematology and transplantation include autoimmune aplastic anemia, transplant rejection (in general), and graft-versus-host disease (bone marrow transplant rejection). In embodiments where the antigen is a human allogeneic transplant antigen, the specific sequences may be selected from: subunits of various MHC class I and MHC class II haplotype proteins (e.g., donor / recipient differences identified in tissue cross-matching), as well as single amino acid polymorphisms on a small number of blood group antigens, including RhCE, Kell, Kidd, Duffy, and Ss. Such compositions may be individually prepared for a given donor / recipient pair.

[0068] In embodiments where the antigen is an autoantigen, the specific antigen (and the autoimmune disease with which they are associated) may be selected from: In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6-phosphatase 2 (IGRP or islet-specific glucose-6-phosphatase catalytic subunit-related protein), insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2 (IA-2); other antigens These include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter type 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST G protein-coupled receptor 1-5. It should be noted that insulin is an example of an antigen that can be characterized as both an autoantigen and a therapeutic protein antigen. For example, rHu insulin and bovine insulin are therapeutic protein antigens (subject to unwanted immune attack), whereas endogenous human insulin is an autoantigen (subject to unwanted immune attack). Since endogenous human insulin is not available for use in pharmaceutical compositions, recombinant forms are utilized in the compositions of the present disclosure. Human insulin, including exogenously derived forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P01308): MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN (SEQ ID NO:5). GAD-65, including exogenously derived forms, 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 PRLIAFTSEH 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 (sequence number: 6). IGRP, including exogenously derived forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT QN9QR9): MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFFIYFPLCFQFNQTVGTKMIWVAVIGDWLNLIFKWILFGHRPYWWVQETQIYPNHSSPCLEQFPTTCETGPGSPSGHAMGASCVWYVMVTAALSHTVCGMDKFSITLHRLTWSFLWSVFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHTPGIQTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPIAKKWCANPDWIHIDTTPFAGLVRNLGVLFGLGFAINSEMFLLSCRGGNNYTLSFRLLCALTSLTILQLYHFLQIPTHEEHLFYVLSFCKSASIPLTVVAFIPYSVHMLMKQSGKKSQ (SEQ ID NO: 7). In autoimmune diseases of the thyroid, including Hashimoto's thyroiditis and Graves' disease, the major antigens include thyroglobulin (TG), thyroid peroxidase (TPO), and thyroid-stimulating hormone receptor (TSHR); other antigens include sodium iodide symporter (NIS) and megalin. In thyroid-associated eye disease and dermatoses, in addition to thyroid autoantigens including TSHR, the antigen is the insulin-like growth factor 1 receptor. In hypoparathyroidism, the major antigen is the calcium-sensing receptor. In Addison's disease, the major antigens include 21-hydroxylase, 17-hydroxylase, and P450 side-chain cleavage enzyme (P450scc); other antigens include ACTH receptor, P450c21, and P450c17. In premature ovarian failure, the major antigens include the FSH receptor and -enolase. In autoimmune hypophysitis or pituitary autoimmune diseases, the major antigens include pituitary specific protein factors (PGSF) 1a and 2; another antigen includes type 2 iodothyronine deiodinase. In multiple sclerosis, the major antigens include myelin basic protein (“MBP”), myelin oligodendrocyte glycoprotein (“MOG”) and myelin proteolipid protein (“PLP”). MBP, including exogenously derived forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P02686): MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELSRTTSEDNEVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKYLATASTMDHARGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 8). MOG, including exogenously derived forms useful in the compositions of the present disclosure, has the following sequence (UNIPROT Q16653): MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF (SEQ ID NO:9). PLP, including exogenously derived forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P60201): MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF (SEQ ID NO: 10). Peptides / epitopes useful in the disclosed compositions for treating multiple sclerosis include some or all of the following sequences, individually in compositions of formula 1, together in mixtures of compositions of formula 1, or fused in one or more compositions of formula 2: MBP13-32: KYLATASTMDHARHGFLPRH (SEQ ID NO: 11); MBP83-99: 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 PLP139-154:HCLGKWLGHPDKFVGI (SEQ ID NO:17). In rheumatoid arthritis, major antigens include collagen II, immunoglobulin binding proteins, fragment crystallizable regions of immunoglobulin G, double-stranded DNA, and native 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 major antigen is H+,K+-ATPase. In pernicious anemia, the primary antigen is intrinsic factor. In celiac disease, the primary antigens are tissue transglutaminase and the native and deamidated forms of gluten or gluten-like proteins such as alpha-, gamma-, and omega-gliadins, glutenins, hordeins, secalins, and avenins. For example, the primary antigen in celiac disease is alpha gliadin, and one skilled in the art will recognize that alpha gliadin is altered in the body to be more immunogenic through deamidation by tissue glutaminase by converting the glutamine of alpha gliadin to glutamic acid. Thus, while alpha gliadin is the original foreign food antigen, once it is altered in the body to become more immunogenic, it can be characterized as an autoantigen. In vitiligo, the major antigens are tyrosinase and tyrosinase-related proteins 1 and 2. MART1, Melanoma antigen recognized by T cells 1, MelanA, including exogenously derived forms useful in the compositions of the present disclosure, has the following sequence (UNIPROT Q16655): MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP (SEQ ID NO: 18). Tyrosinase, including exogenously obtained forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P14679): (Sequence number: 19). The melanocyte protein PMEL, gp100, including exogenously derived forms useful in the compositions of the present disclosure, has the following sequence (UNIPROT P40967): (Sequence number: 20). In myasthenia gravis, the primary antigen is the acetylcholine receptor. In pemphigus vulgaris and variant forms, the major antigens are desmogleins 3, 1 and 4; other antigens include pemphaxin, desmocollin, plakoglobin, perplakin, desmoplakin, and the acetylcholine receptor. In bullous pemphigoid, the major antigens include BP180 and BP230; other antigens include plectin and laminin 5. In Dühring's dermatitis herpetiformis, the major antigens include endomysial and tissue transglutaminase. In epidermolysis bullosa acquisita, the major antigen is collagen VII. In systemic sclerosis, major antigens include matrix metalloproteinases 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 major antigen is U1snRNP. In Sjögren's syndrome, the major 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, major antigens include nuclear proteins, including the “Smith antigen”, SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone proteins and double-stranded DNA, against which autoantibodies are made during the disease process. · In Goodpasture's syndrome, the major antigens include glomerular basement membrane proteins, including collagen IV. In rheumatic heart disease, the major antigen is cardiac myosin. In polyglandular autoimmune syndrome type 1, antigens include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochrome P450 1A2 or 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and the type 1 interferons interferon alpha, beta, and omega. In neuromyelitis optica, the major antigen is AQP4. Aquaporin 4, including exogenously obtained forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P55087): MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTIWGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV (SEQ ID NO:21). · In uveitis, the major antigens include retinal S-antigen or “S-arrestin” and interphotoreceptor retinoid-binding protein (IRBP) or retinol-binding protein 3. S-arrestin, including exogenously derived forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P10523): MAASGKTSKS EPNHVIFKKI SRDKSVTIYL GNRDYIDHVS QVQPVDGVVL VDPDLVKGKK VYVTLTCAFR YGQEDIDVIG LTFRRDLYFS RVQVYPPVGA ASTPTKLQES LLKKLGSNTY PFLLTFPDYL PCSVMLQPAP QDSGKSCGVD FEVKAFATDS TDAEEDKIPK KSSVRLLIRK VQHAPLEMGP QPRAEAAWQF FMSDKPLHLA VSLNKEIYFH GEPIPVTVTV TNNTEKTVKK IKAFVEQVAN VVLYSSDYYV KPVAMEEAQE KVPPNSTLTK TLTLLPLLAN NRERRGIALD GKIKHEDTNL ASSTIIKEGI DRTVLGILVS YQIKVKLTVS GFLGELTSSE VATEVPFRLM HPQPEDPAKE SYQDANLVFE EFARHNLKDA GEAEEGKRDK NDVDE (sequence number: 22). IRBP, including exogenously derived forms, useful in the compositions of the present disclosure has the following sequence (UNIPROT P10745):

[0069] In embodiments where the antigen is a foreign antigen, such as a food antigen, to which an unwanted immune response may be developed, the specific antigen may be: · From peanuts: conarachin (Ara h 1), allergen II (Ara h 2), peanut agglutinin, conglutinin (Ara h 6); Conarachin has a sequence identified as, for example, UNIPROT Q6PSU6 · From apple: 31 kda major allergen / disease tolerance protein homologue (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1); From milk: alpha-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), kiwellin (Act d 5); · From egg white: ovomucoid, ovalbumin, ovotransferrin, and lysozyme; From egg yolk: livetin, apovitillin, and vosvetin; · From mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4); · From celery: profilin (Api g 4), a high molecular weight glycoprotein (Api g 5); From shrimp: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), fast tropomyosin isoform; From wheat and / or other cereals: high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma- and omega-gliadins, hordeins, secalins and / or avenins; Peptides / epitopes useful in the compositions of the present disclosure for treating celiac disease include some or all of the following sequences, individually in compositions of formula 1, together in mixtures of compositions of formula 1, or fused in one or more compositions of formula 2: DQ-2 related, native alpha-gliadin “33-mer”: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 24) DQ-2 related, deamidated alpha-gliadin “33-mer”: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 25) DQ-8 related, alpha-gliadin: QQYPSGQGSFQPSQQNPQ (SEQ ID NO: 26) DQ-8 related, omega-gliadin (wheat, U5UA46): QPFPQPEQPFPW (SEQ ID NO: 27) From strawberries: the main strawberry allergen Fra a 1-E (Fra a 1); and · From banana: Profilin (Mus xp 1).

[0070] In embodiments where the antigen is a foreign antigen, such as an animal, plant or environmental antigen, to which an unwanted immune response is developed, the specific antigen may be, for example, cat, mouse, dog, horse, bee, dust, tree and solidago altissima, including proteins or peptides from: weed (including ragweed allergens amb a 1, 2, 3, 5, and 6, and Amb t 5; pigweed Che a 2 and 5; and other weed allergens Par j 1, 2, and 3, and Par o 1); grass (contains major 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 grasses (including curly dock, lambs quarters, pigweed, plantain, sorghum, and sagebrush), grasses (including bermudagrass, johnsongrass, kentucky grass, orchard grass, morning glory, and timothy grass), and trees (including catalpa, elm, hickory, olive, pecan, sycamore, and walnut); dust (including major allergens from Dermatophagoides species, e.g. Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21 and 23; from Dermatophagoides species, e.g. Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18, 22 and 24; from tropical Dermatophagoides species, e.g. Blo t 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 19 and 21; further including allergen Eur m 2 from Euroglyphus maynei, allergen Tyr p 13 from Tyrophagus putrescentiae and allergens Bla g 1, 2 and 4; and Per a 1, 3 and 7 from cockroaches); · pets (including cats, dogs, rodents, and livestock; major cat allergens include Fel d 1 to 8, feline IgA, BLa g 2, and feline albumin; major dog allergens include Can f 1 to 6, and dog albumin); Bee stings, containing the major allergens API m 1 to 12; and Fungi, including Aspergillus and Penicillium species, as well as allergens from Alternaria alternata, Davidiella tassiana, and Trichophyton rubrum species.

[0071] As will be appreciated by one of skill in the art, patients can be tested to identify antigens against which they have developed an unwanted immune response, and proteins, peptides, etc. can be developed based on those antigens and incorporated as X in the compositions of the present disclosure.

[0072] Sialylated antigens, antibodies, and antibody fragments

[0073] The following are examples of antigens, antibodies, and antibody fragments with sialylation that can be removed to leave the glycosylation specifically targeted to the ASGPR: follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), luteinizing hormone (LH), osteopontin, thyroid stimulating hormone (TSH), agalsidase alfa, agalsidase beta (FABRAZYME®; Genzyme), epoetin alfa and epoetin beta, follitropin alfa (GONAL-F®; Merck / Serono) and follitropin beta (FOLLISTIM®; Schering-Plough), insulin growth factor binding protein 6 (IGFBP-6), lutropin alfa (LUVERIS®; Merck / Serono), transforming growth factor agonist (AGG), and thyroid hormone agonist (THG). Factor beta1, antithrombin (ATryn® / TROMBATE-III®; Genzyme / Tarecris Biotherapeutics), thyroid-stimulating hormone alpha (THYROGEN®; Genzyme), lenograstim, sargramostim (LEUKINE®; Genzyme), interleukin-3, pro-urokinase, lymphotoxin, C1-esterase inhibitor (Berinert®; CSL), IgG-like antibodies, interferon beta, activated coagulation factor VII (NOVOSEVEN®; Novo Nordisk), activated coagulation factor VIII (moroctocog alfa), activated coagulation factor IX (nonacog alfa) (BENEFIX®; Wyeth), and p55 tumor necrosis receptor 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 were previously hyperglycosylated and can be desialylated for hepatocyte-ASGPR targeting include: interferon alpha and gamma, luteinizing hormone, Fv antibody fragments, asparaginase, cholinesterase, darbepoetin alfa (AraNESP®; Amgen), thrombopoietin, leptin, FSH, IFN-α2, serum albumin, and corifollitropin alfa.

[0074] Proteins with glycans that do not normally terminate in sialic acid, including proteins produced in bacteria or yeast (such as arginase, some insulins, and uricase), will be immune to desialylation.

[0075] One of skill in the art will recognize that publicly available references such as UNIPROT disclose the presence and location of sites for desialylation on most, if not all, antigens, antibodies, antibody fragments and ligands of interest.

[0076] Antibody and Peptide Ligands

[0077] In the embodiment that utilizes antibody, antibody fragment or ligand, such moiety is selected to specifically bind to the circulating protein or peptide or antibody that is targeted, and causes the liver uptake of the circulating targeted moiety, possibly as an adduct with the targeting moiety, ultimately causing the clearance and inactivation of the circulating targeted moiety.For example, liver-targeted factor VIII will bind to and clear circulating factor VIII antibody.Those skilled in the art will be familiar with the procedures for identifying such moieties.

[0078] Linker

[0079] Linkers utilized in the compositions of the present disclosure ("Y" in Formula 1) can include N-hydroxysuccinimidyl linkers, maleimide linkers, vinylsulfone linkers, pyridyldithiol-poly(ethylene glycol) linkers, pyridyldithiol linkers, n-nitrophenyl carbonate linkers, NHS-ester linkers, nitrophenoxypoly(ethylene glycol) ester linkers, and the like.

[0080] One particular group of linkers has the formula Y'-CMP, where Y' represents the remaining portion of the linker and R 9 and Z are as defined. More particularly, in the linker group having the formula Y'-CMP, R 9 The substituent is an ethylacetamide group, and even more particularly, an ethylacetamide conjugated to C1 of N-acetylgalactosamine. [ka]

[0081] Dithiol-containing linkers, and most particularly disulfanylethyl carbamate-containing linkers (named with the free amine of X, otherwise designated "disulfanylethyl ester" without the free amine of X), are particularly advantageous in the compositions of the present disclosure because of their ability to cleave and release the antigen in its original form once inside the cell, for example as described below (where Y' represents the remaining portion of the linker and X' and Z are as defined).

[0082] [ka]

[0083] In particular with respect to the linkers illustrated below in formulas Ya to Yp: The left bracket “(” indicates the bond between X and Y; The right or bottom bracket “)” indicates the bond between Y and Z; n is an integer representing a mixture containing about 1 to 100, particularly about 8 to 90 (e.g., 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 particularly about 40 to 80 (e.g., 39, 40, 43, 45, 46, 48, 50, 52, 53, 55, 57, 60, 62, 65, 66, 68, 70, 73, 75, 78, 80 or 81) ethylene glycol groups, where the mixture typically encompasses the integer specified as n±10%; p is an integer representing a mixture comprising about 2 to 150, particularly about 20 to 100 (e.g., 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 105) and more particularly 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 mixture typically encompasses the integers specified as p±10%; q is an integer representing a mixture including about 1 to 44, particularly about 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22), and more particularly about 4 to 12 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13), where the mixture typically encompasses the integers specified as q±10%; and R 8 is -CH 2 -("Methyl") or -CH 2 -CH 2 -C(CH 3 )(CN)-("1-cyano-1-methyl-propyl" or "CMP"). The following structure of formula 1: [ka] As explained in 9 is a direct bond or -CH 2 -CH 2-NH-C(O)-(ethylacetamide group or "EtAcN"), where the EtAcN group is shown and the remainder of the linker is designated Y': and Z is galactose, galactosamine or N-acetylgalactosamine conjugated at C1.

[0084] [ka]

[0085] [ka]

[0086] [ka]

[0087] [ka]

[0088] [ka]

[0089] [ka]

[0090] [ka]

[0091] [ka]

[0092] [ka]

[0093] [ka]

[0094] [ka]

[0095] [ka]

[0096] [ka]

[0097] [ka] (Linkers of formula Yn can be synthesized via certain precursors that give Yn that are particularly suitable for conjugation to hydrophobic antigens.)

[0098] [ka]

[0099] [ka]

[0100] The linkers shown above as formulae Yh to Yn are synthesized as isomers that are utilized without separation. For example, the linkers of formulae Yh, Yi, Yj and Yn may be a mixture of 8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8yl and 8,9-dihydro-3H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8yl structures as illustrated below: [ka] The linkers of formula Yk, YL and Ym may be a mixture of 8,9-dihydro-1H-dibenzo[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 as illustrated below: [ka] The presence of such isomeric mixtures does not impair the functionality of compositions utilizing such linkers.

