Preparation of active highly phosphorylated human lysosomal sulfatase enzymes and uses thereof

Abstract

The invention relates to the preparation of an active highly phosphorylated human lysosomal sulfatase enzyme and an application thereof. The present invention provides, inter alia, compositions of active highly phosphorylated lysosomal sulfatase enzymes, pharmaceutical compositions thereof, methods of producing and purifying such lysosomal sulfatase enzymes, and their use in the diagnosis, prevention or treatment of diseases and disorders, including, inter alia, lysosomal storage disorders caused by or associated with a lack of lysosomal sulfatase enzymes.

Description

[0001] This application claims priority to the following prior applications: U.S. Provisional Patent Application No. 61 / 022,179, filed January 18, 2008; U.S. Provisional Patent Application No. 61 / 099,373, filed September 23, 2008; and U.S. Provisional Patent Application No. 61 / 110,246, filed October 31, 2008, the descriptions of which are incorporated herein by reference. Invention Field

[0002] This invention relates to the fields of cell and molecular biology and medicine, and particularly to the preparation of highly phosphorylated human lysosomal sulfatase and its application in controlling lysosomal storage diseases associated with lysosomal sulfatase deficiency. Specifically, this invention relates to the preparation of highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and its application in controlling mucopolysaccharidosis type IVa (MPS IVa or Molgio A syndrome) and other lysosomal storage diseases associated with GALNS deficiency. Background Technology

[0003] A deficiency of specific lysosomal enzymes, crucial for the degradation of cellular waste within lysosomes, leads to lysosomal storage disorders (LSD). This deficiency results in the accumulation of undegraded "storage material" within lysosomes, causing lysosomal swelling and dysfunction, ultimately leading to cell and tissue damage. Numerous lysosomal enzymes have now been identified and are associated with related diseases. Once the missing enzyme is identified, treatment can be simplified to the single problem of effectively delivering a replacement enzyme to the patient's affected tissues.

[0004] One approach to treating lysosomal storage disorders is intravenous enzyme replacement therapy (ERT) (Kakkis, Expert Opin. Investig. Drugs 11(5):675-685, 2002). ERT utilizes the vascular system to carry enzymes from a single site of administration to most tissues. Once the enzyme is widely distributed, it must be taken up into cells. The unique characteristics of lysosomal enzymes are the basis for their uptake into cells. Lysosomal enzymes are a separate class of glycoproteins defined by the phosphate at the 6-position of the terminal mannose residue. Receptors found on most cell surfaces bind with high affinity and specificity to mannose-6-phosphate (Munier-Lehmann et al., Biochem. Soc. Trans. 24(1):133-136, 1996; Marnell et al., J. Cell. Biol. 99(6):1907-1916, 1984). Each polypeptide chain of the mannose-6-phosphate receptor (MPR) has two mannose-6-phosphate binding sites (Tong et al., J. Biol. Chem. 264:7962-7969, 1989). This receptor directs the uptake of enzymes from the blood to tissues and then mediates intracellular orientation to lysosomes.

[0005] Large-scale production of lysosomal enzymes involves expression in mammalian cell lines. The goal is to make the recombinant enzymes primarily secreted into the surrounding growth environment for collection and downstream processing. In an ideal system for large-scale production of lysosomal enzymes, the enzyme is efficiently phosphorylated and then directed primarily to the cell surface (i.e., secretion), rather than primarily to the lysosome. As mentioned above, this distribution of phosphorylated lysosomal enzymes is exactly the opposite of what occurs in normal cells. Cell lines for the production of lysosomal enzymes have been developed to maximize the level of mannose-6-phosphate in the enzyme molars, but are characterized by low specific productivity. In vitro efforts to produce lysosomal enzymes containing high levels of the mannose-6-phosphate moiety have yielded paradoxical success (Canfield et al., U.S. Patent No. 6,537,785). The in vitro enzymes exhibit high levels of mannose-6-phosphate as well as high levels of unmodified terminal mannose. Competition for lysosomal enzymes between mannose-6-phosphate and mannose receptors leads to the need for high doses of enzyme to produce potency, and also results in higher immunogenicity that is detrimental to the treated subjects.

[0006] Sulfatases constitute a unique subclass of lysosomal enzymes. Sulfatases cleave sulfates from a variety of substrates, including, for example, steroids, carbohydrates, proteoglycans, and glycolipids. All known eukaryotic sulfatases contain a cysteine ​​residue at their catalytic site. Sulfatase activity requires post-translational modification of this cysteine ​​residue to form a C-type nucleotide. α-Formylglycine (FGly). Post-translational activation of cysteine ​​to FGly occurs within the endoplasmic reticulum, immediately after translation, before sulfatase targeting the lysosome (Dierks et al., Proc. Natl. Acad. Sci. USA 94:11963-11968, 1997). The formylglycine-producing enzyme catalyzing this reaction is sulfatase modifier 1 (SUMF1). The importance of this unique post-translational modification is highlighted because mutations in SUMF1 impair FGly formation in lysosomal sulfatases, leading to multiple sulfatase deficiency (MSD) in humans (Diez-Ruiz et al., Annu. Rev. Genomics Hum. Genet. 6:355-379, 2005).

[0007] Therefore, the efficacy of lysosomal sulfatase products depends on the level of mannose-6-phosphate and the presence of active enzymes in the product.

[0008] Therefore, there is a need in the art for efficient production systems to produce on a large scale therapeutically effective, active, highly phosphorylated lysosomal sulfatases that can control lysosomal storage disorders caused by or associated with the deficiency of such lysosomal sulfatases. Invention Overview

[0009] This invention relates to the discovery that when an intron acidification-deficient CHO-K1 cell line derivative (designated G71) is engineered to express recombinant human sulfatase modifier 1 (SUMF1), the modified G71 cell portion produces a large amount of highly phosphorylated recombinant lysosomal sulfatase by preventing the loss of material from the lysosomal compartments of the producing cell line. In one embodiment, the END3 complementary group cell lines provided by this invention co-express recombinant human SUMF1 and recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), resulting in a high yield of highly phosphorylated enzyme. Exemplary cell lines are G71, G71S, and derivatives thereof that retain the desired characteristic of G71, namely, the ability to produce large amounts of highly phosphorylated recombinant lysosomal sulfatase. Such applications of END3 complementary group modified CHO-K1 cell lines co-expressing recombinant human SUMF1 and recombinant lysosomal sulfatase are particularly useful for the preparation of highly phosphorylated lysosomal sulfatase for the control of lysosomal storage diseases via enzyme replacement therapy (ERT).

[0010] In a first aspect, the present invention is characterized by a novel method for producing highly phosphorylated recombinant human lysosomal sulfatase or its biologically active fragments, mutants, variants or derivatives thereof in END3 complementary CHO cell lines or derivatives thereof, in quantities suitable for their therapeutic applications. In a broad implementation, the method includes the following steps: (a) culturing CHO-derived END3 complementary group cells or derivatives thereof; (b) preparing a first mammalian expression vector capable of expressing active highly phosphorylated recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof in CHO-derived END3 complementary group cells or derivatives thereof; (c) preparing a second mammalian expression vector capable of expressing recombinant human sulfatase modification factor 1 (SUMF1) or a biologically active fragment, mutant, variant, or derivative thereof in CHO-derived END3 complementary group cells or derivatives thereof; (d) transfecting the CHO-derived END3 complementary group cells or derivatives thereof with the first and second expression vectors; (e) selecting and cloning transfectants of CHO-derived END3 complementary group cells or derivatives thereof expressing active highly phosphorylated recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof; and (f) optimizing cell culture methods to produce highly phosphorylated recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof. The recombinant human lysosomal sulfatases were selected from: arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS).

[0011] The method includes the following steps: transfecting cDNA encoding all or part of lysosomal sulfatase and cDNA encoding all or part of human SUMF1 into CHO-derived END3 complementary cells or derivatives thereof. In some embodiments, the first and second expression vectors capable of expressing the encoded active, highly phosphorylated recombinant human lysosomal sulfatase and human SUMF1, respectively, are simultaneously transfected into CHO-derived END3 complementary cells or derivatives thereof. In some embodiments, the first and second expression vectors are sequentially transfected into CHO-derived END3 complementary cells or derivatives thereof. In some embodiments, cDNA encoding full-length human lysosomal sulfatase is used, while in other embodiments, cDNA encoding a biologically active fragment, mutant, variant, or derivative thereof is used. In some embodiments, cDNA encoding full-length human SUMF1 is used, while in other embodiments, cDNA encoding a biologically active fragment, mutant, variant, or derivative thereof is used. In some embodiments, human lysosomal sulfatase and human SUMF1 cDNA are simultaneously or sequentially delivered into CHO-derived END3 complementary cells or derivatives thereof using multiple expression vectors. In some embodiments, human lysosomal sulfatase and human SUMF1 cDNA are simultaneously or sequentially delivered into CHO-derived END3 complementary cells or derivatives thereof using a single expression vector. In a preferred embodiment, the CHO-derived END3 complementary cells or derivatives thereof are G71 cell lines, G71S cell lines, or G71 or G71S derivatives.

[0012] In a preferred embodiment, the method includes generating an active, highly phosphorylated recombinant human lysosomal sulfatase, such as arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), or N-acetylgalactosamine-6-sulfatase (GALNS), from an END3 complementary group CHO cell line or a derivative thereof. In a particularly preferred embodiment, the method includes generating an active, highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), from an END3 complementary group CHO cell line or a derivative thereof. END3 complementary group cell lines are any modified CHO cell lines that retain the characteristics of END3 complementary group cell lines, such as intron acidification-deficient cells. In a preferred embodiment, the CHO-derived END3 complementary group cell line or a derivative thereof is a G71 cell line, a G71S cell line, or a G71 or G71S derivative.

[0013] In a second aspect, the present invention provides intron acidification-deficient mammalian cell lines characterized by the ability to produce highly phosphorylated recombinant human lysosomal sulfatase, the production of which is sufficient for therapeutic utilization of lysosomal sulfatase. In a preferred embodiment, the present invention provides CHO-K1-derived END3 complementary cell lines designated as G71, G71S, or derivatives thereof, which produce large quantities of highly phosphorylated recombinant human lysosomal sulfatase, thereby enabling large-scale production of such therapeutic lysosomal sulfatase. In a more preferred embodiment, the cell line expresses and secretes at least about 0.5, preferably at least about 0.75, more preferably at least about 1.0, and even more preferably at least about 1.25 picograms / cell / day of recombinant human lysosomal sulfatase.

[0014] END3 complementary cell lines are modified CHO cell lines that retain the characteristics of END3 complementary cell lines, such as intron acidification-deficient ones. In one embodiment, the END3 complementary CHO cell line is derived from G71 or a derivative thereof and comprises (a) an expression vector for recombinant human sulfatase modification factor 1 (SUMF1) and (b) an expression vector for recombinant human lysosomal sulfatase, wherein the recombinant human lysosomal sulfatase is selected from: arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS). In a preferred embodiment, the END3 complementary CHO cell line comprises an expression vector for recombinant human N-acetylgalactosamine-6-sulfatase (GALNS). In a more preferred embodiment, the END3 complementary CHO cell line expresses and secretes recombinant human GALNS. In another preferred embodiment, the END3 complementary CHO cell line is selected from: clone 4, clone 5, clone C6, clone C2, clone C5, clone C7, clone C10, clone C11, and clone C30. In a more preferred embodiment, the END3 complementary CHO cell line is clone C2. In another preferred embodiment, the END3 complementary CHO cell line is selected from those suitable for suspension growth.

[0015] In a third aspect, the present invention provides a recombinant human lysosomal sulfatase produced according to the method of the present invention, wherein the amount of the lysosomal sulfatase is sufficient for therapeutic application. The lysosomal sulfatase may be a full-length protein, or a fragment, mutant, variant, or derivative thereof. In some embodiments, the lysosomal sulfatase of the present invention, or a fragment, mutant, variant, or derivative thereof, may be modified as needed to improve its stability or pharmacokinetic properties (e.g., PEGylation, mutagenesis, fusion, conjugation). In a preferred embodiment, the enzyme is a lysosomal sulfatase, a fragment of human lysosomal sulfatase having the biological activity of a native sulfatase, or a polypeptide substantially homologous to the amino acid sequence of human lysosomal sulfatase. In some embodiments, the lysosomal sulfatase is a protein of human or mammalian sequence, origin, or source. In other embodiments, the lysosomal sulfatase is the enzyme whose deficiency leads to human diseases such as metachromatic leukodystrophy or MLD (i.e., arylsulfatase A (ARSA)), Mar-Lan II syndrome or MPS VI (i.e., arylsulfatase B (ARSB)), Hunt syndrome or MPS II (i.e., iduronic acid-2-sulfatase (IDS)), Schennfeld A syndrome or MPS IIIa (i.e., sulfonamide / heparin-N-sulfatase (SGSH)), Schennfeld D syndrome or MPS IIId (i.e., N-acetylglucosamine-sulfatase (G6S)), and Morcho A syndrome or MPS IVa (i.e., N-acetylgalactosamine-6-sulfatase (GALNS)). In a particularly preferred embodiment, the lysosomal sulfatase is the enzyme whose deficiency leads to Morcho A syndrome or MPS IVa (i.e., N-acetylgalactosamine-6-sulfatase (GALNS)). In another particularly preferred embodiment, the lysosomal sulfatase is an enzyme whose deficiency is associated with human diseases such as multiple sulfatase deficiency or MSD (i.e., N-acetylgalactosamine-6-sulfatase (GALNS)).

[0016] Lysosomal sulfatases can also be of human or mammalian sequence origin. In other embodiments of the invention, the amino acid sequence of the lysosomal sulfatase is identical in various respects to the corresponding portion of the amino acid sequence of a human or mammalian lysosomal sulfatase. In other embodiments, the polypeptide portion is a native human or mammalian lysosomal sulfatase. In other embodiments, the lysosomal sulfatase polypeptide is substantially homologous to the amino acid sequence of a native human or mammalian lysosomal sulfatase over at least about 25, 50, 100, 150, or 200 amino acids (i.e., the amino acid sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical). In other embodiments, the lysosomal sulfatase is administered to a human.

[0017] In a preferred embodiment, the lysosomal sulfatase is a highly phosphorylated recombinant human lysosomal sulfatase produced by an intron acidification-deficient cell line, such as a CHO-derived END3 complementary group cell line. The END3 complementary group cell line is any modified CHO cell line that retains the characteristics of an END3 complementary group cell line, such as an intron acidification-deficient cell line. In a preferred embodiment, the CHO-derived END3 complementary group cell line or a derivative thereof is a G71 cell line, a G71S cell line, or a G71 or G71S derivative.

[0018] In a more preferred embodiment, the recombinant human lysosomal sulfatase has a high level of phosphorylated oligosaccharides (i.e., more than about 0.25, preferably more than 0.5, more preferably more than about 0.75, di-phosphorylated oligomannose chains per protein chain). In an even more preferred embodiment, the enzyme is a highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

[0019] In a more preferred embodiment, a high percentage (i.e., at least about 50%, preferably at least about 70%, more preferably at least about 90%, and even more preferably at least about 95%) of the active site cysteine ​​residues of the recombinant human lysosomal sulfatase are converted to C α -Formylglycine (FGly). In an even more preferred embodiment, the enzyme is active recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

[0020] In a more particularly preferred embodiment, the recombinant human lysosomal sulfatase has a high level of phosphorylated oligosaccharides (i.e., more than about 0.25, preferably more than 0.5, more preferably more than about 0.75, di-phosphorylated oligomannose chains per protein chain) and a high percentage (i.e., at least about 50%, preferably at least about 70%, more preferably at least about 90%, and even more preferably at least about 95%) of active site cysteine ​​residues converted to C α -Formylglycine (FGly). In the most particularly preferred embodiment, the enzyme is highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

[0021] In a fourth aspect, the present invention provides a method for producing and purifying recombinant human lysosomal sulfatase using the method of the present invention. In a preferred embodiment, lysosomal sulfatase is purified using a dual-column method comprising at least five purification steps: (dye-ligand chromatography, such as Blue-Sepharose, and anion exchange chromatography, such as SE Hi-Cap): (1) filtering the collection, i.e., the culture medium of an END3 complementary group CHO cell line or its derivative expressing human sulfatase modifier 1 (SUMF1) and recombinant human lysosomal sulfatase; (2) adjusting the pH of the filtered collection to pH 4.5 (to induce precipitation of contaminating proteins); (3) loading the pH-adjusted filtered collection onto a dye-ligand column, such as a Blue-Sepharose column, washing the column and eluting the lysosomal sulfatase from the column; (4) loading the eluent from the dye-ligand column onto an anion exchange column, such as an SE Hi-Cap column, washing the column and eluting the lysosomal sulfatase from the column; and (5) ultrafiltration and dialysis of the anion exchange eluent. Optionally, the filtered collection from step (1) can be concentrated 10-20 times by ultrafiltration, and then the pH can be adjusted. Optionally, the lysosomal sulfatase from step (5) can be prepared in a preparation buffer. In a particularly preferred embodiment, the lysosome is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

[0022] In another preferred embodiment, lysosomal sulfatase is purified using a three-column method comprising at least the following five purification steps: (capture chromatography, e.g., anion exchange SE Hi-Cap; intermediate chromatography, e.g., dye-ligand CaptoBlueZinc, chelate agarose FF, or Capto attachment; polishing chromatography, e.g., ToyoPearl Butyl 650M, phenyl agarose Hi-Sub, or phenyl agarose Low-Sub): (1) filtration of the collection, e.g., a culture medium of an END3 complementary group CHO cell line expressing human sulfatase modifying factor 1 (SUMF1) and recombinant human lysosomal sulfatase or its derivatives, via a Sarton box (10 kDa, Hydrosart); (2) pH adjustment of the filtered collection to pH 4.5 (to induce precipitation of contaminating proteins); (3) loading the pH-adjusted filtered collection onto a capture column, e.g., a Fractogel EMD SE column. (3) Wash the Hi-CAP(M) anion exchange column and elute the lysosomal sulfatase from the column; (4) Load the eluent from the capture column onto an intermediate column, such as a dye-ligand Capto BlueZinc, chelate agarose FF, or Capto attachment column, wash the column and elute the lysosomal sulfatase from the column; and (5) Load the eluent onto a varnishing column, such as a ToyoPearl Butyl 650M, phenyl agarose Hi-Sub, or phenyl agarose Low-Sub, wash the column and elute the lysosomal sulfatase from the column. Prepare the lysosomal enzyme eluted in step (5) in a preparation buffer. Optionally, ultrafilter the lysosomal sulfatase eluted in step (5) and prepare it in a preparation buffer. Optionally, contact the lysosomal sulfatase from the column in step (4) to pH 3.5 to inactivate the virus at low pH before loading it onto the varnishing column in step (5). In a particularly preferred embodiment, the lysosome is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

[0023] In a fifth aspect, the present invention provides purified, highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or its biologically active mutants, variants, or derivatives, which may be used to treat subjects suffering from lysosomal storage disorders caused by or related to GALNS enzyme deficiency (e.g., mucopolysaccharidosis IVa (MPS IVa) or Molcio A syndrome) or multiple sulfatase deficiency (MSD)). In a preferred embodiment, the purified, highly phosphorylated recombinant human GALNS: (a) has a purity of at least about 90% as determined by Coomassie blue staining under non-reducing conditions; and (b) at least about 90% of the cysteine ​​residue at position 53 is converted to C. α-formylglycine (FGly); and (c) are N-linked glycosylated at asparagine residues at positions 178 and 397, wherein at least about 50% of the oligomannose chain linked to the asparagine residue at position 178 is di-phosphorylated. When SDS-PAGE is performed under reducing conditions, the purified, highly phosphorylated recombinant human GALNS consists of a major band of about 55-60 kDa (i.e., the precursor human GALNS is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% of the visible protein) and minor bands of about 39 kDa and about 19 kDa (i.e., the mature or processed human GALNS is less than about 25%, preferably less than about 15%, more preferably less than about 10%, and even more preferably less than about 5% of the visible protein). In a particularly preferred embodiment, when SDS-PAGE is performed under reducing conditions, the purified, highly phosphorylated recombinant human GALNS consists essentially of a single band of approximately 55-60 kDa (i.e., precursor human GALNS). In one embodiment, the purified, highly phosphorylated recombinant human GALNS can be used to treat MPS IVa or Molcio A syndrome. In one embodiment, the purified, highly phosphorylated recombinant human GALNS can be used to treat MSD.

[0024] In a sixth aspect, the present invention provides a method for treating diseases caused or related to complete or partial deficiency of lysosomal sulfatase. The method includes administering a therapeutic recombinant human lysosomal sulfatase produced by the method of the present invention, wherein the lysosomal sulfatase binds to an MPR receptor, is transported across the cell membrane, enters the cell, and is delivered to the lysosome within the cell.

[0025] In one embodiment, the method includes treating a subject suffering from lysosomal sulfatase deficiency, comprising administering a therapeutically effective amount of the lysosomal sulfatase to the subject in need, wherein the lysosomal sulfatase is a recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof produced by CHO-derived END3 complementary cells or derivatives thereof. In some embodiments, the method includes administering, alone or in combination with a pharmaceutically acceptable carrier, diluent, or excipient, a therapeutic recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof. Preferred embodiments include optimizing the dosage according to the needs of the subject to be treated, preferably a mammal, and most preferably a human, thereby most effectively improving lysosomal sulfatase deficiency.

[0026] Such therapeutic lysosomal sulfatases are particularly useful for treating, for example, patients with lysosomal storage disorders caused by lysosomal sulfatase deficiency, such as patients with: metachromatic leukodystrophy or MLD, mucopolysaccharidosis type VI (MPS VI) or Marr-Lan syndrome, mucopolysaccharidosis type II (MPS II) or Hunter syndrome, mucopolysaccharidosis type IIIa (MPS IIIa) or Selphys A syndrome, mucopolysaccharidosis type IIId (MPS IIId) or Selphys D syndrome, and mucopolysaccharidosis type IVa (MPS IVa) or Morgio A syndrome. In a particularly preferred embodiment, the lysosomal storage disorder is MPS IVa or Morgio A syndrome, and the lysosomal sulfatase is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS). In other embodiments, the present invention also provides pharmaceutical compositions comprising a lysosomal sulfatase that causes a deficiency of lysosomal storage disease and a pharmaceutically acceptable carrier, diluent, or excipient.

[0027] In another embodiment, the method includes treating a subject suffering from a lysosomal storage disorder associated with a deficiency of one or more lysosomal sulfatases, including administering a therapeutically effective amount of lysosomal sulfatase to the subject in need, wherein the lysosomal sulfatase is recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active fragment, mutant, variant, or derivative thereof produced by CHO-derived END3 complementary cells or derivatives thereof. In some embodiments, the method includes administering a therapeutic recombinant human GALNS enzyme or a biologically active fragment, mutant, variant, or derivative thereof, alone or in combination with a pharmaceutically acceptable carrier, diluent, or excipient. In a particularly preferred embodiment, the lysosomal storage disorder is multiple sulfatase deficiency (MSD).

[0028] In a particularly preferred embodiment, the CHO-derived END3 complementary group cells or their derivatives are G71 cell lines, G71S cell lines or their G71 or G71S derivative lines.

[0029] In another embodiment, the present invention provides an enzyme replacement therapy method in which a subject requiring enzyme replacement therapy is given a therapeutically effective amount of lysosomal sulfatase, such that a sufficient amount of lysosomal sulfatase enters the lysosome to prevent or reduce cell damage, wherein the amount of lysosomal sulfatase contained in the lysosomes of the patient's cells is insufficient to prevent or reduce cell damage. Cells may or may not be within the CNS, or may be separated from the blood without passing through capillary walls, the endothelial cells of the capillary walls being tightly closed to prevent the diffusion of active agents through tight junctions.

[0030] In certain embodiments, the present invention provides compositions and pharmaceutical compositions comprising an active recombinant human lysosomal sulfatase having reduced, insufficient, or absent biological activity in a target lysosome, the enzyme being administered to the subject. Preferred active human lysosomal sulfatases include, but are not limited to: arylsulfatase A, arylsulfatase B, iduronate-2-sulfatase, sulfonamide / heparin-N-sulfatase, N-acetylglucosamine-sulfatase, and N-acetylgalactosamine-6-sulfatase. In a preferred embodiment, the N-acetylgalactosamine-6-sulfatase is an active recombinant human lysosomal sulfatase.

[0031] In a preferred embodiment, the present invention provides a method for treating subjects with MPS IVa, Morgio A syndrome, or MSD by administering a therapeutically effective amount of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), wherein the active site of the recombinant human GALNS has a high level of cysteine ​​residues converted to C α -formylglycine (FGly) (i.e., at least about 50%, preferably at least about 70%, more preferably at least about 90%, and even more preferably at least about 95% conversion) and a high level of phosphorylation (i.e., more than about 0.25, preferably more than 0.5, more preferably more than about 0.75 of the di-phosphorylated oligomannose chain per protein chain).

[0032] In a more preferred embodiment, the present invention provides a method for treating subjects with MPS IVa, Morgio A syndrome, or MSD by administering a therapeutically effective amount of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) produced by END3 complementary group cells, wherein the active site of the recombinant human GALNS is highly converted to C α -formylglycine (FGly) (i.e., at least about 50%, preferably at least about 70%, more preferably at least about 90%, and even more preferably at least about 95% conversion) and a high level of phosphorylation (i.e., more than about 0.25, preferably more than 0.5, more preferably more than about 0.75 of the di-phosphorylated oligomannose chain per protein chain).

[0033] In a particularly preferred embodiment, the present invention provides a method for treating subjects with MPS IVa, Moulciano A syndrome, or MSD by administering a therapeutically effective amount of purified, highly phosphorylated recombinant human GALNS, wherein the GALNS has the following characteristics: (a) a purity of at least about 90% as determined by Coomassie blue staining under non-reducing conditions; and (b) at least about 90% of the cysteine ​​residue at position 53 is converted to C. α-formylglycine (FGly); and (c) are N-linked glycosylated at asparagine residues at positions 178 and 397, wherein at least about 50% of the oligomannose chain linked to the asparagine residue at position 178 is di-phosphorylated. When SDS-PAGE is performed under reducing conditions, the purified, highly phosphorylated recombinant human GALNS consists of a major band of about 55-60 kDa (i.e., the precursor human GALNS is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% of the visible protein) and minor bands of about 39 kDa and about 19 kDa (i.e., the mature or processed human GALNS is less than about 25%, preferably less than about 15%, more preferably less than about 10%, and even more preferably less than about 5% of the visible protein). In a more particularly preferred embodiment, when SDS-PAGE is performed under reducing conditions, the purified active highly phosphorylated recombinant human GALNS consists essentially of a single band of about 55-60 kDa (i.e., precursor human GALNS).

