HK40115862A - Glycotargeting therapeutics - Google Patents

Description

Glucose-targeted therapy

This application is a divisional application of the patent application filed on September 16, 2016, with a priority date of September 19, 2015, application number 201680064903.3, entitled "Glucose-Targeted Therapeutic Agent".

Cross-reference to related applications

This application claims the benefit of U.S. Patent Application No. 14/859,292, filed September 19, 2015, entitled “Glucose-Targeted Therapeutic Agent”, and U.S. Patent Application No. 15/185,564, filed June 17, 2016, the entire contents of which are incorporated herein by reference.

Reference sequence list

The sequence list submitted via EFS-Web as an ASCII document is incorporated herein by reference in accordance with 35 U.S.C. §1.52(e). The ASCII document in the sequence list is named ANOK001P1WO_ST25.TXT, created on September 14, 2016, and is 47.4KB in size.

Background of the Invention

Technical Field

Some embodiments disclosed in this invention relate to pharmaceutically acceptable compositions that can be used to treat graft rejection, autoimmune diseases, allergic reactions (e.g., food allergies), and immune responses to therapeutic agents.

Description of related technologies

Various methods have been used to induce tolerance to antigens that elicit undesirable immune responses. Some methods employ antigens that target specific cells. Applications US 2012/0039989, US 2012/0178139, and WO 2013/121296 describe targeting antigens to red blood cells to utilize the role of red blood cells in antigen presentation for tolerance induction.

Invention Overview

While cell-targeting approaches have yielded positive results to date, the possibility of alternative methods remains a concern. Specifically, some embodiments disclosed in this invention involve compositions configured to target one or more cell types in the liver, thereby delivering a desired-tolerance antigen to the targeted cell types and thereby inducing antigen treatment and immune tolerance to the antigen. Antigens disclosed in more detail below may include therapeutic agents, proteins, protein fragments, antigen mimics of proteins or protein fragments (e.g., epitopes). Other types of antigens are discussed in more detail below. In some embodiments, methods and uses of such compositions are also provided.

In some embodiments, compounds comprising Formula 1 are provided:

m is an integer from approximately 1 to 10, X contains molecules with antigenic regions, and Y is a linker part having a formula selected from the following:

Wherein the left parenthesis “(” indicates a bond with X, the right parenthesis or bottom parenthesis and “)” indicate a bond between Y and Z, n is an integer from about 1 to 100, where p is an integer from about 2 to 150, where q is an integer from about 1 to 44, where ^R8 is _–CH2- or –CH2- _CH2 - _C ( _CH3 )(CN)-; and where ^R9 is a direct bond or _–CH2 - _CH2 -NH-C(O)-, and Z contains a liver-targeting moiety. In some embodiments, X is a protein or protein fragment containing an antigenic region. In some embodiments, Z is galactose, while in some embodiments, Z is glucose. In some embodiments, Z is galactosamine, while in some embodiments, Z is glucosamine. In some embodiments, the composition uses an α-anion of glucose or galactose. In some embodiments, the composition uses a β-anion of glucose or galactose. In some embodiments, a mixture of α and β anions is used, including optionally a mixture of glucose and galactose. In some embodiments, Z is N-acetylgalactosamine, while in other embodiments, Z is N-acetylglucosamine. In some embodiments, the composition uses an α-anomeric form of glucosamine or galactosamine. In some embodiments, the composition uses an α-anomeric form of glucosamine or galactosamine. In some embodiments, a mixture of α and β anomeric forms is used, including optionally a mixture of glucosamine and galactosamine. Similarly, in some embodiments, an α-anomeric form, a β-anomeric form, or a combination of α and β anomeric forms of N-acetylgalactosamine or N-acetylglucosamine is used. In various embodiments, any combination of any liver-targeting glucose α or β anomeric forms and any combination of glucose can be employed. In some embodiments, Y comprises _Ym or _Yn , where m is 1-5, n is 75-85, p is 85-95, and q is 2-6. In some embodiments, m is 1-3, n is 79, p is 90, and q is 4. In some embodiments, X is selected from the group consisting of: insulin, proinsulin, preinsulin, gluten, gliadin, myelin, myelin oligodendrocyte glycoprotein and proteolipoprotein, factor VIII, factor IX, asparaginase, uricase, and any of the aforementioned fragments. In some embodiments, antigen X is not a full-length protein. For example, in some embodiments, the antigen is not full-length gliadin, insulin, or proinsulin. In some embodiments, antigen X is not a protein fragment. In some embodiments, m is not greater than 3, n is not greater than 80, p is not greater than 100, and q is not greater than 5.

In some implementations, Y is a connection base portion having the following formula:

In some implementations, Y is a connection base portion having the following formula:

In some implementations, Y is a connection base portion having the following formula:

In some implementations, Y is a connection base portion having the following formula:

In some implementations, combinations of linker bases disclosed in this invention may be used, just as combinations of liver-targeting portions may be employed.

As discussed in more detail below, there are various antigens to which tolerance can be expected. These may include, but are not limited to, exogenous antigens that cause an undesirable immune response when a subject is exposed to them. In some embodiments, an undesirable immune response may be the result of antigen ingestion (e.g., orally or nasally, or via some other mucosal route). These routes may be, for example, the case of food antigens. In some embodiments, the antigen may be administered to the subject intentionally, for example, by administering a therapeutic composition to treat a disease or condition in the subject. In other embodiments, the antigen may be generated by the subject, such as an autoantigen. For example, in some embodiments, X comprises a foreign graft antigen or a tolerogenic portion thereof, against which the graft recipient forms an undesirable immune response. In some embodiments, X comprises a foreign food, animal, plant, or environmental antigen or a tolerogenic portion thereof, against which the patient forms an undesirable immune response. In some embodiments, X comprises a foreign therapeutic agent or a tolerogenic portion thereof, against which the patient forms an undesirable immune response. In some embodiments, X comprises a synthetic autoantigen or a tolerogenic portion thereof, against which the patient forms an undesirable immune response in its endogenous form.

In further details above, in some embodiments, a compound is provided, wherein X is a food antigen. In some such embodiments, X is one or more of the following: conarachidin (Ara h 1), allergen II (Ara h 2), peanut lectin, blue bean protein (Ara h 6), alpha-lactalbumin (ALA), lactoferrin, Pen a 1 allergen (Pen a1), allergen Pen m 2 (Pen m 2), tropomyosin fast isotype, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, seclain, and oat protein. In some embodiments, fragments of any of these antigens and/or mimic epitopes of any of these antigens are also used. In some embodiments, X is selected from the group consisting of: glutenin, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, omega-gliadin, barley gliadin, rye alkaloids, and oat protein and fragments thereof. In some embodiments, X is glutenin or a fragment thereof. In some embodiments, X is gliadin or a fragment thereof.

In some embodiments, a compound is provided wherein X is a therapeutic agent. In some embodiments, X is selected from the group consisting of: factor VII, factor IX, asparaginase, and uricase and fragments thereof. In some embodiments, X is a therapeutic agent selected from the group consisting of: factors VII and IX and fragments thereof. In some embodiments, X is a therapeutic agent selected from the group consisting of: factor VIII or a fragment thereof. In some embodiments, when X is a therapeutic agent, the compound can be used to treat, prevent, reduce, or otherwise mitigate the immune response to a therapeutic agent for hemophilia. As discussed in this invention, mimicry epitopes of any antigenic portion of the above-described antigens can be used in some embodiments.

In some embodiments, X comprises asparaginase or a fragment thereof. In some embodiments, X comprises uricase or a fragment thereof. In several such embodiments, the compound can be used to treat, prevent, reduce, or otherwise mitigate the immune response to an anticancer agent. As discussed herein, mimicry epitopes of any antigenic portion of the above-described antigens can be used in some embodiments.

In some implementations, X is associated with an autoimmune disease. For example, in some implementations, the associated autoimmune disease is one or more of the following: type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, and neuromyelitis optica.

In some embodiments, the autoimmune disease is type 1 diabetes, and X comprises insulin or a fragment thereof. In some embodiments, the autoimmune disease is type 1 diabetes, and X comprises proinsulin or a fragment thereof. In some embodiments, the autoimmune disease is type 1 diabetes, and X comprises pre-proinsulin or a fragment thereof. As discussed in this invention, mimicry epitopes of any antigenic portion of the above-described antigens may be used in some embodiments. In some embodiments, combinations of these antigens may be incorporated into tolerogenic compounds that may help reduce the immune response to autoantigens at multiple points along the insulin pathway.

In some embodiments, the autoimmune disease is multiple sclerosis, and X comprises myelin base protein or a fragment thereof. In some embodiments, the autoimmune disease is multiple sclerosis, and X comprises myelin oligodendrocyte glycoprotein or a fragment thereof. In some embodiments, the autoimmune disease is multiple sclerosis, and X comprises myelin lipoprotein or a fragment thereof. As discussed in this invention, mimicry epitopes of any antigenic portion of the above-described antigens may be used in some embodiments. In some embodiments, combinations of these antigens may be incorporated into tolerogenic compounds that may help reduce the immune response to autoantigens at several points along the enzymatic pathways controlling myelin formation or myelin repair.

In some implementations, the autoimmune disease is rheumatoid arthritis, and X is selected from the group consisting of: fibrinogen, vimentin, type II collagen, α-enolase and fragments thereof.

In some implementations, the autoimmune disease is vitiligo, and X is selected from the group consisting of: Pmel17, tyrosinase, and fragments thereof.

In some implementations, the autoimmune disease is uveitis, and X is selected from the group consisting of: retinaldehyde inhibitory protein and interreceptor receptor-like visual pigment-binding protein (IRBP) and fragments thereof.

In some implementations, the autoimmune disease is pemphigus vulgaris, and X is selected from the group consisting of: desmosome core proteins 3, 1 and 4, pemphaxin, desmosome glycoprotein, plaque protein, perplakin, acetylcholine receptor and fragments thereof.

In some implementations, the autoimmune disease is neuromyelitis optica, and X is aquaporin-4 or a fragment thereof.

As discussed in this invention, mimic epitopes of any antigenic portion of the self-antigen described above (or otherwise disclosed in this invention) may be used in some embodiments.

In some embodiments, the use of the above-described (or otherwise disclosed in this invention) compounds for inducing tolerance to X is also provided.

In some embodiments, the present invention also provides pharmaceutically acceptable compositions comprising the compounds described above (or otherwise disclosed herein). Use of such compositions in inducing tolerance to X is also provided. In some embodiments, the pharmaceutically acceptable composition consists of, or is primarily composed of, compounds in which X is a food antigen, a therapeutic agent, an autoantigen, or a fragment thereof, Y is a linker, and the liver-targeting portion Z is selected from glucose, galactose, glucosamine, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine.

The present invention also provides a method for inducing tolerance to an antigen, enabling a subject to develop an undesirable immune response to the antigen, comprising administering a compound disclosed above (or otherwise disclosed herein). In some embodiments, the compound is administered before the subject is exposed to the antigen. However, in some embodiments, the compound is administered after the subject has been exposed to the antigen. In some embodiments, at least one intravenous administration comprising the compound is administered (e.g., a bolus dose followed by a series of optional maintenance doses).

In some embodiments, the use of the compounds disclosed above (or otherwise disclosed herein) in the preparation of a medicament for inducing tolerance to an antigen or a tolerogenic portion thereof, in which a subject develops an undesirable immune response against the antigen.

In some embodiments disclosed herein, compositions for inducing immune tolerance in a subject, as well as methods and uses of said immune tolerance compositions are provided. In some embodiments, immune tolerance is desirable because the subject develops an undesirable immune response to an antigen. According to embodiments, the antigen can be one or more of a variety of antigens, such as foreign antigens, such as ingested food antigens, or antigenic portions of therapeutic drugs administered to the subject. In other embodiments, the antigen can be an autoantigen that the subject's immune system cannot recognize (or only recognizes as itself to a limited extent) and thus elicits a targeted immune response, leading to an autoimmune disease.

In some embodiments, a composition comprising Formula 1 is provided:

Where m is an integer from approximately 1 to 10, X contains food antigens, therapeutic agents, autoantigens, fragments of any such antigens, or mimic epitopes of any such antigens, and Y is a linker portion having the following formula:

Wherein: the left parenthesis “(” indicates a bond with X, the right parenthesis or bottom parenthesis and “)” indicate a bond between Y and Z, n is an integer of about 70 to 85, where p is an integer of about 85 to 95; where q is an integer of about 1 to 10, where R ⁸ is –CH ₂ – or –CH ₂ -CH ₂ -C(CH ₃ )(CN)–; and where R ⁹ is a direct bond or –CH ₂ -CH ₂ -NH–C(O)–; and Z contains a liver-targeting portion containing glucose or galactose. In some embodiments, m is 1-3, n is 79, p is 90, and q is 4. In some embodiments, X is selected from the group consisting of: insulin, proinsulin, preinsulin, glutenin, gliadin, myelin, myelin oligodendrocyte glycoprotein and proteolipoprotein, factor VIII, factor IX, asparaginase, uricase, and any of the foregoing fragments. In some embodiments, the composition comprises antigen X, linker Y, and liver-targeting moiety Z; is composed of antigen X, linker Y, and liver-targeting moiety Z; or is substantially composed of antigen X, linker Y, and liver-targeting moiety Z.

In some embodiments, compounds comprising Formula 1 are provided:

Where m is an integer from approximately 1 to 10, X is selected from the following group: insulin, proinsulin, preinsulin, gluten, gliadin, myelin, myelin oligodendrocyte glycoprotein and proteolipoprotein, factor VIII, factor IX, asparaginase, uricase and any of the aforementioned fragments, and Y is a linker portion having the following formula:

Wherein, the left parenthesis “(” indicates a bond with X, the right parenthesis or bottom parenthesis and “)” indicate a bond between Y and Z, n is an integer of about 70 to 85, where p is an integer of about 85 to 95, where q is an integer of about 1 to 10, where R ⁸ is –CH ₂ – or –CH ₂ -CH ₂ -C(CH ₃ )(CN)–; and where R ⁹ is a direct bond or –CH ₂ -CH ₂ -NH–C(O)–, and Z contains a liver-targeting portion which contains a glycoside. In some embodiments, m is 1-3, n is 79, p is 90, and q is 4. In some embodiments, Z is selected from the group consisting of glucose, glucosamine, galactose, galactosamine, N-acetylgalactosamine, and N-acetylglucosamine.

In some embodiments, 2,5-dioxopyrrolidone-1-ylpropyl carbonate linker and/or 2-(ethyl dithioalkyl)ethylcarbamate linker may be used.

In some embodiments, a composition comprising a compound of formula 1 is provided:

in:

m is an integer from approximately 1 to 10;

X contains antigens against which the patient would form an undesirable immune response, wherein the antigen is a food antigen, a therapeutic agent, an autoantigen, or a fragment of any such antigen.

Y is a linking basis part having a formula selected from the following group:

Wherein the left parenthesis “(” indicates a key to X, where the right “)” indicates a key to Z, where the bottom “)” indicates a key to Z, where n is an integer from about 1 to about 80, where q is an integer from about 1 to about 4, where p is an integer from about 1 to about 90, where R ₈ is –CH ₂ – or –CH ₂ -CH ₂ -C(CH ₃ )(CN)–, and Z contains one or more liver-targeting portions that specifically target hepatocytes expressing sialic acid glycoprotein receptors.

In some embodiments of the present invention, m is 1 to 4, and Y is a connection base portion having the following formula:

Furthermore, Z includes a liver-targeting portion, which contains one or more of the following: galactose, galactosamine, or N-acetylgalactosamine.

In some implementations, m resolves to integers from 1 to 4, and Y is the linking basis part having the following expression:

Furthermore, Z contains a liver-targeting component, which includes one or more of the following: glucose, glucosamine, or N-acetylglucosamine.

In some embodiments, compositions of formula 1 (X-[-Y---Z] _m ) are provided, wherein m is an integer from about 1 to 100, X comprises an antigen to which the patient forms an undesirable immune response, or a tolerogenic portion thereof, or X comprises an antibody, antibody fragment, or ligand that specifically binds to a circulating protein or peptide or antibody involved for reasons such as graft rejection, immune response to a therapeutic agent, autoimmune disease, hypersensitivity, and/or allergic reactions, Y comprises a linker portion, and Z comprises a liver-targeting portion. In some embodiments, Z comprises galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine.

In some embodiments, Y is selected from N-hydroxysuccinamidyl linkers, maleimide linkers, vinyl sulfone linkers, pyridyl di-thiol-poly(ethylene glycol) linkers, pyridyl di-thiol linkers, n-nitrophenyl carbonate linkers, NHS-ester linkers, and nitrophenoxy poly(ethylene glycol) ester linkers. In some embodiments, Y comprises an antibody, antibody fragment, peptide or other ligand that specifically binds to X, a dithioalkyl ethyl ester, or a structure represented by one of the formulas Ya to Yp.

Or Y has a portion represented by the formula Y’-CMP:

In such embodiments, the left bracket "(" indicates the bond between X and Y, the right bracket or bottom bracket and ")" indicate the bond between Y and Z, n is an integer from about 1 to 100, q is an integer from about 1 to 44, R ⁸ is–CH _2– or –CH ₂ -CH ₂ -C(CH ₃ )(CN)–, Y' represents the remainder of Y; and W represents a polymer with the same W ¹ group, or W is a copolymer or random copolymer of the same or different W ¹ and W ² groups, wherein:

And where p is an integer from 2 to about 150, ^R9 is a direct bond, _–CH2 - _CH2 -NH–C(O)– or _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)-, where t is an integer from 1 to 5; and ^R10 is an aliphatic group, an alcohol or an aliphatic alcohol. In one such embodiment, m is 1 to 3, Y is represented by the formula Ym, where ^R8 is _–CH2 - _CH2 -C( _CH3 )(CN)–, and W is represented by a block copolymer of ^W1 and ^W2 , where ^R9 is _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)-, t is 1, and ^R10 is 2-hydroxypropyl; and Z comprises a liver-targeting moiety comprising one or more of the following: galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, N-acetylglucosamine. In some embodiments, Z is a β-anomeric form of the corresponding sugar.

In several other embodiments, compositions for inducing tolerance to an antigen to which a subject develops an undesirable immune response are provided, the compositions comprising compounds of formula 1 (Formula 1(X-[-Y---Z] _m ), where m is an integer from about 1 to 10, X comprises an antigen to which the patient develops an undesirable immune response, wherein the antigen is a food antigen, a therapeutic agent, an autoantigen, or a fragment of any such antigen, and Y is a linker portion having a formula selected from the group consisting of:

Wherein the left bracket "(" indicates a bond with X, the right bracket or bottom bracket and ")" indicate a bond between Y and Z, n is an integer from about 1 to 100, where p is an integer from about 2 to 150, where q is an integer from about 1 to 44, where R ⁸ is –CH ₂ – or –CH ₂ -CH ₂ -C(CH ₃ )(CN)–, and where R ⁹ is a direct bond or –CH ₂ -CH ₂ -NH–C(O)–, and Z contains galactose, galactosamine or N-acetylgalactosamine.

In some embodiments of such compositions, m is 1 to 3, and Y is a connecting base portion having the following formula:

Wherein CH ₂ -CH ₂ -NH–C(O)–; and Z comprises a liver-targeting moiety comprising one or more of the following: galactose, galactosamine, or N-acetylgalactosamine. In some embodiments, Z is a selected moiety of β-anomeric derivatives.

As discussed above, in some implementations, X is a self-antigen, and the undesired immune response is an autoimmune response.

This invention discloses various autoantigens, but in several specific embodiments, X is a myelin oligodendrocyte glycoprotein or myelin lipoprotein. In such embodiments, the unintended immune response experienced by the subject is associated with multiple sclerosis. In another embodiment, X is insulin, proinsulin, or preproinsulin, and the unintended immune response is associated with diabetes. It should be understood that association with multiple sclerosis, diabetes, or other autoimmune diseases does not necessarily require a formal diagnosis of such autoimmune disease, but may be associated with common symptoms or characteristics of a particular autoimmune disease.

In other embodiments, as discussed in this invention, undesirable immune responses may arise against therapeutic agents, such as protein drugs or drugs derived from non-human and/or non-mammalian species. For example, in some embodiments, X is a therapeutic agent, such as factor VIII, factor IX, or other hemostatic inducers. In such embodiments, the undesirable immune response is against the agent, and the associated disease is hemophilia, which cannot be improved due to an autoimmune response (in the absence of the composition). However, after administration of the composition, hemophilia may improve because the composition helps induce tolerance to the agent, reduces the response to the agent, and allows for a reduction in hemophilia symptoms. In other embodiments, X is a therapeutic agent, such as asparaginase and uricase. As discussed above, undesirable immune responses may arise from the administration of such agents because they are derived from non-human sources. The ability of the compositions disclosed in this invention to induce tolerance to these agents allows these agents to continue to be used by subjects requiring treatment, while reducing, mitigating, eliminating, or otherwise improving side effects arising from the immune response.

In some embodiments, X is a food antigen. Many food antigens are known to cause allergic reactions upon ingestion; however, in some embodiments, X is selected from the group consisting of: peanut globulin (Ara h 1), allergen II (Ara h 2), peanut lectin, blue bean protein (Ara h 6), α-lactalbumin (ALA), lactoferrin, Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, rye alkaloids, and oat protein. In some embodiments, treatment with the compositions disclosed in this invention, wherein X is a food antigen, allows the subject to have a significantly reduced immune response to the antigen, for example, many peanut allergies are so severe that exposure to peanut dust or peanut oil can cause an allergic reaction. In some implementations, the treatment reduces and/or eliminates the response to such incidental exposure to the antigen. In other implementations, the treatment allows the subject to ingest food from the antigen source while generating a limited or no immune response.

In some embodiments, administration of the composition to a subject results in a greater proliferation of antigen-specific T cells compared to the proliferation of antigen-specific T cells induced by antigen administration alone. In such embodiments, the proliferation of antigen-specific T cells indicates enhanced delivery of the antigen (via the composition) to a molecular processing mechanism that processes self/non-self antigens, relative to antigen administration alone. In other words, targeted delivery is effective in such embodiments. In other embodiments, administration of the compounds disclosed in this invention results in greater expression of exhaustion markers or apoptosis markers on antigen-specific T cells compared to the expression of exhaustion markers or apoptosis markers on antigen-specific T cells induced by antigen administration alone. This result indicates a specific reduction in the activity of T cells against the antigen of interest and/or the deletion of T cells against the antigen of interest. In some embodiments, these molecular markers of tolerance induction are precursors to a reduction or improvement in the symptoms of an immune response previously experienced by a subject upon exposure to the antigen.

In some embodiments, Z comprises a liver-targeting moiety, which is a carbohydrate. In some embodiments, the carbohydrate is a short-chain carbohydrate. In some embodiments, Z is a sugar. In some embodiments, Z is galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine. In some embodiments, immune tolerance induction is greater when glucose, glucosamine, or N-acetylglucosamine is used as Z. In other embodiments, enhanced immune tolerance induction can be achieved when the liver-targeting moiety is a sugar and the sugar is in a β-anomeric configuration. In some embodiments, Z is galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine, and is conjugated to Y at its C1, C2, or C6.

The present invention also provides methods for inducing tolerance to antigens that, when administered alone (e.g., without the currently disclosed compositions), result in an unfavorable immune response. According to embodiments, such methods involve administration before or after exposure to the antigen. In some embodiments, pre-exposure administration exerts a prophylactic effect, which in some embodiments substantially avoids or significantly reduces the immune response. Administration of the composition can be performed via a variety of methods, including but not limited to intravenous, intramuscular, oral, transdermal, or other infusion routes. Administration can be daily, weekly, multiple times a day, or as needed (e.g., before anticipated exposure).

The present invention also provides the use of the compositions disclosed herein for treating undesirable immune responses after exposure to an antigen. As discussed herein, such use can be for preventative effects and/or for reducing symptoms arising from prior exposure to the antigen (or prior adverse immune effects, such as those in an autoimmune setting). For example, the present invention provides the use of compositions according to Formula 1 for treating undesirable side effects resulting from exposure to a therapeutic antigen, exposure to a food antigen, or adverse effects of an immune response against an autoantigen. The compositions disclosed herein are suitable for administration to a patient for such purposes, for example, via oral, intravenous, intramuscular, or other suitable routes. In some embodiments, the use of the compositions disclosed herein unexpectedly results in a reduction, elimination, or improvement of adverse immune responses to the antigen of interest.

This invention provides additional compositions and methods of using thereof. For example, in some embodiments, pharmaceutically acceptable compositions are provided for inducing tolerance to therapeutic proteins in subjects with defects in the production of functionally similar natural proteins. These pharmaceutically acceptable compositions comprise compounds of formula 1 (X-[-Y---Z] _m ), where m is an integer from about 1 to 10, X comprises an antigenic protein or protein fragment, Y is a linker portion having a formula selected from the group consisting of: formula Ya, formula Yc, formula Ym, formula Yn, where a left bracket "(" indicates a bond with X, a right bracket or bottom bracket and ")" indicate a bond between Y and Z, n is an integer from about 1 to 100, where p is an integer from about 2 to 150, where q is an integer from about 1 to 44, where ^R8 is _–CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-, and where ^R9 is a direct bond or _–CH2 - _CH2 -NH–C(O)–, and Z contains galactose, galactosamine, or N-acetylgalactosamine.

In some embodiments of the composition, m is 1 to 3, and Y is a connecting base portion having the following formula:

Wherein CH ₂ -CH _2- NH–C(O)–, and Z comprises a liver-targeting moiety comprising one or more of the following: glucose, glucosamine, N-acetylglucosamine, galactose, galactosamine, or N-acetylgalactosamine. In some embodiments, galactose, galactosamine, or N-acetylgalactosamine is a β-anomeric compound. In some embodiments, a combination of galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine is used.

The present invention also provides pharmaceutically acceptable compositions for inducing tolerance to therapeutic proteins in subjects with defects in the production of functionally similar natural proteins, said compositions comprising compounds of formula 1 (X-[-Y---Z] _m ), wherein m is an integer from about 1 to 10, X comprises an antigenic protein or protein fragment, Y is a linker portion having a formula selected from the group consisting of: formula Ya, formula Yc, formula Ym, or formula Ym, wherein a left bracket "(" indicates a bond with X, wherein a right bracket ")" indicates a bond with Z, wherein a bottom bracket ")" indicates a bond with Z, wherein n is an integer from about 1 to about 80, wherein q is an integer from about 1 to about 4, wherein p is an integer from about ₁ to about 90, wherein ^R8 is –CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-, and wherein W represents a polymer of formula ^W1 or ^W2 or W is a copolymer of formula ^W1 or ^W2 , wherein:

Wherein ^R9 is a direct bond, _–CH2 - _CH2 -NH–C(O)– or _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)-, where t is an integer from 1 to 5; ^R10 is an aliphatic group, an alcohol, or an aliphatic alcohol; and Z comprises glucose, glucosamine, N-acetylglucosamine, galactose, galactosamine, or N-acetylgalactosamine. In some embodiments, galactose, galactosamine, or N-acetylgalactosamine is a β-anomeric compound. In some embodiments, a combination of galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine is used. In some embodiments of the composition, m is 1 to 3, Y is represented by the formula Ym, wherein ^R8 is _–CH2 - _CH2 -C( _CH3 )(CN)–, and W is represented by a block copolymer of ^W1 and ^W2 , wherein ^R9 is _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)-, t is 1, and ^R10 is 2-hydroxypropyl; and Z comprises a liver-targeting moiety comprising one or more of the following: glucose, glucosamine, N-acetylglucosamine, galactose, galactosamine, or N-acetylgalactosamine. In some embodiments, galactose, galactosamine, or N-acetylgalactosamine is a β-anomeric compound. In some embodiments, a combination of galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine is used.

In some embodiments, X comprises an antigenic region of myelin, myelin oligodendrocyte glycoprotein, or myelin lipoprotein. In other embodiments, X comprises an antigenic region of factor VIII, factor IX, insulin, uricase, PAL, or asparaginase. In still other embodiments, X comprises a foreign antigen, such as conarachidin (Ara h 1), allergen II (Ara h 2), peanut lectin, blue bean protein (Ara h 6), α-lactalbumin (ALA), lactoferrin, Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isoform, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, rye alkaloid, and oat protein.

Additionally, the present invention provides compositions comprising compounds of formula 1 (X-[-Y---Z]m), wherein m is an integer from about 1 to 100, X comprises an antigen against which the patient forms an undesirable immune response, or a tolerogenic portion thereof, or X comprises an antibody, antibody fragment, or ligand that specifically binds to a circulating protein or peptide or antibody that is involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason, Y comprises a linker portion, and Z comprises a liver-targeting portion.

In some embodiments, Z is galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine. In some embodiments, combinations of galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine may also be used. Furthermore, in some embodiments, galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine is optionally a β-anomer. In some embodiments, Z is conjugated to Y at its C1, C2, or C6 position.

In some embodiments, Y is selected from N-hydroxysuccinic acid amide linkages, maleimide linkages, vinyl sulfone linkages, pyridyl di-thiol-poly(ethylene glycol) linkages, pyridyl di-thiol linkages, n-nitrophenyl carbonate linkages, NHS-ester linkages, and nitrophenoxy poly(ethylene glycol) ester linkages. In some embodiments, Y comprises an antibody, antibody fragment, peptide or other ligand that specifically binds to X, a dithioalkyl ethyl ester, a structure represented by one of formulas Ya to Yp, or Y has a portion represented by formula Y’-CMP.

Where the left bracket "(" indicates the bond between X and Y, the right bracket or bottom bracket and ")" indicate the bond between Y and Z, n is an integer from about 1 to 100, q is an integer from about 1 to 44, ^R8 is _–CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-, Y' represents the remainder of Y, and W represents a polymer with the same ^W1 group, or W is a copolymer or random copolymer with the same or different ^W1 and ^W2 groups, wherein:

Where p is an integer from 2 to about 150, ^R9 is a direct bond, _–CH2 - _CH2 -NH–C(O)– or _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)-, t is an integer from 1 to 5; and ^R10 is an aliphatic group, an alcohol or an aliphatic alcohol.

In some such embodiments, n is about 40 to 80, p is about 10 to 100, q is about 3 to 20, ^R8 is _–CH2 - _CH2 -C( _CH3 )(CN)–, and when R9 is _–CH2 - _CH2 -NH–C(O)–, Z is glucose, galactose, N-acetylgalactosamine, or N-acetylglucosamine conjugated at its C1, and when W is a copolymer, R10 is 2-hydroxypropyl. In some embodiments, Y comprises formula Ya, Yb, Yc, Yf, Yg, Yh, Yi, Yk, Ym, or Yn. In some embodiments, Y comprises formula Ya, Yb, Yc, Ym, or Yn. Still in other embodiments, Y comprises formula Ya, Yb, Yc, Ym, or Yn.

In some embodiments, X comprises a foreign graft antigen against which the graft recipient forms an undesirable immune response, a foreign food, animal, plant, or environmental antigen against which the patient forms an undesirable immune response, a foreign therapeutic agent against which the patient forms an undesirable immune response, or a synthetic autoantigen against which the patient forms an undesirable immune response, or a tolerogenic portion thereof, and specific examples of various antigens are disclosed herein.

The present invention also provides a method for treating undesirable immune responses to antigens by administering an effective amount of a composition to a mammal requiring such treatment, the composition comprising a compound of formula 1 (X-[-Y---Z]m), wherein m is an integer from about 1 to 100, X comprises an antigen against which the patient forms an undesirable immune response, or a tolerogenic portion thereof, or X comprises an antibody, antibody fragment, or ligand that specifically binds to a circulating protein or peptide or antibody that is involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason, Y comprises a linker portion, and Z comprises a glucosylated liver-targeting portion.

In several such embodiments, X comprises an antigen against which the patient forms an undesirable immune response, or a tolerogenic portion thereof, and Y comprises an antibody, antibody fragment, peptide or other ligand that specifically binds to X, a dithioalkyl ethyl ester, a structure represented by one of formulas Ya to Yp, or Y having a portion represented by formula Y'-CMP, wherein a left bracket "(" indicates a bond between X and Y, a right bracket or bottom bracket and ")" indicate a bond between Y and Z, n is an integer from about 1 to 100, q is an integer from about 1 to 44, ^R8 is _–CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-, Y' represents the remaining portion of Y, and W represents a polymer of the same ^W1 group, or W is a copolymer or random copolymer of the same or different ^W1 and ^W2 groups, wherein:

Where p is an integer from 2 to about 150, ^R9 is a direct bond, _–CH2 - _CH2 -NH–C(O)– or _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)-, t is an integer from 1 to 5, and ^R10 is an aliphatic group, alcohol, or aliphatic alcohol. In several embodiments of such treatment methods, X comprises an antibody, antibody fragment, or ligand, and the composition is administered to clear circulating proteins or peptides or antibodies that specifically bind to X, said circulating proteins or peptides or antibodies being involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason.

In another embodiment, X comprises an antibody, an antibody fragment, or a ligand, and the composition is administered in an effective amount that reduces the concentration of antibodies in the patient's blood that are involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason by at least 50% w/w, measured over a period of about 12 to about 48 hours after the administration.

In several such treatment implementations, the composition is administered to induce tolerance in the patient with respect to antigen fraction X.

In some embodiments, X includes a foreign graft antigen against which the graft recipient forms an undesirable immune response, a foreign food, animal, plant, or environmental antigen against which the patient forms an undesirable immune response, a foreign therapeutic agent against which the patient forms an undesirable immune response, or a synthetic autoantigen against an endogenous form of the synthetic autoantigen, or a tolerogenic portion thereof.

Some embodiments disclosed in this invention provide compositions comprising compounds of formula 1:

in:

m is an integer approximately from 1 to 100;

X contains an antigen against which the patient develops an undesirable immune response, or a tolerogenic portion thereof; or

X contains antibodies, antibody fragments, or ligands that specifically bind to circulating proteins or peptides or antibodies, which are involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason.

Y contains a connection base portion; and

Z includes a liver-targeting component.

Z may also comprise galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine, for example, conjugated to Y at its C1, C2, or C6. N-acetylglucosamine and glucose bind to different lectin receptors than N-acetylgalactosamine and galactose. In the embodiments described below, experimental data (and the full disclosure of this application) indicate that selecting Z as N-acetylglucosamine results in an elevated level of regulatory T cell response compared to the level achieved with N-acetylgalactosamine. In some embodiments, unexpectedly, this leads to enhanced induction of immune tolerance and/or clearance of antigens from the subject's blood.

Y can be selected from: N-hydroxysuccinic acid amide group, maleimide group, vinyl sulfone group, pyridyl di-thiol-poly(ethylene glycol) group, pyridyl di-thiol group, n-nitrophenyl carbonate group, NHS-ester group and nitrophenoxy poly(ethylene glycol) ester group.

Y may also include: antibodies, antibody fragments, peptides or other ligands that specifically bind to X; dithioalkyl ethyl esters; structures represented by one of the formulas Ya to Yp:

Or Y has a portion represented by the formula Y’-CMP:

in:

The left bracket "(" indicates the key between X and Y;

A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z;

n is an integer from approximately 1 to 100;

q is an integer from approximately 1 to 44;

^R8 is _–CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-;

Y’ represents the remainder of Y (e.g., HS-PEG); and

W represents polymers with the same ^W1 group, or W is a copolymer (preferably a random copolymer) with the same or different ^W1 and ^W2 groups, wherein:

in:

p is an integer from 2 to approximately 150;

^R9 is a direct bond, _–CH2 - _CH2 -NH–C(O)– (i.e., an ethyl acetamino group or “EtAcN”) or _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)- (i.e., a PEGylated ethyl acetamino group or “Et-PEGt _- AcN”).

t is an integer from 1 to 5 (especially 1 to 3, and more especially 1 or 2); and

R ¹⁰ is an aliphatic group, an alcohol, or an aliphatic alcohol. In some embodiments, R ¹⁰ is a C_{_f}alkyl or C_{_f}alkylOH_{_g} , where f is independently an integer from 0 to 10, and g is independently an integer from 0 to 10. In some embodiments, R ¹⁰ is 2-hydroxypropyl.

