Targeted delivery of therapeutic enzymes

Abstract

The present invention relates to the field of biotechnology, more specifically to the delivery of therapeutic enzymes applicable to medicine. The present invention relates to compounds containing a therapeutic enzyme and a transport element coupled to each other directly or by a linker, the transport element being a Fab fragment of immunoglobulin IgG specific for an insulin receptor epitope, and to the use of the compounds for the manufacture of pharmaceutical compositions for treating diseases, as well as the use of the compounds for the treatment and prevention of diseases, in particular lysosomal storage diseases, in particular enzymatic deficiencies characteristic of the respective lysosomal storage diseases, such as mucopolysaccharidosis, in particular type I and type II mucopolysaccharidosis.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, more specifically to the delivery (transportation) of therapeutic enzymes applicable to pharmaceuticals. The present invention relates to a compound comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to the insulin receptor epitope, and to the use of the compound for producing pharmaceutical compositions for treating diseases, and to the use of the compound for the treatment and prevention of diseases, in particular lysosomal storage disorders, and especially for the treatment and prevention of enzyme deficiencies characteristic of each lysosomal storage disorder, such as mucopolysaccharidosis, in particular type I and type II mucopolysaccharidosis. [Background technology]

[0002] The use of enzymes in therapy has been known in the field for many years. Modern medicine widely uses therapeutic enzymes in various medical fields due to their high activity and specificity. Currently, the following directions of enzyme therapy are emerging (Kazanskaya NF et al., 1984): 1) Elimination of enzyme deficiencies to compensate for congenital or acquired dysfunction; 2) Removal of non-viable degenerated structures, cells, and tissue fragments; 3) Dissolution of blood clots; 4) Combination therapy for malignant neoplasms; 5) Detoxification of the body.

[0003] The use of therapeutic enzymes to eliminate enzyme deficiencies, either congenital or acquired, has been practiced for many years. Treating congenital enzyme deficiencies, of which over 150 have been described, is a significant challenge in substitution therapy. Genetic disorders, such as glycogen storage diseases, lipidosis, mucopolysaccharidosis, and other lysosomal storage disorders, are primarily treated by intravenous administration of recombinant analogs of the corresponding natural enzymes.

[0004] Lysosomal storage disorders are a group of rare (orphan) hereditary metabolic disorders caused by the absence or dysfunction of lysosomal enzymes involved in the degradation of complex molecules.

[0005] Currently (Novikov PV, 2014), the following lysosomal storage disorders have been identified: 1) Mucopolysaccharidosis; 2) Lipidosis (sphingolipidosis - GM1 and GM2 gangliosidosis, Gaucher disease, galactosialidosis, Faber granulomatosis, leukodystrophy, Niemann-Pick disease type A and B); 3) Mucolipidosis; 4) Glycoproteinosis (fucosidosis, sialidosis, mannosidosis, type II glycogen storage disorder - Pompe disease, Danon disease, etc.); 5) Neuronal ceroid lipofuscinosis; 6) Other storage disorders (Niemann-Pick disease type C, Wolmann disease, cholesterol storage disorder, cystinosis, primate disease, dysosomal dysostosis, etc.).

[0006] Mucopolysaccharidosis (MPS) is a group of nine metabolic disorders (I-IX, or 14 if intermediate forms are included) caused by more than 40 genetic disorders, leading to the absence or dysfunction of many lysosomal enzymes involved in the hydrolytic breakdown of glycosaminoglycans (mucopolysaccharides). Glycosaminoglycans are oligosaccharides involved in the formation of bone, cartilage, ligaments, cornea, skin, connective tissue, and synovial fluid (Burrow TA et al, 2013).

[0007] In patients with mucopolysaccharidosis, there is a deficiency or absence of at least one of the 11 enzymes involved in glycosaminoglycan catabolism, which leads to the gradual accumulation of these carbohydrates in cells and body fluids, including connective tissue, over a long period of time. As a result, the persistent accumulation of mucopolysaccharides leads to cell damage, tissue and organ dysfunction, which is most often evident in hypertension-hydrocephalus syndrome, hepatosplenomegaly, cardiovascular failure, osteoarthritis, and central nervous system (CNS) dysfunction, including severe cognitive impairment and dementia (Table 1) (Burrow TA et al, 2013).

[0008] Table 1. Characteristics of various MPS syndromes [Table 1] TIFF0007887076000002.tif143169

[0009] Type I mucopolysaccharidosis is caused by a deficiency in the lysosomal enzyme, α-L-iduronidase. This deficiency leads to the accumulation of mucopolysaccharides, particularly dermatan sulfate, in tissues and organs. Excessive accumulation of dermatan sulfate leads to the progressive development of many morphological abnormalities in tissues and organs. Type I mucopolysaccharidosis is characterized by an autosomal recessive pattern of inheritance.

[0010] To date, two effective methods for treating type I MPS have been developed: hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT).

