WO2008089339A2 - Oligosaccharide conjugates for cellular targeting - Google Patents

Abstract

This disclosure relates to conjugation of therapeutic proteins (e.g., lysosomal enzymes) with oligosaccharides comprising mannose-6-phosphate. Conjugation of proteins with oligosaccharides comprising mannose-6-phosphate results in protein-oligosaccharide conjugates that are targeted to the lysosome by mannose-6-phosphate receptors. The proteins of the invention may be used in enzyme replacement therapy of, e.g., lysosomal storage disorders.

Description

OLIGOSACCHARIDE CONJUGATES FOR CELLULAR TARGETING

This application claims the benefit of U.S. Provisional Application No. 60/885,457, filed January 18, 2007, the disclosure of which is incorporated herein by reference. [0001] The invention relates to conjugation of therapeutic proteins with oligosaccharides, and more particularly, to the conjugation of lysosomal enzymes with oligosaccharides. The invention further relates to methods of treating lysosomal storage disorders using such conjugates.

Lysosomal Storage Disorders [0002] Lysosomal storage disorders (LSDs; see, e.g., Table 1 infra) are a class of rare metabolic disorders, comprising over forty genetic diseases involving deficiencies in the activity of lysosomal hydrolases. A hallmark feature of LSDs is the abnormal accumulation of lysosomal metabolites. This accumulation leads to the formation of large numbers of distended lysosomes that disrupt intracellular architecture and result in LSD disease pathology.

[0003] LSDs can be treated by administration of the active version of the enzyme deficient in the patient, a process termed enzyme replacement therapy (ERT). For example, Pompe disease, an LSD, can be treated by ERT with Myozyme® (Genzyme Corporation, Cambridge, MA), a recombinant version of human GAA (rhGAA) that is now approved in the United States and the European Union.

[0004] Pompe disease, also known as Glycogen Storage Disease Type II, glycogenosis type II, and acid maltase deficiency, is an autosomal recessive neuromuscular disorder resulting from a metabolic deficiency of the lysosomal enzyme acid α-glucosidase (GAA). That deficiency in GAA activity results in lysosomal glycogen accumulation throughout the body, particularly in skeletal and cardiac muscle, resulting in myofibril damage and impaired muscle function.

[0005] Although Pompe disease can be viewed along a spectrum of severity, historically the disease has been categorized into an infantile-onset form and a late-onset form. The infantile form progresses rapidly, and typically results in death in the first two years of life from cardiorespiratory failure. The late-onset form typically progresses more slowly than the infantile form. On average, late-onset Pompe patients require a wheelchair approximately eleven years after the first symptoms of the disease appear, and a ventilator after approximately fifteen years. The most common cause of death in the late-onset disease population (i.e., adult and juvenile Pompe patients) is respiratory failure.

[0006] In a second example, Fabry disease, an LSD, can be treated with Fabrazyme® (Genzyme Corporation, Cambridge, MA), a recombinant human α-galactosidase A (AGAL). Fabry disease, or Anderson-Fabry disease, is a rare, X-linked, lysosomal storage disorder marked by a deficiency of AGAL. Fabry disease results in the accumulation of globotriaosylceramide and other neutral glycosphingolipids in the lysosomes of visceral tissues and endothelial, perithelial, and muscle cells. Accumulation of the neutral glycosphingolipids in the vasculature results in narrowing and dilatation of the blood vessels, and ultimately to ischemia and infarction.

[0007] In a third example, recombinant human acid sphingomyelinase (rhASM) produced in Chinese hamster ovary (CHO) cells is currently under development as a therapeutic for Niemann-Pick disease. Niemann-Pick disease is an autosomal recessive LSD having four subtypes, A, B, C, and D. Niemann-Pick types A and B result from a deficiency of acid sphingomyelinase (ASM), leading to the accumulation of lipid substances such as sphingomyelin in the cells of the spleen, liver, lungs, and, in some cases, the bone marrow and lymph nodes. Such accumulation causes these cells to malfunction.

[0008] Although the A and B forms of Niemann-Pick disease characterized by a lack of acid sphingomyelinase can be viewed along a spectrum of severity, historically this form of the disease has been categorized into an infantile-onset (type A) and a juvenile-onset (type B) form. Niemann-Pick type A causes enlargement of the liver and spleen, severe brain damage by six months of age, and usually causes death before eighteen months of age. Niemann-Pick type B causes enlargement of the liver and spleen, pulmonary difficulties, and often results in ataxia and peripheral neuropathy. Niemann-Pick type B patients often are treated with lipid-lowering drugs, antibiotics, and supplemental oxygen, but those treatments have limited benefits. Accordingly, effective treatments are urgently needed for patients afflicted with Niemann-Pick disease and other LSDs. Oligosaccharide Targeting for ERT

[0009] In enzyme replacement therapy for lysosomal storage disorders, replacement enzymes bearing oligosaccharides containing a terminal mannose-6- phosphate (M6P) are administered and taken up by target cells through cell surface associated cation independent M6P receptor (CI-MPR)-mediated endocytosis. Consequently, the therapeutic efficacy of an administered enzyme is linked, in part, to the level of exposed M6P. (One exception is ERT for Gaucher disease, in which cellular uptake occurs through a mannose receptor.)

[0010] In many instances, recombinant replacement enzymes are not expressed with reproducibly high levels of M6P. For example, rhGAA typically contains approximately one mole of M6P per mole of protein, resulting in a relatively low affinity for the M6P receptor. However, significant differences in phosphorylation levels between lysosomal enzymes expressed in CHO cells have been reported, even within a single cell line. Zhao et al., Protein Expression Purif. 19:202-211 (2000). [0011] Poorly phosphorylated proteins are not internalized efficiently by the

CI-MPR on cell surfaces, and therefore may not be internalized by cells and directed to the lysosome where they function. For example, phosphorylated GAA isolated from bovine testis is taken up 200-fold more efficiently by cultured human fibroblasts than is poorly phosphorylated GAA isolated from human placenta. Reuser et al., Exp. Cell Res. 155:178-189 (1984). It is therefore therapeutically important to develop methods of consistently producing phosphorylated replacement enzymes.

[0012] Several approaches to increase the M6P content of GAA and other lysosomal enzymes have been proposed. U.S. Patent Nos. 6,534,300; 6,670,165; and 6,861 ,242 concern the use of recombinant GlcNAc-phosphotransferase and phosphodiester oGlcNAcase for the enzymatic phosphorylation of terminal mannose residues. Alternatively, lysosomal enzymes can be expressed in cells expressing Pro-Λ/-Acetylglucosamine-1 -Phosphodiester α-N-Acetyl Glucosimanidase, as described in U.S. Patent No. 6,800,472.

[0013] An alternative approach is described in U.S. Patent Application Pub. No. 2002/0137125. In this work, periodate or galactose oxidase oxidation of glycoprotein carbohydrates result in carbonyl compounds, which are then chemically conjugated with an oligosaccharide functionalized at the reducing end with a hydrazide group to yield a hydrazone. For example, rhGAA conjugated with a bis- M6P oligosaccharide by this method was found to effectively reduce skeletal and cardiac muscle glycogen when administered to the Pompe KO mouse. Moreover, the conjugate was also reported to have a higher intrinsic potency than unmodified rhGAA, which may be attributed to the increased levels of M6P.

[0014] Therefore, there exists a need for new methods to modify therapeutic agents, such as replacement enzymes, to improve their targeting and/or to increase cellular uptake.