In some embodiments, specific linkers are preferred. For example, in some embodiments, unexpectedly, a potent tolerability endpoint is generated based on the linker of Ym. In other embodiments, unexpectedly, a potent tolerability endpoint is generated based on the linker of Yn. In yet another embodiment, a formulation of F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀₎ achieves a particularly potent tolerability-related endpoint. In some embodiments, combinations of these linkers lead to synergistic results and a further unexpected increase in the induction of immune tolerance.

In another aspect above, n is about 40 to 80, p is about 10 to 100, q is about 3 to 20, ^R8 is _–CH2 - _CH2 -C( _CH3 )(CN)–; and when ^R9 is _–CH2 - _CH2 -NH–C(O)–, Z is galactose or N-acetylgalactosamine conjugated at its C1.

In another aspect mentioned above, Y includes formulas Ya, Yb, Yh, Yi, Yk, Ym, or Yn, particularly formulas Ya, Yb, Ym, or Yn.

X may also include: a foreign graft antigen against which the graft recipient forms an undesirable immune response; a foreign food, animal, plant, or environmental antigen against which the patient forms an undesirable immune response; a foreign therapeutic agent against which the patient forms an undesirable immune response; or a synthetic autoantigen against which the patient forms an undesirable immune response, or a tolerogenic portion thereof.

The present invention also relates to a method for treating an undesirable immune response to an antigen, which is carried out by administering an effective amount of a composition to a mammal requiring such treatment, the composition comprising a compound of formula 1 as disclosed herein. In some such methods, the composition may be administered to eliminate circulating proteins or peptides or antibodies that specifically bind to antigen portion X, which are involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason. An effective amount of the composition may be administered that reduces the concentration of antibodies in the patient's blood involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason by at least 50% w/w, as measured over a time period between about 12 and about 48 hours after the administration. The composition may be administered to induce tolerance in the patient with respect to antigen portion X.

In addition, the present invention also relates to the following embodiments.

1. Compounds containing Formula 1:

in:

m is an integer from approximately 1 to 10;

X contains a protein or protein fragment containing an antigenic region;

Y is a linking basis part having a formula selected from the following group:

in:

The left parenthesis "(" indicates the key with X;

A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z;

n is an integer from approximately 1 to 100;

The p that exists is an integer from approximately 2 to 150;

The q that exists is an integer from approximately 1 to 44;

The ^R8 present therein is _–CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-; and

The R ⁹ present therein is a direct bond or –CH ₂ -CH ₂ -NH–C(O)–; and Z contains the liver-targeting portion.

2. The compound of embodiment 1, wherein Z is galactose or glucose.

3. The compound of embodiment 1, wherein Z is galactosamine or glucosamine.

4. The compound of embodiment 1, wherein Z is N-acetylgalactosamine or N-acetylglucosamine.

5. The compound of embodiment 1, wherein Y is a linker portion having the following formula:

6. The compound of embodiment 1, wherein Y is a linker portion having the following formula:

7. The compound of embodiment 1, wherein Y is a linker portion having the following formula:

8. The compound of embodiment 1, wherein Y is a linker portion having the following formula:

9. The compound of embodiment 1, wherein X is a food antigen selected from the group consisting of: peanut globulin (Ara h 1), allergen II (Ara h 2), peanut lectin, blue bean protein (Ara h 6), α-lactalbumin (ALA), lactoferrin, Pena 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isotype, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, rye alkaloids, and oat protein and fragments thereof.

10. The compound of embodiment 9, wherein X is selected from the group consisting of: glutenin, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, rye alkaloids, and oat protein and fragments thereof.

11. The compound of embodiment 10, wherein X is selected from the group consisting of: glutenin, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, and Ω-gliadin and fragments thereof.

12. The compound of embodiment 11, wherein X is gluten or a fragment thereof.

13. The compound of embodiment 11, wherein X is gliadin or a fragment thereof.

14. The compound of embodiment 1, wherein X is a therapeutic agent selected from the group consisting of factor VII, factor IX, asparaginase, and uricase and fragments thereof.

15. The compound of embodiment 14, wherein X is a therapeutic agent selected from the group consisting of factor VII and factor IX and fragments thereof.

16. The compound of embodiment 15, wherein X is a therapeutic agent selected from the group consisting of factor VIII or a fragment thereof.

17. The compound of embodiment 14, wherein X comprises asparaginase or a fragment thereof.

18. The compound of embodiment 14, wherein X comprises uricase or a fragment thereof.

19. The compound of embodiment 1, wherein X is associated with an autoimmune disease.

20. The compound of embodiment 19, wherein the autoimmune disease is selected from the group consisting of: type 1 diabetes, multiple sclerosis, rheumatoid arthritis, vitiligo, uveitis, pemphigus vulgaris, and neuromyelitis optica.

21. The compound of embodiment 20, wherein the autoimmune disease is type 1 diabetes, and X comprises insulin or a fragment thereof.

22. The compound of embodiment 20, wherein the autoimmune disease is type 1 diabetes, and X comprises proinsulin or a fragment thereof.

23. The compound of embodiment 20, wherein the autoimmune disease is type 1 diabetes, and X comprises proinsulin or a fragment thereof.

24. The compound of embodiment 20, wherein the autoimmune disease is multiple sclerosis, and X comprises myelin or a fragment thereof.

25. The compound of embodiment 20, wherein the autoimmune disease is multiple sclerosis, and X comprises myelin oligodendrocyte glycoprotein or a fragment thereof.

26. The compound of embodiment 20, wherein the autoimmune disease is multiple sclerosis, and X comprises myelin lipoprotein or a fragment thereof.

27. The compound of embodiment 20, wherein the autoimmune disease is rheumatoid arthritis, and X is selected from the group consisting of fibrinogen, vimentin, type II collagen, α-enolase and fragments thereof.

28. The compound of embodiment 20, wherein the autoimmune disease is vitiligo, and X is selected from the group consisting of Pmel17, tyrosinase and fragments thereof.

29. The compound of embodiment 20, wherein the autoimmune disease is uveitis, and X is selected from the group consisting of: retinaldehyde inhibitory protein and interreceptor receptor-like visual pigment binding protein (IRBP) and fragments thereof.

30. The compound of embodiment 20, wherein the autoimmune disease is pemphigus vulgaris, and X is selected from the group consisting of: desmosome core proteins 3, 1 and 4, pemphigus annexin, desmosome glycoprotein, plaque protein, periplastic protein, desmosomal plaque protein, acetylcholine receptor and fragments thereof.

31. The compound of embodiment 20, wherein the autoimmune disease is neuromyelitis optica, and X is aquaporin-4 or a fragment thereof.

32. Use of the compound according to any one of embodiments 1 to 31 for inducing tolerance to X.

33. A composition comprising a compound of any one of embodiments 1 to 8.

34. The composition according to embodiment 33 is used to induce tolerance to X.

35. A method for inducing tolerance in a subject to an antigen that enables the subject to form an undesirable immune response, comprising administering a compound according to any one of embodiments 1 to 31.

36. The method of embodiment 35, wherein the compound is administered before the subject is exposed to the antigen.

37. The method of embodiment 35, wherein the compound is administered after the subject has been exposed to the antigen.

38. The method of embodiment 35, wherein the administration comprises at least one intravenous administration of the compound.

39. Use of a compound according to any one of embodiments 1 to 31 in the preparation of a medicament, wherein the medicament is used to induce tolerance to an antigen or a tolerogenic portion thereof, wherein a subject develops an undesirable immune response against the antigen.

40. A composition of any one of embodiments 1 to 8, wherein X comprises a foreign graft antigen or a tolerogenic portion thereof against which the graft recipient forms an undesirable immune response.

41. A composition of any one of embodiments 1 to 8, wherein X comprises a foreign food, animal, plant or environmental antigen or a tolerogenic portion thereof to which the patient forms an undesirable immune response.

42. A composition of any one of embodiments 1 to 8, wherein X comprises an exogenous therapeutic agent or a tolerogenic portion thereof to which the patient forms an undesirable immune response.

43. A composition of any one of embodiments 1 to 8, wherein X comprises a synthetic autoantigen or a tolerogenic portion thereof, to which the patient develops an undesirable immune response against an endogenous form of the synthetic autoantigen.

44. Compounds containing Formula 1:

in:

m is an integer from approximately 1 to 10;

X contains a protein or protein fragment that contains an antigenic region of a therapeutic protein selected from the following group: factor VIII, factor IX, insulin, uricase, PAL, and asparaginase.

Y is a connecting base part selected from the following group:

in:

The left parenthesis "(" indicates the key with X;

The right parenthesis “)” indicates the key with Z;

The parentheses “)” at the bottom indicate the key to Z;

There exist n that are integers from approximately 1 to approximately 80;

There exist q that are integers from approximately 1 to approximately 4;

The p that exists is an integer from approximately 1 to approximately 90;

The R8 present therein is –CH2– or –CH2-CH2-C(CH3)(CN)–; and

The presence of W in polymers representing W1 or W2 groups or

W is a copolymer of formula W1 or W2, wherein:

in:

R9 is a direct bond, –CH2-CH2-NH–C(O)– or -CH2-CH2-(O-CH2-CH2)t-NH-C(O)-;

t is an integer from 1 to 5;

R10 is an aliphatic group, an alcohol, or an aliphatic alcohol; and

Z contains one or more liver-targeting components.

45. The compound of embodiment 44, wherein the one or more liver-targeting portions comprise one or more of the following: galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine.

46. The compound of embodiment 44 or 45, wherein the one or more liver-targeting portions comprise one or more portions having a formula selected from the group consisting of:

47. The compound of any one of embodiments 44 to 46, wherein Y is selected from the group consisting of: formula Ya, formula Yb, formula Yc, formula Ym or formula Yn.

48. The compound of any one of embodiments 44 to 47, wherein Y is selected from the group consisting of: formula Ya, formula Yc, and formula Ym.

49. A compound of any one of embodiments 44 to 48, wherein X comprises an antibody, antibody fragment, or ligand that specifically binds to a circulating protein or peptide or antibody, said circulating protein or peptide or antibody being involved in graft rejection, immune response to a therapeutic agent, autoimmune disease, hypersensitivity, and/or allergic reactions for such reason.

50. The composition of any one of embodiments 44 to 49, wherein Z is conjugated with Y at C1, C2 or C6.

51. The composition of any one of embodiments 44 to 50, wherein Y is selected from N-hydroxysuccinic acid amide group, maleimide group, vinyl sulfone group, pyridyl di-thiol-poly(ethylene glycol) group, pyridyl di-thiol group, n-nitrophenyl carbonate group, NHS-ester group, and nitrophenoxy poly(ethylene glycol) ester group.

52. A pharmaceutically acceptable composition for inducing tolerance to a therapeutic protein in a subject with a defect in the production of a functionally similar natural protein, said pharmaceutically acceptable composition comprising a compound of any one of embodiments 44 to 51.

53. Use of any compound or composition of any one of embodiments 23 to 31 for treating an undesirable immune response to an antigen.

54. The compound or composition of any one of embodiments 44 to 52 is used to prepare a medicament for treating an undesirable immune response against an antigen.

55. Compounds of Formula 1:

in:

m is an integer from approximately 1 to 10;

X contains a protein or protein fragment containing an antigenic region;

Y is a connecting base part selected from the following group:

Or Y has a portion represented by the formula Y’-CMP:

in:

The left parenthesis “(” indicates the key between X and Y;

A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z;

n is an integer from approximately 1 to 100;

q is an integer from approximately 1 to 44;

R8 is –CH2– or –CH2-CH2-C(CH3)(CN)–;

Y’ represents the remainder of Y; and

W represents polymers with the same W1 group, or W is a copolymer or random copolymer with the same or different W1 and W2 groups, wherein:

in:

p is an integer from 2 to approximately 150;

R9 is a direct bond, –CH2-CH2-NH–C(O)– or -CH2-CH2-(O-CH2-CH2)t-NH-C(O)-;

t is an integer from 1 to 5; and

R10 is an aliphatic group, an alcohol, or an aliphatic alcohol.

56. The compound of embodiment 54, wherein:

n is approximately 40 to 80;

p is approximately 10 to 100;

q is approximately 3 to 20;

R8 is –CH2-CH2-C(CH3)(CN)–;

When R9 is –CH2-CH2-NH–C(O)–, Z is one of the following: galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine; and

When W is a copolymer, R10 is 2-hydroxypropyl.

57. The compound of embodiment 55 or 56, wherein Y comprises formula Ya, formula Yb, formula Yc, formula Yf, formula Yg, formula Yh, formula Yi, formula Yk, formula Ym or formula Yn.

58. The composition of any one of embodiments 55 to 57, wherein Y comprises formula Ya, formula Yb, formula Yc, formula Ym or formula Yn.

59. The composition of any one of embodiments 55 to 58, wherein Y comprises formula Ya, formula Yb, formula Yc, formula Ym or formula Yn.

60. The composition of any one of embodiments 55 to 59, wherein X comprises:

Foreign graft antigens that trigger an undesirable immune response in the graft recipient;

Foreign food, animal, plant, or environmental antigens that trigger an undesirable immune response in patients;

The patient responds to exogenous therapeutic agents that elicit an undesirable immune response; or

The patient synthesizes autoantigens, and against the endogenous form of these synthesized autoantigens, an undesirable immune response is generated.

Or its tolerogenic components.

61. A composition comprising a compound of any one of embodiments 55 to 60.

62. Use of the composition of any one of embodiments 55 to 61 for treating an undesirable immune response.

63. Use of the compounds of Formula 1 for treating undesirable immune responses against antigens:

in:

m is an integer approximately from 1 to 100;

X contains an antigen against which the patient develops an undesirable immune response, or a tolerogenic portion thereof; or

X includes antibodies, antibody fragments, or ligands that specifically bind to circulating proteins or peptides or antibodies, which are involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason.

Y contains a connection base portion; and

Z contains a glucosylated liver-targeting component;

The treatment is performed by administering an effective amount of the pharmaceutical composition to a mammal in need of such treatment.

64. The use of embodiment 63, wherein:

X contains an antigen against which the patient develops an undesirable immune response, or a tolerogenic portion thereof; and

Y includes:

Antibodies, antibody fragments, peptides, or other ligands that specifically bind to X;

Dithioalkyl ethyl ester;

Structures represented by one of the equations Ya to Yp:

Or Y has a portion represented by the formula Y’-CMP:

in:

The left bracket "(" indicates the key between X and Y;

A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z;

n is an integer from approximately 1 to 100;

q is an integer from approximately 1 to 44;

R8 is –CH2– or –CH2-CH2-C(CH3)(CN)–;

Y’ represents the remainder of Y; and

W represents polymers with the same W1 group, or W is a copolymer or random copolymer with the same or different W1 and W2 groups, wherein:

in:

p is an integer from 2 to approximately 150;

R9 is a direct bond, –CH2-CH2-NH–C(O)– or -CH2-CH2-(O-CH2-CH2)t-NH-C(O)-;

t is an integer from 1 to 5; and

R10 is an aliphatic group, an alcohol, or an aliphatic alcohol.

65. Use of embodiment 63 or 64, wherein X comprises the antibody, antibody fragment or ligand, and the composition is administered to eliminate circulating proteins or peptides or antibodies that specifically bind to X, the circulating proteins or peptides or antibodies being involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity and/or allergic reactions for this reason.

66. Use of any one of embodiments 63 to 65, wherein X comprises an antibody, an antibody fragment, or a ligand, and the composition is administered in an effective amount that reduces the concentration of antibodies in the patient’s blood that are involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, hypersensitivity, and/or allergic reactions for this reason by at least 50% w/w, as measured over a time period between about 12 and about 48 hours after the administration.

67. Use of any one of embodiments 63 to 66, wherein the composition is administered to induce tolerance of the patient to antigen portion X.

68. The use of any one of embodiments 63 to 67, wherein X comprises:

Foreign graft antigens that trigger an undesirable immune response in the graft recipient;

Foreign food, animal, plant, or environmental antigens that trigger an undesirable immune response in patients;

The patient responds to exogenous therapeutic agents that elicit an undesirable immune response; or

The patient synthesizes autoantigens, and against the endogenous form of these synthesized autoantigens, an undesirable immune response is generated.

Or its tolerogenic components.

69. Compounds containing Formula 1:

in:

m is an integer from approximately 1 to 10;

X contains food antigens, therapeutic agents, autoantigens, fragments of any such antigen, or mimic epitopes of any such antigen;

Y is the connection base part with the following formula:

in:

The left parenthesis "(" indicates the key with X;

A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z;

n is an integer approximately between 70 and 85;

The p that exists is an integer between approximately 85 and 95;

The q that exists is an integer from approximately 1 to 10;

The R8 present therein is –CH2– or –CH2-CH2-C(CH3)(CN)–; and

The R9 present therein is a direct bond or –CH2-CH2-NH–C(O)–; and

Z contains a liver-targeting component, which contains glucose or galactose.

70. The compound of embodiment 69, wherein:

m is 1-3,

n is 79.

p is 90, and

q is 4.

71. The compound of embodiment 70, wherein X is selected from the group consisting of: insulin, proinsulin, pre-proinsulin, gluten, gliadin, myelin, myelin oligodendrocyte glycoprotein and proteolipoprotein, factor VIII, factor IX, asparaginase, uricase and any of the aforementioned fragments.

72. Compounds containing Formula 1:

in:

m is an integer from approximately 1 to 10;

X is selected from the following group: insulin, proinsulin, preinsulin, gluten, gliadin, myelin, myelin oligodendrocyte glycoprotein and proteolipoprotein, factor VIII, factor IX, asparaginase, uricase and any of the aforementioned fragments;

Y is the connection base part with the following formula:

in:

The left parenthesis "(" indicates the key with X;

A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z;

n is an integer approximately between 70 and 85;

The p that exists is an integer between approximately 85 and 95;

The q that exists is an integer from approximately 1 to 10;

The R8 present therein is –CH2– or –CH2-CH2-C(CH3)(CN)–; and

The R9 present therein is a direct bond or –CH2-CH2-NH–C(O)–; and

Z contains a liver-targeting component, which includes a glycoside.

73. The compound of embodiment 69, wherein:

m is 1-3,

n is 79.

p is 90, and

q is 4.

74. The compound of embodiment 73, wherein Z is selected from the group consisting of glucose, glucosamine, galactose, galactosamine, N-acetylgalactosamine, and N-acetylglucosamine.

Brief description of the attached diagram

Figures 1A-1D are a series of diagrams showing the different cellular uptake of galactose conjugates. Figure 1A shows that F1aA-PE- _m4 - _n80 (Gal-PE) preferentially targets PE to hepatic sinusoidal endothelial cells (LSEC). Figure 1B shows that F1aA-PE- _m4 - _n80 (Gal-PE) preferentially targets PE to hepatic Kupffer cells (KC). Figure 1C shows that F1aA-PE- _m4 - _n80 (Gal-PE) preferentially targets PE to hepatocytes. Figure 1D shows that F1aA-PE- _m4 - _n80 (Gal-PE) preferentially targets PE to other antigen-presenting cells (APCs) in the liver. * = P < 0.05.

Figure 2 shows the proliferation of OT-I CD8+ T cells in mice treated with F1aA-OVA- _m4 - _n80 (Gal-OVA), OVA, or saline (i.e., untreated), with the largest proliferation observed in the Gal-OVA-treated group.

Figures 3A-3B are a series of graphs relating to marker expression on T cells. Figure 3A shows the percentage of OT-I CD8 ⁺ T cells expressing PD-1 (“PD1+”) in proliferating T cell generations treated with saline, OVA, or F1aA-OVA- _{m4 - n80} (GAL-OVA), with the highest PD-1 levels in the gal-OVA treatment group. Figure 3B shows the percentage of OT-I CD8+ T cells expressing phosphatidylserine (stained “annexin V ₊ ”) in proliferating T cell generations treated with saline, OVA, or F1aA-OVA-m4 ^-n80 (GAL-OVA), with the highest annexin V+ cell levels in the gal-OVA treatment group.

Figure 4 shows that the galactose conjugate [F1aA-OVA- _m4 - _n80 (Gal-OVA)] reduces the immunogenicity of OVA, as determined by the OVA-specific antibody titer (shown as Ab titer ^log⁻¹ ).

Figure 5 shows that repeated administration of F1aA-OVA- _m4 - _n80 (Gal-OVA) over time depletes OVA-specific antibodies from mouse serum.

Figures 6A-6F show data related to the reduction of OVA-specific immune responses. Figure 6A shows the immune response in mice challenged with OVA and LPS. Figure 6B shows the immune response in mice treated with OVA, while Figure 6C shows the immune response in untreated mice. Figures 6D and 6E (respectively) show that F1aA-OVA- _m4 - _n80 (mGal-OVA; 6D) and F1b-OVA- _m1 - _n44 - _p34 (pGal-OVA; 6E) can reduce OVA-specific immune responses in draining lymph nodes after intradermal challenge with OVA and adjuvant LPS. Figure 6F is from the parent design and does not form part of this invention.

Figures 7A-7B show the characterization of F1aA-OVA- _m4 - _n80 and F1b-OVA- _m1 - _n44 - _p34 . Figure 7A shows the size exclusion HPLC curves of F1aA-OVA- _m4 - _n80 (hollow triangle), F1b-OVA- _m1 - _n44 - _p34 (solid circle), and unconjugated OVA (solid line). A leftward shift indicates an increase in molecular weight. Figure 7B shows polyacrylamide gels demonstrating the increased molecular weight after OVA conjugation: (1.) unconjugated OVA, (2.) F1aA-OVA- _m4 - _n80 , and (3.) F1b-OVA- _m1 - _n44 - _p34 .

Figures 8A-8B show data related to decreased antigen-specific immune responses following administration of F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ ) [labeled as OVA-p(Glu-HPMA) and shown as solid circles] or F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ [labeled as OVA-p(Gal-HPMA) and shown as solid rhombuses]. Figure 8A shows the OTI CD8+ T cell population (CD3e ⁺ /CD8α ⁺ /CD45.2 ⁺⁾ quantified from draining lymph nodes (groin and popliteal) 4 days after antigen challenge in CD45.1 ⁺ mice. Flow cytometry analysis of OT-II CD8+ T cells was performed. A significant reduction in OT-II CD8 ⁺ T cells was detected after administration of OVA-p (Gal-HPMA) and OVA-p (Glu-HPMA). Figure 8B shows flow cytometry analysis of the OT-II CD4+ T cell population (CD3e ⁺ /CD4 ⁺ /CD45.2 ⁺ ) quantified from draining lymph nodes (groin and popliteal) in CD45.1+ mice 4 days after antigen challenge. A significant reduction in OT-II CD4+ T cells was detected after administration of OVA-p (Gal-HPMA) and OVA-p (Glu-HPMA). *＝P<0.05, **＝P<0.01;#＝P<0.05,##＝P<0.01(# indicates significance compared to untreated animals).

Figures 9A-B show data related to the increase in antigen-specific regulatory T cells in lymph nodes and spleen of mice after antigen challenge. Figure 9A shows the F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q 4 -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ ) [labeled as OVA-p(Glu-HPMA) and shown as solid circles] and F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ ] collected from lymph nodes in CD45.1+ mice ₄ days after antigen challenge. Increased flow cytometry detection induced by [labeled OVA-p(Gal-HPMA) and shown as solid rhombuses]. Figure 9B shows the corresponding analysis of spleens from mice treated with OVA-p(Glu-HPMA) or OVA-p(Gal-HPMA) compared to animals treated with OVA or saline (i.e., challenge). * = P < 0.05, ** = P <0.01; *** = P <0.001;# = P < 0.01, ## = P <0.01;### = P < 0.001 (# indicates significance compared to untreated animals).

Figure 10 shows flow cytometry data related to the decrease in the percentage of antigen-specific effector cells (IFNγ+OTICD8+ T cells (CD3e+CD8α+CD45.2+IFNγ+) 4 days after antigen challenge in CD45.1+ mice. The data were obtained using F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ [labeled as OVA-p(Glu-HPMA) and shown as solid circles] or F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA _60] . Mice treated with the [labeled OVA-p(Gal-HPMA) and shown as solid diamonds] conjugate produced significantly fewer IFNγ+OTI CD8+ T cells after antigen challenge compared to animals treated with OVA or saline (i.e., challenge). * = P < 0.01, ** = P <0.01;## = P < 0.01 (# indicates significance compared to untreated animals).

Figures 11A-11B show data related to T cell depletion and regulation in an OTII adoptive transfer model where OTII cells (CD4 ⁺ T cells from CD45.2 ⁺ mice) were adoptively transferred into CD45.1 ⁺ acceptors, which were administered with either F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ [“OVA-p(Gal ^- HPMA)”] or F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA _60] Treatment with either [“OVA-p(Glu-HPMA)”] or unlinked OVA [“OVA”] was used to induce a T-regulated response and prevent subsequent responses to vaccine-mediated antigen challenge. On day 0, ³ x 10⁵ CFSE-labeled OTI and 3 x ^10⁵ CFSE-labeled OTII cells were adoptively transferred to CD45.1. ⁺ Mice (n = 8 mice per group). The tolerable dose or control dose was administered on days 1, 4, and 7. In one regimen, OVA was administered at the following doses: 2.5 μg on day 1, 2.5 μg on day 4, and 16 μg on day 7. In another regimen, the same total dose was achieved at the following doses: 7 μg on day 1, 7 μg on day 4, and 7 μg on day 7. Similarly, the same total dose was achieved at 2.5 μg on day 1, 2.5 μg on day 4, and 16 μg on day 7, or 7 μg on day 1, 7 μg on day 4, and 7 μg on day 7. In the other groups, pGal-OVA and pGlu-OVA were administered separately, with all doses based on equivalent OVA doses. In the final group, saline was administered on the same day. On day 14, recipient mice were then challenged by intradermal injection of OVA (10 μg) adjuvanted with lipopolysaccharide (50 ng). Characterization of draining lymph nodes was completed on day 19 to allow determination of whether actual attrition occurred and whether regulatory T cells were induced from adoptive transfer cells. Figure 11A shows the number of OTII cells present after challenge, and Figure 11B shows the frequency of FoxP3 ⁺ CD25 ⁺ cells (a marker of regulatory T cells). * and # indicate p < 0.05, ** and ## indicate p < 0.01, and ### indicate p < 0.001.

Figures 12A-12B show data related to T cell depletion and regulation in an OTI adoptive transfer model where OTI cells (CD8 ⁺ T cells from CD45.2 ⁺ mice) were adoptively transferred to CD45.1 ⁺ acceptors, which were administered with either F1m' ^- OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ [“OVA-p(Gal-HPMA)”] or F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA _60] Treatment with either [“OVA-p(Glu-HPMA)”] or unlinked OVA [“OVA”] was used to induce a T-regulated response and prevent subsequent responses to vaccine-mediated antigen challenge. Both 3 x ^10⁵ CFSE-labeled OTI and 3 x ^10⁵ CFSE-labeled OTII cells were adoptively transferred to CD45.1 on day 0. ⁺ Mice (n = 8 mice per group). Tolerable or control doses were administered on days 1, 4, and 7. In one regimen, OVA was administered at the following doses: 2.5 μg on day 1, 2.5 μg on day 4, and 16 μg on day 7. In another regimen, OVA was administered at the following doses: 7 μg on day 1, 7 μg on day 4, and 7 μg on day 7, achieving the same total dose. Similarly, in other groups, pGal-OVA and pGlu-OVA were administered at the same doses of 2.5 μg on day 1, 2.5 μg on day 4, and 16 μg on day 7, or 7 μg on day 1, 7 μg on day 4, and 7 μg on day 7. OVA, all doses based on equivalent OVA doses. In the final group, saline was administered on the same day. Then, on day 14, recipient mice were challenged by intradermal injection of OVA (10 μg) adjuvanted with lipopolysaccharide (50 ng). Characterization of draining lymph nodes was completed on day 19 to allow determination of whether actual attrition occurred and whether regulatory T cells responded to antigen re-exposure via their cytokine expression. Figure 12A shows the number of OTII cells present after challenge, and Figure 12B shows the frequency of IFNγ-expressing cells (which lacked an indicator of non-responsiveness). * and # indicate p < 0.05, ** and ## indicate p < 0.01.

Figure 13 shows the data related to blood glucose levels. Mice were treated with conjugates (or saline) of F1m'-P31-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ [labeled P31-p(Glu-HPMA)], F1m'-P31-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ [labeled P31-p(Gal-HPMA)]). Animals receiving P31-p(Glu-HPMA) or P31-p(Gal-HPMA) maintained normal blood glucose levels for up to 42 days, while animals treated with P31 or saline developed rapid hyperglycemia within 5-10 days, demonstrating that the conjugates disclosed in this invention protect mice from T-cell-induced autoimmune diabetes.

Figure 14 shows data associated with the development of spontaneous diabetes in non-obese diabetic (NOD) mice. Mice treated with F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀₎ are shown as solid squares. Mice treated with F1c'-insulin-Bm ₁ -n ₄ -p _90- CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ are shown as solid triangles. Mice treated with saline are shown as solid rhombuses. Treatment with the compound of Formula 1 reduced the incidence of diabetes onset in NOD mice compared to saline-treated animals.

Figures 15A-15B show data related to the biodistribution of the model antigen OVA linked to synthetic glycopolymers, illustrating liver uptake under conditions of restricted spleen uptake. A. Fluorescence signals of perfused livers collected from animals treated with OVA (1) or OVA (2-5) conjugated with various glycopolymers. B. Fluorescence images of spleens collected from animals treated with OVA (1) or OVA (2-5) conjugated with various glycopolymers. The formulations are as follows: 1. OVA, 2. OVA-p(Galβ-HPMA), 3. OVA-p(Gal-HPMA), 4. OVA-p(Gluβ-HPMA), 5. OVA-p(Glu-HPMA).

Figures 16A through 16F show data related to the experiments comparing the linker base portion. OVA-p(Gal-HPMA), OVA-p(Glu-HPMA), OVA-p(Galβ-HPMA), and OVA-p(Gluβ-HPMA) conjugates were synthesized, and their ability to induce antigen-specific T cell non-responsiveness and eliminate T cell populations causing long-term memory was tested. Figure 16A shows a schematic of the treatment regimen used in the 7-day experiment. Figure 16B shows the percentage of proliferating OTI spleen T cells, as determined by CSFE dilution. Figure 16C shows the percentage of annexin V+ OTI T cells in the spleen of animals treated with OVA-glycopolymer conjugates or free OVA. Figure 16D shows the percentage of PD-1+ spleen OTI cells. Figure 16E shows the percentage of T memory cells in the OTI population, where T memory cells are defined as CD62L+ and CD44+. Figure 16F shows the percentage of OTI cells expressing CD127. Of particular note was the unexpectedly enhanced potency of compositions using the β-conformation of glucose or galactose compared to the α-conformation. * = P < 0.05, ** = P < 0.01; # = P < 0.05, ## = P < 0.01, and ### = P < 0.001 (# indicates significance compared to animals treated with OVA alone).

Figure 17 shows data related to the development of diabetes symptoms in the untreated, control, and experimental groups.

Figure 18 shows the various treatment groups and experimental timelines used in experiments related to assessing long-acting tolerance induction in metastatic T cells.

Figures 19A-19B show data related to the composition of T cells in lymph nodes. Figure 19A shows the percentage of OTI cells remaining after antigen challenge in various treatment groups (as a percentage of total CD8 ⁺ cells). Figure 19B shows the percentage of OTII cells remaining after antigen challenge in various treatment groups (as a percentage of total CD4 ⁺ cells).

Figure 20 shows the various treatment groups and experimental timelines used in experiments related to assessing long-acting tolerance induction in endogenous T cells.

Figures 21A-21B show data related to T cell composition in lymph nodes. Figure 21A shows the percentage of OTI cells remaining after antigen challenge in various treatment groups (as a percentage of total CD8 ⁺ cells). Figure 21B shows the percentage of OTII cells remaining after antigen challenge in various treatment groups (as a percentage of total CD4 ⁺ cells).

Figure 22 shows an experimental design for evaluating the ability of compositions as disclosed in this invention to preventively reduce antibody responses.

Figure 23 shows experimental data relating to the amount of anti-asparaginase antibodies in mice from various treatment groups.

Figures 24A and 24B illustrate experimental designs and compositions for evaluating tolerance to myelin oligodendrocyte glycoprotein (MOG). Figure 24A shows the experimental protocol for immunizing donor mice and treating recipient mice. Figure 24B shows an example of a tolerogenic composition according to some embodiments disclosed herein.

Figures 25A and 25B show experimental data related to tolerance induction against MOG obtained using the first concentration of the tolerogenic composition. Figure 25A shows data related to delay in disease onset in various treatment groups. Figure 25B shows data related to reduction in weight loss in various treatment groups.

Figures 26A and 26B show experimental data related to tolerance induction against MOG obtained with other concentrations of the tolerogenic composition. Figure 26A shows data related to delay in disease onset in various treatment groups. Figure 26B shows data related to reduction in weight loss in various treatment groups.

Figures 27A through 27E show experimental data related to the biodistribution of the pGal and pGlu compositions with the β conformation. Figure 27A shows LSECs targeted to the liver via various conjugates. Figure 27B shows Kupffer cells targeted to the liver via various conjugates. Figure 27C shows CD11c+ cells targeted to the liver via various conjugates. Figure 27D shows hepatocytes targeted to the liver via various conjugates. Figure 27E shows stellate cells targeted to the liver via various conjugates.

Invention Details

Two known asialic acid glycoprotein receptors (“ASGPRs”) are expressed on hepatocytes and sinusoidal endothelial cells (or “LSECs”). Various forms of other galactose/galactosamine/N-acetylgalactosamine receptors can be found in a variety of cell types [e.g., dendritic cells, hepatocytes, LSECs, and Kupffer cells]. While the molecular and cellular targets of glucose, glucosamine, and N-acetylglucosamine can differ from those of their corresponding galactose isomers, it has been found that in some cases, the corresponding compounds of Formula 1, where Z is the glucosylated moiety, are equally effective, and in some embodiments disclosed in this invention, they are unexpectedly effective. Dendritic cells are considered “specialized antigen-presenting cells” because their primary function is to present antigens to the immune system for generating an immune response. While some cells within the liver are known to present antigens, the liver is more widely known to be involved in tolerogenicity. The liver is understood to be a tolerogenic organ. For example, in the case of multi-organ grafts, when the liver is one of the transplanted organs, a lower rate of rejection has been reported. LSECs are much newer in the literature; therefore, their role in inducing tolerance and/or mitigating inflammatory immune responses is not widely recognized or fully understood. However, it is becoming increasingly clear that they can also play a significant role in the induction of antigen-specific tolerance.

One of the most striking features of the surface of red blood cells is their glycosylation, meaning the presence of a significant number of glycosylated proteins. In fact, blood group glycoproteins (e.g., blood group glycoprotein A) have been utilized as targets for red blood cell binding. Blood group glycoproteins are proteins with numerous covalently linked sugar chains, terminated by sialic acid. As red blood cells grow and mature and are cleared, the terminal sialic acid of their blood group glycoproteins tends to be lost, leaving N-acetylgalactosamine at the free end. N-acetylgalactosamine is selectively received by ligands associated with hepatocyte-associated ASGPR, leading to the binding of N-acetylgalactosamine-containing substances by hepatocytes and their subsequent uptake and processing in the liver.

To date, those skilled in the art have understood that glycosylation of therapeutic agents leading to liver targeting should be avoided due to the poor circulating half-life of therapeutic agents resulting from first-pass clearance by the liver. For the same reason, some monoclonal antibodies require specific glycosylation at Asn297 to achieve optimal binding to their Fc receptor. It has now been surprisingly discovered, and is disclosed herein, that galactosylation and glucosylation can be used in a manner that induces tolerance.