[0011] HSCT treats only the severe form of type I MPS, Hurler syndrome. This treatment allows for the correction of α-L-idulonidase deficiency, subsequently leading to a significant improvement in the patient's condition, although some severe complications of this disease do not completely disappear. HSCT should be performed as early as possible, before the onset of severe neurological impairment. Despite the improvement in the patient's condition, HSCT is a complex, multi-stage, and costly procedure with a high risk of serious post-transplant complications.

[0012] ERT is safe, well-tolerated by patients, does not cause serious adverse events, and leads to the degradation of non-hydrolyzable substrates. The rare possible reaction to administration of this drug is the formation of antibodies against the injected protein, but these are not permanent and are usually rapidly alleviated by standard medication. The principle of ERT is based on hydrolyzing the accumulated substrate and restoring enzyme activity to a sufficient level to prevent further accumulation.

[0013] The drug Aldrazyme (INN: laronidase) (Genzyme, USA) is intended for use in patients with the three clinical variants of type I mucopolysaccharidosis (type IH, IH / S, and IS, or Hurler, Hurler-Schaye, and Schaye syndromes), as well as in children waiting for HSCT (while searching for related / unrelated donors) and during the 3–6 months post-HSCT period until the child's condition stabilizes. In addition, Aldrazyme is indicated for patients with Hurler syndrome after HSCT if levels of the donor enzyme α-L-iduronidase remain low.

[0014] Laronidase (Bayomarin Pharmaceutical Inc., USA) is a recombinant human α-L-iduronidase produced using recombinant DNA technology with Chinese hamster ovary cell cultures. After intravenous administration, laronidase rapidly leaves the systemic circulation and is absorbed by cells via the mannose-6-phosphate receptor (M6PR), entering lysosomes. This drug is administered intravenously once a week over a period of 3-4 hours (100 units / kg body weight).

[0015] Laronidase is a recombinant protein with a molecular weight of approximately 83 kDa and 628 amino acid residues. Its molecule contains six sections that bind via amino acids to oligosaccharides of different structures, including those containing mannose-6-phosphate (M6P) residues (Rodney JY Ho, Biotechnology and biopharmaceuticals: transforming proteins and genes into drugs. - Wiley-Blackwell, 2013, p. 380). The laronidase molecule does not penetrate the blood-brain barrier (a physiological barrier between peripheral circulation and the brain formed by tight junctions in the plasma membrane of endothelial cells of brain capillaries, creating a tight barrier that restricts the transfer of even very small molecules into the brain; hereafter also referred to as the BBB), and therefore does not delay or prevent the development of central nervous system (CNS) damage characteristic of type I mucopolysaccharidosis (Pastores GM et al, 2007).

[0016] Mucopolysaccharidosis type II (MPS-II, Hunter syndrome) is the only type of mucopolysaccharidosis that has an X-linked recessive mode of inheritance, unlike all other types of mucopolysaccharidosis that have an autosomal recessive-like mode of inheritance. MPS II is heterogeneous according to clinical signs of a progressive lysosomal storage disorder resulting from deficiency of the enzyme iduronate-2-sulfatase (IDS). This enzyme is normally involved in the breakdown of mucopolysaccharides, dermatan sulfate, and heparan sulfate by hydrolytic removal of O-linked sulfate groups. Thus, in the case of MPS II, the main pathogenic mechanism is associated with the progressive accumulation of dermatan sulfate and heparan sulfate in lysosomes.

[0017] The most common clinical symptoms of MPS-II are characteristic of mental retardation, macroglossia, cranioskeletosis, alopecia, dental abnormalities, restrictive lung disease, hepatosplenomegaly, cardiac valve pathology, osteoarticular pathology, and severe short stature. Progressive neurological disorders are also frequently observed, which are accompanied by hydrocephalus and increased intracranial pressure. Usually, from the teenage years to the twenties, death most frequently occurs due to respiratory and / or heart failure. It is important to emphasize that this disease occurs with involvement of the nervous system in the pathological process, including cognitive decline.

[0018] Today, there are two main ways to treat MPS II, namely ERT and symptomatic therapy (including the use of hepatoprotective agents, cardiovascular and anti-inflammatory agents, vitamins, and agents to improve antioxidant protection). HSCT for the treatment of MPS II is not effective, unlike for mucopolysaccharidosis type I.

[0019] Elaprase (INN: idursulfase) (Shayer, USA) is a pharmaceutical product corresponding to recombinant human iduronate-2-sulfatase. This enzyme is involved in the hydrolysis of the C2-sulfate ester bond of iduronate residues, which are part of the glycosaminoglycans (mucopolysaccharides) dermatan sulfate and heparan sulfate (Burrow T. A. et al., 2013). The usual dosing regimen of Elaprase is 0.5 mg / kg body weight once a week, and the administration is carried out by a 3-hour intravenous infusion (the infusion time may be gradually reduced to 1 hour).

[0020] Idursulfatase contains two disulfide bonds and eight N-linked glycosylation sites occupied by complex high-mannose oligosaccharides. The presence of M6P residues in the oligosaccharide chain enables the recombinant enzyme to bind to the M6P receptor on the target cell surface, which leads to the internalization of this enzyme into the cell and into the lysosome, where the degradation of lysosomal mucopolysaccharides is ensured (Muenzer J., et al., Genet Med 2006 Aug;8(8):465-473).