[0101] Galactosylated moiety

[0102] The galactosylation moiety utilized in the composition of the present disclosure serves to target the composition to hepatocytes (e.g., bind specifically to hepatocytes) and can be selected from the following: galactose, galactosamine, or N-acetylgalactosamine. It has been reported that ASGPR affinity can be maintained while modifying either the C3 / C4-diol anchor side of galactose (Mamidyala, Sreeman K., et al., J. Am. Chem. Soc. 2012, 134, 1978-1981), and thus the conjugation points are specifically C1, C2 and C6.

[0103] Particular galactosylation moieties include galactose conjugated at C1 or C6, galactosamine conjugated at C2, and N-acetylgalactosamine conjugated at C6. Other particular galactosylation moieties include galactose conjugated at C2, more particularly CH 2 R 9 Yet another particular galactosylating moiety comprises an N-acetylgalactosamine conjugated to a linker having a substituent. 9 It comprises galactose, galactosamine or N-acetylgalactosamine conjugated to a linker having a substituent.

[0104] ASGPR targeting antibody

[0105] The ASGPR-specific antibodies utilized in the compositions of the present disclosure also serve to target the compositions of the present disclosure to hepatocytes and can be selected from commercially available products, for example: anti-asialoglycoprotein receptor 1 antibody (ab42488) from Abcam plc, Cambridge, UK and ASGPR 1 antibody (ab42489) from Santa Cruz Biotechnology, Inc., Dallas, Texas. 1 Alternatively, such antibodies may be used with any of the many published sequences, such as Dom26h-196-61, a single domain anti-ASGPR antibody: EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSS (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 sequences with conservative substitutions can be expressed. The above-referenced US patent applications disclose liver-targeting molecules such as DOM26h-196-61 for delivery of certain therapeutic agents, including interferon (interferon alpha 2, interferon alpha 5, interferon alpha 6, or consensus interferon), ribavirin, nexavar / sorafenib, erbitux / cetuximab, avastatin / bevacizumab, and herceptin / trastuzumab for the treatment of liver disease. Compositions of matter corresponding to Formula 2 utilizing DOM26h-196-61 or another liver targeting molecule described in US 2013 / 0078216 include within their scope interferon (interferon alpha 2, interferon alpha 5, interferon alpha 6, or consensus interferon), ribavirin, nexavar / sorafenib, erbitux / cetuximab, avastatin / bevacizumab, and herceptin / trastuzumab.

[0106] Novel sequences for antibodies, antibody fragments, or peptides that specifically target ASGPR can be discovered using a variety of methods well known to those skilled in the art, including, but not limited to, vaccination techniques, hybridoma techniques, library display techniques (including bacterial and phage platforms), endogenous repertoire screening techniques, directed evolution, and rational design.

[0107] nomenclature

[0108] The 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 a cyclobutyl moiety (shown in place of the antigen for illustrative purposes), Y is of formula Ya, m is 1, n is 4, and Z is N-acetylgalactosamin-2-yl: [ka] can be named (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-dithiahenicosan-21-ylidene)triaz-1-yn-2-ium chloride, and thus the corresponding composition of the disclosure, where X is tissue transglutaminase, can be named (Z)-(21-(tissue transglutaminase)-1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-ylidene)triaz-1-yn-2-ium chloride. A corresponding composition of the disclosure where X' is tissue transglutaminase, m is 2, n is 4, and Z' is N-acetylgalactosamin-2-yl can be designated (3Z)-((tissue transglutaminase)-1,3-dihydrobis(1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-yl-21-ylidene))bis(triaz-1-yn-2-ium) chloride.

[0109] For simplicity, the composition of Formula 1 may be represented by X and an entity corresponding to one of Formulas 1a through 1p (as illustrated in the reaction scheme), followed by the variables m, n, p, and / or q, R 8 , R 9and by reference to a description of integers to identify the galactosyl moiety and position to which it is conjugated. Under this system, a composition of formula 1a, where X is ovalbumin, m is 2, n is 4, and Z is N-acetylgalactosamin-2-yl, may be named using an alternative nomenclature system. 2 -n 4 -2NAcGAL”.

[0110] Similarly, a composition of formula 1 [ka] teeth, [ka] Isomers: [ka] teeth, [ka] (Bold highlighting has been added for convenience in identifying discrepancies from the proper name). Using the adopted nomenclature system for this disclosure, both isomers may be designated "F1n-insulin-m 1 -n 1 -p 1 -q 4 -CMP-EtAcN-1NAcGAL”, where CMP is R 8 indicates 1-cyano-1-methyl-propyl, and EtAcN indicates R 9 indicates that Z" is N-acetylgalactosamine conjugated at C1. The absence of the abbreviation EtAcN before the name of Z indicates that R 9 would show that is a direct bond.

[0111] In the composition of Formula 2, the left to right orientation should not be construed as specifying the order of N to C unless specifically indicated to the contrary. For example, m' is 1, m" is 0, X is ovalbumin, and Y is Gly. 3 The compound of formula 2 is OVA-Gly, 3 It can be named Ser-DOM and read as encompassing both: N-OVA-Gly 3 Ser-DOM-C and N-DOM-Gly 3 Ser-OVA-C For example, m' is 1, m" is 0, X is ovalbumin, and Y is Gly. 3 A composition of formula 2 where Ser is and Z is anti-ASGPR Dom26h-196-61 (with a purification tag attached via an additional linker) may 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 Here, the C'-terminal Gly 3 The Ser-6xHis group represents an amino acid sequence that facilitates isolation and purification.

[0112] m' is 1, m" is 1, each X is Factor VIII, and each Y is Gly. 3 The composition of formula 2, where Z is anti-ASGPR Dom26h-196-61 (with a purification tag attached via an additional linker), is 3 Ser-DOM-Gly 3 Ser-FVIII-Gly 3 Ser-6xHis and includes both of the following: N-FVIII-Gly 3 Ser-DOM-Gly3 Ser-FVIII-Gly 3 Ser-6xHis-C and N-6xHis-Gly 3 Ser-FVIII-Gly 3 Ser-DOM-Gly 3 Ser-FVIII-C.

[0113] The composition of formula 2, wherein m' is 3, m" is 0, 1, the three X antigens are deamidated alpha-gliadin "33-mer" (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26) and omega-gliadin (SEQ ID NO:27), respectively, each Y is a linker with an immunoproteasome cleavage site ("IPC"), and Z is anti-ASGPR Dom26h-196-61, is: It may be named deamidated alpha-gliadin "33-mer"-IPC-alpha-gliadin-IPC-omega-gliadin-IPC-DOM.

[0114] Preparation of the compositions of the present disclosure

[0115] For example, compositions of formula 1 can be prepared by adapting 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 respect to Schemes 1 to 14. Other synthetic approaches will be apparent to those skilled in the art.

[0116] Equation 101 (below) is an alternative representation of X, [ka] Here, R 1 , a free surface amino group (-NH) located on the three-dimensional structure of X to make it accessible for conjugation to a linker. 2) or a thiol (-SH) moiety, and X' represents the remainder of X excluding the free amino group as identified [(X" is used in the reaction schemes to represent the remainder of X excluding the free thiol group]. Depending on the identity of X, at least one (N-terminal amine) will be present, and multiple R 1 Groups (mainly derived from lysine residues or cysteine ​​residues involved in disulfide bonds) can be present.

[0117] The variables utilized in the schemes are as defined above and additionally include the following, which should be understood not to have any specific meaning of these terms except in relation to a particular scheme or process: R 2 is OH or a protecting group; R 3 OH, NH 2 , NHAc, a protecting group or an NH-protecting group; R 4 is OH or a protecting group; R 5 is OH or a protecting group; R 6 is OH or a protecting group; Z' is C1- or C6-conjugated galactose, C2-conjugated galactosamine, or C6-conjugated N-acetylgalactosamine; R 8 -CH 2 or -CH 2 -CH 2 -C(CH 3 )(CN)-; and R 9 is a direct bond and Z″ is N-acetylgalactosamine conjugated at C2; R 9is an ethylacetamide group, and Z″ is galactose, galactosamine or N-acetylgalactosamine conjugated at C1.

[0118] Synthesis reaction parameters

[0119] The terms "solvent", "inert organic solvent" or "inert solvent" mean a solvent that is inert under the conditions of the reaction being described in conjunction with it, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ("THF"), dimethylformamide ("DMF"), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine, and the like. Unless specified to the contrary, the solvents used in the reactions of the present disclosure are inert organic solvents.

[0120] The term "qs" means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to a desired volume (i.e., 100%).

[0121] If desired, the compounds and intermediates described herein can be isolated and purified by any suitable separation or purification procedure, such as, for example, filtration, extraction, crystallization, column chromatography, thin 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 procedures. Specific examples of suitable separation and isolation procedures can be obtained by reference to the examples below. However, other equivalent separation or isolation procedures can also be used.

[0122] Unless otherwise specified (including the examples), all reactions are conducted at standard atmospheric pressure (about 1 atmosphere) and ambient (or room) temperature (about 20° C.), at about pH 7.0-8.0.

[0123] Characterization of the reaction products is by conventional means, such as proton and carbon NMR, mass spectrometry, size exclusion chromatography, infrared spectroscopy, and gel electrophoresis.

[0124] Scheme 1 illustrates the preparation of a composition of formula 1, where Z can be galactose, galactosamine, or N-acetylgalactosamine. In that regard, and as defined above, Z′ as utilized in Scheme 1 represents galactose conjugated at C1 and C6 and corresponding to the following structure according to formula 1: [ka] , conjugated at C2 and corresponding to the following structure according to formula 1: [ka] and N-acetylgalactosamine conjugated at C6 and corresponding to the following structure according to formula 1: [ka] Includes:

[0125] Reaction Scheme 1

[0126] [ka]

[0127] [ka]

[0128] [ka]

[0129] [ka]

[0130] [ka]

[0131] As described above in Scheme 1, step 1, surface thiol groups can be generated on an antigen, antibody, antibody fragment, or ligand having a free surface amino group (Formula 101') by contact with Traut's reagent (Formula 102) at about pH 8.0 for about 1 hour to give Formula 103' from which unreacted Traut's reagent has been removed, e.g., via centrifugal size exclusion chromatography. The two structures shown below illustrate the product of Scheme 1, step 1, respectively showing the first identified free surface amino group on X (i.e., Formula 103', where X' represents the remainder of X excluding the identified free surface amino group), and omitting the free surface amino group (i.e., Formula 103). This is similar to the distinction described between X and Formula 101. A convention followed in subsequent schemes. [ka]

[0132] In Scheme 1, Step 2, pyridyldithiol-poly(ethylene glycol)-NHS ester (Formula 104) is reacted with galactosamine (Formula 105, where R 3 NH 2 and R 2 , R 4 , R5 and R6 are OH) at about pH 8 with stirring for about 1 hour to provide the corresponding pyridyldithiol-poly(ethylene glycol)-sugar of Formula 106A, which can be used without further purification.

[0133] In Scheme 1, Step 3, 4,4'-dithiodipyridine (Formula 107) is contacted with a thiol-poly(ethylene glycol)propanoic acid (Formula 108) to give the corresponding pyridyldithiol-poly(ethylene glycol)propanoic acid (Formula 109).

[0134] In Scheme 1, Step 4, an acid of formula 109 is reacted with R 2 is OH, and R 3 , R 4 , R5 and R6 are protecting groups ("PG"), where R 6 is OH and R 2 , R 3 , R 4 and R 5 is PG, or where R 6 is N-acetyl, and R 2 , R 3 , R 4 and R 5 is PG to give the corresponding pyridyldithiol-poly(ethylene glycol)-sugars of formula 106B, 106C and 106D, which can be used after deprotection.

[0135] In Scheme 1, Step 5, to a stirred solution of the product of Step 1 (Formula 103') is added an excess (corresponding to the value of m) of the product of Step 2 or Step 4 (Formula 106, i.e., 106A, 106B, 106C or 106D) for about 1 hour, followed by centrifugal size exclusion chromatography to remove any free remaining reactants, to give the corresponding products according to Formula 1a, Formula 1aA, Formula 1aB, Formula 1aC and Formula 1aD, respectively.

[0136] Compositions corresponding to formula 1a may be named, for example, respectively, as follows: "F1aA-X'-m m -n n " or "F1a-X'-m m -n n -2NGAL” "F1aB-X'-m m -n n " or "F1a-X'-m m -n n -1GAL” "F1aC-X'-m m -n n " or "F1a-X'-m m -n n -6GAL” "F1aD-X'-m m -n n " or "F1a-X'-m m -n n -6NAcGAL” Each is made as a product utilizing intermediates according to formulae 106A-D.

[0137] Turning now to Schemes 2-14, and for purposes of the nomenclature used therein, unless otherwise noted, Z" represents N-acetylgalactosamine conjugated at C2: [ka] Or it refers to galactose, galactosamine or N-acetylgalactosamine conjugated at C1. It should be noted that C1 conjugated to the composition needs to be protected during synthesis, for example by cyclizing the amine at the C3 hydroxyl and subsequently deprotecting the incorporation of the protected galactosamine into the adjacent linker moiety.

[0138] Poly(galactose methacrylic acid) reactants of formulas 201, 401, 501, 601, 701, 803 and 1401 can be prepared by methacrylation of galactose, for example, by contacting galactosamine and methacrylic anhydride, followed by reversible addition-fragmentation chain transfer (RAFT) polymerization with the corresponding RAFT agent in the presence of azobisisobutyronitrile (AIBN) in a suitable solvent, starting with freeze-thaw cycles, followed by heating at about 60-80° C., preferably 70° C., for about 5-8, preferably about 6 hours. The polymer can be precipitated in a lower alkanol, preferably methanol.

[0139] Reaction Scheme 2

[0140] [ka]

[0141] As illustrated in Scheme 2, an antigen, antibody, antibody fragment or ligand having free surface thiol groups, for example as described with respect to Scheme 1, step 1 (formula 103'), prepared is contacted with an excess (corresponding to the value of m) of pyridyldithiol-poly(ethylene glycol) of Formula 201 for about 1 hour to obtain the corresponding product according to Formula 1b.

[0142] The compositions of formula 1b may be named as follows: "F1b-X'-m m -n n -p p -2NAcGAL” or “F1b-X'-m 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, is designated "F1b-uricase-m 1 -n 4 -p 4 -2NAcGAL" or "30-(uricase)-3,5,7,9-tetramethyl-12-oxo-1-phenyl-1-thioxo-3,5,7,9-tetrakis((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)carbamoyl)-13,16,19,22-tetraoxa-2,25,26-trithiatriacontan-30-iminium".

[0143] Reaction Scheme 3

[0144] [ka]

[0145] As illustrated in Scheme 3, an antigen, antibody, antibody fragment or ligand having a naturally occurring free surface thiol group (cysteine) [Formula 101″ which corresponds to Formula 101, and which explains that as that term is subsequently used herein, X″ represents X exclusive of the identified free surface thiol group] is contacted with an excess (corresponding to the value of m) of pyridyldithiol-poly(ethylene glycol) of Formula 201 to obtain the corresponding product according to Formula 1c.

[0146] Compositions corresponding to formula 1c may be named as follows: "F1c-X'-m m -n n -p p -2NAcGAL” or “F1c-X'-m m -n n -p p -EtAcN-Z”.

[0147] Reaction Scheme 4

[0148] [ka]

[0149] As illustrated in Scheme 4, an antigen, antibody, antibody fragment or ligand having a native free surface thiol group of formula 101″ is contacted with an excess (corresponding to the value of m) of a pyridyldithiol of formula 401 to give the corresponding product according to formula 1d.

[0150] Compositions corresponding to formula 1d may be named as follows: "F1d-X'-m m -p p -2NAcGAL” or “F1d-X'-m m -p p -EtAcN-Z”.

[0151] Reaction Scheme 5

[0152] [ka]

[0153] As illustrated in Scheme 5, an antigen, antibody, antibody fragment or ligand having a native free surface amino group of formula 101' is contacted with an excess (corresponding to the value of m) of n-nitrophenyl carbonate of formula 501 to give the corresponding product according to formula 1e.

[0154] Compositions corresponding to formula 1e may be named as follows: "F1e-X'-m m -p p -2NAcGAL” or “F1e-X'-m m -p p -EtAcN-Z”.

[0155] Reaction Scheme 6

[0156] [ka]

[0157] As illustrated in Scheme 6, an antigen, antibody, antibody fragment or ligand having native free surface amino groups of formula 101' is contacted with an excess (corresponding to the value of m) of an n-nitrophenyl carbonate poly(ethylene glycol) ester of formula 601 to give the corresponding product according to formula 1f.

[0158] Compositions corresponding to formula 1f may be named as follows: "F1f-X'-m m -n n -p p -2NAcGAL” or “F1f-X'-m m -n n -p p -EtAcN-Z”.

[0159] Reaction Scheme 7

[0160] [ka]

[0161] As illustrated in Scheme 7, an antigen, antibody, antibody fragment or ligand having native free surface amino groups of formula 101′ is contacted with an excess (corresponding to the value of m) of an NHS-ester poly(ethylene glycol) ester of formula 701 to give the corresponding product according to formula 1g.