In some embodiments, the present invention provides therapeutic compositions that are targeted to the liver, particularly hepatocytes, LSECs, Kupffer cells, and/or astrocytes, more particularly hepatocytes and/or LSECs (and taken up by them), and even more specifically, specifically bind to ASGPR. Two mechanisms by which liver-targeted therapy promotes treatment are tolerization and clearance. Tolerization utilizes the liver's role in clearing apoptotic cells and processing their proteins for recognition as "self" by the immune system, and in sampling peripheral proteins for immune tolerance. Clearance utilizes the liver's role in blood purification through the rapid removal and breakdown of toxins, peptides, etc. Targeting these compositions to the liver is accomplished by: glycosylation (e.g., galactose, galactosamine, and N-acetylgalactosamine, particularly conjugated at _C1 , C2, or C6, although some embodiments involve conjugation at other or any carbons in the molecule), glucosylation (e.g., glucose, glucosamine, and N-acetylglucosamine, particularly conjugated at _C1 , C2, or C6, although some embodiments involve conjugation at other or any carbons in the molecule), or by desialylation of the peptide to which such liver targeting is desired. The galactosylated or glucosylated moieties can be chemically conjugated to or recombinantly fused to the antigen, while desialylation exposes the galactose-like moieties on the antigenic peptide. Antigens can be endogenous (autoantigens) or exogenous (foreign antigens), including but not limited to: exogenous graft antigens to which the transplant recipient forms an undesirable immune response (e.g., graft rejection); foreign food, animal, plant, or environmental antigens to which the patient forms an undesirable immune response (e.g., allergic or hypersensitivity); therapeutic agents to which the patient forms an undesirable immune response (e.g., hypersensitivity and/or reduced therapeutic activity); autoantigens to which the patient forms an undesirable immune response (e.g., autoimmune diseases); or tolerogenic motifs (e.g., fragments or epitopes); these compositions can be used to induce tolerance to the antigen. Alternatively, galactosylation or other liver-targeting motifs can be conjugated to antibodies, antibody fragments, or ligands that specifically bind to circulating proteins or peptides or antibodies that are involved in graft rejection, immune responses to therapeutic agents, autoimmune diseases, and/or allergic reactions (as discussed above); these compositions can be used to clear circulating proteins, peptides, or antibodies. Therefore, according to embodiments, the compositions of the present invention can be used to treat undesirable immune responses, such as graft rejection, immune responses to therapeutic agents, autoimmune diseases, and/or allergic reactions. Pharmaceutical compositions are also provided comprising a therapeutically effective amount of the composition of the present invention, mixed with at least one pharmaceutically acceptable excipient. In another aspect, the present invention provides a method for treating undesirable immune responses, such as graft rejection, responses to therapeutic agents, autoimmune diseases, or allergic reactions.

definition

As used in this specification, the following words and phrases are generally intended to have the meanings listed below, unless otherwise indicated in the context in which they are used.

The singular forms “a,” “an,” and “the/that” include plural references unless the context clearly indicates otherwise.

The term “about” when used in conjunction with a numerical value is intended to cover a range of values, the lower limit of which is typically, for example, 5-10% smaller than the indicated value, and the upper limit of which is typically, for example, 5-10% larger than the indicated value. It also includes any numerical values within a publicly disclosed range, including the listed endpoints.

As used in this invention, "antigen" is any substance that acts as a target of a receptor for an adaptive immune response, such as a T-cell receptor, major histocompatibility complex class I and II, a B-cell receptor, or an antibody. In some embodiments, the antigen may be derived from within the body (e.g., "self," "autologous," or "endogenous"). In other embodiments, the antigen may be derived from outside the body ("non-self," "exogenous," or "foreign"), for example, through inhalation, ingestion, injection, or transplantation, percutaneous delivery, etc. In some embodiments, exogenous antigens may be biochemically modified within the body. Exogenous antigens include, but are not limited to, food antigens, animal antigens, plant antigens, environmental antigens, therapeutic agents, and antigens present in allogeneic grafts.

As used in this invention, "antigen-binding molecule" refers to a molecule containing an antibody variable region that provides binding to an epitope (or, in some embodiments, specific binding), particularly a protein such as an immunoglobulin molecule. The antibody variable region can be present in, for example, an intact antibody, an antibody fragment, and a recombinant derivative of an antibody or antibody fragment. As used in this invention, the term "antigen-binding fragment" (or "binding portion") 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 (but are not limited to) "Fv", "Fab", and "F(ab') ₂ " regions, "single-domain antibody (sdAB)", "nanobody", "single-chain Fv (scFv)" fragments, "tandem scFv" (V _H AV _L AV _H BV _L B), "biantibody", "tribodies" or "tribodies", "single-chain biantibody (scDb)", and "bispecific T-cell adaptor (BiTE)".

As used in this invention, "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 made to the side chains or ends, for example, altering the amino terminus or carboxyl terminus. In some embodiments, modifications can be used to create chemical groups that can be readily used to link the polypeptide to other materials or attach it to therapeutic agents.

The term "comprising," synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unstated elements or method steps. The phrase "consisting of..." excludes any unspecified element, step, or ingredient. The phrase "substantially consisting of..." limits the scope of the described subject matter to the specified materials or steps and those that do not substantially affect its basis and novel features. It should be understood that wherever the term "comprising" is used to describe an embodiment, other similar embodiments described with the terms "consisting of..." and/or "substantially consisting of..." are also provided. When used as transitional phrases in the claims, each should be interpreted separately and within the appropriate legal and factual context (e.g., "comprising" is considered more of an open-ended phrase, "consisting of..." more exclusive, and "substantially consisting of..." reaching an intermediate level).

Generally, "conservative changes" can be made to amino acid sequences without altering their activity. These changes are called "conservative substitutions" or mutations; that is, one amino acid can be substituted with an amino acid belonging to a group of amino acids with a specific size or characteristic. Substitutions in an amino acid sequence can be selected from other members of the same class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are not expected to materially affect apparent molecular weight or isoelectric point, as determined by polyacrylamide gel electrophoresis. Conservative substitutions also include substituting one optical isomer of the sequence for another optical isomer; specifically, substituting an L amino acid for one or more residues of the sequence with a D amino acid. Furthermore, all amino acids in the sequence can undergo DL isomer substitution. Exemplary conservative substitutions include, but are not limited to, substituting Lys for Arg and vice versa to maintain a positive charge; substituting Glu for Asp and vice versa to maintain a negative charge; substituting Ser for Thr to maintain free OH; and substituting Gln for Asn to maintain free _NH₂ . Another type of conservative substitution occurs where, if chemical derivatization is desired, an amino acid with the desired chemical reactivity is introduced to confer a reaction site for the chemical conjugation reaction. Such amino acids include, but are not limited to, Cys (for inserting a thiol group), Lys (for inserting a primary amine), Asp and Glu (for inserting a carboxylic acid group), or specific non-canonical amino acids containing ketone, azide, alkyne, olefin, and tetrazine side chains. Conservative substitution or addition of amino acids carrying free _-NH₂ or -SH can be particularly advantageous for chemical conjugation with the linker and galactosylation moiety of Formula 1. Furthermore, in some cases, point mutations, deletions, and insertions of polypeptide sequences or corresponding nucleic acid sequences can be performed without loss of polypeptide or nucleic acid fragment function. 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 (including any number of substitutions among those listed). Variations usable in this invention may exhibit changes in a total number of amino acid sequences up to 200 (e.g., up to 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 200, including any number among those listed) (e.g., exchanges, insertions, deletions, N-terminal truncation, and/or C-terminal truncation). In some embodiments, the number of changes is greater than 200. Additionally, in some embodiments, variants include polypeptide sequences or corresponding nucleic acid sequences that exhibit a degree of functional equivalence to a reference (e.g., an unmodified or native sequence). In some embodiments, variants exhibit approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 97%, approximately 98%, or approximately 99% functional equivalence to the unmodified or native reference sequence (and any degree of functional equivalence among those listed). To conform to standard polypeptide nomenclature J. Biol. Chem., (1969), 243, 3552-3559, the amino acid residues described herein are represented by single-letter amino acid identifiers or three-letter abbreviations. All amino acid residue sequences are represented in this invention by general formulas with left and right orientations in a conventional amino-to-carboxyl direction.

The term "effective amount" or "therapeutic effective amount" refers to the amount of the composition of the present invention sufficient to achieve the treatment as defined below when administered to a mammal requiring such treatment. This amount will vary depending on the subject being treated and the nature of the disease, the subject's weight and age, the severity of the disease, the specific composition of the present invention selected, the dosing regimen to be followed, the timing of administration, the method of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

An epitope, also known as an antigenic determinant, is a segment of a macromolecule (e.g., a protein) that is recognized by the adaptive immune system, such as by antibodies, B cells, major histocompatibility complex molecules, or T cells. An epitope is a portion or segment of a macromolecule capable of binding an antibody or its antigen-binding fragment. In this context, the term "binding" specifically refers to specific binding. In the context of some embodiments of the invention, it is preferred that the term "epitaxe" refer to a segment of a protein or polymer recognized by the immune system.

The term galactose refers to a monosaccharide existing in both open-chain and cyclic forms, with D- and L-isomers. In the cyclic form, there are two anomers, α and β. In the α form, the C1 alcohol group is axially positioned, while in the β form, the C1 alcohol group is equatorially positioned. Specifically, "galactose" refers to a cyclic six-membered pyranose, more specifically, the D-isomer and even more specifically the α-D type (α-D-galactopyranose), whose formal name is (2R,3R,4S,5R,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol. Glucose is a diastereomer of galactose; its formal name is (2R,3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol. The structures and numbering of galactose and glucose are shown, and two non-limiting examples of stereochemical representation are given.

The term "galactosylated moiety" refers to a specific type of liver-targeting moiety. Galactosylated moieties include, but are not limited to, galactose, galactosamine, and/or N-acetylgalactosamine residues. "Glucosylated moieties" refer to another specific type of liver-targeting moiety, and include, but are not limited to, glucose, glucosamine, and/or N-acetylglucosamine.

The term "liver-targeting portion" refers to a portion capable of directing, for example, a polypeptide to the liver. The liver contains different types of cells, including but not limited to hepatocytes, sinusoidal epithelial cells, Kupffer cells, stellate cells, and/or dendritic cells. Typically, a liver-targeting portion directs a polypeptide to one or more of these cells. On the surface of the corresponding hepatocyte, there are receptors that recognize and specifically bind to the liver-targeting portion. Liver targeting can be achieved through: chemical conjugation of an antigen or ligand to a galactosylated or glucosylated portion; desialylation of the antigen or ligand to expose the underlying galactosyl or glucosyl portion; or specific binding of an endogenous antibody to an antigen or ligand, wherein the antigen or ligand is desialylated to expose the underlying galactosyl or glucosyl portion; conjugation to a galactosylated or glucosylated portion. Naturally occurring desialylated proteins are not covered within the scope of some embodiments of the invention.

The “numerical values” and “ranges” provided for various substituents are intended to cover all integers within the range. For example, when n is defined as an integer representing a mixture comprising about 1 to 100, particularly about 8 to 90, more particularly about 40 to 80 ethylene glycol groups (where mixtures typically cover integers specified as n ± about 10% (or smaller integers from 1 to about 25, ± 3)), it should be understood that n can be an 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, 35, 37, 4 0, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83, 85, 88, 90, 95, 99, 100, 105, or 110, or any of those listed, including the endpoints of the range), and the disclosed mixtures cover 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. Wherever used, the terms “about” and “±10%” or “±3” for combinations shall be understood to disclose and provide specific support for equivalent ranges.

The terms “optional” or “optionally” refer to the event or situation described below that may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur.

A peptide that specifically binds to a particular target is called a "ligand" of that target.

"Polypeptide" is a term referring to a chain of amino acid residues, whether or not it has post-translational modifications (e.g., phosphorylation or glycosylation), and/or complexed with other polypeptides, and/or synthesized with nucleic acids and/or carbohydrates or other molecules as a multi-subunit complex. Therefore, proteoglycans are also referred to as polypeptides in this invention. Long polypeptides (having more than about 50 amino acids) are called "proteins." Short polypeptides (having less than about 50 amino acids) are called "peptides." Depending on their size, amino acid composition, and three-dimensional structure, some polypeptides may be referred to as "antigen-binding molecules," "antibodies," "antibody fragments," or "ligands." Polypeptides can be produced by many methods, many of which are well known in the art. For example, polypeptides can be obtained by extraction (e.g., from isolated cells), by expressing recombinant nucleic acids encoding the polypeptide, or by chemical synthesis. Polypeptides can be produced, for example, by recombinant techniques and by introducing an expression vector encoding the polypeptide into host cells for expressing the polypeptide (e.g., by transformation or transfection).

As used in this invention, "pharmaceutical-acceptable carrier" or "pharmaceutical-acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption-retarding agents, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is covered unless any conventional media or reagent is incompatible with the active ingredient. Additional active ingredients may also be added to the composition.

As used in this invention with respect to polypeptides, the term "purified" refers to a polypeptide that has been chemically synthesized and is therefore substantially free from contamination by other polypeptides, or has been separated or isolated from most other cellular components (e.g., other cellular proteins, polynucleotides, or cellular components) that naturally accompany it. An example of a purified polypeptide is a polypeptide that is at least 70% (by dry weight) free of proteins and naturally occurring organic molecules associated with it. Therefore, formulations of purified polypeptides can be, for example, at least 80%, at least 90%, or at least 99% (by dry weight) polypeptides. Polypeptides may also be engineered to contain tagged sequences (e.g., multihistidine tags, myc tags, or other affinity tags) that facilitate purification or labeling (e.g., capture onto an affinity matrix, visualization under a microscope). Therefore, unless otherwise indicated, a purified composition containing a polypeptide refers to a purified polypeptide. The term "isolated" indicates that the polypeptide or nucleic acid of the present invention is not in its natural environment. Therefore, the isolated products of the present invention may be contained in the culture supernatant, partially enriched, generated from a heterologous source, cloned in a vector, or prepared with a vehicle, etc.

The term "random copolymer" refers to the product of the simultaneous polymerization of two or more monomers in a mixture, wherein the probability of finding a given monomer unit at any given site in the polymer chain does not depend on the nature of the adjacent units at said site (Bernoullian distribution). Therefore, when the variable group identified as _Wp represents a random copolymer, the chain can contain any sequence of up to about 150 ^W1 and ^W2 groups, such as ^-W1 - ^W2 - ^W1 - ^W2- ; ^-W2 -W1- ^W2 -W1-; ^{-W1- W1} - ^W1 -W2-; -W1- ^{W1 - W2-} ; -W1- ^{W1-W2 - W2- ; -W1} - ^W2 - ^W2 - ^W1- ; ^-W1 - ^W2 - ^W1 - ^{W2 - W2 -W1- ;} -W1- ^W1 - ^W2 - ^W2 - ^W1 -W2 ^{- W1-} ; and ^{W2 -W2 - W1} - ^W2 - ^W1 - ^W1 - ^W1 - ^W2 - ^W2 - ^W1 - ^W2 - ^{W1- ; -W1} ; infinitely, where Z is attached to each ^W1 group, and the ^W1 and ^W2 groups themselves can be the same or different.

The term "sequence identity" is used in relation to comparisons of polypeptide (or nucleic acid) sequences. Specifically, this expression refers to the percentage of sequence identity relative to a corresponding reference polypeptide or corresponding reference polynucleotide, such as 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%. Specifically, the polypeptide in question and the reference polypeptide exhibit the specified sequence identity over a continuous segment of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids, or over the entire length of the reference polypeptide.

"Specific binding," as the term is commonly used in biology, refers to molecules that bind to a target with a relatively high affinity compared to non-target tissues, and generally involves various non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and so on. Specific binding interactions characterize antibody-antigen binding, enzyme-substrate binding, and some protein-receptor interactions; although such molecules may sometimes bind to tissues other than their specific target, high-affinity binding pairs can still fall within the definition of specific binding if such non-target binding is insignificant.

The term "treatment" or "treatment" refers to any treatment of a disease or ailment in mammals, including:

To prevent or avoid disease or symptom, that is, to prevent the development of clinical symptoms;

Suppressing a disease or symptom, that is, preventing or inhibiting the development of clinical symptoms; and/or

To alleviate a disease or symptom, that is, to reduce clinical symptoms.

The term "unintended immune response" refers to a subject's immune system response that is undesirable under given circumstances. An immune system response is undesirable if it does not lead to the prevention, mitigation, or cure of a disease or condition, but instead causes, enhances, or worsens the condition or disease, or is otherwise associated with the induction or exacerbation of the condition or disease. Typically, if the immune system's response targets an inappropriate target, the response causes, enhances, or worsens the disease. Exemplary undesirable immune responses include, but are not limited to, graft rejection, immune responses to therapeutic agents, autoimmune diseases, and allergic reactions or hypersensitivity.

The term "variant" should be understood as a protein (or nucleic acid) that differs from the protein from which it is derived in one or more alterations in its length, sequence, or structure. The polypeptide from which a protein variant is derived is also called the parent polypeptide or polynucleotide. The term "variant" includes "fragments" or "derivatives" of the parent molecule. Typically, a "fragment" is smaller in length or size than the parent molecule, while a "derivative" exhibits one or more differences in its sequence or structure compared to the parent molecule. Modified molecules are also included, such as, but not limited to, post-translational modified proteins (e.g., glycosylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids, such as methylated DNA. The term "variant" also includes mixtures of different molecules, such as, but not limited to, RNA-DNA hybrids. As used in this invention, naturally occurring and artificially constructed variants should be understood to be covered by the term "variant." Additionally, variants used in this invention may also be derived from homologs, orthologs, paralogs, or artificially constructed variants of the parent molecule, provided that the variant exhibits at least one biological activity of the parent molecule, such as being functionally active. Variants can be characterized by a certain degree of sequence identity with the parent polypeptide from which they are derived. More specifically, in the context of this invention, protein variants may exhibit at least 80% sequence identity with their parent polypeptide. Preferably, the sequence identity of the protein variant lies in a continuous segment of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids. As discussed above, in some embodiments, variants exhibit about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% functional equivalence (and any degree of functional equivalence among those listed) compared to an unmodified or native reference sequence.

Composition

One aspect of the present invention relates to compositions, pharmaceutical preparations, and treatment methods employing such compositions, as represented by Formula 1:

in:

m is an integer from about 1 to 100, especially from about 1 to 20, and most preferably from 1 to about 10;

X is the antigenic part that the patient is responding to in order to form an undesirable immune response, especially foreign antigens or autoantigens, or tolerogenic parts of such antigenic parts (e.g., fragments or epitopes).

Y is an antibody, antibody fragment, peptide, or other ligand that specifically binds to X via a linker base or direct bond; and

Z is the liver-targeting region, particularly the galactosylation or glucosylation region.

In Formula 1, the value of m will depend on the nature of X, as each antigen, antibody, antibody fragment, or ligand will have a number and density of sites (primarily N-terminal amines, lysine residues, and cysteine residues) that can bind to linkers, galactosylated moieties, or glucosylated moieties. Antigens with a limited number of such sites can be derivatized at the N- or C-terminus, for example, by adding lysine or cysteine residues (optionally via cleavable linkers, particularly those with immunosomal cleavage sites). Generally, it is preferable to provide a sufficient degree of galactosylation/glucosylation in the compositions of Formula 1 to promote hepatocyte uptake. The pharmaceutical formulations and methods of the present invention can use mixtures of compositions of Formula 1, each carrying a different X moiety (e.g., several epitopes associated with a particular undesirable immune response).

The composition of Formula 1 comprises a sub-genus, wherein X is an exogenous graft antigen against which the transplant recipient forms an undesirable immune response (e.g., graft rejection); an exogenous food, animal, plant, or environmental antigen against which the patient forms an undesirable immune response (e.g., allergic or hypersensitivity); an exogenous therapeutic agent against which the patient forms an undesirable immune response (e.g., hypersensitivity and/or reduced therapeutic activity); or an autoantigen against which the patient forms an undesirable immune response (e.g., autoimmune disease); wherein Y is a linker of Formulas Ya to Yp; and/or wherein Z is galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine, as shown in Formulas 1a to 1p as described below by reference reaction protocols.

In another embodiment, in the composition of Formula 1, X may be an antibody, antibody fragment, or ligand that specifically binds to a circulating protein or peptide or antibody that is involved in graft rejection, immune response to a therapeutic agent, autoimmune disease, hypersensitivity, and/or allergic reactions for this reason.

antigen

In the composition of Formula 1, the antigen used as X can be a protein or peptide. For example, the antigen can be a complete or partial therapeutic agent, a full-length graft protein or a peptide thereof, a full-length autoantigen or a peptide thereof, a full-length allergen or a peptide thereof, and/or a nucleic acid, or a mimic of the above antigens. The inclusion of any particular antigen in the listed categories or its association with any particular disease or reaction does not preclude that the antigen from being considered part of another class or associated with another disease or reaction.

The antigens used in the practice of this invention may be one or more of the following:

● Therapeutic agents are proteins, peptides, antibodies, and antibody-like molecules, including antibody fragments and fusion proteins containing antibodies and antibody fragments. These include human, non-human (e.g., mouse), and non-natural (i.e., engineered) proteins, antibodies, chimeric antibodies, humanized antibodies, and non-antibody binding scaffolds such as fibronectin, DARPins, and knottins.

● Human allogeneic graft antigens (HAGs) can trigger an undesirable immune response in the transplant recipient.

● Autoantigens, which elicit unintended autoimmune responses. Those skilled in the art will understand that while autoantigens are of endogenous origin in patients with autoimmune diseases, the peptides used in the disclosed compositions are typically exogenously synthesized (in contrast to those purified and concentrated from a source).

● Patients may develop foreign antigens, such as food, animal, plant, and environmental antigens, against which they have experienced an undesirable immune response. Those skilled in the art will understand that while therapeutic proteins may also be considered foreign antigens due to their exogenous origin, such therapeutic agents are described as belonging to a different group for clarity in the description of this invention. Similarly, plant or animal antigens can be edible and considered food antigens, and environmental antigens can be derived from plants. However, they are all foreign antigens. For simplicity, no attempt has been made to describe, distinguish, and define all such potentially overlapping groups, as those skilled in the art will understand the antigens available in the compositions of this invention, particularly based on the detailed description and examples.

Antigens can be complete proteins, parts of complete proteins, peptides, etc., and can be derivatized (as discussed above) for attachment to linkers and/or galactosylated moieties, can be variants and/or can contain conserved substitutions, particularly conserved substitutions that maintain sequence identity, and/or can be desialylated.

In embodiments where the antigen is a therapeutic protein, peptide, antibody, or antibody-like molecule, the specific antigen may be selected from: abatacept, abciximab, adalimumab, adenosine deaminase, ado-trastuzumab emtansine, agalsidase alfa, agalsidase beta, aldeslukin, and alglucerase. Alglucosidase α, α-1-protease inhibitor, Anakinra, Anistreplase (anisoylated plasminogen streptokinase activator complex), Antithrombin III, Antithymocyte globulin, Alteplase Ateplase, bevacizumab, bivalirudin, botulinum toxin type A, botulinum toxin type B, C1 esterase inhibitor, canakinumab, carboxypeptidase G2 (glutaminase and voraxaze), cetolizumab pegol, cetuximab, collagenase, rattlesnake immune Fab ( Crotalidae Immune Fab), Darbepoetin-α, Denosumab, Digoxin Immune Fab, Dornase Alfa, Eculizumab, Etanercept, Factor VIIa, Factor VIII, Factor IX, Factor XI, Factor XIII, Fibrinogen, Filgrastim, Galsulfase, Golime Monoclonal antibody (Golimumab), Historelin acetate, Hyaluronidase, Idursulphase, Imiglucerase, Infliximab, Insulin [including recombinant human insulin (“rHu insulin”) and bovine insulin], Interferon-α2a, Interferon-α2b, Interferon-β1a, Interferon-β1b, Interferon-γ1b, Ipilimumab, L-arginase, L-asparaginase, L- Methionase, lactase, laronidase, lepirudin/hirudin, mecasermin, mecasermin rinfabate, methoxynatalizumab, octreotide, ofatumumab, oprelvekin, pancreatic amylase Mylase, pancreatic lipase, papain, Peg-asparaginase, Peg-doxorubicin HCl, PEG-epoetin-β, Pegfilgrastim, Peg-interferon-α2a, Peg-interferon-α2b, Pegloticase, Pegvisomant, Phenylalanine ammonia lyase (PAL), protein C, and rapu Rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, type 1 alpha interferon, ustekinumab, and vW factor. Therapeutic proteins can be obtained from natural sources (e.g., concentration and purification) or synthesized, such as through recombinant synthesis, and include antibody therapeutics, typically IgG monoclonal antibodies or fragments or fusions.

Specific therapeutic proteins, peptides, antibodies, or antibody-like molecules include abciximab, adalimumab, agalsidase α, agalsidase β, interleukin, aglucosidase α, factor VIII, factor IX, infliximab, insulin (including rHu insulin), L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine aminolysin (PAL), or brevisterase (uricase), and IgG monoclonal antibodies, typically in their various forms.

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

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

In implementation schemes where the antigen is an autoantigen, the specific antigen (and the autoimmune diseases associated with them) may be selected from:

● In type 1 diabetes, several major antigens have been identified: insulin, proinsulin, preinsulin, glutamate decarboxylase-65 (GAD-65 or glutamate 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 2β (IA-2β); other antigens include ICA69, ICA12 (SOX-13), and carboxypeptidase H. Imogen 38, GLIMA38, chromogranin-A, HSP-60, carboxypeptidase E, peripheral proteins, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreas/pancreas-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreaticoduodenal homology box 1, dystrophiamyotonica kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST G protein-coupled receptors 1-5. It should be noted that insulin is an example of an antigen that can be characterized as both an autoantigen and a therapeutic protein antigen. For example, rHu insulin and bovine insulin are therapeutic protein antigens (which are targets of undesirable immune attack), while endogenous human insulin is an autoantigen (which is a target of undesirable immune attack). Since endogenous human insulin cannot be used in pharmaceutical compositions, recombinant forms are utilized in compositions of some embodiments of the present invention.

○ Human insulin, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROT P01308):

○GAD-65, comprising an exogenously obtained form that can be used in the compositions of the present invention, having the following sequence (UNIPROT Q05329):

○IGRP, including exogenously obtained forms that can be used in the compositions of the present invention, having the following sequence (UNIPROTQN9QR9):

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

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

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

● In autoimmune hypophysitis, or pituitary autoimmune diseases, the main antigens include pituitary-specific protein factors (PGSF) 1a and 2; another antigen is type 2 iodinated thyroxine deiodinase.

● In multiple sclerosis, the main antigens include myelin basic protein (“MBP”), myelin oligodendrocyte glycoprotein (“MOG”), and myelin lipid protein (“PLP”).

○MBP, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROTP02686):

○MOG, including exogenously obtained forms used in the compositions of the present invention, having the following sequence (UNIPROTQ16653):

○PLP, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROTP60201):

○ The peptides/epitopes that can be used to treat multiple sclerosis in the compositions of the present invention include some or all of the following sequences, which are present individually in the compositions of Formula 1 or together in mixtures of the compositions of Formula 1:

■MBP13-32: KYLATASTMDHARHGFLPRH (SEQ ID NO: 7);

■MBP83-99: ENPWHFFKNIVTPRTP (SEQ ID NO: 8);

■MBP111-129: LSRFSWGAEGQRPGFGYGG (SEQ ID NO: 9);

■MBP146-170: AQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 10);

■MOG1-20: GQFRVIGPRHPIRALVGDEV (SEQ ID NO: 11);

■MOG35-55: MEVGWYRPPFSRWHLYRNGK (SEQ ID NO: 12); and

■PLP139-154: HCLGKWLGHPDKFVGI (SEQ ID NO: 13).

● In rheumatoid arthritis, the main antigens include type II collagen, immunoglobulin-binding proteins, crystallizable regions of immunoglobulin G fragments, double-stranded DNA, and native and citrullinated forms of proteins involved in the pathology of rheumatoid arthritis, including fibrin/fibrinogen, vimentin, collagen I and II, and α-enolase.

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

●In pernicious angemisus, 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 α-, γ-, and Ω-gliadin, glutenin, barley gliadin, rye alkaloids, and oat protein. Those skilled in the art will understand that, for example, while the primary antigen in celiac disease is α-gliadin, it is transformed in vivo into a more immunogenic form through deamidation by tissue transglutaminase, which converts glutamine in α-gliadin to glutamate. Therefore, although α-gliadin is originally a foreign food antigen, once it is modified in vivo to become more immunogenic, it can be characterized as an autoantigen.

● In vitiligo, the main antigens are tyrosinase and tyrosinase-associated proteins 1 and 2.

○MART1, a melanoma antigen recognized by T cells 1, Melan-A, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROT Q16655):

○ Tyrosinase, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROT P14679):

○ Melanocyte protein PMEL, gp100, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROT P40967):

●In myasthenia gravis, the primary antigen is the acetylcholine receptor.

● In pemphigus vulgaris and its variants, the major antigens are desmoglein 3, 1, and 4; other antigens include pemphaxin, desmocollin, plakoglobin, perplakin, desmoplakin, and acetylcholine receptors.

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

● In Duhring herpetiformis, the main antigens include endothelial and tissue transglutaminase.

●In acquired epidermolysis bullosa acquisita, the major antigen is collagen VII.

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

●In mixed connective tissue diseases, the main antigen is U1snRNP.

● In Sjogren's syndrome, the primary antigens are nuclear antigens SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose) polymerase and topoisomerase, muscarinic receptor, and Fc-γ receptor IIIb.

● In systemic lupus erythematosus, the main antigens include nucleoproteins, including the "Smith antigen", SS-A, high-mobility box 1 (HMGB1), nucleosomes, histones, and double-stranded DNA (which generate autoantibodies against during the course of the disease).

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

●In rheumatic heart disease, the primary antigen is cardiac myosin.

●In type 1 autoimmune polyendocrine syndrome, antigens include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine sulfinate decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, liver P450 cytochrome P450 1A2 and 2A6, SOX-9, SOX-10, calcium sensing receptor protein, and type 1 interferons interferon α, β, and Ω.

● In neuromyelitis optica, the main antigen is AQP4.

○ Aquaporin-4, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROT P55087):

● In uveitis, the main antigens include retinal S-antigen or "S-inhibitor protein" and interphotoreceptor retinoid binding protein (IRBP) or retinol binding protein 3.

○S-inhibitory protein, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROT P10523):

○IRBP, including the exogenously obtained form used in the compositions of the present invention, having the following sequence (UNIPROTP10745):

In some implementations where the antigen is a foreign antigen against which an undesirable immune response can be elicited, such as a food antigen, the specific antigen may be:

●Derived from peanuts: conarachigoglobulin (Ara h 1), allergen II (Ara h 2), peanut lectin, blue bean protein (Ara h 6);

○ Conjugated arachidonic acid, for example, has a sequence identified as UNIPROT Q6PSU6.

●From Apple: 31kda major allergen/disease resistance protein homolog (Mal d 2), lipid transport protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1);

●From milk: α-lactalbumin (ALA), lactoferrin; From kiwi: actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), kiwellin (Act d 5);

●From egg white: ovalbumin, ovalbumin, ovaltransferrin, and lysozyme;

●From egg yolk: vitellin, apovitillin, and vosvetin;

●From mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transporter (Sin a 3), and inhibitory protein (Sin a 4);

●From celery: Inhibitory protein (Api g 4), high molecular weight glycoprotein (Api g 5);

●From shrimp: Pen a 1 allergen, Pen m 2 allergen, fast isoform of tropomyosin;

●Derived from wheat and/or other grains: high molecular weight glutenin, low molecular weight glutenin, α-, γ- and Ω-gliadin, barley gliadin, rye alkaloids, and/or oat protein;

○ The peptides/epitopes that can be used to treat celiac disease in the compositions of the present invention include some or all of the following sequences, which are present individually in the compositions of Formula 1 or together in mixtures of the compositions of Formula 1:

■DQ-2 related, natural α-gliadin "33-mer":

■DQ-2 related, deacylated α-gliadin "33-mer":

■DQ-8 related, α-gliadin:

■DQ-8 related, Ω gliadin (wheat, U5UA46):

●From strawberry: The main strawberry allergen is Fra a 1-E (Fra a 1), and

●From bananas: Inhibitory protein (Mus xp 1).

In embodiments where the antigen is a foreign antigen against which an undesirable immune response is generated (such as against animal, plant, and environmental antigens), the specific antigen may be, for example, cat, mouse, dog, horse, bee, dust, tree, and goldenrod, including proteins or peptides derived from the following:

●Weeds (including ragweed allergens amb a, 1, 2, 3, 5, and 6, and Amb t 5; quinoa Che a 2 and 5; and other weed allergens Par j 1, 2, 3, and Par o 1);

● Herbs (including 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 a 1; Pha a 1; Poa p 1 and 5);

● Pollen from the following: ragweed and other weeds (including curly dock, lambsquarters, pigweed, plantain, sheep sorrel, and sagebrush), grasses (including Bermuda, Johnson, Kentucky, Orchard, sweet vernal, and Timothy grass), and trees (including catalpa, elm, hickory, olive, pecan, sycamore, and walnut);

● Dust (including major allergens from species house dust mites (Dermatophagoides pteronyssinus), such as Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21, and 23; from species house dust mites (Dermatophagoides farina), such as Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18, 22, and 24; from The species *Blomia tropicalis*, such as Blo t 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 19, and 21; and allergens from *Euroglyphus maynei* (Eur m 2), *Tyr p 13* from *Tyrophagus putrescentiae* (Tyr p 13), and *Blag 1, 2, and 4*; and *Per a 1, 3, and 7* from cockroaches.

● Pets (including cats, dogs, rodents, and farm animals; major feline allergens include Fel d1 to 8, feline IgA, BLa g2, and feline albumin; major dog allergens include Can f1 to 6, and canine albumin);

● Bee stings, including major allergens Apim 1 to 12; and

● Fungi, including allergens derived from the following species: species of the genera Aspergillus and Penicillium, as well as species of the genera Alternaria alternata, Davidiellatassiana, and Trichophyton rubrum.

As those skilled in the art will understand, patients can be tested to identify antigens against which an undesirable immune response has been generated, and proteins, peptides, etc., can be developed based on these antigens and incorporated as X into the compositions of the present invention.

Sialization of antigens, antibodies, and antibody fragments

The following are examples of antigens, antibodies, and antibody fragments that specifically target ASGPR and can be removed, leaving behind glycosylated sialylated fragments: follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), luteinizing hormone (LH), osteopontin, thyroid-stimulating hormone (TSH), agalsidase α, agalsidase β (Genzyme), erythropoietin α and erythropoietin β, follicle-stimulating hormone α (Merck/Serono) and follicle-stimulating hormone β (Schering-Plough), insulin-binding factor 6 (IGFBP-6), luteinizing hormone α (Merck/Serono), transforming growth factor β1, and antithrombin (Genzyme). yme/Talecris Biotherapeutics), thyroid-stimulating hormone α (Genzyme), lenograstim, sargramostim (Genzyme), interleukin-3, prourokinase, lymphotoxin, C1 esterase inhibitor (CSL), IgG-like antibody, interferon β, coagulation factor VIIa (Novo Nordisk), coagulation factor VIII (moroctocog alfa), coagulation factor IX (nonacog alfa) (Wyeth) and p55 tumor necrosis receptor fusion protein. (See: Byrne et al., Drug Discovery Today, Vol 12, No.7/8, pp. 319-326, April 2007 and Sola et al., BioDrugs. 2010; 24(1):9-21). Pharmaceutically relevant proteins that have previously been highly glycosylated and can be desialylated for hepatocyte-ASGPR targeting include: interferon α and γ, luteinizing hormone, Fv antibody fragment, asparaginase, cholinesterase, dabepoetin α (Amgen), thrombopoietin, leptin, FSH, IFN-α2, serum albumin, and chorionic follicle-stimulating hormone α (corifollitropin alfa).

Proteins with glycans that do not normally terminate with sialic acid, including proteins produced in bacteria or yeast (such as arginase, some insulin and uricase), are not adapted for desialylation.

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

Antibodies and peptide ligands

In embodiments utilizing antibodies, antibody fragments, or ligands, such moieties are selected to specifically bind to a targeted circulating protein or peptide or antibody, resulting in hepatic uptake of the circulating target moieties, which may act as adducts of the target moieties, ultimately leading to clearance and inactivation of the circulating target moieties. For example, liver-targeting factor VIII will bind to and clear circulating anti-factor VIII antibodies. Methods for identifying such moieties are well known to those skilled in the art.

Connecting base

The linker used in the compositions of the present invention (“Y” in Formula 1) may include an N-hydroxysuccinamidyl linker, a maleimide linker, a vinyl sulfone linker, a pyridyl dithiol-poly(ethylene glycol) linker, a pyridyl dithiol linker, an n-nitrophenyl carbonate linker, an NHS-ester linker, a nitrophenoxy poly(ethylene glycol) ester linker, and so on.