[0021] When ERT is performed, the average life expectancy of patients with Hunter syndrome increases several-fold. Nevertheless, Elaprase also does not penetrate the blood-brain barrier, and as a result, when ERT is performed with Elaprase, patients die at the age of 20 as a result of neurodegeneration. Typically, patients administered Elaprase experience significant learning difficulties in their teens and even require helpers in their daily lives (da Silva E. M. et al., 2016; Wraith J.E. et al., 2008).

[0022] Therefore, a common disadvantage of all existing drugs used for ERT of type I and type II mucopolysaccharidoses is their inability to penetrate from the blood-brain barrier into the central nervous system, which limits the effectiveness of the use of these drugs in patients with nervous system damage associated with mucopolysaccharidoses, including type I and type II mucopolysaccharidoses.

[0023] For many lysosomal storage disorders, it is necessary to improve the internalization of enzymes into cells in specific peripheral tissues (e.g., the diaphragmatic muscle in Pompe disease, the liver and spleen in Hunter's disease, and the kidney in Fabry disease) (Hawkes C. et al., 2004). [Overview of the Initiative]

[0024] Therefore, there is an urgent need to develop therapeutic compounds that can be used to treat lysosomal storage disorders such as type I or type II MPS, exhibit high activity, and have improved transportability to lysosomes in tissue cells of various organs, including nerve tissue cells, while retaining the functional properties of the corresponding therapeutic enzymes, thereby enabling significant improvements in the quality of life and life expectancy of patients with type I and type II MPS.

[0025] The above objective has been successfully achieved in the present invention, which relates to a compound intended for the treatment of lysosomal storage disorders, comprising a therapeutic enzyme and a transport element coupled directly or by a linker, wherein the specified transport element is a Fab fragment of IgG immunoglobulin specific to the insulin receptor epitope. The present invention is based on the discovery that the transport element, a Fab fragment of IgG immunoglobulin specific to the insulin receptor epitope, coupled directly or by a linker to the therapeutic enzyme, unexpectedly leads to an improved ability of the enzyme to be transported to lysosomes in a variety of organs and tissues, including lysosomes in nerve tissue cells, while simultaneously maintaining a high level of enzymatic activity of the compound. Thanks to this unexpected effect, advances have been made in the field of enzyme replacement therapy for lysosomal storage disorders such as type I and type II mucopolysaccharidosis, resulting in increased duration and improved quality of life for subjects suffering from lysosomal storage disorders and requiring therapy. The compounds of the present invention also provide an expansion of the arsenal of drugs used for the treatment and prevention of diseases, particularly lysosomal storage disorders, such as mucopolysaccharidosis, and especially type I and type II mucopolysaccharidosis.

[0026] Furthermore, compared to therapeutic enzymes without the transport element, compounds containing a transport element—a Fab fragment of IgG immunoglobulin specific to the insulin receptor epitope, directly or via a linker—show an unexpected increase in enzymatic activity; and the introduction of compounds containing a transport element—a Fab fragment of IgG immunoglobulin specific to the insulin receptor epitope, directly or via a linker—to the therapeutic enzyme provides a higher degree of activity substitution of the therapeutic enzyme in the human brain compared to compounds containing the Mab fragment.

[0027] This summary of the present invention provides a brief description of the invention to briefly outline its subject matter and essence. This summary is provided with full awareness that it should not be used to interpret or limit the scope or content of the claims.

[0028] The first subject of the present invention is a compound intended for the treatment of lysosomal storage disorders, comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to the insulin receptor epitope.

[0029] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to an insulin receptor epitope and capable of transporting this enzyme across the blood-brain barrier.

[0030] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to an insulin receptor epitope, capable of transporting the enzyme across the blood-brain barrier, and the therapeutic enzyme and the transport element are coupled to each other by a linker. The linker may be a peptide linker containing one or more amino acids. The linker may be a peptide linker containing one or more amino acids selected from glycine, serine, and leucine.

[0031] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG and the immunoglobulin IgG is IgG1, IgG2, or IgG4.

[0032] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG and the immunoglobulin IgG is IgG1.

[0033] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG1, comprising the light chain (SEQ ID NOs: 2, 8, 9, 10, and 11) and heavy chain (SEQ ID NOs: 3) of immunoglobulin IgG1 toward the insulin receptor. In certain embodiments, the subject of the present invention is a compound in which the transport element is a Fab fragment of immunoglobulin IgG1, comprising the light chain, SEQ ID NOs: 2, and heavy chain, SEQ ID NOs: 3, of immunoglobulin IgG1 toward the insulin receptor.

[0034] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the therapeutic enzyme is used in the treatment of the lysosomal storage disorder, including the treatment of the enzyme deficiency characteristic of the lysosomal storage disorder.

[0035] In certain embodiments, the present invention provides compounds for the treatment of lysosomal storage disorders, comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the therapeutic enzyme is used to treat a lysosomal storage disorder comprising a neurological component, the therapeutic enzyme being for the treatment of an enzyme deficiency characteristic of the lysosomal storage disorder.