[0162] Compositions corresponding to formula 1g may be named as follows: "F1g-X'-m m -p p -2NAcGAL” or “F1g-X'-m m -p p -EtAcN-Z”

[0163] Reaction Equation 8

[0164] [ka]

[0165] [ka]

[0166] As illustrated in Scheme 8, step 1, an antigen, antibody, antibody fragment or ligand having a native free surface amino group of Formula 101' is contacted with an excess (corresponding to the value of m) of an amine-reactive linker for click chemistry of Formula 801 to give the corresponding product according to Formula 802.

[0167] In Scheme 8, Step 2, the product of formula 802 is then contacted with an equivalent amount (again corresponding to the value of m) of a galactose(amine) polymer of formula 803 to give the corresponding isomeric product according to formula 1h. The two isomers described above result in non-specific cyclization of the azide of formula 803 with the triple bond of formula 802. Such non-specific cyclization occurs in the synthesis of other compositions where Y is selected from formulas Yh to Yn, but will not be described in each instance.

[0168] Compositions corresponding to formula 1h may be named as follows: "F1h-X'-m m -n n -p p -q q -2NAcGAL” or “F1h-X'-m m -n n -p p -q q -EtAcN-Z”.

[0169] Reaction Scheme 9

[0170] [ka]

[0171] [ka]

[0172] As illustrated in Scheme 9, step 1, an antigen, antibody, antibody fragment or ligand bearing a native free surface thiol group of Formula 101″ is contacted with an excess (corresponding to the value of m) of a thiol-reactive linker for click chemistry of Formula 901 to give the corresponding product according to Formula 902″.

[0173] In Scheme 9, Step 2, the product of Formula 902" is then contacted with an equivalent amount of a galactose(amine) polymer of Formula 803 (again corresponding to the value of m) to give the corresponding isomeric product according to Formula 1i.

[0174] Compositions corresponding to formula 1i may be named as follows: "F1i-X'-m m -n n -p p -q q -2NAcGAL” or “F1i-X'-m m -n n -p p -q q -EtAcN-Z”.

[0175] Following the procedures described for Scheme 9 and substituting starting material 101″ with a compound of formula 103′ (derivatized with Traut's reagent) provides the corresponding isomeric product of formula 1j, as shown below.

[0176] [ka]

[0177] Compositions corresponding to formula 1j may be named as follows: "F1j-X'-m m -n n -p p -q q -2NAcGAL” or “F1j-X'-m m -n n -p p -q q -EtAcN-Z”.

[0178] Reaction Scheme 10

[0179] [ka]

[0180] [ka]

[0181] As illustrated in Scheme 10, step 1, an antigen, antibody, antibody fragment or ligand bearing a native free surface thiol group of Formula 101″ is contacted with an excess (corresponding to the value of m) of a thiol-reactive linker for click chemistry of Formula 1001 to give the corresponding product according to Formula 1002.

[0182] In Scheme 10, Step 2, the product of Formula 1002 is then contacted with an equivalent amount (again corresponding to the value of m) of a galactose(amine) polymer of Formula 803 to give the corresponding isomeric product according to Formula 1k.

[0183] Compositions corresponding to formula 1k may be named as follows: "F1k-X'-m m -n n -p p -q q -2NAcGAL” or “F1k-X'-m m -n n -p p -q q -EtAcN-Z”.

[0184] Following the procedures described for Scheme 10 and substituting starting material 101″ with a compound of formula 103′ (derivatized with Traut's reagent) provides the corresponding isomeric product of formula 1L, as shown below.

[0185] [ka]

[0186] Compositions corresponding to Formula 1L may be named as follows: "F1L-X'-m m -n n -p p -q q -2NAcGAL” or “F1L-X'-m m -n n -p p -q q -EtAcN-Z”.

[0187] Reaction Scheme 11

[0188] [ka]

[0189] [ka]

[0190] [ka]

[0191] [ka]

[0192] [ka]

[0193] [ka]

[0194] [ka]

[0195] As illustrated in Scheme 11, step 1, galactose, protected galactosamine, or N-acetyl-D-galactosamine (Formula 1101, where R 3 and R 4 is OH and R 3 is an NH-protecting group (e.g., R 4 or R 3 is NHAc and R 4 (wherein is OH) is contacted with 2-chloroethan-1-ol, followed by cooling and dropwise addition of acetyl chloride. The solution is allowed to warm to room temperature and then heated to 70° C. for several hours. Ethanol is added to the crude product and the resulting solution is stirred under carbon, then filtered, followed by solvent removal to give the corresponding product of formula 1102.

[0196] As illustrated in Scheme 11, Step 2, the product of formula 1102 is added to an excess of sodium azide and heated to 90° C. for several hours, followed by filtration and subsequent solvent removal to give the corresponding product of formula 1103.

[0197] As illustrated in Scheme 11, Step 3, the product of Formula 1103 is added to a solution of palladium on carbon and ethanol and stirred under hydrogen gas (3 atm) for several hours, followed by filtration and subsequent solvent removal to give the corresponding product of Formula 1104.

[0198] As illustrated in Scheme 11, Step 4, the product of formula 1104 is added to a solution of methacrylic anhydride, triethylamine is added, and the reaction is stirred for 2 hours, followed by solvent removal and isolation to give the corresponding product of formula 1105.

[0199] As illustrated in Scheme 11, step 5, an azide-modified uRAFT agent (Formula 1106) is added to a solution of the product of Formula 1105 with azobisisobutyronitrile, subjected to four freeze-degassing cycles, and then stirred at 70° C. After several hours, the corresponding polymer product of Formula 1107 is precipitated by addition of a lower alkanol, followed by solvent removal. 3 is an NH-protecting group (e.g., R 4 (cyclized by), at which point the protecting group is removed.

[0200] As illustrated in Scheme 11, step 6, an antigen, antibody, antibody fragment or ligand having a native free surface amino group of Formula 101' is added to a pH 8.0 buffer and contacted with an excess (corresponding to the value of m) of a dioxopyrrolidine of Formula 1108 with stirring. After 1 hour, unreacted Formula 1108 is removed and the resulting product of Formula 1109 is used without further purification.

[0201] As illustrated in Scheme 11, Step 7, the product of Formula 1107 is added to a pH 8.0 buffer and added to the product of Formula 1109. After stirring for 2 hours, excess Formula 1107 is removed to give the corresponding isomeric product of Formula 1m.

[0202] Substitution of the product of Formula 1105 with N-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)methacrylamide in step 5 and continuing with steps 6 and 7 provides the corresponding isomeric product of Formula 1m, where Z″ is N-acetylgalactosamine conjugated at C2.

[0203] Compositions corresponding to formula 1m may be named as follows: "F1m-X'-m m -n n -p p -q q -EtAcN-Z", where Z" is 1GAL, 1NGAL or 1NAcGAL, or "F1m-X'-m m -n n -p p -q q -2NAcGAL”.

[0204] Reaction Scheme 12 [ka]

[0205] The synthetic approach of Scheme 12 is particularly suitable for hydrophobic antigens, antibodies, antibody fragments and ligands (eg, insulin) due to the use of organic solvents.

[0206] As illustrated in Scheme 12, step 1, an antigen, antibody, antibody fragment or ligand having a native free surface amino group of formula 101' is dissolved in an organic solvent (e.g., DMF) containing triethylamine. To this is added an amount of a compound of formula 1201 (corresponding to the value of m), followed by stirring and adding t-butyl methyl ether. The corresponding product of formula 1202 is recovered as a precipitate.

[0207] The product of formula 1202 is resuspended in an organic solvent and an amount (corresponding to the value of m) of formula 1107 (e.g., obtained as described for reaction formula 11) is added, followed by stirring. The reaction product is precipitated via addition of dichloromethane, followed by filtration and solvent removal. Purification (e.g., resuspension in PBS, followed by centrifugal size exclusion chromatography) gives the corresponding isomeric product of formula 1n.

[0208] Compositions corresponding to formula In may be named as follows: "F1n-X'-m m -n n -p p -q q -EtAcN-Z", where Z" is 1GAL, 1NGAL or 1NAcGAL, or "F1m-X'-nm-n n -p p -q q -2NAcGAL”.

[0209] Reaction Scheme 13

[0210] [ka]

[0211] [ka]

[0212] In Scheme 13, Step 1, a nitrophenoxycarbonyl-oxyalkyldithiol-poly(ethylene glycol)-NHS ester (Formula 1301) is contacted with galactose, galactosamine, or N-acetylgalactosamine (Formula 105) to give the corresponding product of Formula 1302, along with the other two illustrated products, from which the desired nitrophenoxycarbonyldithiol-poly(ethylene glycol)-carboxyethylgalactose, galactosamine, or N-acetylgalactosamine of Formula 1302 is isolated prior to proceeding to the next step.

[0213] As illustrated in Scheme 13, step 2, an antigen, antibody, antibody fragment or ligand having a native free surface amino group of formula 101' is contacted with an excess (corresponding to the value of m) of the product of formula 1302 to give the corresponding product according to formula 1o.

[0214] Compositions corresponding to formula 1o may be named as follows: "F1o-X'-m m -n n -Z'”.

[0215] Reaction Scheme 14

[0216] [ka]

[0217] As illustrated in Scheme 14, an antigen, antibody, antibody fragment or ligand having native free surface amino groups (Formula 101') is contacted with an excess (corresponding to the value of m) of a pyridyldithiol-poly(ethylene glycol)-NHS ester of Formula 1401 to give the corresponding product according to Formula 1p.

[0218] Compositions corresponding to formula 1p may be named as follows: "F1p-X'-m m -n n -p p -2NAcGAL” or “F1p-X'-m m -n n -p p -EtAcN-Z”.

[0219] Preparation of fusion proteins

[0220] The fusion protein composition of formula 2 can be expressed through artificially acceptable methodologies using commercially available mammalian, bacterial, yeast, or insect cell expression vectors, as well as published, discovered, or designed gene sequences. The sequences encoding X, Y, and Z together with the tag sequence can be cloned into an expression vector, for example, into the mammalian expression vector pSecTag A, where the fusion protein is inserted C-terminally into the Ig kappa chain secretion leader sequence. A variety of other cloning techniques, including site-directed mutagenesis and variations of the QuikChange protocol (Geiser, et al.), are available and well known to those skilled in the art. In mammalian cells, for example in human embryonic kidney (HEK293) cells or Chinese hamster ovary (CHO) cells, the fusion protein can be transiently expressed by transient transfection with the vectors described above using polyethyleneimine. For example, the transfected cells are cultured in an appropriate medium (e.g., FreeStyle 293 medium, Life Technologies) supplemented with valproic acid or DMSO for about 7 days, after which the cells are removed by centrifugation and the culture supernatant is collected and sterilized by filtration.

[0221] Alternatively, 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 E. coli, Corynebacterium, or Pseudomonas fluorescens by using compatible media (e.g., LB, 2XYT, SOB, SOC, TB, and other broths), supplements (e.g., glycerol, glucose, and other supplements), and appropriate growth and expression conditions. Expression systems in yeast can generally use Saccharomyces cerevisiae and Pichia pastoris, or other organisms in the genera Saccharomyces, Pichia, Kluyveromyces, and Yarrowia, as well as less commonly used organisms such as the filamentous fungi Aspergillus, Trichoderma, or Myceliophthora thermophila C1. Insect expression systems can utilize baculovirus-infected insect cells or non-lytic insect cell expression to achieve protein expression levels in high quantities; the most common insect cells include, but are not limited to, Sf9 and Sf21 (derived from Spodoptera frugiperda), Hi-5 (derived from Triticum aureum), and Schneider 2 and 3 (derived from Drosophila melanogaster).

[0222] By affinity chromatography (e.g., HisTrap Ni 2+The expressed fusion protein products can be purified from the culture supernatant using a Sepharose column (for His-tagged fusion proteins) followed by other chromatographic purification steps such as size exclusion chromatography (for example, using a Superdex75 column, GE Healthcare) or ion exchange chromatography. Protein purity can be verified, for example, by Coomassie Brilliant Blue staining of SDS-PAGE gels and Western blotting (for example, anti-6xHis tag Western blotting for His-tagged fusion proteins). Protein concentration can be determined using the Beer-Lambert law, for example, by measuring absorbance at 280 nm using a NanoDrop 2000 (Thermo Scientific). Molecular weights and extinction coefficients can be estimated from 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) following the manufacturer's instructions.

[0223] Preparation of desialylated antigens, antibodies, antibody fragments and ligands

[0224] Commercially available neuraminidase (also known as acetylneuraminyl hydrolase or sialidase) enzyme or sulfuric acid can be used to produce desialylated proteins via artificially acceptable methodologies. For enzymatic desialylation of a protein of interest, the protein can be incubated with neuraminidase at 37° C. for 1 hour, or longer as needed. For chemical desialylation by acid hydrolysis, the protein of interest can be treated with 0.025 N sulfuric acid at 80° C. for 1 hour, or longer as needed. The protein can then be desialylated by immobilized metal ion affinity chromatography (e.g., HisTrap Ni). 2+The desialylated protein can be purified from the reaction mixture (e.g., using a Sepharose column, GE Healthcare) followed by size exclusion chromatography (e.g., using a Superdex75 column, GE Healthcare). Protein purity can be verified, for example, by Coomassie Brilliant Blue staining of SDS-PAGE gels and anti-6xHis tag western blotting. Desialylation can be verified, for example, by lectin-based protein detection, sialic acid content in western blots or colorimetric quantification of sialic acid content, for example, using commercially available kits (e.g., Abcam, Prozyme, or Sigma). The desialylated protein concentration can be determined using the Beer-Lambert law, for example, by measuring absorbance at 280 nm using a NanoDrop 2000 (Thermo Scientific). The molecular weight and extinction coefficient can be estimated from 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) following the manufacturer's instructions.

[0225] Specific processes and final steps

[0226] The compound of formula 103' is contacted with an excess (corresponding to the value of m) of a compound of formula 106 to give the corresponding product of formula 1a.

[0227] The compound of formula 103' is contacted with an excess (corresponding to the value of m) of a compound of formula 201 to give the corresponding product of formula 1b.

[0228] A compound of formula 802, 902 or 1002 is contacted with an excess (corresponding to the value of m) of a compound of formula 803 to give the corresponding product of formula 1h, 1i or 1k, respectively.

[0229] A compound of formula 1109 is contacted with an excess (corresponding to the value of m) of a compound of formula 1107 to give the corresponding product of formula 1m, particularly where n is about 80, p is about 30, q is about 4, and m, which is an antigenic function, is about 2 to 10.

[0230] A compound of formula 1202 is contacted with an excess (corresponding to the value of m) of a compound of formula 1107 to give the corresponding product of formula 1n, particularly where n is about 1, p is about 30, q is about 4, and m is about 2 to 10, which is a function of the antigen.

[0231] Specific Composition

[0232] As non-limiting examples, preferred specific groups for the compositions, pharmaceutical formulations, methods of manufacture and uses of the present disclosure are the following combinations and permutations of the substituent groups of Formula 1 (each subgrouped in order of increasing preference): X is a foreign transplant antigen to which the transplant patient develops an unwanted immune response, a foreign antigen to which the patient develops an unwanted immune response, a therapeutic protein to which the patient develops an unwanted immune response, a self-antigen to which the patient develops an unwanted immune response, or a tolerogenic portion thereof. X is a therapeutic protein to which the patient develops an undesirable immune response selected from the following: abatacept, abciximab, adalimumab, adenosine deaminase, ado-trastuzumab emtansine, agalsidase alfa, agalsidase beta, aldesleukin, alglucerase, alglucosidase alfa, alpha-1-protease inhibitor, anakinra, anistreplase (anisoylated plasminogen streptokinase activating complex), antithrombin III, antithymocyte globulin, alteplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitor, canakinumab, carboxypeptidase G2 (glucarpidase and voraxaze), certolizumab Pegol, cetuximab, collagenase, Crotalidae immune Fab, darbepoetin-α, denosumab, digoxin immune Fab, dornase alfa, eculizumab, etanercept, activated factor VII, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histrelin acetate, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin (rHu insulin and bovine insulin), interferon-alpha 2a, interferon-alpha 2b, interferon-beta 1a, interferon-beta 1b, interferon-gamma 1b, ipilimumab, L-arginase, L-asparaginase, L-methionase, lactase, laronidase, lepirudin / hirudin, mecasermin, mecasermin rinfavert, methoxyofatumumab, natalizumab, octreotide, oprelvekin, pancreatic amylase, pancreatic lipase, papain, PEG-asparaginase, PEG-doxorubicinHCl, PEG-epoetin-β, pegfilgrastim, Peg-interferon-α2a, Peg-interferon-α2b, pegloticase, pegvisomant, phenylalanine ammonia-lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, type 1 alpha-interferon, ustekinumab, and vW factor. Specifically, X is abciximab, adalimumab, agalsidase alfa, agalsidase beta, aldesleukin, alglucosidase alfa, factor VIII, factor IX, infliximab, L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL), or rasburicase (uricase). In particular, X is factor VIII, factor IX, uricase, PAL, or asparaginase. · X is an autoantigenic polypeptide selected for treating type 1 diabetes, childhood multiple sclerosis, juvenile rheumatoid arthritis, celiac disease, or alopecia universalis. In particular, X is an autoantigenic polypeptide of choice for treating new onset type 1 diabetes, childhood multiple sclerosis, or celiac disease. X is a foreign antigen to which the patient develops an unwanted immune response: Contains conarachin (Ara h 1), derived from peanuts. those derived from wheat, including native alpha-gliadin “33-mer” (SEQ ID NO:24), deamidated alpha-gliadin “33-mer” (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26) and omega-gliadin (SEQ ID NO:27); Those of feline origin, including Fel d 1A (UNIPROT P30438) and feline albumin (UNIPROT P49064). From dog, including Can f 1 (UNIPROT O18873) and canine albumin (UNIPROT P49822). · X is a foreign transplant antigen, such as a human leukocyte antigen protein, to which the transplant patient develops an unwanted immune response. X is an antibody, antibody fragment or ligand that specifically binds to a circulating protein or peptide or antibody that causes transplant rejection, an immune response to a therapeutic agent, an autoimmune disease, and / or an allergy. In particular, X binds to an endogenous circulating protein or peptide or to an antibody. Y is a linker selected from the following: 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. In particular, 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 of formula Ya, of formula Yb, of formula Ym or of formula Yn. 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. More particularly, 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 Z is conjugated to Y via an ethylacetamide group. More particularly, wherein Z is conjugated to Y at its C1. More particularly, where R 8 is a CMP. More particularly, where R 8 is a CMP. In particular, where R 8 is a CMP. Z is galactose, galactosamine, or N-acetylgalactosamine. In particular, where Z is galactose or N-acetylgalactosamine conjugated at C1, C2 or C6. In particular, where Z is galactose or N-acetylgalactosamine conjugated at C1 or C2. More particularly, wherein Z is N-acetylgalactosamine conjugated at C1.