[0015] The invention provides protein-oligosaccharide conjugates in which the oligosaccharide comprises M6P, and is connected to the sulfur atom of a cysteine residue. The protein may be an enzyme, e.g., a lysosomal enzyme such as any one of the lysosomal hydrolases listed in Table 1. In illustrative embodiments, the lysosomal hydrolase is GAA or ASM. In some embodiments, the oligosaccharide comprises one or more terminal and/or penultimate M6P residues. In illustrative embodiments, the oligosaccharide is a synthetic biantennary hexamannosyl oligosaccharide having two terminal M6P residues.

[0016] In the conjugates of the invention, the oligosaccharide is connected to the sulfur atom of a cysteine residue of the protein through covalent bonds other than those in the peptide backbone of the protein. The reducing end of the oligosaccharide may be connected directly to the sulfur atom of a cysteine residue of the protein. In certain embodiments, the reducing end of the oligosaccharide may be connected to the sulfur atom of a cysteine residue through a disulfide or thioether linkage. In particular embodiments, the reducing end of the oligosaccharide may be connected to the sulfur atom of a cysteine residue through a linker comprising, for example, chemical groups such as, e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amido, ester, keto, ether, thioether, or amino.

[0017] The invention provides a first general method of making the conjugates of the invention, comprising: (a) providing a protein having at least one cysteine thiol group;

(b) providing an oligosaccharide comprising mannose-6-phosphate and having a thiol-reactive group (e.g., a disulfide-forming reagent or an alkylating reagent) at the reducing end; and (c) reacting the thiol group of the protein and the thiol-reactive group of the oligosaccharide, thereby yielding a protein-oligosaccharide conjugate. [0018] The invention further provides a second general method of making the conjugates of the invention, comprising:

(a) providing a protein having at least one cysteine thiol group; (b) reacting the thiol group of the protein with an activating reagent to produce a protein having at least one thiol-reactive group;

(c) providing an oligosaccharide comprising mannose-6-phosphate and having a thiol group at the reducing end; and

(d) reacting the thiol-reactive group of the protein and the thiol group of the oligosaccharide, thereby yielding a protein-oligosaccharide conjugate. [0019] This disclosure also provides methods of using the conjugates of the invention, including methods of treating lysosomal storage disorders such as disorders listed in Table 1. The methods of treatment include administration of a lysosomal enzyme-oligosaccharide conjugate of the invention to a mammal having a lysosomal storage disorder. In certain embodiments, the lysosomal storage disorder is one of the LSDs listed in Table 1. For example, in certain embodiments, the lysosomal storage disorder is Pompe disease, Fabry disease, or Niemann-Pick disease (such as, e.g., Niemann-Pick type B disease). This disclosure further provides the use of a conjugate of the invention for treating a lysosomal storage disorder in a subject in need thereof, and in the manufacture of a medicament for treating a lysosomal storage disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 depicts one embodiment of conjugation method 1 described herein. In this embodiment, protein 1 , having at least one cysteine residue, is reacted with oligosaccharide 2, comprising mannose-6-phosphate and having a reducing end disulfide-forming reagent, to yield protein-oligosaccharide conjugate 3. In Figure 1a, the disulfide-forming reagent is a disulfide group. In Figure 1b, the disulfide-forming reagent is a thiosulfate group. In Figures 1-3, the

symbol ' — ' represents a protein, the symbol * — ' represents a linker, the symbol f OS

¹ — ' represents an oligosaccharide comprising mannose-6-phosphate and having a reducing end thiol-reactive group or reducing end thiol group, and R represents an optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl group. [0021] Figure 2 depicts another embodiment of conjugation method 1 described herein. In this embodiment, protein 1 , having at least one cysteine residue, is reacted with oligosaccharide 2, comprising mannose-6-phosphate and having a reducing end alkylating reagent, to yield protein-oligosaccharide conjugate 3. In Figure 2a, the alkylating agent is an alkyl halide or alkyl sulfonate, and X is a halide or sulfonate group. In Figure 2b, the alkylating agent is an α,β-unsaturated carbonyl group.

[0022] Figure 3 depicts one embodiment of conjugation method 2 described herein. In this embodiment, protein 1 , having at least one thiol group, is reacted with an activating reagent to yield a protein having an activated thiol group 2. Protein 2 is reacted with oligosaccharide 3, comprising mannose-6-phosphate and having a reducing end thiol group, to yield protein-oligosaccharide conjugate 4. In Figure 3a, the activating agent is a disulfide. In Figure 3b, the activating agent is a sulfenyl chloride. [0023] Figure 4 depicts an illustrative embodiment of conjugation method

1 , as described in Figure 1 a, wherein the protein is the lysosomal enzyme acid α-glucosidase (GAA).

[0024] This invention provides conjugates of proteins with oligosaccharides comprising mannose-6-phosphate, wherein the oligosaccharide is connected to the sulfur atom of a cysteine residue. The protein may be, e.g., an enzyme, such as a lysosomal hydrolase, including, e.g., those listed in Table 1. For example, the protein may be acid α-glucosidase, α-galactosidase A, or acid sphingomyelinase. These conjugates may have higher affinity for M6P receptors as compared to unconjugated proteins. [0025] This invention further provides general methods for the conjugation of proteins with oligosaccharides comprising mannose-6-phosphate, thereby yielding protein-oligosaccharide conjugates. These methods are specific for cysteine thiol groups, and encompass a number of variations, as described below. One potential advantage of such methods is that they do not require oxidation of the protein in question, and thus preserve the native protein and oligosaccharide structure (e.g., by avoiding oxidation of methionine). Protein

[0026] These conjugation methods are broadly applicable to any pure protein, partially purified protein, or fragment thereof having at least one thiol group, including isolated proteins and recombinantly or synthetically produced proteins. The terms "pure," "purified", and "isolated" refer to a molecule that is substantially free of its natural environment. For instance, a pure protein is substantially free of cellular material and/or other proteins from the cell or tissue source from which it is derived. The term refers to preparations that are, for example, at least 70% to 80%, 80% to 90%, 90 to 95%; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

[0027] A protein may be conjugated through the side chain thiol group (also known as a sulfhydryl or mercaptan group) of one or more cysteine residues. The thiol group may be, for example, on the surface of the protein or in the interior of the protein, such as, e.g., in a solvent-accessible cavity. The number of thiol groups, or of reactive thiol groups, may be, e.g., at least 1 , 2, 3, 4, 5, 6, 8, 10, 20, 30, 40, or 50. The number of thiol groups, or of reactive thiol groups, may be an odd number, such as, e.g., 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, or 25. For example, human GAA has one reactive cysteine thiol group.

[0028] In some embodiments, not all cysteine residues of a protein have thiol groups that may react with a thiol-reactive group. For example, the side chain of a cysteine residue may form a disulfide with the side chain of another cysteine residue. Thiol groups may be made accessible to a thiol-reactive group by the partial or complete reduction of disulfide groups in a protein. For example, disulfides may be reduced by the addition of dithiothreitol, 2-mercaptoethanol, 2- mercaptoethylamine, glutathione, tris-(2-carboxyethyl)phosphine) (TCEP), or tris-(2-cyanoethyl)phosphine. It may be possible to selectively reduce solvent- exposed disulfides by reaction with TCEP, or to reduce solvent-exposed and buried disulfides by reacton with tris-(2-cyanoethyl)phosphine. See, e.g., The Handbook - A Guide to Fluorescent Probes and Labeling Technologies, 10^th ed. (Invitrogen Corporation, 2005). [0029] Not all thiol groups of a protein may react with a thiol-reactive group in the presence of less than a stoichiometric amount of the thiol-reactive group. Moreover, some thiol groups may be buried in the interior of the protein, or otherwise less reactive than other thiol groups. In certain embodiments, a thiol group may be made accessible to a thiol-reactive group by the partial or complete unfolding or denaturation of the protein.