[0036] In certain embodiments, the present invention provides compounds for the treatment of lysosomal storage disorders, comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the therapeutic enzyme for lysosomal storage disorders, comprising a neurological component, is selected from the group consisting of iduronate-2-sulfatase and α-L-iduronidase.

[0037] In certain embodiments, the present invention provides compounds for the treatment of lysosomal storage disorders, comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the therapeutic enzyme for lysosomal storage disorders comprising a neurological component is selected from the group consisting of iduronate-2-sulfatase, an iduronate-2-sulfatase fragment having iduronate-2-sulfatase activity, or an iduronate-2-sulfatase analog.

[0038] In certain embodiments, the present invention provides compounds for the treatment of lysosomal storage disorders, comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the therapeutic enzyme for lysosomal storage disorders, comprising a neurological component, is selected from the group consisting of α-L-idulonidase, an α-L-idulonidase fragment having α-L-idulonidase activity, or an α-L-idulonidase analog.

[0039] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the linker is a peptide linker containing one or more amino acids.

[0040] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the linker is a peptide linker containing one or more amino acids selected from glycine, serine, and leucine.

[0041] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is capable of transporting the enzyme to lysosomes in tissue cells.

[0042] In certain embodiments, the present invention provides a compound for the treatment of lysosomal storage disorders comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is capable of transporting the enzyme to lysosomes in nerve tissue cells.

[0043] Another subject of the present invention is a compound for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in subjects having type II mucopolysaccharidosis, represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11 and a second amino acid sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15. In a particular embodiment, the present invention provides a compound for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in subjects having type II mucopolysaccharidosis, represented by the amino acid sequences of SEQ ID NOs: 2 and SEQ ID NOs: 4.

[0044] Another subject of the present invention is a compound for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in subjects having type II mucopolysaccharidosis, which includes neurological components represented by the amino acid sequences of SEQ ID NOs: 2 and SEQ ID NOs: 4.

[0045] Another subject of the present invention is a compound for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in subjects having type II mucopolysaccharidosis, which includes neurological components represented by the amino acid sequences of SEQ ID NOs.2 and SEQ ID NOs.5.

[0046] Another subject of the present invention is a compound for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in subjects having type I mucopolysaccharidosis, as represented by the amino acid sequences of SEQ ID NOs: 2 and SEQ ID NOs: 6.

[0047] Another subject of the present invention is a compound represented by the amino acid sequences of SEQ ID NOs: 2 and SEQ ID NOs: 6 for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in subjects having type I mucopolysaccharidosis.

[0048] Another subject of the present invention is a compound represented by the amino acid sequences of SEQ ID NOs: 2 and SEQ ID NOs: 6 for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in subjects having type I mucopolysaccharidosis including neurological components.

[0049] Another subject of the present invention is a compound represented by the amino acid sequences of SEQ ID NOs: 2 and SEQ ID NOs: 7 for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in subjects having type I mucopolysaccharidosis including neurological components.

[0050] Furthermore, the present invention relates to the use of the compound, comprising therapeutic enzymes and transport elements coupled to each other, either directly or by a linker, for preparing a pharmaceutical composition containing an effective amount of the compound and a pharmaceutically acceptable carrier.

[0051] Furthermore, the present invention relates to the use of a compound containing a therapeutic enzyme and a transport element, either directly or coupled to each other by a linker, for the treatment or prevention of a subject having a lysosomal storage disorder, comprising administering an effective amount of the compound to the subject.

[0052] In certain embodiments, the present invention relates to the use of compounds comprising therapeutic enzymes and transport elements, either directly or coupled to each other by a linker, for the treatment or prevention of enzyme deficiency in subjects having lysosomal storage disorders, wherein the lysosomal storage disorder is a mucopolysaccharidosis, and the compounds are administered to the subject in an effective amount, preferably in combination with a pharmaceutically acceptable carrier.

[0053] In certain embodiments, the present invention relates to the use of compounds comprising a therapeutic enzyme and a transport element, either directly or coupled to each other by a linker, for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in subjects having lysosomal storage disorders, wherein the lysosomal storage disorder is a type II mucopolysaccharidosis involving neurological components, and the compounds are administered to the subject in an effective amount, preferably in combination with a pharmaceutically acceptable carrier.

[0054] In certain embodiments, the present invention relates to the use of compounds comprising a therapeutic enzyme and a transport element, either directly or coupled to each other by a linker, for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in subjects having lysosomal storage disorders, wherein the lysosomal storage disorder is a type I mucopolysaccharidosis comprising neurological components, and the compounds are administered to the subject in an effective amount, preferably in combination with a pharmaceutically acceptable carrier.

[0055] Furthermore, the present invention relates to the use of a compound containing a therapeutic enzyme and a transport element, either directly or coupled to each other by a linker, for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in subjects having a lysosomal storage disorder, wherein the lysosomal storage disorder is type II mucopolysaccharidosis, the compound is indicated by amino acid sequence, SEQ ID NO: 2 and SEQ ID NO: 4, and the use is for the preparation of a pharmaceutical composition containing an effective amount of the compound and a pharmaceutically acceptable carrier.