[0034] In some implementations, the subject has MPS IVa or Morquio A syndrome. In some implementations, the subject has MSD.

[0035] The application of the highly phosphorylated lysosomal sulfatase of the present invention in the preparation of medicaments for treating the above-mentioned lysosomal storage diseases is also considered, wherein the enzyme is preferably prepared by the method of the present invention.

[0036] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the above-described highly phosphorylated recombinant human lysosomal sulfatase and one or more pharmaceutically acceptable carriers, diluents, or excipients, the enzyme being used to treat diseases caused or related to complete or partial deficiency of such lysosomal sulfatases. In a preferred embodiment, the pharmaceutical composition comprises a highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active fragment, mutant, variant, or derivative thereof produced by the methods of the present invention, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Such pharmaceutical compositions are suitable for administration via several routes, such as intrathecal, parenteral, topical, intranasal, inhalation, or oral administration. In a preferred embodiment, the pharmaceutical composition is suitable for parenteral administration. Embodiments falling within this scope are characterized by the ability to be administered in vivo to cells affected by lysosomal enzyme deficiency, containing a nucleic acid sequence encoding a full-length lysosomal sulfatase or a fragment, mutant, variant, or derivative thereof.

[0037] In another aspect, the present invention provides a method for detecting lysosomal sulfatase activity, comprising (a) culturing chondrocytes from a patient with lysosomal sulfatase deficiency, such as a patient with Molcio syndrome, under conditions that promote chondrocyte differentiation maintenance; (b) exposing the chondrocytes to a lysosomal sulfatase that degrades keratin sulfate; and (c) detecting the keratin sulfate level in the cells, wherein a decrease in the keratin sulfate level in cells exposed to the lysosomal sulfatase compared to cells not exposed to the lysosomal sulfatase indicates lysosomal sulfatase activity. In some embodiments, the lysosomal sulfatase is N-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, the cells are cultured using a medium containing insulin-like growth factor 1 (IGF1), transforming growth factor β (TGF-β), transferrin, insulin, and ascorbic acid. In some embodiments, keratin sulfate is detected using confocal microscopy or by binding to an anti-keratin sulfate assay. The method can be carried out using any lysosomal sulfatase, including naturally occurring or recombinant human enzymes, or fragments or variants thereof, including variants containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the precursor human enzyme, without a signal sequence, or the mature form thereof.

[0038] In another aspect, the present invention provides a cell assay for detecting the activity of recombinant human lysosomal enzymes that degrade natural substrates. The method comprises (a) culturing isolated human cells lacking the lysosomal enzyme under conditions of accumulation of the natural substrate of the lysosomal enzyme; (b) contacting the cells with the lysosomal enzyme; (c) lysing the cells; (d) adding an enzyme to the cell lysate, said enzyme being: (i) specific to the natural substrate, and (ii) cleaving a small oligosaccharide on the natural substrate; (e) labeling the small oligosaccharide with a detectable portion; (f) optionally separating the labeled small oligosaccharide; (g) detecting the labeled small oligosaccharide; and (h) determining the activity of the lysosomal enzyme in degrading the natural substrate by comparing (i) and (ii): (i) the amount of labeled small oligosaccharide in cells contacted with the lysosomal enzyme and (ii) the amount of labeled small oligosaccharide in cells not contacted with the lysosomal enzyme, wherein a decrease in the amount of labeled small oligosaccharide in (h)(i) compared to (h)(ii) indicates the activity of the lysosomal enzyme in degrading the natural substrate. In one embodiment, the small oligosaccharide is a monosaccharide, disaccharide, or trisaccharide. In some embodiments, the oligosaccharide is a disaccharide. In some embodiments, the lysosomal enzyme is selected from: arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, the lysosomal enzyme is α-L-iduronidase (IDU). In some embodiments, the lysosomal enzyme is acid α-glucosidase (GAA). In some embodiments, the lysosomal enzyme is β-glucuronidase (GUSB). In some embodiments, the lysosomal enzyme is β-galactosidase (GLB1).

[0039] Suitable human cells for cell assays include any human cell lacking the lysosomal enzyme being tested, thus capable of accumulating the natural substrate of that lysosomal enzyme. For example, cells naturally exhibiting complete or partial defects in activity can be used, such as reduced activity by 30%, 50%, 70%, 80%, 90%, 95%, or more. Cells expressing mutant enzymes with reduced activity, or cells obtained from patients with lysosomal storage disorders, such as mucopolysaccharidosis, can be used. Cells recombinantly altered to knock out or reduce the activity of lysosomal enzymes can be used, for example, by introducing mutations into the coding gene or its promoter or other regulatory regions. Cells treated to reduce the activity of lysosomal enzymes can be used, for example, by treatment with antisense or RNAi to reduce enzyme expression.

[0040] Those skilled in the art can select suitable enzymes that cleave (digest) oligosaccharides from carbohydrates and that have "specificity" (i.e., primary digestion) for the natural substrates of lysosomal enzymes. For example, to detect the activity of GALNS or GLB1 (an enzyme that degrades keratin sulfate), the enzyme in step (d) can be keratinase II or any enzyme that primarily acts on keratin sulfate. As another example, to detect IDU, ARSB, IDS, or GUSB (an enzyme that degrades dermatan sulfate), the enzyme in step (d) can be chondroitinase ABC or any enzyme that primarily acts on dermatan sulfate. As another example, to detect IDU, IDS, SGHS, G6S, or GUSB (an enzyme that degrades heparan sulfate), the enzyme in step (d) can be heparinoid I or heparinoid II, or both. As another example, to detect GAA (an enzyme that degrades glycogen), the enzyme in step (d) can be α-amylase or any enzyme that primarily acts on glycogen.

[0041] This cellular method can detect lysosomal enzyme activity with high sensitivity. In some embodiments, lysosomal enzyme activity can also be detected when the concentration of lysosomal enzymes is as low as about 10 nM, or about 5 nM, or about 1 nM, or about 0.75 nM, or about 0.5 nM, or about 0.25 nM, or about 0.1 nM, or about 0.05 nM, or about 0.01 nM, or about 0.005 nM, or about 1 pM, or about 0.5 pM.

[0042] Other features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments illustrating preferred embodiments of the invention are provided for illustrative purposes only, as those skilled in the art will understand, based on this description, various changes and modifications falling within the scope and spirit of the invention. Brief description of the attached diagram

[0043] Figure 1 The nucleotide sequence describing human sulfatase modifying factor 1 (SUMF1) (SEQ ID NO:1).

[0044] Figure 2 The amino acid sequence describing human sulfatase modifying factor 1 (SUMF1) (SEQ ID NO:2).

[0045] Figure 3 The nucleotide sequence describing human N-acetylgalactosamine-6-sulfatase (GALNS) (SEQ ID NO:3).

[0046] Figure 4 The amino acid sequence of human N-acetylgalactosamine-6-sulfatase (GALNS) is described (SEQ ID NO:4). Processed GALNS does not contain the N-terminal 26-amino acid signal peptide.

[0047] Figure 5 Describe the structure and characteristics of processed human N-acetylgalactosamine-6-sulfatase (GALNS).

[0048] Figure 6 Human N-acetylgalactosamine-6-sulfatase (GALNS) is expressed in G71S cells co-transfected with human sulfatase-modifying factor 1 (SUMF1) and human GALNS expression vector. (A) Viable GALNS of G71S clones are screened in 96-wells. (B) G71S clone GALNS productivity, in picograms / cells / day.

[0049] Figure 7 A schematic diagram of the WAVE bioreactor controller for the large-scale production of G71S cells expressing human N-acetylgalactosamine-6-sulfatase (GALNS) and its variants.

[0050] Figure 8 The stability of purified human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity is shown when stored at 4 °C (diamond) or -70 °C (triangle).

[0051] Figure 9 The image shows the purification of human N-acetylgalactosamine-6-sulfatase (GALNS) sequentially by (A) blue agarose 6 fast flow chromatography and (B) Fractogel SE Hi-CAP chromatography. Purity was determined by Coomassie blue staining with SDS-PAGE (left) and Western blotting using anti-GALNS (IVA) antibody (right).

[0052] Figure 10 Human N-acetylgalactosamine-6-sulfatase (GALNS) was purified by ultrafiltration / dialysis (UF / DF), Fractogel SE Hi-Cap chromatography, Zn-chelate agarose chromatography, and ToyoPearl Butyl 650M chromatography. Purity was determined by Coomassie blue staining with SDS-PAGE (top left) and by Western blotting using anti-GALNS antibody (top right), anti-cathepsin L antibody (bottom left), and anti-CHOP (Chinese hamster ovary cell protein) (bottom right).

[0053] Figure 11 A dose-dependent decrease in the amount of dermatan sulfate substrate was observed in IDU-treated GM01391 cells.

[0054] Figure 12 A dose-dependent decrease in dermatin sulfate substrate levels was observed in ARSB-treated GM00519 cells.

[0055] Figure 13The image shows cultured synovial cells taking up unlabeled (circles) or human N-acetylgalactosamine-6-sulfatase (GALNS) coupled with A488 (squares) or A555 (triangles). Invention Details

[0056] This invention relates to a method that enables the large-scale production of recombinant lysosomal sulfatase to meet the demand for a highly active phosphorylated lysosomal sulfatase product that effectively targets lysosomes and is therefore therapeutically effective.

[0057] The therapeutic efficacy of lysosomal enzyme preparations depends on the level of mannose-6-phosphate in the preparation. Phosphate is added to the target glycoprotein via post-translational modifications in the endoplasmic reticulum and early Golgi apparatus. Folded lysosomal enzymes exhibit a unique tertiary determinant cluster recognized by oligosaccharide-modifying enzymes. This cluster consists of a specially separated set of lysine residues found on most lysosomal enzymes but lacking primary sequence homology. The modifying enzyme, UDP-GlcNAc phosphotransferase, binds to the protein determinant cluster and adds GlcNAc-1-phosphate to the 6-position of the terminal mannose residue on the oligosaccharide near the binding site; then, a second enzyme, phosphodiester α-GlcNAc enzyme, cleaves the GlcNAc-phosphate bond to yield the mannose-6-phosphate-terminal oligosaccharide (Canfield et al., US Patent No. 6,537,785). The purpose of mannose-6-phosphate is to divert lysosomal enzymes from the secretory pathway to the intracellular lysosomal pathway. The MPR in the outer part of the Golgi apparatus binds to an enzyme carrying mannose-6-phosphate, thereby directing the enzyme to the lysosome rather than the cell surface.

[0058] Besides the presence of mannose-6-phosphate labeling on the lysosomal enzyme oligosaccharide, the lysosomal orientation of the enzyme depends on the acidification of transport integuments located at the trans Golgi stack. The acidic environment within these integuments is quenched by diffusing basic molecules, leading to the release of vesicle integuments, including lysosomal enzymes, into the extracellular environment (Braulke et al., Eur. J. Cell Biol. 43(3):316-321, 1987). Acidification requires a specific vacuolar ATPase embedded within the integument membrane (Nishi et al., Nat. Rev. Mol. Cell Biol. 3(2):94-103, 2002). The inactivation of this ATPase is expected to enhance the secretion of lysosomal enzymes, thereby impairing lysosomal orientation. Defective cell lines for the preparation of vacuolar ATPases are estimated to nonproductively divert recombinant enzymes that prevent phosphorylation to the intracellular lysosomal compartment.

[0059] In 1984, mutants of Chinese hamster ovary (CHO) cells with specific defects in intron acidification were generated and characterized (Park et al., Somat. CellMol. Genet. 17(2):137-150, 1991). CHO-KI cells were chemically mutagenized, and survival was selected in the presence of toxins and at elevated temperatures. Intron acidification is required for these toxins to fully exhibit their lethality (Marnell et al., J. Cell. Biol. 99(6):1907-1916, 1984). In a previous study, a mixture of two toxins with different mechanisms of action was selected to avoid selecting for toxin-specific tolerance. The principle was that the likelihood of a sporadic mutation leading to tolerance to a particular toxin was low, and the likelihood of simultaneous sporadic mutations of two completely different toxins was also low. Selection was performed at high temperatures to facilitate temperature-sensitive mutations. This genetic screening yielded two mutants, one of which was designated G.7.1 (G71), which was resistant to the toxin at high temperatures. The damage in G71 was not due to the uptake of the two toxins or their mechanism of action, but rather to the clone's inability to acidify integuments at high temperatures. This inability was also observed at permissible temperatures (34°C), but to a lesser extent. G71 cells were also found to be auxotrophic for iron at high temperatures, although they could normally take up transferrin from the culture medium (Timchak et al., J. Biol. Chem. 261(30):14154-14159, 1986). Since transferrin only releases iron at low pH, the auxotrophic nature of iron, despite normal transferrin uptake, indicates a failure in integument acidification. Another study demonstrated that the acidification defect occurred primarily in integuments rather than lysosomes (Stone et al., J. Biol. Chem. 262(20):9883-9886, 1987). The data for G71 are consistent with the conclusion that mutations lead to instability of vacuolar ATPases responsible for integument acidification. Instability was most pronounced at high temperatures (39.5°C), but was partially observed even at lower temperatures (34°C). Studies of transporting two endogenous lysosomal enzymes, cathepsin D and α-glucosidase, into G71 cells (Park et al., Somat. Cell Mol. Genet. 17(2):137-150, 1991) showed that both enzymes were quantitatively secreted at high temperatures, while glycosylation of both enzymes was unaffected. Secretion of phosphorylated acid α-glucosidase increased significantly at unpermitted temperatures.

[0060] The therapeutic efficacy of lysosomal sulfatase preparations depends not only on the level of mannose-6-phosphate but also on the presence of an active enzyme in the preparation. All known sulfatases contain a cysteine ​​residue at their catalytic site; this cysteine ​​residue is post-translationally modified to form C... α-Formylglycine (FGly) is used to activate the enzyme. This post-translational activation of the cysteine ​​to FGly, catalyzed by sulfatase modifier 1 (SUMF1), occurs immediately after translation on the unfolded sulfatase within the endoplasmic reticulum, prior to targeting the sulfatase to the lysosome (Dierks et al., Proc. Natl. Acad. Sci. USA 94:11963-11968, 1997). The importance of this unique post-translational modification is highlighted because mutations in SUMF1 impair FGly formation in lysosomal sulfatases, leading to multiple sulfatase deficiency (MSD) in humans (Diez-Ruiz et al., Annu. Rev. Genomics Hum. Genet. 6:355-379, 2005).

[0061] Therefore, the ability of G71 cells with endosome acidification deficiency, i.e. mutant CHO cells, to co-express recombinant human sulfatase-modifying enzyme (SUMF1) and human lysosomal sulfatase provides a mechanism for the large-scale production of active, highly phosphorylated recombinant human lysosomal sulfatase for controlling lysosomal storage diseases caused by or associated with such lysosomal sulfatase deficiency.

[0062] I. Definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide general definitions for many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd edition, 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker, ed., 1988); THE GLOSSARY OF GENETICS, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); Hale and Marham, THE HARPERCOLLINSDICTIONARY OF BIOLOGY (1991).

[0064] The publications, patent applications, patents, and other references cited in this article are incorporated into this article to the same extent as the article itself.

[0065] Unless otherwise expressly stated in the text, it should be noted that the singular forms “a,” “an,” and “the” used in this specification and the appended claims include the plural forms.

[0066] Unless otherwise specified, the following terms as used herein have the meanings attributable to them.

[0067] "Allele variants" refer to two or more polymorphic forms of a gene occupying the same genetic locus. Mutations naturally produce allele variants, which can lead to phenotypic polymorphism in a population. Gene mutations can be silent (i.e., no change in the encoded polypeptide) or can encode a polypeptide with altered amino acid sequence. "Allele variants" also refer to the cDNA of mRNA transcripts derived from genetic allele variants and the proteins they encode.

[0068] "Amplification" refers to any method of copying a polynucleotide sequence and thus expanding it into a large number of polynucleotide molecules, such as through reverse transcription, polymerase chain reaction, and ligase chain reaction.

[0069] The first sequence is said to be an "antense sequence" relative to the second sequence if: its sequence is a polynucleotide-specific hybridization of the first sequence or its sequence is a polynucleotide of the second sequence.

[0070] “cDNA” refers to DNA in single-stranded or double-stranded form that is complementary to or identical to mRNA.

[0071] This article uses conventional markers to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5' direction. The 5' to 3' direction of adding nucleotides to nascent RNA transcripts is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the "coding strand"; the sequence on the DNA strand with the same sequence as the mRNA transcribed from that DNA and located at the 5' end of the RNA transcript in the 5' direction is called the "upstream sequence"; the sequence on the DNA strand with the same sequence as the RNA and located at the 3' end of the coding RNA transcript in the 3' direction is called the "downstream sequence".

[0072] "Complementarity" refers to the morphological compatibility or mutual matching of the interacting surfaces of two polynucleotides. Therefore, the two molecules can be described as complementary, and furthermore, their contact surface features are complementary to each other. The first polynucleotide is complementary to the second polynucleotide if its nucleotide sequence is identical to the nucleotide sequence of its polynucleotide chaperone. Therefore, a polynucleotide with the sequence 5'-TATAC-3' is complementary to a polynucleotide with the sequence 5'-GTATA-3'. If the complementary sequence of a nucleotide sequence is substantially identical to the reference nucleotide sequence, then the nucleotide sequence is "substantially complementary" to the reference nucleotide sequence.

[0073] "Conservative substitution" refers to the substitution of a functionally similar amino acid for a polypeptide. The following six groups each contain amino acids that are conserved substitutes for each other:

[0074] 1) Alanine (A), Serine (S), Threonine (T);

[0075] 2) Aspartic acid (D), glutamic acid (E);

[0076] 3) Asparagine (N), glutamine (Q);

[0077] 4) Arginine (R), Lysine (K);

[0078] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0079] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0080] When the term "fragment" is used to refer to a polypeptide, it means a polypeptide shorter than the full-length polypeptide due to truncation at the N-terminus, C-terminus, or both ends of the protein, and / or the deletion of internal portions or regions of the protein. Fragments of polypeptides can be prepared by any method known in the art.

[0081] When the term "mutant" is used to refer to a polypeptide, it means a polypeptide in which one or more amino acids in a protein are replaced by different amino acids. Amino acid substitutions can be either conserved substitutions as defined above or non-conserved substitutions. Mutant polypeptides can be prepared using methods known in the art.

[0082] When the term "derivative" is used to refer to a polypeptide, it means a polypeptide that has been chemically modified by techniques such as, but not limited to, pervasive proteination, labeling (e.g., using radionuclides or various enzymes), covalent polymerization (e.g., PEGylation, i.e., derivatization with polyethylene glycol), and insertion or substitution of amino acids, such as ornithine, that are not normally produced in human proteins through chemical synthesis. Derivative polypeptides can be prepared by methods known in the art.

[0083] When the term “derivative line” is used to refer to a cell line, it means a cell line that is a descendant of a parent cell line; for example, the term includes cells that have been passaged or subcloned from parent cells and retain the desired characteristics, the descendants of parent cell lines that have retained the desired characteristics through mutation and selection, and the descendants of parent cell lines that have been altered to contain different expression vectors or different exogenously added nucleic acids.

[0084] "Detection" refers to determining the presence, absence, or content of an analyte in a sample, which may include quantitative determination of the analyte content in the sample or per cell of the sample.

[0085] "Detectable portion" or "label" refers to a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical methods. For example, useful labels include... 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., those routinely used in ELISA), biotin-streptavidin, dioxigenin, haptens and proteins with available antiserum or polyclonal antibodies, or nucleic acid molecules with sequences complementary to the target. The detectable moiety often produces a detectable signal, such as a radioactive, chromogenic, or fluorescent signal, which can be used to quantify the amount of the bound detectable moiety in the sample. The detectable moiety can be covalently incorporated into or attached to primers or probes, or via ionic bonds, van der Waals forces, or hydrogen bonds, for example, by incorporating radioactive nucleotides or biotinylated nucleotides recognized by streptavidin. The detectable moiety can be detected directly or indirectly. Indirect detection may include binding a second direct or indirect detectable moiety to the detectable moiety. For example, the detectable moiety can be a ligand for a binding partner, such as biotin, which is a binding partner of streptavidin, or a nucleotide sequence that is a binding partner of a complementary sequence that can specifically hybridize with it. The binding partner itself can be detectable, for example, by labeling the antibody itself with a fluorescent molecule. The binding partner can also be indirectly detectable; for example, a nucleic acid with a complementary nucleotide sequence can be part of a branched DNA molecule and can then be detected by hybridization with other labeled nucleic acid molecules (see, for example, Fahrlander et al., Bio / Technology 6:1165, 1988). The signal can be quantified by, for example, scintillation counting, densitography, or flow cytometry.

[0086] "Diagnosis" refers to the identification of the presence or nature of a pathological condition. Diagnostic methods vary in specificity and selectivity. While a specific diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides evidence that aids in the diagnosis.

[0087] The term "effective amount" refers to a dose sufficient to produce the desired outcome for a subject's health condition, pathological condition, or disease, or sufficient for diagnostic purposes. The desired outcome may include subjective or objective improvement in the recipient from that dose. "Therapeutic effective amount" refers to a dosage sufficient to produce the desired health benefit.

[0088] “Encoding” refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules having a specified nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a specified amino acid sequence and the resulting biological characteristics during biological processes. Therefore, if a gene produces mRNA that is transcribed and translated into a protein in a cell or other biological system, then that gene encodes a protein. The coding and non-coding strands of a gene or cDNA that serve as a template for transcription may be referred to as encoding the protein or other product of that gene or cDNA, wherein the nucleotide sequence of the coding strand is identical to the mRNA sequence and is typically provided in a sequence listing. Unless otherwise specified, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0089] "Equivalent dose" refers to a dose containing the same amount of active agent.

[0090] "Expression control sequence" refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and / or translation) of nucleotide sequences operablely linked to it. "Operationally linked" refers to a functional relationship between two parts, where the activity of one part (e.g., the ability to regulate transcription) affects the transcription of another part (e.g., the transcription of a sequence). Expression control sequences may include, for example, but not limited to: promoter sequences (e.g., inducible or constitutive), enhancers, transcription terminators, start codons (i.e., ATGs), intron splicing signals, and stop codons.

[0091] "Expression vector" refers to a vector containing a recombinant polynucleotide containing an expression control sequence operatively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as granules, plasmids (e.g., naked plasmids or those contained in liposomes) incorporating recombinant polynucleotides, and viruses.

[0092] "Highly phosphorylated," "highly phosphorylated," and "highly phosphorylated oligosaccharide" refer to a lysosomal sulfatase preparation in which at least 50% of the lysosomal sulfatase binds to a cation-independent mannose-6-phosphate acceptor via phosphorylated oligosaccharides. The binding is further characterized by sensitivity to mannose-6-phosphate competition. Highly phosphorylated lysosomal sulfatase can also be described as a lysosome having at least 0.25, preferably at least 0.5, and more preferably at least 0.75 di-phosphorylated oligosaccharide chains per protein chain.

[0093] The term "biphosphorylated oligosaccharide chain" as used in this article refers to a mannose-containing oligosaccharide chain whose N-link is attached to an asparagine residue in a lysosomal sulfatase and contains two mannose-6-phosphate residues. Biphosphorylated oligomannose chains typically have seven mannose residues, namely mannose bisphosphate 7 (BPM7), which are attached to two GlcNAc residues, and then to an asparagine residue in a lysosomal sulfatase.

[0094] "Active," "activated," and "highly activated" refer to a lysosomal sulfatase preparation in which at least 50%, preferably at least 70%, more preferably at least 90%, and even more preferably at least 95% of the cysteine ​​residues at the active site of the protein are post-translational modified to C. α -Formylglycine (FGly).

[0095] "Highly phosphorylated activity" refers to a lysosomal sulfatase product in which at least 50%, preferably at least 70%, more preferably at least 90%, and even more preferably at least 95% of the cysteine ​​residues at the protein's active site are post-translational modified to C. α -formylglycine (FGly), wherein each protein chain has at least 0.25, preferably at least 0.5, more preferably at least 0.75 di-phosphorylated oligosaccharide chains.

[0096] The term "biologically active" refers to a fragment, mutant, variant, or derivative of a polypeptide (i.e., enzyme) that retains at least a basic amount (e.g., at least about 50%, preferably at least about 70%, more preferably at least about 90%) of one or more biological activities of the full-length polypeptide. When referring to lysosomal sulfatase, the biologically active fragment, mutant, variant, or derivative retains at least a basic amount of sulfatase activity (i.e., cleavage of sulfates from their target substrate). When referring to sulfatase modification factor 1 (SUMF1), the biologically active fragment, mutant, variant, or derivative retains at least a basic amount of formylglycine-producing activity (i.e., modification of the cysteine ​​residue at the active site of lysosomal sulfatase to C...). α -Formylglycine (FGly)).

[0097] When the terms "purity" or "pure" are used to refer to polypeptides, they refer to the amount of the analyzed polypeptide compared to any contaminant that can be detected by a specific method. For the recombinant lysosomal sulfatase of the present invention, the sulfatase preparation can be separated by SDS-PAGE electrophoresis under reducing or non-reducing conditions, followed by Coomassie blue or silver staining, or by HPLC chromatography (e.g., C4 reversed-phase (RP)) or by any other chromatographic separation, such as size exclusion (SEC), to determine the "purity". Using these methods, the purified recombinant lysosomal sulfatase of the present invention has a purity of at least about 80%, preferably at least about 90%, more preferably at least about 95%, and even more preferably at least about 98% or 99%.