[0030] If a protein does not naturally contain a cysteine residue, or does not naturally contain a cysteine residue having a reactive thiol group, such a cysteine residue may be introduced into the protein, e.g., using standard site-directed mutagenesis techniques. For example, a protein of the invention may differ from a naturally occurring protein by an addition (insertion or substitution) of one or more (e.g., 2, 3, 4, 5, or more) cysteine residues, relative to the naturally occurring protein.

[0031] In certain embodiments, the protein is a therapeutic protein, and may be targeted to the lysosome by conjugation with an oligosaccharide comprising mannose-6-phosphate (M6P). For example, the protein may be a lysosomal enzyme, including an ERT enzyme. The enzyme may be a lysosomal hydrolase, including those listed in Table 1. In certain embodiments, the lyosomal hydrolase is chosen from, e.g., α-glucosidase, α-galactosidase A, and acid sphingomyelinase.

Table 1 : Examples of LSDs and Corresponding Lysosomal Hydrolases

[0032] In certain embodiments, the protein may be a glycoprotein, such as a glycoprotein having at least 1 , 2, 3, 4, 5, or more glycosylated amino acid residues. In other embodiments, the protein may have 1 , 2, 3, 4, 5 or more consensus sites for /V-linked or O-linked glycosylation, each of which may or may not be glycosylated.

[0033] In other embodiments, the protein may be an enzyme that has optimal activity, as measured by an activity assay, at a pH ranging from 1-7, such as, e.g., 1-3, 2-5, 3-6, 4-5, 5-6, or 4-6. For example, the lysosomal enzyme may have a pH optimum at a pH ranging betweeen 3-5. [0034] In particular embodiments, the protein may be a ligand for a receptor. For example, the protein may bind to a receptor that recognizes a sugar such as, e.g., mannose or mannose-6-phosphate. In some embodiments, the protein may bind to, e.g., the asialoglycoprotein receptor, the cation-dependent mannose-6-phosphate receptor, the insulin-like growth factor I I/cation-independent mannose-6-phosphate receptor, or the macrophage mannose receptor.

[0035] In certain embodiments, the protein, when conjugated to an oligosaccharide comprising mannose-6-phosphate, is internalized more efficiently by a target cell (e.g., via CI-MPR-mediated endocytosis) than is the corresponding unconjugated protein. For example, the conjugated protein may be internalized more efficiently than the unconjugated protein by, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% (w/w) in a given time period. In other embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold (w/w) as much of the conjugated protein may be internalized, relative to the unconjugated protein, in a given time period. The referenced time period may be, for example, 10, 30, 45 min or 1 , 2, 3, 5, 6, 12, 24, 48, 72 hours or more. Oligosaccharide

[0036] The methods of the invention are applicable to a broad range of oligosaccharides comprising mannose-6-phosphate and having a thiol-reactive group or thiol group connected, optionally through the intermediacy of one or more chemical groups, to the reducing end of the oligosaccharide (a "reducing end group"). As used herein, the term "reducing end" of an oligosaccharide refers to the anomeric carbon through which the oligosaccharide could be attached to a protein by a glycosidic linkage. One example is depicted below.

[0037] The thiol-reactive group or thiol group at the reducing end of the oligosaccharide may be connected to the reducing end of the oligosaccharide by an optional linker. A linker may comprise chemical groups such as, e.g., optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, amido, ester, keto, ether, thioether, or amino. The optional linker may be, for example, polyethyleneglycol (PEG).

[0038] In particular embodiments, the oligosaccharide may be the M6P- containing hexasaccharide of Formula I depicted below.

Formula [0039] The oligosaccharide may be isolated from a natural source or may be prepared by chemical synthesis. An oligosaccharide isolated from a natural source may be homogeneous or may be a heterogeneous mixture of related oligosaccharides. In some embodiments, the oligosaccharide may be prepared by chemical modification of an oligosaccharide isolated from a natural source ("semi- synthesis"). In some embodiments, the oligosaccharide may have the chemical structure of a naturally occurring oligosaccharide.

[0040] The oligosaccharide may be, e.g., a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, or a larger oligosaccharide. The oligosaccharide may be mono-, bi-, tri-, tetra-, or pentaantennary in structure. The oligosaccharide may contain 0, 1 , 2, 3, 4, or more branch points.

[0041] In some embodiments, the oligosaccharide may comprise a monosaccharide, which may be a penultimate monosaccharide or a terminal monosaccharide, that is recognized by a particular receptor. In some aspects, the oligosaccharide may comprise a terminal galactose, mannose, M6P, glucose, GIcNAc, or sialic acid residue. The oligosaccharide may, in some embodiments, contain at least 1 , 2, 3, 4, 5, 6, or 7 terminal M6P residues.

[0042] In illustrative embodiments, the oligosaccharide is a synthetic biantennary hexamannosyl oligosaccharide of Formula Il having two terminal M6P

residues, where * — ' depicts an optional linker, and Y depicts a thiol-reactive group at the reducing end of the oligosaccharide. In illustrative embodiments, the thiol- reactive group of Formula Il is, e.g., a disulfide-forming reagent, an alkylating reagent, or an activating reagent.

Formula Il

[0043] The oligosaccharide of Formula Il can be described as α-D-(M6P)- (1 →2)-α- D-Man-(1→6)-α- D-Man-(1→6)-[α- D-(M6P)-(1 →2)-α- D-Man-(1→3)]-β- D-

Man-1 -( * — ' -Y), where *^• — ' represents a linker and Y is a thiol-reactive group. Conjugates

[0044] The invention provides a protein-oligosaccharide conjugate comprising a protein, such as, e.g., a lysosomal hydrolase, and an oligosaccharide comprising mannose-6-phosphate, wherein the oligosaccharide is connected directly or through one or more optional chemical groups to the sulfur atom of a cysteine residue.

[0045] In some embodiments, the conjugate provided herein comprises a lysosomal enzyme, such as a lysosomal hydrolase listed in Table 1. For example, in some embodiments, the lysosomal enzyme is chosen from, e.g., acid α-glucosidase, α-galactosidase A, or acid sphingomyelinase. In some embodiments, the conjugate provided herein comprises a lysosomal hydrolase, such as a lysosomal hydrolase listed in Table 1 , and an oligosaccharide comprising mannose-6-phosphate. Conjugation methods

[0046] This invention provides conjugation methods for the synthesis of protein-oligosaccharide conjugates comprising a protein and an oligosaccharide comprising mannose-6-phosphate, wherein the oligosaccharide is connected directly or through one or more optional chemical groups to the sulfur atom of a cysteine residue. Cysteine residues are relatively rare, comprising on average only about 2% of protein amino acids. See, e.g., Biochemistry, 2^nd ed., John Wiley & Sons: New York, 1995. As a consequence of the scarcity of cysteine residues, modification of cysteines provides a greater degree of specificity than many other protein conjugation methods.