[0056] Furthermore, the present invention relates to the use of a compound containing a therapeutic enzyme and a transport element, either directly or linked to each other, for the treatment or prevention of enzyme deficiency (iduronate-2-sulfatase deficiency) in a subject having a lysosomal storage disorder, wherein the lysosomal storage disorder is type II mucopolysaccharidosis, the compound is indicated by its amino acid sequence, SEQ ID NO: 2, and SEQ ID NO: 4, and the use is for the treatment or prevention of enzyme deficiency in the subject.

[0057] In other embodiments, the approach of the present invention may be used to treat other lysosomal storage disorders, particularly other types of mucopolysaccharidosis, such as type III mucopolysaccharidosis (Sanfilippo syndrome), including types IIIA, IIIB, IIIC, and IIID mucopolysaccharidosis; type IV mucopolysaccharidosis (Morcchio syndrome), including types IVA and IVB mucopolysaccharidosis; type VI mucopolysaccharidosis (Maroto-Lamy syndrome); type VII mucopolysaccharidosis (Sly syndrome); and type IX mucopolysaccharidosis. In these cases, instead of α-L-iduronidase or iduronic acid-2-sulfatase, other therapeutic enzymes are used as appropriate therapeutic enzymes for patients with the corresponding type of mucopolysaccharidosis who experience deficiency, such as: heparan sulfamidase for type IIIA mucopolysaccharidosis, N-acetylglucosaminidase for type IIIB mucopolysaccharidosis, heparan-α-glucosaminide-N-acetyltransferase for type IIIC mucopolysaccharidosis, N-acetylglucosamine-6-sulfatase for type IIID mucopolysaccharidosis, galactose-6-sulfate sulfatase for type IVA mucopolysaccharidosis, β-galactosidase for type IVB mucopolysaccharidosis, N-acetylgalactosamine-4-sulfatase for type VI mucopolysaccharidosis, β-glucuronidase for type VII mucopolysaccharidosis, and hyaluronidase for type IX mucopolysaccharidosis. The amino acid sequences of these therapeutic enzymes can be obtained using any of the many computer programs known in the art, such as BLAST or FASTA. Both BLAST and FASTA have offline and online search capabilities (see Ausubel et al., 1999 ibid, pp. 7-58 - 7-60, and the National Center for Biotechnology Information website on the National Institutes of Health website).

[0058] The approach proposed in this invention may also be used in the treatment of lysosomal storage disorders not associated with mucopolysaccharidosis by using, in another embodiment of the invention, another lysosomal enzyme that is deficient in patients with the corresponding lysosomal storage disorder, instead of α-L-iduronidase or iduronate-2-sulfatase. In certain non-limited embodiments of the invention, the proposed approach may be used, in particular, for the treatment of amino acid metabolic disorders, e.g., cystinosis; carbohydrate metabolic disorders, e.g., glycogen storage disorders, especially Pompe disease; sphingolipid metabolic disorders and other lipid storage disorders, especially GM2 gangliosidosis including Sandhoff disease and Tay-Sachs disease; other gangliosidosis, especially GM1 gangliosidosis and mucolipidosis IV; other sphingolipidosis, especially Fabry disease, Gaucher disease, Krabbe disease, Niemann-Pick disease, Faber syndrome, and metachromatic white matter. It may be used for the treatment of dystrophy and polysulfatase deficiency; lipofuscinosis in the nerves, especially Batten, Bierszowski-Jansky, Cuffs, Spielmeyer-Voigt disease; other lipid storage disorders, especially Wolmann disease; and for the treatment of glycoprotein metabolic disorders, including type II mucolipidosis (I-cell disease) and type III mucolipidosis (Haller pseudolipodystrophy); and for the treatment of glycoprotein degradation disorders, including aspatylglucosamineuria, fucosidosis, mannosidosis, and sialidosis.

[0059] In these cases, instead of α-L-iduronidase or iduronate-2-sulfatase, the amino acid sequence of the corresponding enzyme, which is deficient in patients with the corresponding lysosomal storage disorder, is coupled to the transport element, either directly or via a linker.

[0060] The most preferred embodiments of the present invention are described below. However, these preferred embodiments are provided solely for illustrative purposes of the present invention and not to limit the scope of the claims.

[0061] The present invention is illustrated by the following figures. [Brief explanation of the drawing]

[0062] [Figure 1] This shows a representative autoradiogram of a male cynomolgus monkey recorded 2 hours after a single intravenous administration of [125I]-HIR-Fab-IDS at a nominal dose level of 0.0020 mg / kg body weight. [Figure 2] This shows a representative autoradiogram of male cynomolgus monkeys recorded 2 hours after a single intravenous administration of [125I]-HIR-Mab-IDS at a nominal dose level of 0.0015 mg / kg body weight. [Figure 3] This shows a representative autoradiogram of male cynomolgus monkeys recorded 2 hours after a single intravenous administration of [125I]-IDS (control) at a nominal dose level of 0.0010 mg / kg body weight. [Modes for carrying out the invention]

[0063] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by a person skilled in the art (e.g., molecular genetics, nucleic acid chemistry, protein chemistry, biochemistry, organic chemistry, immunology, microbiology, genetics, etc.).