[0098] The term "precursor" or "precursor form" refers to the recombinant lysosomal sulfatase form secreted by mammalian cells. That is, it lacks a signal sequence but also lacks certain modifications, such as internal protein cleavage that typically occurs in lysosomes. The terms "mature," "mature form," "processed," or "processed form" refer to the form of recombinant lysosomal sulfatase that is normally present in lysosomes. For the recombinant lysosomal sulfatase of the present invention, the sulfatase preparation can be separated by SDS-PAGE electrophoresis under reducing conditions and then stained with Coomassie blue or silver, or by HPLC chromatography (e.g., C4 reversed-phase (RP)) or by any other chromatographic separation, such as size exclusion (SEC), to determine the "precursor" or "precursor form" and the "mature," "mature form," "processed," or "processed form." Using these methods, the purified recombinant lysosomal sulfatase of the present invention contains at least about 75%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% of the "precursor" or "precursor form." Alternatively, using these methods, the purified recombinant lysosomal sulfatase of the present invention comprises at least about 25%, preferably at least about 15%, more preferably at least about 10%, and even more preferably at least about 5% of a "mature," "mature form," "processed," or "processed form." In some embodiments, only the "precursor" or "precursor form" is detected, i.e., the sulfatase preparation consists essentially of a single detectable band when SDS-PAGE is performed under reducing conditions, or is essentially a single peak when analyzed by HPLC.

[0099] With respect to two or more polynucleotide or peptide sequences, the terms "identical" or "percentage of identical" refer to two or more sequences or subsequences being identical or having a certain percentage of identical nucleotide or amino acid residues when compared and aligned to the maximum correspondence using one of the following sequence comparison algorithms or by visual inspection.

[0100] A "connector" refers to a molecule that covalently, or through ionic bonds, van der Waals forces, or hydrogen bonds, connects two other molecules. For example, a complementary sequence is hybridized at the 5' end and another complementary sequence is hybridized at the 3' end, thereby connecting two non-complementary nucleic acid molecules.

[0101] "Low-level phosphorylation" or "hypophosphorylation" refers to a lysosomal sulfatase product in which the half-maximum concentration taken up into fibroblasts is greater than 10 nM or the lysosomal sulfatase fraction bound to the mannose-6-phosphate acceptor column is less than about 25%.

[0102] The term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, polypeptides or polypeptide sequences present in organisms (including viruses) are naturally occurring, can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory.

[0103] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in target animals, including humans and mammals. A pharmaceutical composition contains a pharmaceutically effective amount of therapeutic lysosomal sulfatase, and also contains one or more pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutical compositions include compositions containing an active ingredient and an inert component constituting a carrier, diluent, or excipient, as well as any product directly or indirectly obtained from the combination, compounding, or aggregation of any two or more components, or the dissociation of one or more components, or other types of reactions or interactions of one or more components. Therefore, the pharmaceutical compositions of the present invention include any composition prepared by mixing the lysosomal sulfatase of the present invention with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0104] "Pharmaceutically acceptable carriers, diluents, or excipients" refers to any standard drug carrier, diluent, buffer, and excipient, such as, but not limited to, phosphate-buffered saline, 5% glucose aqueous solution, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents and / or adjuvants. Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co., Easton, 1995), describes suitable drug carriers, diluents, or excipients and formulations. Preferred drug carriers, diluents, or excipients depend on the intended route of administration of the active agent. Typical routes of administration include, for example, but not limited to: enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal) injection, or local, transdermal, or transmucosal administration.

[0105] "Pharmaceutically acceptable salts" are salts that can be formulated into lysosomal sulfatases for pharmaceutical applications, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonium or organic amines.

[0106] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include nucleotides containing non-naturally occurring bases linked to nucleotides other than naturally occurring phosphodiester bonds, or those containing bases linked by linkages other than phosphodiester bonds. Therefore, nucleotide analogs include, for example, but not limited to, phosphate thioides, phosphate dithioides, phosphate triesters, aminophosphates, boranophosphates, methyl phosphonates, chiral methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to a polynucleotide. The term "oligonucleotide" generally refers to a short polynucleotide, typically no more than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T".

[0107] "Polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, its associated naturally occurring structural variants and synthetic non-natural analogs. For example, polypeptides can be synthesized using an automated polypeptide synthesizer. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. This article uses conventional markers to depict polypeptide sequences: the left-hand end of the polypeptide sequence is the amino-terminus; the right-hand end of the polypeptide sequence is the carboxyl-terminus.

[0108] A primer is a polynucleotide that specifically hybridizes to a specified polynucleotide template and provides a starting point for the synthesis of a complementary polynucleotide. This type of synthesis occurs when a polynucleotide primer is placed under conditions of induced synthesis, i.e., in the presence of a nucleotide, a complementary polynucleotide template, and a polymerization reagent, such as DNA polymerase. Primers are typically single-stranded, but can be double-stranded. Primers are usually deoxyribonucleic acid (DNA), but a variety of synthetic and naturally occurring primers are available for many applications. Primers are designed to be complementary to the template with which they hybridize in order to serve as a starting point for synthesis, but do not need to reflect the exact sequence of the template. In this case, the specific hybridization of the primer with the template depends on the stringency of the hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive, or fluorescent components, and can be used as a detectable part.

[0109] When the term "probe" is used to refer to a polynucleotide, it refers to a polynucleotide that specifically hybridizes to a specified sequence of another polynucleotide. The probe specifically hybridizes to its complementary target polynucleotide, but does not need to reflect the exact complementary sequence of the template. In such cases, the specific hybridization of the probe and target depends on the stringency of the hybridization conditions. The probe can be labeled, for example, with chromogenic, radioactive, or fluorescent components and used as the detectable portion.

[0110] "Preventative" treatment is the administration of medication to subjects who do not show signs of disease or only show early signs in order to reduce the likelihood of developing symptoms. The compounds of this invention can be administered as preventative treatment to reduce the likelihood of developing symptoms or, if they do, minimize their severity.

[0111] "Recombinant polynucleotides" refer to polynucleotides that have naturally unlinked sequences. Amplified or assembled recombinant polynucleotides can be contained in a suitable vector, which can then be used to transform suitable host cells. The host cell containing the recombinant polynucleotide is called a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide." Recombinant polynucleotides can also perform non-coding functions (e.g., promoters, origins of replication, ribosome binding sites, etc.).

[0112] "Specific hybridization," "selective hybridization," or "selective hybridization" refers to the phenomenon where, under strict conditions, when a specific nucleotide sequence is present in a complex mixture (such as total cellular DNA or RNA), nucleic acid molecules preferentially bind to that sequence to form a double strand or hybridize.

[0113] The term "stringent conditions" refers to conditions under which a probe preferentially hybridizes to its target sequence, while hybridizing with other sequences to a low degree or not at all. For nucleic acid hybridization experiments, such as Southern and Northern hybridization, "stringent hybridization" and "stringent hybridization washing conditions" are sequence-dependent and vary under different environmental parameters. For extensive guidance on nucleic acid hybridization, see Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York. High-stringent hybridization and washing conditions are typically chosen to be approximately 5°C lower than the thermal desorption temperature (Tm) of the specific sequence at a given ionic strength and pH. This Tm is the temperature at which a probe achieves a 50% perfect hybridization match with the target sequence (at a given ionic strength and pH). Very stringent conditions are chosen to be equal to the Tm of the specific probe.

[0114] For Southern or Northern blot membranes, an example of stringent hybridization conditions for complementary nucleic acids with more than 100 complementary residues is 42°C, 50% formalin containing 1 mg heparin, and overnight hybridization. An example of highly stringent wash conditions is 72°C, 0.15 M NaCl, approximately 15 minutes. An example of stringent wash conditions is 65°C, 0.2X SSC wash, 15 minutes (see Sambrook et al. for a description of the buffer). A less stringent wash is often performed before a highly stringent wash to remove background probe signals. For example, for duplexes with more than 100 nucleotides, an exemplary moderately stringent wash is 1x SSC, 45°C, 15 minutes. For example, for duplexes with more than 100 nucleotides, an exemplary less stringent wash is 4-6x SSC, 40°C, 15 minutes. A signal-to-noise ratio of 2x (or higher) than observed irrelevant probes in a specific hybridization assay generally indicates the detection of specific hybridization.

[0115] The “object” of diagnosis or treatment is a human or a non-human animal, including mammals or primates.

[0116] For the purposes of comparing two nucleic acids or polypeptides, the phrase "substantially homologous" or "substantially identical" generally means that two or more sequences or subsequences have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue similarity when compared and aligned to the maximum correspondence using one of the following sequence comparison algorithms or by visual inspection. Substantially identical sequences are preferably present in a sequence region of at least about 50 residues, more preferably in a region of at least about 100 residues, and most preferably substantially identical in at least about 150 residues. In the most preferred embodiment, the sequences are substantially identical along the entire length of one or both of the compared biopolymers.

[0117] For sequence comparisons, a reference sequence is typically used as the comparison sequence with the test sequence. When employing a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence similarity between the test sequence and the reference sequence based on the specified program parameters.

[0118] The best alignment for sequence comparison can be achieved through methods such as Smith and Waterman’s local homology algorithm (Adv. Appl. Math. 2:482, 1981), Needleman and Wunsch’s homology alignment algorithm (J. Mol. Biol. 48:443, 1970), Pearson and Lipman’s similarity retrieval method (Proc. Nat'l. Acad. Sci. USA 85:2444, 1988), computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or visual observation.

[0119] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to generate multiple sequence alignments from a set of related sequences, displaying relationships and the percentage of sequence similarities. It also plots trees and dendograms showing the clustering relationships used to generate the alignments. PILEUP utilizes the simplified progressive alignment method of Feng and Doolittle (J.Mol.Evol.35:351-360, 1987). The method used is similar to that described by Higgins and Sharp (CABIOS 5:151-153, 1989). The procedure can align up to 300 sequences, each up to 5,000 nucleotides or amino acids in length. This multiple alignment method starts with pairwise alignment of the two most similar sequences, generating two clusters of aligned sequences. This cluster can then be aligned with the next most related sequence or the cluster of aligned sequences. Two sequence clusters can be aligned by simply extending the pairwise alignment of two individual sequences. The final alignment is achieved through a series of progressive pairwise alignments. The program is run by specifying the amino acid or nucleotide coordinates of a particular sequence and the sequence comparison region, along with specified program parameters. For example, the following parameters can be used to compare a reference sequence with other test sequences to determine the percentage of sequence similarity: default vacancy weight (3.00), default vacancy length weight (0.10), and weighted terminal vacancy. Another algorithm that can be used to generate multiple sequence alignments is Clustal W (Thompson et al., Nucleic Acids Research 22:4673-4680, 1994).

[0120] Another example of an algorithm suitable for determining sequence identity and sequence similarity percentage is the BLAST algorithm, described in Altschul et al., J.Mol.Biol.215:403-410, 1990. Software for BLAST analysis is publicly available from the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) in the query sequence by identifying short characters of length W that match or satisfy certain positive threshold scores T when aligned with characters of the same length in a sequence database. T is called the adjacent character score threshold (Altschul et al., J.Mol.Biol.215:403-410, 1990). These initial adjacent character hits serve as seeds to initiate a search for longer HSPs containing them. The character hits then extend in both directions for each sequence until the cumulative alignment score increases. The cumulative score of the nucleotide sequence is calculated using parameters M (reward score for matching residue pairs; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acids, a scoring matrix can be used to calculate the cumulative score. Extension in each direction is stopped when the selected word is: the cumulative alignment score decreases by X compared to the maximum obtained value; the cumulative score approaches or falls below zero due to the accumulation of one or more negatively scored residues; or either sequence end is reached. The BLAST algorithm parameters W, T, and X determine the alignment sensitivity and speed. The BLASTN program (for nucleotide sequences) uses default parameters: word length (W) 11, expected value (E) 10, M = 5, N = -4, and compares two strands. For amino acid sequences, the BLASTP program uses default parameters: word length (W) 3, expected value (E) 10, and uses the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

[0121] In addition to calculating the percentage of sequence similarity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One similarity detection provided by the BLAST algorithm is the minimum probability sum (P(N)), which indicates the probability that a match will occasionally occur between two nucleotide or amino acid sequences. For example, if the minimum probability sum when comparing the tested nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0122] Another indicator that two nucleic acid sequences or polypeptides are substantially identical is the immunological cross-reactivity between the polypeptide encoded by the first nucleic acid and the polypeptide encoded by the second nucleic acid, as described below. Therefore, when the difference between two peptides lies only in conserved substitutions, one polypeptide is usually substantially identical to the other. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize under the stringent conditions described herein.

[0123] "Substantially pure" or "isolated" means that the target substance is the dominant substance present (i.e., more abundant, in molar terms, than any other macromolecule in the composition), and a complementaryly pure portion is a composition in which the target substance constitutes at least about 50% (in molar terms) of all present macromolecules. A substantially pure composition typically means that about 80% to 90% or more of the macromolecules present in the composition are the purified substances of interest. If the composition consists essentially of a single macromolecule, the target substance is purified to be substantially homogeneous (contaminating substances cannot be detected in the composition by conventional detection methods). For the purposes of this definition, solvents, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ions are not considered macromolecules. In some embodiments, the lysosomal sulfatase of the present invention is substantially pure or isolated. In some embodiments, the lysosomal sulfatase of the present invention is substantially pure or isolated relative to the macromolecular starting material used in its synthesis. In some embodiments, the pharmaceutical composition of the present invention comprises a substantially pure or isolated therapeutic lysosomal sulfatase mixed with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0124] "Therapeutic" treatment is the administration of medication to a subject exhibiting signs or symptoms in order to reduce or eliminate the signs or symptoms of a disease. Signs or symptoms can be biochemical, cellular, histological, functional, subjective, or objective. The lysosomal sulfatase of the present invention can be administered as a therapeutic treatment or for diagnostic purposes.

[0125] "Therapeutic index" refers to the dosage range (dosage and / or timing) above the minimum therapeutic dose and below the unacceptable toxicity dose.

[0126] "Treatment" refers to preventive treatment, therapeutic treatment, or diagnostic treatment.

[0127] As used herein, the term "unit dosage form" refers to a physically discontinuous unit suitable for use as a single dose in humans and animals, each unit containing a predetermined amount of the lysosomal sulfatase of the present invention, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients, the predetermined amount being calculated to produce the desired effect. The specifications of the novel unit dosage forms of the present invention depend on the specific lysosomal sulfatase used, the effect to be achieved, and the relevant pharmacodynamic properties of each lysosomal sulfatase in the host.

[0128] II. Production of lysosomal sulfatase

[0129] On one hand, the present invention is characterized by a novel method for generating highly active phosphorylated lysosomal sulfatases, the amount of which produced is sufficient for therapeutic application. This method is typically characterized by transforming suitable cell lines with cDNA encoding a biologically active fragment, mutant, variant, or derivative of human sulfatase modification factor 1 (SUMF1) or thereof, and cDNA encoding a full-length lysosomal sulfatase or thereof, a biologically active fragment, mutant, variant, or derivative. Those skilled in the art can prepare expression constructs other than those specifically described herein to optimize the production of such lysosomal sulfatases in suitable transformed cell lines. Furthermore, it is readily apparent to those skilled in the art that fragments encoding biologically active fragments, variants, mutants, or derivatives of naturally occurring SUMF1 or lysosomal sulfatases, which have the same or similar biological activity as the naturally occurring full-length enzyme.

[0130] host cells

[0131] The host cells used to produce recombinant lysosomal sulfatases are endosome acidification-deficient cell lines, characterized by their ability to produce such lysosomal sulfatases in quantities sufficient for therapeutic application. This invention provides a CHO-K1-derived END3 complementary cell line, designated G71. This invention also provides a G71 cell line suitable for serum-free suspension culture, designated G71S. This invention also provides derivative lines of the G71 and G71S cell lines, which have undergone further subcloning or contain different expression vectors.

[0132] This article refers to cells containing and expressing DNA or RNA encoding recombinant proteins as genetically modified cells. Similarly, mammalian cells containing and expressing DNA or RNA encoding recombinant proteins are referred to as genetically modified mammalian cells. DNA or RNA can be introduced into cells using known transfection methods, such as, but not limited to: electroporation, microinjection, microparticle bombardment, calcium phosphate precipitation, modified calcium phosphate precipitation, cationic lipid treatment, photoporation, fusion methods, receptor-mediated transfer, or 1,5-dimethyl-1,5-diazadecylmethylene polymethyl bromide precipitation. Alternatively, DNA or RNA can be introduced via viral vector infection. Methods for producing cells, including mammalian cells, that express DNA or RNA encoding recombinant proteins are described in: co-pending patent applications U.S. Serial No. 08 / 334,797, entitled "In Vivo Protein Production and Delivery System for Gene Therapy," Richard F. Selden, Douglas A. Treco, and Michael W. Heartlein (filed November 4, 1994); U.S. Serial No. 08 / 334,455, entitled "In Vivo Production and Delivery of Erythropoietin or Insulinotropin for Gene Therapy," Richard F. Selden, Douglas A. Treco, and Michael W. Heartlein (filed November 4, 1994); and U.S. Serial No. 08 / 231,439, entitled "Targeted Introduction of DNA Into..." "Primary or Secondary Cells and Their Use for Gene Therapy", Douglas A. Treco, Michael W. Heartlein, and Richard F. Selden (filed April 20, 1994). The teachings of each of these applications are incorporated herein by reference in full.

[0133] In a preferred embodiment, the host cells used to produce recombinant lysosomal sulfatase are intron acidification-deficient cell lines, characterized by their ability to produce such lysosomal sulfatase in a quantity sufficient for therapeutic application. In a preferred embodiment, the present invention provides a CHO-K1-derived END3 complementary cell line designated G71 and a G71 cell line designated G71S suitable for serum-free suspension culture, both of which co-express human sulfatase modifying factor 1 (SUMF1) and recombinant lysosomal sulfatase, thus enabling the production of high yields of highly phosphorylated lysosomal sulfatase as defined, thereby enabling large-scale production of therapeutic lysosomal sulfatase. In the most preferred embodiment, the G71 or G71S cell line or its derivatives express and secrete recombinant lysosomal sulfatase in quantities of at least about 0.5, preferably at least about 0.75, more preferably at least about 1.0, and even more preferably at least about 1.25 picograms / cell / day.

[0134] Vectors and nucleic acid constructs

[0135] Nucleic acid constructs for expressing recombinant proteins, i.e., human sulfatase-modifying factor 1 (SUMF1) or lysosomal sulfatase, or both, can be expressed extrachromosomally (in episomes) in transfected mammalian cells or randomly or randomly integrated into the genome of recipient cells via homologous recombination at preselected target sites. In addition to the recombinant protein-coding sequence, the extrachromosomal expression construct contains sequences sufficient for expression of the protein in cells and optionally sequences for construct replication. It typically contains a promoter, recombinant protein-coding DNA, and a polyadenylation site. The placement of the recombinant protein-coding DNA in the construct allows its expression to be controlled by the promoter. The construct may optionally contain additional components, such as one or more of the following: splicing sites, enhancer sequences, selectable marker genes under the control of a suitable promoter, amplifiable marker genes under the control of a suitable promoter, and matrix attachment regions (MARs) or other elements known in the art that enhance expression of the inserted region.

[0136] In those embodiments where the DNA construct is integrated into the cellular genome, it only needs to contain a recombinant protein-coding nucleic acid sequence. It may optionally contain promoter and enhancer sequences, one or more polyadenylation sites, nucleic acid sequences encoding one or more selectable markers, matrix attachment regions (MARs) or other elements known in the art that enhance expression in the inserted region, and / or DNA homologous to the genomic DNA in the recipient cell, thereby targeting the DNA integration into a selected site in the genome (target DNA or DNA sequence).

[0137] Cell culture methods

[0138] Mammalian cells containing recombinant protein-encoding DNA or RNA are cultured under conditions suitable for cell growth and DNA or RNA expression. Cells expressing the recombinant protein can be identified using known methods and those described herein, and the recombinant protein can be isolated and purified using known methods and those described herein, wherein the recombinant protein production may or may not be amplified. Identification can be performed, for example, by screening genetically modified mammalian cells that exhibit a phenotype indicating the presence of DNA or RNA encoding the recombinant protein, such as PCR screening, screening by Southern blotting analysis, or screening for recombinant protein expression. Cells containing the incorporated recombinant protein-encoding DNA can be selected by incorporating an optional marker into the DNA construct and then culturing transfected or infected cells containing the optional marker gene under conditions suitable for the survival of only those cells expressing the optional marker gene. The introduced DNA construct can be further amplified by culturing genetically modified mammalian cells containing the amplifiable marker gene under suitable conditions (e.g., at drug concentrations where only cells containing multiple copies of the amplifiable marker gene can survive).

[0139] As described herein, genetically modified mammalian cells encoding recombinant proteins can be identified by detecting the expression products. For example, mammalian cells expressing highly phosphorylated lysosomal sulfatases can be identified by sandwich enzyme immunoassay. Antibodies can directly target the active agent moiety.

[0140] variants of lysosomal sulfatase

[0141] In some embodiments, mutants or variants of highly phosphorylated lysosomal sulfatases can be prepared, which are useful in a variety of fields where highly phosphorylated lysosomal sulfatases can be utilized. The amino acid sequence mutants or variants of the polypeptide can be substitution, insertion, or deletion mutants or variants. Deletion mutants or variants lack one or more residues in the native protein that are not essential for function or immunogenic activity. Common types of deletion mutants or variants lack secretion signaling sequences or signaling sequences that guide protein binding to specific cellular regions. Insertion mutants or variants typically involve the addition of material at a non-terminal site of the polypeptide. This can include the insertion of an immunoreactive epitope or simply the insertion of a single residue. Terminal addition, also known as fusion proteins, is discussed below.

[0142] Variants may be substantially homologous to or substantially identical to the unmodified lysosomal sulfatases described above. Preferred variants are those that retain at least some of the biological activities of lysosomal sulfatases, such as highly phosphorylated lysosomal sulfatases. Other preferred variants include variants of N-acetylgalactosamine-6-sulfatase polypeptides that retain at least some of the sulfatase activities of human N-acetylgalactosamine-6-sulfatase.

[0143] Substitutional mutants or variants typically replace one wild-type polypeptide amino acid at one or more sites within a protein with another, and can be engineered to modulate one or more properties of the polypeptide, such as, but not limited to, stability against protein hydrolysis cleavage, without losing other functions or properties. Such substitutions are preferably conserved, i.e., replacing an amino acid with one that is similar in shape and charge. Conserved substitutions are well known in the art and include, for example, the following replacements: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0144] This invention considers, in one aspect, the generation of glycosylation site mutants or variants, wherein the O- or N-linked glycosylation sites of lysosomal sulfatases are mutated. Such mutants or variants generate important information related to the biological activity, physical structure, and substrate binding potential of highly phosphorylated lysosomal sulfatases. Some aspects consider other mutants or variants that could generate highly phosphorylated lysosomal sulfatase polypeptides that retain biological activity but have increased or decreased substrate binding activity. Therefore, mutations in the active site or catalytic region are particularly considered to generate protein mutants or variants with altered substrate binding activity. In such embodiments, the sequence of the highly phosphorylated lysosomal sulfatase is compared with the sequences of other related enzymes, and selected residues are specifically mutated.

[0145] To encode the amino acid at the putative N-terminus of a mature protein as amino acid 1, exemplary mutations that may be employed include, for example, replacing all or some of the potentially glycosylated asparagine in recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), including positions 178 and 397 (see [link to relevant documentation]). Figure 5 ).

[0146] Mutations at or near the active site of lysosomal sulfatases can improve substrate binding. Given that such mutations are exemplary, those skilled in the art will recognize that other mutations can be made into the enzyme sequence to provide additional structural and functional information about the protein and its activity.

[0147] To construct mutants or variants, such as those described above, those skilled in the art can employ well-known standard techniques. In particular, N-terminal deletions, C-terminal deletions, internal deletions, and random and point mutations are considered.

[0148] N-terminal and C-terminal deletions are deletion mutagenesis that utilize, for example, the presence of a suitable single restriction site near the end of a C- or N-terminal region. DNA is cleaved at this site, and the cleaved ends are degraded with nucleases such as BAL31, exonuclease III, DNase I, and S1 nuclease. Rejoining the two ends produces a range of DNA sequences with deletions of various sizes around the restriction site. The appropriate biological function, such as enzymatic activity, of the protein expressed by such mutants can be tested using standard techniques in the art and techniques described in the specification. Similar techniques can be applied to internal deletion mutants using two appropriately positioned restriction sites, thereby precisely defining the deletion to be made and the ends to be rejoined.

[0149] Partial digestion mutants have also been considered. In such examples, those skilled in the art utilize a “frequent cutter” that cuts DNA at many locations depending on the length of the reaction time. Thus, by varying the reaction conditions, it is possible to generate a series of mutants of different sizes, whose activity can then be screened.

[0150] DNA sequences can also be cut using DNase I to perform random insertion mutations, such as inserting nucleotide extensions encoding amino acids 3, 6, 9, or 12 and then ligating them to the ends. Once such mutations are made, the various activities exhibited by wild-type proteins in these mutants can be screened.

[0151] Point mutagenesis can also be used to specifically identify which amino acid residues are crucial for specific activities related to the biological activity of lysosomal sulfatases. Therefore, those skilled in the art can produce single-base changes in the DNA strand, resulting in altered codons and missense mutations.

[0152] The amino acids of specific proteins can be altered to produce equivalent, or even improved, second-generation molecules. Such alterations include replacing a given amino acid in a protein without significantly losing its ability to interact with structures such as the antigen-binding region of an antibody, a substrate molecule, or a binding site on a receptor. Since the biological function and activity of a protein are determined by its interacting abilities and properties, certain amino acid substitutions can be made in the protein sequence and its DNA coding sequence, yet proteins with similar properties can be obtained. Therefore, various alterations can be made in the DNA sequence of genes without significantly losing their biological utility or activity, as described below.

[0153] In preparing such modified proteins, the hydropathic index of the amino acids should be considered. It is generally accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn determines its interaction with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Hydropathic indices are assigned to each amino acid based on its hydrophobicity and charge characteristics (by reference Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982, incorporated herein by reference). Amino acids can often be substituted with other amino acids having similar hydropathic indices or scores, yet proteins with similar biological activities are still obtained; that is, proteins with equivalent biological functions are still obtained.

[0154] Furthermore, similar amino acid substitutions can be made effectively based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, describes how the highest local average hydrophilicity of a protein, controlled by the hydrophilicity of adjacent amino acids, is correlated with the protein's biological properties. Therefore, one amino acid can be substituted for another amino acid with a similar hydrophilicity value, while still yielding a biologically and immunologically equivalent protein.

[0155] Exemplary amino acid substitutions that can be used in this invention include, but are not limited to: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and exchanges of valine, leucine, and isoleucine. Other such substitutions are well known to those skilled in the art, taking into account the need to retain some or all of the biological activity while altering the secondary structure of the protein.