[0047] These methods are broadly applicable for conjugation of proteins with targeting oligosaccharides. These methods effect conjugation under conditions that do not result in oxidation of non-cysteine amino acids of the protein. Therefore, proteins conjugated to oligosaccharides by these methods are free of oxidative changes to other amino acid side chains and sugar residues. In embodiments wherein the conjugated oligosaccharide contains a terminal monosaccharide that is recognized by a particular receptor, the protein-oligosaccharide conjugate may have improved affinity for the cognate receptor.

[0048] Particular embodiments of conjugation method 1 are depicted in Figures 1 , 2, and 4. Conjugation method 1 comprises;

(a) providing a protein having at least one cysteine thiol group;

(b) providing an oligosaccharide comprising mannose-6-phosphate and having a thiol-reactive group at the reducing end; and (c) reacting the thiol group of the protein and the thiol-reactive group of the oligosaccharide, thereby yielding a protein-oligosaccharide conjugate. An illustrative embodiment of conjugation method 1 is depicted in Figure 4.

[0049] Conjugation method 2 comprises: (a) providing a protein having at least one cysteine thiol group;

(b) reacting the thiol group of the protein with an activating reagent to produce a protein having at least one thiol-reactive group;

(c) providing an oligosaccharide comprising mannose-6-phosphate and having a thiol group at the reducing end; and (d) reacting the thiol-reactive group of the protein with the thiol group of the oligosaccharide, thereby yielding a protein-oligosaccharide conjugate. [0050] In some embodiments, the oligosaccharide having a thiol-reactive group at the reducing end further comprises one or more optional chemical groups between the reducing end of the oligosaccharide and the thiol-reactive group. Linkers

[0051] A wide variety of linkers are possible. A linker may comprise, for example, chemical groups such as, e.g., alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amido, ester, keto, ether, thioether, or amino. One or more carbon atoms (e.g., methylene or methine groups) of a linker may be substituted by a heteroatom such as, e.g., sulfur, oxygen, or nitrogen. In certain embodiments, a linker may comprise an amide group.

[0052] One or more hydrogen atoms of a linker may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido.

[0053] In certain aspects a linker may comprise a linear or branched saturated radical having at least one carbon atom, such as 1-20 carbon atoms, 1 -12,

1-10, or 1-6 carbon atoms, or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, unless otherwise specified.

Thiol-Reactive Groups

[0054] A. Disulfide-Forming Reagents. A cysteine thiol group may react with a disulfide-forming reagent at the reducing end of an oligosaccharide to form a protein-oligosaccharide conjugate comprising a disulfide.

[0055] A thiol-reactive group may be a symmetric or asymmetric disulfide.

A thiol group can react with a symmetric or asymmetric disulfide by disulfide exchange (see Figure 1 a). In embodiments wherein the thiol-reactive group is a symmetric disulfide, a large molar excess of the symmetric disulfide may be added to drive the reaction to completion.

[0056] In other embodiments, a thiol-reactive group may be an asymmetric

(or "mixed") disulfide, RS-SR', where R and R' are not equivalent. For example, if R' represents an oligosaccharide comprising mannose-6-phosphate and an optional linker, then R may be, e.g., 2-nitrophenyl, 2-pyridyl, 3-nitro-2-pyridyl,

5-nitro-2-pyridyl, or 6-nitro-2-pyridyl, each of which would result in the formation of a stable leaving group upon reaction with a protein thiol or thiolate group. See, e.g.,

Rabanal et al., Tet. Lett. 37:1347-1350 (1996) and Wynn et al., Meth. Enzymol.

251 :351 -356 (1995). In particular embodiments, R may be 6-nitro-2-pyridyl. A molar excess of the asymmetric disulfide may be used to drive the reaction to completion. [0057] A thiol-reactive group may be a thiosulfate, as depicted in Figure 1 b. Thiols generally react stoichiometrically and rapidly with thiosulfates to yield the corresponding disulfide. See, e.g., Wynn et al., Meth. Enzymol. 251 :351-356 (1995). [0058] B. Alkylating Reagents. A cysteine thiol group may react with an alkylating reagent at the reducing end of an oligosaccharide to form a protein- oligosaccharide conjugate comprising a thioether.

[0059] An alkylating reagent may be an alkyl halide (e.g., an alkyl chloride, an alkyl bromide, or an alkyl iodide), an alkyl sulfonate (e.g., an alkyl tosylate, an alkyl mesylate, or an alkyl triflate), or a strained ring (e.g., an epoxide or aziridine). In certain embodiments, a thiol reactive group may comprise an α-halo carbonyl moiety, such as, e.g., an α-iodo carbonyl moiety. In other embodiments, an alkylating reagent, such as, e.g., a benzylic halide, may comprise an aromatic group. A cysteine thiol group can react with an alkyl halide, alkyl sulfonate, or three- membered ring by nucleophilic substitution. An example is depicted in Figure 2a. [0060] An alkylating reagent may be an α,β-unsatu rated carbonyl compound. For example, a thiol-reactive group may contain a maleimide or acetamide group. A cysteine thiol group can react with an α,β-unsaturated carbonyl compound by conjugate (Michael) addition, as depicted in Figure 2b.

[0061] C. Activating Reagents. A cysteine thiol group may react with an activating reagent to form a thiol-reactive group, such as a mixed disulfide. The protein containing the mixed disulfide can then be reacted with an oligosaccharide having a reducing end thiol to form a protein-oligosaccharide conjugate comprising a disulfide, as depicted in Figures 3a and 3b.

[0062] An activating reagent may be a disulfide, e.g., 2,2'-dipyridyl disulfide, 2,2'-dinitrophenyl disulfide, Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid)), 4,4'-dipyridyl disulfide, or dithiazolyl disulfide. See, e.g., Wynn et al., Meth. Enzymol. 251 :351-356 (1995) and Faulstich et al., Meth. Enzymol. 251 :357-366 (1995).

[0063] An activating reagent may also be a sulfenyl halide such as, e.g., 2,4-dinitrophenylsulfenyl chloride or 3-nitro-2-pyridylsulfenyl chloride. See, e.g., Faulstich et al., Meth. Enzymol. 251 :357-366 (1995). Methods of Treatment

[0064] Methods of targeting proteins to the lysosome by conjugation with oligosaccharides comprising mannose-6-phosphate are provided by the disclosure. In some embodiments, this disclosure provides the use of a conjugate of the invention for treating a lysosomal storage disorder in a subject in need thereof.

[0065] In certain embodiments, the invention provides a method for treating a lysosomal storage disorder, such as a lysosomal storage disorder named in Table 1 , in a subject by administering a therapeutically effective amount of the metabolically deficient enzyme as a conjugate with an oligosaccharide comprising mannose-6-phosphate. In one embodiment, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising at least one of the conjugates described herein.

[0066] In certain embodiments, conjugates of the invention may be administered with one or more other therapies. The one or more other therapies may be administered concurrently with (including concurrent administration as a combined formulation), before, or after the administration of the conjugates of the invention.

[0067] In some embodiments, a patient may be treated (before, after, or during treatment with a conjugate of the invention) with an antipyretic, antihistamine, and/or immunosuppressant. In some embodiments, a patient may be treated with an antipyretic, antihistamine, and/or immunosuppressant prior to treatment with an oligosaccharide-glycoprotein conjugate of the invention in order to decrease or prevent infusion associated reactions. For example, patients may be pretreated with one or more of acetaminophen, azathioprine, cyclophosphamide, cyclosporin A, methotrexate, mycophenolate mofetil, oral steroids, or rapamycin.