[0064] As used herein, the term “compound” is understood in its broadest sense to mean at least one molecule, such as a conjugate, fusion protein, protein construct, or protein complex.

[0065] As used herein, the term "contains" refers to a compound that contains the listed elements without excluding others.

[0066] In this specification, the terms "therapeutic enzyme" and "enzyme" are synonymous and refer to an enzyme used to treat diseases resulting from a deficiency, defect, or dysfunction of that enzyme, and which can be treated by an ERT, enzyme administration, or the like in a subject suffering from the disease. In particular, the enzyme may be, but is not limited to, an enzyme used to treat diseases that may result from a deficiency, insufficiency, or dysfunction of lysosomal enzymes.

[0067] The therapeutic enzymes contained in the compounds of this disclosure include, without limitation, β-glucosidase, β-galactosidase, galactose-6-sulfatase, acid ceramidase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, β-hexosaminidase A, β-hexosaminidase B, heparin-N-sulfatase, α-D-mannosidase, β-glucuronidase, N-acetylgalactosamine-6-sulfatase, lysosomal acid lipase, α-N-acetyl-D-glucosaminidase (NAGLU), glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, and acetyl-CoA-glucosaminide-N-acetyltran This includes any enzymes that have therapeutic effects against lysosomal storage disorders, including spherase, N-acetylglucosamine-6-sulfatase, galactosamine-6-sulfatase (GALN), hyaluronidase, α-fucosidase, β-mannosidase, α-neuraminidase (sialidase), N-acetylglucosamine-1-phosphotransferase, mucolipin-1, α-N-acetyl-galactosaminidase, N-aspartyl-β-glucosaminidase, LAMP-2 (lysosome-associated membrane protein 2), cystinosine, sialin, ceramidase, acid P-glucosidase, galactosylceramidase, NPC1 (Niemann-Pick disease protein type C1), cathepsin A, SUMF-1 (sulfatase modifier-1), lysosomal acid lipase (LIPA), and tripeptidyl peptidase 1.

[0068] In particular, therapeutic enzymes include enzymes such as agalsidase, imiglucerase, galsulfase, iduronate-2-sulfatase, and α-L-iduronidase. Most preferably, the therapeutic enzyme is iduronate-2-sulfatase or α-L-iduronidase.

[0069] However, this specification may also include any therapeutic enzyme, without limitation, regardless of the type or origin of the enzyme.

[0070] In this disclosure, the terms “iduronate-2-sulfatase,” as well as “idulsulfatase,” “IDS,” and “I2S,” typically refer to recombinant analogs of iduronate-2-sulfatase, a lysosomal enzyme that hydrolyzes the O-linked sulfate groups of mucopolysaccharides, dermatan sulfate, and heparan sulfate. In the present invention, the term “iduronate-2-sulfatase” may be used interchangeably with the term “idulsulfatase.”

[0071] In the context of this disclosure, the terms “HIR-Fab-IDS,” as well as “rIDS-FAB-HI,” “rHI-FAB-IDS,” “rIDS-FAB-HIR,” and “rHIR-FAB-IDS,” are synonymous and refer to hybrid recombinant proteins of iduronate-2-sulfatase covalently linked to a Fab fragment of a monoclonal antibody against the human insulin receptor. As used herein, “HIR-Fab-IDS” means iduronate-2-sulfatase accompanied by a monoclonal antibody fragment against the human insulin receptor, but is not limited to these. In certain (non-limited) embodiments of the present invention, an HIR-Fab-IDS variant is represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15. In certain embodiments of the present invention, HIR-Fab-IDS is represented by the first amino acid sequence of SEQ ID NOs: 2 and the second amino acid sequence of SEQ ID NOs: 4.

[0072] In the context of this disclosure, “HIR-Mab-IDS” is a recombinant iduronate-2-sulfatase fusion protein covalently linked to a full-length monoclonal antibody against the human insulin receptor. In certain (but not limited) embodiments of the present invention, a variant of HIR-Mab-IDS is the variant described in U.S. Patent Document US8834874 B2, 16.09.2014. In certain embodiments of the present invention, HIR-Mab-IDS is the amino acid sequence of a product of ArmaGen Technologies Inc. described in Project AGT-182.

[0073] In this disclosure, the terms “α-L-idulonidase,” “idulonidase,” “laronidase,” and “IDUA” refer to enzymes involved in the hydrolysis of glycosaminoglycans such as dermatan sulfate and heparan sulfate. As used herein, the term “α-L-idulonidase” may be used interchangeably with the term “laronidase.” Izuronidase deficiency (genetically determined deficiency) occurring in type I mucopolysaccharidosis leads to the gradual accumulation of glycosaminoglycans, heparan sulfate, and dermatan sulfate in the body's cells and tissues.