[0233] Each of the groups and subgroups described above are individually preferred and can be combined to describe further preferred aspects of the present disclosure, such as, but not limited to, the following: · X is an autoantigenic polypeptide selected for treating type 1 diabetes, childhood multiple sclerosis, juvenile rheumatoid arthritis, celiac disease, or alopecia universalis. In particular, wherein X is an autoantigenic polypeptide selected for treating new onset type 1 diabetes, childhood multiple sclerosis or celiac disease. In particular, wherein Y is a linker selected from the following: of formula Ya, of formula Yb, of formula Yh, of formula Yi, of formula Yk, of formula Ym, of formula Yn, of formula Yo and of 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. In particular, 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 of formula Ya, of formula Yb, of formula Ym or of formula Yn. 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. More particularly, 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 particularly, wherein Z is conjugated to Y via an ethylacetamide group. More particularly, wherein Z is conjugated to Y via an ethylacetamide group. In particular, where Z is conjugated to Y via an ethylacetamide group. In particular, where Z is galactose, galactosamine or N-acetylgalactosamine. In particular, where Z is galactose or N-acetylgalactosamine conjugated at C1, C2 or C6. More particularly, wherein Z is a galactose or N-acetylgalactosamine conjugated at C1 or C2. Even more particularly, wherein Z is N-acetylgalactosamine conjugated at C1. In particular, where Z is galactose, galactosamine or N-acetylgalactosamine. In particular, where Z is galactose or N-acetylgalactosamine conjugated at C1, C2 or C6. In particular, where Z is galactose or N-acetylgalactosamine conjugated at C1 or C2. More particularly, wherein Z is N-acetylgalactosamine conjugated at C1. In particular, wherein Y is a linker selected from the following: of formula Ya, of formula Yb, of formula Yh, of formula Yi, of formula Yk, of formula Ym, of formula Yn, of formula Yo and of 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. More particularly, 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 of formula Ya, of formula Yb, of formula Ym or of formula Yn. More particularly, 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, wherein n is from 40 to 80±10%, p is from 30 to 40±10% and q is from 4 to 12±3. More particularly, wherein Z is conjugated to Y via an ethylacetamide group. In particular, where Z is galactose, galactosamine or N-acetylgalactosamine. In particular, where Z is galactose or N-acetylgalactosamine conjugated at C1, C2 or C6. More particularly, wherein Z is a galactose or N-acetylgalactosamine conjugated at C1 or C2. More preferably, wherein Z is N-acetylgalactosamine conjugated at C1. m is an integer from about 1 to 100. m is 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 about 1 to 20. m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. More particularly, m is about 10. m is 9, 10, or 11. n is an integer representing a mixture of about 1 to 100 inclusive. n is 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 between about 8 and 90. In particular, n is 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 particularly, n is between about 40 and 80. More particularly, n is 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83 or 88. n represents a mixture encompassing 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. In particular, n represents a mixture encompassing the ranges 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82, 85-90 and 82-99. More particularly, n represents a mixture encompassing the ranges 36-44, 42-50, 46-57, 60-82 and 75-85. p is an integer representing a mixture of about 2 to 150 inclusive. p is 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. In particular, where n is an integer representing a mixture of about 1 to 100, inclusive. In particular, n is 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 particularly, where n is from about 8 to 90. More particularly, n is 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 particularly, where n is from about 40 to 80. Even more particularly, n is 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83 or 88. More particularly, p is 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 110. In particular, where n is an integer representing a mixture of about 1 to 100, inclusive. In particular, n is 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 particularly, where n is from about 8 to 90. More particularly, n is 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 particularly, where n is from about 40 to 80. Even more particularly, n is 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83 or 88. More particularly, p is 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44. In particular, where n is 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 particularly, where n is from about 8 to 90. More particularly, n is 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 particularly, where n is from about 40 to 80. Even more particularly, n is 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83 or 88. q is an integer ranging from about 1 to 44 inclusive. q is 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.

[0234] As non-limiting examples, preferred specific groups for the compositions, pharmaceutical formulations, methods of manufacture and uses of the present disclosure are the following combinations and permutations of the substituent groups of Formula 2 (each subgrouped in order of increasing preference): X is a therapeutic protein against which the patient develops an undesirable immune response, selected from the following: abatacept, abciximab, adalimumab, adenosine deaminase, ado-trastuzumab emtansine, agalsidase alfa, agalsidase beta, aldesleukin, alglucerase, alglucosidase alfa, alpha-1-protease inhibitor, anakinra, anistreplase (anisoylated plasminogen streptokinase activating complex), antithrombin III, antithymocyte globulin, alteplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitor, canakinumab, carboxypeptidase G2 (glucarpidase and voraxaze), certolizumab Pegol, cetuximab, collagenase, Crotalidae immune Fab, darbepoetin-α, denosumab, digoxin immune Fab, dornase alfa, eculizumab, etanercept, activated factor VII, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histrelin acetate, hyaluronidase, idursulfase, imiglucerase, infliximab, insulin, interferon-α2 a, interferon-α2b, interferon-β1a, interferon-β1b, interferon-γ1b, ipilimumab, L-arginase, L-asparaginase, L-methioninase, lactase, laronidase, lepirudin / hirudin, mecasermin, mecasermin rinfavert, methoxyofatumumab, natalizumab, octreotide, oprelvekin, pancreatic amylase, pancreatic lipase, papain, PEG-asparaginase, PEG-doxorubicinHCl, PEG-epoetin-β, pegfilgrastim, Peg-interferon-α2a, Peg-interferon-α2b, pegloticase, pegvisomant, phenylalanine ammonia-lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, type 1 alpha-interferon, ustekinumab, and vW factor; m'+m" equals 1 and Z is DOM. With the proviso that excluded from the scope of Formula 2 are interferons (interferon alpha 2, interferon alpha 5, interferon alpha 6, or consensus interferon), ribavirin, nexavar / sorafenib, erbitux / cetuximab, avastatin / bevacizumab, and herceptin / trastuzumab if the interferon is another liver-targeted molecule described in US 2013 / 0078216 or US 26h-196-61. · Specifically, X is abciximab, adalimumab, agalsidase alfa, agalsidase beta, aldesleukin, alglucosidase alfa, factor VIII, factor IX, infliximab, L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL), or rasburicase (uricase). In particular, where X is Factor VIII, Factor IX, uricase, PAL or asparaginase. · X is an autoantigenic polypeptide selected for treating type 1 diabetes, childhood multiple sclerosis, juvenile rheumatoid arthritis, celiac disease, or alopecia universalis. In particular, wherein X is an autoantigenic polypeptide selected for treating new onset type 1 diabetes, childhood multiple sclerosis or celiac disease. X is a foreign antigen to which the patient develops an unwanted immune response Contains conarachin (Ara h 1), derived from peanuts. those derived from wheat, including native alpha-gliadin “33-mer” (SEQ ID NO:24), deamidated alpha-gliadin “33-mer” (SEQ ID NO:25), alpha-gliadin (SEQ ID NO:26) and omega-gliadin (SEQ ID NO:27); Those of feline origin, including Fel d 1A (UNIPROT P30438) and feline albumin (UNIPROT P49064). From dog, including Can f 1 (UNIPROT O18873) and canine albumin (UNIPROT P49822). · X is a foreign transplant antigen, such as a human leukocyte antigen protein, to which the transplant patient develops an unwanted immune response. X is an antibody, antibody fragment or ligand that specifically binds to a circulating protein or peptide or antibody that causes transplant rejection, an immune response to a therapeutic agent, an autoimmune disease, and / or an allergy. In particular, where X binds to an endogenous circulating protein or peptide or an antibody. Y is for Gly 3 It's Ser. Z is anti-ASGPR Dom26h-196-61 or a conservative substitution. m'+m" is equal to 1 or 2 and X is a full-length protein comprising a protein therapeutic. m'+m" is equal to 1 or 2, where m' is 1 and m" is 0 or 1. m'+m" is equal to 2 to about 10, where X is a group of autoantigenic peptides (epitopes) well known to be associated with a particular autoimmune disease or as a treatment for a genetically diverse target population. m'+m" is equal to about 4 to 7, where 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 equal to 7 and X is, 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).

[0235] Similar to the discussion above regarding Formula 1, each of the groups and subgroups for Formula 2 described above are individually preferred and can be combined to describe further preferred embodiments of the present disclosure.

[0236] Availability, Testing and Administration

[0237] General usefulness

[0238] The compositions of the present disclosure find use in a variety of applications, including the treatment of transplant rejection, immune responses to therapeutic agents, autoimmune diseases, and food allergies, as will be appreciated by those of skill in the art.

[0239] In a preferred embodiment, the compositions of the present disclosure are used to modulate, in particular downregulate, unwanted antigen-specific immune responses.

[0240] The compositions of the present disclosure are useful for binding to and clearing circulating specific undesirable proteins that cause autoimmunity and related pathologies, allergies, inflammatory immune responses, and anaphylaxis, including antibodies (i.e., antibodies that are not exogenous and administered to the patient) that are endogenously produced in the patient, peptides, and the like.

[0241] In the present disclosure, antigen is targeted to liver for presentation via antigen-presenting cells to specifically downregulate immune system, or for clearance of undesirable circulating protein.This is different from the previous use of liver targeting, for example, as described in US 2013 / 0078216, where the purpose of liver targeting molecule such as DOM26h-196-61 is to deliver therapeutic agent to treat liver disease, such as fibrosis, hepatitis, cirrhosis and liver cancer.

[0242] The present disclosure provides compositions and methods for treating unwanted immune responses to self-antigens and foreign antigens, including, but not limited to: foreign transplant antigens to which a transplant patient develops an unwanted immune response (e.g., transplant rejection), foreign antigens to which a patient develops an unwanted immune response (e.g., allergic or hypersensitivity), therapeutic agents to which a patient develops an unwanted immune response (e.g., hypersensitivity and / or reduced therapeutic activity), and self-antigens to which a patient develops an unwanted immune response (e.g., autoimmune disease).

[0243] Autoimmune disease conditions that can be treated using the methods and compositions provided herein include, but are not limited to, acute disseminated encephalomyelitis (ADEM); acute interstitial allergic nephritis (drug allergy); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; alopecia areata; alopecia universalis; ankylosing spondylitis; juvenile arthritis; psoriatic arthritis; rheumatoid arthritis; atopic dermatitis; autoimmune aplastic anemia; autoimmune gastritis; autoimmune hepatitis; autoimmune hypophysitis; autoimmune oophoritis; autoimmune orchitis; polyglandular autoimmune syndrome type 1; polyglandular autoimmune syndrome type 2; autoimmune thyroiditis; Behcet's disease; bronchiolitis obliterans; bullous pemphigoid; celiac disease; Churg-Strauss syndrome; chronic inflammatory demyelinating polyneuropathy; cicatricial pemphigoid; Crohn's disease; Coxsackie syndrome; Key myocarditis;Dermatitis herpetiformis;Diabetes mellitus (type 1);Erythema nodosum;Epidermolysis bullosa acquisita;Giant cell arteritis (temporal arteritis);Giant cell myocarditis;Goodpasture's syndrome;Graves' disease;Guillain-Barré syndrome;Hashimoto's encephalopathy;Hashimoto's thyroiditis;IgG4-related sclerosing disease;Lambert-Eaton syndrome;Mixed connective tissue disease;Much-Habermann disease;Multiple sclerosis;Myasthenia gravis;Ophthalmology Neurotis; neuromyelitis optica; pemphigus vulgaris and variants; pernicious anemia; pituitary autoimmune disease; polymyositis; postpericardiotomy syndrome; premature ovarian failure; primary biliary cirrhosis; primary sclerosing cholangitis; psoriasis; rheumatic heart disease; Sjogren's syndrome; systemic lupus erythematosus; systemic sclerosis; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vitiligo; and Wegener's granulomatosis.

[0244] Particular groups of autoimmune disease conditions that can be treated using the methods and compositions provided 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; polyglandular autoimmune syndrome type 1; bullous pemphigoid; celiac disease; Coxsackie myocarditis; Juhring dermatitis herpetiformis; diabetes mellitus (type 1); epidermolysis bullosa acquisita; giant cell myocarditis; Goodpasture's syndrome; Graves' disease; Hashimoto's thyroiditis; mixed connective tissue disease; multiple sclerosis; myasthenia gravis; neuromyelitis optica; pernicious anemia; pemphigus vulgaris and variant types; pituitary autoimmune diseases; premature ovarian failure; rheumatic heart disease; systemic sclerosis; Sjogren's syndrome; systemic lupus erythematosus; and vitiligo.

[0245] In embodiments utilizing antigens such as food antigens that elicit an unwanted immune response, treatments can be provided for reactions to, for example: peanuts, apples, milk, egg whites, egg yolks, mustard, celery, shrimp, wheat (and other grains), strawberries, and bananas.

[0246] One skilled in the art will appreciate that patients may be tested to identify foreign antigens to which they have developed an unwanted immune response, and compositions of the present disclosure may be developed based on the antigens.

[0247] test

[0248] To establish the usefulness of the compositions and methods of the present disclosure, the specificity of binding to antigen-presenting cells in the liver (especially binding to hepatocytes, specifically ASGPR) should be determined first. In the compositions of the present disclosure, this can be achieved, for example, by utilizing a marker, such as the fluorescent marker phycoerythrin ("PE"). The composition is administered to appropriate experimental subjects. A control, such as unconjugated PE, or vehicle (saline) is administered to another group of subjects. The composition and control are allowed to circulate for 1 to 5 hours, after which the subject's spleen and liver are harvested and measured for fluorescence. The specific cells that are identified as fluorescent can be subsequently identified. When tested in this manner, the compositions of the present disclosure show higher levels of concentration in antigen-presenting cells in the liver compared to unconjugated PE or vehicle.

[0249] In response to administration of a composition of the present disclosure incorporating a well-known antigen, such as ovalbumin ("OVA"), compared to administration of the antigen alone or simply vehicle, OT-I CD8 + The efficacy in immunomodulation can be tested by measuring the proliferation of the cells (implanted into a host mouse). When tested in this manner, the compositions of the present disclosure show increased CD8 + We show increased OT1 cell proliferation, indicating T cell cross-priming. To distinguish T cells expanding to a functional effector phenotype from those that are expanded and deleted, we analyzed the proliferation of OT-I CD8 + T cells can be phenotypically analyzed for molecular signatures of exhaustion [such as programmed death-1 (PD-1), FasL, and others], as well as annexin V as a hallmark of apoptosis and therefore elimination. OT-I CD8 +T cells can also be evaluated for their responsiveness to antigen challenge with adjuvant to demonstrate functional non-responsiveness and thus immune tolerance to the antigen.To do so, cells are analyzed for inflammatory signatures after administration of the composition of the present disclosure to host mice followed by antigen challenge.When tested in this manner, the composition of the present disclosure suppresses very low (e.g., background) levels of inflammatory OT-I CD8 T cells to OVA. + It is demonstrated that it induces a T cell response and thus immune tolerance.

[0250] Humoral immune responses can be tested by administering compositions of the present disclosure incorporating a well-known antigen, such as OVA, and measuring the resulting levels of antibodies, compared to administering the antigen alone or simply the vehicle. When tested in this manner, the compositions of the present disclosure show very low (e.g., background) levels of antibody formation in response to their administration and to administration of the vehicle, and significantly higher levels of antibody formation in response to administration of the antigen.

[0251] The effectiveness of tolerizing to an antigen can be tested as described above for humoral immune response, where a group of subjects is challenged with the antigen alone several weeks after treatment with the composition of the present disclosure, and the level of antibodies to the antigen is then measured. When tested in this manner, the composition of the present disclosure shows a lower level of antibody formation in response to antigen challenge in groups pretreated with such compositions compared to groups not pretreated.

[0252] Disease-focused 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, as well as 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 negative control, followed by blood glucose measurement. Successful treatment corresponds to a mechanistic proof of concept for the possibility of treating diabetes in humans, or an approach to the treatment of other autoimmune diseases. (See, e.g., Anderson and Bluestone, Annu. Rev. Immunol. 2005;23:447-85.)

[0253] Administration

[0254] The compositions of the present disclosure are administered at a therapeutically effective dose, e.g., a dose sufficient to provide treatment for the disease conditions previously described. Administration of the compounds of the present disclosure, or pharma- ceutically acceptable salts thereof, can be via any of the accepted modes of administration for drugs that serve similar utilities.