[0156] Another variant considered for preparing the peptides of this invention is the use of peptide mimics. Mimics are peptide-containing molecules that mimic elements of the secondary structure of proteins. See, for example, Johnson et al., “Peptide Turn Mimetics,” in Biotechnology and Pharmacy, eds. Pezzuto et al., C&H (Chapman and Hall), New York (1993). The principle behind using peptide mimics is that the presence of the peptide backbone in proteins primarily serves to orient amino acid side chains to facilitate molecular interactions, such as antibody-antigen interactions. Peptide mimics are expected to engage in molecular interactions similar to those of natural molecules. These principles, along with those described above, can be combined to engineer second-generation molecules that possess many of the natural properties of lysosomal sulfatases, but with altered or even enhanced characteristics.

[0157] Modification of lysosomal sulfatases by glycosylation

[0158] Compared to the parent peptide, variants of highly phosphorylated lysosomal sulfatases with modified glycosylation patterns can also be produced, such as by the deletion of one or more carbohydrate moieties and / or the addition of one or more glycosylation sites not present in the native peptide.

[0159] Glycosylation is typically N-linked or O-linked. N-linked glycosylation refers to the carbohydrate moiety being attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Therefore, an N-linked glycosylation site can be added to a polypeptide by altering its amino acid sequence, thus giving it one or more of these tripeptide sequences. O-linked glycosylation sites refer to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. O-linked glycosylation sites can be added by inserting or substituting one or more serine or threonine residues into the original polypeptide sequence.

[0160] Domain conversion

[0161] The various parts of the lysosomal sulfatase protein share significant sequence homology. Mutations that alter the function of the lysosomal sulfatase polypeptide can be identified. These studies are likely important for at least two reasons. First, they reasonably anticipate the presence of other homologs, allelic variants, and mutants of this gene in relevant species such as rats, rabbits, monkeys, gibbons, chimpanzees, apes, baboons, cattle, pigs, horses, sheep, and cats. Isolating these homologs, variants, and mutants, and combining them with other analyses, could identify certain active and functional domains. Second, this will provide a starting point for further mutational analysis of the aforementioned molecules. One approach to developing this information is "domain switching."

[0162] Domain switching involves generating recombinant molecules from dissimilar but related peptides. For example, by comparing the sequences of lysosomal sulfatases, such as N-acetylgalactosamine-6-sulfatase, with those of similar lysosomal sulfatases from another source, functionally significant regions in these molecules can be predicted. Relevant domains of these molecules can then be switched to determine the criticality of these regions for enzyme function and their role in lysosomal storage diseases. An added value of these molecules is that these “chimeras” may differ from the native molecules but may provide the same or even enhanced functions.

[0163] Further analysis of mutations and their projected effects on secondary structure, based on the many lysosomal sulfatases currently identified, will enhance this understanding. Mutants that switch domains between lysosomal sulfatases are expected to provide useful information about the structure-activity relationships of these molecules and the polypeptides that interact with them.

[0164] Fusion protein

[0165] In addition to the mutations described above, this invention also considers generating specialized types of insert variants, termed fusion proteins. These molecules typically possess all or most of the native molecule, linked at the N- or C-terminus to all or part of a second polypeptide. For example, fusions often utilize leader sequences from other species to enable recombinant protein expression in a heterologous host. Another useful fusion involves the addition of immunologically active domains, such as antibody epitopes, to facilitate fusion protein purification. The presence of cleavage sites at or near the fusion binding site aids in the removal of the foreign polypeptide after purification. Other useful fusions include sites that link functional domains, such as enzyme, glycosylation domains, cell-targeting signaling sites, or transmembrane active sites.

[0166] This invention can utilize a variety of commercially available fusion protein expression systems. Particularly useful systems include, but are not limited to: the glutathione S-transferase (GST) system from Pharmacia, Piscataway, NJ; the maltose-binding protein system from NEB, Beverley, MA; the FLAG system from IBI, New Haven, CT; and the 6xHis system from Qiagen, Chatsworth, CA. These systems produce recombinant peptides carrying only a small number of additional amino acids, unlikely to affect the antigenicity of the recombinant peptide. For example, the FLAG and 6xHis systems only add short sequences, both known to have poor antigenicity, which does not adversely affect the folding of the peptide into its native conformation. Another potentially useful N-terminal fusion is the fusion of a Met-Lys dipeptide into the N-terminal region of a protein or peptide. Such fusions can beneficially increase protein expression or activity.

[0167] A particularly useful construct may be one in which an active, highly phosphorylated lysosomal sulfatase polypeptide or fragment thereof is fused to a hapten, thereby enhancing the immunogenicity of the lysosomal sulfatase fusion construct. This can be used to generate antibodies against the active, highly phosphorylated lysosomal sulfatase, enabling the detection of the protein. In other embodiments, fusion constructs that enhance the targeting of lysosomal sulfatase-related compositions to specific sites or cells can be prepared.

[0168] The invention includes heterologous peptides with desired properties, such as other fusion constructs that target lysosomal sulfatases to specific organs, tissues, or cell types. In a preferred embodiment, a fusion construct containing a bone-targeting peptide, such as a fusion construct fused to six aspartic residues (6xAsp or 6D) of a lysosomal sulfatase, can target the enzyme to specific sites in bone.

[0169] Other fusion constructs incorporating heterologous peptides with desired properties, such as Ig constant regions, have also been considered to extend the serum half-life of the targeting antibody or fragment thereof. Other fusion systems produce peptide hybrids, in which case the fusion partner of the desired peptide is preferably cleaved. In one embodiment, the fusion partner is linked to a recombinant, highly phosphorylated lysosomal sulfatase peptide via a peptide sequence containing a specific recognition sequence of the protease. Examples of suitable sequences are those recognized by the tobacco etch virus protease from Life Technologies, Gaithersburg, MD, or by Factor Xa from New England Biolabs, Beverley, MA.

[0170] derivative

[0171] As described above, derivatives refer to polypeptides chemically modified by techniques such as, but not limited to, ubiquitous proteination, labeling (e.g., with radionuclides or various enzymes), covalent polymer linkage, such as pegification (derived from polyethylene glycol), and chemically synthesized amino acids, such as the insertion or substitution of ornithine. Derivatives of lysosomal sulfatases can also be used as therapeutic agents and can be produced by the methods of this invention.

[0172] Polyethylene glycol (PEG) can be attached to lysosomal sulfatases produced by the method of the present invention to provide a longer in vivo half-life. The PEG group can have any convenient molecular weight and can be linear or branched. The average molecular weight of PEG is preferably from about 2 kilodaltons (“kDa”) to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, and most preferably from about 5 kDa to about 10 kDa. The PEG group is generally attached to the lysosomal sulfatase of the present invention via acylation or reductive alkylation, whereby the reactive group of the PEG moiety (e.g., aldehyde, amino, thiol, or ester group) is attached to the reactive group of the protein moiety (e.g., aldehyde, amino, or ester group). The PEG moiety can be added to the polypeptide of interest using techniques well known in the art. See, for example, International Publication No. WO 96 / 11953 and U.S. Patent No. 4,179,337.

[0173] The lysosomal sulfatase peptide is linked to PEG in an aqueous phase, which can be easily monitored by reversed-phase analytical HPLC. The PEGylated peptide can be easily purified by preparative HPLC, and characterized by analytical HPLC, amino acid analysis, and laser desorption / resorption spectroscopy.

[0174] markers

[0175] In some embodiments, therapeutic lysosomal sulfatases are labeled to facilitate detection. A “label” or “detectable portion” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical methods. For example, labels suitable for use in this invention include, but are not limited to, radiolabels (e.g., 32 P), fluorophores (e.g., fluorescein), electron densifiers, enzymes (e.g., those routinely used in ELISA), biotin, digitoxin, or haptens, and proteins that can be formulated into detectable (e.g., by incorporating a radiolabeled substance into a hapten or peptide) or used to detect antibodies that specifically react with a hapten or peptide.

[0176] Examples of markers suitable for use in this invention include, but are not limited to: fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), and radioactive markers (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colorimetric markers such as colloidal gold, colored glass, or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

[0177] The label can be directly or indirectly coupled to the desired portion of lysosomal sulfatase using methods well known in the art. In one embodiment, the label is preferably covalently bound to lysosomal sulfatase using an isocyanate reagent coupled with the active substance of the present invention. In one aspect of the invention, the label can be coupled to lysosomal sulfatase using the bifunctional isocyanate reagent of the present invention to form a label-lysosomal sulfatase conjugate without an active agent. The label-lysosomal sulfatase conjugate can be used as an intermediate in the synthesis of the label-conjugate of the present invention, or can be used to detect lysosomal sulfatase conjugates. As described above, various labels can be used, selected according to the desired sensitivity, ease of coupling with the desired portion of lysosomal sulfatase, stability requirements, available instruments, and processing specifications. Non-radioactive labels are often linked indirectly. Ligand molecules (e.g., biotin) are typically covalently bound to lysosomal sulfatase. The ligand is then bound to another molecule (e.g., streptavidin), which is either detectable on its own or covalently bound to a signaling system, such as a detectable enzyme, fluorescent compound, or chemiluminescent compound.

[0178] The lysosomal sulfatase of the present invention can also be directly coupled to a signal-generating compound, for example, coupled to an enzyme or a fluorophore. Suitable enzymes for use as labels include, but are not limited to: hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidotases, particularly peroxidases. Suitable fluorescent compounds, i.e., fluorophores, for use as labels include, but are not limited to: fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Other examples of suitable fluorophores include, but are not limited to: eosin, TRITC-amine, quinine, fluorescein W, acridine yellow, rhodamine sulfonyl chloride, erythroscein, ruthenium (tris, dipyridyl salt), Texas red, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, etc. Suitable chemiluminescent compounds for use as labels include, but are not limited to: fluorescein and 2,3-dihydrophthalazine dione, such as luminol. A summary of various labels or signal-generating systems that can be used in the methods of the present invention is available in U.S. Patent No. 4,391,904.

[0179] Devices for detecting labeled substances are well known to those skilled in the art. Therefore, for example, if the labeled substance is radioactive, the detection device includes a scintillation counter or photographic film, such as that used in autoradiography. If the labeled substance is fluorescent, it can be detected by exciting a fluorescent dye with light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be visually detected using electronic detectors, such as charge-coupled devices (CCDs) or photomultiplier tubes. Similarly, enzyme labels can be detected by providing a suitable substrate for the enzyme and detecting the reaction products. Colorimetric or chemiluminescent labels can be easily detected by observing the associated color of the label. Other labeling and detection systems suitable for the methods of this invention will be readily apparent to those skilled in the art. Modifiers and ligands of such labels can be used to diagnose diseases or health conditions.

[0180] In a preferred embodiment, the method includes the step of generating an active, highly phosphorylated lysosomal sulfatase from an endosome-defective cell line. In a particularly preferred embodiment, the method includes generating an active, highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) from a CHO cell line G71 or a derivative thereof. Generating a lysosomal sulfatase, such as, but not limited to, GALNS, includes the steps of: (a) generating a G71 or G71 derivative line co-expressing a recombinant human lysosomal sulfatase, such as N-acetylgalactosamine-6-sulfatase (GALNS), and recombinant human sulfatase modification factor 1 (SUMF1); (b) culturing a cell line co-expressing human lysosomal sulfatase and SUMF1; and (c) scaling up the cell line co-expressing human lysosomal sulfatase and SUMF1 to a bioreactor to generate lysosomal sulfatase. In a preferred embodiment, human lysosomal sulfatase, such as N-acetylgalactosamine-6-sulfatase (GALNS), and human SUMF1 cDNA are subcloned into a mammalian expression vector, essentially as described below.

[0181] For cell line development, G71 or G71S (G71 clones suitable for serum-free suspension culture) were co-transfected with human GALNS mammalian expression vector, human SUMF1 mammalian expression vector, and selectable marker genes, and stable transformants were selected. After the first round of subcloning, stable transfectants were used to select cell lines using fluorescent substrates and named accordingly. Cell-specific yields (picograms of product / cell) of G71 or G71S cell lines in microcarrier-containing spinners or suspension cultures were analyzed. The optimal cell lines for human GALNS production were identified and scaled up in bioreactors to produce preclinical materials.

[0182] In another embodiment, the present invention provides a cell assay for detecting the activity of recombinant human lysosomal enzymes in degrading natural substrates. The method includes (a) culturing isolated human cells deficient in the lysosomal enzyme under conditions of natural substrate accumulation; (b) contacting the cells with the lysosomal enzyme; (c) lysing the cells; (d) adding an enzyme that is (i) specific to the natural substrate and (ii) cleaves a small oligosaccharide from the natural substrate to the cell lysate; (e) labeling the small oligosaccharide with a detectable fraction; (f) optionally isolating the labeled small oligosaccharide; (g) detecting the labeled small oligosaccharide; and (h) determining the activity of the lysosomal enzyme in degrading natural products by comparing the content of the labeled small oligosaccharide in (i) cells contacted with the lysosomal enzyme with the content of the labeled small oligosaccharide in (ii) cells not contacted with the lysosomal enzyme, wherein a decrease in (h)(i) indicates a decrease in the activity of the lysosomal enzyme in degrading natural substrates compared to (h)(ii). In one embodiment, the small oligosaccharide is a monosaccharide, disaccharide, or trisaccharide. In a related embodiment, the small oligosaccharide is a disaccharide.

[0183] In some embodiments, the lysosomal enzyme is selected from: arylsulfatase B (ARSB), iduronate-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS). In some embodiments, the lysosomal enzyme is α-L-iduronate (IDU). In some embodiments, the lysosomal enzyme is acid α-glucosidase (GAA). In some embodiments, the lysosomal enzyme is β-glucuronidase (GUSB). In some embodiments, the lysosomal enzyme is β-galactosidase (GLB1).

[0184] Suitable human cells for cell assays include any human cell lacking the lysosomal enzyme being tested, thus capable of accumulating the natural substrate of that lysosomal enzyme. For example, cells naturally exhibiting complete or partial defects in activity can be used, such as reduced activity by 30%, 50%, 70%, 80%, 90%, 95%, or more. Cells expressing mutant enzymes with reduced activity, or cells obtained from patients with lysosomal storage disorders, such as mucopolysaccharidosis, can be used. Cells recombinantly altered to knock out or reduce the activity of lysosomal enzymes can be used, for example, by introducing mutations into the coding gene or its promoter or other regulatory regions. Cells treated to reduce the activity of lysosomal enzymes can be used, for example, by treatment with antisense or RNAi to reduce enzyme expression.

[0185] Those skilled in the art can select suitable enzymes that cleave (digest) oligosaccharides from carbohydrates and that have "specificity" (i.e., primary digestion) for the natural substrates of lysosomal enzymes. For example, to detect the activity of GALNS or GLB1 (an enzyme that degrades keratin sulfate), the enzyme in step (d) can be keratinase II or any enzyme that primarily acts on keratin sulfate. As another example, to detect IDU, ARSB, IDS, or GUSB (an enzyme that degrades dermatan sulfate), the enzyme in step (d) can be chondroitinase ABC or any enzyme that primarily acts on dermatan sulfate. As another example, to detect IDU, IDS, SGHS, G6S, or GUSB (an enzyme that degrades heparan sulfate), the enzyme in step (d) can be heparinoid I or heparinoid II, or both. As another example, to detect GAA (an enzyme that degrades glycogen), the enzyme in step (d) can be α-amylase or any enzyme that primarily acts on glycogen.

[0186] This cellular method can detect lysosomal enzyme activity with high sensitivity. In some embodiments, lysosomal enzyme activity can also be detected when the concentration of lysosomal enzymes is as low as about 10 nM, or about 5 nM, or about 1 nM, or about 0.75 nM, or about 0.5 nM, or about 0.25 nM, or about 0.1 nM, or about 0.05 nM, or about 0.01 nM, or about 0.005 nM, or about 1 pM, or about 0.5 pM.

[0187] II. Purification of lysosomal sulfatase

[0188] Bioreactor material containing recombinant human GALNS was sterile filtered through a 0.2 μm filter and stored at 4 °C. The bioreactor material was either directly loaded onto a trapping column or concentrated 10- or 20-fold by ultrafiltration before loading. The pH of the bioreactor material or concentrated bioreactor material was adjusted to pH 4.5, and then loaded onto a blue agarose column. The material was washed sequentially with 20 mM acetic acid / phosphate, 50 mM NaCl, pH 4.5 and 20 mM acetic acid / phosphate, 50 mM NaCl, pH 6.0, followed by elution with 20 mM acetic acid / phosphate, 100 mM NaCl, pH 7.0. The blue agarose column eluent was then loaded onto a Fractogel SE Hi-Cap and washed sequentially with 20 mM acetic acid / phosphate, 50 mM NaCl, pH 5.0 and 20 mM acetic acid / phosphate, 50 mM NaCl, pH 5.5, followed by elution with a gradient of 20 mM acetic acid / phosphate, 50–350 mM NaCl, pH 5.5. The Fractogel SE Hi-Cap eluent was prepared with 10 mM NaOAc, 1 mM NaH₂PO₄, 0.005% Tween-80, pH 5.5.

[0189] Alternatively, the bioreactor material containing recombinant human GALNS can be concentrated 20-fold by ultrafiltration before being loaded onto a capture column. The concentrated bioreactor material is then adjusted to pH 4.5 and loaded onto a Fractogel SE Hi-Cap column. It is washed sequentially with 10 mM acetic acid / phosphate, 50 mM NaCl, pH 4.5 and 10 mM acetic acid / phosphate, 50 mM NaCl, pH 5.0, followed by elution with 10 mM acetic acid / phosphate, 140 mM NaCl, pH 5.0. The Fractogel SE Hi-Cap column eluent is then adjusted to 500 mM NaCl, pH 7.0 and loaded onto a Zn-chelated agarose (Zn-IMAC) column. The eluent is washed with 10 mM acetic acid / phosphate, 125 mM NaCl, 10 mM imidazole, pH 7.0, followed by elution with 10 mM acetic acid / phosphate, 125 mM NaCl, 90 mM imidazole, pH 7.0. The Zn-chelate agarose (Zn-IMAC) column eluent was adjusted to pH 3.5 for low-pH virus inactivation, and then adjusted to 10 mM acetic acid / phosphate, 2 M NaCl, pH 5.0. This solution was then loaded onto a ToyoPearl Butyl 650M column and washed with 10 mM acetic acid / phosphate, 2 M NaCl, pH 5.0, followed by elution with 10 mM acetic acid / phosphate, 0.7 M NaCl, pH 5.0. The ToyoPearl Butyl 650M eluent was ultrafiltered, dialyzed against 20 mM acetic acid, 1 mM phosphate, 150 mM NaCl, pH 5.5, and then prepared with 20 mM acetic acid, 1 mM phosphate, 150 mM NaCl, 0.01% Tween-20, pH 5.5.

[0190] The purification of recombinant human GALNS is described in detail below, and an improved method for purifying recombinant human GALNS according to the above protocol is also described in detail below.

[0191] As described in Example III, recombinant human GLANS enzymes were expressed in G71S cells and purified as described in Example V. The purified recombinant human GLANS of this invention can be compared with other GLANS recordings. Masue et al. (J. Biochem. 110:965-970, 1991) described the purification and characterization of human placental GLANS. They found that the purified enzyme had a molecular weight of 120 kDa, composed of 40 kDa and 15 kDa polypeptides, the latter being proven to be a glycoprotein. Therefore, the GLANS enzyme of Masue et al. appears to correspond to... Figure 5The processing method described. Bielicki et al. (Biochem. J. 279:515-520, 1991) described the purification and characterization of human liver GALNS. When analyzed by SDS-PAGE, the molecular weight of the enzyme was 70 kDa under non-reducing conditions and 57 kDa, 39 kDa, and 19 kDa under reducing conditions. Bielicki et al. (Biochem. J. 311:333-339, 1995) described the purification and characterization of recombinant human GALNS from Chinese hamster ovary cells. SDS-PAGE revealed that the purified enzyme had a molecular weight of 58-60 kDa under non-reducing conditions and 55-57 kDa, 39 kDa, and 38 kDa under reducing conditions. Therefore, the GALNS enzyme described by Bielicki et al. appears to correspond to... Figure 5 The enzyme is a mixture of its pre-processing (precursor) and processed forms. In contrast, the recombinant human GALNS enzyme of the present invention is essentially composed entirely of the enzyme's precursor form (see...). Figure 9 ), or mainly (i.e., at least about 85%) the precursor form of the enzyme (see Figure 10 ).

[0192] IV. Lysosomal sulfatase and lysosomal storage diseases

[0193] Lysosomal sulfatase is at least a majority (e.g., at least about 50%, preferably at least about 75%, more preferably at least about 90%), substantially all, or all of any fragment, mutant, variant, or derivative thereof of the full-length enzyme or which retains the enzyme’s therapeutic or biological activity (e.g., sulfatase activity).

[0194] In some embodiments, the lysosomal sulfatase is an enzyme that, if not expressed or produced, or if its expression or production is substantially reduced, will cause disease, including but not limited to lysosomal storage diseases. In some embodiments, the lysosomal sulfatase is an enzyme that, if not expressed or produced, or if its expression or production is substantially reduced, may not cause disease, but its absence or reduced expression or production is associated with disease, including but not limited to lysosomal storage diseases. The lysosomal sulfatase is preferably derived from or obtained from humans.

[0195] In the treatment of lysosomal storage diseases, lysosomal sulfatases are preferably enzymes that cause lysosomal storage diseases if they are not expressed or not produced, or if their expression or production is substantially reduced. Alternatively, in the treatment of lysosomal storage diseases, lysosomal sulfatases are enzymes whose absence or substantial reduction in expression or production is associated with the disease, but whose absence or substantial reduction in expression or production may not itself cause the disease. Lysosomal sulfatases are preferably derived from or obtained from humans.

[0196] The enzyme is preferably a lysosomal sulfatase, such as arylsulfatase A (ARSA) (Genbank accession number NP_000478 (isotype a), Genbank accession number NP_001078897 (isotype b) and its precursor variant), arylsulfatase B / N-acetylglucosamine-4-sulfatase (ARSB) (Genbank accession number P15848), and iduronate-2-sulfatase (IDS) (Genbank accession number NP_0001). 93 (isotype a), Genbank accession number NP_006114 (isotype b)), sulfonamide / heparin-N-sulfatase (SGSH) (Genbank accession number NP_000190), N-acetylglucosamine-sulfatase (G6S) (Genbank accession number NP_002067), and galactosyl-6-sulfatase / N-acetylgalactosamine-6-sulfatase (GALNS) (Genbank accession number NP_000503). A table of lysosomal storage diseases and the lysosomal sulfatases that are deficient in them and can be used as therapeutic agents is shown below:

[0197]

[0198] In a preferred embodiment, the lysosomal sulfatase is a recombinant human lysosomal sulfatase produced by an intron acidosis-deficient cell line. In a more preferred embodiment, the recombinant human lysosomal sulfatase is active and has a high level of phosphorylated oligosaccharides, as specified in the "Definitions". In the most preferred embodiment, the lysosomal sulfatase is a highly active phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

[0199] Therefore, lysosomal storage disorders that can be treated or prevented using the methods of the present invention include, but are not limited to: metachromatic leukodystrophy or MLD, Marr-Lan II syndrome or MPS VI, Hunter syndrome or MPS II, Schennfeld A syndrome or MPS IIIa, Schennfeld D syndrome or MPS IIId, and Morquio A syndrome or MPS IVa. In a particularly preferred embodiment, the lysosomal sulfatase is the enzyme whose deficiency leads to Schennfeld A syndrome or MPS IVa. In another particularly preferred embodiment, the lysosomal sulfatase is the enzyme whose deficiency is associated with human lysosomal storage disorders, such as multiple sulfatase deficiency or MSD.

[0200] Therefore, according to the table above, for each disease, the lysosomal sulfatase preferably includes the specific active lysosomal sulfatase lacking in the disease. For example, for methods involving MPSII, the preferred enzyme is iduronic acid-2-sulfatase. For methods involving MPS IIIA, the preferred enzyme is sulfonamide / heparin-N-sulfatase. For methods involving MPS IIID, the preferred enzyme is N-acetylglucosamine-6-sulfatase. For methods involving MPS IVA, the preferred enzyme is galactosyl-6-sulfatase / N-acetylgalactosamine-6-sulfatase. For methods involving MPSVI, the preferred enzyme is N-acetylgalactosamine-4-sulfatase. For methods involving metachromatic leukodystrophy (MLD), the preferred enzyme is arylsulfatase A. For methods involving multiple sulfatase deficiency (MSD), the enzyme may be arylsulfatase A, arylsulfatase B / N-acetylglucosamine 4-sulfatase, iduronic acid-2-sulfatase, sulfonamide / heparin-N-sulfatase, N-acetylglucosamine-sulfatase, or galactose-6-sulfatase / N-acetylgalactosamine-6-sulfatase, with galactose-6-sulfatase / N-acetylgalactosamine-6-sulfatase being the preferred enzyme.

[0201] V. Mucopolysaccharidosis IVA (Morchio syndrome, MPSIVA)

[0202] Mucopolysaccharidosis type IVA (Morchio syndrome, MPS IVA) is a hereditary, autosomal recessive disorder of the mucopolysaccharidosis disorder family. Morchio syndrome is caused by a deficiency of lysosomal enzymes required to degrade two glycosaminoglycans (GAGs), keratin sulfate (KS), and chondroitin-6-sulfate (C6S). Specifically, MPS IVa is characterized by a deficiency of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS) and the secretion of KS into the urine. The lack of GALNS leads to an abnormal accumulation of large amounts of mucopolysaccharides in hyaline cartilage (a major component of bone tissue). All patients have systemic skeletal developmental abnormalities. Other symptoms vary in severity from patient to patient and may include hearing loss, cataracts, spinal instability, valvular heart disease, and respiratory problems.

[0203] GALNS hydrolyzes the sulfate ester bond of galactose-6-sulfate on KS and the sulfate ester bond of N-acetylgalactosamine-6-sulfate on C6S. Human GALNS is expressed as a 55-60 kDa precursor protein with only two potential asparagine-linked glycosylation sites. Mannose-6-phosphate (M6P) is part of the oligosaccharide present on the GALNS molecule. Receptors on the lysosomal cell surface recognize M6P, thus being crucial for efficient uptake of GALNS.