[0068] In some embodiments, patients may be treated with one or more of acetaminophen, azathioprine, cyclophosphamide, cyclosporin A, methotrexate, mycophenolate mofetil, oral steroids, or rapamycin at or about, e.g., t = 0 (the time of administration of the conjugate of the invention) and/or t = 12, 24, 36, 48, 60, 72, 96, 120, and 144 hours for, e.g., the first 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more incidences of treatment with a conjugate of the invention. For example, in some embodiments a patient with Fabry disease or Pompe disease may be treated with methotrexate (e.g., with 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 8, 10, 12, 15, 25, 30, 35, 40, 50, 60, 70, 80 mg/kg methotrexate, or more) at or about, e.g., t = 0, 24, and 48 hours for, e.g., the first 1 , 2, 3, 4, 5, 6, 7, 8 weeks of treatment with a conjugate of the invention. In some embodiments, immune tolerance toward conjugates of the invention may be induced in a patient with a lysosomal storage disorder such as, e.g., mucopolysaccharidosis I, by treatment with cyclosporin A and azathioprine. For example, the patient may be treated with cyclosporine A and azathioprine as described in Kakkis et al., Proc. Natl. Acad. Sci. U.S.A. 101 :829-834 (2004).

[0069] In some embodiments, a patient may be treated (before, after, or during treatment with a conjugate of the invention) with e.g., small molecule therapy and/or gene therapy, including small molecule therapy and gene therapy directed toward treatment of a lysosomal storage disorder. Small molecule therapy may comprise administration of one or more compounds described in, e.g., U.S. Patent Application Publication Nos. 2003/0050299, 2003/0153768; 2005/0222244; and 2005/0267094. Gene therapy may be performed as described in, e.g., U.S. Patent Nos. 5,952,516; 6,066,626; 6,071 ,890; and 6,287,857 and U.S. Patent Application Publication No. 2003/0087868.

[0070] The terms "treatment," "therapeutic method," and their cognates refer to both therapeutic treatment and prophylactic/preventative measures. Thus, those in need of treatment may include individuals already having a particular lysosomal storage disease as well as those at risk for the disease (i.e., those who are likely to ultimately acquire the disorder or certain symptoms of the disorder).

[0071] A therapeutic method results in the prevention or amelioration of symptoms or an otherwise desired biological outcome, and may be evaluated by improved clinical signs or delayed onset of disease, increased activity of the metabolically defective enzyme, and/or decreased levels of the accumulated substrate of the metabolically defective enzyme.

[0072] The conjugates of the present invention are useful to treat various lysosomal storage disorders in humans or animals. For example, administration of the conjugates can be used to increase the deficient enzymatic activity in a patient, for example, by at least 10%. The increased enzymatic activity may be determined by, e.g., a reduction in clinical symptoms or by an appropriate clinical or biological assay.

[0073] GAA conjugates may be administered for the treatment of Pompe disease (also known as acid α-glucosidase deficiency, acid maltase deficiency, glycogen storage disease type II, glycogenosis II, and lysosomal α-glucosidase deficiency). Increased GAA activity may be determined by biochemical (see, e.g., Zhu et al., J. Biol. Chem. 279: 50336-50341 (2004)) or histological observation of reduced lysosomal glycogen accumulation in, e.g., cardiac myocytes, skeletal myocytes, or skin fibroblasts. GAA activity may also be assayed in, e.g., a muscle biopsy sample, in cultured skin fibroblasts, in lymphocytes, and in dried blood spots. Dried blood spot assays are described in e.g., Umpathysivam et al., CHn. Chem. 47:1378-1383 (2001) and Li et al., CHn. Chem. 50:1785-1796 (2004). Treatment of Pompe disease may also be assessed by, e.g., serum levels of creatinine kinase, gains in motor function (e.g., as assessed by the Alberta Infant Motor Scale), changes in left ventricular mass index as measured by echocardiogram, and cardiac electrical activity, as measured by electrocardiogram. Administration of GAA conjugates may result in a reduction in one or more symptoms of Pompe disease such as cardiomegaly, cardiomyopathy, daytime somnolescence, exertional dyspnea, failure to thrive, feeding difficulties, "floppiness," gait abnormalities, headaches, hypotonia, organomegaly (e.g., enlargement of heart, tongue, liver), lordosis, loss of balance, lower back pain, morning headaches, muscle weakness, respiratory insufficiency, scapular winging, scoliosis, reduced deep tendon reflexes, sleep apnea, susceptibility to respiratory infections, and vomiting.

[0074] In another aspect, conjugates of α-galactosidase A with oligosaccharides comprising M6P are administered for the treatment of Fabry disease. Fabry disease, or Anderson-Fabry disease, is a rare, X-linked, lysosomal storage disorder marked by a deficiency of α-galactosidase A, and results in accumulation of globotriaosylceramide (GL3) and other neutral glycosphingolipids in the lysosomes of visceral tissues and endothelial, perithelial, and muscle cells. Accumulation of the neutral glycosphingolipids in the vasculature results in narrowing and dilatation of the blood vessels, and ultimately to ischemia and infaraction.

[0075] Administration of α-galactosidase A conjugates may result in a reduction in one or more clinical symptoms of Fabry disease including, e.g., acroparesthesia, angina, angiokeratoma, arrythmia, ataxia of gait, burning and/or tingling pain in the hands and feet, cataracts, cold intolerance, conduction abnormalities, corneal whorling, coronary artery disease, dementia, depression, diarrhea, dilated cardiac chambers, dizziness, cardiomegaly, cardiomyopathy, diplopia, dysarthria, fatigue, fever with elevated erythrocyte sedimentation rate, hearing problems, heart disease, heart valve problems, heat intolerance, hemiataxia, hemiparesis, hypohidrosis, impaired sweating, infaraction, ischemia, joint pain, kidney disease, left ventricular hypertrophy, lenticular abnormalities, lenticular opacity, lipiduria, muscle weakness, myocardial infarction, nausea, nystagmus, pain (e.g., intense pain radiating throughout the body), polydipsia, proteinuria, post- prandial pain, renal failure, retinal abnormalities, ringing in ears, stomach pain, ST-T wave changes, stroke, uremia, valvular disease, vertigo, vomiting, and weakness. Administration of α-galactosidase A conjugates may result in increased α-galactosidase A activity in, e.g., plasma, tears, leukocytes, biopsied tissues, or cultured skin fibroblasts. Administration of α-galactosidase A conjugates may also result in a histologic finding of a reduction (e.g., of at least 10%) or lack of increase of birefringent lipid globules. It may also result in a decrease in lipid globules in urinary sediment, improved renal function as measured by serum creatinine levels or creatinine clearance, and reduced proteinuria. Administration of α-galactosidase A conjugates may also result in a reduction in GL3 inclusions in the capillary endothelium of the kidney, heart, and skin. Additional assays for measuring efficacy of treatment for Fabry disease can be found in, e.g., MacDermott et al., J. Med. Genet. 38:750-760 (2001).

[0076] In yet another aspect, conjugates of acid sphingomyelinase are administered for treatment of Niemann-Pick disease, or acid sphingomyelinase deficiency. Administration of acid sphingomyelinase conjugates may result in a reduction in one or more clinical symptoms of Niemann-Pick disease including, e.g., abnormal cholesterol levels, abnormal lipid levels, ataxia, blood abnormalities, cherry red spots in the eye, frequent lung infections, growth retardation, hepatosplenomegaly, low numbers of platelets, lymphadenopathy, peripheral neuropathy, problems with lung function, shortness of breath, skin pigmentation changes, or xanthomas. In some embodiments, conjugates may be administered intracranially.