[0255] Although human dosage levels have not yet been optimized for the compounds of the present disclosure, these can be estimated at doses of about 10 μg to 100 μg administered initially for mice. In general, individual human doses are about 0.01 to 2.0 mg / kg body weight, preferably about 0.1 to 1.5 mg / kg body weight, and most preferably about 0.3 to 1.0 mg / kg body weight. Treatment can be administered for a period of one or several days, and can be repeated at intervals of several days, one or several weeks, or one or several months. Administration can be as a single dose (e.g., as a bolus) or as an initial bolus, followed by continuous infusions of the remaining portion of the full dose over time, e.g., one to seven days. The amount of active compound administered will, of course, depend on any or all of the following: the subject and disease state being treated, the severity of the affliction, the mode and schedule of administration, and the judgment of the prescribing physician. It will be appreciated that the amount administered will also depend on the molecular weight of the antigen, antibody, antibody fragment, or ligand, as well as the size of the linker.

[0256] The compositions of the present disclosure can be administered either alone or in combination with other pharma- ceutically acceptable excipients. All typical routes of administration are contemplated, with the present invention preferably providing a liquid dosage form suitable for injection. The formulation typically includes a conventional pharmaceutical carrier or excipient and the composition of the present disclosure or a pharma-ceutically acceptable salt thereof. In addition, these compositions can include other medicinal agents, pharmaceutical agents, carriers, etc., including, but not limited to, therapeutic proteins, peptides, antibodies or antibody-like molecules corresponding to the antigen (X) utilized in the compositions of the present disclosure, as well as other active agents that can act as immunomodulators and, more specifically, have inhibitory effects on B cells, including antifolates, immunosuppressants, cytostatics, mitotic inhibitors, and antimetabolites, or combinations thereof.

[0257] Generally, depending on the intended mode of administration, a pharma- ceutically acceptable composition will contain from about 0.1% to 95%, preferably from about 0.5% to 50%, by weight of the compositions of the present disclosure, with the remainder being suitable pharmaceutical excipients, carriers, etc. Dosage forms or compositions containing active ingredient in the range of 0.005% to 95%, the balance consisting of non-toxic carriers, may be prepared.

[0258] Liquid pharma- ceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc. The active composition of the present disclosure (e.g., lyophilized powder) and any pharmaceutical adjuvants in a carrier, such as, for example, water (water for injection), saline, aqueous dextrose, glycerin, glycol, ethanol, etc. (except galactose), thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, stabilizers, solubilizers, pH buffering agents, etc., such as, for example, sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, and triethanolamine oleate, osmolytes, amino acids, sugars and carbohydrates, proteins and polymers, salts, surfactants, chelating agents and antioxidants, preservatives, and specific ligands. Actual methods of preparing such dosage forms are well known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd Edition, 2012. The composition or formulation to be administered will, in any event, contain a quantity of active compound in an amount effective to treat the condition of the subject being treated.

[0259] Working Example The following examples serve to more fully describe the manner of use of the disclosure described above, as well as the best manner envisioned for carrying out various aspects of the disclosure. These examples should in no way be construed as limiting the true scope of the disclosure, but are instead presented for illustrative purposes. All references cited herein are incorporated by reference in their entirety.

[0260] Example 1 F1aA-OVA-m 4 -n 80 (or F1a-OVA-m 4 -n 80 -2NGAL)

[0261] Example 1A. Formula 103' where X' is OVA and m is 4

[0262] In an endotoxin-free tube, OVA (5.0 mg, 0.00012 mmol) was added to 100 μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg of Traut's reagent was dissolved in 100 μl of pH 7.0 PBS, and then 16 μl (0.00119 mmol) of the Traut's reagent solution thus obtained was added to the stirred solution of OVA with continued stirring. After 1 h, the excess Traut's reagent was removed using a centrifugal size-exclusion column to obtain the corresponding product of formula 103'.

[0263] Example 1B. Formula 106A where n is 80

[0264] In an endotoxin-free tube, galactosamine (10.0 mg, 0.04638 mmol) was dissolved in 100 μl of pH 8.0 PBS containing 5 mM EDTA with stirring. Pyridyldithiol-poly(ethylene glycol)-NHS ester (n is 80, Formula 104) (16.23 mg, 0.00464 mmol) dissolved in 100 μl of pH 7.0 PBS was added to the stirred solution of galactosamine. After 1 hour, the resulting pyridyldithiol-poly(ethylene glycol)-N-acetylgalactosamine (Formula 106A) was ready for use without further purification.

[0265] Example 1C. Formula 1aA where X' is OVA, m is 4, and n is 80 (and Z' is C2 galactosamine)

[0266] The purified OVA-trout conjugate of formula 103' prepared in Example 1A was added directly to the stirred product of formula 106A prepared in Example 1B. After 1 hour, the reaction mixture was passed through a centrifugal size exclusion column to purify the product of formula 1a. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product. (See FIG. 5.)

[0267] Example 1D. Other Compounds of Formula 103'

[0268] By following the procedure described in Example 1A and substituting OVA with: Abciximab, Adalimumab, Agalsidase alfa, Agalsidase beta, Aldesleukin, Alglucosidase alfa, · 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), MART1 (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), · Konarakin (UNIPROT Q6PSU6), Natural alpha-gliadin “33-mer” (SEQ ID NO:24), Deamidated alpha-gliadin “33-mer” (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), The following corresponding compounds of formula 103' are obtained, where: X is abciximab and m is 10; X is adalimumab and m is 11; X is agalsidase alfa and m is 14; X is agalsidase beta and m is 14; X is aldesleukin and m is 6; X is alglucosidase alfa 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 is rasburicase and m is 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 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 is conarachin and m is 21; X is the native alpha-gliadin “33-mer” (SEQ ID NO:24) and m is 1; X is deamidated alpha-gliadin “33-mer” (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 is Fel d 1 and m is 4; X is feline albumin and m is 16; X is Can f 1 and m is 6, X is canine albumin and m is 23; and X is an RhCE example and m is 10.

[0269] Example 1E. Other Compounds of Formula 1aA

[0270] By following the procedure described in Example 1C and substituting the compound of formula 103′, for example as obtained in Example 1D, the following corresponding compounds of formula 1aA are obtained: F1aA-abciximab-m 10 -n 80 , F1aA-adalimumab-m 11 -n 80 , F1aA-Agalsidase alpha-m 14 -n 80 , F1aA-Agalsidase beta-m 14 -n 80 , F1aA-Aldesleukin-m 6 -n 80 , F1aA-alglucosidase alpha-m 13 -n 80 , · F1aA-factor VIII-m 100 -n 80 , · F1aA-Factor IX-m 18 -n 80 , F1aA-L-asparaginase-m 5 -n 80 , F1aA-Laronidase-m 7 -n 80 , F1aA-Octreotide-m 1 -n 80 , F1aA-phenylalanine ammonia-lyase-m 12 -n 80 , F1aA-rasburicase-m 12 -n 80 , F1aA-insulin-m 2 -n 80 , F1aA-GAD-65-m 8 -n 80 , F1aA-IGRP-m7 -n 80 , F1aA-MBP-m 6 -n 80 , F1aA-MOG-m 5 -n 80 , F1aA-PLP-m 8 -n 80 , F1aA-MBP13-32-m 1 -n 80 , F1aA-MBP83-99-m 1 -n 80 , F1aA-MBP111-129-m 1 -n 80 , F1aA-MBP146-170-m 2 -n 80 , F1aA-MOG1-20-m 1 -n 80 , F1aA-MOG35-55-m 2 -n 80 , F1aA-PLP139-154-m 3 -n 80 , F1aA-MART1-m 4 -n 80 , F1aA-tyrosinase-m 8 -n 80 , F1aA-PMEL-m 5 -n 80 , F1aA-Aquaporin 4-m 4 -n 80 , F1aA-S-arrestin-m 12 -n 80 , F1aA-IRBP-m 21 -n 80 , F1aA-Conarakin-m 21 -n 80, F1aA-Native alpha-gliadin “33-mer”-m 1 -n 80 , F1aA-deamidated alpha-gliadin “33-mer”-m 1 -n 80 , F1aA-alpha-gliadin-m 1 -n 80 , F1aA-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-m 6 -n 80 , F1aA-Canine Albumin-m 23 -n 80 , and F1aA-RhCE-m 10 -n 80 .

[0271] Example 1F. Other Compounds of Formula 106A

[0272] By following the procedure described in Example 1B and substituting pyridyldithiol-poly(ethylene glycol)-NHS ester (formula 104, where n is 80) with: Equation 104, where n is 12; Equation 104, where n is 33; Equation 104, where n is 40; Equation 104, where n is 43; Equation 104, where n is 50; Equation 104, where n is 60; Equation 104, where n is 75, and Equation 104, where n is 80; The following corresponding compounds of formula 106A are obtained, wherein: n is 12, n is 33, n is 40, n is 43, n is 50, n is 60, n is 75, and n is 84.

[0273] Example 1G. Other Compounds of Formula 1aA

[0274] By following the procedure described in Example 1E and substituting the compound of formula 106A with the compound obtained in Example 1F, the corresponding compounds of formula 1aA, where n is 12, 33, 40, 43, 50, 60, 75 and 84, are obtained: F1aA-insulin-m 2 -n 12 , F1aA-insulin-m 2 -n 33 , F1aA-insulin-m 2 -n 40 , F1aA-insulin-m 2 -n 43 , F1aA-insulin-m 2 -n 50 , F1aA-insulin-m 2 -n 60 , F1aA-insulin-m 2 -n 75 , and F1aA-insulin-m 2 -n 84 And so on.

[0275] Example 2

[0276] F1b-OVA-m 1 -n 4 -p 34 -2NAcGAL

[0277] Example 2A. Formula 103' where X' is ovalbumin and m is 1

[0278] In an endotoxin-free tube, OVA (6.5 mg, 0.000155 mmol) was added to 200 μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg of Traut's reagent was dissolved in 100 μl of pH 7.0 PBS, and 43 μl (0.00310 mmol) of the thus obtained Traut's reagent solution was added to the stirred solution of OVA with continued stirring. After 1 h, unreacted Traut's reagent was removed using a centrifugal size-exclusion column to obtain the product of formula 103'.

[0279] Example 2B. X' is ovalbumin, m is 1, n is 4, p is 34, and R 9 Formula 1b, where Z″ is a direct bond and Z″ is 2NAcGAL

[0280] In a microcentrifuge tube, poly(galactosamine methacrylate)-(pyridyl disulfide) (Formula 201) (20.0 mg, 0.0020 mmol) was solubilized in 50 μl of pH 8.0 PBS containing 5 mM EDTA. To this was added the purified OVA-trout product from Example 2A, followed by stirring for 1 hour. The product of Formula 1b was purified from a centrifugal size exclusion column by passing the reaction mixture through it. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product. (See FIG. 5.)

[0281] Example 2C. Other compounds of formula 1b

[0282] By following the procedure described in Example 2B and substituting the compound of formula 103′, for example as obtained in Example 1D, the following corresponding compounds of formula 1b are obtained: F1b-abciximab-m 10 -n 4 -p 34 -2NAcGAL, F1b-adalimumab-m11 -n 4 -p 34 -2NAcGAL, F1b-agalsidase alpha-m 14 -n 4 -p 34 -2NAcGAL, F1b-agalsidase beta-m 14 -n 4 -p 34 -2NAcGAL, F1b-Aldesleukin-m 6 -n 4 -p 34 -2NAcGAL, F1b-alglucosidase alpha-m 13 -n 4 -p 34 -2NAcGAL, · F1b-factor VIII-m 100 -n 4 -p 34 -2NAcGAL, · F1b-factor IX-m 18 -n 4 -p 34 -2NAcGAL, F1b-L-asparaginase-m 5 -n 4 -p 34 -2NAcGAL, F1b-Laronidase-m 7 -n 4 -p 34 -2NAcGAL, F1b-Octreotide-m 1 -n 4 -p 34 -2NAcGAL, F1b-phenylalanine ammonia-lyase-m 12 -n 4 -p 34 -2NAcGAL, F1b-rasburicase-m 12 -n 4 -p 34 -2NAcGAL, F1b-insulin-m 2 -n4 -p 34 -2NAcGAL、 · F1b-GAD-65-m 8 -n 4 -p 34 -2NAcGAL、 · F1b-IGRP-m 7 -n 4 -p 34 -2NAcGAL、 · F1b-MBP-m 6 -n 4 -p 34 -2NAcGAL、 · F1b-MOG-m 5 -n 4 -p 34 -2NAcGAL、 · F1b-PLP-m 8 -n 4 -p 34 -2NAcGAL、 · F1b-MBP13-32-m 1 -n 4 -p 34 -2NAcGAL、 · F1b-MBP83-99-m 1 -n 4 -p 34 -2NAcGAL、 · F1b-MBP111-129-m 1 -n 4 -p 34 -2NAcGAL、 · F1b-MBP146-170-m 2 -n 4 -p 34 -2NAcGAL、 · F1b-MOG1-20-m 1 -n 4 -p 34 -2NAcGAL、 · F1b-MOG35-55-m 2 -n 4 -p 34 -2NAcGAL、 · F1b-PLP139-154-m 3 -n 4 -p 34 -2NAcGAL、 F1b-MART1-m 4 -n 4 -p 34 -2NAcGAL, F1b-tyrosinase-m 8 -n 4 -p 34 -2NAcGAL, F1b-PMEL-m 5 -n 4 -p 34 -2NAcGAL, F1b-Aquaporin 4-m 4 -n 4 -p 34 -2NAcGAL, F1b-S-arrestin-m 12 -n 4 -p 34 -2NAcGAL, F1b-IRBP-m 21 -n 4 -p 34 -2NAcGAL, F1b-Conarakin-m 21 -n 4 -p 34 -2NAcGAL, F1b-Native alpha-gliadin “33-mer”-m 1 -n 4 -p 34 -2NAcGAL, F1b-deamidated alpha-gliadin “33-mer”-m 1 -n 4 -p 34 -2NAcGAL, F1b-alpha-gliadin-m 1 -n 4 -p 34 -2NAcGAL, F1b-omega-gliadin-m 1 -n 4 -p 34 -2NAcGAL, F1b-Fel d 1-m 4 -n 4 -p 34 -2NAcGAL, F1b-Feline Albumin-m 16 -n 4 -p 34 -2NAcGAL, F1b-Can f 1-m 6 -n 4 -p 34 -2NAcGAL, F1b-Canine Albumin-m 23 -n 4 -p 34 -2NAcGAL, and F1b-RhCE-m 10 -n 4 -p 34 -2NAcGAL.

[0283] Example 3

[0284] F1f-OVA-m 1 -n 4 -p 33 -2NAcGAL

[0285] Example 3A. X' is ovalbumin, m is 1, n is 4, p is 33, and R 9 Formula 1f, where Z″ is a direct bond and Z″ is 2NAcGAL

[0286] In an endotoxin-free tube, OVA (4.0 mg, 0.0000952381 mmol) was added to 0.1 ml of pH 7.4 PBS and vortexed. Separately, poly-(N-acetylgalactosamine)-p-nitrophenyl carbonate (33.0 mg, 0.002380952 mmol) of formula 601, where n is 4 and p is 33, was added to 100 μl of pH 7.5 PBS and vortexed until dissolved. The two solutions were combined and the mixture was vigorously stirred for 1 hour. The mixture was then collected and dialyzed against pH 7.4 PBS for 3 days (30 kDa molecular weight cutoff) to obtain the product of formula 1f.

[0287] Example 4

[0288] F1g-PVA-m 1 -p90 -2NAcGAL

[0289] Example 4A. X' is ovalbumin, m is 1, p is 90, and R 9 Formula 1g, where Z″ is a direct bond and Z″ is 2NAcGAL

[0290] In an endotoxin-free tube, OVA (5.0 mg, 0.000119048 mmol) was added to 0.2 ml of pH 7.4 PBS and stirred. To the stirred solution was added 75 mg (0.00297619 mmol) of poly(galactosamine methacrylate)-NHS (Formula 701) dissolved in 0.4 ml of pH 7.4 PBS. The mixture was stirred for 2 hours. The mixture was then collected and dialyzed against pH 7.4 PBS for 3 days (30 kDa molecular weight cutoff) to obtain the product of Formula 1g.

[0291] Example 5

[0292] F1h-OVA-m 2 -n 45 -p 55 -q 4 -2NAcGAL

[0293] Example 5A. Formula 802' where X' is ovalbumin, m is 2, and n is 45

[0294] In an endotoxin-free tube, OVA (3.0 mg, 0.0000714286 mmol) was added to 50 μl of pH 8.0 PBS containing 15 mM EDTA and stirred. Dibenzocyclooctyne-PEG-(p-nitrophenyl carbonate) (Formula 801) (5.265 mg, 0.002142857 mmol) dissolved in DMF was added to the OVA solution and stirred for 1 h. Excess dibenzocyclooctyne-PEG-(p-nitrophenyl carbonate) was removed using a centrifugal size exclusion column to obtain the product of Formula 802'.

[0295] Example 5B. X' is ovalbumin, m is 2, n is 45, p is 55, q is 4, and R 8 But, CH 2 and R 9 Formula 1h, where Z″ is a direct bond and Z″ is 2NAcGAL

[0296] 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 of pH 7.4 PBS and the product of Example 5A was added with stirring. After 1 hour, the resulting product of Formula 1h was purified by centrifugal size exclusion chromatography.