[0204] Similar to all sulfatases, GALNS requires processing by the formylglycine-activating enzyme (FGE) encoded by the sulfatase modification factor 1 (SUMF1) gene to acquire activity. This activation step involves post-translational modification of the cysteine ​​residue at the active site to C. α -Formylglycine (FGly), overexpression of recombinant sulfatase can lead to lower specific activity of sulfatase production (i.e., a mixture of activated and non-activated sulfatases) and lower titers (i.e., degradation and / or non-secretion of non-activated sulfatases).

[0205] The object of this invention is to provide a highly phosphorylated human N-acetylgalactosamine-6-sulfatase that can be used to treat Morgio syndrome and other diseases associated with N-acetylgalactosamine-6-sulfatase deficiency, such as multiple sulfatase deficiency (MSD). This highly phosphorylated human N-acetylgalactosamine-6-sulfatase can localize to tissues where KS and C6S accumulate, has sufficient M6P levels for effective uptake, a sufficiently high percentage of FGly for enzyme activity, and a high production level.

[0206] It should be understood that the methods of the present invention described herein are applicable to the production of other lysosomal sulfatases, such as arylsulfatase A (ARSA), arylsulfatase B / N-acetylglucosamine-4-sulfatase (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), and N-acetylglucosamine-sulfatase (G6S), which can be used to treat lysosomal storage disorders caused by or characterized by their deficiency.

[0207] VI. Pharmaceutical composition and administration

[0208] The lysosomal sulfatase of the present invention can be administered via various routes. For oral products, the lysosomal sulfatase can be used alone or in combination with suitable additives to prepare tablets, powders, granules, or capsules, such as conventional additives like sucrose, mannose, corn starch, or potato starch; binders like microcrystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin; disintegrants like corn starch, potato starch, or sodium carboxymethyl cellulose; lubricants like talc or magnesium stearate; and, if desired, may contain diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0209] Injectable articles can be formulated by dissolving or suspending the lysosomal sulfatase of the present invention in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, or emulsifying therein; if desired, conventional additives may be included, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0210] The lysosomal sulfatase of the present invention can be prepared as an aerosol for inhalation administration. The lysosomal sulfatase of the present invention can be formulated into a pressurized, acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, etc.

[0211] Furthermore, the lysosomal sulfatase of the present invention can be formulated into suppositories by mixing with various base materials, such as emulsified base materials or water-soluble base materials. The lysosomal sulfatase of the present invention can be administered rectally via suppositories. The suppositories may contain a carrier that melts at body temperature but is solid at room temperature, such as cocoa butter, carbon wax, and polyethylene glycol.

[0212] Unit dosage forms of the lysosomal sulfatase of the present invention, for oral or rectal administration, such as syrups, elixirs, and suspensions, are available, wherein each dosage unit, such as a teaspoon, a tablespoon, a tablet, or a suppository, contains a predetermined amount of lysosomal sulfatase containing the active agent. Similarly, unit dosage forms for injection or intravenous administration may contain lysosomal sulfatase, for example, a solution formulated with sterile water, ordinary saline, or another pharmaceutically acceptable carrier.

[0213] In practical applications, the lysosomal sulfatase of the present invention can be closely mixed as an active ingredient with one or more pharmaceutically acceptable carriers, diluents, or excipients using conventional pharmaceutical mixing techniques. Depending on the preferred form of the product required for administration, such as oral or parenteral (including intravenous) administration, the carrier, diluent, or excipient can take various forms. When preparing oral dosage forms of lysosomal sulfatase compositions, any commonly used pharmaceutical medium can be used. In the case of oral liquid products, such as suspensions, elixirs, and solutions, water, glycerin, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used; in the case of oral solid products, such as powders, hard and soft gelatin capsules and tablets, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, etc., may be used, with solid oral products being preferred over liquid products.

[0214] For methods of transdermal drug delivery, see Remington Pharmaceutical Sciences, 17th edition (Gennaro et al., eds., Mark Publishing, 1985). Dermal or skin patches are the preferred method for transdermal delivery of the lysosomal sulfatase of the present invention. The patch preferably provides an absorption enhancer, such as DMSO, to increase the absorption of the lysosomal sulfatase. Other methods of transdermal drug delivery are described in U.S. Patent Nos. 5,962,012, 6,261,595, and 6,261,595, each incorporated herein by reference in its entirety.

[0215] Pharmaceutically acceptable excipients, such as carriers, adjuvants, transporters, or diluents, are commercially available. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tension modifiers, stabilizers, and humectants, are also commercially available.

[0216] In all respects, lysosomal sulfatase compositions include, but are not limited to, compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), pulmonary (nasal or oral inhalation), or nasal administration; however, the most suitable route of administration in any given case depends in part on the nature and severity of the condition being treated and the nature of the active ingredient. Exemplary routes of administration are oral and intravenous. Lysosomal sulfatase compositions can be conveniently produced in unit dosage forms and can be prepared by any method well known in the pharmaceutical field.

[0217] Tablets and capsules represent the most advantageous oral unit dosage forms due to their ease of administration, a situation clearly utilizing solid drug carriers. Tablets can be coated using standard aqueous or non-aqueous techniques if desired. The percentage of active lysosomal sulfatase in these compositions is, of course, variable and can be conveniently taken as approximately 2% to approximately 60% per unit weight.

[0218] The lysosomal sulfatase of the present invention can be administered by encapsulating or attaching to a viral envelope or vesicle, or by incorporating it into a cell. Vesicles are typically spherical and often lipid-based microcellular particles. Liposomes are vesicles formed by a double membrane. Suitable vesicles include, but are not limited to, monolayered vesicles and multilayered lipid vesicles or liposomes. Such vesicles and liposomes can be prepared using standard techniques, such as those described in U.S. Patent No. 4,394,448, from various lipid or phospholipid compounds, such as phosphatidylcholine, phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, glycolipids, gangliosides, etc. Lysosomal sulfatase can be administered intracellularly using such vesicles or liposomes, and can be delivered to target organs. Encapsulation can also be used to achieve controlled release of the lysosomal sulfatase of interest (see, for example, U.S. Patent No. 5,186,941).

[0219] The lysosomal sulfatase composition can be administered by diluting it into the bloodstream, or preferably by any route of administration at least outside the blood-brain barrier. Peripheral administration is preferred, and intravenous or cardiac catheterization is most preferred. Intravenous and intracarotid injection are also possible. The lysosomal sulfatase composition can be administered locally or regionally, for example intraperitoneally, subcutaneously, or intramuscularly. In one aspect, the lysosomal sulfatase composition is administered in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0220] Those skilled in the art will readily recognize that dosage levels vary functionally with the specific lysosomal sulfatase, the severity of symptoms, and the subject's sensitivity to side effects. Those skilled in the art can readily determine the optimal dosage for a given lysosomal sulfatase using various methods, including but not limited to dose-response and pharmacokinetic assessments in patients, test animals, and in vitro.

[0221] The dosage to be administered may also depend on individual needs, the desired effect, the specific lysosomal sulfatase used, and the chosen route of administration. The dosage range of lysosomal sulfatase is from about 0.2 picomoles / kg to about 20 nanomoles / kg, with a preferred range of 2 picomoles / kg to 2 nanomoles / kg, and a particularly preferred range of 2 picomoles / kg to 200 picomoles / kg. Alternatively, the dosage range of lysosomal sulfatase may be from 0.01 to 1000 mg / kg. A preferred range may be from 0.1 to 100 mg / kg, and a particularly preferred range of 0.1 to 10 mg / kg. These dosages may be affected by, for example but not limited to, the specific lysosomal sulfatase, the form of the pharmaceutical composition, the route of administration, and the site of action of the specific lysosomal sulfatase.

[0222] The lysosomal sulfatases of the present invention can be used in animals, and particularly in humans, for therapeutic, preventative, and diagnostic interventions. The lysosomal sulfatases can exhibit preferential accumulation in specific tissues. Preferred medical indications for diagnostic applications include, for example, any condition related to a target organ of interest (e.g., lung, liver, kidney, spleen).

[0223] The method can be used to treat a variety of diseases. In some embodiments, particular interest is found in diseases where a lysosomal sulfatase with the desired activity has been previously identified, but which has not been adequately delivered to the target site, region, or compartment, to produce a completely satisfactory therapeutic outcome. Using such lysosomal sulfatases, the efficacy and therapeutic index of lysosomal sulfatases can be enhanced by employing the method to produce highly active phosphorylated sulfatases.

[0224] Treatment encompasses any outcome beneficial to the subject of administration of lysosomal sulfatase, including a reduced likelihood of illness, prevention of disease, slowing, terminating, or reversing disease progression, or alleviating symptoms associated with the host's disease. This relief or benefit is used broadly and refers at least to the treated pathological condition, such as a reduction in the magnitude of related inflammation and pain parameters. Therefore, treatment also includes complete inhibition, such as prevention of occurrence, or termination, such as termination of the pathological condition or at least the associated symptoms, such that the host no longer suffers from the pathological condition or at least is no longer tormented by its characteristic symptoms.

[0225] The method can treat a variety of hosts or objects. Such hosts are generally “mammals,” a term that is widely used to describe organisms in the class Mammalia, including the following orders: carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host is a human.

[0226] The invention has now been generally described, and the invention will be more readily understood with reference to the following examples, which provide exemplary protocols for the production and purification of highly phosphorylated lysosomal sulfatases and their use in the treatment of lysosomal storage diseases. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should, of course, be taken into account. Example

[0227] Example 1

[0228] Human sulfatase modifying factor 1 (SUMF1) and human N-acetylgalactosamine-6-sulfatase

[0229] mammalian expression vector of (GALNS)

[0230] The goal is to construct a mammalian expression vector that is suitable for producing sufficient amounts of phosphorylated, active lysosomal sulfatases in stably transfected cells.

[0231] A full-length human sulfatase-modifying factor 1 (SUMF1) cDNA encoding a 374-amino acid polypeptide (see US Patent Application Nos. US20005 / 0123949, published June 9, 2005, and US2004 / 0229250, published November 8, 2004, both incorporated herein by reference in their entirety) was cloned into the mammalian expression vector cDNA4 from Invitrogen, Carlsbad, CA, containing a human CMV enhancer-promoter and a multiple cloning site. The presence of bovine growth hormone polyadenylation sequences ensured effective transcription termination. The selection markers were the zero-mycin tolerance gene controlled by the EM-7 promoter and the early polyadenylation sequence of SV40. The resulting plasmid was named pcDNA4SUMF1. The sequences of the human SUMF1 polynucleotide (SEQ ID NO:1) and polypeptide (SEQ ID NO:2) are shown below. Figure 1 and Figure 2 .

[0232] A full-length human N-acetylgalactosamine-6-sulfatase (GALNS) cDNA encoding a 522-amino acid polypeptide containing a 26-amino acid signal peptide (see Tomatsu et al., Biochem. Biophys. Res. Commun. 181(2):677-683, 1991) was cloned into the BioMarin mammalian expression vector pCIN, which contains a human CMV enhancer-promoter and multiple cloning site linked to the rabbit β-globin IVS2 intron. The presence of a bovine growth hormone polyadenylation sequence ensured effective transcription termination. The selection marker was a neomycin phosphotransferase gene carrying a point mutation to reduce enzyme efficiency. The weakened marker was further impeded with a weak HSV-tk promoter. The resulting plasmid was named pCIN 4A. The sequences of the human GALNS polynucleotide (SEQ ID NO:3) and polypeptide (SEQ ID NO:4) are shown in the figure. Figure 3 and Figure 4 .

[0233] To increase the expression levels of SUMF1 and GALNS, scaffold / matrix junction (MAR) elements (see Mermod et al., U.S. Patent No. 7,129,062) were cloned into SUMF1 and GALNS expression plasmids.

[0234] BMAR SUMF1 was prepared by digesting Selexis P<1_68X_X NcoI with BamHI and HincII to fill MAR fragments, and then inserting the released MAR fragments into pcDNA4 SUMF1 digested with BglII and NruI.

[0235] PMARSUMF1 was prepared by digesting Cyrex's P<1_68NcoI-filled (MAR)SV40EGFP with HindIII and XbaI to remove the EGFP gene, and then inserting the SUMF1 gene released from pcDNA4 SUMF1 by digestion with HindIII and XbaI.

[0236] BMAR 4A was prepared by digesting BMAR SUMF1 with PmeI and SpeI to remove the SUMF1 gene, and then inserting the GALNS gene released from pCIN 4A by digestion with PmeI and SpeI.

[0237] PMAR 4A was prepared by digesting Silex's P<1_68NcoI-filled (MAR)SV40EGFP with HindIII and XbaI to remove the EGFP gene, followed by inserting the GALNS gene released from pCIN 4A by HindIII and XbaI digestion.

[0238] The full-length human GALNS cDNA was cloned into the mammalian expression vector pcDNA4 from Ingenie Biosciences in Carlsbad, California. pCDNA4 SUMF1 was digested with HindIII and XbaI to remove SUMF1 cDNA, and pCIN 4A was digested with HindIII and XbaI to isolate GALNS cDNA. The GALNS cDNA HindIII / XbaI fragment was ligated into the pcDNA4 vector HindIII / XbaI fragment. The resulting plasmid was named pcDNA4-4A.

[0239] Restriction mapping was performed using enzymes obtained from the New England Biolabs to confirm the integrity of the GALNS gene in the pCIN 4A, BMAR, and pCDNA4-4A expression vectors. The PMAR 4A expression vector was not mapped.

[0240] The structure of the fully processed form of human N-acetylgalactosamine-6-sulfatase (GALNS) is described in Figure 5 GALNS is expressed as a 522-amino acid polypeptide containing a 26-amino acid signal peptide sequence. The 496-amino acid GALNS polypeptide is secreted as a pre-enzymatically processed (precursor) form of approximately 55-60 kDa. In active GALNS, the cysteine ​​residue at position 53 (corresponding to position 79 of the full-length GALNS polypeptide) in the precursor or fully processed GALNS polypeptide is converted to C by sulfatase modifier 1 (SUMF1). α -Formylglycine (FGly). In lysosomes, GALNS is cleaved after position 325 of the fully processed GALNS peptide, yielding GALNS peptide fragments of approximately 40 kDa and 19 kDa. These GALNS peptides are linked by disulfide bonds between cysteine ​​(C) residues at positions 282 and 393 of the fully processed GALNS peptide. There are two standard N-linked glycosylation sites at positions 178 and 397 of the fully processed GALNS peptide. Bis-phosphorylated mannose 7 (BPM7), containing two mannose-6-phosphate residues, is found at N178, not N397.

[0241] Example II

[0242] G71S cell line co-expressing human sulfatase modifying factor 1 (SUMF1) and human N-acetylgalactosamine-6-sulfatase (GALNS)

[0243] The goal is to develop cell lines that produce active lysosomal sulfatases with increased phosphorylation levels.

[0244] G71 cells (Rockford K. Draper) are directly derived from CHO-K1 (ATCC CCL-61). Regarding the acidification of integrons, the G71 cell line is a temperature-sensitive mutant of CHO-K1, and it has been observed that it differs in the total protein secretion of several enzymes and phosphorylation of mannose residues at elevated temperatures (Park et al., Somat. Cell Mol. Genet. 17(2):137-150, 1991; Marnell et al., J. Cell. Biol. 99(6):1907-1916, 1984).

[0245] G71 cells were maintained at 34°C in BioWhitaker UltraCHO medium supplemented with 2.5% fetal bovine serum, 2 mM glutamine, gentamicin and amphotericin B.

[0246] To facilitate the use of cell lines for protein production, a protocol was adopted to pre-acclimate adherent G71 cells to serum-free growth medium by adapting them to high-density serum-free suspension culture (Sinacore et al., Mol. Biotechnol. 15(3):249-257, 2000), thereby generating a serum-free suspension culture-adapted cell line, G71S. Alternatively, after stable transfection as described below, adherent G71 cells can be adapted to serum-free growth medium as outlined by Sinacore et al.

[0247] Following the MARtech II protocol described by Cellex, paired combinations of human SUMF1 and human GALNS expression vectors (Example I), pcDNA4 SUMF1+pCIN4 4A, BMAR SUMF1+BMAR 4A, or PMAR SUMF1+PMAR 4A, were transformed into G71S cells grown in a medium supplemented with an antibiotic-antifungal solution (100 IU penicillin, 10 mg streptomycin, 25 μg amphotericin B, Cellgro). The transfectant pools were grown in UltraCHO medium (Cambrex) supplemented with 5% γ-irradiated fetal bovine serum (FBS, JRH), 200 μg / mL G418 (AG Scientific), and 200 μg / mL zero-myomycin (Ingenium), and cloned in 96-well plates using the same growth medium via limiting dilutions. Clonal growth was monitored using cell screening (Innovatis) imaging. Active GALNS were screened for all clones using an enzyme capture activity ELISA (see Example IV). Cell yield was calculated by dividing the enzyme capture activity ELISA value of GALNS activity by the daily cell growth (VC, Beckman Coulter counter).

[0248] 202 G71S clones were prepared and viable GALNS were screened: 86 clones were co-transfected with pcDNA4 SUMF1 + pCIN 4A, 65 clones were co-transfected with BMAR SUMF1 + BMAR 4A, and 51 clones were co-transfected with PMAR SUMF1 + PMAR 4A. Clones were first selected based on the high levels of viable GALNS in the 96-well tissue culture plates. Figure 6 A). GALNS activity was detected using an enzyme capture activity ELISA, expressed as ng / mL (y-axis). The x-axis shows three co-transfection conditions used for SUMF1 and GALNS expression: hCMV promoter, MAR-free; hCMV promoter, MAR-containing; and SV40 promoter, MAR-containing. Each column represents one clone from each population. Cell density was not indicated in this 96-well clone screening, and this figure does not show all co-transfected G71S clones.

[0249] The G71S clone that produced the highest activity of GALNS was selected for yield analysis. Figure 6 B). Daily cell yield is expressed in picograms / cells / day. Daily cell yield is obtained by dividing GALNS activity by the cell density for that day. This figure shows the yield at 5x10⁻¹². 5On day 4 (96 hours) after cell / flask seeding, GALNS of clones were detected using an enzyme capture activity ELISA, in picograms / cell / day (y-axis). Positive controls consisted of BHK and CHO clones (BioMarin) expressing GALNS. Each vertical column represents one clone. The pCIN 4A clone produced active GALNS, but only slightly above the experimental background.

[0250] Cloning analysis using 96-well screening and 4-day yield assays demonstrated that co-transfection with expression vectors containing MAR elements improved G71S clone productivity compared to co-transfection with expression vectors lacking MAR elements. Clones co-transfected with BMAR 4A and BMAR SUMF1 showed rapid merge production, rapid clone growth, and twice the amount of active GALNS produced compared to the highest-yielding PMAR 4A clone, and up to 10-fold higher compared to CHO 4A and BHK 4A clones lacking MAR elements.

[0251] G71S clones expressing GALNS were adapted to serum-free growth media following the protocol outlined by Sinacore et al. (Mol. Biotechnol. 15(3):249-257, 2000). Complete adaptation was performed in the presence of two selectable reagents (200 μg / mL of nifedipine and 200 μg / mL of neomycin). G71S clones expressing GALNS cultured in T-flasks were aliquoted into: (1) 125 mL flasks containing Cambrex UltraCHO medium and 5% FBS (batch number 8L2242); (2) 125 mL flasks containing JRH 302M medium (production medium) and 5% FBS; and (3) T-flasks as stock (UltraCHO, 5% FBS). Once suspension culture was established, adherent cells were discarded, and FBS supply was discontinued. When growth returned to >0.5 (1 / day), after 3 passages, and with a survival rate >95%, the FBS concentration was reduced by 50%. Cells were maintained for at least 3 passages at any given FBS concentration. Once adapted to growth in 2.5% FBS, cells were added directly to serum-free medium. Cells were stored in fresh medium containing 10% (v / v) DMSO. Experimental thawing was performed to ensure cell survival during freezing. Two G71S clones expressing GALNS, transfected with BMAR 4A+BMAR SUMF1, were passaged approximately 15 times to adapt them to serum-free suspension culture. GALNS-expressing clones transfected with pcDNA4SUMF1+pCIN 4A were also isolated and adapted to serum-free culture.

[0252] Except for selection using 200 μg / mL zero-mycin (Ingenieur), essentially as described above, paired combinations of human SUMF1 and human GALNS expression vectors (Example I), pcDNA4 SUMF1+pCDNA4-4A, were transformed into G71S cells. Six GALNS expression clones, C2, C5, C7, C10, C11, and C30, were isolated and adapted for serum-free suspension culture, essentially as described above.

[0253] Example III

[0254] Large-scale culture of G71S cell lines expressing human N-acetylgalactosamine-6-sulfatase (GALNS)

[0255] The aim was to detect the enzyme yield of a G71S clone expressing human N-acetylgalactosamine-6-sulfatase (GALNS). Serum-adapted G71S cell lines co-expressing human SUMF1 and human GALNS were cultured on a large scale, and the yield of active GALNS enzymes was assessed.

[0256] Because the G71S host cell line adapts to serum-free suspension culture relatively quickly, it was determined that production could be carried out in a WAVE bioreactor operated by perfusion. The WAVE bioreactor offers great flexibility in inoculation volume because it allows for direct scale-up in the bag, thereby reducing the risk of contamination and accelerating material production. Figure 7 This diagram shows a schematic of the WAVE bioreactor apparatus. The diagram illustrates the loading chamber in a perfusion configuration, where the volume of culture medium in the bag is monitored by measuring the bag weight and adjusting the feed and collection rates to maintain an ideal volume. In 10L bags, the pH is also controlled at the desired set point using probes inserted into the bag.

[0257] G71S clones 4 and 5 expressing GALNS produced material in 1L increments. Culture pH was not controlled during these operations. The operational limit for the WAVE bags was a throughput of 3 container volumes per day (VV / day). To prevent any material inactivation, the target cell specific perfusion rate (CSPR) was 0.3 nanoliters / cell / day, resulting in an average residence time of 8 hours for the GALNS. Therefore, the cell density in the bags was maintained at approximately 10⁻¹² x 10⁻¹². 6 Cells / mL. The growth rates of G71S clones 4 and 5 expressing GALNS were 0.16 and 0.20, respectively. Cells were released directly from the bag to maintain target cell density.

[0258] The pH of the collection solution was adjusted to 5.5–6.5 to maintain enzyme activity, as GALNS has been shown to be stable at pH 6. This was achieved by periodically adding 5 v / v of pH 4.0 sodium citrate buffer to the collection solution leaving the reactor. The adjusted collection solution was stored at 4°C before downstream processing. The average titers of the two G71S clones expressing GALNS, 4 and 5, were approximately 4.2 mg / L, with a corresponding unit productivity of approximately 1.25 picograms / cell / day.

[0259] The G71S clone expressing GALNS, C2, C5, C6, C7, C10, C11 and C30 were similarly cultured on a large scale, and the production of active GALNS enzymes was evaluated.

[0260] Example IV

[0261] Detection of the concentration and activity of human N-acetylgalactosamine-6-sulfatase (GALNS)

[0262] Develop an enzyme-linked immunosorbent assay (ELISA) to detect the concentration and activity of G71S clones that co-express human SUMF1 and human N-acetylgalactosamine-6-sulfatase (GALNS).

[0263] Enzyme capture activity ELISA

[0264] After being captured by anti-GALNS-specific antibodies bound to an ELISA plate, enzyme capture activity ELISA was used to detect the activity of GALNS enzymes on the solid phase.

[0265] Buffer. Buffer A (Carbonated Buffer): Dissolve 3.09 g Na₂CO₃ and 5.88 g NaHCO₃ in 900 mL of deionized (DI)H₂O, then add DIH₂O to a final volume of 1000 mL. Check if the pH is between 9.4 and 9.6, then filter sterilize. To completely coat one 96-well microtiter plate with 100 μL per well, dilute 19 μL of anti-GALNS antibody into a test tube (12 mL). Buffer B (ELISA Blocking Buffer and Serial Dilution Buffer): 1x acidic PBS, 0.05% Tween-20, and 2% BSA, adjusted to pH 6.5 with acetic acid. Buffer B WWash buffer: 100 mM NaOAc and 0.05% Tween-20, adjusted to pH 6.5 with acetic acid. Buffer C (Substrate buffer): 25 mM sodium acetate, 1 mM NaCl, 0.5 mg / mL desalted BSA and 0.01% sodium azide, adjusted to pH 4.0 with glacial acetic acid. Buffer D (β-galactosidase buffer): 300 mM disodium hydrogen phosphate, 0.1 mg / mL BSA, 0.01% sodium azide and 0.01% Tween-20, adjusted to pH 7.2 with phosphate. Buffer E (Termination buffer): 350 mM glycine and 440 mM carbonate buffer, adjusted to pH 10.7 with 6 M NaOH.

[0266] Reagents. Anti-GALNS IgG antibody: Polyclonal rabbit antibody is a G protein purified from serum. Total protein = 3.17 mg / mL (BCA) in D-PBS. Store sample (19 μL) at -20°C for single use. 4MU-Gal-6-S substrate (solid; 440 MW): 100 mM stock solution prepared with DI water, stored at 4°C. β-galactosidase (Sigma G-4155): Dilute to 12 μg / mL with buffer D before use.

[0267] Protocol: Anti-GALNS antibody binding to plates: Nunc MaxiSorp ELISA plates (Nalge / Nunc International, Fisher, No. 12-565-135) were coated with anti-GALNS at a final protein concentration of 5 μg / mL using buffer A. To prepare this solution, thaw one 19 μL aliquot of the sample and centrifuge briefly (10 seconds) to collect the liquid. Transfer all 19 μL to 12 mL of buffer A. Mix vigorously by inverting, then pour into a reservoir and plate using a multichannel pipette (100 μL / well). Cover the plate and incubate overnight at 4°C. Removal of unbound anti-GALNS antibody: Using buffer B... WWash the plate three times with overflow. Block: Block the plate with buffer B (320 μL / well), then cover the plate and incubate at 37°C for 1 hour. Prepare a series of dilutions of purified GALNS standard and test sample (unknown) during the blocking step: Dilute the standard to the high end of the linear range of the assay (128 ng / mL, row A) with buffer B, then perform serial dilutions (2-fold) in row BG of a 96-well plate. Row H is the buffer blank (i.e., without GALNS enzyme). First prepare 500 μL of buffer B at a concentration of 128 ng / mL. Then perform 2-fold serial dilutions with buffer B (250 μL added to 250 μL) until a concentration of 2 ng / mL is achieved. Remove the blocking buffer: Discard buffer B after the blocking step. Bind GALNS enzyme and test sample to anti-GALNS antibody: Load the serially diluted standard and test sample at 100 μL / well onto the plate (in duplicate). Cover the plate and incubate at 37°C for 1 hour. Remove GALNS inhibitors: with buffer B W Overflow the plate three times. Add GALNS substrate (first reaction): Prepare sufficient final substrate solution to load 100 μL per well (prepare within 1 hour before use). Dilute 4MU-Gal-6-S stock solution (100 mM) to 1 mM with buffer C. Load 100 μL per well. Cover the plate and incubate at 37°C for 30 minutes. Add β-galactosidase (second reaction): Add 50 μL of 12 μg / ml β-galactosidase prepared with buffer D to each well. Cover the plate and incubate at 37°C for 15 minutes. Terminate the reaction: Add 100 μL of buffer E (stop buffer) to each well to ionize the released 4MU. Transfer to fluoroplate: Transfer 200 μL (8 wells at a time) of the 250 μL from each well of the ELISA plate to a black untreated flat-bottomed microtiter plate (fluoroplate, Costa No. 3915). Fluorescence readings: The plate was read using a Gemini plate reader from Molecular Devices Corporation with the SOFTmax PRO program (366nm excitation, 446nm emission, 435nm cutoff).