[0077] An alternative embodiment relates to treatment of mucopolysaccharidosis I (including, e.g., Hurler and Hurler-Scheie forms of MPS I) with conjugates comprising α-L-iduronidase. Administration of α-L-iduronidase conjugates may result in a reduction in one or more clinical symptoms of MPS I including, e.g., aortic regurgitation, aortic stenosis, carpal tunnel syndrome, chronic rhinitis, conductive hearing loss, constipation, corneal clouding, developmental delay, diarrhea, distended abdomen, dorsolumbar kyphosis, gibbus deformity of the back, hepatosplenomegaly, hydrocephalus, inguinal hernia, kyphosis, mental retardation, mitral regurgitation, mitral stenosis, night-blindness, open-angle glaucoma, poor hand function, progressive arthropathy, recurrent respiratory infections, respiratory insufficiency, retinal degeneration, scoliosis, sensorineural hearing loss, severe back pain, rhinorrhea, sleep apnea, spinal cord compression, thenar atrophy, umbilical hernia, and upper airway complications. Pharmaceutical Compositions

[0078] This disclosure provides the use of a conjugate of the invention in the manufacture of a medicament for treating a lysosomal storage disorder. It also provides pharmaceutical compositions comprising an oligosaccharide-protein conjugate of the invention. In some embodiments, the pharmaceutical compositions of the invention comprise a conjugate of an oligosaccharide comprising at least one M6P and a lysosomal enzyme.

[0079] Pharmaceutical compositions of the invention may comprise one or more suitable pharmaceutical excipients. Standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art (see, e.g., 2005 Physicians' Desk Reference®, Thomson Healthcare: Montvale, NJ, 2004; Remington: The Science and Practice of Pharmacy, 20th ed., Gennado et al., Eds. Lippincott Williams & Wilkins: Philadelphia, PA, 2000. The compositions may or may not contain preservatives. In some embodiments, pharmaceutical compositions comprising α-galactosidase A conjugates may comprise one or more excipients such as, e.g., mannitol, sodium phosphate monobasic monohydrate, and/or sodium phosphate dibasic heptahydrate. In other embodiments, pharmaceutical compositions comprising conjugates of α-glucosidase may comprise one or more excipients such as, e.g., mannitol, polysorbate 80, sodium phosphate dibasic heptahydrate, and sodium phosphate monobasic monhydrate.

[0080] The pharmaceutical composition may comprise any of the conjugates described herein either as the sole active compound or in combination with another compound, composition, or biological material. For example, the pharmaceutical composition may also comprise one or more small molecules useful for the treatment of a LSD and/or a side effect associated with the LSD. In some embodiments, the composition may comprise miglustat and/or one or more compounds described in, e.g., U.S. Patent Application Publication Nos. 2003/0050299, 2003/0153768; 2005/0222244; 2005/0267094.

[0081] The formulation of pharmaceutical compositions may vary depending on the intended route of administrations and other parameters (see, e.g., Rowe et al. Handbook of Pharmaceutical Excipients, 4th ed., APhA Publications, 2003.) In some embodiments, the composition may be a sterile, non-pyrogenic, white to off-white lyophilized cake or powder to be administered by intravenous injection upon reconstitution with Sterile Water for Injection, USP. [0082] Administration of a pharmaceutical composition of the invention is not limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intracranial, intramedullary, intraarticular, intramuscular, intrathecal, or intraperitoneal injection), transdermal, or oral (for example, in capsules, suspensions, or tablets). Administration to an individual may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition.

[0083] The conjugates described herein are administered in therapeutically effective amounts. Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition in the subject. The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in vitro (i.e., cell cultures) or in vivo (i.e., experimental animal models), e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (or therapeutic ratio), and can be expressed as the ratio LD50/ED50. Conjugates that exhibit therapeutic indices of at least 1 , 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 20 are described herein. Conjugates that exhibit a large therapeutic index are preferred.

[0084] The data obtained from in vitro assays and animal studies, for example, can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with low, little, or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any conjugate used in the present invention, the therapeutically effective dose can be estimated initially from in vitro assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test conjugate which achieves a half-maximal inhibition of symptoms) as determined in in vitro experiments. Levels in plasma may be measured, for example, by high performance liquid chromatography or by an appropriate enzymatic activity assay. The effects of any particular dosage can be monitored by a suitable bioassay of endpoints.

[0085] Unless otherwise indicated, conjugates of the invention may be administered at a dose of approximately from 1 mg/kg to 500 mg/kg, depending on the severity of the symptoms and the progression of the disease. For example, proteinaceous compounds may be administered by slow intravenous infusion in an outpatient setting every, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days, or by, e.g., weekly, biweekly, monthly, or bimonthly administration. The appropriate therapeutically effective dose of a compound is selected by a treating clinician and would range approximately from 1 mg/kg to 500 mg/kg, from 1 mg/kg to 10 mg/kg, from 1 mg/kg to 1 mg/kg, from 10 mg/kg to 1 mg/kg, from 10 mg/kg to 100 mg/kg, from 100 mg to 1 mg/kg, and from 500 mg/kg to 5 mg/kg.

[0086] For example, conjugates of α-galactosidase A may be administered by intravenous infusion at a dose of, e.g., 1.0 mg/kg body weight every two weeks at an infusion rate of, e.g., less than or equal to 10, 13, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 mg/hour). For example, conjugates of α-glucosidase may be administered intravenous injection at a dose of, e.g., 20 mg/kg or 40 mg/kg every two weeks, over approximately, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some embodiments, the rate of administration of α-glucosidase may be started at, e.g., 1 mg/kg/hr and then increased by, e.g., 2 mg/kg/hr every 30 minutes, after establishing patient tolerance to the infusion rate, until a maximum of, e.g., 7 mg/kg/hr. Additionally, examples of specific dosages may be found in the Physicians' Desk Reference®.

EXAMPLES

[0087] Conjugation of recombinant human lysosomal enzymes (LEs) with oligosaccharides comprising mannose-6-phosphate, such as, e.g., oligosaccharides of Formula II, by the methods of the invention to produce lysosomal enzyme conjugates (LECs) is expected to result in LECs having improved affinity for the M6P receptor. Conjugation of LEs with oligosaccharides comprising mannose-6- phosphate is not expected to affect the intracellular processing or pharmacokinetic properties of the LECs relative to the unconjugated LEs. Example 1 : Conjugation of Lysosomal Hydrolases with Oligosaccharides of Formula I

[0088] General Considerations. Deprotonated thiolate anions are generally more reactive than protonated thiols, and it may therefore be advantageous, in certain embodiments, to perform conjugation reactions under moderately basic conditions. However, in embodiments where a thiol-reactive group is also an amine-reactive group, such as, e.g., an alkylating agent, it is often desirable that the reaction be conducted under conditions such that cysteine side chain thiol groups are modified selectively over, e.g., lysine side chain amine groups. This may be achieved at a pH at which a significant fraction of cysteines are

deprotonated, and at which lysine side chain amine groups are largely protonated and unreactive (e.g., at pH 7.0-8.0).