A specific set of linkers includes linkers of the following formula Y'-CMP (where Y' indicates the remainder of the linker, and ^R9 and Z are as defined). More specifically, in the group including linkers of formula Y'-CMP, in some embodiments, the ^R9 substituent is an ethylacetamino group, and even more specifically, the ethylacetamino group is C1 conjugated with N-acetylgalactosamine or N-acetylglucosamine.

A linker containing a dithiol, particularly a linker containing a dithioalkylcarbamate (the free amine in the nomenclature includes X, or is named “dithioalkyl ethyl ester” without including X), is particularly advantageous in this composition because, once inside the cell, it has the ability to cleave the antigen and release it in its original form, for example, as shown below (where Y’ indicates the remainder of the linker, and X’ and Z are as defined).

Specifically, regarding the linking base shown below in formulas Ya to Yp: the left bracket "(" indicates the key between X and Y; the right bracket or bottom bracket ")" indicates the key between Y and Z;

n is an integer representing a mixture comprising 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), and 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, wherein the mixture typically covers an integer specified as n ± 10%;

p is an integer representing a mixture of 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), wherein the mixture typically covers an integer specified as p ± 10%;

q is an integer representing a mixture containing approximately 1 to 44, particularly approximately 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 approximately 4 to 12 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13), wherein the mixture typically covers an integer specified as q ± 10%; and

^R8 is _–CH2 -(“methyl”) or _–CH2 - _CH2 -C( _CH3 )(CN)–(“1-cyano-1-methyl-propyl” or “CMP”).

Y’ represents the remainder of Y (e.g., HS-PEG); and

W represents polymers with the same ^W1 group, or W is a copolymer (preferably a random copolymer) with the same or different ^W1 and ^W2 groups, wherein:

in:

p is an integer from 2 to approximately 150;

^R9 is a direct bond, _–CH2 - _CH2 -NH–C(O)– (i.e., ethyl acetamino group or “EtAcN”) or _-CH2 - _CH2- (O- _CH2 - _CH2 ) _t- NH-C(O)- (i.e., PEGylated ethyl acetamino group or “Et-PEG _t -AcN”).

t is an integer from 1 to 5 (especially 1 to 3, and more especially 1 or 2); and

R ¹⁰ is an aliphatic group, an alcohol, or an aliphatic alcohol, particularly N-(2-hydroxypropyl)methacrylamide; and

Z (not shown) is galactose, glucose, galactosamine, glucosamine, N-acetylgalactosamine, or N-acetylglucosamine.

From formula Ya to Yp

(This can be achieved by making Yn particularly suitable for linker groups of some precursor synthetic Yn conjugated to hydrophobic antigens.)

The linkers shown above with formulas Yh to Yn are synthesized into isomers that can be used without separation. For example, the linkers of formulas Yh, Yi, Yj, and Yn are mixtures of the structures 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 shown below:

The linkers of formulas Yk, YL, and Ym will be a mixture of the structures shown below: 8,9-dihydro-1H-dibenzo[3,4:7,8]cyclooct[1,2-d][1,2,3]triazol-8-yl and 8,9-dihydro-1H-dibenzo[3,4:7,8]cyclooct[1,2-d][1,2,3]triazol-9-yl.

The presence of such isomer mixtures does not impair the functionality of compositions employing such linker groups.

Liver-targeted portion

The galactosylation moiety used in the compositions of the present invention is used to target the composition to hepatocytes (e.g., specifically bind to hepatocytes) and can be selected from: galactose, galactosamine, or N-acetylgalactosamine. The glucosylation moiety used in the compositions of the present invention is used to target the composition to hepatocytes (e.g., specifically bind to hepatocytes or LSECs) and can be selected from: glucose, glucosamine, or N-acetylglucosamine. It has been reported that ASGPR affinity can be retained on either side of the C3/C4-diol anchorage of modified galactose (Mamidyala, Sreeman K., et al., J. Am. Chem. Soc. 2012, 134, 1978-1981), therefore, in some embodiments, the conjugation sites used are particularly located at C1, C2, and C6.

Specific liver-targeting moieties include galactose or glucose conjugated at C1 or C6, galactosamine or glucosamine conjugated at C2, and N-acetylgalactosamine or N-acetylglucosamine conjugated at C6. Other specific liver-targeting moieties include N-acetylgalactosamine or N-acetylglucosamine conjugated at C2, more particularly conjugated with a linker carrying an ^R9 substituent as _CH2 . Other specific liver-targeting moieties also include galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine conjugated at C1, more particularly conjugated with a linker carrying an ^R9 substituent as an ethylacetamino group.

Nomenclature

Compositions of Formula 1 can be named using a combination of IUPAC and conventional names. For example, a compound corresponding to Formula 1 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-dithiotecosano-21-ylidene)triaza-1-yne-2-cationic chloride ((Z)-(21-cyclobutyl-1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino) l)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-ylidene)triaz-1-yn-2-ium chloride), where X is the 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-acetylgalactosamine-2-yl or N-acetylglucosamine-2-yl:

Therefore, the corresponding composition of the present invention, in which 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-dithiotecosano-21-ylidene)triaza-1-yne-2-cationic chloride. Where X’ is tissue transglutaminase, m is 2, n is 4, and Z’ is N-acetylgalactosamine-2-yl or N-acetylglucosamine-2-yl, the corresponding composition of the present invention can be named (3Z)-((tissue transglutaminase)-1,3-dimethylbis(-1-oxo-1-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-4,7,10,13-tetraoxo-16,17-dithiococoane-21-yl-21-yl))bis(triaza-1-yne-2-cation)chloride((3Z)-((t issuetransgultaminase)-1,3-diylbis(1-oxo-1-((2,4,5-trihydroxy-6-(hydroxymethyl)tetra hydro-2H-pyran-3 -yl)amino)-4,7,10,13-tetraoxa-16,17-dithiahenicosan-21-yl-21-ylidene))bis(triaz-1-yn-2-ium)chloride).

To simplify processing, compositions of Formula 1 can be named using an alternative nomenclature system by referring to X and corresponding to one of Formulas 1a to 1p (as shown in the reaction scheme), followed by referencing the integers of variables m, n, p, and/or q, ^R8 , ^R9 , and identifying the galactosylated moiety and the position where it is conjugated. In some embodiments, the W of the copolymer compound is specified by a letter followed by a "Y group" (e.g., F1c') and includes the number and identification of the comonomer. Under this system, a composition of Formula 1a in which X is ovalbumin, m is 2, n is 4, and Z is N-acetylgalactosamine-2-yl can be named "F1a-OVA- _m2 - _n4-2NAcGAL ". The corresponding composition of Formula 1a in which X is ovalbumin, m is 2, n is 4, and Z is N-acetylglucosamine-2-yl can be named "F1a-OVA- _m2 - _n4-2NAcGLU ".

Similarly, the following compounds of Formula 1:

It can be named "2-((2-(((3-(3-(22-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbothio)thio)-3,6,9,12-tetraoxo-20-aza-eicosyl)-3,9-dihydro-8H-dibenzo-[b,f][1,2,3]triazolo[4,5-d]acoxin-8-yl)-3-oxopropyl)carbamoyl)oxy)ethyl)dithio)ethyl insulin carboxylate. Isomer:

It can be named "2-((2-(((3-(1-(22-(3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-cyano-16,18-dimethyl-13,19-dioxo-18-((phenylcarbothio)thio)-3,6,9,12-tetraoxo-20-azadocosyl)-1,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]acoxin-8-yl)-3-oxopropyl)carbamoyl)-oxy)ethyl)dithio)ethyl insulin carboxylate" (bold text added for emphasis to facilitate identification of differences between the formal names). Using the nomenclature system suitable for this invention, these two isomers can be named “F1n-insulin _m1 - _n1 - _p1 - _q4 -CMP-EtAcN-1NAcGAL” (or “F1n-insulin- _m1 - _n1 - _p1 - _q4 -CMP-EtAcN-1NAcGLU”, since the stereochemistry of the sugar ring is not shown), where CMP indicates that ^R8 is 1-cyano-1-methyl-propyl, EtAcN indicates that ^R9 is ethylacetamido, and 1NAcGAL indicates that “Z” is an N-acetylgalactosamine conjugated at C1. The abbreviation EtAcN is missing before Z to indicate that ^R9 is a direct bond.

The following compositions of Formula 1 exemplify compounds in which W is a copolymer:

It can be named 2-(2-(2-(QQYPSGQGSFQPSQQNPQGGGSC-thioalkyl)ethoxy)ethoxy)ethoxy)ethyl 6,10-di((2-(2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)carbamoyl)-4-cyano-13-((2-hydroxypropyl)amino)-8-((2-hydroxypropyl)carbamoyl)-4,6,8,10,12-pentylmethyl-13-oxo-12-((phenylcarbothio)thio)tridecanoic acid. Using a naming system suitable for this invention, the compound can be named "F1c'-DQ8-related α-gliadin- _m1 - _n4 - _p4 -CMP-poly-( _EtPEG1AcN - _1NAcGLU2 - _HPMA2 )".

Preparation of the composition of the present invention

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

Equation 101 (below) is an alternative representation of X.

^R1 is a free surface amino ( _-NH2 ) or thiol (-SH) moiety located on the three-dimensional structure of X, thus accessible for conjugation with a linker, and X' represents the remainder of X other than the identified free amino group [(in the reaction scheme, X” is used to represent the remainder of X excluding the free thiol group). Depending on the characteristics of X, there will be at least one (N-terminal amine), and there may be multiple ^R1 groups (mainly derived from lysine residues or cysteine residues not involved in disulfide bonding), as indicated by m, which range from about 1 to 100, more typically 1 or about 4 to 20, and most typically an integer from 1 to about 10.

The variables used in the reaction scheme are as defined above, and additionally include the following items, which should be understood to have the following meanings with respect to a particular reaction scheme or step, in the absence of any other specific instructions.

●R ² is an OH group or a protecting group;

●R ³ is OH, NH ₂ , NHAc, a protecting group, or an NH protecting group;

●R ⁴ is an OH group or a protecting group;

●R ⁵ is an OH group or a protecting group;

●R ⁶ is an OH group or a protecting group;

●Z’ is galactose or glucose conjugated at C1 or C6, galactosamine or glucosamine conjugated at C2, and N-acetylgalactosamine or N-acetylglucosamine conjugated at C6.

●R ⁸ is -CH _2- or -CH ₂ -CH ₂ -C(CH ₃ )(CN)--; and

●R ⁹ is a direct bond and Z” is an N-acetylgalactosamine conjugated at C2; or

●R ⁹ is an ethyl acetamino group or a PEGylated ethyl acetamino group, and Z” is a galactose, glucose, galactosamine, glucosamine, N-acetylgalactosamine or N-acetylglucosamine conjugated at C1.

Synthesis reaction parameters

The terms “solvent,” “inert organic solvent,” or “inert solvent” refer to solvents that are inert under the reaction conditions described in connection with this invention [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform, methylene dichloro (or dichloromethane), diethyl ether, methanol, pyridine, etc.]. Unless otherwise specified, the solvents used in the reactions of this invention are inert organic solvents.

The term "q.s" refers to the amount added sufficient to achieve the desired function, for example, to bring the solution to the desired volume (i.e., 100%).

As needed, the separation and purification of the compounds and intermediates described in this invention can be carried out by any suitable separation or purification procedure, such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, size exclusion chromatography, high-performance liquid chromatography, recrystallization, sublimation, rapid protein liquid chromatography, gel electrophoresis, dialysis, or combinations of these procedures. Specific examples of suitable separation and purification procedures can be found in the embodiments below. However, other equivalent separation and purification procedures may of course be used.

Unless otherwise specified (including in the examples), all reactions were carried out at standard atmospheric pressure (about 1 atmosphere) and ambient (or room temperature) (about 20°C) at a pH of about 7.0-8.0.

The reaction products can be characterized by conventional methods, such as proton and carbon NMR, mass spectrometry, size exclusion chromatography, infrared spectroscopy, and gel electrophoresis.

Reaction Scheme 1 illustrates the preparation of the composition of Formula 1, wherein Z can be galactose, glucose, galactosamine, glucosamine, N-acetylgalactosamine, or N-acetylglucosamine. In this respect, and as defined above, Z' used in Reaction Scheme 1 encompasses galactose or glucose conjugated at C1 and C6 and corresponding to the following structures according to Formula 1:

At C2, a galactosamine or glucosamine is conjugated and corresponds to the following structure according to Formula 1:

And N-acetylgalactosamine or N-acetylglucosamine conjugated at C6 and corresponding to the following structures according to Formula 1:

Reaction Scheme 1

As shown above in reaction scheme 1, step 1, surface thiol groups can be generated on an antigen, antibody, antibody fragment, or ligand (formula 101’) having a free surface amino group as follows: Formula 103’ is obtained by contacting with Traut reagent (formula 102) at pH approximately 8.0 for approximately 1 hour, from which unreacted Traut reagent is removed, for example, by size exclusion chromatography. The two structures shown below illustrate the product of reaction scheme 1, step 1, which respectively show the free surface amino group initially found on X (i.e., formula 103’, where X’ represents the remainder of X other than the identified free surface amino group) and omit the free surface amino group (i.e., formula 103). This makes the distinction shown between X and formula 101 parallel. In subsequent reaction schemes, conversion immediately follows.

In reaction scheme 1, step 2, pyridyl dithiol-poly(ethylene glycol)-NHS ester (formula 104) is contacted with galactosamine or glucosamine (formula 105, wherein ^R3 is _NH2 and ^R2 , ^R4 , ^R5 and ^R6 are OH) at approximately pH 8 for approximately 1 hour with stirring to obtain the corresponding pyridyl dithiol poly(ethylene glycol)-saccharide of formula 106A, which can be used without further purification.

In reaction scheme 1, step 3, 4,4'-dithiodipyridine (Formula 107) is contacted with thiol poly(ethylene glycol)propionic acid (Formula 108) to obtain the corresponding pyridyl dithiol-poly(ethylene glycol)propionic acid (Formula 109).

In reaction scheme 1, step 4, the acid of formula 109 is contacted with the protected galactose or N-acetylgalactosamine of formula 105 to obtain the corresponding pyridyl dithiol-poly(ethylene glycol)-sugars of formulas 106B, 106C, and 106D, which can be used after deprotection, wherein ^R2 is OH and ^R3 , ^R4 , ^R5 , and ^R6 are protecting groups (“PG”), wherein ^R6 is OH and ^R2 , ^R3 , ^R4 , R5, and ^R6 are PG, or wherein ^R6 is N-acetyl and ^R2 , ^R3 , ^{R4 ,} and ^R5 are PG.

In reaction scheme 1, step 5, 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) is added to the stirred solution of the product of step 1 (formula 103’) for about 1 hour, and then any free residual reactants are removed by size exclusion chromatography by centrifugation, thereby producing the corresponding products according to formula 1a, namely formula 1aA, formula 1aB, formula 1aC and formula 1aD.

For products prepared using intermediates according to formula 106A-D, the compositions corresponding to formula 1a can be named, for example, 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"

"F1aA-X'-m _m -n _n " or "F1a-X'-m _m -n _n -2NGLU"

"F1aB-X'-m _m -n _n " or "F1a-X'-m _m -n _n -1GLU"

"F1aC-X'-m _m -n _n " or "F1a-X'-m _m -n _n -6GLU"

“F1aD-X'-m _m -n _n ” or “F1a-X'-m _m -n _n -6NAcGLU”.

Reaction schemes 2-14 illustrate the preparation of compounds, wherein W is a polymer of the same ^W1 group. For the purposes of the nomenclature used in this invention, unless otherwise expressly stated, "Z" refers to N-acetylgalactosamine or N-acetylglucosamine conjugated at C2:

This may refer to galactose, glucose, galactosamine, glucosamine, N-acetylgalactosamine, or N-acetylglucosamine conjugated at C1. It should be noted that, according to some embodiments, to improve yield, the C1-conjugated composition can be protected during synthesis, for example by cyclizing the amine with a C3 hydroxyl group, and then deprotected after the protected galactosamine is incorporated into the adjacent portion of the linker.

Poly(galactosyl methacrylate) and poly(glucose methacrylate) reactants of formulas 201, 401, 501, 601, 701, 803, and 1401 can be prepared by esterifying galactosyl methacrylate or glucose, for example, by contacting galactosamine or glucosamine with methacrylic anhydride, followed by reversible addition-fragmentation chain transfer (RAFT) polymerization in a suitable solvent in the presence of azobisisobutyronitrile (AIBN) using the corresponding RAFT reagent, starting from a freeze-thaw cycle, and then heating at about 60-80°C, preferably 70°C, for about 5-8 hours, preferably about 6 hours. The polymer can be precipitated in lower alkanols, preferably methanol.

Reaction Scheme 2

As shown in reaction scheme 2, an antigen, antibody, antibody fragment or ligand (formula 103’) having free surface thiol groups prepared as described in step 1, for example, as in reference reaction scheme 1, is contacted with an excess (corresponding to the m value) of pyridyl dithiol-poly(ethylene glycol) of formula 201 for about 1 hour to produce the corresponding product according to formula 1b.

The composition in Formula 1b can be named as follows: “F1b-X'-m _m -n _n -p _p -2NAcGAL”, “F1b-X'-m _m -n _n -p _p -2NAcGLU”, 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, can be named “F1b-uricase-m ₁ -n ₄ -p ₄ -2NAcGAL” or “30-(uricase)-3,5,7,9-tetramethyl-12-oxo-1-phenyl-thio(thioxo)-3,5,7,9-tetra((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)carbamoyl)-13,16,19,22-tetraoxo-2,25,26-trithiotriacontane-30-iminium ion”.

Reaction scheme 3

As shown in reaction scheme 3, an antigen, antibody, antibody fragment, or ligand having a natural free surface thiol group (cysteine) [Formula 101", corresponding to Formula 101 and showing "X", as the term will be used thereafter, indicates X other than the identified free surface thiol] is contacted with an excess (corresponding to the m value) of pyridyl dithiol-poly(ethylene glycol) of Formula 201 to produce the corresponding product according to Formula 1c.

Compositions corresponding to Formula 1c can be named as follows: “F1c-X'-m _m -n _n -p _p -2NAcGAL”, “F1c-X'-m _m -n _n -p _p -2NAcGLU”, or “F1c-X'-m _m -n _n -p _p -EtAcN-Z”.

Reaction scheme 4

As shown in reaction scheme 4, an antigen, antibody, antibody fragment or ligand of formula 101” having a natural free surface thiol group is contacted with an excess (corresponding to the m value) of pyridyl dithiol of formula 401 to produce the corresponding product according to formula 1d.

The composition corresponding to Formula 1d can be named as follows: "F1d-X'-m _m -p _p -2NAcGAL", "F1d-X'-m _m -p _p -2NAcGLU" or "F1d-X'-m _m -p _p -EtAcN-Z".

Reaction scheme 5

As shown in reaction scheme 5, an antigen, antibody, antibody fragment or ligand having a natural free surface amino group of formula 101’ is contacted with an excess (corresponding to the m value) of n-nitrophenyl carbonate of formula 501 to produce the corresponding product according to formula 1e.

Compositions corresponding to Formula 1e can be named as follows: “F1e-X'-m _m -p _p -2NAcGAL”, “F1e-X'-m _m -p _p -2NAcGLU”, or “F1e-X'-m _m -p _p -EtAcN-Z”.

Reaction scheme 6

As shown in reaction scheme 6, an antigen, antibody, antibody fragment or ligand of formula 101’ having a natural free surface amino group is contacted with an excess (corresponding to the m value) of n-nitrophenyl carbonate poly(ethylene glycol) ester of formula 601 to produce the corresponding product according to formula 1f.

Compositions corresponding to Formula 1f can be named as follows: “F1f-X'-m _m -n _n -p _p -2NAcGAL”, “F1f-X'-m _m -n _n -p _p -2NAcGLU”, or “F1f-X'-m _m -n _n -p _p -EtAcN-Z”.

Reaction Scheme 7

As shown in reaction scheme 7, an antigen, antibody, antibody fragment or ligand of formula 101’ having natural free surface amino groups is contacted with an excess (corresponding to the m value) of NHS ester poly(ethylene glycol) ester of formula 701 to produce the corresponding product according to formula 1g.

Compositions corresponding to Formula 1g can be named as follows: “F1g-X'-m _m -p _p -2NAcGAL”, “F1g-X'-m _m -p _p -2NAcGLU”, or “F1g-X'-m _m -p _p -EtAcN-Z”.

Reaction Scheme 8

As shown in step 1 of reaction scheme 8, an antigen, antibody, antibody fragment or ligand of formula 101’ having a natural free surface amino group is contacted with an excess (corresponding to the m value) of an amine reactive linker of formula 801 for Click chemistry to produce the corresponding product according to formula 802.

In reaction scheme 8, step 2, the product of formula 802 is then contacted with an equivalent (again corresponding to the m value) of the galactose (amine) polymer of formula 803 to produce the corresponding isomer product according to formula 1h. The two isomers shown above are produced by the nonspecific cyclization of the azide of formula 803 with the triple bond of formula 802. Such nonspecific cyclization occurs in the synthesis of other compositions in which Y is selected from formulas Yh to Yn, but is not shown in every case.

The composition corresponding to formula 1h can be named as follows:

"F1h-X'-m _m -n _n -p _p -q _q -2NAcGAL", "F1h-X'-m _m -n _n -p _p -q _q -2NAcGLU" or "F1h-X'-m _m -n _n -p _p -q _q -EtAcN-Z".

Reaction Scheme 9

As shown in step 1 of reaction scheme 9, an antigen, antibody, antibody fragment or ligand of formula 101” having a natural free surface thiol group is contacted with an excess (corresponding to the m value) of a thiol reactive linker of formula 901 for Click chemistry to produce the corresponding product according to formula 902”.

In reaction scheme 9, step 2, the product of formula 902 is then contacted with an equivalent amount (again corresponding to the m value) of galactose (amine) polymer of formula 803 to produce the corresponding isomer product according to formula 1i.

The composition corresponding to formula 1i can be named as follows:

"F1i-X'-m _m -n _n -p _p -q _q -2NAcGAL""F1i-X'-m _m -n _n -p _p -q _q -2NAcGLU" or "F1i-X'-m _m -n _n -p _p -q _q -EtAcN-Z".

By following the procedure described in reaction scheme 9, but replacing the starting material 101” with a compound of formula 103’ (derivatively derived with Traut reagent), the corresponding isomer product corresponding to formula 1j shown below was obtained.

The composition corresponding to formula 1j can be named as follows:

"F1j-X'-m _m -n _n -p _p -q _q -2NAcGAL", "F1j-X'-m _m -n _n -p _p -q _q -2NAcGLU" or "F1j-X'-m _m -n _n -p _p -q _q -EtAcN-Z".

Reaction Scheme 10

As shown in step 1 of reaction scheme 10, an antigen, antibody, antibody fragment or ligand having a natural free surface thiol group of formula 101” is contacted with an excess (corresponding to the m value) of a thiol reactive linker of formula 1001 for Click chemistry to produce the corresponding product according to formula 1002.

In reaction scheme 10, step 2, the product of formula 1002 is then contacted with an equivalent (again corresponding to the m value) galactose (amine) polymer of formula 803 to produce the corresponding isomer product according to formula 1k.

Compositions corresponding to Formula 1k can be named as follows: “F1k-X'-m _m -n _n -p _p -q _q -2NAcGAL”, “F1k-X'-m _m -n _n -p _p -q _q -2NAcGLU”, or “F1k-X'-m _m -n _n -p _p -q _q -EtAcN-Z”.

By following the procedure described in reaction scheme 10, but replacing the starting material 101” with a compound of formula 103’ (derivatively derived with Traut reagent), the corresponding isomer product corresponding to formula 1L shown below was obtained.

Compositions corresponding to Formula 1L can be named as follows: “F1 _L -X'-m _m -n _n -p _p -q _q -2NAcGAL”, “F1 _L -X'-m _m -n _n -p _p -q _q -2NAcGLU”, or “F1 _L -X'-m _m -n _n -p _p -q _q -EtAcN-Z”.

Reaction Scheme 11

As shown in step 1 of reaction scheme 11, galactose, protected galactosamine, or N-acetyl-D-galactosamine (Formula 1101, wherein ^R3 and ^R4 are OH, ^R3 is an NH protecting group (e.g., cyclized with ^R4 ), or ^R3 is NHAc and ^R4 is OH) is contacted with 2-chloroethane-1-ol, followed by cooling and dropwise addition of acetyl chloride. The solution is warmed 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 in the presence of carbon, then filtered, followed by removal of the solvent to produce the corresponding product of Formula 1102.

As shown in step 2 of reaction scheme 11, the product of formula 1102 is added to an excess of sodium azide and heated to 90°C for several hours, then filtered, and the solvent is removed to produce the corresponding product of formula 1103.

As shown in step 3 of reaction scheme 11, the product of formula 1103 is added to a solution of palladium on carbon and ethanol and stirred for several hours under hydrogen (3 atm), then filtered, and the solvent is removed to produce the corresponding product of formula 1104.

As shown in step 4 of reaction scheme 11, the product of formula 1104 is added to a solution of methacrylate anhydride. Triethylamine is added, and the reaction is stirred for 2 hours. The solvent is then removed and the mixture is separated to produce the corresponding product of formula 1105.

As shown in step 5 of reaction scheme 11, the azide-modified uRAFT agent (formula 1106) is added together with azobisisobutyronitrile to a solution of the product of formula 1105, and four free-pump-thaw cycles are performed, followed by stirring at 70°C. After several hours, the corresponding polymer product of formula 1107 is precipitated by adding a lower alkanol, followed by removal of the solvent. If ^R3 is an NH protecting group (e.g., cyclized with ^R4 ), the protecting group is removed at this point.

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

As shown in step 7 of reaction scheme 11, the product of formula 1107 is added to a buffer solution at pH 8.0, and the product of formula 1109 is added to the buffer solution. After stirring for 2 hours, excess formula 1107 is removed to produce the corresponding isomer product of formula 1m.

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

The composition corresponding to formula 1m can be named as follows:

"F1m-X'-m _m -n _n -p _p -q _q- EtAcN-Z", where Z" is 1GAL, 1NGAL, 1NAcGAL, "F1m-X'-m _m -n _n -p _p -q _q- 2NAcGAL" or "F1m-X'-m _m -n _n -p _p -q _q- 2NAcGLU" (or the corresponding 1GAL, 1GLU, 1NGAL, 1NGLU, 1NAcGAL or 1NAcGLU compound).

Reaction Scheme 12

Because of the use of organic solvents, the synthetic method of reaction scheme 12 is particularly suitable for hydrophobic antigens, antibodies, antibody fragments, and ligands (e.g., insulin).

As shown in step 1 of reaction scheme 12, an antigen, antibody, antibody fragment, or ligand of formula 101' having a natural free surface amino group is dissolved in an organic solvent containing triethylamine (e.g., DMF). A certain amount (corresponding to the m value) of the compound of formula 1201 is added, followed by stirring and the addition of tert-butyl methyl ether. The corresponding product of formula 1202 is recovered as a precipitate.

The product of formula 1202 is resuspended in an organic solvent, and a certain amount (corresponding to the m value) of formula 1107 (e.g., obtained as described with reference to reaction scheme 11) is added, followed by stirring. The reaction product is precipitated by adding dichloromethane, then filtered and the solvent is removed. Purification (e.g., resuspending in PBS) is then performed, followed by size exclusion chromatography to produce the corresponding isomer of formula 1n.

The compositions corresponding to formula 1n can be named as follows:

"F1n-X'-m _m -n _n -p _p -q _q- EtAcN-Z", where Z" is 1GAL, 1NGAL, 1NAcGAL, 1GLU, 1NGLU, 1NAcGLU, or "F1m-X'-n _m -n _n -p _p -q _q- 2NAcGAL" or "F1m-X'-n _m -n _n -p _p -q _q- 2NAcGLU".

Reaction Scheme 13

In reaction scheme 13, step 1, nitrophenoxycarbonyl-oxoalkyldithiol-poly(ethylene glycol)-NHS ester (Formula 1301) is contacted with galactose, galactosamine, or N-acetylgalactosamine (Formula 105) to obtain the corresponding product of Formula 1302. The desired nitrophenoxycarbonyldithiol-poly(ethylene glycol)carboxyethyl galactose, galactosamine, or N-acetylgalactosamine of Formula 1302 is then isolated from the product, and subsequent steps are performed.

As shown in step 2 of reaction scheme 13, an antigen, antibody, antibody fragment or ligand having free surface amino groups of formula 101’ can be contacted with an excess (corresponding to the m value) of the product of formula 1302 to produce the corresponding product according to formula 1o.

The composition corresponding to formula 1o can be named as follows:

“F1o-X'-m _m -n _n -Z'.

Reaction Scheme 14

As shown in reaction scheme 14, an antigen, antibody, antibody fragment or ligand (formula 101’) having free surface amino groups is contacted with an excess (corresponding to the m value) of pyridyl-dithiol-poly(ethylene glycol)-NHS ester of formula 1401 to produce the corresponding product according to formula 1p.

Compositions corresponding to Formula 1p can be named as follows: “F1p-X'-m _m -n _n -p _p -2NAcGAL”, “F1p-X'-m _m -n _n -p _p -2NAcGLU”, or “F1p-X'-m _m -n _n -p _p -EtAcN-Z”.

Reaction schemes 15-18 show the preparation of compounds, wherein W is a copolymer of the same or different ^W1 and ^W2 groups.

Reaction Scheme 15

As shown in step 1 of reaction scheme 15, galactose or glucose (formula 1101, where ^R3 and ^R4 are OH), protected galactosamine or protected glucosamine (formula 1101, where ^R3 is an NH protecting group, for example, cyclized with ^R4 ) or N-acetyl-D-galactosamine or N-acetyl-D-glucosamine (formula 1101, where ^R3 is NHAc and ^R4 is OH) is contacted with 2-(poly-(2-chloroethoxy)ethoxy)ethanol-1-ol of formula 1501 (where t is 1 to 5), followed by cooling and dropwise addition of acetyl chloride. The solution is warmed 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 in the presence of carbon, then filtered, followed by removal of the solvent to produce the corresponding product of formula 1502.

As shown in step 2 of reaction scheme 15, the product of formula 1502 is added to an excess of sodium azide and heated to 90°C for several hours, then filtered, and the solvent is removed to produce the corresponding product of formula 1503.

As shown in step 3 of reaction scheme 15, the product of formula 1503 is added to a solution of palladium on carbon and ethanol and stirred for several hours under hydrogen (3 atm), then filtered, and the solvent is removed to produce the corresponding product of formula 1504.

As shown in step 4 of reaction scheme 15, the product of formula 1504 is added to a solution of methacrylic anhydride. Triethylamine is added, and the reaction mixture is stirred for 2 hours, then the solvent is removed and the mixture is separated to produce the corresponding product of formula 1505. Alternatively, pentafluorophenyl methacrylate (or another acrylate agent) can be used to prepare the corresponding product of formula 1505. In some embodiments, the product of formula 1504 is added to DMF. Triethylamine (e.g., an organic base) is added, and the mixture is cooled (e.g., using an ice bath to 4°C). Subsequently, pentafluorophenyl methacrylate (or another acrylate agent) is added (e.g., dropwise with constant stirring). After a period of time (e.g., 30 minutes), the cooling (e.g., ice bath) is removed, and the reaction mixture is stirred at room temperature for a period of time (e.g., 4 hours). In some embodiments, the solvent is then removed. In some embodiments, the product is purified using rapid chromatography.

As shown in step 5 of reaction scheme 15, the azide-modified uRAFT agent of formula 1106 and the methacrylamide of formula 1506, together with azobisisobutyronitrile, are added to a solution of the product of formula 1505, subjected to four freeze-thaw pump cycles, and then stirred at 70°C. After several hours, the corresponding random copolymer product of formula 1507 is precipitated by adding a lower alkanol or acetone, followed by solvent removal. If ^R3 is an NH protecting group (e.g., cyclized with ^R4 ), the protecting group is removed at this point.

As shown in step 6 of reaction scheme 15, the product of formula 1507 is added to a buffer solution at pH 8.0, to which the product of formula 1109 (prepared, for example, as described in reference reaction scheme 11) is added. After stirring for 2 hours, excess formula 1109 is removed to produce the corresponding isomer random copolymer product of formula 1m’.

By adding more than one methacrylamide of formula 1505 (e.g., glucose and galactose methacrylamide, or two or more methacrylamides with different t values) and/or two or more methacrylamides of formula 1506 in step 5, and continuing to step 6, the corresponding product of formula 1m' having a mixture of ^R3 and/or PEG (“t”) and/or ^R10 groups is obtained, i.e., the compound of formula 1, wherein W is a random copolymer of different ^W1 and ^W2 groups.

The composition corresponding to formula 1m’ can be named as follows:

"F1m'-X'-m _m -n _n -p _p -q _q -R ⁸ -poly-(W ¹ _t Z" _W1p -ran-W ² _W2p )".

Reaction Scheme 16

As shown in step 1 of reaction scheme 16, the compound of formula 1601 is contacted with the compounds of formulas 1505 and 1506 under conditions similar to those in step 5 of reaction scheme 15 to provide the corresponding compound of formula 1602.

In some embodiments, the following synthesis is performed to form compound 1601a (embodiments of 1601):

In some embodiments, t is an integer from about 1 to about 10 or from about 1 to about 5. In some embodiments, oligoethylene glycol (1650) is reacted with p-toluenesulfonyl chloride (or some other reagent capable of functionalizing 1650 with a leaving group) to form oligoethylene glycol mono-p-toluenesulfonic acid (1651) (or some other oligoethylene glycol functionalized with a leaving group). In some embodiments, compound 1651 can be reacted with potassium thioacetate to form compound 1652. In some embodiments, compound 1652 is reacted with 2,2-dithiodipyridine to form compound 1653. In some embodiments, compound 1653 is coupled with compound 1654 to form compound 1601a.

As shown in step 2 of reaction scheme 16, the compound of formula 1602 is contacted with the compound of formula 101” under conditions similar to those in step 6 of reaction scheme 15 to provide the corresponding compound of formula 1c’.

The composition corresponding to formula 1c’ can be named as follows:

“F1c'-X'-m _m -n _n -p _p -R ⁸ -聚-(W ¹ _t Z”-ran-W ² )”.

Reaction Scheme 17

As shown in step 1 of reaction scheme 17, the compound of formula 600’ is contacted with the compounds of formulas 1505 and 1506 under conditions similar to those in step 5 of reaction scheme 15 to provide the corresponding compound of formula 601’.

As shown in step 2 of reaction scheme 17, the compound of formula 601’ is contacted with the compound of formula 101’ under conditions similar to those in step 6 of reaction scheme 15 to provide the corresponding compound of formula 1f’.

The composition corresponding to formula 1f’ can be named as follows:

"F1f'-X'-m _m -n _n -p _p -R ⁸ -poly-(W ¹ _t Z"-ran-W ² )".

Reaction Scheme 18

As shown in step 1 of reaction scheme 18, the compound of formula 700’ is contacted with the compounds of formulas 1505 and 1506 under conditions similar to those in step 5 of reaction scheme 15 to provide the corresponding compound of formula 701’.

As shown in step 2 of reaction scheme 18, the compound of formula 701’ is contacted with the compound of formula 101’ under conditions similar to those in step 6 of reaction scheme 15 to provide the corresponding compound of formula 1g’.

The composition corresponding to formula 1g’ can be named as follows:

“F1g'-X'-m _m -p _p -R ⁸ -聚-(W ¹ _t Z”-ran-W ² )”.

Specific processing and final steps

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

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

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

The compound of formula 1109 is contacted with an excess (corresponding to the value of m) of the compound of formula 1107 to obtain the corresponding product of formula 1m, wherein n is about 80, p is about 30, q is about 4, and m as an antigen function is about 2 to 10.

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

The compound of formula 1507 is contacted with the compound of formula 1109 to obtain the corresponding product of formula 1m', wherein n is about 4, p is about 90, q is about 4, t is about 1 or 2, R ³ is NHAc, R ⁴ is OH, R ⁸ is CMP, R ¹⁰ is 2-hydroxypropyl, and m as an antigenic function is about 1 to 10.