[0297] Example 6

[0298] F1j-OVA-m 10 -n 45 -p 55 -q 4 -2NAcGAL

[0299] Example 6A. Formula 103' where X' is ovalbumin and m is 10

[0300] In an endotoxin-free tube, OVA (5.0 mg, 0.00019 mmol) was added to 50 μl of pH 8.0 PBS containing 15 mM EDTA and stirred. Separately, 1 mg of Traut's reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl (0.0019 mmol) of the thus obtained Traut's reagent solution was added to the stirred solution of OVA with continued stirring. After 1 h, unreacted Traut's reagent was removed using a centrifugal size-exclusion column to obtain the product of formula 103'.

[0301] Example 6B. Formula 902" Wherein X' is ovalbumin, m is 10, and n is 45

[0302] 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 obtained in Example 6A and stirred for 1 hour. Excess dibenzocyclooctyne-PEG-(pyridyl disulfide) was removed using centrifugal size exclusion chromatography to obtain the product of Formula 902″.

[0303] Example 6C. X' is ovalbumin, m is 10, n is 45, p is 55, q is 4, and R 8 But, CH 2 and R 9 Formula 1j, where Z″ is a direct bond and Z″ is 2NAcGAL

[0304] 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 of pH 7.4 PBS and the product of Example 6B was added with stirring. After 1 h, the resulting product of Formula 1j was purified by centrifugal size exclusion chromatography (excess p(GMA)-N3 was removed). Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

[0305] Example 7

[0306] F1L-OVA-m 2 -n 80 -p 55 -q 4 -2NAcGAL

[0307] Example 7A. Formula 1002, Wherein X' is ovalbumin, m is 2, and n is 80

[0308] 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 a purified OVA solution of Formula 103' (where X' is ovalbumin and m is 2), prepared for example as described in Example 6A, and stirred for 1 hour. Excess dibenzocyclooctyne-PEG-(pyridyl disulfide) was removed using centrifugal size exclusion chromatography to obtain the product of Formula 1002.

[0309] Example 7B. X' is ovalbumin, m is 2, n is 80, p is 55, q is 4, and R 8 But, CH 2 and R 9 Formula 1L, where Z″ is a direct bond and Z″ is 2NAcGAL

[0310] 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 of pH 7.4 PBS and the product of Example 7A was added with stirring. After 1 h, the resulting product of Formula 1L was purified by centrifugal size exclusion chromatography (excess poly(galactosamine methacrylate)-N3 is removed). Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

[0311] Example 8

[0312] Preparation of poly(galactosamine methacrylic acid) polymers.

[0313] Example 8A. Galactosamine Methacrylate

[0314] To the stirred galactosamine hydrochloride (2.15 g, 10.0 mmol) was added 0.5 M sodium methoxide (22 ml, 11.0 mmol). After 30 min, methacrylic anhydride (14.694 g, 11.0 mmol) was added and stirred continuously for 4 h. The resulting galactosamine methacrylate was loaded onto silica gel via rotary evaporation and purified via column chromatography using DCM:MeOH (85:15).

[0315] Example 8B. Formula 201 where n is 4 and p is 30

[0316] 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 of 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 under 12 ml of methanol and excess solvent was removed under reduced pressure.

[0317] Example 9

[0318] F1aA-PE-m 3 -n 80 Preparation of

[0319] Example 9A. Formula 103' where X' is phycoerythrin

[0320] In an endotoxin-free tube, phycoerythrin ("PE") (purchased from Pierce) (200 μl, 0.000004 mmol) was added to 50 μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg of Taut's reagent was dissolved in 100 μl of pH 7.0 PBS, and 2 μl (0.00013 mmol) of the thus obtained Traut's reagent solution was added to the stirred solution of PE with continued stirring. After 1 hour, the excess Traut's reagent was removed using a centrifugal size exclusion column to obtain the product of formula 103'.

[0321] Example 9B. Formula 106A where n is 80

[0322] In an endotoxin-free tube, galactosamine (7.0 mg, 0.03246 mmol) was dissolved in 100 μl of pH 8.0 PBS containing 5 mM EDTA with stirring. Pyridyldithiol-poly(ethylene glycol)-NHS ester (n is 80, Formula 104) (16.23 mg, 0.00464 mmol) dissolved in 50 μl of pH 7.0 PBS was added to the stirred solution of galactosamine. After 1 hour, the resulting product of Formula 106A was ready to be used without further purification.

[0323] Example 9C. Formula 1a, Wherein X' is phycoerythrin, m is 3, n is 80, and Z' is galactosamine

[0324] The purified PE-Trout conjugate prepared in Example 9A was added directly to the stirred product of Formula 106A prepared in Example 9B. After 1 hour, the reaction mixture was passed through a centrifugal size exclusion column to purify the product of Formula 1a. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

[0325] Example 10

[0326] OVA-DOM

[0327] 10A. Preparation of expression vectors

[0328] The mammalian cell expression vector pSecTag A was purchased from Life Technologies. The gene encoding the anti-ASGPR domain antibody, Dom26h-196-61, referred to herein as "DOM", was purchased from the provider GenScript as a codon-optimized sequence for protein expression in human cells. The mammalian expression vector pSecTag A was engineered by site-directed mutagenesis following a variation of the QuikChange protocol (Geiser, et al.) to incorporate the Ig κ chain secretion leader sequence OVA, DOM, and the flexible linker Gly at the C-terminus. 3 The sequences encoding the Ser and 6xHis tags were cloned.

[0329] 10B. m' is 1, m" is 0, X is ovalbumin, and Y is Gly 3 Expression and purification of Formula 2 where Ser is and Z is anti-ASGPR Dom26h-196-61

[0330] HEK293 cells were transiently transfected with a modified pSecTag A vector, prepared, for example, as described in Example 10A, using polyethylenimine. The 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 culture supernatant was collected and sterilized by filtration. HisTrap Ni 2+ The OVA / DOM fusion protein of formula 2 was purified from the culture supernatant by immobilized metal ion affinity chromatography using a Sepharose column (GE Healthcare), followed by size exclusion chromatography using a Superdex75 column (GE Healthcare), which has the following generalized structure: N-OVA-Gly 3 Ser-DOM-Gly 3 Ser-6xHis-C and having the following amino acid sequence: GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPI NFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKL TEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSHHHHH (SEQ ID NO: 28).

[0331] Protein purity was verified by Coomassie Brilliant Blue staining of SDS-PAGE gels and anti-6xHis tag western blotting. Protein concentration was determined using the Beer-Lambert law, for which the absorbance at 280 nm was measured using a NanoDrop 2000 (Thermo Scientific) and the molecular weight (57.3 kDa) and extinction coefficient (57,090 M -1 cm -1 ) was deduced from the amino acid sequence of the 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.

[0332] 10C. Other Compositions of Formula 2

[0333] By following the procedures described in Examples 10A and 10B and substituting the pSecTag A vector, the following fusion protein of formula 2 was obtained accordingly: N-DOM-Gly 3 Ser-OVA-Gly 3 Ser-6xHis-C having the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSGSIGAASME FCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQAR ELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH (SEQ ID NO: 29); and N-OVA-Gly 3 Ser-DOM-Gly 3 Ser-OVA-Gly 3 Ser-6xHis-C and having the following amino acid sequence: GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASHKRHEHTRFDSWGQGTLVTVSSGGGSGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH(SEQ ID NO: 30).

[0334] 10D. Other compositions of Formula 2

[0335] Following the procedures described in Examples 10A and 10B, and using a Gly for the linker with an immunoproteasome cleavage site ("IPC") 3 By substituting the Ser vector, and the OVA vector for: 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), or a vector for: Deamidated alpha-gliadin "33-mer" (SEQ ID NO:25) alpha-gliadin (SEQ ID NO:26), and omega-gliadin (SEQ ID NO:27), A fusion protein of formula 2 is obtained: MBP13-32-IPC-MBP83-99-IPC-MBP111-129-IPC-MBP146-170-IPC-MOG1-20-IPC-MOG35-55-IPC-PLP139-154-IPC-DOM, and Deamidated alpha-gliadin “33-mer”-IPC-alpha-gliadin-IPC-omega-gliadin-IPC-DOM.

[0336] Example 11

[0337] Desialylated OVA

[0338] 11A. Preparation of expression vectors

[0339] The mammalian cell expression vector pSecTag A was purchased from Life Technologies. pSecTag A was engineered by site-directed mutagenesis following a variation of the QuikChange protocol (Geiser, et al.) to engineer an Ig κ chain secretion leader sequence at the C-terminus, OVA, a flexible linker Gly, and a 5'-terminal nucleotide sequence at the C-terminus. 3 The sequences encoding the Ser and 6xHis tags were cloned.

[0340] 11B. OVA Expression and Purification

[0341] HEK293 cells were transiently transfected with a modified pSecTag A vector, prepared, for example, as described in Example 10A, using polyethylenimine. The 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 culture supernatant was collected and sterilized by filtration. HisTrap Ni 2+ OVA protein was purified from the culture supernatant by immobilized metal ion affinity chromatography using a Sepharose column (GE Healthcare), followed by size exclusion chromatography using a Superdex75 column (GE Healthcare), which has the following structure: N'-OVA Gly 3 Ser-6xHis-C' and having the following amino acid sequence: GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSPGGGSHHHHHH (SEQ ID NO: 31)

[0342] Protein purity was verified by Coomassie Brilliant Blue staining of SDS-PAGE gels and anti-6xHis tag western blotting. Protein concentration was determined using the Beer-Lambert law, for which the absorbance at 280 nm was measured using a NanoDrop 2000 (Thermo Scientific) and the molecular weight (43.8 kDa) and extinction coefficient (31,525 M -1 cm -1 ) was predicted from the amino acid sequence of the protein using the ExPASy ProtParam tool.

[0343] 11C. Desialylation

[0344] OVA was desialylated by incubation with neuraminidase (New England Biolabs) for 1 hour at 37° C. Immobilized metal ion affinity chromatography (e.g., HisTrap Ni 2+ Desialylated OVA was purified from the reaction mixture by chromatography (eg, using a Sepharose column, GE Healthcare) followed by size exclusion chromatography (eg, using a Superdex75 column, GE Healthcare).

[0345] Protein purity was verified by Coomassie Brilliant Blue staining of SDS-PAGE gels and anti-6xHis tag western blotting. Desialylation was verified by Sambucus nigra lectin-based detection of protein sialic acid content in western blots (Vector Biolabs) and by a sialic acid-mediated fluorescent assay (Prozyme). Desialylated protein concentrations were determined using the Beer-Lambert law as described above for pre-desialylated proteins. Endotoxin levels are measured using the HEK-Blue TLR4 reporter cell line (Invivogen) according to the manufacturer's instructions.

[0346] Example 12

[0347] Liver Distribution

[0348] 12A. For example, as described in Example 9, F1aA-PE-m 3 -n 80 A 30 μg / 100 μl solution was prepared for injection in sterile saline.

[0349] One of three groups (3 per group) of C57 black 6 mice were administered F1aA-PE-m 3 -n 80 Two other groups of mice received an equivalent amount of phycoerythrin in 100 μl of saline or saline vehicle. Three hours after administration, the livers and spleens of these animals were harvested and the level of cellular fluorescence in these organs was determined by flow cytometry as an index of cellular PE content.

[0350] As shown in Figure 1, F1aA-PE-m 3 -n 80Liver-derived sinusoidal endothelial cells (LSECs), hepatocytes, Kupffer cells (KCs), and other antigen-presenting cells (APCs) from mice treated with F1aA-PE-m showed at least a three-fold increase in fluorescence compared to animals that received PE solution. No detectable differences were identified in fluorescence in splenocytes collected from the three groups. These results support the conclusion that F1aA-PE-m 3 -n 80 confirms that it has sufficient specificity to bind to antigen-presenting cells in the liver.

[0351] 12B. Following the procedure described in Example 12A, F1aA-PE-m 3 -n 80 to compound F1b-PE-m, which is prepared by substitution of X in Examples 2B, 3, 4, 5B, 6B, 7B, 19G, 19L, 20B and 20F, as described, for example, in Example 9. 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, F1j-PE-m 3 -n 45 -p 55 -q 4 -2NAcGAL, F1L-PE-m 3 -n 80 -p 55 -q 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 -q4 -CMP-2NHAc and F1n-PE-m 3 -n 33 -p 30 -q 8 -CMP-2OH to give compounds F1aA-PE-m 3 -n 80 and compound 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, F1j-PE-m 3 -n 45 -p 55 -q 4 -2NAcGAL, F1L-PE-m 3 -n 80 -p 55 -q 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-m 3 -n 33 -p 30 -q 8 -CMP-2OH is confirmed to have sufficient specificity to bind to antigen presenting cells in the liver.

[0352] Example 13

[0353] Antigen-specific OT1 CD8 +T cell proliferation

[0354] 13A. Synthetic F1aA-OVA-m, e.g., as described in Example 1 4 -n 80 was prepared as a 10 μg / 100 μl saline solution for injection. 6 OT-1 T cells were fluorescently labeled and adoptively transferred via tail vein injection into three groups of CD45.2 mice (5 per group). The following day (i.e., day 1), each of the three groups of mice was injected with 10 μg of F1aA-OVA-m 4 -n 80 , OVA or saline was administered via tail vein injection. On day 6, the animals were sacrificed and the percentage of splenic proliferative OT-1 cells was determined via fluorescence-activated cell sorting.

[0355] The results of the study (see Figure 2) showed that F1aA-OVA-m 4 -n 80 The percentage of proliferating OTI T cells in mice treated with Gal-OVA (Fig. 2) was significantly greater than the percentage of proliferating OTI cells in the spleens of mice treated with OVA or saline (Fig. 2). The increase in OTI cell proliferation was due to the F1aA-OVA-m 4 -n 80 Vs. increased CD8 in animals treated with other therapies + These results demonstrate T cell cross-priming. In accordance with the results of Example 12, 4 -n 80 4 shows that the ability of IgG1 to target antigens to the liver increases OVA presentation by antigen-presenting cells in the liver to OVA-specific OTI T cells.

[0356] 13B. To distinguish T cells expanding towards a functional effector phenotype from those expanding and depleting, we used proliferating OTI CD8 +T cells were analyzed for annexin V, a hallmark of apoptosis and therefore elimination, as well as the exhaustion marker programmed death-1 (PD-1). As shown in Figure 3, F1aA-OVA-m 4 -n 80 ("Gal-OVA" in Figure 3) showed a much higher amount of annexin V than soluble OVA. + and PD-1 + Proliferative OTI CD8 + T cells were induced.

[0357] 13C. Following the procedures described in Examples 13A and 13B, and 4 -n 8 For example, by substituting the resulting compounds of formula 1 described in Examples 3A, 4A, 5B, 6C, 7B and 19G, the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G produce much higher amounts of annexin V than soluble OVA. + and PD-1 + Proliferative OTI CD8 + It has been shown to induce T cells.

[0358] 13D. Following the procedures described in Examples 13A and 13B, and 4 -n 8 by the resulting compounds of formulas 1 and 2 described, for example, in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and by replacing OVA with the antigen corresponding to X (or X' or X"), the compounds from Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, respectively, produce a much higher number of annexin V than the soluble antigen X. + and PD-1 + Proliferative OTI CD8 + It has been shown to induce T cells.

[0359] Example 14 F1aA-OVA-m 4 -n 8 does not induce OVA-specific antibody responses

[0360] 14A. F1aA-OVA-m 4 -n 8 To evaluate the humoral immune response to F1aA-OVA-m 4 -n 8 Mice were treated with weekly intravenous injections of either F1aA-OVA or OVA, and the levels of OVA-specific antibodies in the blood were then measured. On days 0, 7, and 14 of the experiment, mice were administered an intravenous injection of 100 μl of saline containing one of the following: 1.) 6 μg of OVA; 2.) 6 μg of F1aA-OVA-m 4 -n 8 3.) 30 μg of OVA; 4.) 30 μg of F1aA-OVA-m 4 -n 8 , or 5.) saline alone. Each group contained 5 mice. On day 19, the mice were bled via cheek puncture and the titer of OVA-specific antibodies in the blood of each mouse was determined via ELISA. The results of this study showed that mice treated with 6 and 30 μg of OVA showed increased OVA-specific antibody titers, but both 6 and 30 μg of F1aA-OVA-m 4 -n 8 Mice treated with ("Gal-OVA" in FIG. 4) were shown to have blood titers similar to saline-treated mice (i.e., 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 of OVA had mean antibody titers of 0.75 and 0.25, respectively.

[0361] 14B. Following the procedure described in Example 14A, and using F1aA-OVA-m 4 -n 8 By substituting, for example, the resulting compounds of formula 1 described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it is shown that mice treated with compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G have similar OVA-specific antibody titers as mice treated with saline.

[0362] 14C. Following the procedure described in Example 14A, and using F1aA-OVA-m 4 -n 8 By substituting the resulting compounds of formulas 1 and 2, e.g., as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and by substituting the antigen corresponding to X (or X' or X") for OVA, respectively, it is shown that mice treated with compounds from Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F have similar antigen X-specific antibody titers as mice treated with saline.