[0074] "HIR-Fab-IDUA," as well as "rIDUA-FAB-HI," "rHI-FAB-IDUA," "rIDUA-FAB-HIR," and "rHIR-FAB-IDS," are fusion recombinant protein α-L-iduronidases covalently linked to a Fab fragment of a monoclonal antibody against the human insulin receptor. In certain (but not limited) embodiments of the present invention, the HIR-Fab-IDUA variant is represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid sequence selected from SEQ ID NOs: 6, 7.

[0075] HIR-Mab-IDUA is a fusion recombinant α-L-iduronidase protein covalently linked to a full-length monoclonal antibody against the human insulin receptor.

[0076] The therapeutic enzymes that may be included in the compounds of the present invention may be in the form of a fragment consisting of a part of an enzyme, or, without limitation, an enzyme analog in which a modification has occurred selected from the group consisting of substitutions, additions, deletions, modifications and combinations of specific amino acids, as long as they have the same enzyme activity as the natural form of the corresponding therapeutic enzyme. In certain (not limited) embodiments of the present invention, an enzyme fragment having the activity of the natural form of the corresponding enzyme may be used. An enzyme analogue is, without limitation, an enzyme having different glycosylation characteristics and degrees of glycosylation due to the expression of a known enzyme in a different host, and an enzyme having various degrees of substitution of specific amino acid residues of the corresponding enzyme relative to the standard sequence, but not 100% substitution. In private (but not limited to) embodiments of the present invention, analogues of α-L-iduronidase, as known from patents US5932211 A, 08 / 03 / 1999, US6153188 A, 11 / 28 / 2006, and US6541254 B1, 04 / 01 / 2003, as well as analogues of iduronic acid-2-sulfatase, may be used. A person of the average skill in the art will understand that other fragments and analogues of the enzymes α-L-iduronidase and iduronic acid-2-sulfatase, having the innate activity of the corresponding enzymes, which are currently known or may become known in the art, may be used.

[0077] Enzymes can be produced by conventional methods of the art, for example, in animal cells, Escherichia coli (E. coli), yeast, insects, plants, and living animals, by genetic recombination using a variety of expression vectors well known to those skilled in the art. The production methods are not limited to these and include other methods for producing enzymes known to those skilled in the art. For an unspecified number of such methods for producing enzymes in the cells of various organisms, and an unspecified number of expression vectors, see Sambrook et al., “Molecular Cloning: A Laboratory Manual” - CSH Press, Cold Spring Harbor, 1989, “Current Protocols in Molecular Biology,” John Wiley & Sons, New York, 2001. In a preferred embodiment of the present invention, the enzyme is produced in mammalian cells. In the most preferred embodiment of the present invention, the enzyme is produced in Chinese hamster ovary cells.

[0078] In certain preferred embodiments of the present invention, the enzyme may be a commercially available enzyme. Furthermore, the enzyme may contain an amino acid sequence having at least 80%, more specifically 90%, and more specifically 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the enzyme or its analogue, and the enzyme may be obtained from a microorganism using recombinant technology, or may be purchased from a commercially available source without limitation.

[0079] In this disclosure, the term “homology” means the degree of similarity to the amino acid sequence or amino acid-coding nucleotide sequence of a wild-type protein, and covers sequences having the above degree of sequence similarity, expressed as a percentage, to the amino acid sequence or nucleotide sequence of the present invention. Homology may be determined by comparing two given sequences with the naked eye, or by using a bioinformatics algorithm that enables homology analysis by aligning the sequences in question for comparison. The homology between two given amino acid sequences may be expressed as a percentage. Effective automated algorithms are available in the GAP, BESTFIT, FASTA, and TFASTA software modules of the Wisconsin Genetics software package (Genetics Computer Group, Madison, WI, USA). Alignment algorithms automated in these modules include the sequence alignment algorithms of Needleman and Wunsch, Pearson and Lipman, and Smith and Waterman. Other algorithms that may be used for sequence alignment and homology determination are automated in the programs FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W. The amino acid and nucleotide sequences encoding enzymes and their analogues can be obtained from well-known databases such as GenBank NCBI, though not limited to these.

[0080] In some embodiments, the compound transport element comprises an IgG1 immunoglobulin Fab fragment consisting of a first amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO: 2.

[0081] In certain embodiments, the compound transport element comprises an IgG1 immunoglobulin Fab fragment consisting of a first amino acid sequence that is at least 80% identical to SEQ ID NO: 2.

[0082] In some embodiments, compounds represented by a first amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO: 2 and a second amino acid sequence that is at least identical to SEQ ID NO: 4 are used in subjects with type II mucopolysaccharidosis for the treatment or prevention of lysosomal enzyme deficiency.

[0083] In certain embodiments, a compound having a first amino acid sequence that is at least 80% identical to SEQ ID NO: 2 and a second amino acid sequence that is at least identical to SEQ ID NO: 4 is used in subjects with type II mucopolysaccharidosis for the treatment or prevention of lysosomal enzyme deficiency.