The compound of formula 101” is contacted with the compound of formula 1602 to obtain the corresponding product of formula 1c', wherein n is about 4, p is about 90, t is about 1 or 2, R ³ is NHAc, R ⁴ is OH, R ⁸ is CMP, R ¹⁰ is 2-hydroxypropyl, and m as an antigenic function is about 1 to 10.

The compound of formula 101' is contacted with the compound of formula 601' to obtain the corresponding product of formula 1f', wherein n is about 4, p is about 90, t is about 1 or 2, R ³ is NHAc, R ⁴ is OH, R ⁸ is CMP, R ¹⁰ is 2-hydroxypropyl, and m as an antigenic function is about 1 to 10.

The compound of formula 101' is contacted with the compound of formula 701' to obtain the corresponding product of formula 1g', wherein n is about 4, p is about 90, t is about 1 or 2, R ³ is NHAc, R ⁴ is OH, R ⁸ is CMP, R ¹⁰ is 2-hydroxypropyl, and m as an antigenic function is about 1 to 10.

Specific Compositions

As a non-limiting example, the preferred specific groups in the compositions, pharmaceutical preparations, preparation methods, and uses of the present invention are the following combinations and arrangements of the substituent groups of Formula 1 (divided into subgroups in preferred ascending order):

●X is a foreign graft antigen against which the transplant recipient forms an undesirable immune response; a foreign antigen against which the patient forms an undesirable immune response; a therapeutic protein against which the patient forms an undesirable immune response; an autoantigen against which the patient forms an undesirable immune response, or a tolerogenic portion thereof.

●X is a therapeutic protein selected from the following group that induces an undesirable immune response in patients: abatacept, abciximab, adalimumab, adenosine deaminase, Ado-trastuzumab-metanetin, agalsidase α, agalsidase β, interleukin, vidarcephalin, aglucosidase α, α-1-protease inhibitor, anarabinose, aniplasmin (anisyl-plasminogen-streptokinase activator complex), antifibrinogen III, antithymocyte globulin, alteplase, bevacizumab, bivalirudin, botulinum toxin type A, B... Botulinum toxin, C1 esterase inhibitor, kanamycin, carboxypeptidase G2 (glutapeptide and voraxaze), cetuzumab, cetuximab, collagenase, rattlesnake immunotherapy Fab, dabepoetin-α, denosumab, digoxin immunotherapy Fab, deoxyribonuclease-α, icoshinone, etanercept, factor VIIa, factor VIII, factor IX, factor XI, factor XIII, fibrinogen, filgrastim, galsulfase, golimumab, histidine Linacetate, hyaluronidase, Idursulphase, imiglucerase, infliximab, insulin [including "rHu insulin" and bovine insulin], interferon-α2a, interferon-α2b, interferon-β1a, interferon-β1b, interferon-γ1b, ipilimumab, L-arginase, L-asparaginase, L-methionine, lactase, laronidase, lepirudin/hirudin, mecaseemine, mecaseemine-linfepe, methoxy-olfamomumab, natalizumab, octreotide, olprexol, pancreatic amylase Enzymes, pancreatic lipase, papain, Peg-asparaginase, Peg-doxorubicin HCl, PEG-erythropoietin-β, pefexine, Peg-interferon-α2a, Peg-interferon-α2b, pegologenase, pevisole, phenylalanine aminolysin (PAL), protein C, rapburicase (uricase), sacrocodilease, salmon calcitonin, saxaglastine, streptokinase, tenepase, teriparatide, atlizumab, trastuzumab, type 1 alpha-interferon, ustekinumab, and vW factor.

○ In particular, X is abciximab, adalimumab, agalsidase α, agalsidase β, interleukin, aglucosidase α, factor VIII, factor IX, infliximab, L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine aminolyase (PAL), or raburicase (uricase).

■ Specifically, X is factor VIII, factor IX, uricase, PAL, or asparaginase.

●X is an autoantigen polypeptide that is selected for the treatment of type 1 diabetes, pediatric multiple sclerosis, juvenile rheumatoid arthritis, celiac disease, or alopecia universalis.

○ In particular, X is an autoantigen polypeptide, which is selected for the treatment of newly diagnosed type 1 diabetes, pediatric multiple sclerosis, or celiac disease.

●X is a foreign antigen that triggers an unwanted immune response in the patient:

○ Derived from peanuts, including conarachigoglobulin (Ara h 1)

○ Derived from wheat, including natural α-gliadin "33-mer" (SEQ ID NO:20), deamidated α-gliadin "33-mer" (SEQ ID NO:21), α-gliadin (SEQ ID NO:22) and Ω-gliadin (SEQ ID NO:23).

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

○Derived from dogs, including Can f 1 (UNIPROT O18873) and canine albumin (UNIPROT P49822).

●X is a foreign graft antigen, such as human leukocyte antigen protein, against which the transplant recipient forms an undesirable immune response.

●X is an antibody, antibody fragment, or ligand that specifically binds to a circulating protein or peptide or antibody that causes graft rejection, an immune response to a therapeutic agent, an autoimmune disease, and/or an allergic reaction.

○ In particular, X binds to endogenous circulating proteins, peptides, or antibodies.

●Y is a linking base, which is selected from formulas Ya, Yb, Yh, Yi, Yk, Ym, Yn, Yo, and Yp.

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

● Specifically, n is 40 to 80 ± 10%, p is 30 to 40 ± 10%, and q is 4 to 12 ± 3. ○ Specifically, Y is of formula Ya, formula Yb, formula Ym, or formula Yn.

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

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

●In particular, Z is conjugated to Y via an ethyl acetamino group.

●More specifically, Z is joined to Y at its C1 position.

More specifically, R ⁸ is CMP.

●More specifically, R ⁸ is CMP.

●Specifically, R ⁸ is CMP.

●Y is a linking basis selected from the following group: formula Yc, formula Yf, formula Yg and formula Ym.

In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

■In particular, t is 1 or 2

●More specifically, where t is 1.

■In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

●Z is galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, or N-acetylglucosamine.

○ In particular, Z is a galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

■In particular, Z is a galactose or N-acetylgalactosamine conjugated at C1 or C2.

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

○ In particular, Z is glucose or N-acetylglucosamine conjugated at C1, C2 or C6.

■In particular, Z is glucose or N-acetylglucosamine conjugated at C1 or C2.

●More specifically, Z is an N-acetylglucosamine conjugated at C1.

Each of the above groups and subgroups is preferred, and they can be combined to describe further preferred aspects of the invention, for example, but not as a limitation, as follows:

●X is an autoantigen polypeptide that is selected for the treatment of type 1 diabetes, pediatric multiple sclerosis, juvenile rheumatoid arthritis, celiac disease, or alopecia universalis.

○ In particular, X is an autoantigen polypeptide, which is selected for the treatment of newly diagnosed type 1 diabetes, pediatric multiple sclerosis, or celiac disease.

■ Specifically, Y is a linking base selected from formulas Ya, Yb, Yc, Yf, Yg, Yh, Yi, Yk, Ym, Yn, Yo, and Yp.

●In particular, _Wp is a ^W1 polymer, ^R9 is Et-PEG _t -AcN, or a random copolymer, where ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

○ In particular, t is 1 or 2.

More specifically, where t is 2.

More specifically, where t is 1.

○ In particular, p is approximately 90.

More specifically, _Wp is a random copolymer and includes about 30 ^W1 and 60 ^W2 comonomers.

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

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

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

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

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

●Even more specifically, Z is conjugated to Y via an ethyl acetamino group.

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

○ In particular, Z is conjugated to Y via an ethyl acetamino group.

●In particular, Z is galactose, galactosamine, or N-acetylgalactosamine.

○ In particular, Z is a galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

More specifically, Z is a galactose or N-acetylgalactosamine conjugated at C1 or C2.

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

●In particular, Z is glucose, glucosamine, or N-acetylglucosamine.

Specifically, Z is glucose or N-acetylglucosamine conjugated at C1, C2 or C6.

More specifically, Z is glucose or N-acetylglucosamine conjugated at C1 or C2.

●Even more specifically, Z is an N-acetylglucosamine conjugated at C1.

■In particular, Y is a linking basis selected from the following group: formula Yc, formula Yf, formula Yg and formula Ym.

●In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

○ In particular, t is 1 or 2.

More specifically, where t is 1.

○In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

■In particular, Y is a linking basis selected from the following group: formula Yc and formula Ym.

●In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t- AcN, and ^R10 is 2-hydroxypropyl.

○ In particular, t is 1 or 2.

More specifically, where t is 1.

○In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

■ In particular, Z is galactose, galactosamine, or N-acetylgalactosamine.

●In particular, Z is a galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

○ In particular, Z is a galactose or N-acetylgalactosamine conjugated at C1 or C2.

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

■ In particular, Z is glucose, glucosamine, or N-acetylglucosamine.

●In particular, Z is glucose or N-acetylglucosamine conjugated at C1, C2 or C6.

More specifically, Z is glucose or N-acetylglucosamine conjugated at C1 or C2.

Even more specifically, Z is an N-acetylglucosamine conjugated at C1.

○In particular, Y is a linking basis selected from the following group: formula Ya, formula Yb, formula Yh, formula Yi, formula Yk, formula Ym, formula Yn, formula Yo and formula Yp.

■In particular, Y is a linking basis selected from the following group: formula Yc, formula Yf, formula Yg and formula Ym.

●In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

○ In particular, t is 1 or 2.

More specifically, where t is 1.

○In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

■In particular, Y is a linking basis selected from the following group: formula Yc and formula Ym.

●In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

○ In particular, t is 1 or 2

More specifically, where t is 1.

○In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

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

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

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

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

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

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

○ In particular, Z is galactose, galactosamine, or N-acetylgalactosamine.

■In particular, Z is a galactose or N-acetylgalactosamine conjugated at C1, C2 or C6.

●More specifically, Z is a galactose or N-acetylgalactosamine conjugated at C1 or C2.

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

●More specifically, where Y is a linking basis selected from the following group: formula Yc, formula Yf, formula Yg and formula Ym.

In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

■ In particular, t is 1 or 2.

●More specifically, where t is 1.

■In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

○ In particular, Z is glucose, glucosamine, or N-acetylglucosamine.

■In particular, Z is glucose or N-acetylglucosamine conjugated at C1, C2 or C6.

●More specifically, Z is glucose or N-acetylglucosamine conjugated at C1 or C2.

○More preferably, Z is an N-acetylglucosamine conjugated at C1.

●More specifically, where Y is a linking basis selected from the following group: formula Yc, formula Yf, formula Yg and formula Ym.

In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

■ In particular, t is 1 or 2.

●More specifically, where t is 1.

■In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

●More specifically, where Y is a linking basis selected from the following group: formula Yc and formula Ym.

In particular, _Wp is a random copolymer, ^R9 is Et-PEG _t -AcN and ^R10 is 2-hydroxypropyl.

■ In particular, t is 1 or 2.

●More specifically, where t is 1.

■In particular, p is about 90 and includes about 30 W ¹ and 60 W ² comonomers.

●m is an integer from approximately 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.

○ Specifically, m is approximately 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 specifically, m is approximately 10.

●m is 9, 10, or 11.

●n represents a mixture of integers from approximately 1 to 100.

○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.

■ Specifically, n is approximately 8 to 90.

■ Specifically, 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 specifically, n is approximately 40 to 80.

●More specifically, n is 37, 40, 41, 45, 50, 54, 55, 59, 60, 65, 70, 75, 80, 82, 83 or 88.

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

■ Specifically, n represents a mixed integer covering the ranges 7-13, 6-14, 15-25, 26-30, 42-50, 46-57, 60-82, 85-90, and 82-99.

●More specifically, n represents a mixed integer covering the ranges of 36-44, 42-50, 46-57, 60-82, and 75-85.

○p represents a mixed integer ranging from approximately 2 to 150.

■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, n represents a mixture of integers from approximately 1 to 100.

● Specifically, 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 specifically, n is approximately 8 to 90.

More specifically, 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 specifically, where n is approximately 40 to 80.

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

●More specifically, 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, n is an integer representing a mixture of approximately 1 to 100.

■ Specifically, 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 specifically, where n is approximately 8 to 90.

●More specifically, 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 specifically, where n is approximately 40 to 80.

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

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

■ 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 specifically, where n is approximately 8 to 90.

●More specifically, 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 specifically, where n is approximately 40 to 80.

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

○q represents a mixture of integers from approximately 1 to 44.

■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.

Utility, testing and application

General utility

The compositions of the present invention can be used in a variety of applications, as those skilled in the art will understand, including graft rejection, immune responses to therapeutic agents, autoimmune diseases, and the treatment of food allergies.

In a preferred embodiment, the compositions of the present invention are used to modulate (in particular downregulate) an undesirable antigen-specific immune response.

In another embodiment, the compositions of the present invention can be used to bind to and eliminate unwanted circulating specific proteins, including endogenously generated antibodies in patients (i.e., exogenous antibodies not administered to the patients), peptides, etc., which induce autoimmunity and related pathologies, allergic reactions, inflammatory immune responses, and anaphylactic reactions.

In some embodiments of the invention, antigens are targeted to the liver for presentation via antigen-presenting cells, thereby specifically downregulating the immune system or clearing unwanted circulating proteins. This differs from previous uses of liver targeting, such as those described in US2013/0078216, where liver-targeting molecules like DOM26h-196-61 were intended to deliver therapeutic agents to treat liver diseases such as fibrosis, hepatitis, cirrhosis, and liver cancer.

According to some embodiments, the present invention provides compositions and methods for treating undesirable immune responses against self-antigens and foreign antigens, wherein the foreign antigens include, but are not limited to: foreign graft antigens against which a graft recipient forms an undesirable immune response (e.g., graft rejection), foreign antigens against which a patient forms an undesirable immune response (e.g., allergic or hypersensitivity), therapeutic agents against which a patient forms an undesirable immune response (e.g., hypersensitivity and/or reduced therapeutic activity), and self-antigens against which a patient forms an undesirable immune response (e.g., autoimmune diseases).

Autoimmune disease states that can be treated using the methods and compositions provided by this invention 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; and rheumatoid arthritis. Autoimmune rheumatoid; atopic dermatitis; autoimmune aplastic anemia; autoimmune gastritis; autoimmune hepatitis; autoimmune hypophysitis; autoimmune oophoritis; autoimmune orchitis; Autoimmune polyendocrine syndrome type 1; Autoimmune polyendocrine syndrome type 2; autoimmune thyroiditis; Behcet's disease; obliterative bronchiolitis; bullous pemphigoid; celiac disease; Churg-Strauss syndrome; chronic inflammatory demyelinating polyneuropathy; cicatricial pemphigoid; Crohn's disease; Coxsackie myocarditis; Dermatitis herpetiformis. rpetiformis Duhring; type 1 diabetes mellitus; erythema nodosum; epidermolysis bullosa acquisita; giant cell arteritis (temporal arteritis); giant cell myocarditis; Goodpasture's syndrome; Graves' disease; Guillain-Barré syndrome; Hashimoto's encephalitis; Hashimoto's thyroiditis; IgG4-related sclerosing disease; Lambert-Eaton syndrome. Mixed connective tissue disease; Mucha-Habermann disease; multiple sclerosis; myasthenia gravis; optic neuritis; neuromyelitis optica; pemphigus vulgaris and its variants; pernicious angemisus; pituitary autoimmune diseases; polymyositis; postpericardiotomy syndrome; premature ovarian failure; primary bile 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.

A specific group of autoimmune disease states that can be treated using the methods and compositions provided in this invention include, but are not limited to: acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; psoriatic arthritis; rheumatoid arthritis; autoimmune aplastic anemia; autoimmune hypophysitis; autoimmune gastritis; type 1 autoimmune polyendocrine syndrome; bullous pemphigoid; celiac disease; Coxsackie myocarditis; Dolin herpesoid dermatitis; diabetes mellitus (type 1); acquired epidermolysis bullosa; giant cell myocarditis; pulmonary hemorrhage nephritis syndrome; Graves' disease; Hashimoto's thyroiditis; mixed connective tissue disease; multiple sclerosis; myasthenia gravis; neuromyelitis optica; pernicious angemisus; pemphigus vulgaris and its variants; pituitary autoimmune diseases; premature ovarian failure; rheumatic heart disease; systemic sclerosis; Sjögren's syndrome; systemic lupus erythematosus; and vitiligo.

In implementation schemes that utilize antigens such as food antigens that induce undesirable immune responses, treatment can be provided for responses to, for example: peanuts, apples, milk, egg whites, egg yolks, mustard greens, celery, shrimp, wheat (and other grains), strawberries, and bananas.

Those skilled in the art will understand that patients can be tested to identify foreign antigens against which an undesirable immune response has been formed, and the compositions of the present invention can be developed based on said antigens.

test

In determining the efficacy of the compositions and methods of the present invention, the specificity of binding to antigen-presenting cells in the liver (particularly to hepatocytes and especially to ASGPR) should initially be determined. This can be accomplished, for example, by incorporating a marker (such as the fluorescent marker phycoerythrin (“PE”)) into the compositions of the present invention. The compositions are administered to suitable experimental subjects. Controls, such as unconjugated PE or a carrier (saline), are administered to subjects in other groups. The compositions and controls are allowed to cycle for a period of 1 to 5 hours, after which the spleen and liver of the subjects are harvested and fluorescence is measured. The specific cells in which fluorescence is found can then be identified. When tested in this manner, the compositions of the present invention show higher concentration levels in antigen-presenting cells of the liver compared to unconjugated PE or a carrier.

The efficiency of immunomodulation can be tested by measuring the proliferation of OT-I CD8 ⁺ cells (transplanted into host mice) in response to the application of the composition of the present invention incorporating a known antigen (such as ovalbumin (“OVA”)) compared to the application of a single antigen or a carrier alone. When tested in this manner, the composition of the present invention shows an increase in OT-I cell proliferation compared to the single antigen or carrier alone, demonstrating increased cross-priming of CD8 ⁺ T cells. To distinguish between T cells expanded to a functional effector phenotype and those expanded and deleted, phenotypic analysis of proliferating OT-ICD8 ⁺ T cells can be performed targeting depleted molecular tags [such as programmed death-1 (PD-1), FasL, etc.] and annexin V, a marker of apoptosis and thus deletion. The responsiveness of OT-I CD8 ⁺ T cells to antigen challenge in the presence of adjuvants can also be assessed to demonstrate functional non-responsiveness to antigens and, therefore, immune tolerance. For this purpose, the composition of the present invention is administered to host mice, followed by antigen challenge, and then the inflammatory tags of the cells are analyzed. When tested in this manner, the compositions of the present invention exhibit very low (e.g., background) levels of inflammatory OT-I CD8 ⁺ T cell response to OVA compared to the control group, thus demonstrating immune tolerance.

Humoral immune responses can be tested by administering the composition of the present invention incorporating a known antigen, such as OVA, compared to administering a single antigen or a carrier only, and by measuring the resulting antibody levels. When tested in this manner, the compositions of the present invention show very low (e.g., background) levels of antibody formation, which are significantly higher in response to antigen administration than in response to administration of the composition and the carrier.

The efficiency of tolerance to an antigen can be assessed using a humoral immune response test, as described above, in which, several weeks after treatment with the composition of the present invention, a group of subjects is challenged with the antigen alone, and then the level of antibodies against the antigen is measured. When tested in this manner, the composition of the present invention shows a lower level of antibody formation in response to antigen challenge in the pretreated group compared to the untreated group.

Disease-focused experimental models are well known to those skilled in the art and include autoimmune and tolerable NOD (or non-obese diabetic) mouse models and EAE (experimental autoimmune encephalomyelitis) models for human inflammatory demyelinating diseases and multiple sclerosis. Specifically, NOD mice develop spontaneous autoimmune diabetes (similar to human type 1a diabetes). The NOD mouse group is treated with a test compound or a negative control, followed by measurement of blood glucose. Successful treatment corresponds to mechanistic evidence of the possibility of treating human diabetes or other autoimmune diseases. (See, for example, Anderson and Bluestone, Annu. Rev. Immunol. 2005; 23:447-85).

application

The compositions of the invention shall be administered at a therapeutically effective dose, for example, a dose sufficient to provide treatment for the previously described disease state. The compounds of the invention or their pharmaceutically acceptable salts may be administered by any acceptable mode of administration of an agent used to achieve a similar effect.

Although human dosage levels for the compounds of the present invention have not yet been optimized, these can initially be extrapolated from doses of about 10 μg to 100 μg administered to mice. Typically, the individual human dose is 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 over periods 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 remainder of the full dose over a period of time, such as 1 to 7 days. The amount of active compound administered will, of course, depend on any or all of the following: the subject being treated and the disease state, the severity of the illness, the manner and schedule of administration, and the judgment of the prescribing physician. It should also be understood that the amount administered will depend on the molecular weight of the antigen, antibody, antibody fragment, or ligand, and the size of the linker.

The compositions of the present invention can be administered alone or in combination with other pharmaceutically acceptable excipients. While all typical routes of administration (e.g., oral, topical, transdermal, injection (intramuscular, intravenous, or intra-arterial)) are considered, it is currently preferred to provide liquid dosage forms suitable for injection. Formulations will typically comprise conventional pharmaceutical carriers or excipients and the compositions of the present invention or pharmaceutically acceptable salts thereof. Furthermore, these compositions may include other medical agents, pharmaceuticals, carriers, etc., including but not limited to therapeutic proteins, peptides, antibodies, or antibody-like molecules corresponding to the antigen (X) used in the compositions of the present invention, and other active agents that can act as immunomodulators, more specifically, that can have an inhibitory effect on B cells, including antifolate agents, immunosuppressants, cytostatics, mitotic inhibitors, and antimetabolites, or combinations thereof.

Generally, depending on the intended administration method, a pharmaceutically acceptable composition will contain about 0.1% to 95%, preferably about 0.5% to 50% by weight of the composition of the present invention, with the remainder being suitable pharmaceutical excipients, carriers, etc. Dosage forms or compositions containing the active ingredient in the range of 0.005% to 95% and with the balance made up by a non-toxic carrier can be prepared.

For example, a pharmaceutically applicable liquid composition can be prepared by dissolving or dispersing the active ingredient of the present invention (e.g., lyophilized powder) and optional pharmaceutical adjuvant in a carrier, such as, for example, water (water for injection), saline, aqueous dextran, glycerol, glycol, ethanol, etc. (excluding galactose), thereby forming a solution or suspension. If desired, the pharmaceutical composition to be applied may also contain small amounts of non-toxic excipients, such as wetting agents, emulsifiers, stabilizers, solubilizers, pH buffers, etc., such as sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate and triethanolamine oleate, etc., osmolyte, amino acids, sugars and carbohydrates, proteins and polymers, salts, surfactants, chelating agents, antioxidants, preservatives, and specific ligands. The actual methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22nd edition, 2012. In any case, the composition or formulation to be administered contains an amount of active compound that is effective in treating the symptoms of the subject being treated.

Example

The following embodiments are provided to more fully describe the methods disclosed above, and to set forth the best modes of consideration for carrying out various aspects of the invention. It should be understood that these embodiments are in no way intended to limit the true scope of the invention, but are presented for illustrative purposes. All references cited in this invention are incorporated herein by reference in their entirety.

Example 1

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

1A. Equation 103’, where X’ is OVA and m is 4

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 reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl (0.00119 mmol) of the resulting Traut reagent solution was added to the stirred OVA solution while continuously stirring. After 1 hour, excess Traut reagent was removed using a size exclusion column to provide the corresponding product of Formula 103’.

1B. Equation 106A, where n is 80

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. Pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104, where n is 80) (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 pyridyl dithiol-poly(ethylene glycol)-N-acetylgalactosamine (Formula 106A) was ready for use without further purification.

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

The purified OVA-Traut conjugate of formula 103’ prepared in Example 1A was directly added to the stirred product of formula 106A prepared in Example 1B. After 1 hour, the product of formula 1a was obtained by purifying the reaction mixture by centrifugation through a size exclusion column. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product (see Figure 5).

1D. Other compounds of formula 103'

By following the procedure described in Example 1A and replacing the OVA as follows:

●Abciximab,

●Adalimumab,

●Agarase α,

● Agargase β,

●Adefolate,

●Aglucosidase α,

●Factor VIII,

●Factor IX,

●L-Asparaginase,

● Laronine enzyme

●Octreotide,

●Phenylanine aminolysin,

●Raburiase,

●Insulin (SEQ ID NO:1),

●GAD-65 (SEQ ID NO:2),

●IGRP (SEQ ID NO:3)

●MBP (SEQ ID NO:4),

●MOG (SEQ ID NO:5),

●PLP (SEQ ID NO:6),

●MBP13-32 (SEQ ID NO:7),

●MBP83-99 (SEQ ID NO:8),

●MBP111-129(SEQ ID NO:9),

●MBP146-170(SEQ ID NO:10),

●MOG1-20 (SEQ ID NO:11),

●MOG35-55(SEQ ID NO:12),

●PLP139-154(SEQ ID NO:13),

●MART1 (SEQ ID NO:14),

●Tyrosinase (SEQ ID NO:15),

●PMEL (SEQ ID NO:16),

●Aquaporin-4 (SEQ ID NO:17),

●S-repressor protein (SEQ ID NO:18),

●IRBP (SEQ ID NO:19),

●Contains peanut globulin (UNIPROT Q6PSU6),

●Natural α-gliadin "33-mer" (SEQ ID NO:20),

●Deacylated α-gliadin "33-mer" (SEQ ID NO:21),

●α-Glucolylein (SEQ ID NO:22),

●Ω-Glucolyl (SEQ ID NO:23),

●Fel d 1A (UNIPROT P30438),

● Feline albumin (UNIPROT P49064),

●Can f 1(UNIPROT O18873),

●Dog albumin (UNIPROT P49822), and

●RhCE (UNIPROT P18577) yields the following corresponding compounds of formula 103’, wherein:

●X is abciximab and m is 10.

●X is adalimumab and m is 11.

●X is agarase α and m is 14.

●X is agarase β and m is 14.

●X is interleukin and m is 6.

●X is α-glucosidase, and m is 13.

●X is a factor of VIII and m is 100.

●X is a factor of 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 aminolysin and m is 12.

●X is raburicase and m is 12

●X is insulin (SEQ ID NO:1) and m is 2.

●X is GAD-65 (SEQ ID NO:2) and m is 8.

●X is IGRP (SEQ ID NO:3) and m is 7.

●X is a MBP (SEQ ID NO:4) and m is 6.

●X is a MOG (SEQ ID NO:5) and m is 5.

●X is PLP (SEQ ID NO: 6) and m is 8.

●X is MBP13-32 (SEQ ID NO:7) and m is 1.

●X is MBP83-99 (SEQ ID NO:8) and m is 1.

●X is MBP111-129 (SEQ ID NO:9) and m is 1.

●X is MBP146-170 (SEQ ID NO:10) and m is 2.

●X is MOG1-20 (SEQ ID NO:11) and m is 1.

●X is MOG35-55 (SEQ ID NO:12) and m is 2.

●X is PLP139-154 (SEQ ID NO:13) and m is 3.

●X is MART1 (SEQ ID NO:14) and m is 4.

●X is tyrosinase (SEQ ID NO:15) and m is 8.

●X is PMEL (SEQ ID NO:16) and m is 5.

●X is aquaporin-4 (SEQ ID NO:17) and m is 4.

●X is an S-repressor protein (SEQ ID NO:18) and m is 12.

●X is an IRBP (SEQ ID NO: 19) and m is 21.

●X is conaragenin and m is 21.

●X is a natural α-gliadin "33-mer" (SEQ ID NO:20) and m is 1,

●X is a deacetylated α-gliadin "33-mer" (SEQ ID NO:21) and m is 1,

●X is α-gliadin (SEQ ID NO:22) and m is 1.

●X is Ω-gliadin (SEQ ID NO:23) 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.

●X is RhCE and m is 10.

1E. Other compounds of formula 1aA

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 α-m ₁₄ -n ₈₀ ,

●F1aA-agalsidase β-m ₁₄ -n ₈₀ ,

●F1aA-Adelineate- _m6 - _n80 ,

●F1aA-glucosidase α-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-laronylase-m ₇ -n ₈₀ ,

●F1aA-octreotide- _m1 - _n80 ,

●F1aA-phenylalanine aminolysin- _m12 - _n80 ,

●F1aA-raburicase-m ₁₂ -n ₈₀ ,

●F1aA-insulin- _m2 - _n80 ,

●F1aA-GAD-65-m ₈ -n ₈₀ ,

●F1aA-IGRP-m ₇ -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- _m4 - _n80 ,

●F1aA-S-inhibitory protein- _m12 - _n80 ,

●F1aA-IRBP-m ₂₁ -n ₈₀ ,

●F1aA-conaragenin- _m21 - _n80 ,

●F1aA - Natural α-Gliadin "33-mer" - m ₁ - n ₈₀ ,

●F1aA-deacylated α-gliadin "33-mer" - m ₁ - _{n 80} ,

●F1aA-α-gliadin-m ₁ -n ₈₀ ,

●F1aA-Ω-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-dog albumin- _m23 - _n80 , and

●F1aA-RhCE-m ₁₀ -n ₈₀ .

1F. Other compounds of formula 106A

By following the procedure described in Example 1B and replacing the following items with pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104, where n is 80):

●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,

1G. Other compounds of formula 1aA

Following the procedure described in Example 1E and replacing the compound of Formula 106A with the compound obtained in Example 1F, the corresponding compound of Formula 1aA is obtained, where n is 12, 33, 40, 43, 50, 60, 75, and 84, as follows:

●F1aA-insulin- _m2 - _n12 ,

●F1aA-insulin- _m2 - _n33 ,

●F1aA-insulin- _m2 - _n40 ,

●F1aA-insulin- _m2 - _n43 ,

●F1aA-insulin- _m2 - _n50 ,

●F1aA-insulin- _m2 - _n60 ,

●F1aA-insulin- _m2 - _n75 , and

●F1aA-insulin- _m2 - _n84 .

Other compounds of formula 1aA, 1H.

Similarly, the corresponding compound of formula 1aA was obtained by following the procedure described in Examples 1A-G and replacing galactosamine with the compound glucosamine, wherein Z’ is C2 glucosamine.

Example 2

F1b-OVA-m ₁ -n ₄ -p ₃₄ -2NAcGAL

2A. Equation 103’, where X’ is ovalbumin and m is 1

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 reagent was dissolved in 100 μl of pH 7.0 PBS, and 43 μl (0.00310 mmol) of the resulting Traut reagent solution was added to the stirred solution of OVA under continuous stirring. After 1 hour, unreacted Traut reagent was removed using a size exclusion column to provide the product of formula 103’.

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

In a microcentrifuge tube, poly(galactosamine methacrylate)-(pyridyl disulfide) (Formula 201) (20.0 mg, 0.0020 mmol) was dissolved in 50 μl of pH 8.0 PBS containing 5 mM EDTA. The purified OVA-Traut product from Example 2A was added, followed by stirring for 1 hour. The reaction mixture was purified by centrifugation using a size exclusion column to obtain the product of Formula 1b. Characterization (UHPLC SEC, gel electrophoresis) confirmed the product's identity (see Figure 5).

2C. Other compounds of formula 1b

By following the procedure described in Example 2B and replacing 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-m ₁₁ -n ₄ -p _34-2NAcGAL ,

●F1b-Agarosylase α- _m14 - _n4 - _p34-2NAcGAL ,

●F1b-Agarosylase β- _m14 - _n4 - _p34-2NAcGAL ,

●F1b-Adedileukin- _m6 - _n4 - _p34-2NAcGAL ,

●F1b-glucosidase α- _m13 - _n4 - _p34-2NAcGAL ,

●F1b-Factor VIII-m ₁₀₀ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-factor IX-m ₁₈ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-L-asparaginase- _m5 - _n4 - _p34-2NAcGAL ,

●F1b-laronylase- _m7 - _n4 - _p34-2NAcGAL ,

●F1b-octreotide- _m1 - _n4 - _p34-2NAcGAL ,

●F1b-phenylalanine aminolysin- _m12 - _n4 - _p34-2NAcGAL ,

●F1b-raburicase- _m12 - _n4 - _p34-2NAcGAL ,

●F1b-insulin- _m2 - _n4 - _p34-2NAcGAL ,

●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- _m8 - _n4 - _p34-2NAcGAL ,

●F1b-PMEL-m ₅ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-aquaporin 4-m ₄ -n ₄ -p _34-2NAcGAL ,

●F1b-S-inhibitory protein-m ₁₂ -n ₄ -p _34-2NAcGAL ,

●F1b-IRBP-m ₂₁ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-conaragenin- _m21 - _n4 - _p34-2NAcGAL ,

●F1b - Natural α-gliadin "33-mer" - m ₁ - n ₄ - p ₃₄ - 2NAcGAL,

●F1b-deacylated α-gliadin "33-mer" - m ₁ - n ₄ - p ₃₄ - 2NAcGAL,

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

●F1b-Ω-Gliadin-m ₁ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-Fel d 1-m ₄ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-feline albumin- _m16 - _n4 - _p34-2NAcGAL ,

●F1b-Can f 1-m ₆ -n ₄ -p ₃₄ -2NAcGAL,

●F1b-dog albumin- _m23 - _n4 - _p34-2NAcGAL , and

●F1b-RhCE-m ₁₀ -n ₄ -p ₃₄ -2NAcGAL.

1D. Other compounds of formula 1b

Similarly, by following the procedures described in Examples 2B-C and replacing the compound poly(glucosamine methacrylate)-(pyridyl disulfide) or poly(galactosamine methacrylate)-(pyridyl disulfide), the corresponding compound of formula 1b is obtained, wherein Z” is 2-NAcGLU.

Example 3

F1f-OVA-m ₁ -n ₄ -p ₃₃ -2NAcGAL

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

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

3B. Formula 1f, where X' is ovalbumin, and m is 1, n is 4, p is 33, R ⁹ is a direct bond, and Z” is 2NAcGLU

Similarly, by following the procedure of Example 3A and replacing poly(n-acetylgalactosamine)-p-nitrophenyl carbonate with poly(n-acetylglucosamine)-p-nitrophenyl carbonate, the corresponding compound of Formula 1f was obtained, where Z” is 2NAcGLU.

Example 4

F1g-PVA-m ₁ -p ₉₀ -2NAcGAL

4A. Formula 1g, where X' is ovalbumin, and m is 1, p is 90, R ⁹ is a direct bond, and Z” is 2NAcGAL3A

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. 75 mg (0.00297619 mmol) of poly(galactosamine methacrylate)-NHS (Formula 701) dissolved in 0.4 mL of pH 7.4 PBS was added to the stirred solution. The mixture was allowed to stir for 2 hours. The mixture was then collected and dialyzed against pH 7.4 PBS for 3 days (30 kDa molecular weight cutoff) to provide 1 g of the product.

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

Similarly, by following the procedure of Example 4A and replacing poly(galactosamine methacrylate)-NHS with poly(glucosamine methacrylate)-NHS, the corresponding compound of formula 1g was obtained, wherein Z” is 2NAcGLU.

Example 5

F1h-OVA-m ₂ -n ₄₅ -p ₅₅ -q ₄ -2NAcGAL

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

In an endotoxin-free tube, OVA (3.0 mg, 0.0000714286 mmol) was added to 150 μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Dibenzocyclooctyn-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 hour. Excess dibenzocyclooctyn-PEG-(p-nitrophenyl carbonate) was removed using a size exclusion column to provide the product of Formula 802'.

5B. Formula 1h, where X' is ovalbumin, m is 2, n is 45, p is 55, q is 4, ^R8 is _CH2 , and ^R9 is a direct bond. Z” is 2NAcGAL

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

5C. Formula 1h, where X' is ovalbumin, m is 2, n is 45, p is 55, q is 4, ^R8 is _CH2 , ^R9 is a direct bond, and Z” is 2NAcGLU

Similarly, by following the procedure of Example 5B and replacing poly(galactosamine methacrylate)-NHS with poly(glucosamine methacrylate)-NHS, the corresponding compound of formula 1h was obtained, where Z” is 2NAcGLU.

Example 6

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

6A. Equation 103’, where X’ is ovalbumin and m is 10.