[0268] GALNS ELISA

[0269] The concentration of GALNS enzyme in cell culture conditioned medium or other process samples was detected by sandwich immunoassay, GALNS ELISA.

[0270] Buffer. Buffer A (Carbonated Buffer): Dissolve 3.09 g Na₂CO₃ and 5.88 g NaHCO₃ in 900 mL of deionized (DI)H₂O, then add DIH₂O to a final volume of 1000 mL. Check if the pH is between 9.4 and 9.6, then filter sterilize. To completely coat one 96-well microtiter plate with 100 μL per well, dilute 19 μL of anti-GALNS antibody into a 12 mL tube. Buffer B (ELISA Blocking Buffer and Serial Dilution Buffer): 1x acidic PBS, 0.05% Tween-20, and 2% BSA, adjusted to pH 6.5 with acetic acid. Buffer B W (Washing buffer): 100 mM NaOAc and 0.05% Tween-20, adjusted to pH 6.5 with acetic acid. Buffer F (Termination buffer): 2N H2SO4: 600 mL total, add 100 mL 12N H2SO4 and 500 mL LmilliQ water.

[0271] Reagents. Anti-GALNS IgG antibody: Rabbit polyclonal antibody is a G protein purified from serum. Total protein = 3.17 mg / mL (BCA) in D-PBS. Store sample (19 μL) at -20°C for single use. HRP-conjugated detection antibody (RIVAH): Dilute the final conjugated antibody 1:100 with D-PBS / 1% BSA. Store sample at -20°C in 120 μL for single use. TMB EIA substrate kit (BioRad, catalog number 172-1067).

[0272] Protocol: Anti-GALNS antibody binding to plates: Nunc MaxiSorp ELISA plates (NN International, Fisher, No. 12-565-135) were coated with anti-GALNS at a final protein concentration of 5 μg / mL using buffer A. To prepare this solution, thaw one 19 μL aliquot of the sample and centrifuge briefly (10 seconds) to collect the liquid. Transfer all 19 μL to 12 mL of buffer A. Mix vigorously by inverting, then pour into a reservoir and plate using multichannel pipettes (100 μL / well). Cover the plate and incubate at 37°C (convection incubator) for 2 hours. No heating block is required. Removal of unbound anti-GALNS antibody: using buffer B... WOverflow to wash the plate three times. Block: Block the plate with buffer B (320 μL / well), then cover the plate and incubate at 37°C for 1 hour. Prepare a series of dilutions of purified GALNS standard and test sample (unknown) during the blocking step: Dilute the standard to the high end of the linear range of the assay (40 ng / mL, row A) with buffer B, then perform serial dilutions (2-fold) in row BG of a 96-well plate. Row H is the buffer blank (i.e., without GALNS enzyme). First prepare 500 μL of buffer B at a concentration of 40 ng / mL. Then perform 2-fold serial dilutions with buffer B (250 μL added to 250 μL) until a concentration of 0.625 ng / mL is achieved. Remove the blocking buffer: After the blocking step, discard buffer B. Bind GALNS enzyme and test sample to anti-GALNS antibody: Load the serially diluted standard and test sample at 100 μL / well onto the plate (in duplicate). Cover the plate and incubate at 37°C for 1 hour. Washing: with buffer B W Wash the plate three times with overflow. For antibody-conjugate detection: thaw one portion of the RIVAH antibody sample (120 μL) and centrifuge briefly (10 seconds) to collect the liquid. Transfer all 120 μL to 11.9 mL of buffer B and mix vigorously by inverting the tube. Pour into a reservoir, then add 100 μL / well using a multichannel pipette. Cover the plate and incubate at 37°C for 30 minutes. Wash: with buffer B. W Wash the plate three times with overflow. TMB substrate: Prepare the final substrate solution by mixing 1.2 mL of solution B with 10.8 mL of solution A. Pour into the reservoir, then add 100 μL / well using a multichannel pipette. Cover the plate and incubate at 37°C for 15 minutes. Stop solution: Aspirate 12 mL of 2N H₂SO₄ stop solution into the reservoir, and add 100 μL / well using a multichannel pipette. Gently tap to mix. Read A450: Read the plate using a plate reader.

[0273] GALNS specific activity test

[0274] The GALNS specific activity assay uses GALNS-specific substrates to detect the enzyme activity of GALNS in solution.

[0275] Buffer solutions. MilliQ H2O is used for all buffer solutions. Dilution Buffer (DB): For 1L DB, dissolve 1.74mL acetic acid, 0.75g sodium acetate, 233.6mg NaCl, 2mL 50% Tween-20, and 10mL 1% sodium azide in MilliQ H2O. If the pH is below 3.95, adjust the pH to 4.0 + / - 0.5 with 0.1M NaOH; if the pH is above 4.05, adjust the pH to 4.0 + / - 0.5 with 0.1M acetic acid. The final concentration is: 19.5mM acetic acid, 5.5mM sodium acetate, 1mM NaCl, 0.1% Tween-20, and 0.01% sodium azide. Phosphate Buffer (PB): For 1L PB, dissolve 13.9g NaH2PO4-H2O and 55g NaHPO4-7H2O in MilliQ H2O and adjust the pH to 7.2. The final concentration is 300mM NaPi. Termination Buffer (SB): For 1 L SB, dissolve 26.2 g glycine and 46.6 g sodium carbonate in MilliQ H2O, and adjust the pH to 10.6 with NaOH. Test Buffer (AB): Dilute 4 MU-Gal-6S stock solution 1:50 with DB (final amount 2 mM). β-Galactosidase Buffer (βGB): Prepare with 25 μg / mL β-galactosidase in 300 mM NaPi, pH 7.2.

[0276] Reagents: 4MU-Gal-6S: 100mM, prepared in H2O (Toronto Research Chemicals, catalog number M334480). β-Galactosidase: Sigma G-4155. 4-Methylumbelliferone (4MU standard): Sigma M-1381 (10mM stock solution, prepared in DMSO).

[0277] Protocol. Serial dilution of GALNS enzyme. For purified and prepared GALNS (approximately 1.5 mg / mL), first dilute the sample 1:10,000 in a low protein adhesion microcentrifuge tube containing DB (USA Scientific catalog number 1415-2600), then perform serial dilutions 1:1. Place 100 μL of DB in a low protein-binding 96-blank. Transfer 100 μL of GALNS sample into the first well. Now serially dilute (1:1) along the plate (AG on a 96-well plate). Do not add sample to well H (blank). The linear range of this assay is 1–75 ng / mL. Prepare a 4MU standard curve using the same method. Dilute 10 mM 4MU stock solution prepared with DB 1:100 with DMSO. Add 50 μL of 50 μM 4MU to the first well to start the 4MU standard curve, then perform serial dilutions. Add 50 μL of substrate prepared with AB (DB-prepared 2 mM 4 MU-galactose-6S) to a 96-well fluorescent plate. Incubate the substrate at 37°C for 10 minutes. Add 50 μL of GALNS serial dilution buffer and 4 MU standard to 50 μL of substrate prepared with AB. Incubate at 37°C for 30 minutes (this first reaction removes sulfate from the substrate). Quench the first reaction and initiate the second reaction by adding 50 μL of β-galactosidase (β-galactosidase stock solution diluted to 25 μg / mL with βGB). Phosphate inhibits GALNS, and increasing the pH also terminates the GALNS reaction. The resulting pH is now within the optimal pH range for β-galactosidase. Incubate the second reaction at 37°C for 15 minutes. Add 100 μL of 4 MU released by SB ionization. Read Ex355 Em460 using a 96-well fluorescent plate reader. Enzyme activity calculation (37℃, pH 4.0 buffer): 1 unit = released μmol 4MU / min; activity = μmol 4MU / min / mL; specific activity = μmol 4MU / min / mg. Protein concentration calculation: using the extinction coefficient of GALNS (1 mg / mL = 1.708 absorbance units at 280 nm).

[0278] Example V

[0279] Purified human N-acetylgalactosamine-6-sulfatase (GALNS)

[0280] The aim was to obtain a large quantity of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS). Stable transfected G71 cells co-expressing human SUMF1 and human GALNS were cultured under bioreactor conditions, and the active GALNS enzyme was purified from the cell culture medium.

[0281] The liquid chromatography equipment is the APBA Explorer 900 system (Amersham Pharmacia Biotech AKTAexplorer 900 system), using Unicorn control software.

[0282] Protein analysis methods followed standard procedures including SDS-PAGE, Coomassie blue staining (B101-02-COOM), Western blotting, and the Bradford assay. Purification was assessed by activity yield, and the purity of the GALNS product was visually evaluated using SDS-PAGE. Processing impurities were detected by Western blotting using anti-GALNS antibody. Protein concentration was determined using the Bradford assay. An extinction coefficient of 1.708 was used to measure A... 280 To detect the concentration of the finally purified GALNS protein.

[0283] Chromatography resins: Blue agarose 6FF (GE Healthcare, lot number 306346) and Fractogel SE Hi-Cap (Merck KgaA, FC040894449).

[0284] GALNS enzyme activity assay. The specific activity of GALNS was determined using the small fluorescent substrate 4-methylumbelliferyl-6-S-GAL (4-MU-6-S-GAL). The GALNS specific activity assay involves a two-step reaction, in which the addition of β-galactosidase is necessary for the release of the fluorescent tag after GALNS has been incubated with the substrate for a certain period. Detection was performed using a fluorescent plate reader.

[0285] Equilibrate the 10DG desalting column (BioRadiation Company) with equilibration buffer (EQB, 50mM NaOAc, 10mM NaCl, pH 5.8). MilliQ H2O was used for all buffers. 3 mL of purified GALNS (0.5–2 mg / mL) was loaded onto the desalting column, eluted, and 4 mL of sample was collected in separate tubes using EQB. Protein concentration was calculated using the extinction coefficient of GALNS (1 mg / mL = 1.708 absorbance units at 280 nm).

[0286] Desalted GALNS samples were serially diluted (1:1) with dilution buffer (DB, 50 mM NaOAc, 1 mM NaCl, pH 4.0 + 0.5 mg / mL BSA). Before use, the BSA stock solution was desalted, and then 50 mg / mL BSA stock solution (not exceeding 5% CV) was loaded onto a G25 column equilibrated with milliQ H2O. 100 μL of desalted GALNS sample was transferred to the first row of a low-protein-binding 96-well plate, followed by serially diluted GALNS samples along the plate (row AG on the 96-well plate). 100 μL of DB was transferred to the last well (H). The linear range of this assay was 200 ng / mL at the top and 3–200 ng / mL in the range of linearity. The same method was used to prepare a standard curve for 4-methylumbelliferone (4MU) (Sigma M-1381, 10 mM stock solution, prepared in DMSO). Transfer 50 μL of 4 MU from 100 μL of serially diluted GALNS buffer to a new 96-well fluorescent plate (black plate). Add 50 μL of 2 mM 4 MU-galactose-6S (prepared with milliQ H2O) to the sample and incubate at 37 °C for 30 min. Quench the first reaction and add 50 μL of β-galactosidase (Sigma G-4155, diluted to 12 μg / mL with 300 mM NaPi, pH 7.2) to start the second reaction, incubating at 37 °C for 15 min. Add 100 μL of stop buffer (glycine / carbonate, pH 10.6) to ionize the released 4 MU. Read the plate using a 96-well fluorescent plate reader (excitation 355 nm, emission 460 nm). One unit is defined as the release of 1 μmol 4 MU / min. Enzyme activity is given in μmol 4 MU / min / mL, and specific activity is given in μmol 4 MU / min / mg. All operations were performed at 37°C in pH 4.0 buffer.

[0287] First purification method. The first purification method includes an ultrafiltration (UF) step, followed by a 2-column purification method.

[0288] 1. Collection and filtration (HF): Bioreactor materials are sterile filtered through a 0.2μm filter.

[0289] 2. Ultrafiltration (UF): Bioreactor materials are concentrated 10-20 times through ultrafiltration using a 30kD Sartocon membrane.

[0290] 3. pH 4.5 adjustment: At room temperature, the concentrated bioreactor material (UF(20X)) was adjusted to pH 4.5 using pH adjustment buffer (1.75M NaOAc, pH 4.0), sterilely filtered, and then loaded onto a blue agarose column.

[0291] 4. Blue agarose 6-speed flow (FF): UF (20X) adjusted to pH 4.5 was loaded onto a blue agarose column, as shown in Table 1 and... Figure 9 A shows the elution of GALNS protein.

[0292] Table 1: Blue agarose 6-velocity flow chromatography

[0293]

[0294]

[0295] *CV: Column volume. Flow rate = 92 cm / h

[0296] 5. Fractogel SE Hi-Cap: Adjust the eluent from the blue agarose column to pH 4.3 and load it onto the Fractogel SE Hi-Cap column, as shown in Table 2. Figure 9 B shows the elution of GALNS protein.

[0297] Table 2: Fractogel SE Hi-Cap Chromatography

[0298] step CV* buffer solution balance 5 20mM acetic acid / phosphoric acid, 50mM NaCl, pH 4.3 load Adjust the pH of the blue agarose eluent to 4.3 and dilute it 1:1 with MQ water. Washing 1 5 20mM acetic acid / phosphoric acid, 50mM NaCl, pH 5.0 Wash 2 5 20mM acetic acid / phosphoric acid, 50mM NaCl, pH 5.5 Washout 20 20mM acetic acid / phosphoric acid, 50-350mM NaCl gradient, pH 5.5 Regeneration 1 5 20mM acetic acid / phosphoric acid, 500mM NaCl, pH 5.5 regeneration 2 5 20mM acetic acid / phosphoric acid, 50mM NaCl, pH 4.3 deal with 5 0.5N NaOH, 0.5 hours regeneration 3 4 <![CDATA[H2O]]> store 3 20% EtOH

[0299] *CV: Column volume. Flow rate = 150 cm / h

[0300] GALNS protein in the eluent was collected in fractions, and the shoulder before elution and the tail after elution were discarded.

[0301] 6. Final UF / HF: As described above, the eluent from the Fractogel SE Hi-CAP column is concentrated by ultrafiltration and then aseptically filtered.

[0302] Preparation: The purified GALNS protein was prepared using 10 mM NaOAc, 1 mM NaH2PO4, 0.005% Tween-80, and pH 5.5.

[0303] Stability studies. The stability of the final purified GALNS at each temperature was monitored as a function of time by storing small amounts of GALNS samples at 4°C and -70°C. At certain time points, samples were rapidly thawed in a 37°C water bath before activity was tested. Figure 8 The purified GALNS were shown to be stable for at least 79 days when stored in the preparation buffer at 4°C and -70°C.

[0304] Results of the first purification method. Table 3 shows the purification rates of the three GALNS protein products produced by G71S clone 4 in a suspension culture bioreactor. In all cases, the purity was approximately 95% as visually assessed by SDS-PAGE.

[0305] Table 3: Purification rate of human N-acetylgalactosamine-6-sulfatase (GALNS) from G71S clone 4 in a WAVE reactor

[0306]

[0307] Figure 9 The image shows SDS-PAGE of GALNS protein separated sequentially by (A) blue agarose 6-speed flow chromatography and (B) Fractogel SE Hi-CAP chromatography. The gels were stained with Coomassie blue (left) or anti-GALNS antibody (right). For Western blotting, anti-GALNS rabbit antibody was diluted 1:5000, and the secondary antibody was anti-alkaline phosphatase rabbit antibody. The apparent molecular weight of the GALNS protein on SDS-PAGE is approximately 55-60 kDa, consistent with the expected size of the pre-processed (precursor) form secreted by the enzyme, which lacks a 26-amino acid signal peptide and post-cleavage at position 325.

[0308] N-terminal characterization. The N-terminus of purified GALNS was determined by LC / MS. The N-terminal sequence was APQPPN, which corresponds to the predicted N-terminus of the secreted form of GALNS lacking the 26-amino acid signal peptide (comparative). Figure 4 and Figure 5 (The human GALNS polypeptide sequence is shown).

[0309] The second purification method includes an ultrafiltration / percolation (UF / DF) step followed by a 3-column purification method.

[0310] 1. Ultrafiltration (UF / DF): Bioreactor material is concentrated 20 times by ultrafiltration / percolation at pH 5.5 using a 30kD Sartocon membrane.

[0311] 2. pH 4.5 adjustment: At room temperature, the concentrated bioreactor material (UF / DF(20X)) was adjusted to pH 4.5 using pH adjustment buffer (1.75M NaOAc, pH 4.0), sterilely filtered, and then loaded onto a Fractogel EMD SE Hi-Cap column.

[0312] 3. Fractogel EMD SE Hi-Cap: Load UF / DF (20X) adjusted to pH 4.5 onto a Fractogel EMDSE Hi-Cap column and wash sequentially with 10 mM acetic acid / phosphate, 50 mM NaCl, pH 4.5 and 10 mM acetic acid / phosphate, 50 mM NaCl, pH 5.0. Elute GALNS protein with 10 mM acetic acid / phosphate, 140 mM NaCl, pH 5.0.

[0313] 5. Zn-chelated agarose FF: Adjust the elution buffer from the Fractogel EMD SE Hi-Cap column to 500 mM NaCl, pH 7.0, load it onto a Zn-chelated agarose FF (Zn-IMAC) column, wash with 10 mM acetic acid / phosphate, 125 mM NaCl, 10 mM imidazole, pH 7.0, and elute GALNS protein with 10 mM acetic acid / phosphate, 125 mM NaCl, 90 mM imidazole, pH 7.0.

[0314] 6. pH 3.5 adjustment: Adjust the elution buffer of the Zn-chelated agarose FF column containing GALNS protein to pH 3.5 to inactivate the virus at low pH, and then adjust to 10 mM acetic acid / phosphate, 2 M NaCl, pH 5.0.

[0315] 7. ToyoPearl Butyl 650M: Load the low-pH-adjusted elution buffer from the Zn-chelated agarose FF column onto a ToyoPearl Butyl 650M column, wash with 10 mM acetic acid / phosphate, 2 M NaCl, pH 5.0, and elute GALNS protein with 10 mM acetic acid / phosphate, 0.7 M NaCl, pH 5.0.

[0316] 8. Final UF / HF: The ToyoPearl Butyl 650M eluent was subjected to ultrafiltration and percolation in 20 mM acetic acid, 1 mM phosphoric acid, 150 mM NaCl, pH 5.5.

[0317] Preparation. Purified GALNS protein was prepared in 10 mM NaOAc / HOAc, 1 mM NaH2PO4, 150 mM NaCl, 0.01% Tween-20, pH 5.5.

[0318] Results of the second purification method. Table 4 shows the recovery rate of GALNS protein produced by G71S clone C2 in a suspension culture bioreactor using this second purification method. The purity of the formulated GALNS enzyme (i.e., both the precursor and mature or processed forms) was approximately 98% as determined by C3 RP-HPLC. The percentage of the precursor form of GALNS enzyme was approximately 85% as determined by SDS-capillary gel electrophoresis.

[0319] Table 4: Recovery rate of human N-acetylgalactosamine-6-sulfatase (GALNS) from G71S clone C2

[0320] Methods and Steps Recovery rate (%) pH adjustment 96 Fractogel SE Hi-Cap column 98 Zn-IMAC column 89 Low pH virus inactivation 89 ToyoPearl Butyl 650M Column 99 Preparation 99 total 70

[0321] Figure 10SDS-PAGE of GALNS enzymes separated by ultrafiltration / percolation (UF / DF), Fractogel SE Hi-CAP chromatography, Zn-chelate agarose FF chromatography, and ToyoPearl Butyl 650M chromatography. Gels were stained with Coomassie blue (top left), anti-GALNS antibody (top right), anti-cathepsin L (bottom left), and anti-CHO protein (CHOP, bottom right). For Western blotting, anti-GALNS rabbit polyclonal antibody was diluted 1:5000 with anti-rabbit AP conjugate as the secondary antibody; anti-cathepsin L goat polyclonal antibody was diluted 1:1000 with anti-goat HRP conjugate as the secondary antibody; and anti-CHOP rabbit polyclonal antibody was diluted 1:1000 with anti-rabbit HRP conjugate as the secondary antibody. The apparent molecular weight of the precursor GALNS enzyme on SDS-PAGE is approximately 55-60 kDa, while the apparent molecular weight of the mature or processed form of GALNS enzyme on SDS-PAGE is approximately 39 kDa and approximately 19 kDa, respectively.

[0322] Summary of the first purification method. GALNS enzymes were purified using a modified standard method (see Table 5). Bioreactor-collected material was sterile filtered through a 0.2 μm filter, maintained at 4°C, and then loaded onto a blue agarose trap column. The filtered bioreactor was either directly loaded or concentrated up to 15-fold by ultrafiltration. This purification method needed improvement because downstream purification steps, SP agarose chromatography, and subsequent phenyl agarose chromatography did not produce sufficiently pure GALNS. Replacing the two downstream purification columns with SE Hi-Cap chromatography to obtain a 2-column purification method significantly improved the purity of the final material, with the total GALNS yield increasing significantly from approximately 22% to approximately 80%. C4-RP chromatography determined the GALNS enzyme (which is essentially composed of the precursor form, see Table 5). Figure 9 The purity of the purified GALNS enzyme was roughly estimated to be >95%, and it remained stable in the preparation buffer for more than 79 days at 4°C and -70°C.

[0323] Table 5: First-person purification method for N-acetylgalactosamine-6-sulfatase (GALNS)

[0324] step conventional methods Improved methods 1 HF(1X) HF(1X) 2* UF(5X) UF(15X) 3 pH 4.5 adjustment pH 4.5 adjustment 4 Blue-Agarose 6FF Blue-Agarose 6FF 5 SP agarose SE Hi-Cap 6 Phenylacetose Hi-Sub Ultimate UF / DF 7 Final UF / DF

[0325] *This step is optional.

[0326] Summary of the second purification method. The GALNS enzyme was also purified using the second purification method (see Table 6). C4-RP chromatography determined the total yield of GALNS to be approximately 70%, while the GALNS enzyme (including precursor and mature or processed forms, see Table 6) yielded a yield of approximately 70%. Figure 10 The purity of the substance is roughly estimated to be about 97%.

[0327] Table 6: Purification method of second-person N-acetylgalactosamine-6-sulfatase (GALNS)

[0328] step method 1 HF(1X) 2 UF / DF(20X) 3 pH 4.5 adjustment 4 SE Hi-Cap 5 Zn-chelated agarose 6 pH adjustment to 3.5 7 ToyoPearl Butyl 650M 8 Ultimate UF / DF

[0329] These experiments demonstrate that the above-described scheme for preparing recombinant lysosomal sulfatase provides an effective method for the large-scale production of highly purified enzymes, particularly the pre-processed (precursor) form secreted by human N-acetylgalactosamine-6-sulfatase (GALNS).

[0330] Example VI

[0331] Characterization of purified human N-acetylgalactosamine-6-sulfatase (GALNS)

[0332] Compared to those observed in common mammalian cell lines, the G71 cell line produces proteins (e.g., lysosomal enzymes) with higher levels of hypermannose phosphorylation and correspondingly lower levels of unphosphorylated hypermannose oligosaccharides. A comparison was made between the lysosomal sulfatases (e.g., recombinant human N-acetylgalactosamine-6-sulfatase (GALNS)) numbered with high levels of diphosphorylated hypermannose oligosaccharides described herein and the molecules obtained by Canfield et al. (US Patent 6,537,785), which contain no complex oligosaccharides and exhibit only hypermannose oligosaccharides.

[0333] To determine the level of unphosphorylated high-mannose on lysosomal sulfatases, those skilled in the art can use exoglycosidase sequencing of released oligosaccharides ("FACE sequencing") to identify the percentage of unphosphorylated high-mannose oligosaccharide chains. On a normal batch-release FACE distribution gel, unphosphorylated high-mannose co-migrates with specific oligosaccharide complexes (e.g., oligomannose 6 and a fully sialylated biantennary complex). Unphosphorylated high-mannose is then distinguished from other oligosaccharides by enzymatic sequencing.

[0334] To determine whether purified lysosomal sulfatases (e.g., recombinant human N-acetylgalactosamine-6-sulfatase (GALNS)) expressed in G71S cells have increased phosphorylation, the level of mannose-6-phosphate (M6P) on the lysosomal sulfatase and the enzyme’s ability to bind to the M6P receptor (MPR) were measured.

[0335] Recombinant human GALNS enzymes were analyzed by fluorescence-assisted carbohydrate electrophoresis (FACE) and MPR-agarose resin chromatography. The FACE system was used to separate, quantify, and determine the oligosaccharide sequences released from the glycoprotein using polyacrylamide gel electrophoresis. Reliable measures of phosphorylation level per mole of protein were obtained from the relative intensity of the oligomannose 7-bisphosphate (O7P) band on FACE (Hague et al., Electrophoresis 19(15):2612-20, 1998) and the percentage of residual activity on the MPR column (Cacia et al., Biochemistry 37(43):15154-61, 1998).

[0336] The specific activity of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) was determined using the small fluorescent substrate 4-methylumbelliferyl ketone-6-S-GAL (4MU-Gal-6S) at 37℃. Using this assay, the specific activity of purified GALNS was 165 μmol / min / mg (0.165 U / mg).