[0363] Example 15

[0364] F1aA-OVA-m 4 -n 8 Deplete OVA-specific antibodies

[0365] 15A. We immunized mice with different OVA-antibody blood titers (each mouse had a titer between 0 and 4.5) with 20 μg of F1aA-OVA-m in 100 μl saline solution. 4 -n 8 Mice were treated with intravenous injection of F1aA-OVA-m on days 0, 5, 7, 12, and 14. 4 -n 8 The mice were given an intravenous injection of (F1aA-OVA-m 4 -n 8 (The injection of F1aA-OVA-m is labeled as "Gal-OVA" and shown as a green arrow on the x-axis of FIG. 5). 4 -n 8 To determine the ability of F1aA-OVA-m to deplete serum OVA-specific antibodies, mice were bled on day -1 to establish initial antibody titers, and then subsequently immunized with each F1aA-OVA-m on days 2, 6, 9, 13, and 16. 4 -n 8 Blood samples were taken after injection of F1aA-OVA-m. The antibody titers of each mouse were determined via ELISA. The results of this study showed that F1aA-OVA-m4 -n 8 shows that the first F1aA-OVA-m 4 -n 8 One day after injection (i.e., day 2), mice with positive OVA-antibody titers experience a 5- to 100-fold decrease in serum antibody levels (Figure 5). Our results show that, although antibody titers increased for certain mice over the course of the 19-day experiment, titer levels never reached the initial antibody titers measured on day -1, and subsequent F1aA-OVA-m 4 -n 8 These results indicate that a dose of F1aA-OVA-m is effective in reducing these transient increases in antibody titers. 4 -n 8 demonstrates the specificity of binding to serum OVA-specific antibodies and the kinetics required to deplete OVA-specific serum antibodies.

[0366] 15B. Following the procedure described in Example 15A, and using F1aA-OVA-m 4 -n 8 By substituting, for example, for the resulting compounds of formula 1 described in Examples 3A, 4A, 5B, 6C, 7B and 19G, the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G demonstrate the specificity of binding to serum OVA-specific antibodies and the kinetics required to deplete OVA-specific serum antibodies.

[0367] 15C. Following the procedure described in Example 15A, and using F1aA-OVA-m 4 -n 8By substituting, for example, the resulting compounds of formula 1 and 2 described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and by substituting the antigen corresponding to X (or X' or X") for OVA, respectively, the compounds from Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F are shown to have specificity for binding to serum antigen X-specific antibodies and the kinetics required for depleting antigen X-specific serum antibodies.

[0368] Example 16

[0369] OT-1 challenge to tolerance models

[0370] 16A. Using OTI challenge in an established tolerance model (Liu, Iyoda, et al., 2002), F1aA-OVA-m 4 -n 8 (mGal-OVA), F1b-OVA-m 1 -n 4 -p 34 (pGal-OVA), and N-DOM-Gly 3 Ser-OVA-Gly 3 The ability of Ser-6xHis-C (Dom-OVA) to prevent immune responses to subsequent vaccine-mediated antigen challenge was also demonstrated following challenge with a highly potent bacterial adjuvant (i.e., lipopolysaccharide). + (CD45.2 + ) T cells are CD45.1 + Mice were adoptively transferred (n=5 mice per group) and on days 1 and 6 received 233 nmol F1aA-OVA-m in 100 μl saline. 4 -n 8 , F1b-OVA-m 1 -n 4 -p 34 , N-DOM-Gly 3 Ser-OVA-Gly 3Either Ser-6xHis-C, or soluble OVA was administered intravenously. After a further 9 days, recipient mice were then challenged intradermally with OVA (10 μg) adjuvanted with lipopolysaccharide (LPS) (50 ng) to allow for potential elimination of transitional T cells. Characterization of the draining lymph nodes 4 days after challenge allowed determination of whether elimination had indeed occurred.

[0371] 16B. F1aA-OVA-m 4 -n 8 , F1b-OVA-m 1 -n 4 -p 34 , and N-DOM-Gly 3 Ser-OVA-Gly 3 Intravenous administration of Ser-6xHis-C significantly reduced OTI CD8 expression in draining lymph nodes compared with mice treated with unmodified OVA prior to antigen challenge with LPS. + This caused a profound decrease in T cell density, indicating deletional tolerance. 4 -n 8 (mGal-OVA), F1b-OVA-m 1 -n 4 -p 34 (pGal-OVA), and N-DOM-Gly 3 Ser-OVA-Gly 3 Draining lymph nodes from mice treated with either Ser-6xHis-C (Dom-OVA) or Ser-6xHis-C (Dom-OVA) had over 9-fold fewer OTI CD8 + T cells and contained over 43-fold fewer cells than challenged control mice that did not receive an intravenous injection of antigen; the response in splenocytes was similar. These results indicate that F1aA-OVA-m 4 -n 8 , F1b-OVA-m 1 -n 4 -p 34 , and N-DOM-Gly 3 Ser-OVA-Gly 3We demonstrate that Ser-6xHis-C attenuated OVA-specific immune responses following adjuvanted OVA challenge.

[0372] 16C. Following the procedures described in Examples 16A and B, and 4 -n 8 , F1b-OVA-m 1 -n 4 -p 34 , and N-DOM-Gly 3 Ser-OVA-Gly 3 By substituting Ser-6xHis-C into the resulting compounds of formula 1, e.g., as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, the compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G are shown to reduce OVA-specific immune responses following adjuvanted OVA challenge.

[0373] 16D. Following the procedure described in Examples 16A and B, and using F1aA-OVA-m 4 -n 8 , F1b-OVA-m 1 -n 4 -p 34 , and N-DOM-Gly 3 Ser-OVA-Gly 3 By substituting Ser-6xHis-C with the resulting compounds of formulas 1 and 2, e.g., as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and by substituting OVA with an antigen corresponding to X (or X' or X"), respectively, the compounds from Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F are shown to reduce antigen X-specific immune responses following adjuvanted antigen X challenge.

[0374] Example 17

[0375] Pharmacokinetics

[0376] 17A. OVA and the fusion protein N-DOM-Gly were incubated in a 35-well plate for 1 h at 4°C for 2 h according to the manufacturer's instructions.3 Ser-OVA-Gly 3 Ser-6xHis-C and N-OVA-Gly 3 Ser-DOM-Gly 3 Ser-6xHis-C (prepared, for example, as described in Example 10) was labeled with IRDye 800CW (LI-COR Biosciences). Unreacted dye was removed using Zeba desalting columns (Thermo Scientific). 50 μg of labeled protein in 100 μl of PBS per dose was administered intravenously or subcutaneously to C57BL / 6 mice (5 mice per group). Blood samples were collected from the tip of the tail into heparin-coated capillary tubes at time points = 0, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 96 hours post-injection. The samples were stored at 4°C and protected from light until analysis.

[0377] On the day of analysis, blood samples were centrifuged to remove cellular components. Plasma was transferred to new capillary tubes and scanned using an Odyssey infrared imaging system (LI-COR Biosciences). Signals were acquired in the 800 nm channel. Using the image-processing program Image J (National Institutes of Health), each sample was approximated 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.

[0378] Fluorescence signal versus time (normalized to fluorescence at time point = 0) for OVA and the fusion proteins DOM-OVA and OVA-DOM, with curves that fit a double exponential decay pattern in Figure 8, show that the ASGPR-targeted OVA fusion protein is cleared from the circulation more rapidly than unmodified OVA.

[0379] 17B. Following the procedure described in Example 17A, and using N-DOM-Gly 3 Ser-OVA-Gly 3Substitution of the Ser-6xHis-C fusion protein and OVA into asialoFactor VIII and Factor VIII, respectively, demonstrates that asialoFactor VIII is cleared from the circulation more rapidly than unmodified Factor VIII.

[0380] Example 18

[0381] Distribution in the body

[0382] 18A. OVA and N-DOM-Gly were incubated in 200 mL of 1 ... 3 Ser-OVA-Gly 3 Ser-6xHis-C fusion protein (prepared, for example, as described in Example 10) was labeled with Alexa Fluor 647 (Life Technologies). Unreacted dye was removed using Zeba desalting columns (Thermo Scientific). 50 μg of labeled protein in 100 μl of PBS per dose was administered intravenously or subcutaneously to C57BL / 6 mice (5 mice per group). Two hours after injection, mice were incubated with CO 2 Mice were euthanized by asphyxiation. Blood, heart, intestine, kidney, liver, embryo, stomach, spleen, and remaining carcasses were imaged using an IVIS Spectrum Imaging System (Caliper Life Science). Data were acquired and analyzed using Living Image software (Caliper Life Science). Single cell suspensions of liver and spleen were prepared and stained with fluorescently conjugated antibodies against MHC class II, CD1d, CD3, CD4, CD8 alpha, CD11b, CD11c, CD14, CD19, CD45, CD123, and / or Lin in 0.1% (w / v) BSA in PBS. Samples were analyzed using an LSR II flow cytometer and FACS Diva software (BD Biosciences).

[0383] Histograms showing the fluorescence signal density (photons / weight) for each organ and each protein are shown for N-DOM-Gly 3 Ser-OVA-Gly 3FIG. 1 shows that in animals injected with Ser-6xHis-C fusion protein, the highest fluorescent signal was in the liver compared to those treated with unmodified OVA.

[0384] N-DOM-Gly 3 Ser-OVA-Gly 3 Histograms showing the percentage of Ser-6xHis-C and OVA-positive cells were obtained from N-DOM-Gly 3 Ser-OVA-Gly 3 1 shows that animals injected with the Ser-6xHis-C fusion protein have a significantly higher percentage of positive hepatocytes compared to those treated with unmodified OVA.

[0385] 18B. Following the procedure described in Example 18A, and using N-DOM-Gly 3 Ser-OVA-Gly 3 Substitution of Ser-6xHis-C fusion protein and OVA for asialoFactor VIII and Factor VIII, respectively, shows that animals injected with asialoFactor VIII have a significantly higher percentage of positive hepatocytes compared to those treated with unmodified Factor VIII.

[0386] 18C. Following the procedure described in Example 18A, and using N-DOM-Gly 3 Ser-OVA-Gly 3 By substituting the Ser-6xHis-C fusion protein for the resulting compounds of formula 1, e.g., as described in Examples 3A, 4A, 5B, 6C, 7B and 19G, it is shown that animals injected with compounds from Examples 3A, 4A, 5B, 6C, 7B and 19G have a significantly higher percentage of positive hepatocytes compared to those treated with OVA.

[0387] 18D. Following the procedures described in Examples 18A and B, and 4 -n 8 , F1b-OVA-m 1 -n 4 -p 34, and N-DOM-Gly 3 Ser-OVA-Gly 3 By replacing Ser-6xHis-C with the resulting compounds of formulas 1 and 2, e.g., as described in Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, and by replacing OVA with the antigen corresponding to X (or X' or X"), it is shown that animals injected with compounds from Examples 1E, 1G, 2C, 10D, 19I, 19L, 20B, 20D and 20F, respectively, have a significantly higher percentage of positive hepatocytes compared to those treated with antigen X.

[0388] Example 19

[0389] F1m-OVA-m 2 -n 80 -p 30 -q 4 -CMP-2NHAc

[0390] 19A.R. 3 is NHAc and R 4 Formula 1102 where is OH

[0391] N-Acetyl-D-Galactosamine (R 3 is NHAc and R 4 Formula 1101) (5 g, 22.6 mmol), where is OH, 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 was allowed to reach room temperature and then heated to 70° C. After 4 hours, unreacted chloroethanol was removed under reduced pressure. 100 ml of ethanol was added to the crude product and the resulting solution was stirred under carbon for 2 hours. The solution was filtered and the solvent was removed under reduced pressure. The corresponding product of Formula 1102, N-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide, was used without further purification.

[0392] 19B.R 3 is NHAc and R4 Formula 1103 where is OH

[0393] N-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (2 g, 7.4 mmol) prepared in Example 19A was added to a stirred solution (100 ml) of DMF and sodium azide (4 g, 61.5 mmol). The solution was heated at 90° C. for 12 hours and then filtered. Residual solvent was removed under reduced pressure and the crude product was purified via 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-3-yl)acetamide.

[0394] 19C.R. 3 is NHAc and R 4 Formula 1104 where is OH

[0395] N-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (2 g, 6.9 mmol) prepared in Example 19B was added to a solution of palladium on carbon and ethanol (50 ml). The solution was stirred under hydrogen gas (3 atm) for 4 hours. The resulting solution was filtered and residual solvent was removed under reduced pressure to give the corresponding product of formula 1104, N-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide, which was used without further purification.

[0396] 19D.R 3 is NHAc and R 4 Formula 1105 where is OH

[0397] N-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (1.0 g, 3.78 mmol) prepared in Example 19C 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 was stirred at room temperature for 2 hours. After 2 hours, 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 via flash chromatography.

[0398] 19E. p is 30, q is 4, and R 3 is NHAc, and R 4 is OH and R 8 is the CMP formula 1107

[0399] The azide-modified uRAFT agent 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 (579 mg, 1.74 mmol) and azobisisobutyronitrile (2.2 mg, 0.0116 mmol) prepared in Example 19D in DMF. The reaction mixture was subjected to 4 freeze-degas 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 via addition of methanol. The solvent was decanted from the solids, the solids were collected, and the remaining solvent was removed via reduced pressure.

[0400] 19F. Formula 1109, in which X' is OVA, m is 2, and n is 80

[0401] Ovalbumin (5 mg, 0.00012 mmol) was added to 100 μl of sodium phosphate buffer (pH 8.0) and stirred. To this solution was added 5 mg of the compound of formula 1108, n=80. After 1 hour, unreacted compound of formula 1108 was removed from the solution via centrifugal size exclusion chromatography. The resulting buffer solution containing the corresponding product of formula 1109 was used in the next reaction without further purification.

[0402] 19G. X' is OVA, m is 2, n is 80, p is 30, q is 4, and R 3 is NHAc and R 8 is the CMP equation 1m

[0403] To the solution prepared in Example 19F, 100 μl of sodium phosphate buffer (pH 8.0) containing 10 mg of the product of formula 1107 prepared in Example 19E was added. The reaction was stirred for 2 hours, after which excess formula 1107 was removed via centrifugal size exclusion chromatography to obtain the corresponding isomeric product of formula 1m in the solution, which was used in biological studies without further purification. 3 The substituent is indicated in the name of the title compound as 2NHAc.

[0404] 19H. Other compounds of formula 1109

[0405] By following the procedure described in Example 19F and substituting OVA with: Abciximab, Adalimumab, Agalsidase alfa, Agalsidase beta, Aldesleukin, Alglucosidase alfa, · 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), MART1 (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), · Konarakin (UNIPROT Q6PSU6), Native alpha-gliadin “33-mer” (SEQ ID NO:24), Deamidated alpha-gliadin “33-mer” (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), The following corresponding compounds of formula 1109 are obtained, where n is 80: X is abciximab and m is 10; X is adalimumab and m is 11; X is agalsidase alfa and m is 14; X is agalsidase beta and m is 14; X is aldesleukin and m is 6; X is alglucosidase alfa 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 is rasburicase and m is 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 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 is conarachin and m is 21; X is the native alpha-gliadin “33-mer” (SEQ ID NO:24) and m is 1; X is deamidated alpha-gliadin “33-mer” (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 is Fel d 1 and m is 4; X is feline albumin and m is 16; X is Can f 1 and m is 6, X is canine albumin and m is 23, and X is an RhCE example and m is 10.

[0406] 19I. Other compounds of formula 1m

[0407] By following the procedure described in Example 19G and substituting the compound of formula 1109, for example as obtained in Example 19H, the following corresponding compounds of formula 1m are obtained: F1m-abciximab-m 10 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-adalimumab-m 11 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Agalsidase alpha-m 14 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Agalsidase beta-m 14 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Aldesleukin-m 6 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Alglucosidase alpha-m 13 -n 80 -p 30 -q 4 -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 -q 4 -CMP-2NHAc, F1m-Laronidase-m7 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Octreotide-m 1 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-phenylalanine ammonia-lyase-m 12 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-rasburicase-m 12 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-insulin-m 2 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-GAD-65-m 8 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-IGRP-m 7 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-MBP-m 6 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-MOG-m 5 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-PLP-m 8 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-MBP13-32-m 1 -n 80 -p30 -q 4 -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-m 1 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-MOG35-55-m 2 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-PLP139-154-m 3 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-MART1-m 4 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-tyrosinase-m 8 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-PMEL-m 5 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Aquaporin 4-m 4 -n 80 -p 30 -q 4-CMP-2NHAc, F1m-S-arrestin-m 12 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-IRBP-m 21 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Conarakin-m 21 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Natural alpha-gliadin “33-mer”-m 1 -n 80 -p 30 -q 4 -CMP-2NHAc, F1m-Deamidated alpha-gliadin “33-mer”-m 1 -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, F1m-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, F1m-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.

[0408] 19J. p is 30, q is 8, and R 3 is OH and R 4 is OH and R 8 is the CMP formula 1107

[0409] By following the procedure described in Example 19E except substituting galactose for N-acetyl-D-galactosamine and using an azide-modified uRAFT agent of formula 1106 where q is 8, p is 30, q is 8, and R 3 is OH and R 4 is OH and R 8 The compound of formula 1107 is obtained, where is CMP.

[0410] 19K. Formula 1109, in which n is 62 and X' and m are those of Example 19H.

[0411] By following the procedure described in Example 19F, substituting the compound described in Example 19H for OVA and utilizing the compound of formula 1108 where n is 62, the corresponding compound of formula 1109 where n is 62 is obtained.