[0089] In certain embodiments, disulfide bonds in the protein may be reduced so as to provide free thiol groups for reaction with a thiol-reactive group. For example, disulfide groups may be reduced by the addition of a molar excess of a reducing agent such as dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine, glutathione, tris-(2-carboxyethyl)phosphine), or tris-(2-cyanoethyl)phosphine. Thiol containing reducing groups such as, e.g., dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine, and glutathione should be removed prior to reaction of the

protein with a thiol-reactive group, so as not to compete with cysteine thiols. Excess reducing agent may be removed by, e.g., dialysis or gel filtration or reversed phase HPLC. The protein may be maintained in a reduced state by performing further manipulations under anaerobic conditions. For example, buffers may be degassed and the solutions stored under an inert atmosphere (e.g., nitrogen or argon).

Moreover, metal chelators such as EDTA may be added to buffers to remove metal ions that may catalyze cysteine re-oxidation. [0090] Conjugation. The LE is dissolved (e.g., at 50-100 μM) in a suitable buffer at pH 7.0-7.5 (e.g., phosphate, Tris, or HEPES). Oligosaccharide (equimolar or 1.1 -100-fold molar excess, in a 1 -10 mM stock in degassed aqueous buffer or in a suitable organic solvent such as, e.g., dimethylsulfoxide or acetonitrile) is added. The mixture is stirred, bubbled, or otherwise agitated at room temperature or at 4 ⁰C. Progress of the reaction may be monitored by, e.g., measuring levels of free thiol (e.g., by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid), also known as Ellman's reagent), or by HPLC monitoring of aliquots of the reaction mixture. The reaction may be quenched by lowering the pH or by the addition of excess thiol (e.g., 1 ,2- ethanedithiol, 2-mercaptoethanol, or glutathione). The conjugate is then purified prior to use, e.g., by gel filtration or reversed phase HPLC.

Example 2: In Vitro Characterization of Lysosomal Enzyme Conjugates (LECs) [0091] Detection of M6P. The extent of oligosaccharide conjugation is measured by assaying LECs for binding to a M6P receptor column to which LEs lacking M6P do not bind. For example, five micrograms of LEC are loaded onto a pre-equilibrated CI-MPR-Sepharose column (the column is prepared by coupling Cl- MPR isolated from bovine serum to NHS-Sepharose), which is then washed with equilibration buffer and eluted with a 0-20% linear gradient of equilibration buffer containing 5 mM M6P. The column is then washed with equilibration buffer containing 5 mM M6P to elute any LEC still bound to the column. Fractions are collected and assayed for enzymatic activity.

[0092] Western Blot Analysis. Samples are boiled in 6x Laemmli sample buffer (350 mM Tris-HCI (pH 6.8), 0.1 mM Bromphenol Blue, 350 mM sodium dodecylsulfate, 9% glycerol) + β-mercaptoethanol and separated by SDS-PAGE. Proteins are transferred to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane with a semidry blot apparatus. The membrane is blocked in TBS-T (2% BSA, 50 mM Tris, 150 mM NaCI₁ 0.05% Tween 20) for 1 hour, and then incubated with a suitable primary antibody (e.g., mouse, goat or rabbit anti-LE antibody) at 5 μg/ml in 2% BSA TBS-Tween for 1 hour at room temperature. The membrane is washed for twenty minutes three times with TBS-T, followed by application of a horseradish peroxidase (HRP)-conjugated secondary antibody (e.g., anti-mouse, anti-goat, or anti-rabbit secondary antibody) at a 1 : 15,000 dilution in 2% BSA TBS-T for 1 hour at room temperature. The membrane is then washed for twenty minutes three times with TBS-T and protein bands detected by the development of a chromophore using an HRP detection system (e.g., visual or chemiluminescence).

[0093] Specific Activity. Enzymatic activity is measured using a spectrometric (e.g., UV/vis or fluorescence) assay in black 96-well microtiter plates using a suitable substrate. For example, substrates may comprise the fluorophore 4-methylumbeHiferone (4MU). Dilutions of the LEC are added in triplicate to a microtiter plate. Substrate is added to each sample. The 96-well black plate is incubated in a 37 ⁰C incubator for 30 minutes. The release of product is detected spectrophotometrically, and compared to standard curves generated by measuring the absorbance or fluorescence of a known quantity of the product. The reaction is quenched by the addition of 125 μl_ of 1.0 M glycine pH 10.5 to all wells. The protein concentration is measured by UV spectroscopy at 280 nm using an appropriate extinction coefficient.

Example 3: In Vitro Characterization of GAA Conjugates

[0094] Specific Activity. GAA activity is measured using a fluorometric assay in black 96-well microtiter plates using 4-methylumbelliferyl-α-D-glucoside as a substrate. Dilutions of GAA are added in triplicate to a microtiter plate. 4-methylumbelliferyl-α-D-glucoside is added to each sample. The 96-well plate is incubated in a 37 ⁰C incubator for 30 minutes. The release of product is detected fluorometrically, and compared to standard curves generated by measuring the absorbance or fluorescence of a known quantity of the product. The reaction is quenched by the addition of 125 μL of 1.0 M glycine pH 10.5 to all wells. The protein concentration is measured by UV spectroscopy at 280 nm using an extinction coefficient of 1.51 mg/ml cm^"1. The specific activity is defined as nmol product released/hr/mg.

[0095] Internalization By L6 Myoblasts. Cells (ATCC CRL-1458) are seeded into 6-well plates at 5.0x10⁵ cells/well in growth media (DMEM + 10% FBS) and grown to confluency. Cells are incubated with 0-25 nM GAA (LEC or unconjugated rhGAA) for 16 hours in Ham's F-10 + 10% HI-FBS + 3 mM Pipes pH 6.7, as in Reuser et al., Exp. Cell Res. 155:178-189 (1984). Non-internalized GAA is inactivated by the addition of 0.2 ml 1.5 M Tris pH 9.0 for 30 minutes. Media pH is then adjusted to approximately 6.8, and cells are washed 2x with DPBS. Cells are lysed with 0.25% Triton X-100 for 1 hour at 37 ⁰C. Lysates are centrifuged at 1800Og for 5 minutes and tested for specific activity.

[0096] Determination of Enzymatic Parameters. Aliquots of diluted GAA (4 μL) are transferred to 0.5 mL microcentrifuge tubes containing 496 μL of bovine liver glycogen covering a range of concentrations, 0-40 mg/ml, mixed, transferred to low volume glass inserts (-400 μL) and equilibrated for 15 minutes at 37 ⁰C prior to LC analysis to determine the concentration of glucose released. The change in glucose over time is used to calculate the rate of glycogen hydrolysis at different substrate concentrations. Enzymatic parameters are calculated by standard Michaelis-Menton equations. [0097] Intracellular Processing of GAA Conjugates. Intracellular processing of is measured by incubating cultured rat L6 myoblasts with the LEC for 16 hours. Cells are harvested, washed and lysed with detergent. Cell lysates are analyzed on a 4-20% SDS-PAGE gel, transferred to PVDF membrane and blotted

using anti-GAA antibody.

Example 4: In Vivo Characterization of GAA Conjugates in Pompe Knockout Mouse

[0098] GAA Activity Determination in Serum Samples. Mice are anesthetized using isoflurane, and a blood sample is collected via tail clip for background GAA level determination. While anesthetized, the mice are administered a 10 mg/kg bolus injection of GAA via tail vein. As the mice recover from anesthesia, blood samples are collected at the following time points: 2, 5, 10, 15, 30, 60, 120, 240, and 360 minutes. Each blood sample is collected from the clipped tail into heparanized capillary tubes and transferred into pre-labeled 0.5 ml microcentrifuge tube. If reduced blood flow requires that the tail be clipped again, the mouse is anesthetized. The samples are immediately placed onto ice and then centrifuged at 14,000 rpm for 5 minutes. The plasma is transferred to a clean 0.5 ml microcentrifuge tube and stored at -80 ⁰C.