[0337] Human serum stability. The in vitro serum stability of GALNS was determined. Human serum (Sigma H-4522) was aseptically filtered through a 0.2 μm PES filter. 4 mL of the filter-sterilized human serum was incubated for 1 hour at 37°C under a 10% CO2 atmosphere in a T-25 cell culture flask (pH 7.4 at this point). + 0.1). Add 0.4 mL of desalted purified GALNS (using a Bio-RAD 10DG column to desalt 2 mg / mL purified GALNS into PBS) to pre-incubated human serum or PBS containing 0.5 mg / L BSA. At specified time points (e.g., 0, 1, 3.5, 7.5, and 26 hours), take 100 μL of sample and add it to 900 mL of quenching buffer (QB, 50 mM NaOAc, pH 5.6 + 150 mM NaCl + 0.5 mg / mL BSA + 0.001% Tween-80). Store the sample at 4°C until GALNS enzyme activity is measured.

[0338] GALNS enzyme activity was detected by enzyme capture activity ELISA. The in vitro serum half-life of purified GALNS was estimated to be 217 hours by extrapolating the exponential decay curve of % residual GALNS enzyme activity.

[0339] GALNS was used to take up synovial cells (chondrocytes). The ability of GALNS to take up synovial cells (chondrocytes) was measured.

[0340] Chondrocytes (ATCC number CRL-1832) were cultured in 12-well plates at 37°C and 5% CO2 using growth medium (Ham F12 + 10% FBS). Four 12-well plates were required for the analysis of three sample uptakes. The purified GALNS samples and GALNS references were diluted to 1 μM with acPBS / BSA (acidic PBS + 200 μg / mL BSA). Uptake dilution profiles of the GALNS samples and references were prepared from the 1 μM stock solution: 50.5 μL (1 μM rhASB) was diluted in 5 mL of uptake assay diluent (UAD, DMEM + 2 mM L-glutamine + 0.5 mg / mL BSA) to obtain 10 nM GALNS samples and references, which were then serially diluted 2-fold with UAD to 5, 2.5, 1.25, 0.62, 0.31, and 0.16 nM. Aspirate the growth medium from each 12-well culture dish containing confluent chondrocytes. Add 1 mL of serially diluted UAD (blank) or GALNS sample or reference to each well and incubate at 37°C, 10% CO2 for 4 hours. Aspirate the uptake medium, tilt each culture dish to completely remove the medium, and wash each well once with 1 mL PBS. Aspirate the PBS and add 0.5 mL of trypsin / EDTA (0.25% trypsin / 0.1% EDTA (Mediatech 25-053-CI, batch number 25053025)) to each well to dissociate the chondrocytes. After dissociation from the plate, aliquot the chondrocytes into pre-chilled microcentrifuge tubes (30 tubes in total). Cool the trypsin-treated chondrocytes and then pellet them at low speed using a microcentrifuge (4000 rpm, 3 minutes). Completely aspirate the trypsin, wash the cell pellet with 1 mL PBS, and repeat the microcentrifugation and aspiration steps once. Add 200 μL of cell lysis buffer (CLB, 50 mM sodium acetate, pH 5.6 + 0.1% Triton X-100) to each tube. Resuspend the cell pellet by pulse-vortex shaking three times. After resuspending, store the cell lysis mixture overnight at -80°C, or analyze directly.

[0341] Thaw cell lysates at room temperature and transfer them to ice with thawed water. Vortex the cell lysates to resuspend any visible solids, then centrifuge at 14 rpm for 10 minutes at 4°C using a microcentrifuge to precipitate insoluble substances. Transfer the supernatant to a set of fresh tubes and discard the precipitate. Perform a GALNS activity assay on the supernatant. Typically, a 7-point dilution profile (serial 2-fold dilutions from 10 nM to 0.16 nM) is performed, with the expected K0... 摄取 The protein is fairly evenly distributed on both sides. The molar concentration of the starting sample is calculated using only the protein molecular weight.

[0342] Based on single-site ligand binding, the Kd uptake of purified GALNS into synovial cells was 4.9 nM.

[0343] Mannose-6-phosphate (M6P) receptor plate binding assay. The ability of GALNS to bind to the mannose-6-phosphate (M6P) receptor was determined using a plate binding assay. FluoroNunc high-binding plates were coated with 4 μg / mL M6P receptor. The coated plates were washed twice with 250 μL / well washing buffer (WB, TBS + 0.05% Tween 20), and non-specific binding was blocked with 200 μL / well blocking and dilution buffer (BDB, Pierce SuperBlock buffer, batch CA46485). The plates were incubated at room temperature (RT) for 1 hour. During this blocking step, purified GALNS samples (stored at 4°C, 0.5–2 mg / mL for 2 weeks) were diluted to 10 nM with BDB, and then serially diluted with dilution buffer (DB, 50 mM NaOAc, 1 mM NaCl, pH 4.0 + 0.5 mg / mL BSA) (250 μL + 250 μL) to 5, 2.5, 1.25, 0.62, 0.31, and 0.16 nM. The blocked plates were washed with Western blotting (WB) as described above, and the diluted GALNS samples were aliquoted into each well at 100 μL / well in duplicate and incubated at room temperature for 1 hour. During this incubation step, 2 mM of active substrate was prepared by diluting 0.1 mL of 100 mM 6S-galactose-4MU stock solution (stored in H2O, -20°C) with 5 mL of DB and preheated in a 37°C water bath. After incubation, the plate was washed twice with the above WB, 100 μL of diluted substrate was added, and the specific activity of GALNS was measured.

[0344] Using this experiment, based on single-site ligand binding, the purified Kd of GALNS-bound M6P receptor was 2.4 nM.

[0345] Column binding assay of mannose-6-phosphate (M6P) receptor. The ability of GALNS to bind to the mannose-6-phosphate (M6P) receptor was determined using a column binding assay. M6P receptor columns were prepared according to the manufacturer's instructions. M6P receptors were purchased from Peter Lobel's laboratory. The column resin was NHS activated resin (Bio-RAD Affi-Gel 15), and the column volume was 0.7 mL. The M6P receptor column was equilibrated with 10 column volumes (CV) of equilibration buffer (EQ, acidic PBS, pH 6.0, containing 5 mM β-glycerophosphate, 0.05% Tween 20, 5 mM glucose-1-phosphate, and 0.02% NaN3) at a flow rate of 0.25 mL / min. 6 μg of purified GALNS (per 200 μl) was loaded onto the M6P receptor column at a flow rate of 0.1 mL / min. Unbound GALNS was washed off the column with 10 CV EQ at a flow rate of 0.25 mL / min. A 0-100% elution buffer gradient (EL, acidic PBS, pH 6.0, containing 5 mM β-glycerol, 0.05% Tween 20, 5 mM mannose-6-phosphate, and 0.02% NaN3) (10 CV) was used, followed by elution of bound GALNS with 2 CV 100% EL. The column was then reequilibrated with 3 CV EQ.

[0346] The percentage of purified GALNS binding to the M6P receptor was determined to be 56% using the GALNS ELISA.

[0347] Capillary electrophoresis (CE) analysis of total oligosaccharides. To determine the mannose-6-phosphorylation level on GALNS, the N-linked carbohydrate characterization of total oligosaccharides on GALNS was determined by capillary electrophoresis (CE) as described by Ma et al. (Anal. Chem. 71(22):5185-5192, 1999). This method utilizes PNG enzyme F to cleave asparagine N-linked oligosaccharides. The cleaved oligosaccharides were separated, derivatized with a fluorescent dye, and applied to a G10 spin column to remove excess dye. The purified, fluorescently labeled oligosaccharides were separated by electrophoresis, and the peaks were quantified using MDQ-CE software (32Karat version 7.0).

[0348] Using this assay, the concentrations of bis-phosphorylated mannose 7 (BPM7), monophosphorylated mannose 6 (BMP6), and sialic acid-containing oligosaccharides in purified GALNS were 0.58 mol / mol enzyme, 0.08 mol / mol enzyme, and undetectable, respectively. The percentage of GALNS protein containing BPM7 was estimated to be 29%.

[0349] Characterization of bis-phosphorylated mannose 7 (BPM7) oligosaccharides on GALNS were determined. The asparagine (Asn) residue at position 178 is a glycosylated N-linked to BPM7. The Asn residue at position 397 is also a glycosylated N-linked to BPM7, but it was found to be primarily an oligomannose-type sugar.

[0350] Hydroxyapatite affinity chromatography. An in vitro bone model was developed to determine the bone-targeting ability of GALNS. A 4 mg / mL HTP-DNA grade hydroxyapatite (Bioradiation Company) suspension was prepared and equilibrated in DBS + 50 μg / mL BSA, pH 7.4. After adding 50 μg / mL BSA, the purified GALNS was desalted in DBS, pH 7.4. The final concentration of desalted GALNS was approximately 2 mg / mL, which was serially diluted in 96-well plates with DBS + 50 μg / mL BSA, pH 7.4. 50 μL of serially diluted GALNS was transferred to a 96-well filter plate (Millipore MSGVN2210, hydrophilic PVDF, low protein binding, 22 μm pore size). 50 μL of the hydroxyapatite suspension was added to the wells of the filter plate containing the serially diluted GALNS, and incubated at 37°C for 1 hour with gentle shaking. Vacuum filter the plate.

[0351] The supernatant of the vacuum filter plate was analyzed by HPLC or by the above-mentioned GALNS enzyme activity assay. The Kd of purified GALNS to p-hydroxyapatite was 3-4.0 μM.

[0352] G71S cell lines expressing human sulfatase modifying factor 1 (SUMF1) produce an activated amount (i.e., the active site cysteine ​​residue is converted to C) α -formylglycine (FGly) has higher levels of lysosomal sulfatase.

[0353] To determine whether the purified recombinant lysosomal sulfatase (i.e., human N-acetylgalactosamine-6-sulfatase (GALNS)) co-expressed with SUMF1 in G71S cells showed increased activation, the translocation of cysteine ​​residues to FGly at the active site on the purified lysosomal sulfatase was measured.

[0354] GALNS activation. The percentage of activated GALNS was determined by LC / MS (TFA), i.e., the conversion of cysteine ​​(Cys) residues at the active site to C. α Percentage conversion of α-formylglycine (FGly). The TIC / 1000 values ​​for Cys, FGly, and Gly were 39, 1840, and 183, respectively, indicating that approximately 90% of the purified GALNS were in the active (i.e., FGly) form.

[0355] Summary. Table 7 shows a summary of the characteristics of recombinant GALNS expressed in G71S clone 4 cells. Table 8 shows a summary of the characteristics of recombinant GALNS expressed in G71S clone C2 cells.

[0356] Table 7: Characterization of human N-acetylgalactosamine-6-sulfatase (GALNS) produced by G71S clone 4

[0357]

[0358]

[0359] Table 8: Characterization of human N-acetylgalactosamine-6-sulfatase (GALNS) produced by G71S clone C2

[0360] Test Category GALNS Specific activity: activity / protein 6.4 U / mg Purity was determined by C4-RP. 97％ Size determined by SEC 115kDa (homogeneous dimer) Intake: Fibroblasts 3.4nM titer 6.4 mg / L (4 batches tested) M6P receptor platelet binding 5.7nM M6P content as determined by CE: % Total Carbohydrates 34.5％ M6P content: moles of M6P / moles of GALNS 0.69

[0361] These results demonstrate that the purified recombinant human GALNS exhibits high levels of activation and mannose-6-phosphate phosphorylation. Therefore, G71S cells co-expressing SUMF1 and lysosomal sulfatase (i.e., GALNS) efficiently produce highly phosphorylated lysosomal sulfatases. The increased levels of mannose residues on these lysosomal sulfatases lead to increased MPR uptake on the cell.

[0362] Example VII

[0363] Uptake and activity of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) in in vitro Morchion chondrocytes

[0364] To evaluate the uptake of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) by lysosomes of Morchion chondrocytes in vitro and the ability of GALNS to degrade keratin sulfate (KS).

[0365] Patients with mucopolysaccharidosis type IVa (MPS IVa or Molcio A syndrome) exhibit reduced GALNS activity in chondrocytes and show lysosomal accumulation of KS. An in vitro model of MPS IVa was established using chondrocytes isolated from iliac crest biopsies of MPS IVa patients. However, primary chondrocytes dedifferentiated and lost their chondrocyte characteristics during culture. Therefore, culture conditions were established to induce in vitro differentiation of chondrocytes.

[0366] Chondrocytes isolated from MPS IVa patients, termed MQCHs, were cultured in alginate beads (chondrocyte growth medium, Lonza, catalog number CC-3225) in the presence of IGF-1, TGF-β, transferrin, insulin, and ascorbic acid. The medium was changed twice weekly during the 6–15 week experimental duration. These culture conditions induced chondrocyte phenotypic expression and differentiation. Quantitative RT-PCR analysis of RNA isolated from MQCH cell cultures detected the expression of chondrocyte markers in these MQCH cells, including sex-determining region Y-box 9 (Sox 9), collagen II, collagen X, chondrocyte oligomeric matrix proteins, and aggregated proteoglycan mRNA. These cultured MQCH cells also produced extracellular matrix.

[0367] Confocal microscopy was performed to confirm KS accumulation in MQCH cells. MQCH cells cultured for 8 weeks were treated with trypsin, cytospinned, fixed with acetone, and frozen for later use. After thawing, the cells were rehydrated and stained with anti-KS monoclonal antibody (Chemicon) and Alexa-488 (green) conjugated with goat anti-rabbit antibody as the first and second antibodies, respectively. MQCH cells showed punctate intracellular staining, consistent with lysosomal KS accumulation.

[0368] To determine whether MQCH cells could absorb purified recombinant human GalNS into lysosomes and degrade KS, 6-week cultures of MQCH cells were seeded with 10 nM recombinant human GalNS twice a week for 9 weeks. GalNS uptake and KS clearance were detected by confocal microscopy. The primary antibodies used were: (a) anti-GALNS rabbit polyclonal antibody and anti-lysosome-associated membrane protein-1 (LAMP-1) monoclonal antibody, or (b) anti-KS monoclonal antibody and anti-LAMP-1 polyclonal antibody. The secondary antibodies used were: Alexa-488 (green) conjugated antibody to detect anti-GALNS or anti-KS antibodies, or Alexa-555 or -594 (red) conjugated antibody to detect anti-LAMP-1 antibodies. MQCH cell preparations were mounted with a mounting medium containing stained DAPI nuclei.

[0369] In GALNS-treated MQCH cells, significant co-localization of GALNS enzyme, KS, lysosomal markers, and LAMP-1 was observed. Intracellular KS levels decreased in MQCH cells after contact with recombinant human GALNS.

[0370] GalNS uptake was also detected using a GalNS enzyme capture ELISA and a GalNS specific activity ELISA, both described in Example IV above. Normal human chondrocytes (NHKC) expressing GalNS were used as a positive control. As shown in Tables 9 and 10, untreated MQCH cells showed no detectable GalNS enzyme or activity, while MCQH cells treated with 10 nM GalNS for 9 weeks exhibited significant GalNS enzyme and activity.

[0371] Table 9: ELISA capture using GALNS enzyme in MQCH cells

[0372] MQCH cells NHKC Unprocessed <![CDATA[N.D. a ]]> <![CDATA[0.12 b ]]> 10nM GALNS, 9 weeks 3.99 0.88

[0373] a was not detected; b ng GALNS antigen / μg total protein

[0374] Table 10: GALNS specific activity assay using MQCH cells

[0375] MQCH cells NHKC Unprocessed <![CDATA[N.D. a ]]> <![CDATA[2.76 b ]]> 10nM GALNS, 9 weeks 3.68 5.15

[0376] a was not detected; b GALNS activity / ng antigen

[0377] These results demonstrate that Molcio chondrocytes take up purified recombinant human GALNS into lysosomes, and that recombinant human GALNS can degrade lysosomal KS in vitro. These Molcio chondrocytes can be used as an in vitro efficacy model to test lysosomal sulfatases, such as GALNS, that degrade KS.

[0378] Example VIII

[0379] Activity of recombinant human lysosomal enzymes in degrading natural substrates in in vitro cell experiments

[0380] In vitro cell assays were developed to detect the activity of recombinant human lysosomal enzymes, such as lysosomal sulfatases, in degrading natural substrates.

[0381] The activity of recombinant human lysosomal enzymes, such as lysosomal sulfatases (GALNS, see Example 4), is typically detected using artificially produced fluorescent substrates via cell-free in vitro assays. However, the detected enzyme activity depends on the size of the artificial substrate, i.e., the number of monosaccharide units. Furthermore, enzyme activity is detected in an environment that does not reflect in vivo conditions. Therefore, cell-free in vitro assays do not take into account either the ability of lysosomal enzymes to cleave native substrates or their ability to take up into target cells and localize to lysosomes.

[0382] In vitro cell assays were developed to detect the activity of two recombinant human lysosomal enzymes, α-L-idurosidase (IDU) and arylsulfatase B (ARSB), in degrading their natural substrates, namely, intracellular dermatan sulfate (DS). DS contains distinct sulfated iduronate β(1–3)–N-acetyl-galactosamine β(1–4) disaccharide units.

[0383] ARSB-deficient GM00519 human fibroblasts or IDU-deficient GM01391 human fibroblasts were cultured in 12-well plates until confluence, and the cultures were maintained for 3-6 weeks after confluence to accumulate intracellular DS.

[0384] Then, the confluent GM00519 or GM01391 cells were exposed to saturated doses of recombinant human ARSB (10 nM) or recombinant human IDU (25 nM) for 4–5 days. Untreated and lysosomal sulfatase-treated cells were collected, lysed, and centrifuged.

[0385] The residual DS content in cells was determined to detect lysosomal enzyme activity in cell lysates by the following steps: (1) lysing cells; (2) specifically digesting the DS-containing substrate into a disaccharide using chondroitin ABC lyase (EC 4.2.2.4) in the cell lysates; (3) labeling the DS disaccharide with a fluorescent dye (e.g., 2-amino-acridone, AMAC); (4) separating the DS disaccharide (e.g., by capillary zone electrophoresis, CZE); and (5) detecting the labeled DS disaccharide (e.g., by laser-induced fluorescence, LIF). Such methods are described, for example, in Zinellu et al., Electrophoresis 2:2439-2447, 2007 and Lamari et al., J. Chromatogr. B 730:129-133, 1999 (see Volpi et al., Electrophoresis 29:3095-3106, 2008 for a review).

[0386] Table 11 shows the percentage of DS degradation in ARSB-treated GM00519 cells determined by detecting the content of N-acetylgalactosamine-4-sulfatase (4S disaccharide) (the main DS disaccharide). Similar results were obtained using IDU-treated GM01391 cells.

[0387] Table 11: Elimination of DS by ARSB in in vitro cell experiments

[0388]

[0389] The percentage of degradation was calculated by detecting the area under the curve of 4S disaccharide in a CZE-LIF scan of ARSB-treated lysates compared to untreated cells.

[0390] The above experiments show that target cells take up recombinant human ARSB and IDU, and then localize them to lysosomes, where they degrade their natural substrate, namely intracellular DS.

[0391] Dosage studies were conducted to determine the concentration at which IDU became the limiting rate in this cell assay. GM01391 cells were cultured in 12-well plates. Four weeks after confluence, cells were exposed to various concentrations of IDU ranging from 0.8 nM to 25 nM for 6 or 26 hours. Cell lysates were prepared as described above. IDU did not become the limiting rate at concentrations below 1 nM.

[0392] In the second dose study, GM01391 cells were exposed to various concentrations of IDU (0.01–0.2 nM) for 2 days 3 weeks after confluence. Cell lysates were prepared and treated as described above. In this experiment, a known amount of the internal standard monosaccharide, GlcNAc-6S, was incorporated into the cell lysates as a recovery control during processing. Figure 11 As shown, a dose-dependent decrease in DS substrate content was observed in IDU-treated GM01391 cells.

[0393] In a similar dose-response study, GM00519 cells were exposed to various ARSBs (0.001–0.06 nM) for 5 days at 3 weeks post-confluence. Cell lysates were prepared and treated as described above. In this experiment, a known amount of the internal standard monosaccharide, GlcNAc-6S, was incorporated into the cell lysates as a recovery control during processing. Figure 12 As shown, a dose-dependent decrease in DS substrate content was observed in ARSB-treated GM00519 cells.

[0394] An in vitro cell assay was developed to detect the activity of recombinant human lysosomal sulfatase GALNS in degrading its natural substrate, namely, intracellular keratin sulfate (KS) substrates.

[0395] GALNS-deficient MQCH cells were cultured as described in Example 7 above and treated with 1 or 10 nM non-recombinant human GALNS. After treatment, MQCH cell lysates were prepared and digested with keratinase II (EC 3.2.1), which degrades larger KS oligosaccharides into KS disaccharides. KS disaccharides were labeled with AMAC as described above for DS disaccharides, separated by CZE, and detected by LIF. GlcNAc-6S and KS monosaccharides were added to the cell lysates as internal standards for recovery during processing. The contents of the two characteristic KS disaccharides, Gal6S-GlcNAc6S and Gal-GlcNAc6S, were measured and corrected for by the recovery of GlcNAc6S. Table 12 shows the percentage of KS degradation in GALNS-treated MQCH cells determined by detecting the contents of the two characteristic KS disaccharides.

[0396] Table 12: GALNS elimination of KS in in vitro cell experiments

[0397] Gal6S-GlcNAc6S Gal-GlcNAc6S 1NM GALNS <![CDATA[85.7 a ]]> <![CDATA[78.5 b ]]> 10nM GALNS 88.6 81.5

[0398] a, b. The percentage of degradation was calculated by detecting the area under the curve (AUC) of Gal6S-GlcNAc6S and Gal-GlcNAc6S in CZE-LIF scans of GALNS-treated lysates compared with untreated MQCH cells, and adjusting the AUC of the control GlcNAc6S to be added.

[0399] The above experiments show that target cells take up recombinant human GALNS and then localize to lysosomes, where GALNS degrades its natural substrate, namely intracellular KS.

[0400] In summary, these results demonstrate that the activities of recombinant human lysosomal enzymes ARSB, IDU, and GALNS in degrading their natural substrates can be detected and quantified using in vitro cell assays. It should be understood that it is not difficult to improve these in vitro cell assays to detect and quantify the activities of other lysosomal sulfatases and various recombinant lysosomal enzymes.

[0401] Example IX

[0402] Delivery of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) to specific tissues

[0403] The ability of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), expressed and purified in G71 cells, to be delivered to specific tissues in mice that were affected by or associated with GALNS deficiency was evaluated.

[0404] The highly specific distribution of keratin sulfate gives rise to a highly characteristic phenotype of mucopolysaccharidosis type IVa (MPS IVa) or Molquiao syndrome. Keratin sulfate is primarily found in cartilage (connecting bone growth surfaces, heart valves, larynx, and nasal septum) and the cornea, the tissues in which keratin sulfate accumulation is observed in MPS IVA patients. Therefore, for N-acetylgalactosamine-6-sulfatase (GALNS), which is lacking in MPS IVA or Molquiao syndrome, the delivery of GALNS enzymes to the growth surfaces of long bones, heart valves, cornea, larynx, and nose is crucial. To observe these specific tissues as targets of poor vascularization, the delivery of fluorescent GALNS was investigated in mice.

[0405] Two immunohistochemical staining methods were tested in mice: (1) human GALNS conjugated with Alexa 488 and (2) unconjugated human GALNS. Human GALNS were conjugated to Alexa 488 using the Molecular Probes Alexa Fluor488C5 maleimide labeling kit (A-10254). The maleimide conjugation chemistry method yielded a 1:1 label-to-protein ratio.

[0406] To confirm that the fluorescent tag does not interfere with GALNS uptake, immunohistochemical experiments were performed using cultured synovial cells (ATCC number CRL-1832). A standard uptake assay was used to compare unconjugated and conjugated GALNS (GALNS-A488 or GALNS-A555). Cells were incubated with GALNS enzyme for 1 hour, followed by α-L-idurosidase (IDU) chase for 2 hours. The results showed that Alexa 488 conjugation did not interfere with cellular uptake. Figure 13 The estimated Kd is shown for GALNS, GALNS-A488, and GALNS-A555. Uptake was detected by antigen ELISA of cell lysates, not enzyme activity, because labeling inactivates the enzyme. The Kd determined to be approximately equal for uncoupled and coupled GALNS enzymes.

[0407] To determine the stability of the fluorescent tag once GALNS enzymes are incorporated into cells, immunostaining was performed on uncoupled and coupled GALNS. The primary antibody used for staining was a purified rabbit anti-GALNS antibody at a concentration of 1 μg / mL. All images were acquired using a Leica IRE2 wide-field epi-fluorescence microscope with MetaMorph software. Due to out-of-plane light, detecting colocalization in these images required deconvolution of the image stack. Deconvolution was performed using the AutoQuant / AutoDeblur imaging software with the theoretical point spread function (blind algorithm).

[0408] Immunostaining showed good overlap with the signal amplified on the GALNS-A488 material. The increased sensitivity was observed because both the first and second antibodies were polyclonal.

[0409] To determine whether the GALNS enzyme targets lysosomes, cultured synovial cells were immunostained with a molecular probe lysosomal tracer or another enzyme localized to lysosomes. The lysosomal tracer appeared to show some overlap with the GALNS-488 enzyme; however, the staining was uneven. Treatment with recombinant human N-acetylgalactosamine-4-sulfatase (rhASB) (a lysosomal enzyme) for 2 hours did indeed show some co-localization with GALNS.

[0410] The above experiments show that cells take up the GALNS-A488 enzyme and locate it in lysosomes, and this enzyme can be used to determine its biodistribution in vivo.

[0411] Two in vivo studies were conducted. The first preliminary study involved a single bolus injection (10 mg / kg) into the tail vein of normal Balb / c mice, followed by multiple injections (5) of 10 mg / kg into the tail vein of normal Balb / c mice every other day for the second study. The experimental plans for the first and second studies are described in Tables 13 and 142, respectively.