In an endotoxin-free tube, OVA (5.0 mg, 0.00019 mmol) was added to 150 μl of pH 8.0 PBS containing 5 mM EDTA and stirred. Separately, 1 mg of Taut reagent was dissolved in 100 μl of pH 7.0 PBS, and 16 μl (0.00119 mmol) of the resulting Taut reagent solution was added to the stirred solution of OVA while continuously stirring. After 1 hour, unreacted Taut reagent was removed using a size exclusion column to provide the product of formula 103’.

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

Dibenzocyclooctyn-PEG-(pyridinyl 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 dibenzocyclooctyn-PEG-(pyridinyl disulfide) was removed by size exclusion chromatography to provide the product of Formula 902.

6C. Formula 1j, where X' is ovalbumin, m is 10, n is 45, p is 55, q is 4, ^R8 is _CH2 , and ^R9 is a direct bond. Z” is 2NAcGAL.

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

6D. Formula 1j, where X' is ovalbumin, m is 10, n is 45, p is 55, q is 4, ^R8 is _CH2 , ^R9 is a direct bond, and And Z” is 2NAcGLU

Similarly, by following the procedure of Example 6C and replacing poly(galactosamine methacrylate)-NHS with poly(glucosamine methacrylate)-NHS, the corresponding compound of Formula 1j was obtained, where Z” is 2NAcGLU.

Example 7

F1L-OVA-m ₂ -n ₈₀ -p ₅₅ -q ₄ -2NAcGAL

7A. Equation 1002, where X’ is ovalbumin, m is 2, and n is 80.

Dibenzocyclooctyn-PEG-(pyridinyl 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 dibenzocyclooctyn-PEG-(pyridinyl disulfide) was removed by size exclusion chromatography to provide the product of Formula 1002.

7B. Formula 1L, where X' is ovalbumin, m is 2, n is 80, p is 55, q is 4, ^R8 is _CH2 , and ^R9 is a direct bond. Z” is 2NAcGAL

Poly(galactosamine methacrylate)-N3 (Formula 803, where p is 55, q is 4 and Z” is N-acetylgalactosamine) (36 mg, 0.00238 mmol) was dissolved in 150 μl of pH 7.4 PBS, and the product of Example 7A was added with stirring. After 1 hour, the product of Formula 1L was purified by size exclusion chromatography by centrifugation (to remove excess poly(galactosamine methacrylate)-N3). Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

7C. Formula 1L, where X' is ovalbumin, m is 2, n is 80, p is 55, q is 4, ^R8 is _CH2 , and ^R9 is a direct bond. Z” is 2NAcGLU

Similarly, by following the procedure of Example 7B and replacing poly(galactosamine methacrylate)-NHS with poly(glucosamine methacrylate)-NHS, the corresponding compound of formula 1jL was obtained, where Z” is 2NAcGLU.

Example 8

Preparation of poly(galactosamine methacrylate) polymer

8A. Galactosamine methacrylate

0.5 M sodium methoxide (22 mL, 11.0 mmol) was added to the stirred galactosamine hydrochloride (2.15 g, 10.0 mmol). After 30 minutes, methacrylic anhydride (14.694 g, 11.0 mmol) was added, and stirring was continued for 4 hours. The obtained galactosamine methacrylate was loaded onto silica gel by rotary evaporation and purified by column chromatography using DCM:methanol (85:15).

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

Galactose methacrylate (600 mg, 2.43 mmol), ethyl 2-(2-(2-(2-(pyridin-2-yldithio)ethoxy)ethoxy)2-(phenylcarbothio)thio)ethyl 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 four freeze-thaw cycles, followed by stirring at 70 °C for 6 hours. The desired polymer product of formula 201 was precipitated in 12 mL of methanol, and excess solvent was removed under reduced pressure.

8C. Equation 201, where n is 4 and p is 30.

Similarly, by following the procedure of Example 8B and replacing galactose methacrylate with glucose methacrylate, the corresponding poly(glucosamine methacrylate) polymer was obtained.

Example 9

Preparation of F1aA-PE- _m3 - _n80

9A. Formula 103’, where X’ is phycoerythrin

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. Independently, 1 mg of Traut reagent was dissolved in 100 μl of pH 7.0 PBS, and 2 μl (0.00013 mmol) of the resulting Traut reagent solution was added to the stirred solution of PE and stirred continuously. After 1 hour, excess Traut reagent was removed using a size exclusion column to provide the product of formula 103’.

9B. Equation 106A, where n is 80

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. Pyridyl dithiol-poly(ethylene glycol)-NHS ester (Formula 104, where n is 80) (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 for use without further purification.

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

The purified PE-Traut conjugate prepared in Example 9A was directly added to the stirred product of Formula 106A prepared in Example 9B. After 1 hour, the product of Formula 1a was obtained by purifying the reaction mixture by centrifugation using a size exclusion column. Characterization (UHPLC SEC, gel electrophoresis) confirmed the identity of the product.

9D. Formula 1a, where X’ is phycoerythrin, m is 3, n is 80, and Z’ is glucosamine.

Similarly, by following the procedures of Examples 9B and C and replacing galactosamine with glucosamine, the corresponding compound of Formula 1a was obtained, where Z” is glucosamine.

Example 10

liver distribution

10A. For example, F1aA-PE- _m3 - _n80 is prepared as described in Example 9. A 30 μg/100 μl solution in sterile saline is prepared for injection.

F1aA-PE- _m3 - _n80 solution (30 μg) was administered via tail vein injection to one group of three C57 black 6 mice (n=3 per group). The other two groups of mice received an equal volume of phycoerythrin or a saline carrier in 100 μl of saline. Three hours after administration, the livers and spleens of these animals were collected, and the cellular fluorescence levels in these organs were determined by flow cytometry as an indicator of cellular PE content.

As shown in Figures 1A-1D, compared with animals receiving PE solution, _sinusoidal endothelial _cells (LSEC) (1A), hepatocytes (1C), Kupffer cells (KC) (1B), and other antigen-presenting cells (APCs) (1D) from the livers of mice treated with F1aA-PE-m3-n80 showed at least a three-fold increase in fluorescence. No detectable differences in fluorescence were found in spleen cells harvested from the three groups. These results confirm that F1aA-PE- _m3 - _n80 has sufficient specificity for binding to antigen-presenting cells in the liver.

10B. By following the procedure described in Example 10A and replacing F1aA-PE- _m3 - _n80 with the following compounds: F1b-PE- _m3 - _n4 - _p34-2NAcGAL , F1f-PE- _m3 - _n4 - _p33-2NAcGAL , F1g-PE- _m3 - _p90-2NAcGAL , F1h-PE- _m3 - _n45 - _p55 - _q4-2NAcGAL , F1j-PE- _m3 - _n45 - _p55 - _q4-2NAcGAL , F1L-PE- _m3 - _n80 - _p55 - _q4-2NAcGAL , F1m-PE- _m3 - _n80 - _p30 - _q4 -CMP-2NHAc, F1m-PE- _m3 - _n62 -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 (prepared, for example, by replacing X in Examples 2B, 3, 4, 5B, 6B, 7B, 15G, 15L, 16B, and 16F, respectively, as described with reference to Example 9), confirm that compounds F1aA-PE-m ₃ -n ₈₀ and compounds F1b-PE-m ₃ -n ₄ -p _34-2NAcGAL , F1f-PE-m ₃ -n ₄ -p _33-2NAcGAL , F1g-PE-m ₃ -p _90-2NAcGAL , and 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 have sufficient specificity for binding to antigen-presenting cells in the liver.

10C. By following the procedures described in Examples 10A and 10B and replacing the galactosylating compounds with the corresponding glucosylating compounds, it was confirmed that the glucosylating compounds had sufficient specificity for binding to antigen-presenting cells in the liver.

Example 11

Proliferation of antigen-specific OT1 CD8 ⁺ T cells

11A. F1aA-OVA- _m4 - _n80 , synthesized for example as described in Example 1, was prepared as a 10 μg/100 μl saline solution for injection. On day 0, 106 fluorescently labeled OT-1T cells were adoptively transferred to three groups of CD45.2 mice (n=5 per group) via tail vein injection. On the following day (i.e., day 1), 10 μg of F1aA-OVA- _m4 - _n80 , OVA, or saline was administered to each of the three groups of mice via tail vein injection. On day 6, the animals were sacrificed, and the percentage of OT-1 cells proliferating in the spleen was determined by fluorescently activated cell sorting.

Results from this study (see Figure 2) show that the percentage of proliferating OTI T cells in mice treated with F1aA-OVA- _m4 - _n80 (“Gal-OVA” in Figure 2) was significantly greater than the percentage of proliferating OTI cells in the spleen of mice treated with OVA or saline (“untreated” in Figure 2). This increased OTI cell proliferation indicates increased cross-priming of CD8+ T cells in animals treated with F1aA-OVA- _m4 - _n80 compared to other therapies. Consistent with results from Example 12, these results suggest that F1aA-OVA- _m4 - _n80 can target the antigen to the liver, thereby increasing OVA presentation by antigen-presenting cells in the liver to OVA-specific OTI T cells.

11B. To distinguish between T cells expanded into functional effector phenotypes and those expanded and deleted, the phosphatidylserine exposure of proliferating OTI CD8 ⁺ T cells can be analyzed by binding to annexin V, a marker of apoptosis and thus deletion, and the exhaustion marker programmed death-1 (PD-1). As shown in Figures 3A-3B, F1aA-OVA- _m4 - _n80 (“Gal-OVA” in Figures 3A-3B) induces a much higher number of annexin-V ⁺ and PD-1 ⁺ proliferating OTI CD8 ⁺ T cells than soluble OVA. These data demonstrate that, according to some embodiments disclosed herein, coupling antigens against which tolerance is induced with linkers and liver-targeting moieties as disclosed herein results in an unexpectedly enhanced generation of T cells capable of becoming immune functional cells.

11C. By following the procedures described in Examples 11A and 11B below, and replacing F1aA-OVA- _m4 - _n8 with compounds of Formula 1 obtained, for example, as described in Examples 3A, 4A, 5B, 6C, 7B, and 19G, it was shown that the compounds from Examples 3A, 4A, 5B, 6C, 7B, and 19G induced a much higher number of annexin-V ⁺ and PD-1 ⁺ proliferating OTI CD8 ⁺ T cells than soluble OVA.

11D. By following the procedures described in Examples 11A and 11B, and replacing F1aA-OVA-m4-n8 with compounds of Formulas 1 and 2 obtained, for example, as described in Examples 1E, 1G, 2C, 15I, 15L, 16B, _16D , and _16F , and replacing OVA with an antigen corresponding to X (or X' or X”), it was shown that the compounds from Examples 1E, 1G, 2C, 15I, 15L, 16B, 16D, and 16F induced a much higher number of annexin-V ⁺ and PD-1 ⁺ proliferating OTI CD8 ⁺ T cells than the soluble antigen X.

11E. By following the procedures described in Examples 11A-D and replacing the galactosylating compounds with the corresponding glucosylating compounds, it was confirmed that the glucosylating compounds induced a much higher number of annexin-V ⁺ and PD-1 ⁺ proliferating OTI CD8 ⁺ T cells than the soluble antigen X.

Example 12

F1aA-OVA- _m4 - _n8 does not induce an OVA-specific antibody response.

12A. To assess the humoral immune response to F1aA-OVA- _m4 - _n8 , we treated mice with weekly intravenous injections of either F1aA-OVA- _m4 - _n8 or OVA, and then measured the levels of OVA-specific antibodies in the blood. On days 0, 7, and 14 of the experiment, mice were administered 100 μl of saline containing one of the following: 1) 6 μg OVA; 2.) 6 μg F1aA-OVA- _m4 - _n8 ; 3.) 30 μg OVA; 4.) 30 μg F1aA-OVA- _m4 - _n8 ; or 5.) saline alone. Each group contained 5 mice. On day 19, mice were bled by buccal puncture, and the titer of OVA-specific antibodies in the blood of each mouse was determined by ELISA. The results of this study showed that while treatment of mice with 6 μg and 30 μg OVA increased OVA-specific antibody titers, mice treated with 6 μg and 30 μg F1aA-OVA- _m4 - _n8 (“Gal-OVA” in Figure 4) had blood titers similar to those of mice treated with saline (i.e., vector-treated animals) (Figure 4). For example, mice treated with 6 μg and 30 μg OVA had mean antibody titers of 3.5 and 2.5, respectively; however, mice treated with 6 μg and 30 μg OVA had mean antibody titers of 0.75 and 0.25, respectively. Therefore, these data demonstrate that conjugation of the desired antigen to which the immunization is targeted with a linker and a liver-targeting moiety according to some embodiments disclosed in this invention results in significantly less antigen-specific antibody production. Therefore, these data demonstrate that the immune response to liver-delivered antigens via the compositions disclosed in this invention is reduced.

12B. By following the procedure described in Example 12A and replacing F1aA-OVA- _m4 - _n8 with compounds of Formula 1 obtained, for example, as described in Examples 3A, 4A, 5B, 6C, 7B and 15G, mice treated with compounds from Examples 3A, 4A, 5B, 6C, 7B and 15G showed similar OVA-specific antibody titers to mice treated with saline.

12C. By following the procedure described in Example 12B and replacing F1aA-OVA- _m4 - _n8 with compounds of Formula 1 obtained, for example, in Examples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F, and replacing OVA with an antigen corresponding to X (or X' or X”), mice treated with compounds from Examples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F had antigen X-specific antibody titers similar to those of mice treated with saline.

12D. By following the procedures described in Examples 12A-C and replacing the galactosylating compounds with the corresponding glucosylating compounds, it was confirmed that the glucosylating compounds had antigen X-specific antibody titers similar to those of mice treated with saline.

Example 13

F1aA-OVA-m ₄ -n ₈ reduces OVA-specific antibodies

13A . Mice with different blood titers of OVA antibodies (0 to 4.5 per mouse) were treated intravenously with 20 μg of F1aA-OVA- _m4 - _n8 dissolved in 100 μl saline. Mice were intravenously injected with F1aA-OVA- _m4 - _n8 on days 0, 5, 7, 12, and 14 (F1aA-OVA- _m4 - _n8 injections were labeled “Gal-OVA” and shown as green arrows on the x-axis of Figure 5). To determine the ability of F1aA-OVA- _m4 - _n8 to reduce serum OVA-specific antibodies, mice were exsanguinated on day -1 to establish an initial antibody titer, followed by subsequent exsanguinations after each injection of F1aA-OVA- _m4 - _n8 on days 2, 6, 9, 13, and 16. Antibody titers per mouse were determined by ELISA. Results from this study demonstrate that F1aA-OVA- _m4 - _n8 can reduce serum antibody levels in mice. For example, one day after the first F1aA-OVA- _m4 - _n8 injection (i.e., day 2), mice with positive OVA antibody titers experienced a 5- to 100-fold decrease in serum antibody levels (Figure 5). These results show that although antibody titers did increase in some mice during the 19-day experiment, titer levels never reached the initial antibody titers measured on day -1, and subsequent administration of F1aA-OVA- _m4 - _n8 effectively reduced these transient increases in antibody titers. These results demonstrate that F1aA-OVA- _m4 - _n8 possesses the specificity for binding to serum OVA-specific antibodies and the kinetics required to reduce OVA-specific serum antibodies.

13B. By following the procedure described in Example 13A and replacing F1aA-OVA- _m4 - _n8 with compounds of Formula 1 obtained, for example, in Examples 3A, 4A, 5B, 6C, 7B and 15G, it is shown that the compounds from Examples 3A, 4A, 5B, 6C, 7B and 15G have the specificity to bind to serum OVA-specific antibodies and the kinetics required to reduce OVA-specific serum antibodies.

13C. By following the procedure described in Example 13A and replacing F1aA-OVA-m _{4 -n 8} with compounds of Formula 1 obtained, for example, in Examples 1E, 1G, 2C, 10D, 15I, 15L, 16B, 16D and 16F, and replacing OVA with an antigen corresponding to X (or X' or X”), it is shown that the compounds from Examples 1E, 1G, 2C, 15I, 15L, 16B _, 16D and 16F have the specificity to bind serum X-specific antibodies and the kinetics required to reduce antigen X-specific serum antibodies.

13D. By following the procedures described in Examples 13A-C and replacing the galactosylating compounds with the corresponding glucosylating compounds, it was confirmed that the glucosylating compounds possess the specificity for binding serum antigen X-specific antibodies and the kinetics required for reducing antigen X-specific serum antibodies.

Example 14

OT-1 attack and tolerance model

14A. Using an established OTI challenge-and-tolerance model (Liu, Iyoda, et al., 2002), the ability of F1aA-OVA- _m4 - _n8 (mGal-OVA) and F1b-OVA- _m1 - _n4 - _p34 (pGal-OVA) to prevent subsequent immune responses to vaccine-mediated antigen challenges—even to challenges involving very strong bacterial-derived adjuvants (i.e., lipopolysaccharides) was demonstrated. For tolerance, 100 μl of 233 nmol of F1aA ^- OVA ^- _m4 - _n8 , F1b-OVA- _m1 - _n4 - _p34 , or soluble OVA in saline was administered intravenously on days 1 and 6 following adoptive transfer of OTI CD8+ (CD45.2+) T cells to CD45.1 ⁺ mice (n = 5 mice per group). Nine days later, after potential deletion of transmissible T cells, recipient mice were challenged by intradermal injection of OVA (10 μg) with lipopolysaccharide (LPS) (50 ng) as an adjuvant. Draining lymph nodes were characterized four days post-challenge to determine whether actual deletion had occurred.

14B . Intravenous administration of F1aA-OVA- _m4 - _n8 and F1b-OVA- _m1 - _n4 - _p34 resulted in a dramatic reduction in the OTI CD8 ⁺ T cell population in draining lymph nodes compared to mice treated with unmodified OVA prior to antigen challenge with LPS, demonstrating deletion tolerance. For example, Figures 6A-6F show that draining lymph nodes from mice treated with F1aA-OVA- _m4 - _n8 (mGal-OVA) and F1b-OVA- _m1 - _n4 - _p34 (pGal-OVA) contained more than 9-fold fewer OTI CD8 ⁺ T cells compared to OVA-treated mice, and more than 43-fold fewer than challenged control mice that did not receive intravenous antigen; responses in spleen cells were similar. These results demonstrate that F1aA-OVA- _m4 - _n8 and F1b-OVA- _m1 - _n4 - _p34 alleviate the OVA-specific immune response following adjuvanted OVA challenge, thus confirming that the compositions disclosed in this invention are suitable for inducing immune tolerance. Regarding characterization, Figure 7 shows the characterization of F1aA-OVA- _m4 - _n80 and F1b-OVA- _m1 - _n44 - _p34 .

14C. By following the procedures described in Examples 14A and 14B, and replacing F1aA-OVA- _m4 - _n8 and F1b-OVA- _m1 - _n4 - _p34 with compounds of Formula 1 obtained, for example, as described in Examples 3A, 4A, 5B, 6C, 7B, and 15G, the compounds from Examples 3A, 4A, 5B, 6C, 7B, and 15G were shown to reduce the OVA-specific immune response following OVA challenge with adjuvant.

14D. By following the procedures described in Examples 14A and 14B, and by replacing F1aA-OVA-m4-n8 and F1b-OVA- _m1 - _n4 - _p34 with compounds of Formula 1 obtained, for example, as described in Examples 1E, 1G, 2C, _15I , 15L, 16B, _16D and 16F, and by replacing OVA with an antigen corresponding to X (or X' or X”), the compounds from Examples 1E, 1G, 2C, 15I, 15L, 16B, 16D and 16F showed that the compounds reduced the antigen X-specific immune response after challenge with adjuvanted antigen X.

14E. By following the procedures described in Examples 14A-D and replacing the galactosylating compounds with the corresponding glucosylating compounds, it was confirmed that the glucosylating compounds reduced the antigen X-specific immune response after antigen X challenge with adjuvant.

Example 15

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

15A. Formula 1102, where ^R3 is NHAc and ^R4 is OH.

N-acetyl-D-galactosamine (Formula 1101, where ^R3 is NHAC and ^R4 is OH) (5 g, 22.6 mmol) was added to a stirred solution (200 mL) of chloroethanol at room temperature. The solution was cooled to 4 °C, and acetyl chloride was added dropwise. The solution was warmed to 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 for 2 hours in the presence of carbon. The solution was filtered, and the solvent was removed under reduced pressure. The product N-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide of the corresponding Formula 1102 was used without further purification.

15B. Formula 1103, where ^R3 is NHAc and ^R4 is OH.

The N-(2-(2-chloroethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (2 g, 7.4 mmol) prepared in Example 15A was added to a stirred solution of DMF (100 mL) and sodium azide (4 g, 61.5 mmol). The solution was heated to 90 °C for 12 hours and then filtered. The residual solvent was removed under reduced pressure, and the crude product was purified by rapid chromatography (10% MeOH in dichloromethane) to give the product N-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide of the corresponding formula 1103.

15C. Formula 1104, where ^R3 is NHAc and ^R4 is OH.

The N-(2-(2-(2-azidoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (2 g, 6.9 mmol) prepared in Example 15B was added to a solution of palladium on carbon and ethanol (50 mL). The solution was stirred under hydrogen (3 atm) for 4 hours. The resulting solution was filtered and residual solvent was removed under reduced pressure to provide the 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.

15D. Equation 1105, where ^R3 is NHAc and ^R4 is OH.

The N-(2-(2-aminoethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (1.0 g, 3.78 mmol) prepared in Example 15C 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 mixture was stirred at room temperature for 2 hours. After 2 hours, excess solvent was removed under reduced pressure, and the product of formula 1105, N-(2-((3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)methacrylamide, was separated by rapid chromatography.

15E. Equation 1107, where p is 30, q is 4, ^R3 is NHAC, ^R4 is OH, and ^R8 is CMP.

Azide-modified uRAFT agent of formula 1106 (where q is 4) (28 mg) was added to 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 DMF. The reaction mixture was subjected to four freeze-thaw pump cycles and then stirred at 70 °C. After 12 hours, the polymer product of formula 1107 was precipitated from the reaction mixture by adding methanol, where p is 30 and q is 4. The solvent was discarded from the solids and the solids were collected, and the residual solvent was removed by vacuum.

15F. Equation 1109, where X’ is OVA, m is 2, and n is 80.

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

15G. Equation 1m, where X' is OVA, m is 2, n is 80, p is 30, q is 4, ^R3 is NHAC, and ^R8 is CMP.

The solution prepared in Example 15F was added to 100 μl of sodium phosphate buffer (pH 8.0), which contained 10 mg of the product of formula 1107 prepared in Example 15E. The reaction mixture was stirred for 2 hours, and then excess formula 1107 was removed by size exclusion chromatography to provide the corresponding isomer of formula 1m in solution, which could be used for biological research without further purification. The R ³ substituent in the title compound name is shown as 2NHAc.

Other compounds of formula 1109 (15H)

By following the scheme described in Example 15F and replacing the OVA with the following:

●Abciximab,

●Adalimumab,

●Agarase α,

● Agargase β,

●Adefolate,

●Aglucosidase α,

●Factor VIII,

●Factor IX,

●L-Asparaginase,

● Laronyl enzyme,

●Octreotide,

●Phenylanine aminolysin

●Raburiase,

●Insulin (SEQ ID NO:1),

●GAD-65 (SEQ ID NO:2),

●IGRP (SEQ ID NO:3)

●MBP (SEQ ID NO:4),

●MOG (SEQ ID NO:5),

●PLP (SEQ ID NO:6),

●MBP13-32 (SEQ ID NO:7),

●MBP83-99 (SEQ ID NO:8),

●MBP111-129 (SEQ ID NO:9),

●MBP146-170(SEQ ID NO:10),

●MOG1-20 (SEQ ID NO:11),

●MOG35-55 (SEQ ID NO:12),

●PLP139-154(SEQ ID NO:13),

●MART1 (SEQ ID NO:14),

●Tyrosinase (SEQ ID NO:15),

●PMEL (SEQ ID NO:16),

●Aquaporin-4 (SEQ ID NO:17),

●S-repressor protein (SEQ ID NO:18),

●IRBP (SEQ ID NO:19),

●Contains peanut globulin (UNIPROT Q6PSU6),

●Natural α-gliadin "33-mer" (SEQ ID NO:20),

●Deacylated α-gliadin "33-mer" (SEQ ID NO:21),

●α-Glucolylein (SEQ ID NO:22),

●Ω-Glucolyl (SEQ ID NO:23),

●Fel d 1A (UNIPROT P30438),

● Feline albumin (UNIPROT P49064),

●Can f 1(UNIPROT O18873),

●Dog albumin (UNIPROT P49822), and

●RhCE (UNIPROT P18577),

The following corresponding compounds of formula 1109 (where n is 80) are obtained:

●X is abciximab, and m is 10.

●X is adalimumab, and m is 11.

●X is agalsidase α, and m is 14.

●X is agalsidase β, and m is 14.

●X is interleukin, and m is 6.

●X is α-glucosidase, and m is 13.

●X is factor VIII, and m is 100.

●X is a factor of 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 aminolysin, and m is 12.

●X is raburicase, and m is 12.

●X is insulin (SEQ ID NO:1), and m is 2,

●X is GAD-65 (SEQ ID NO:2), and m is 8.

●X is IGRP (SEQ ID NO:3), and m is 7.

●X is a MBP (SEQ ID NO:4), and m is 6.

●X is a MOG (SEQ ID NO:5), and m is 5.

●X is PLP (SEQ ID NO: 6), and m is 8.

●X is MBP13-32 (SEQ ID NO:7), and m is 1,

●X is MBP83-99 (SEQ ID NO:8), and m is 1.

●X is MBP111-129 (SEQ ID NO:9), and m is 1,

●X is MBP146-170 (SEQ ID NO:10), and m is 2.

●X is MOG1-20 (SEQ ID NO:11), and m is 1,

●X is MOG35-55 (SEQ ID NO:12), and m is 2.

●X is PLP139-154 (SEQ ID NO:13), and m is 3.

●X is MART1 (SEQ ID NO:14), and m is 4.

●X is tyrosinase (SEQ ID NO:15), and m is 8.

●X is PMEL (SEQ ID NO:16), and m is 5.

●X is aquaporin 4 (SEQ ID NO: 17), and m is 4.

●X is an S-repressor protein (SEQ ID NO: 18), and m is 12.

●X is an IRBP (SEQ ID NO: 19), and m is 21.

●X is conaragenin, and m is 21.

●X is the natural α-gliadin "33-mer" (SEQ ID NO:20), and m is 1,

●X is a deacylated α-gliadin "33-mer" (SEQ ID NO:21), and m is 1,

●X is α-gliadin (SEQ ID NO:22), and m is 1,

●X is Ω-gliadin (SEQ ID NO:23), 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 RhCE and m is 10.

15I. Other compounds of formula 1m

By following the procedure described in Example 15G and replacing the compound of formula 1109, for example, as obtained in Example 15H, 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-Agarasase α-m ₁₄ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

●F1m-Agarasase β-m ₁₄ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

●F1m-Adedileukin- _m6 - _n80 - _p30 - _q4 -CMP-2NHAc,

●F1m-glucosidase α-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-Laronylase- _{m 7} -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

●F1m-Octreotide-m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

●F1m-phenylalanine aminolyase-m ₁₂ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

●F1m-Raburicase-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 -p ₃₀ -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-inhibitor protein-m ₁₂ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

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

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

●F1m-Natural α-Gliadin "33-mer"-m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

●F1m-Deacylated α-gliadin "33-mer" -m ₁ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc,

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

●F1m-Ω 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-Cat 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-dog albumin-m ₂₃ -n ₈₀ -p ₃₀ -q ₄ -CMP-2NHAc, and

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

15J. Equation 1107, where p is 30, q is 8, ^R3 is OH, ^R4 is OH and ^R8 is CMP.

By following the procedure described in Example 15A, and replacing N-acetyl-D-galactosamine with galactose, and following the procedure described in Example 15E (except for using an azide-modified uRAFT agent of Formula 1106 (where q is 8), a compound of Formula 1107 was obtained, wherein p is 30, q is 8, ^R3 is OH, ^R4 is OH and ^R8 is CMP.

15K. Equation 1109, where n is 62, and where X’ and m are as in Example 19H.

By following the procedure described in Example 15F, replacing the OVA with the compound described in Example 15H and utilizing the compound of Formula 1108 (where n is 62), the corresponding compound of Formula 1109 is obtained, where n is 62.

15L. Other compounds of formula 1m

By following the procedure described in Example 15G and replacing the compound of formula 1107 with the compound obtained in Example 15J and replacing the compound of formula 1109 with the compound obtained in Example 15K, the following corresponding compounds of formula 1m were 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-Agarasase α-m ₁₄ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

●F1m-Agarosidase β-m ₁₄ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

●F1m-Adedileukin- _m6 - _n62 - _p30 - _q8 -CMP-2OH,

●F1m-glucosidase α-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-Laronylase- _{m 7} -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

●F1m-octreotide-m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

●F1m-phenylalanine aminolysin-m ₁₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

●F1m-Raburicase-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-inhibitory protein-m ₁₂ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

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

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

●F1m-Natural α-Gliadin "33-mer" -m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

●F1m-Deacylated α-gliadin "33-mer" -m ₁ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH,

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

●F1m-Ω-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-Cat 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-dog albumin-m ₂₃ -n ₆₂ -p ₃₀ -q ₈ -CMP-2OH, and

●F1m-RhCE-m ₁₀ -n ₆₂ -p ₃₀ - _{q 8} -CMP-2OH.

Other compounds of formula 1m (15M)

By following the procedures described in Examples 15A-L and replacing galactosamine or galactose with glucosamine or glucose respectively, the corresponding glucosylated compounds of Formula 1m were obtained.

Example 16

F1n-insulin- _m2 - _n1 - _p30 - _q4 -CMP-2NHAc

16A. Equation 1202, where X’ is insulin, m is 2 and n is 1.

Recombinant human insulin (5 mg) was added to 100 μL of DMF containing 10 μL of triethylamine and stirred until the insulin dissolved. 10 mg (0.0161 mmol) of the linker precursor of formula 1201 was added to the solution, where n is 1, and the reaction mixture was stirred. After 1 hour, 1.3 mL of tert-butyl methyl ether was added to separate the corresponding product of formula 1202, which was recovered as a precipitate. Residual DMF and tert-butyl methyl ether were removed under reduced pressure. The identity of the product was confirmed by characterization by liquid chromatography, mass spectrometry, and polyacrylamide gel electrophoresis. The modified insulin product of formula 1202 was used without further purification.

16B. Equation 1n, where X' is insulin, m is 2, n is 1, p is 30, q is 4, and ^R8 is CMP.

The product of formula 1202 obtained in Example 16A was resuspended in 100 μl of DMF. The polymer product of formula 1107 obtained in Example 15E (10 mg) was added, and the reaction mixture was stirred for 1 hour. After 1 hour, the product was precipitated by adding dichloromethane (1.3 ml). The product was filtered, and residual solvent was removed under reduced pressure. The crude product was then resuspended in 500 μl of PBS, and low molecular weight components were removed by size exclusion chromatography to provide the corresponding isomer of formula 1n. The identity of the product was confirmed by characterization by liquid chromatography, mass spectrometry, and polyacrylamide gel electrophoresis. The modified insulin product of formula 1202 was used without further purification.

16C. Other compounds of formula 1202

By following the procedure described in Example 16A and replacing insulin with the following:

●Abciximab,

●Adalimumab,

●Agarase α,

● Agargase β,

●Adefolate,

●Aglucosidase α,

●Factor VIII,

●Factor IX,

●L-Asparaginase,

● Laronyl enzyme,

●Octreotide,

●Phenylanine aminolysin

●Raburiase,

●GAD-65 (SEQ ID NO:2),

●IGRP (SEQ ID NO:3)

●MBP (SEQ ID NO:4),

●MOG (SEQ ID NO:5),

●PLP (SEQ ID NO:6),

●MBP13-32 (SEQ ID NO:7),

●MBP83-99 (SEQ ID NO:8),

●MBP111-129 (SEQ ID NO:9),

●MBP146-170(SEQ ID NO:10),

●MOG1-20 (SEQ ID NO:11),

●MOG35-55 (SEQ ID NO:12),

●PLP139-154(SEQ ID NO:13),

●MART1 (SEQ ID NO:14),

●Tyrosinase (SEQ ID NO:15),

●PMEL (SEQ ID NO:16),

●Aquaporin-4 (SEQ ID NO:17),

●S-repressor protein (SEQ ID NO:18),

●IRBP (SEQ ID NO:19),

●Contains peanut globulin (UNIPROT Q6PSU6),

●Natural α-gliadin "33-mer" (SEQ ID NO:20),

●Deacylated α-gliadin "33-mer" (SEQ ID NO:21),

●α-Glucolylein (SEQ ID NO:22),

●Ω gliadin (SEQ ID NO:23),

●Fel d 1A (UNIPROT P30438),

● Feline albumin (UNIPROT P49064),

●Can f 1(UNIPROT O18873),

●Dog albumin (UNIPROT P49822), and

●RhCE (UNIPROT P18577),

The following corresponding compounds of formula 1202 (where n is 1) are obtained:

●X is abciximab, and m is 10.

●X is adalimumab, and m is 11.

●X is agalsidase α, and m is 14.

●X is agalsidase β, and m is 14.

●X is interleukin, and m is 6.

●X is α-glucosidase, and m is 13.

●X is factor VIII, and m is 100.

●X is a factor of 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 aminolysin, and m is 12.

●X is raburicase, and m is 12.

●X is GAD-65 (SEQ ID NO:2), and m is 8.

●X is IGRP (SEQ ID NO:3), and m is 7.

●X is a MBP (SEQ ID NO:4), and m is 6.

●X is a MOG (SEQ ID NO:5), and m is 5.

●X is PLP (SEQ ID NO: 6), and m is 8.

●X is MBP13-32 (SEQ ID NO:7), and m is 1,

●X is MBP83-99 (SEQ ID NO:8), and m is 1.

●X is MBP111-129 (SEQ ID NO:9), and m is 1,

●X is MBP146-170 (SEQ ID NO:10), and m is 2.

●X is MOG1-20 (SEQ ID NO:11), and m is 1,

●X is MOG35-55 (SEQ ID NO:12), and m is 2.

●X is PLP139-154 (SEQ ID NO:13), and m is 3.

●X is MART1 (SEQ ID NO:14), and m is 4.

●X is tyrosinase (SEQ ID NO:15), 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 an S-repressor protein (SEQ ID NO:22), and m is 12.

●X is an IRBP (SEQ ID NO: 19), and m is 21.

●X is conaragenin, and m is 21.

●X is the natural α-gliadin "33-mer" (SEQ ID NO:20), and m is 1,

●X is a deacylated α-gliadin "33-mer" (SEQ ID NO:21), and m is 1,

●X is α-gliadin (SEQ ID NO:22), and m is 1,

●X is Ω-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 RhCE and m is 10.

16D. Other compounds of formula 1n

By following the procedure described in Example 16B and replacing, for example, the compound of formula 1202 obtained as in Example 16C, 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-Agarasase α- _m14 - _n1 - _p30 - _q4- CMP-2NHAc,

●F1n-Agarasase β- _m14 - _n1 - _p30 - _q4- CMP-2NHAc,

●F1n-Adedileukin- _m6 - _n1 - _p30 - _q4- CMP-2NHAc,

●F1n-glucosidase α- _m13 - _n1 - _p30 - _q4 -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- _m5 - _n1 - _p30 - _q4 -CMP-2NHAc,

●F1n-Laronylase- _m7 - _n1 - _p30 - _q4 -CMP-2NHAc,

●F1n-Octreotide- _m1 - _n1 - _p30 - _q4 -CMP-2NHAc,

●F1n-phenylalanine aminolysin- _m12 - _n1 - _p30 - _q4 -CMP-2NHAc,

●F1n-Laburicase- _m12 - _n1 - _p30 - _q4 -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- _m8 - _n1 - _p30 - _q4 -CMP-2NHAc,

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

●F1n-aquaporin-4- _m4 - _n1 - _p30 - _q4 -CMP-2NHAc,

●F1n-S-inhibitory protein-m ₁₂ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

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

●F1n-conaragenin- _m21 - _n1 - _p30 - _q4 -CMP-2NHAc,

●F1n - Natural α-Gliadin "33-mer" - m ₁ - n ₁ - p ₃₀ - _{q 4} - CMP-2NHAc,

●F1n-Deacylated α-gliadin "33-mer" - _{m 1} - n ₁ - _{p 30} - q _{4 -} CMP-2NHAc,

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

●F1n-Ω-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-Cat albumin-m ₁₆ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc,

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

●F1n-dog albumin- _m23 - _n1 - _p30 - _q4 -CMP-2NHAc, and

●F1n-RhCE-m ₁₀ -n ₁ -p ₃₀ -q ₄ -CMP-2NHAc.