[0100] Plasma samples are diluted 1 :100, 1 :1000, and 1 :10,000 in 200 mM potassium acetate, 1.0 mg/ml BSA, 0.02% sodium azide, pH 4.0. Twenty five microliters of the diluted plasma is transferred in triplicate to a 96 well black sample

plate. An equal volume of 1.0 mM 4-methylumbelliferyl-α-D-glucoside in 200 mM

potassium acetate, 0.02% sodium azide, pH 4.0 is added to each sample. A five point 4-methylumbelliferyl curve (10 - 0.16 nmol/mL) is included on each plate. Each plate is incubated for 60 minutes at 37 ⁰C and the reaction is quenched by the addition of 125 μl_ of 1.0 M glycine, pH 10.5 to all wells. Fluorescence is measured (360 nm ex, 455 nm em, and 435 nm cutoff) and converted to nmol/ml/hr using the standard curve. The activity numbers are converted to nmol 4-MU released/L plasma. Fluorescence levels (nmol/L) for each pre-dose sample is then subtracted from each time point to remove any non-specific fluorescence.

[0101] Clearance of Glycogen From Pompe KO Mouse Tissues. Pompe KO mice are injected via the tail vein with varying doses of GAA. Mice are administered three weekly doses of about 10 mg/kg, 20 mg/kg, or 50 mg/kg and euthanized two weeks after the last treatment. Tissues including the heart, diaphragm and skeletal muscles are collected and stored at -80⁰C until further analysis.

[0102] The glycogen content in the various muscles of the Pompe mice is assayed by measuring the difference in the amount of glucose released from a boiled tissue homogenate following digestion in presence or absence of Aspergillus niger amyloglucosidase, e.g., as described in Amalfitano et al., Proc. Natl. Acad. Sci. U.S.A. 96:8861-8866 (1999). Glucose levels are assayed using the Amplex Red glucose assay kit (Molecular Probes, Eugene, OR), according to the manufacturer's instructions. Bovine liver glycogen (Sigma Chemical Co.) is used as a standard. [0103] Glycogen content is measured using periodic acid Schiff (PAS) staining followed by computer-assisted histomorphometric analysis (Metamorph) as described in Raben et al., MoI. Genet. Metab. 80:159-169 (2003)). Higher enzyme levels are expected to be detected in the skeletal muscle and heart tissues in animals administered Neo-GAA2 relative to untreated mice and to mice treated with rhGAA. [0104] Tissue samples are stained for lysosomal glycogen followed by analysis of tissues by high resolution light microscopy (HRLM). Lysosomal glycogen appears as discrete, purple beaded structures scattered throughout each myocyte. These glycogen-containing structures are expected to decrease in size and number upon administration of the oligosaccharide conjugated lysosomal enzyme (LEC).

Example 5: In Vitro Characterization of ASM Conjugates

[0105] Specific Activity. ASM activity is measured using an assay for

ASM activity, e.g., as described in He et al., Anal. Biochem. 314:116-20 (2003). [0106] The specification is most thoroughly understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited and sequences identified by accession or database reference numbers in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[0107] Unless otherwise indicated, all numbers expressing quantities of ingredients, treatment conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0108] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A lysosomal enzyme-oligosaccharide conjugate comprising a lysosomal enzyme and an oligosaccharide comprising mannose-6-phosphate, wherein the oligosaccharide is connected to the sulfur atom of a cysteine residue.

2. The conjugate of claim 1 , wherein the lysosomal enzyme is α-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-glucosidase, acid β-galactosidase, iduronate-2-sulfatase, α-L-iduronidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, arylsulfatase B, arylsulfatase A, /V-acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, acid sphingomyelinase, acid α-glucosidase, β-hexosaminidase B, heparan /V-sulfatase, α-A/-acetylglucosaminidase, acetyl-CoA:α-glucosaminide Λ/-acetyltransferase, /V-acetylglucosamine-6-sulfate sulfatase, α-A/-acetylgalactosaminidase, sialidase, β-glucuronidase, or β-hexosaminidase A.

3. The conjugate of claim 2, wherein the lysosomal enzyme is acid α-glucosidase, α-galactosidase A, acid sphingomyelinase, or α-L-iduronidase.

4. The conjugate of claim 3, wherein the lysosomal enzyme is acid α-glucosidase.

5. The conjugate of claim 1 , wherein the oligosaccharide comprises a tetrasaccharide.

6. The conjugate of claim 1 , wherein the oligosaccharide comprises a terminal or penultimate mannose-6-phosphate.

7. The conjugate of claim 1 , wherein the oligosaccharide is

8. The conjugate of claim 1 , wherein the oligosaccharide is connected to the sulfur atom of a cysteine residue through a disulfide group.

9. The conjugate of claim 1 , wherein the oligosaccharide is connected to the sulfur atom of a cysteine residue through a thioether group.

10. The conjugate of claim 1 , wherein the conjugate further comprises a linker between the reducing end of the oligosaccharide and the sulfur of the thiol group, the linker comprising at least one chemical group chosen from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted amido, ester, keto, ether, thioether, and optionally substituted amino.

11. A method of making a lysosomal enzyme-oligosaccharide conjugate comprising a lysosomal enzyme and an oligosaccharide comprising mannose-6-phosphate, wherein the oligosaccharide is connected to the sulfur atom of a cysteine residue, the method comprising:

(a) providing a lysosomal enzyme having at least one cysteine thiol group; (b) providing an oligosaccharide comprising mannose-6-phosphate and having a thiol-reactive group at the reducing end; and (c) reacting the thiol group of the lysosomal enzyme and the thiol-reactive group of the oligosaccharide, thereby yielding a lysosomal enzyme-oligosaccharide conjugate.

12. A method of making a lysosomal enzyme-oligosaccharide conjugate comprising a lysosomal enzyme and an oligosaccharide comprising mannose-6-phosphate, wherein the oligosaccharide is connected to the sulfur atom of a cysteine residue, the method comprising:

(a) providing a lysosomal enzyme having at least one cysteine thiol group;

(b) reacting the thiol group of the lysosomal enzyme with an activating reagent to produce a lysosomal enzyme having at least one thiol-reactive group;

(c) providing an oligosaccharide comprising mannose-6-phosphate and having a thiol group at the reducing end; and

(d) reacting the thiol-reactive group of the lysosomal enzyme and the thiol group of the oligosaccharide, thereby yielding a lysosomal enzyme-oligosaccharide conjugate.

13. A lysosomal enzyme-oligosaccharide conjugate prepared according to claim 11 or claim 12.

14. A method of treating a lysosomal storage disorder, comprising administering the conjugate of claim 13, thereby treating the lysosomal storage disorder,

15. The method of claim 14, wherein the lysosomal storage disorder is Pompe disease, Fabry disease, Niemann-Pick disease, or mucopolysaccharidosis I.

16. The method of claim 15, wherein the lysosomal storage disorder is Pompe disease.

17. The method of claim 14, further comprising administering methotrexate to the mammal before, after, or during treatment with the conjugate.

18. Use of a conjugate of any of claims 1 -10 or 13 for treating a lysosomal storage disorder in a subject in need thereof.

19. Use of a conjugate of any of claims 1 -10 or 13 in the manufacture of a medicament for treating a lysosomal storage disorder.