[0412] Table 13: Experimental Design of the First Preliminary Study

[0413] Group total 2-hour time point 24-hour time point PBS control 4 2 2 GALNS-A488 4 2 2 Unmarked GALNS 4 2 2 Unmarked ASB 1 1 0

[0414] Table 14: Experimental Design of the Second Study

[0415] Group total 2-hour time point 4-hour time point 8-hour time point PBS control 2 1 0 1 PBS / Cys control 4 2 0 2 GALNS-A488 9 3 3 3 Unmarked GALNS 6 3 0 3 Unmarked ASB 3 2 0 1

[0416] In the first preliminary study, the heart, liver, and tibia / femoral joint were collected at 2-hour and 24-hour time points. In the second study, the heart, kidney, liver, and bone containing the quadriceps and soleus muscles were collected at 2-hour, 4-hour, and 8-hour time points. In both studies, the heart, kidney, and liver were fixed in 4% paraformaldehyde (PFA) for 4 days, paraffin-embedded, and then sectioned into 7-micrometer-thick sections. In the second study, bone containing muscle was impregnated in 4% PFA for 8 days, decalcified, paraffin-embedded, and sectioned into 7-micrometer-thick sections.

[0417] Images of GALNS-A488-injected mice were acquired using a Zeiss laser scanning confocal microscope. For the analysis in the first preliminary study, one confocal stack was obtained for each heart valve and liver sample for volume analysis. Two confocal stacks were obtained for each growth surface sample for volume analysis. In the second study, one confocal stack was obtained for each heart valve, kidney, and liver sample for volume analysis; two confocal stacks were obtained for each growth surface and zone of rest cartilage (zrc) sample for volume analysis.

[0418] The conclusions of the confocal microscopy study are: (1) possible in vivo detection of fluorescent GALNS; (2) the signal is specific (no background) and localized to lysosomes; (3) evidence of the presence of GALNS in hepatic sinusoidal cells; (5) in the heart, GALNS enzymes are present in the diaphragm and atria, but more importantly, they are clearly visible at the level of the heart valves, and after multiple injections, they are distributed more deeply in the heart valves; (6) at the femoral / tibial junction, GALNS enzymes are present in the mineralized part of bone (epithelium) and bone marrow. GALNS are present on the growth surface. More specifically, GALNS are abundant in chondrocytes in the resting area (or storage cartilage area), present at the beginning of the proliferative zone, and reappear in large quantities in the ossification zone when the growth surface terminates. Although it is difficult to quantify the cumulative effect of multiple injections, the second study appears to show a wider distribution. Table 15 summarizes the confocal microscopy study.

[0419] Table 15: Biodistribution of GALNS in mice

[0420] organize position Femur mineralized area yes marrow yes growth surface yes heart Heart valves yes atrium yes diaphragm yes liver hepatocytes no sinus cells yes

[0421] For secondary staining, the initial steps for the primary antibody against GALNS were optimized. Various tissues were stained with 1:100 to 1:400 dilutions of G protein-purified anti-GALNS rabbit antibody. Initial results from the first study indicated that a 1:100 dilution was optimal for high signal-to-noise ratios. This result was confirmed in a second study. The remaining slides were processed with a 1:100 dilution of the primary antibody and a 1:1000 dilution of the secondary antibody.

[0422] When stained with anti-GALNS antibody purified from G proteins, Balb / c mice given GALNS showed a higher signal than controls (i.e., mice given PBS-Cys). To confirm that the GALNS enzyme is localized in lysosomes, sections were stained with anti-LAMP1 antibody. LAMP1 is a marker for lysosomes. The images showed overlap between anti-LAMP1 and anti-GALNS antibodies, indicating that the GALNS enzyme is localized in lysosomes.

[0423] In summary, these two in vivo studies demonstrate that the biodistribution of GALNS is associated with vascularization, meaning that more vascularized tissues contain more fluorescent signals. More specifically, these studies demonstrate that in Morquio syndrome, GALNS are present at sites of keratin sulfate accumulation, even in areas with poor vascularization.

[0424] Example X

[0425] Role of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and other lysosomal sulfatases in lysosomal sulfatase activity deficient mice

[0426] The effects of the highly phosphorylated human lysosomal sulfatases of the present invention, such as recombinant human N-acetylgalactosamine-6-sulfatase (GALNS), were evaluated in lysosomal sulfatase-deficient mice.

[0427] Recombinant human GALNS protein was expressed and purified in G71S cells. Other recombinant human lysosomal sulfatases can be expressed and purified substantially according to the methods described herein or by methods known in the art.

[0428] Several mouse models of human lysosomal sulfatase deficiency have been described, including: metachromatic leukodystrophy (MLD) (arylsulfatase A deficiency) (Hess et al., Proc. Natl. Acad. Sci. USA 93:14821-14826, 1996), mucopolysaccharidosis type VI (MPS VI) or Marr-Lanner syndrome (arylsulfatase B deficiency) (Evers et al., Proc. Natl. Acad. Sci. USA 93:8214-8219, 1996), and mucopolysaccharidosis type II (MPS II) or Hunter syndrome (iduronate-2-sulfatase deficiency) (Muenzer et al., Acta...). Paediatr.Suppl.91(439):98-99,2002; Cardone et al., Hum.Mol.Genet.15:1225-1236,2006), mucopolysaccharidosis type IIIa (MPS IIIa) or Sangfield A syndrome (sulfonamide / heparin-N-sulfatase deficiency) (Bhaumik et al., Glycobiology9(12):1389-1396,1999), mucopolysaccharidosis type IVa (MPS Mucopolysaccharidosis type IIId (MPS IIId) or Morgio A syndrome (N-acetylgalactosamine-6-sulfatase deficiency) (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003) and multiple sulfatase deficiency (MSD) (sulfatase modifying factor 1 deficiency) (Settembre et al., Proc. Natl. Acad. Sci. USA 104:4506-4511, 2007). Mouse models of mucopolysaccharidosis type IIId (MPS IIId) or Sempelleep D syndrome (N-acetylgalactosamine-6-sulfatase deficiency) have not yet been described.

[0429] The feasibility of enzyme replacement therapy (ERT) as a treatment for lysosomal storage diseases can be evaluated using mouse models of human lysosomal sulfatase deficiency. For example, MPS IVa knockout mice (GALNS) - / - Mice (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003) showed no detectable GALNS enzyme activity and exhibited elevated urinary glucosamine (GAG), i.e., keratin sulfate and chondroitin-6-sulfate. GAG accumulated in various tissues and organs, such as the liver, kidneys, spleen, heart, brain, bone marrow, and cartilage. However, GALNS... - / - The mice did not show skeletal abnormalities associated with the human disease. Another MPS IVa mouse model (GALNS) expressing inactivated human GALNS and mutant inactivated endogenous mouse GALNS was developed. tm (hC79S.mC76S)sluMice; Tomatsu et al., Hum. Mol. Genet. 14:3321-3335, 2005). In the absence of detectable GALNS enzyme activity in mice. tm(hC79S.mC76S)slu In mice, urinary GAG secretion is increased, and GAG accumulates in various tissues, including internal organs, brain, cornea, bone, ligaments, and bone marrow. Lysosomal storage is significant in multiple tissues, with marked bone accumulation. The pathological alterations in GALNS tm(hC79S.mC76S)slu The pathological changes in mice are different from those in GALNS. - / - Observed in mice. However, compared with GALNS - / - Mouse-like, GALNS tm(hC79S.mC76S)slu Mice do not show skeletal abnormalities associated with human disease. Therefore, GALNS can be utilized. - / - Or GALNS tm(hC79S.mC76S)slu The effects of administering recombinant human GALNS to mice on increased urinary GAG and GAG accumulation in tissues were studied.

[0430] For four-week-old GALNS - / - GALNS tm(hC79S.mC76S)slu Or wild-type mice were intravenously injected weekly (n = at least 6 or 8 per group) with various doses of recombinant human GALNS (e.g., 0.1, 0.3, 1, 3, 10 mg / kg) or given the vector pair at 16–20 weeks of age, followed by sacrifice for histological examination. Urine was collected from mice, and urinary GAG secretion was measured as described in (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003). Histopathological examination of various tissues was performed as described in (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003).

[0431] Leveraging GALNS - / - Or GALNS tm(hC79S.mC76S)slu In mice, the recombinant human GALNS of the present invention is expected to reduce: (1) urinary GAG secretion; (2) GAG accumulation in various tissues, such as visceral organs, brain, cornea, bone, ligaments and bone marrow; (3) lysosomal storage in various tissues; and (4) bone storage.

[0432] In a mouse model of multiple sulfatase deficiency (MSD) (SUMF1) - / - The role of recombinant human GALNS was studied in mice (Settembre et al., Proc. Natl. Acad. Sci. USA 104:4506-4511, 2007). Due to SUMF1... - / - Mice that died frequently in early life; these mice were injected with recombinant human GALNS earlier than the aforementioned GALNS.- / - Mice.

[0433] According to methods known in the art, in MLD (ASA) - / - Mice; Hess et al., Proc. Natl. Acad. Sci. USA 93:14821-14826, 1996), MPS VI (As1-s - / - Mice; Evers et al., Proc. Natl. Acad. Sci. USA 93:8214-8219, 1996), MPS II (ids y / - Mice; Cardone et al., Hum. Mol. Genet. 15:1225-1236, 2006), MPS IIIa (Bhaumik et al., Glycobiology 9(12):1389-1396, 1999) and MSD (SUMF1 - / - The effects of other recombinant human lysosomal sulfatases, namely arylsulfatase A, arylsulfatase B, iduronate-2-sulfatase, sulfonamide / heparin-N-sulfatase, and N-acetylgalactosamine-6-sulfatase, were investigated in a mouse model (Settembre et al., Proc. Natl. Acad. Sci. USA 104:4506-4511, 2007).

[0434] Example XI

[0435] Patients with mucopolysaccharidosis type IVA (or Molgio syndrome) or other lysosomal sulfatase deficiencies treated with recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) and other lysosomal sulfatases

[0436] Patients exhibiting clinical signs of lysosomal sulfatase deficiency, such as those diagnosed with mucopolysaccharidosis type IVA (MPS IVa or Molchio syndrome), should be considered for enzyme replacement therapy with a recombinant enzyme, namely human N-acetylgalactosamine-6-sulfatase (GALNS). All patients with lysosomal sulfatase deficiency demonstrate some degree of functional impairment or deterioration in health associated with specific lysosomal storage disorders, exhibiting some clinical evidence of excessive or harmful accumulation of stored substances in their lysosomes within visceral and soft tissues. All MPS IVa patients exhibit clinical evidence of skeletal deformities, short stature, and abnormal gait, and / or accumulation of glucosamine (GAG) in the blood or urine, accompanied by varying degrees of functional impairment.

[0437] Monitoring enzyme levels in patients with lysosomal sulfatase deficiency is preferred to confirm a deficiency or reduction in the activity of this lysosomal sulfatase in their tissues. Patients with lysosomal enzyme activity below 10%, preferably below 5%, more preferably below 2%, and even more preferably below 1%, compared to other normal subjects are suitable candidates for treatment with an appropriate lysosomal sulfatase. Data can be collected to determine the patient's lysosomal sulfatase activity before, during, and after treatment.

[0438] Efficiency is determined by measuring the percentage reduction in urinary excretion of lysosomal sulfatase substrate, glucosamine (GAG), over time. Urinary GAG levels in patients with lysosomal sulfatase deficiency are compared to normal excretion levels and / or levels in untreated patients with the same lysosomal sulfatase deficiency and / or water levels in the same patient before treatment with lysosomal sulfatase. A reduction in the excretion of undegraded GAG exceeding 25%, preferably exceeding 50%, after treatment with lysosomal sulfatase is an effective way to assess an individual's response to treatment.

[0439] The efficacy of assays is reduced based on signs and symptoms of pathology associated with lysosomal storage disorders. Efficacy is determined by tissue biopsy and examination of cells and / or lysosomes to ascertain the extent of GAG reduction in lysosomes, cells, or tissues. Efficacy can be determined through subjective or objective functional assessments (e.g., relief of pain or functional difficulties, increase in muscle strength or energy, increase in cardiac output, exercise endurance, weight, height, or changes in appearance, etc.).

[0440] The pharmaceutical composition comprising recombinant human GALNS expressed and purified in G71S cells is formulated according to methods known in the art. The pharmaceutical composition of the present invention is preferably administered intravenously.

[0441] The basic design of a preliminary clinical study to investigate the effects of recombinant human GALNS on MPSIVa patients includes an open-label, dose-escalation safety / efficacy study in which various doses of the enzyme are administered intravenously to patients at fixed intervals, such as, but not limited to, weekly injections of the enzyme.

[0442] For MPS IVa patients, efficacy is determined by tests such as a reduction in blood or urinary GAG that may be observed within weeks of ERT, an increase in endurance in cardiac, pulmonary and / or motor function tests that may be observed within months of ERT, and / or changes in bone and / or body growth that may be observed within years of ERT.

[0443] Urinary GAG testing can be used to establish appropriate dosing regimens and determine efficacy by detecting the percentage decrease in urinary GAG excretion over time.

[0444] Various endurance tests can be used, including, but not limited to, walking tests (distance walked in 6 or 12 minutes), stair climbing (stairs climbed per minute), and lung / respiratory function, including cardiac function (ECG, echocardiography) and lung function (FVC, FEV1, maximum respiratory flow).

[0445] For young patients undergoing long-term treatment, growth (height) can be measured.

[0446] The lysosomal storage disorders associated with lysosomal sulfatase activity deficiency that can be treated or prevented using the method of this invention are: metachromatic leukodystrophy (MLD), mucopolysaccharidosis type VI (MPS VI) or Marr-Lan II syndrome, mucopolysaccharidosis type II (MPS II) or Hunter syndrome, mucopolysaccharidosis type IIIa (MPS IIIa) or Sempelep A syndrome, mucopolysaccharidosis type IIId (MPS IIId) or Sempelep D syndrome, mucopolysaccharidosis type IVa (MPS IVa) or Molgio A syndrome, or multiple sulfatase deficiency (MSD). For each lysosomal storage disorder, the recombinant lysosomal sulfatase contains a specific lysosomal sulfatase.

[0447] For methods involving MLD, the preferred lysosomal sulfatase is arylsulfatase A. For methods involving MPS VI, the preferred lysosomal sulfatase is arylsulfatase B. For methods involving MPS II, the preferred lysosomal sulfatase is iduronic acid-2-sulfatase. For methods involving MPS IIIA, the preferred lysosomal sulfatase is sulfonamide / heparin-N-sulfatase. For methods involving MPS IIID, the preferred lysosomal sulfatase is N-acetylglucosamine-sulfatase. For methods involving MPS IVA, the preferred lysosomal sulfatase is N-acetylgalactosamine-6-sulfatase. For methods involving MSD, the preferred lysosomal sulfatase is N-acetylgalactosamine-6-sulfatase.

[0448] *****

[0449] Those skilled in the art will anticipate many improvements and modifications to the invention as illustrated in the above exemplary embodiments. Therefore, only the limitations set forth in the appended claims apply to this invention.

Claims

1. A recombinant human lysosomal sulfatase or its biologically active fragment, mutant, variant, or derivative thereof produced in END3 complementary group CHO cells or derivatives thereof, wherein the END3 complementary group CHO cells or derivatives thereof express recombinant human sulfatase modifying factor 1 (SUMF1), wherein the recombinant lysosomal sulfatase or its biologically active fragment, mutant, variant, or derivative thereof has a high level of activation and a high level of phosphorylation.

2. The recombinant human lysosomal sulfatase as described in claim 1, or its biologically active fragment, mutant, variant, or derivative, characterized in that, The recombinant lysosomal sulfatase is selected from: arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS).

3. A recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or its biologically active fragment, mutant, variant, or derivative thereof, produced from END3 complementary group CHO cells or derivatives thereof, wherein the END3 complementary group CHO cells or derivatives thereof express recombinant human sulfatase modifying factor 1 (SUMF1), wherein the recombinant human GALNS has a high level of activation and a high level of phosphorylation.

4. The enzyme according to any one of claims 1-3, characterized in that, The END3 complementary group CHO cells or their derivatives are G71 cell lines or G71 derivatives.

5. A method for producing highly phosphorylated recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof, comprising the following steps: (a) Culture of Chinese hamster ovary (CHO)-derived END3 complementary cells or derivatives thereof; (b) Prepare a first mammalian expression vector capable of expressing the active highly phosphorylated recombinant human lysosomal sulfatase or its biologically active fragments, mutants, variants or derivatives thereof in the END3 complementary group cells or their derivatives; (c) Prepare a second mammalian expression vector capable of expressing recombinant human sulfatase-modifying factor 1 (SUMF1) or its biologically active fragments, mutants, variants or derivatives in the END3 complementary group cells or their derivatives; (d) Transfect the END3 complementary group cells or their derivatives with the first and second expression vectors; (e) Selecting and cloning transfectants of the END3 complementary group cells or derivatives expressing the said highly phosphorylated recombinant human lysosomal sulfatase or its biologically active fragments, mutants, variants, or derivatives; and (f) Optimize cell culture methods to produce the highly phosphorylated recombinant human lysosomal sulfatase or its biologically active fragments, mutants, variants, or derivatives. The recombinant human lysosomal sulfatase mentioned therein is selected from: arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS).

6. The method as described in claim 5, characterized in that, In step (d), the first and second expression vectors are simultaneously transfected into the END3 complementary group cells or their derivatives.

7. The method as described in claim 5, characterized in that, The first and second expression vectors described in step (d) are sequentially transfected into the END3 complementary group cells or their derivatives.

8. The method according to any one of claims 5-7, characterized in that, The END3 complementary group CHO cells or their derivatives are G71 cell lines or G71 derivatives.

9. An active, highly phosphorylated recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof produced by the method of any one of claims 5-8.

10. A pharmaceutical composition comprising the lysosomal sulfatase of claim 9 or a biologically active fragment thereof, mutant, variant or derivative thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

11. A method for producing highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active fragment, mutant, variant, or derivative thereof, comprising the following steps: (a) Culture of Chinese hamster ovary (CHO)-derived END3 complementary cells or derivatives thereof; (b) Prepare a first mammalian expression vector suitable for expressing the active highly phosphorylated recombinant human GALNS or its biologically active fragments, mutants, variants or derivatives thereof in said END3 complementary group cells or their derivatives; (c) Prepare a second mammalian expression vector suitable for expressing recombinant human sulfatase-modifying factor 1 (SUMF1) or its biologically active fragments, mutants, variants or derivatives in the END3 complementary group cells or their derivatives. (d) Transfect the END3 complementary group cells or their derivatives with the first and second expression vectors; (e) Selecting and cloning transfectants of the END3 complementary group cells or derivatives expressing the said highly phosphorylated recombinant human GALNS or its biologically active fragments, mutants, variants, or derivatives; and (f) Optimize cell culture methods to produce the said highly phosphorylated recombinant human GALNA or its biologically active fragments, mutants, variants or derivatives.

12. The method as described in claim 11, characterized in that, In step (d), the first and second expression vectors are simultaneously transfected into the END3 complementary group cells or their derivatives.

13. The method as described in claim 11, characterized in that, The first and second expression vectors described in step (d) are sequentially transfected into the END3 complementary group cells or their derivatives.

14. The method according to any one of claims 11-13, characterized in that, The END3 complementary group CHO cells or their derivatives are G71 cell lines or G71 derivatives.

15. A highly active, highly phosphorylated recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active fragment, mutant, variant, or derivative thereof produced by the method of any one of claims 11-14.

16. A pharmaceutical composition comprising the recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) of claim 15 or a biologically active fragment thereof, mutant, variant or derivative thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

17. A recombinant human GALNS or its biologically active fragment, mutant, variant, or derivative thereof for use in treating subjects suffering from or related to lysosomal storage disorders caused by N-acetylgalactosamine-6-sulfatase (GALNS) deficiency, wherein said GALNS enzyme or its biologically active mutant, variant, or derivative thereof: (a) When performing SDS-PAGE under non-reducing conditions, the purity determined by Coomassie blue staining is at least approximately 90%; (b) At least approximately 90% of the cysteine ​​residue at position 53 is converted to C. α -formylglycine (FGly); and (c) The asparagine residues at positions 178 and 397 are N-linked glycosylated, wherein at least about 50% of the oligomannose chain linked to the asparagine residue at position 178 is bis-phosphorylated.

18. The GALNS enzyme as described in claim 17, characterized in that, The lysosomal storage disease is mucopolysaccharidosis type IVa (MPS IVa) or Morquio A syndrome.

19. The GALNS enzyme as described in claim 17, characterized in that, The lysosomal storage disease is a polysulfatase deficiency.

20. The GALNS enzyme as described in claim 17, characterized in that, When SDS-PAGE was performed under reducing conditions, the GALNS enzyme was determined by Coomassie blue staining to consist of a major band of approximately 55-60 kDa, which constituted at least approximately 75% of the visible protein.

21. The GALNS enzyme as described in claim 17, characterized in that, When SDS-PAGE was performed under reducing conditions, Coomassie blue staining revealed that the GALNS enzyme consisted essentially of a single band of approximately 55-60 kDa.

22. A method of treating a subject with lysosomal sulfatase deficiency, comprising administering a subject requiring the lysosomal sulfatase a therapeutically effective amount of the lysosomal sulfatase, wherein the lysosomal sulfatase is a recombinant human lysosomal sulfatase or a biologically active fragment, mutant, variant, or derivative thereof produced by CHO-derived END3 complementary group cells or derivatives thereof.

23. The method as described in claim 22, characterized in that, The lysosomal sulfatase deficiency is selected from the following groups: metachromatic leukodystrophy (MLD), mucopolysaccharidosis type VI (MPS VI) or Mar-Lan II syndrome, mucopolysaccharidosis type II (MPS II) or Hunter syndrome, mucopolysaccharidosis type IIIa (MPS IIIa) or Sempelep A syndrome, mucopolysaccharidosis type IIId (MPS IIId) or Sempelep D syndrome, and mucopolysaccharidosis type IVa (MPS IVa) or Molchio A syndrome.

24. The method as described in claim 23, characterized in that, The lysosomal sulfatase deficiency is MPS IVa or Morquio A syndrome.

25. A method of treating a subject suffering from lysosomal storage disease associated with a deficiency of one or more lysosomal sulfatases, comprising administering to the subject lacking said one or more lysosomal sulfatases a therapeutically effective amount of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active fragment, mutant, variant, or derivative thereof produced from CHO-derived END3 complementary group cells or derivatives thereof.

26. The method as described in claim 25, characterized in that, The lysosomal storage disease is multiple sulfatase deficiency (MSD).

27. The method according to any one of claims 22-26, characterized in that, The END3 complementary group CHO cells or their derivatives are G71 cell lines or their G71 derivatives.

28. A method of treating a subject suffering from mucopolysaccharidosis type VIa (MPS IVa), Morgio A syndrome, or multiple sulfatase deficiency (MSD), comprising administering to the subject a therapeutically effective amount of purified recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) or a biologically active mutant, variant, or derivative thereof, wherein said GALNS or the biologically active mutant, variant, or derivative thereof: (a) When performing SDS-PAGE under non-reducing conditions, Coomassie blue staining was determined to have a purity of at least approximately 90%; (b) At least approximately 90% of the cysteine ​​residue at position 53 is converted to C. α -formylglycine (FGly); and (c) The asparagine residues at positions 178 and 397 are N-linked glycosylated, wherein at least about 50% of the oligomannose chain linked to the asparagine residue at position 178 is bis-phosphorylated.

29. The method as described in claim 28, characterized in that, The subject suffered from MPS IVa or Morquio A syndrome.

30. The method as described in claim 28, characterized in that, The subject suffers from MSD.

31. The method as described in claim 28, characterized in that, When SDS-PAGE was performed under reducing conditions, the GALNS enzyme was determined by Coomassie blue staining to consist of a major band of approximately 55-60 kDa, which constituted at least approximately 75% of the visible protein.

32. The method as described in claim 28, characterized in that, When SDS-PAGE was performed under reducing conditions, Coomassie blue staining revealed that the GALNS enzyme consisted essentially of a single band of approximately 55-60 kDa.

33. An END3 complementary group CHO cell line derived from G71 or a derivative thereof, wherein the END3 complementary group CHO cell line comprises: (a) Expression vector for recombinant human sulfatase-modifying factor 1 (SUMF1); and (b) An expression vector for recombinant human lysosomal sulfatase, wherein the recombinant human lysosomal sulfatase is selected from: arylsulfatase A (ARSA), arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylgalactosamine-6-sulfatase (GALNS).

34. The END3 complementary group CHO cell line as described in claim 33, characterized in that, The END3 complementary group CHO cell line contains an expression vector for recombinant human N-acetylgalactosamine-6-sulfatase (GALNS).

35. The END3 complementary group CHO cell line as described in claim 34, characterized in that, The END3 complementary group CHO cell line expressed and secreted recombinant human GALNS.

36. The END3 complementary group CHO cell line as described in claim 34, characterized in that, The END3 complementary group CHO cell lines were selected from: clone 4, clone 5, clone C6, clone C2, clone C5, clone C7, clone C10, clone C11 and clone C30.

37. The END3 complementary group CHO cell line as described in claim 34, characterized in that, The END3 complemented group CHO cell line is clone C2.

38. The END3 complementary group CHO cell line as described in claim 34, characterized in that, The END3 complement group CHO cell line is suitable for suspension growth.

39. A method for detecting the activity of recombinant human lysosomal enzymes in degrading natural substrates, comprising: (a) Culture isolated human cells lacking lysosomal enzymes under conditions of accumulation of natural substrates of lysosomal enzymes; (b) Introduce the cell to lysosomal enzymes; (c) Lyse the cell; (d) Add an enzyme to the cell lysate, said enzyme being: (i) a natural substrate-specific enzyme, and (ii) an enzyme that cleaves small oligosaccharides on the natural substrate; (e) Label the oligosaccharide with a detectable portion; (f) Optionally separated labeled oligosaccharides; (g) Detection of labeled oligosaccharides; and (h) The activity of lysosomal enzymes in degrading natural substrates was determined by comparing (i) and (ii): (i) the amount of labeled oligosaccharides in cells exposed to lysosomal enzymes; and (ii) the amount of labeled oligosaccharides in cells not exposed to lysosomal enzymes. The reduction in the amount of labeled oligosaccharides in (h)(i) compared to (h)(ii) indicates the activity of lysosomal enzymes in degrading natural substrates.

40. The method as described in claim 39, characterized in that, The lysosomal enzyme is a lysosomal sulfatase selected from the group consisting of: arylsulfatase B (ARSB), iduronic acid-2-sulfatase (IDS), sulfonamide / heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S), and N-acetylglucosamine-6-sulfatase (GALNS).

41. The method as described in claim 40, characterized in that, The lysosomal enzyme is GALNS.

42. The method as described in claim 39, characterized in that, The lysosomal enzyme is α-L-idurosidase (IDU).

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