16E. Equation 1202, where n is 33, and where X’ and m are as described in Example 20C.

By following the procedure described in Example 16F and replacing insulin with the compound described in Example 16C and using the compound of Formula 1201 (where n is 33), the corresponding compound of Formula 1202 is obtained, where n is 33.

16F. Other compounds of formula 1n

By following the procedure described in Example 16B, and by replacing the compound of formula 1107 with the compound obtained in Example 15J and replacing the compound of formula 1202 with the compound obtained in Example 16E, 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-Agarosidase α-m ₁₄ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-Agarosidase β-m ₁₄ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-Adedileukin- _m6 - _n33 - _p30 - _q8 -CMP-2OH,

●F1n-glucosidase α-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-Laronylase- _m7 - _n33 - _p30 - _q8 -CMP-2OH,

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

●F1n-phenylalanine aminolysin-m ₁₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-Laburicase-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 -p ₃₀ -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 -m4- _n33 - _p30 - _q8 -CMP-2OH,

●F1n-S-inhibitor protein-m ₁₂ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-IRBP-m ₂₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-conaragenin-m ₂₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n - Natural α-Gliadin "33-mer" - m ₁ - n ₃₃ - p ₃₀ - _{q 8} - CMP-2OH,

●F1n-Deacylated α-gliadin "33-mer" -m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-α-gliadin-m ₁ -n ₃₃ -p ₃₀ -q ₈ -CMP-2OH,

●F1n-Ω-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-Cat 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-dog albumin- _m23 - _n33 - _p30 - _q8 -CMP-2OH, and

●F1n-RhCE-m ₁₀ -n ₃₃ -p ₃₀ - _{q 8} -CMP-2OH.

Other compounds of formula 1n in 16G

By following the procedures described in Examples 16A-F and replacing the galactosylation portion with the glucosylation portion, the corresponding glucosylated compounds of Formula 1n were obtained.

Example 17

Equation 1507, where p is 90, t is 1, ^R3 is NHAc and ^R4 is OH

17A. Equation 1502, where t is 1, ^R3 is NHAc and ^R4 is OH.

N-acetyl-D-glucosamine (Formula 1101, where ^R3 is NHAc and ^R4 is OH) (5.0 g, 22.6 mmol) was added to a stirred solution of 2-(2-chloroethoxy)ethanol (50 mL) at room temperature. The solution was cooled to 4 °C, and acetyl chloride was added dropwise. The solution was brought to room temperature and then heated to 70 °C. After 4 hours, the reaction mixture was added to 200 mL of ethyl acetate. The precipitate formed was collected, 100 mL of ethanol was added, and the mixture was stirred for 2 hours in the presence of carbon. The solution was filtered, and the solvent was removed under reduced pressure. The corresponding product of Formula 1502, N-acetyl-D-glucosamine-2-(chloroethoxy)ethanol, was used without further purification.

17B. Equation 1503, where t is 1, ^R3 is NHAc, and ^R4 is OH.

N-acetyl-D-glucosamine-2-(chloroethoxy)ethanol (2.0 g, 6.11 mmol) was added to a stirred solution of DMF (100 mL) and sodium azide (4.0 g, 61.5 mmol). The solution was heated at 90 °C for 12 hours and then filtered. Residual solvent was removed under pressure, and the crude product was purified by rapid chromatography (10% MeOH in dichloromethane) to give the corresponding product N-acetyl-D-glucosamine-2-(azidoethoxy)ethanol of formula 1503.

17C. Equation 1504, where t is 1, ^R3 is NHAc, and ^R4 is OH.

N-acetyl-D-glucosamine-2-(azidoethoxy)ethanol (2.0 g, 5.9 mmol) was added to a solution of palladium on carbon and ethanol (50 mL). The solution was stirred under hydrogen (3 atm) for 4 hours. The resulting solution was filtered, and residual solvent was removed under pressure to provide the corresponding product N-acetyl-D-glucosamine-2-(aminoethoxy)ethanol of formula 1504.

17D. Equation 1505, where t is 1, ^R3 is NHAc, and ^R4 is OH.

N-acetyl-D-glucosamine-2-(aminoethoxy)ethanol (1.0 g, 3.25 mmol) was added to a solution of methacrylic anhydride (0.583 g, 3.78 mmol) in DMF (50 mL). Triethylamine was then added to the solution, and the reaction mixture was stirred at room temperature for 2 hours. After 2 hours, excess solvent was removed under reduced pressure, and the corresponding product of formula 1505 ((2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-2-(2-(2-methacrylamidoethoxy)ethoxy)tetrahydro-2H-pyran-3-yl)carbamic acid was separated by rapid chromatography.

17E. Formula 1507, where p is 90, q is 4, t is 1, ^R3 is NHAc, ^R4 is OH, ^R8 is CMP, and ^R10 is 2-hydroxyl. propyl

A 25 mL Schlenk flask was filled with the compound of formula 1505, the product of Example 17D (272 mg, 0.72 mmol), N-(2-hydroxypropyl)methacrylamide (“HPMA”, used as received from the manufacturer) (211 mg, 1.47 mmol), the azide-modified uRAFT agent of formula 1106 (where q is 4 and R ⁸ is CMP) (10.41 mg, 0.0217 mmol), azobis(isobutyronitrile) (0.98 mg, 0.005 mmol), and 1.2 mL of dimethylformamide. The reaction mixture was subjected to four freeze-pump-thaw degassing cycles, followed by stirring at 70 °C for 20 hours. The corresponding random polymerization product of formula 1507 was recovered by precipitation of the reaction mixture in acetone. Excess acetone was removed under reduced pressure to provide the random polymerization product, which was used without further purification.

17F. Formula 1507, where p is 90, q is 4, t is 1, ^R3 is NHAc, ^R4 is OH, ^R8 is CMP, and ^R10 is 2-hydroxyl. Propyl, using N-acetyl-D-galactosamine

By following the procedures of Examples 17A to 17E and replacing the N-acetyl-D-glucosamine in the procedure of Example 17A with N-acetyl-D-galactosamine, the corresponding galactoside compound of Formula 1507 was obtained.

Compounds of formula 1507, where t is not 1.

By following the procedures of Examples 17A to 17E and substituting the following for 2-(2-chloroethoxy)ethanol-1-ol:

●2-(2-(2-chloroethoxy)ethoxy)ethyl-1-ol, providing the corresponding compound of formula 1507, where t is 2,

●2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethyl-1-ol, providing the corresponding compound of formula 1507, wherein t is 3,

●2-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethoxy)ethyl-1-ol, providing the corresponding compound of formula 1507, wherein t is 4,

●2-(2-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethyl-1-ol, providing the corresponding compound of formula 1507, wherein t is 5, and

●2-(2-(2-(2-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)eth-1-ol, providing the corresponding compound of formula 1507, wherein t is 6.

17H. Compounds of formula 1507 having multiple W ¹ groups, wherein t varies

By following the procedure of Example 17E and replacing the compound of formula 1505 (where t is 1) with each of 0.36 mmol of formula 1505 (where t is 2 and 4) (e.g., by following the procedure of Examples 17A to 17D as described in Example 17F), a corresponding random copolymer of formula 1507 is obtained, having about 15 W ¹ groups when t is 2, 15 W ¹ groups when t is 4, and 60 W ² groups.

17I. Compounds of formula 1507 having a mixture of glucosyl and galactosyl moieties.

By following the procedure of Example 17E and replacing the compound of Formula 1505 with 0.36 mmol each of glucosyl and galactosyl of Formula 1505 (e.g., by following the procedure of Examples 17A to 17D as described in Examples 17D and 17F), a corresponding random copolymer of Formula 1507 was obtained, having about 15 glucosyl ^W1 groups, 15 galactosyl ^W1 groups and 60 ^W2 groups.

Example 17.1

Equation 1507, where t is 1, ^R3 is NHAc, and ^R4 is OH.

17.1A. Formula 1502, where t is 1, ^R3 is NHAc, and ^R4 is OH: 2-(2-(2-chloroethoxy)ethoxy)-α- NAc-galactosamine (1502.1A).

Acetyl chloride (4.35 mL, 61.05 mmol) was added dropwise to an ice-cold solution of NHAc-protected D-galactosamine (10.0 g) in 2-(2'-chloroethoxy)ethanol (40 mL). The mixture was stirred at 4 °C for 15 minutes and then transferred to an oil bath at 70 °C. The reaction was kept mixed under a cooling condenser for 4 hours. After this time, the dark brown solution was cooled and poured into 400 mL of ethyl acetate and dichloromethane (3:1, v/v) to remove excess unreacted chloroethanol. The mixture was placed in a freezer for 30 minutes and then the dark brown viscous precipitate was poured off. The precipitate was dissolved in anhydrous ethanol and activated charcoal was added. The suspension was mixed for 1.5 hours and then filtered through diatomaceous earth and washed with ethanol. In the final step, ethanol was evaporated under vacuum to provide 12.8 g of product (1502.1A) (95.24% yield).

17.1B. Formula 1503, where t is 1, ^R3 is NHAc and ^R4 is OH; 2-(2-(2-azidoethoxy)ethoxy)- α-NAc-galactosamine (1503.1B).

Compound (1502.1A) (5.0 g) was dissolved in 20 mL of N,N-dimethylformamide. Sodium azide (26628-22-8) (5.0 g) was added to the solution. The suspension was placed in an oil bath and stirred overnight at 80 °C. After overnight incubation, the reaction mixture was filtered through diatomaceous earth. The solvent was then evaporated under high pressure to provide an oily brown substance. The final product was purified by rapid chromatography (82.2% yield).

17.1C. Formula 1504, where t is 1, ^R3 is NHAc, and ^R4 is OH; 2-(2-(2-aminoethoxy)ethoxy)- α-NAc-galactosamine (1504.1C).

A suspension of (1503.1B) (5.5 g) and 10% palladium on carbon (approximately 500 mg) in 20 mL of ethanol was hydrogenated in a Shlenk flask at an initial pressure of 2 bar of hydrogen gas. The reduction process was controlled by TLC. After 3 hours, the reduction was complete, and the suspension was filtered through diatomaceous earth (78% yield).

17.1D. Formula 1505, where t is 1, ^R3 is NHAc and ^R4 is OH; α-NAc-galactosamine-amine-methacrylic acid Ester (1505.1D)

The compound (1504.1C) (4.5 g) was dissolved in 10 mL of N,N-dimethylformamide. Triethylamine (3 mL) was added to the solution, and the mixture was cooled to 4 °C. Subsequently, pentafluorophenyl methacrylate (13642-97-2) (4.38 mL) was added dropwise under constant stirring. After 30 minutes, the ice bath was removed, and the reaction mixture was allowed to stir again at room temperature for 4 hours. The solvent was then evaporated, and the residue was adsorbed onto silica gel. The crude material was purified by rapid chromatography (dichloromethane:methanol 95:5, v/v) to provide 3.8 g of NAc-galactosamine monomer (α-NAc-galactosamine-amine-methacrylate (1505.1D)) (64.73% yield).

Tetraethylene glycol mono-p-toluenesulfonate (1651a).

Tetraethylene glycol triethylene glycol (1650a) (112-60-7) (2.5 g) and pyridine (1.0 g) were added to 50 mL of dichloromethane and stirred at 0 °C for 20 min. p-Toluenesulfonyl chloride (98-59-9) (2.37 g) in 15 mL of dichloromethane was then slowly added to the solution. The reaction mixture was then stirred at 0 °C for 2 h, followed by stirring at room temperature for 4 h. After this time, the solvent was evaporated, and the crude product was purified by rapid chromatography (ethyl acetate:hexane 6:4, v/v) to provide 1651a (44% yield).

S-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl] ester (1652a)

A solution of potassium thioacetate (10387-40-3) (10.1 g, 88 mmol) in 100 mL of DMF (1651a) (15.4 g) was added to a suspension of potassium thioacetate (10387-40-3) (10.1 g, 88 mmol) in 650 mL of DMF. The mixture was stirred at room temperature for 1 h, then at 90°C for 4 h. After filtration, the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 × 50 mL) and brine (2 × 50 mL). The aqueous washing solution was extracted again with ethyl acetate (2 × 50 mL), and the combined organic layer was dried with magnesium sulfate and evaporated under reduced pressure to give a yellow oily product 1652a (45% yield).

2-(2-(2-(2-(pyridin-2-yldithioalkyl)ethoxy)ethoxy)ethoxy)ethyl-1-ol (1653a)

Sodium methoxide (0.5 M in 1.40 mL of methanol) was added dropwise to a stirred solution of 1652a (70.9 mg) and 2,2-dithiodipyridine (2127-03-9) (77.4 mg, 0.351 mmol) in 3 mL of anhydrous methanol under an argon atmosphere. After 2 h, the reactants were concentrated to powder with silica, and the crude product was purified by rapid chromatography with silica (1:1 hexane:EtOAc) to provide 1653a as a clear, pale yellow liquid (26.3 mg, 44% yield).

uRAFT agent (1601a)

Compound 1653a (1 g) was added dropwise to a stirred solution of 4-cyano-4-(thiobenzoylthio)pentanoic acid (1.1 g) (201611-92-9), N,N'-dicyclohexylcarbodiimide (538-75-0) (0.5 g), and 4-dimethylaminopyridine (DMAP) (1122-58-3) (0.1 g) in DCM (15 ml). The reaction mixture was stirred at 0°C for 2 h, and then allowed to warm to room temperature. After 3 h, the reaction mixture was filtered through diatomaceous earth, and the solvent was removed by reduced pressure. The final product (1601a) (67% yield) was recovered by rapid chromatography.

pGal(17.1E)

The following conditions are used to provide 17.1E using an α-NAc-galactosamine-amine-methacrylate monomer (e.g., 1505.1D). In some embodiments, an α-NAc-glucosamine-amine-methacrylate monomer (e.g., 1505.2D) may be used instead to provide a glucosamine-based monomer. In some embodiments, a is an integer from about 0 to about 150, from about 1 to about 100, from about 1 to about 50, from about 1 to about 10, or from about 1 to about 5. In some embodiments, b is an integer from about 0 to about 150, from about 1 to about 100, from about 1 to about 50, from about 1 to about 10, or from about 1 to about 5.

Compounds 1601a, 1505.1D, azobisisobutyronitrile (78-67-1), and N-(2-hydroxypropyl)methacrylamide (21442-01-3) were added to DMF (1 ml). The reaction mixture was subjected to four freeze-pump-thaw degassing cycles, followed by stirring at 70°C for 20 h. The polymerization product was recovered from acetone via precipitation. Excess solvent was removed under reduced pressure (55% yield).

Example 17.2

Equation 1507, where t is 1, ^R3 is NHAc and ^R4 is OH

17.2A. Formula 1502, where t is 1, ^R3 is NHAc and ^R4 is OH; 2-(2-(2-chloroethoxy)ethoxy)-α- NAc-glucosamine (1502.2A).

Acetyl chloride (75-36-5) (4.35 mL, 61.05 mmol) was added dropwise to an ice-cold solution of D-glucosamine (7512-17-6) (10.0 g) in 2-(2'-chloroethoxy)ethanol (628-89-7) (40 mL). The mixture was stirred at 4 °C for 15 minutes and then transferred to an oil bath at 70 °C. The reactants were kept mixed under a cooling condenser for 4 hours. After this time, the dark brown solution was cooled and poured into 400 mL of ethyl acetate and dichloromethane (3:1, v/v) to remove excess unreacted chloroethanol. The mixture was placed in a freezer for 30 minutes and then the dark brown viscous precipitate was poured off. The precipitate was dissolved in anhydrous ethanol and activated charcoal was added. The suspension was mixed for 1.5 hours and then filtered through diatomaceous earth and washed with ethanol. In the final step, ethanol was evaporated under vacuum to provide 1502.2A (76% yield).

17.2B. Formula 1503, where t is 1, ^R3 is NHAc and ^R4 is OH; 2-(2-(2-chloroethoxy)ethoxy)-α- NAc-glucosamine (1503.2B).

Compound (1502.2A) (5.0 g) was dissolved in 20 mL of N,N-dimethylformamide. Sodium azide (26628-22-8) was added to the solution. The suspension was placed in an oil bath and stirred overnight at 80 °C. After overnight incubation, the reaction mixture was filtered through diatomaceous earth. The solvent was then evaporated under high pressure to provide an oily brown substance. The final product 1503.2B was purified by rapid chromatography (75.4% yield).

17.2C. Formula 1504, where t is 1, ^R3 is NHAc and ^R4 is OH; 2-(2-(2-aminoethoxy)ethoxy)-α- NAc-glucosamine (1504.2C).

A suspension of (1503.2B) (5.5 g) and 10% palladium on carbon (approximately 500 mg) in 20 mL of ethanol was hydrogenated in a Shlenk flask at an initial pressure of 2 bar of hydrogen. The reduction process was controlled by TLC. After 3 hours, the reaction was complete, and the suspension was filtered through diatomaceous earth to provide 1504.2C (65% yield).

17.2D. Formula 1505, where t is 1, ^R3 is NHAc and ^R4 is OH; α-NAc-glucosamine-amine-methacrylate (1505.2D).

Compound 1504.2C (4.5 g) was dissolved in 10 mL of N,N-dimethylformamide. Triethylamine (3 mL) was added to the solution, and the mixture was cooled to 4 °C. Subsequently, pentafluorophenyl methacrylate (13642-97-2) (4.38 mL) was added dropwise under constant stirring. After 30 minutes, the ice bath was removed, and the reaction mixture was allowed to stir again at room temperature for 4 hours. The solvent was then evaporated, and the residue was adsorbed onto silica gel. The crude material was purified by rapid chromatography (dichloromethane:methanol 95:5, v/v) to provide 3.8 g of NAc-glucosamine monomer 1505.2D (74% yield).

Example 18

Formula 1m', where X' is OVA, m is 1-3, n is 79, p is 90 (30W ¹ + 60W ² ), q is 4, t is 1, R ³ is NHAc, R ⁴ is OH, R ⁸ is CMP, and R ¹⁰ is 2-hydroxypropyl.

18A. Equation 1109, where X’ is OVA, m is 1-3, and n is 79.

A solution of Formula 101’ (where X’ is OVA) (10 mg endotoxin-free ovalbumin) in pH 7.6 PBS was added to a tube containing Formula 1108 (where n is 79) (10 mg). The reaction mixture was allowed to be stirred at room temperature. After 1 hour, any unconjugated Formula 1108 was removed by size exclusion chromatography to provide the corresponding product of Formula 1109, which was used without further purification.

18B. Equation 1m', where X' is OVA, m is 1-3, n is 79, p is 90, q is 4, t is 1, ^R3 is NHAc, ^R4 is OH, ^R8 It is CMP, and R ¹⁰ is 2-hydroxypropyl.

Then, the solution of Formula 1109 obtained in Example 18A was added to an endotoxin-free tube containing Formula 1507 (20 mg) as obtained in Example 17E, and stirred at room temperature to provide the corresponding product of Formula 1m'(“F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ ”), which was purified from the reaction mixture by rapid protein liquid chromatography (FPLC) using a Superdex 200 preparative grade column and used without further purification.

18C. Equation 1m', where X' is OVA, m is 1-3, n is 79, p is 90, q is 4, t is 1, ^R3 is NHAc, ^R4 is OH, ^R8 It is CMP, and R ¹⁰ is 2-hydroxypropyl, using N-acetyl-D-galactosamine.

By following the procedure of Example 18B and replacing the galactoside compound of Formula 1507 obtained as in Example 17F, the corresponding galactoside compound of Formula 1m'(“F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ ”) was obtained.

18D. Other compounds of formula 1m', where X' is OVA, m is 1-3, n is 79, p is 90, q is 4, t is 1, and ^R3 is... NHAc, ^R4 is OH, ^R8 is CMP, and ^R10 is 2-hydroxypropyl.

By following the procedures described in Examples 18A, 18B, and 18C and replacing the OVA with the following:

●Abciximab,

●Adalimumab,

●Agarase α,

● Agargase β,

●Adefolate,

●Aglucosidase α,

●Factor VIII,

●Factor IX,

●L-Asparaginase,

● Laronyl enzyme,

●Octreotide,

●Phenylanine aminolysin

●Rabrizolase,

●GAD-65 (SEQ ID NO:2),

●IGRP (SEQ ID NO:3)

●MBP (SEQ ID NO:4),

●MOG (SEQ ID NO:5),

●PLP (SEQ ID NO:6),

●MBP13-32 (SEQ ID NO:7),

●MBP83-99 (SEQ ID NO:8),

●MBP111-129 (SEQ ID NO:9),

●MBP146-170(SEQ ID NO:10),

●MOG1-20 (SEQ ID NO:11),

●MOG35-55 (SEQ ID NO:12),

●PLP139-154(SEQ ID NO:13),

●MART1 (SEQ ID NO:14),

●Tyrosinase (SEQ ID NO:15),

●PMEL (SEQ ID NO:16),

●Aquaporin-4 (SEQ ID NO:17),

●S-repressor protein (SEQ ID NO:18),

●IRBP (SEQ ID NO:19),

●Contains peanut globulin (UNIPROT Q6PSU6),

●Natural α-gliadin "33-mer" (SEQ ID NO:20),

●Deacylated α-gliadin "33-mer" (SEQ ID NO:21),

●α-Glucolylein (SEQ ID NO:22),

●Ω-Glucolyl (SEQ ID NO:23),

●Fel d 1A(UNIPROT P30438),

● Feline albumin (UNIPROT P49064),

●Can f 1(UNIPROT O18873),

●Dog albumin (UNIPROT P49822), and

●RhCE (UNIPROT P18577),

The following corresponding glucosyl and galactoside compounds of formula 1m’ were obtained:

●X is abciximab, and m is 10.

●X is adalimumab, and m is 11.

●X is agalsidase α, and m is 14.

●X is agalsidase β, and m is 14.

●X is interleukin, and m is 6.

●X is α-glucosidase, and m is 13.

●X is factor VIII, and m is 100.

●X is a factor of 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 aminolysin, and m is 12.

●X’ is raburicase, and m is 12.

●X’ is GAD-65 (SEQ ID NO:2), and m is 8.

●X’ is IGRP (SEQ ID NO:3), and m is 7.

●X’ is MBP (SEQ ID NO:4), and m is 6.

●X’ is MOG (SEQ ID NO:5), and m is 5.

●X’ is PLP (SEQ ID NO: 6), and m is 8.

●X’ is MBP13-32 (SEQ ID NO:7), and m is 1,

●X’ is MBP83-99 (SEQ ID NO:8), and m is 1,

●X’ is MBP111-129 (SEQ ID NO:9), and m is 1,

●X’ is MBP146-170 (SEQ ID NO:10), and m is 2,

●X’ is MOG1-20 (SEQ ID NO:11), and m is 1,

●X’ is MOG35-55 (SEQ ID NO:12), and m is 2,

●X’ is PLP139-154 (SEQ ID NO:13), and m is 3.

●X’ is MART1 (SEQ ID NO:14), and m is 4.

●X’ is tyrosinase (SEQ ID NO:15), and m is 8.

●X’ is PMEL (SEQ ID NO:16), and m is 5,

●X’ is aquaporin-4 (SEQ ID NO:17), and m is 4.

●X’ is an S-repressor protein (SEQ ID NO: 18), and m is 12.

●X’ is IRBP (SEQ ID NO: 19), and m is 21.

●X’ is conaradin, and m is 21.

●X’ is a natural α-gliadin “33-mer” (SEQ ID NO:20), and m is 1,

●X’ is a deacylated α-gliadin “33-mer” (SEQ ID NO:21), and m is 1,

●X’ is α-gliadin (SEQ ID NO:22), and m is 1,

●X’ is Ω-gliadin (SEQ ID NO:23), 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 RhCE and m is 10.

18E. Compounds of formulas 1h’, 1i’, 1j’, 1k’, 1L’ and 1n’

By following the procedures described in Examples 18B, 18C, and 18D and replacing Formula 1109 with the following:

● Formula 802 provides a corresponding random copolymer of Formula 1h’.

● Formula 902 provides a corresponding random copolymer of Formula 1i’.

● Formula 902, prepared using compounds of formula 103’, provides a corresponding random copolymer of formula 1j’.

● Formula 1002 provides a corresponding random copolymer of Formula 1k’.

● Formula 1002, prepared from compounds of formula 103’, provides a corresponding random copolymer of formula 1L’, and

● Formula 1202 provides a corresponding random copolymer of Formula 1n’.

18F. Other compounds of formula 1h’, 1i’, 1j’, 1k’, 1L’, 1m’ and 1n’

By following the procedures described in Examples 18B, 18C, 18D and 18E, and replacing Formula 1507 with compounds prepared as described in Examples 17G, 17H and 17I, corresponding compounds of Formulas 1h’, 1i’, 1j’, 1k’, 1L’, 1m’ and 1n’ are obtained, wherein t is not 1, the compound has a plurality of t groups, and has a mixture of glucosyl and galactosyl moieties.

Example 19

Formula 1c', where X” is insulin-B, m is 1, n is 4, p is 90 (30W ¹ + 60W ² ), t is 1, R ³ is NHAc, R ⁴ is OH, R ⁸ is CMP, and R ¹⁰ is 2-hydroxypropyl

19A. Formula 1602, where n is 4, p is 90, t is 1, ^R3 is NHAc, ^R4 is OH, ^R8 is CMP, and ^R10 is 2-hydroxyl. propyl

Add ((2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-2-(2-(2-methacrylamidoethoxy)ethoxy)tetrahydro-2H-pyran-3-yl)carbamic acid (272 mg, 0.72 mmol) (Formula 1505, for example, prepared as described in Example 17D), HPMA (211 mg, 1.47 mmol) (Formula 1506), a dithiopyridyl functionalized uRAFT agent of Formula 1601 (where n is 4 and R ⁸ is CMP) (12.5 mg, 0.0217 mmol), azobis(isobutyronitrile) (0.98 mg, 0.005 mmol), and 1.2 ml of dimethylformamide to a 25 ml Schlenk flask. The reaction mixture was subjected to four freeze-pump-thaw degassing cycles, and then stirred at 70 °C for 20 hours. The corresponding random polymerization product of formula 1602 (having about 30 ^W1 groups and about 60 ^W2 groups) was recovered by precipitation of the reaction mixture in acetone. Excess acetone was removed under reduced pressure to provide the random polymerization product, which was used without further purification.

19B. Formula 1c', where X” is insulin-B, m is 1, n is 4, p is 90(30W ¹ + 60W ² ), t is 1, R ³ is NHAc, R ⁴ R is OH, ^R8 is CMP, and ^R10 is 2-hydroxypropyl.

The Formula 1602 solution (20 mg) obtained in Example 19A was suspended in 200 μl of dimethylformamide, and an endotoxin-free tube containing insulin-B (1 mg) was added. The mixture was stirred at room temperature for 3 hours to provide the corresponding product of Formula 1c'(“F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ ”). The reaction mixture was then precipitated in acetone and purified from the reaction mixture by rapid protein liquid chromatography (FPLC) using a Superdex 200 preparative grade column, and used without further purification.

19C. Equation 1c', where X” is insulin-B, m is 1, n is 4, p is 90(30W ¹ + 60W ² ), t is 1, R ³ is NHAc, R ⁴ It is OH, ^R8 is CMP, and ^R10 is 2-hydroxypropyl, using N-acetyl-D-galactosamine.

By following the procedures of Examples 19A and 19B and replacing Formula 1505 with ((2S,3S,4S,5S,6S)-4,5-dihydroxy-6-(hydroxymethyl)-2-(2-(2-methacrylamidoethoxy)ethoxy)tetrahydro-2H-pyran-3-yl)carbamic acid, the corresponding galactoside compound of Formula 1c'(“F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ ”) was obtained.

19D. Equation 1c', where X” is P31, m is 1, n is 4, p is 90( ^30W1 + ^60W2 ), t is 1, ^R3 is NHAc, ^R4 is OH, ^R8 is CMP, and ^R10 is 2-hydroxypropyl.

By following the procedures of Examples 19B and 19C and replacing insulin-B with 20 mg P31, the corresponding glucosyl and galactoside compounds of Formula 1c’ were obtained, wherein X” is P31.

Compounds of formula 1f’ and 1g’ (19E)

By following the procedures of Examples 19A and 19B and replacing the uRAFT agent of Formula 1601 with the uRAFT agent of Formula 600’ or 700’, the corresponding compound of Formula 601’ or 701’ is obtained, and then the compound is contacted with the compound of Formula 101’ to provide the corresponding compound of Formula 1f’ or Formula 1g’, respectively.

Example 20

OT-1 attack and tolerance model

20A. As discussed above in Example 14, F1aA-OVA- _m4 - _n8 and F1b-OVA- _m1 - _n4 - _p34 reduce the OVA-specific immune response after adjuvanted OVA challenge.

20B. A total of ³ x 10⁵ CFSE-labeled OTI CD8+ T cells and ³ x 10⁵ CFSE-labeled OTI CD4+ T cells were injected into CD45.1+ recipient mice. On days 1 and 6 post-adoption, mice were intravenously administered saline solutions or saline alone containing OVA, F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ [“OVA-p(Gal-HPMA)”], F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ [“OVA-p(Glu-HPMA)”], or other saline solutions. Each mouse treated with a formulation containing free or conjugated OVA received a molar equivalent of 20 μg of OVA. On day 15 post-adoption, 5 μg of OVA was administered intradermally into the hind leg pad of each mouse in 25 μL of saline solution. Mice were challenged with OVA and 25 ng of ultrapure E. coli LPS (InvivoGen) (Hock method; total dose 10 μg OVA and 50 ng LPS). Mice were sacrificed 4 days post-challenge, and spleen and draining lymph node cells were isolated for restimulation. For flow cytometry analysis of intracellular cytokines, cells were restimulated for 3 h in the presence of 1 mg/mL OVA or 1 μg/mL SIINFEKL peptide (Genscript). Brefidobacterium-A (5 μg/mL; Sigma) was added, and restimulation was continued for another 3 h, followed by staining and flow cytometry analysis.

As shown in Figures 8A-8B, administration of OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) resulted in a significant reduction in the percentage of OT-I cells (in the total CD8+ T cell population) and OT-II cells (in the total CD4+ T cell population). Figure 8A shows that administration of OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) significantly reduced OT-I cells compared to mice receiving only OVA (e.g., unconjugated). The reduction was even greater when compared to mice receiving only OVA and LPS challenge (e.g., saline injection). Notably, treatment with OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) reduced OT-I cell levels to levels not significantly different from untreated mice. Similarly, as shown in Figure 8B, administration of OVA-p (Gal-HPMA) and OVA-p (Glu-HPMA) resulted in a significant reduction in OT-II cells compared to mice receiving unconjugated OVA or challenged alone. These data indicate a reduction in the generation of cells specifically designed to respond to OVA as an antigen, suggesting a decreased immune response to OVA.

Furthermore, administration of OVA-p (Gal-HPMA) and OVA-p (Glu-HPMA) resulted in a significant increase in antigen-specific regulatory T cells in the lymph nodes and spleen of mice. As shown in Figure 9A, treatment with any of these conjugates induced a significant increase in CD25+/FoxP3+ cells in the lymph nodes. Similarly, Figure 9B shows a significant increase in CD25+/FoxP3+ OT-II cells (relative to untreated, challenged (saline only), and OVA-treated animals). These data indicate an upregulation of regulatory T cell production, which in turn indicates that the immune system is negatively regulated (e.g., less responsive, or more tolerant) in its response to OVA.

The data shown in Figure 10 further demonstrate the increased tolerance to the antigen following delivery of the antigen complex with a liver-targeting portion. In this experiment, the percentage of cells expressing interferon-γ (IFNγ) was measured. CD4 and CD8 T cells generate IFNγ after antigen-specific immunization. As shown in Figure 10, mice pre-challenged with saline alone had approximately 60% of their total OTI cells expressing IFNγ. In comparison, mice treated with OVA had approximately 40% of their cells expressing IFNγ. Almost identical to untreated mice, mice treated with OVA-p (Gal-HPMA) and OVA-p (Glu-HPMA) had less than 20% of their OTI cells being IFNγ-positive. This significant reduction in IFNγ indicates a reduction in the mechanism driving antigen-specific immunity. Commonly and in view of the further disclosure of the invention, these data demonstrate that targeting the antigen to the liver can reduce the antigen-specific immune response to said antigen. In particular, targeting with glucose or galactose resulted in a significant shift in the cell population that induces antigen-specific immunity, demonstrating tolerance to the specific antigen.

20C. By following the procedure described in Example 20A or 20B and replacing the test OVA composition with other compositions of Formula 1, followed by challenge with unconjugated antigen X, the treated animals demonstrated tolerance to the specific antigen X.

Example 21

OTI/OTII attack and tolerance model

Using the model from Example 20, and additionally with OTII cells (which are CD4 ⁺ T cells from CD45.2 ⁺ mice, similar to CD8 ⁺ T cells OTI cells), the ability of F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ [“OVA-p(Gal-HPMA)”] and F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ [“OVA-p(Glu-HPMA)”] to induce T-regulated responses and prevent subsequent responses to vaccine-mediated antigen challenge was demonstrated, with different dosing regimens used. On day 0, 3 x ^10⁵ CFSE-labeled OTI cells and 3 x 10⁵ CFSE-labeled OTI cells were administered. ^Five CFSE-labeled OTII cells were adoptively transferred to CD45.1 ⁺ mice (n = 8 mice per group). Tolerogenic or control doses were administered on days 1, 4, and 7. In one regimen, OVA was administered at a dose of 2.5 μg on day 1, 2.5 μg on day 4, and 16 μg on day 7. In another regimen, OVA was administered at a dose of 7 μg on day 1, 7 μg on day 4, and 7 μg on day 7 to achieve the same total dose. Similarly, OVA was administered at a dose of 2.5 μg on day 1, 2.5 μg on day 4, and 16 μg on day 7. In other groups, pGal-OVA and pGlu-OVA were administered at the same doses: 7 μg on day 1, 7 μg on day 4, and 7 μg on day 7, all doses based on OVA equivalents. In the last group, saline was administered on the same day. On day 14, recipient mice were then challenged by intradermal injection of OVA (10 μg) adjuvanted with lipopolysaccharide (50 ng). Characterization of draining lymph nodes was completed on day 19 to allow determination of whether deletion actually occurred and whether regulatory T cells were induced from the adopted cells.

Significant tolerance was induced in the CD4+ T cell compartment, as shown in Figures 11A-11B. In terms of total cell frequency, both OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) administration regimens resulted in equivalently low levels of OTII cells after attack, statistically lower than OVA treatment (* and # indicate p < 0.05, ** and ## indicate p < 0.01), as shown in Figure 11A. When the remaining cells were analyzed by flow cytometry for the presence of transcription factor FoxP3 and receptor CD25, the number of FoxP3+CD25+ cells (markers of T regulatory cells) was statistically significantly increased compared to OVA treatment alone, as shown in Figure 11B. Here, for both OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) treatments, the number of T regulatory cells was significantly higher with the 2.5 μg/2.5 μg/16 μg administration regimen compared to the 7 μg/7 μg/7 μg administration regimen.

Significant tolerance was also induced in the CD8+ T cell compartment, as shown in Figures 12A-12B. In terms of total cell frequency, both OVA-p(Gal-HPMA) and OVA-p(Glu-HPMA) administration regimens resulted in equivalently low levels of OTI cells after attack, statistically lower than OVA treatment (* and # indicate p < 0.05, ** and ## indicate p < 0.01), as shown in Figure 12A. When the remaining cells were analyzed by flow cytometry for IFN-γ expression after re-exposure to the OVA antigen, the frequency of cells expressing this inflammatory cytokine was lower in the 2.5 μg/2.5 μg/16 μg administration regimens compared to the 7 μg/7 μg/7 μg administration regimen, as shown in Figure 12B.