[0412] 19L. Other compounds of formula 1m

[0413] By following the procedure described in Example 19G, substituting the compound of formula 1107 with the compound obtained in Example 19J and the compound of formula 1109 with the compound obtained in Example 19K, the following corresponding compounds of formula 1m are obtained: F1m-abciximab-m 10 -n 62 -p 30 -q 8 -CMP-2OH, F1m-adalimumab-m 11 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Agalsidase alpha-m 14 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Agalsidase beta-m 14 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Aldesleukin-m 6 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Alglucosidase 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 62 -p 30 -q 8 -CMP-2OH, F1m-Octreotide-m 1 -n62 -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 62 -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 -n 62 -p 30 -q 8 -CMP-2OH, F1m-MBP-m 6 -n 62 -p 30 -q 8 -CMP-2OH, F1m-MOG-m 5 -n 62 -p 30 -q 8 -CMP-2OH, F1m-PLP-m 8 -n 62 -p 30 -q 8 -CMP-2OH, F1m-MBP13-32-m 1 -n 62 -p 30 -q 8 -CMP-2OH, F1m-MBP83-99-m 1 -n 62 -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-PLP139-154-m 3 -n 62 -p 30 -q 8 -CMP-2OH, F1m-MART1-m 4 -n 62 -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 62 -p 30 -q 8 -CMP-2OH, F1m-S-arrestin-m 12 -n 62 -p 30 -q 8 -CMP-2OH, F1m-IRBP-m 21 -n62 -p 30 -q 8 -CMP-2OH, F1m-Conarakin-m 21 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Natural alpha-gliadin “33-mer”-m 1 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Deamidated alpha-gliadin “33-mer”-m 1 -n 62 -p 30 -q 8 -CMP-2OH, F1m-alpha-gliadin-m 1 -n 62 -p 30 -q 8 -CMP-2OH, F1m-omega-gliadin-m 1 -n 62 -p 30 -q 8 -CMP-2OH, F1m-Fel d 1-m 4 -n 62 -p 30 -q 8 -CMP-2OH, F1m-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, F1m-Canine Albumin-m 23 -n 62 -p 30 -q 8 -CMP-2OH, and F1m-RhCE-m 10 -n 62 -p30 -q 8 -CMP-2OH.

[0414] Example 20

[0415] F1n-insulin-m 2 -n 1 -p 30 -q 4 -CMP-2NHAc

[0416] 20A. Formula 1202, where X' is insulin, m is 2, and n is 1.

[0417] Recombinant human insulin (5 mg) was added to 100 μl of DMF containing 10 μl of triethylamine and stirred until the insulin was dissolved. To this solution was added 10 mg (0.0161 mmol) of precursor linker of formula 1201 where n is 1 (obtained from ???) and the reaction was stirred. After 1 hour, 1.3 ml of tert-butyl methyl ether was added to isolate the corresponding product of formula 1202, which was collected as a precipitate. Residual DMF and tert-butyl methyl ether were removed under reduced pressure. Characterization via liquid chromatography, mass spectrometry and polyacrylamide gel electrophoresis confirmed the identity of the product. The modified insulin product of formula 1202 was used without further purification.

[0418] 20B. X' is insulin, m is 2, n is 1, p is 30, q is 4, and R 8 is the formula 1n where CMP

[0419] 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 was stirred for 1 hour. After 1 hour, the reaction product was precipitated by adding dichloromethane (1.3 ml). The product was filtered and the remaining solvent was removed under reduced pressure. The crude product was then resuspended in 500 μl of PBS and the low molecular weight components were removed via centrifugal size exclusion chromatography to obtain the corresponding isomeric product of formula 1n. Characterization via liquid chromatography, mass spectrometry and polyacrylamide gel electrophoresis confirmed the identity of the product. The modified insulin product of formula 1202 was used without further purification.

[0420] 20C. Other compounds of formula 1202

[0421] By following the procedure described in Example 19F and substituting the OVA insulin with: Abciximab, Adalimumab, Agalsidase alfa, Agalsidase beta, Aldesleukin, Alglucosidase alfa, · 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), MART1 (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), · Konarakin (UNIPROT Q6PSU6), Native alpha-gliadin “33-mer” (SEQ ID NO:24), Deamidated alpha-gliadin “33-mer” (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), The following corresponding compounds of formula 1202 are obtained, where n is 1: X is abciximab and m is 10; X is adalimumab and m is 11; X is agalsidase alfa and m is 14; X is agalsidase beta and m is 14; X is aldesleukin and m is 6; X is alglucosidase alfa 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 is rasburicase and m is 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 is conarachin and m is 21; X is the native alpha-gliadin “33-mer” (SEQ ID NO:24) and m is 1; X is deamidated alpha-gliadin “33-mer” (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 is Fel d 1 and m is 4; X is feline albumin and m is 16; X is Can f 1 and m is 6, X is canine albumin and m is 23, and X is an RhCE example and m is 10.

[0422] 20D. Other compounds of formula 1n

[0423] By following the procedures described in Example B and substituting the compounds of formula 1202, for example as obtained in Example 20C, the following corresponding compounds of formula 1m are obtained: F1n-abciximab-m 10 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-adalimumab-m 11 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-agalsidase alpha-m 14 -n 1 -p30 -q 4 -CMP-2NHAc, F1n-agalsidase beta-m 14 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-Aldesleukin-m 6 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-alglucosidase alpha-m 13 -n 1 -p 30 -q 4 -CMP-2NHAc, · F1n-factor VIII-m 100 -n 1 -p 30 -q 4 -CMP-2NHAc, · F1n-factor IX-m 18 -n 1 -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-Octreotide-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-rasburicase-m 12 -n 1 -p 30-q 4 -CMP-2NHAc、 · F1n-GAD-65-m 8 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-IGRP-m 7 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MBP-m 6 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MOG-m 5 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-PLP-m 8 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MBP13-32-m 1 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MBP83-99-m 1 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MBP111-129-m 1 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MBP146-170-m 2 -n 1 -p 30 -q 4 -CMP-2NHAc、 · F1n-MOG1-20-m 1 -n 1 -p 30 -q 4 -CMP-2NHAc、 F1n-MOG35-55-m 2 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-PLP139-154-m 3 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-MART1-m 4 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-tyrosinase-m 8 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-PMEL-m 5 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-Aquaporin 4-m 4 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-S-arrestin-m 12 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-IRBP-m 21 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-Conarakin-m 21 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-Native alpha-gliadin “33-mer”-m 1 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-deamidated alpha-gliadin “33-mer”-m 1 -n 1 -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 -n 1 -p 30 -q 4 -CMP-2NHAc, F1n-Feline 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 F1n-RhCE-m 10 -n 1 -p 30 -q 4 -CMP-2NHAc.

[0424] 20E. Formula 1202, in which n is 33 and X' and m are those of Example 20C.

[0425] By following the procedure described in Example 19F, substituting the compound described in Example 20C for insulin and utilizing a compound of formula 1201 where n is 33, the corresponding compound of formula 1202 where n is 33 is obtained.

[0426] 20F. Other compounds of formula 1n

[0427] By following the procedure described in Example 20B, substituting the compound of formula 1107 with the compound obtained in Example 19J and the compound of formula 1202 with the compound obtained in Example 20E, the following corresponding compounds of formula 1n are obtained: F1n-abciximab-m 10 -n 33 -p 30 -q 8 -CMP-2OH, F1n-adalimumab-m 11 -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-alglucosidase 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, F1n-rasburicase-m 12 -n 33 -p 30 -q 8 -CMP-2OH, F1n-GAD-65-m 8 -n 33 -p 30 -q 8 -CMP-2OH, F1n-IGRP-m 7 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MBP-m 6 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MOG-m 5 -n 33 -p 30 -q 8 -CMP-2OH, F1n-PLP-m 8 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MBP13-32-m 1 -n 33 -p30 -q 8 -CMP-2OH, F1n-MBP83-99-m 1 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MBP111-129-m 1 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MBP146-170-m 2 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MOG1-20-m 1 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MOG35-55-m 2 -n 33 -p 30 -q 8 -CMP-2OH, F1n-PLP139-154-m 3 -n 33 -p 30 -q 8 -CMP-2OH, F1n-MART1-m 4 -n 33 -p 30 -q 8 -CMP-2OH, F1n-tyrosinase-m 8 -n 33 -p 30 -q 8 -CMP-2OH, F1n-PMEL-m 5 -n 33 -p 30 -q 8 -CMP-2OH, F1n-Aquaporin 4-m 4 -n 33 -p 30 -q 8 -CMP-2OH, F1n-S-arrestin-m 12 -n 33 -p 30 -q 8 -CMP-2OH, F1n-IRBP-m 21 -n 33 -p 30 -q 8 -CMP-2OH, F1n-Conarakin-m 21 -n 33 -p 30 -q 8 -CMP-2OH, F1n-Native alpha-gliadin “33-mer”-m 1 -n 33 -p 30 -q 8 -CMP-2OH, F1n-deamidated alpha-gliadin “33-mer”-m 1 -n 33 -p 30 -q 8 -CMP-2OH, F1n-alpha-gliadin-m 1 -n 33 -p 30 -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-m23 -n 33 -p 30 -q 8 -CMP-2OH, and F1n-RhCE-m 10 -n 33 -p 30 -q 8 -CMP-2OH.

[0428] Example 21

[0429] Clearance of OVA-specific antibodies from the circulation

[0430] To induce the production of OVA-specific antibodies for subsequent depletion, C57BL / 6 mice were intravenously injected with up to five doses of 10 μg OVA until the titer of anti-OVA IgG in plasma reached 3, as determined by ELISA (Figure 9A). Plasma samples were prepared by centrifugation of blood collected in EDTA-coated tubes at 2000×g for 10 min at room temperature and stored at −20° C. until analysis. Titers were calculated as the log 2 of the highest fold dilution of plasma with detectable anti-OVA IgG. 10 Here, plasma samples were assayed at 10-, 50-, 100-, 500-, 1,000-, 5,000-, 10,000-, and 50,000-fold dilutions, resulting in potential titers of 0, 1, 1.7, 2, 2.7, 3, 3.7, 4, and 4.7. Mice with titers below 3 after 5 doses of 10 μg OVA were given two additional doses of 10 μg OVA adjuvanted with 10 μg CpG-B to boost anti-OVA IgG production and therefore the amount of circulating anti-OVA IgG available for depletion (FIG. 9B).

[0431] To evaluate the efficacy of hepatocyte ASGPR-targeted OVA for anti-OVA antibody clearance, mice with circulating anti-OVA IgG were intravenously injected with saline or saline containing DOM and equivalent molar concentrations of OVA, DOM-OVA, or OVA-DOM. Specifically, mice were treated with equivalent molar concentrations of 10, 50, 100, or 200 μg OVA, as shown in FIG. 9. At time points t=-1 day, +3 hours, +1 day, and +7 days, blood samples were collected for comparable times of treatment injection, and the amount of anti-OVA IgG in the circulation was evaluated by ELISA analysis of plasma. Clearance of anti-OVA IgG from the circulation is expected to reduce the amount of anti-OVA IgG in plasma and therefore reduce the plasma anti-OVA IgG titer as determined by ELISA. In mice immunized with OVA alone and treated with DOM-OVA or OVA-DOM, the titers of anti-OVA IgG decreased to 3 or 2, representing a 1000- or 100-fold reduction in the amount of circulating anti-OVA IgG, respectively, and up to 50-fold more effective than control treatment with saline or DOM and OVA (Figure 9A). In mice immunized with OVA adjuvanted with CpG-B, no clearance of circulating anti-OVA IgG was observed (Figure 9B).

[0432] Example 22

[0433] NOD mice

[0434] Nonobese diabetic (NOD) mice are susceptible to the spontaneous development of autoimmune diabetes, which is the result of an autoimmune response to various pancreatic self-antigens. Diabetes develops in NOD mice as a result of insulitis characterized by infiltration of various leukocytes into the pancreatic islets.

[0435] To evaluate the efficacy of treatments for diabetes, beginning at 6 weeks of age, NOD mice are divided into control or test groups and treated with weekly intravenous injections of the test composition (10 μg) or an inactive control such as saline, respectively, for 18 consecutive weeks.

[0436] Blood glucose concentrations of the mice are measured weekly. Mice that maintain a blood glucose concentration below 300 mg / ml throughout the experiment are considered nondiabetic. Additionally, pancreases of the mice are collected at the end of the study, and T cell infiltration in the pancreas is determined via immunohistochemistry as an assessment of insulitis. Tolerance induction is assessed by depletion of autoantigen-specific CD8 T cells compared to mice treated with saline that develop diabetes. The presence of autoantigen-specific CD8 T cells is determined via ELISpot assay.

[0437] When tested as described above, X is F1m-insulin-m 2 -n 1 -p 30 -q 4 Compositions of formulas 1 and 2, such as insulin, -2NAcGAL, demonstrate efficacy for treating diabetes.

[0438] Although the present disclosure has been described with reference to specific embodiments thereof, it should be understood that various modifications may be made and equivalents may be substituted by those skilled in the art without departing from the true spirit and scope of the disclosure.In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present disclosure.All such modifications are intended to be within the scope of the appended claims.All of the above cited patents and publications are incorporated herein by reference.

Claims

1. 1. A tolerogenic compound comprising a liver-binding moiety and an antigen to which tolerance is desired, Where: the liver-binding moiety and the antigen to which tolerance is desired are recombinantly fused or chemically conjugated; the liver-binding moiety is a moiety that specifically binds to ASGPR expressed on one or more of hepatocytes and liver sinusoidal endothelial cells, and is selected from an ASGPR-targeting antibody, an ASGPR-targeting antibody fragment, an ASGPR-targeting peptide, or an ASGPR-targeting scFv; The antigen to which tolerance is desired is an antigen that elicits an undesirable immune response in a patient exposed to the antigen; The antigen to which tolerance is desired is a food antigen associated with celiac disease, an autoantigen associated with multiple sclerosis or type 1 diabetes, or a transplant antigen; and A tolerogenic compound, which upon administration to a subject, is capable of inducing one or more immune tolerance to an antigen to which tolerance is desired.

2. 2. The compound of claim 1, wherein the antigen to which tolerance is desired is selected from the group consisting of myelin oligodendrocyte glycoprotein, myelin proteolipid protein, myelin basic protein, tolerogenic portions of any of the above antigens, and mimetics of any of the above antigens, and wherein the unwanted immune response is associated with multiple sclerosis.

3. 2. The compound of claim 1, wherein the antigen to which tolerance is desired comprises one or more of high molecular weight glutenin, low molecular weight glutenin, alpha-, gamma- and omega-gliadins, hordeins, secalins, avenins, the major strawberry allergen Fra a 1-E (Fra a 1), profilin (Mus xp 1), tolerogenic portions of any of the above antigens, and mimetics of any of the above antigens.

4. 2. The compound of claim 1, wherein the antigen to which tolerance is desired comprises one or more of alpha-gliadin, gamma-gliadin, omega-gliadin, tolerogenic portions of any of said antigens, and mimetics of any of said antigens.

5. Antigens to which tolerance is desired include insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2β (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, type 2 glucose transporter, hepatocellular carcinoma-entero-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, SST 2. The compound of claim 1, comprising one or more of G-protein coupled receptors 1-5, a tolerogenic portion of any of said antigens, and a mimetic of any of said antigens.

6. 2. The compound of claim 1, wherein the antigen to which tolerance is desired is selected from the group consisting of insulin, proinsulin, preproinsulin, insulinoma-associated protein 2, insulinoma-associated protein 2β, tolerogenic portions of any of said antigens, and mimetics of any of said antigens, and wherein the unwanted immune response is associated with type 1 diabetes.

7. The compound of claim 1 , wherein the antigen to which tolerance is desired comprises a foreign transplant antigen or a tolerogenic portion thereof that elicits an undesirable immune response in the transplant patient.

8. 2. The compound of claim 1, wherein the antigens to which tolerance is desired include one or more subunits of MHC class I and MHC class II haplotype proteins, and one or more of the minor blood group antigens RhCE, Kell, Kidd, Duffy and Ss.

9. 2. The compound of claim 1, wherein the antigen to which tolerance is desired comprises one or more of insulin, proinsulin, preproinsulin, a tolerogenic portion of any of the above antigens, or a mimetic of any one of the above antigens.

10. 5. The compound of claim 1, 3, or 4, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:

24.

11. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising SEQ ID NO:

8.

12. 3. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises SEQ ID NO:

15.

13. 3. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises SEQ ID NO:

16.

14. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising a portion of SEQ ID NO:

9.

15. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising a portion of SEQ ID NO:

10.

16. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:

11.

17. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:

12.

18. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:

13.

19. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:

14.

20. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:

17.

21. The compound of claim 1 or 2, wherein the antigen to which tolerance is desired comprises one or both of a polypeptide comprising the amino acid sequence of SEQ ID NO: 15 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, and the antigen to which tolerance is desired further comprises SEQ ID NO:

12.

22. The compound of claim 1 , 5, or 6, wherein the antigen to which tolerance is desired comprises a polypeptide comprising the tolerogenic portion of SEQ ID NO:

5.

23. The compound of claim 1, 5, or 6, wherein the antigen to which tolerance is desired comprises an amino acid sequence comprising SEQ ID NO:5 and IA-2.

24. A pharma- ceutically acceptable composition comprising a compound according to any one of claims 1 to 10 and a pharma- ceutically acceptable carrier.

25. 13. Use of a compound according to any one of claims 1 to 10 in the manufacture of a medicament for treating an unwanted immune response in a subject against an antigen to which tolerance is desired.

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