Example 22

BDC2.5 Research

22A. CD4+ T cells from transgenic NOD-BDC2.5 mice express the diabetic BDC-2.5-specific regulatory T cell receptor (TCR). BDC2.5 T cells specifically target the pancreatic β-cell autoantigen chromogranin-A. T cells were isolated from the spleen of transgenic NOD-BDC2.5 mice and cultured for 4 days in DMEM supplemented with 10% (vol/vol) FBS, 0.05 mM β-mercaptoethanol, 1% puromycin/streptomycin, and 0.5 μM P31 peptide (a mimic epitope of pancreatic β-cell autoantigen chromogranin-A that stimulates T cells expressing the BDC2.5 T cell receptor). After stimulation with P31, cells were washed with basal DMEM, and purity was analyzed by flow cytometry. 5 × ^10⁶ T cells were intravenously injected into normoglycemic NOD/ShiLtJ mice. At 8 h and 3 days post-adoptive transfer, mice were intravenously administered saline, 10 μg of F1c'-P31-m ₁ -n ₄ -p _{90 -} CMP-poly-(EtPEG ₁ AcN-1NAcGAL _{30 -} ran-HPMA _{60 )} , or an equimolar dose of P31 peptide. Starting on day ₄ , _mice were administered AccuCheck _... The Aviva Roche glucometer was used to measure non-fasting blood glucose levels to detect the onset of diabetes. Mice were considered to have diabetes when their blood glucose readings were ≥300 mg/dL. Mice were euthanized after two hyperglycemic readings. Data from this experiment are shown in Figure 13. As shown, mice receiving saline developed diabetic blood glucose levels within 4–8 days of adoptive transfer. Similarly, mice receiving P31 (unconjugated) developed diabetic blood glucose levels approximately 7–10 days after transfer. In stark contrast, mice receiving F1c'-P31-m ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ or F1c'-P31-m ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA 60) were also euthanized. ₆₀ mice maintained relatively stable blood glucose levels (<200 mg/dl) for more than 40 days.

22B. By following the procedure described in Example 21A and replacing the test composition with other compositions of Formula 1, wherein X is insulin-B or proinsulin, preinsulin, glutamate decarboxylase-65 (GAD-65 or glutamate 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 2β (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E, peripheral protein, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreas/pancreas-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic-duodenal homology frame 1, dystrophic myotonic kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST. G protein-coupled receptors 1-5, such as F1aA-insulin- _m2 - _n80 , F1aA-insulin- _m2 - _n12 , F1aA-insulin- _m2 - _n33 , F1aA-insulin- _m2 - _n40 , F1aA-insulin- _m2 - _n43 , F1aA-insulin- _m2 - _n50 , F1aA-insulin- _m2 - _n60 , F1aA-insulin- _m2 - _n75 , F1aA-insulin- _m2 - _n84 , F1b-insulin- _m2 - _n4 - _p34-2NAcGAL , F1m-insulin- _m2 - _n80 - _p30 - _q4- CMP-2NHAc, and F1m-insulin- _m2 - _n62 - _p30 - _q8. -CMP-2OH, F1n-insulin- _m2 - _n1 - _p30 - _q4 -CMP-2NHAc, F1c'-insulin- _Bm1 - _n4 - _p90 -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ or F1c'-insulin- _Bm1 - _n4 - _p90 -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ , compared with animals receiving saline, resulted in stable blood glucose levels in NOD mice treated with these formulations.

Example 23

NOD mice

23A. Non-obese diabetic (NOD) mice, such as NOD/ShiLt mice, are prone to spontaneously developing autoimmune diabetes, which is the result of an autoimmune response to various pancreatic autoantigens. Diabetes develops in NOD mice due to pancreatitis characterized by infiltration of the islets of Langerhans by various leukocytes. As diabetes progresses, leukocyte infiltration of the islets of Langerhans is present, followed by a significant decrease in insulin production and a corresponding increase in blood glucose levels.

To evaluate the therapeutic efficacy against diabetes, the compositions and treatments provided in this invention were monitored weekly using an AccuCheck Aviva blood glucose meter (Roche) to measure non-fasting blood glucose levels, starting at the onset of diabetes in 5-week-old NOD/ShiLt mice. Starting at 6 weeks of age, mice were randomly assigned to control and test groups (n = 15 for each group) and treated weekly with saline, 10 μg of F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀₎ , or 10 μg of F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ ), respectively. Injections continued for 10 consecutive weeks. The percentage of diabetic-free animals was measured over time. Mice were considered diabetic when two consecutive blood glucose readings were ≥300 mg/dL or one blood glucose reading was ≥450 mg/dL. Mice deemed diabetic were euthanized.

Figure 14 shows the percentage of non-diabetic animals as described above, measured over time. Mice treated with F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀₎ appear as solid squares. Mice treated with F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀₎ appear as solid triangles. Mice treated with saline appear as solid rhombuses. Spontaneous diabetes is understood based on data collected from saline-treated animals as early as 11 weeks of age over time. Prevalence increases over time (shown by the downward trend in the figure), with 60% of the test animals developing diabetes by week 20. As shown in Figure 14, mice treated with F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA) Treatment of NOD mice with ₆₀ or F1c'-insulin-Bm ₁ -n ₄ -p ₉₀ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ reduced the incidence of diabetes flare-ups in NOD mice compared to animals receiving saline. Data demonstrate that administration of insulin conjugated to the linker and liver-targeting moiety as disclosed in this invention successfully reduces the development of type 1 diabetes by decreasing the autoimmune response to various pancreatic autoantigens generated.

23B. By following the procedure described in Example 22A and replacing the test composition with other compositions of Formula 1, wherein X is insulin-B or proinsulin, preinsulin, glutamate decarboxylase-65 (GAD-65 or glutamate 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 2β (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A, HSP-60, carboxypeptidase E, peripheral protein, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreas/pancreas-associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic-duodenal homology frame 1, dystrophic myotonic kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST. G protein-coupled receptors 1-5, such as F1aA-insulin- _m2 - _n80 , F1aA-insulin- _m2 - _n12 , F1aA-insulin- _m2 - _n33 , F1aA-insulin- _m2 - _n40 , F1aA-insulin- _m2 - _n43 , F1aA-insulin- _m2 - _n50 , F1aA-insulin- _m2 - _n60 , F1aA-insulin- _m2 - _n75 , F1aA-insulin- _m2 - _n84 , F1b-insulin- _m2 - _n4 - _p34-2NAcGAL , F1m-insulin- _m2 - _n80 - _p30 - _q4- CMP-2NHAc, and F1m-insulin- _m2 - _n62 - _p30 - _q8. -CMP-2OH, F1n-insulin- _m2 - _n1 - _p30 - _q4 -CMP-2NHAc, F1c'-P31- _m1 - _n4 - _p90 -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ or F1c'-P31- _m1 - _n4 - _p90 -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀₎ , compared with animals receiving saline, showed a reduced incidence of diabetic onset in NOD mice.

Example 24

Biological distribution

To examine the biodistribution of the antigenic sugar polymer conjugates, we treated BALB/c mice with fluorescently labeled OVAs or fluorescently labeled OVAs conjugated with p(Gal-HPMA), p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA). Sugar moieties attached to the p(Gal-HPMA) and p(Glu-HPMA) backbones were attached to the α-conformation polymer at the C1 position, while sugar moieties attached to the p(Galβ-HPMA) and p(Gluβ-HPMA) backbones were attached to the β-conformation polymer at the C1 position. The OVAs were labeled with Dy750. All treatments were administered at 140 μl via tail vein injection. Each animal was treated with an equal volume of the fluorescent conjugate based on fluorescence units. After 3 hours, the animals were euthanized, and the liver of each animal was perfused with saline. The liver and spleen were then harvested and imaged via an IVIS Spectrum system with a suitable filter assembly.

Figure 15 shows representative images of fluorescence signals from the liver (A) and spleen (B) of animals treated with OVA or OVA sugar polymer conjugates. The formulations are as follows: 1.OVA, 2.F1m'-OVA750-m _1-3 -n ₇₉ - _{p 90} - _{q 4} -CMP-poly-(EtPEG ₁ AcN-1NAcGALβ ₃₀ -ran-HPMA ₆₀ ) ["OVA-p(Galβ-HPMA)"], 3.F1m'-OVA750-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ ) ["OVA-p(Gal-HPMA)"], 4.F1m'-OVA750-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLUβ ₃₀ -ran-HPMA ₆₀ [“OVA-p(Gluβ-HPMA)”], 5.F1m'-OVA750-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ )[“OVA-p(Glu-HPMA)”]. Images of livers from animals treated as described above show that the glycopolymer conjugates significantly enhance the delivery of the conjugated antigen to the liver (or spleen) compared to the uptake of the unconjugated antigen. Liver from animals treated with unconjugated OVA showed less fluorescence signal compared to livers from animals treated with OVA conjugated with p(Gal-HPMA), p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA). Additionally, spleen images from animals treated as described above show that conjugating the antigen with the glycopolymer reduces antigen delivery to the spleen. Spleens from animals treated with unconjugated OVA showed significantly more fluorescence signal compared to spleens from animals treated with OVA conjugated with p(Gal-HPMA), p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA). These data are significant because they demonstrate enhanced targeting of the liver and/or spleen by the desired antigen, as demonstrated by the experimental data presented in this invention, leading to a reduced immune response to the antigen (i.e., induced tolerance). According to some embodiments disclosed in this invention, this induced tolerance can treat, reduce, prevent, or otherwise improve undesirable immune responses that, in other cases, have been associated with exposure to the antigen.

Example 25

7-day OTI/OTII phenotypic analysis

To compare the ability of various glycopolymer-antigen conjugates to induce antigen-specific T cell proliferation and to upregulate the expression and presentation of various markers that are unresponsive or deleted on T cells, OVA or conjugates with p(Gal-HPMA), p(Glu-HPMA), p(Galβ-HPMA), or p(Gluβ-HPMA) (with formulations having the following characteristics: F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGAL ₃₀ -ran-HPMA ₆₀ ) [“OVA-p(Gal-HPMA)”]; F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLU ₃₀ -ran-HPMA ₆₀ ) [“OVA-p(Glu-HPMA)”]; F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGALβ ₃₀ -ran-HPMA ₆₀ )["OVA-p(Galβ-HPMA)"];F1m'-OVA-m _1-3 -n ₇₉ -p ₉₀ -q ₄ -CMP-poly-(EtPEG ₁ AcN-1NAcGLUβ ₃₀ -ran-HPMA ₆₀ Intravenous injection of OVA conjugated with [“OVA-p(Gluβ-HPMA)”] was performed on mice that had received an infusion of 400,000 OTI cells labeled with carboxyfluorescein succinimide (CSFE). Animals treated with free or conjugated OVA received 10 μg of OVA on days 1 and 3 of the experiment. The timeline of experimental details is shown in Figure 16A. After 7 days, the mice were sacrificed and their spleen cells were harvested and analyzed by flow cytometry for phenotypic markers of T cell unresponsiveness, deletion, and memory characteristics.

Figure 16B shows that the OVA-glycopolymer conjugate induced greater OTIT cell proliferation compared to the OTI proliferation observed in animals treated with unconjugated OVA. As discussed above, these data further support that, according to some embodiments disclosed herein, the sugar-targeting portion leads to increased antigen-specific T cell proliferation, a key step in inducing tolerance to antigens. Interestingly, animals treated with the OVA-glycopolymer conjugate containing a β-linked sugar induced significantly more proliferation compared to animals treated with a glycopolymer containing the same sugar moiety linked to the polymer via an α-link (e.g., p(Galβ-HPMA) vs. p(Gal-HPMA)). Unexpectedly, this conformational change in one element of the overall composition resulted in enhanced potency in terms of T cell proliferation, which the applicant believes (without resorting to theory) stems from the synergistic interaction between the components of the composition and their respective physiological targets. Furthermore, compared to cells collected from animals treated with OVA, the OTI cell populations collected from animals treated with all OVA-glycopolymer conjugates (except OVA-p(Gal-HPMA)) showed significantly higher expression of the apoptosis marker annexin V+ (see Figure 16C). Consistent with the data discussed above, this indicates a larger percentage of antigen-specific T cells being targeted or in the apoptosis cascade. As shown in Figure 16D, OTI cells collected from animals treated with OVA-glycopolymer conjugates containing β-linked sugars showed increased expression of the T cell depletion marker PD-1 compared to animals treated with glycopolymers containing the same sugar moiety linked to the polymer via α-linking and animals treated with free OVA. To maintain long-term tolerance, treatment must reduce the number of long-acting antigen-specific memory T cells. Figures 16E and 16F show that both OVA-p(Galβ-HPMA) and OVA-p(Gluβ-HPMA) induced a significant reduction in OTI cells expressing memory T cell markers. Both OVA-p (Galβ-HPMA) and OVA-p (Gluβ-HPMA) induced a 5-fold reduction in the number of memory T cells compared to animals treated with free OVA. These data further indicate that compositions as disclosed herein can lead to tolerance to antigens (OVA selected in this invention because it is recognized in the art as the "gold standard" antigen), and in some embodiments, can unexpectedly enhance the induction of tolerance (e.g., through at least part of antigen-specific T cell proliferation, increased annexin V expression on antigen-specific T cells, increased depletion marker expression on antigen-specific T cells, and decreased expression of memory T cells).

Example 26

Study of glucose-conjugated antigen-BDC2.5

This study was conducted to test the ability of the p(Glu)-antigen conjugate to inhibit the formation of CD4 T cell-driven autoimmunity. An autoimmune diabetes mouse model was induced using antigen-specific T cells. Autoimmune diabetes was induced by transferring activated BDC2.5 T cells (which carry the T cell receptor for the diabetes autoantigen chromogranin A) into immunocompromised mice. Recipient mice were then treated with either an epitope mimicking the autoantigen peptide called p31 or with the p31-p(Glu) conjugate.

Protocol: Freshly isolated spleen cells from female BDC2.5 transgenic mice were stimulated for 4 days in DMEM supplemented with 10% FBS, 0.05 mM β-mercaptoethanol, 1% puromycin/streptomycin, and 0.5 μM p31 peptide. Following stimulation, cells were washed with DMEM, resuspended in DMEM, and intravenously injected into glucose-normal NOD/Scid mice. Eight hours after adoptive transfer, mice were intravenously administered saline (untreated), 2 μg p31, or 2 μg p31 as a p31-pGlu conjugate (β-conformation glucose was used in this experiment, but α-conformation may be used in some embodiments). Three days after initial treatment, mice were treated again with saline, 2 μg p31, or 2 μg p31 as a p31-pGlu conjugate. Starting on day 1, non-fasting blood glucose levels were measured in each mouse using an Accucheck Aviva glucometer. Mice were considered to have diabetes when a single blood glucose measurement exceeded 450 mg/dL, or when two consecutive blood glucose readings exceeded 350 mg/dL. All groups contained 7 mice.

Results: As shown in Figure 17, untreated mice began to develop diabetes (as measured by hyperglycemia) within 5 days of spleen cell transfer. Within approximately 7 days, all untreated mice developed hyperglycemia. Mice treated with the control composition (p31; solid circle) began to develop hyperglycemia shortly after the untreated mice. By day 8 post-transfer, all p31-treated mice had developed hyperglycemia. In stark contrast, administration of the p31-p(Glu) conjugate (solid triangle) resulted in mice maintaining normal blood glucose levels for a significantly longer period compared to untreated animals and those treated with p31 peptide alone. At day 15 post-transfer, 100% of p31-pGlu mice still had normal blood glucose levels.

These results demonstrate that the p31-p(Glu) conjugate prevents the formation of hyperglycemia in mice. Therefore, according to some embodiments disclosed in this invention, several conjugates are effective in preventing the formation of diabetes. In other embodiments, the p(Glu) conjugate is effective in reducing the development of pre-existing diabetes, and in yet another embodiment, it is effective in reversing pre-existing diabetes. In yet another embodiment, other mimicry epitopes of the pancreatic β-cell autoantigen chromogranin-A can be used. Furthermore, according to some embodiments disclosed in this invention, chromogranin-A or a fragment thereof, as a full-length protein, can be used as the antigenic portion of the tolerogenic composition. Additionally, other antigens disclosed in this invention related to diabetes can be used. In some embodiments, these include, but are not limited to, insulin, proinsulin, pre-proinsulin, GAD-65, GAD-67, IGRP, IA-2, IA-2β, fragments thereof, or mimicry epitopes thereof. Diabetes-related antigens are disclosed in this invention. Furthermore, in some embodiments, antigens related to other autoimmune diseases, as disclosed in this invention, can be used. In other embodiments, foreign antigens, graft antigens, or other types/classifications may be used, such as chromogranin A as employed in this invention, and this model is used as a non-limiting example of the ability of compositions as disclosed in this invention to induce tolerance.

Example 27

Tolerance memory

The following experiments were conducted to demonstrate that the tolerogenic compositions disclosed in this invention can establish tolerance memory. To determine whether regulatory T cells (Tregs) induced by administration of the tolerogenic compositions disclosed in this invention (using p(Glu) antigen conjugates as a non-limiting example) could establish long-term tolerance after initial therapy, mice were pretreated with an infusion of OTIIT cells, followed by administration of the p(Glu)-OVA conjugate. These mice were then challenged 3 weeks later with an infusion of OVA-specific T cells and antigen challenge. To demonstrate that any tolerance-inducing effect was a result of Tregs, mice treated with p(Glu)-OVA were injected with an anti-CD25 antibody, which showed Treg reduction.

Protocol: On day 0, C57BL/6 mice received an intravenous infusion of 450,000 OTII cells, followed by treatment on days 1 and 4 with saline or 1.5 μg of OVA as free OVA or a p(Glu)-OVA conjugate (a non-limiting example of OVA used as the antigen to be tolerated in this invention; β-conformation glucose was used in this experiment, but in some embodiments, α-conformation may be used). On day 7, mice received a final treatment with saline or 15 μg of OVA as free OVA or a p(Glu)-OVA conjugate. On day 15, half of the p(Glu)-OVA-treated animals were treated with an intraperitoneal injection of 300 μg of anti-CD25 antibody, which had been shown to reduce regulatory T cells (Tregs). On day 29, mice received an infusion of 750,000 OTII T cells and 750,000 OTI T cells. The following day, animals were challenged with 5 μg OVA and 50 ng ultrapure LPS in each of the four footpads. Five days after antigen challenge, mice were sacrificed, and the number of OTI and OTII T cells in the draining lymph nodes was assessed by flow cytometry. (Group n = 5: challenge (i.e., vector-treated animals), OVA, p(Glu)-OVA, p(Glu)-OVA+αCD25). This protocol is schematically illustrated in Figure 18.

Results: The results of this experiment are shown in Figures 19A-19B. Figure 19A shows the remaining OTI T cells (as a percentage of total CD8 ⁺ cells) after antigen challenge. Figure 19B shows the remaining OTII T cells (as a percentage of total CD4 ⁺ cells) after antigen challenge. Mice that received LPS challenge and OVA lacking the tolerogenic composition showed a significant increase in OTI and OTII cells in the draining lymph nodes. Mice that received pGlu-OVA showed a significant reduction in both OTI and OTII cells. The elimination of tolerance induced by pGlu-OVA by administration of anti-CD25 antibody demonstrates the role of Tregs in pGlu-OVA-induced tolerance. In summary, these data indicate that the tolerogenic compositions disclosed in this invention can induce long-term tolerance via the induction of regulatory T cells. In some embodiments, this is advantageous because the reduction (or elimination in some embodiments) of long-term tolerance necessitates continuous administration of the compositions disclosed in this invention. That is, in some embodiments, repeated administration is optionally performed.

Example 28

Tolerable memory (endogenous)

The following experiments were conducted to demonstrate that the tolerogenic compositions disclosed in this invention can establish tolerance memory from endogenous T cells. These experiments were performed similarly to those in Example 27, but the mice did not receive an initial infusion of donor OTII T cells.

To determine whether Tregs derived from an endogenous T cell population could be induced by the p(Glu)-antigen conjugate and exhibit long-term tolerance, mice were treated with the p(Glu)-OVA conjugate. Three weeks later, they were challenged with OVA-specific T cell infusion and antigen challenge. To investigate whether the demonstrated tolerance-inducing effect was a result of endogenous Tregs, mice were treated with p(Glu)-OVA followed by anti-CD25 antibody.

Protocol: On days 0 and 3, BL6/C57 mice received intravenous infusions of saline or 1.5 μg of OVA as free OVA or a p(Glu)-OVA conjugate (β-conformation glucose was used in this experiment, but in some embodiments, α-conformation may be used). On day 6, mice received a final treatment with saline or 15 μg of OVA as free OVA or a p(Glu)-OVA conjugate. On day 14, half of the animals treated with p(Glu)-OVA were treated with an intraperitoneal injection of 300 μg of anti-CD25 antibody. On day 28, mice received infusions of 750,000 OTII and 750,000 OTI T cells. The following day, animals were challenged with 5 μg of OVA and 50 ng of ultrapure LPS in each of the four footpads. Five days post-antigen challenge, mice were sacrificed, and the number of OTI and OTII T cells in the draining lymph nodes was assessed by flow cytometry. (Group n = 5: attack (i.e., animal treated with the vector), OVA, p(Glu)-OVA, p(Glu)-OVA+αCD25). This scheme is schematically shown in Figure 20.

Results: The results of this experiment are shown in Figures 21A-21B. Figure 21A shows the remaining OTI T cells (as a percentage of total CD8 ⁺ cells) after antigen challenge. Figure 21B shows the remaining OTII T cells (as a percentage of total CD4 ⁺ cells) after antigen challenge. Mice that received LPS challenge and OVA lacking the tolerogenic composition showed a significant increase in OTI and OTII cells in the draining lymph nodes. In contrast, mice that received pGlu-OVA showed a significant reduction in both OTI and OTII cells. Elimination of tolerance induced by pGlu-OVA by administration of anti-CD25 antibody demonstrates that Tregs derived from the endogenous T cell population play a significant role in the induction of tolerance via the pGlu-antigen composition. In summary, these data indicate that the tolerogenic compositions disclosed in this invention can induce long-lasting tolerance via the induction of endogenous regulatory T cells. In some embodiments, this is advantageous because the reduction (or elimination in some embodiments) of long-lasting tolerance necessitates continuous administration of the compositions disclosed in this invention. That is, in some embodiments, repeated administration is optionally performed. Furthermore, these data indicate that supplementing the recipient's T cells with exogenous T cells is unnecessary for inducing tolerance. Specifically, the endogenous pool of T cells is sufficient to provide an adequate number of regulatory T cells to lead to long-term tolerance.

Example 29

Prophylactic administration of tolerogenic compositions reduces subsequent antibody production.

As disclosed in this invention, in some embodiments, the tolerogenic composition can be used to induce, treat, prevent, or otherwise improve an immune response against an antigen of interest. Therefore, in some embodiments, the final use of the tolerogenic composition (e.g., prevention vs. treatment) is determined by the current state of the subject prior to administration of the composition. This experiment was conducted to demonstrate that the degree of antibody production against a specific antigen can be reduced via prophylactic administration of the tolerogenic composition disclosed in this invention.

Protocol: The experimental design is shown in Figure 22. For pretreatment, there were two groups: those receiving 15 μg of the tolerogenic composition containing p-Glu-asparaginase (high dose) and those receiving 2.5 μg of p-Glu-asparaginase (low dose) (β-conformation glucose was used in this experiment, but in some embodiments, α-conformation may be used). These pretreatment steps are shown as step "A" in Figure 22. Note that the use of asparaginase as the antigen to which tolerance is desired in this experiment is a non-limiting example. As discussed above, many other antigens, fragments thereof, or their mimic epitopes may also be used depending on the implementation. After pretreatment, or at the start of the experimental protocol for wild-type asparaginase (WT-Asn) and mixed groups, animals were administered 15 μg of asparaginase or a combination of 12.5 μg of asparaginase and 2.5 μg of pGlu-asparaginase. These administrations are shown as step "B" in Figure 22. For all experimental groups, step "C" represents the collection of 10 μL of blood sample.

Results: Figure 23 shows the results of this experiment. The increase in anti-asparaginase antibody titers, which began at approximately day 2 and stabilized at a relative value of 3–4, is the result of asparaginase-only administration. In comparison, pretreatment with either high or low doses of asparaginase significantly reduced anti-asparaginase antibody formation and consistently maintained relatively low antibody titers throughout the 59-day testing protocol. The negative control group (untreated antigen) showed no antigen-specific antibody formation.

These data demonstrate that, in addition to treating or reducing a pre-existing immune response to the antigen of interest, the tolerogenic compositions disclosed in this invention can also be used in some embodiments to prevent an initial immune response. As shown in this experiment, both high and low doses of a sugar-targeted therapeutic agent conjugated to the antigen of interest as a pretreatment treatment improved antibody production against the antigen of interest. In some embodiments, such methods are particularly effective when administering endogenous therapeutic agents to patients. In some such embodiments, depending on the therapeutic agent, the tolerogenic composition does not need to contain the entire therapeutic agent as the antigen of interest (although in some embodiments the entire therapeutic agent is included), but may contain fragments of the therapeutic agent, specific epitopes of the therapeutic agent, or mimicking epitopes of antigenic regions of the therapeutic agent. In some embodiments, the therapeutic agent is a protein drug. Additionally, in some embodiments, longer and/or more frequent pretreatment treatments of the tolerogenic composition can be used to reduce antibody production against the antigen of interest even further. However, in some embodiments, a single pretreatment step may be sufficient.

Example 30

Tolerogenic compositions improve multiple sclerosis

As discussed above, a variety of diseases related to immune responses, including autoimmune responses, can be treated by generating and using tolerogenic compositions as disclosed in this invention. As a non-limiting example of such use in an autoimmune context, this experiment is designed to determine the efficacy of the tolerogenic compositions in the treatment of multiple sclerosis (MS).

MS-related tolerogenic compositions were tested in a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE). The autoantigen used for vaccination in the model was a peptide derived from myelin oligodendrocyte glycoprotein (MOG), amino acid numbers 35-55 (MOG35-55; SEQ ID NO: 24). The autoantigen used for treatment in the model was a slightly longer peptide sequence (MOG30-60; SEQ ID NO: 25) containing the vaccination peptide as defined above. The use of a longer therapeutic sequence ensures processing by antigen-presenting cells, thus reducing the tendency of soluble peptides to bind to the major histocompatibility complex on the cell surface in the absence of uptake by antigen-presenting cells.

Protocol: The experimental protocol is shown in Figure 24A, and the therapeutic tolerogenic composition is shown in Figure 24B. EAE was induced by immunizing donor B6.SJL mice with MOG35-55/CFA. After 11 days, their spleen cells were harvested and restimulated in culture with MOG35-55 peptide, anti-IFNγ antibody, and IL-12 for 3 days. The resulting encephaloinflammatory cells were injected into mice in the recipient group, which then developed MS symptoms. The group size was 10 mice in each group, with half sacrificed at the peak of the disease (day 11) and half sacrificed at the end of the experiment (day 20). Body weight was measured three times a week, and disease status was scored daily in a blinded manner. The control peptide (MOG30-60), the sugar-targeting peptide (pGlu-MOG30-60; (α-conformation glucose was used in this study, but in some embodiments, β-conformation may be used) or the carrier (saline) were administered at doses of 0.8 μg or 4.0 μg on days 0, 3, and 6. A positive potency treatment control (FTY720) was administered daily throughout the study. FTY720 (fingolimod) is a first-in-class sphingosine 1-phosphate (S1P) receptor modulator considered effective in a Phase II clinical trial for MS.

Figure 25A shows the assessment of the ability of the tolerogenic composition to delay disease onset compared to control animals. It is evident that on day 11 (the peak of disease symptoms in the control group), administration of 0.8 μg pGlu-MOG _30-60 (solid triangle) resulted in a significant reduction in EAE disease scores (compared to MOG peptide alone). Figure 25B shows the data related to the reduction in MS-related weight loss. Similar to the EAE disease scores, pGlu-MOG _30-60 showed a significant reduction in weight loss at day 11 of the experiment (Figure 25B; compared to MOG alone). In comparison, the peptide-only group and the control group showed approximately 25% weight loss at the same time points in the experiment.

Figure 26A corresponds to the assessment of disease onset delay using a higher dose of pGlu-MOG _30-60 (4 μg). Unexpectedly, the delay in disease onset was significantly improved (significantly relative to MOG and FTY720 alone), with none of the mice in the treatment group (solid triangle) showing a non-zero EAE disease score until day 14 of the experiment. In contrast, by day 6, every mouse in the other experimental groups showed signs of MS, as evidenced by the increased EAE disease scores. Again, similar to the EAE disease scores, the higher dose of pGlu-MOG _30-60 resulted in a significant reduction in weight loss. By day 11, the weight loss in the pGlu-MOG _30-60 group (closed triangle) was essentially zero, while all other groups had already lost weight (approximately 2.5 g in the FTY720 group and approximately 5 g in the MOG-only and control groups, see Figure 26B). Therefore, the higher dose of pGlu-MOG _30-60 (4 μg) produced a significant (relative to MOG and FTY720 alone) improvement in weight retention, which lasted substantially throughout the experiment.

As discussed above, this specific experiment is a non-limiting example of how tolerogenic compositions according to some embodiments of the present invention can be used to reduce the immune response of a subject to an antigen of interest, wherein the antigen is an antigen associated with the autoimmune disease multiple sclerosis. As previously mentioned, immune tolerance can also be successfully induced according to the present invention using other antigens, autoantigens, therapeutic proteins, fragments or specific epitopes of any of the foregoing substances, or mimicry epitopes of any of the foregoing substances.

Example 31

Biodistribution of pGal and pGluB conjugates

This experiment was conducted to determine the types of hepatocytes targeted by the pGal and pGlu conjugates as disclosed in this invention.

Protocol: Mice were treated via tail vein injection with 100 μg of OVA labeled with the fluorescent dye Dy-649 (OVA649), 100 μg of OVA conjugated with pGluβ (OVA649-pGluβ), or 100 μg of OVA conjugated with pGalβ (OVA649-pGluβ). After 3 h, the mice were sacrificed, and livers were harvested, processed into single-cell suspensions, and separated by density gradient centrifugation. The protein content (e.g., OVA649) of various hepatocyte types was then analyzed by flow cytometry.

Results: The results showed that both OVA649-pGalβ and OVA649-pGluβ conjugates were more effective at targeting hepatic sinusoidal endothelial cells (LSECs) than OVA649 (Figure 27A). LSECs collected from animals treated with OVA649-pGalβ showed a two-fold increase in mean fluorescence intensity (MFI) compared to those collected from animals treated with OVA649, and LSECs collected from animals treated with OVA649-pGluβ showed a 2.5-fold increase in MFI.

The OVA649-pGluβ conjugate also effectively targets Kupffer cells. The percentage of Kupffer cells that took up OVA649-pGluβ in animals treated with OVA649-pGluβ was significantly greater than the percentage of Kupffer cells that took up OVA649 (Figure 27B). Therefore, in some embodiments, the β-conformation of pGlu is used where targeting Kupffer cells is desirable. That is, in some embodiments, the pGal conjugate may also be optionally used.

In addition to Kupffer cells, CD11c+ cells also efficiently take up OVA649-pGluβ. The percentage of CD11c+ cells taking up OVA649-pGluβ is significantly greater than the percentage of CD11c+ cells targeted by OVA649 (Figure 27C). Therefore, in some embodiments, the β-conformation of pGlu is used when it is desirable to target CD11C+. That is, in some embodiments, the pGal conjugate may also be optionally used.

Interestingly, both OVA649-pGluβ and the OVA649-pGalβ conjugates target hepatocytes more effectively than OVA649. See Figure 27D. However, the percentage of hepatocytes that take up OVA649-pGalβ is greater than the percentage of hepatocytes targeted by OVA649-pGluβ. Therefore, in some embodiments, the β-conformation of pGal is used where targeting hepatocytes is desirable. That is, in some embodiments, the pGlu conjugate may also be optionally used.

Interestingly, analyses of the ability of free OVA and OVA-glycopolymer conjugates to target astrocytes showed contradictory results. Figure 27E shows that OVA targets astrocytes more effectively than either OVA-glycopolymer conjugate.

In some embodiments, a combination of pGlu and pGal conjugates may be desired, and the two classes of compositions synergistically interact to target the liver (and various cell types within the liver). Additionally, in some embodiments, mixtures of α and β configurations of conjugates may also be used. OVA is used as a non-limiting example of an antigen to which tolerance is desired, as in other experiments disclosed herein. Based on the disclosures provided herein, other antigens, fragments thereof, and their mimic epitopes disclosed herein may also be conjugated to the sugar-targeting portion, and tolerance to them may be induced. According to some embodiments disclosed herein, this induced tolerance may treat, reduce, prevent, or otherwise improve an undesired immune response that, in other cases, would be related to exposure to the antigen.

Although the invention has been described with reference to its specific embodiments, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt particular circumstances, materials, composition, processes, or one or more process steps to the purpose, spirit, and scope of the invention. All such modifications are intended to be within the scope of the appended claims. All publications, patents, patent applications, Internet addresses, and registry/database sequences (including both polynucleotide and polypeptide sequences) cited are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication, patent, patent application, Internet address, or registry/database sequence is specifically and individually indicated as incorporated by reference.

It is conceivable that various combinations or sub-combinations of the specific features and aspects of the embodiments disclosed above can be made, and still fall within one or more of the scope of this invention. Furthermore, any specific features, aspects, methods, properties, characteristics, qualities, attributes, elements, etc., disclosed in the embodiments can be used in all other embodiments set forth herein. Therefore, it should be understood that various features and aspects of the disclosed embodiments can be combined or substituted with each other to form different modes of the disclosed invention. Therefore, the scope of the invention as disclosed herein is not intended to be limited to the specific disclosed embodiments described above. Furthermore, while the invention is readily adaptable to various modifications and alternatives, specific examples of which have been shown in the accompanying drawings and described in detail herein. However, it should be understood that the invention is not limited to the specific forms or methods disclosed, but rather, the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the various described embodiments and the appended claims. Any method disclosed herein need not be performed in the listed order. The methods disclosed herein include actions taken by a practitioner; however, they may also include any instructions from a third party regarding these actions, whether express or implied. For example, the action such as “administering a sugar-targeted tolerance-inducing composition” includes “instructing the administration of a sugar-targeted tolerance-inducing composition.” Furthermore, when the features or aspects of the invention are described in accordance with the Markush group, those skilled in the art will recognize that the invention is also described in accordance with any individual member or subgroup of the Markush group.

The scope disclosed in this invention also covers any and all repetitions, sub-scopes, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” etc., includes the stated number. Numbers preceded by terms such as “about” or “approximately” include the stated number. For example, “about 10 nanometers” includes “10 nanometers.”

Claims (10)

1. Compounds containing Formula 1: in: m is an integer from approximately 1 to 10; X contains a protein or protein fragment containing an antigenic region; Y is a linker portion having a formula selected from the following: in: The left parenthesis "(" indicates the key with X; A right parenthesis or bottom parenthesis and “)” indicate the key between Y and Z; n is an integer from approximately 1 to 100; The p that exists is an integer from approximately 2 to 150; The q that exists is an integer from approximately 1 to 44; The ^R8 present therein is _–CH2- or _–CH2 - _CH2 -C( _CH3 )(CN)-; and The ^R9 present therein is a direct bond or _–CH2 - _CH2 -NH–C(O)–; and Z includes a liver-targeting component. 2. The compound of claim 1, wherein Z is galactose or glucose. 3. The compound of claim 1, wherein Z is galactosamine or glucosamine. 4. The compound of claim 1, wherein Z is N-acetylgalactosamine or N-acetylglucosamine. 5. The compound of claim 1, wherein Y is a linker portion having the following formula: 6. The compound of claim 1, wherein Y is a linker portion having the following formula: 7. The compound of claim 1, wherein Y is a linker portion having the following formula: 8. The compound of claim 1, wherein Y is a linker portion having the following formula: 9. The compound of claim 1, wherein X is a food antigen selected from the group consisting of: peanut globulin (Ara h 1), allergen II (Ara h 2), peanut lectin, blue bean protein (Ara h 6), α-lactalbumin (ALA), lactoferrin, Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), tropomyosin fast isotype, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, rye alkaloids, and oat protein and fragments thereof. 10. The compound of claim 9, wherein X is selected from the group consisting of: glutenin, high molecular weight glutenin, low molecular weight glutenin, α-gliadin, γ-gliadin, Ω-gliadin, barley gliadin, rye alkaloids, and oat protein and fragments thereof.