[0084] The term "amino acid" refers to any group of carboxy-α-amino acids that are either naturally occurring, i.e., those that can be encoded by nucleic acids directly or in precursor form, or those that are not naturally occurring. Each naturally occurring amino acid is encoded by a nucleic acid consisting of three nucleotides, called a codon or base triple. Each amino acid is encoded by at least one codon.

[0085] As used herein, the term "amino acid" refers to naturally occurring carboxy-α-amino acids, including: alanine (3-letter code: Ala, 1-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Examples of amino acids that do not exist in nature (non-proteinogenic amino acids) include Aad (alpha-aminoadipic acid), Abu (aminobutyric acid), Ach (alpha-aminocyclohexanecarboxylic acid), Acp (alpha-aminocyclopentanecarboxylic acid), Acpc (1-aminocyclopropane-1-carboxylic acid), Aib (alpha-aminoisobutyric acid), and Aic (2-aminoindan-2-carboxylic acid).Also known as 2-2-Aic, 1-1-Aic (1-aminoindan-1-carboxylic acid), (2-aminoindan-2-carboxylic acid), allylglycine (allylGly), alloisoleucine (allo-Ile), Asu (alpha-aminosuberic acid, 2-aminooctanedioic acid), Bip (4-phenyl-phenylalanine-carboxylic acid), BnHP ((2S,4R)-4-hydroxyproline), Cha (beta-cyclohexyl (Lualanine), Cit (Citrulline), Cyclohexylglycine (Chg), Cyclopentylalanine, Beta-Cyclopropylalanine, Dab (1,4-Diaminobutyric Acid), Dap (1,3-Diaminopropionic Acid, p-(3,3-Diphenylalanine-carboxylic Acid), 3,3-Diphenylalanine, Di-n-Propylglycine (Dpg), 2-Furylalanine, Homocyclohexylalanine (HoCha), Homocitrulline ( HoCit), homocycloleucine, homoleucine (HoLeu), homoarginine (HoArg), homoserine (HoSer), hydroxyproline, Lys (Ac), (1)Nal(1-naphthylalanine), (2)Nal(2-naphthylalanine), 4-MeO-Ars(1-amino-4-(4-methoxyphenyl)-cyclohexane-1-carboxylic acid), norleucine (Nle), Nva(norvaline), omatin (om This includes, but is not limited to, atine, 3-Pal(alpha-amino-3-pyridylalanine-carboxylic acid), 4-Pal(alpha-amino-4-pyridylalanine-carboxylic acid), 3,4,5,F3-Phe(3,4,5-trifluorophenylalanine), 2,3,4,5,6,F5-Phe(2,3,4,5,6-pentafluorophenylalanine), Pqa(4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline acetate (CAS 889958-08-1)), pyridylalanine, quinolylalanine, sarcosine (Sar), thiazolylalanine, thienylalanine, Tic(alpha-amino-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), Tic(OH), Tle(tert-butylglycine), and Tyr(Me).

[0086] The term "amino acid sequence" variantA variant refers to a polypeptide having an amino acid sequence that differs to some extent from the polypeptide having the natural sequence of the corresponding therapeutic enzyme. Typically, an amino acid sequence variant will have at least about 70% sequence identity with the polypeptide having the natural sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 80% or more sequence identity with the polypeptide having the natural sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 90% or more sequence identity with the polypeptide having the natural sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 95% or more sequence identity with the polypeptide having the natural sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 98% or more sequence identity with the polypeptide having the natural sequence of the corresponding therapeutic enzyme. The amino acid sequence variant has substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the natural amino acid sequence of the corresponding therapeutic enzyme. Amino acids are represented by their traditional names, one-letter, and three-letter codes.

[0087] In the present invention, the term "first amino acid sequence" refers to the entire amino acid sequence of IgG immunoglobulin, and more particularly to the light chain of IgG immunoglobulin. In particular, in an embodiment not limited to this invention, the first amino acid sequence is an IgG1 immunoglobulin amino acid sequence, an IgG1 immunoglobulin light chain amino acid sequence, or a fragment of an IgG1 immunoglobulin light chain amino acid sequence, selected from SEQ ID NOs: 2, 8, 9, 10, or 11. In a specific embodiment, the first amino acid sequence is the amino acid sequence of the immunoglobulin IgG light chain, or SEQ ID NO: 2.

[0088] The term "second amino acid sequence," as used in this invention, refers to the entire amino acid sequence of IgG immunoglobulin; in particular, to the IgG immunoglobulin heavy chain, the IgG immunoglobulin heavy chain fragment, or the IgG immunoglobulin heavy chain fragment coupled directly or by a linker to an enzymatic sequence. In certain (but not limited) embodiments, the second amino acid sequence is the immunoglobulin IgG1 amino acid sequence, the heavy chain fragment amino acid sequence, SEQ ID NO: 3, and the IgG1 immunoglobulin heavy chain fragment coupled to an iduronic acid-2-sulfatase sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15.

[0089] The term “transport element,” as used in this invention, refers to a substance capable of carrying, transporting, or delivering therapeutic enzymes to lysosomes in cells of various tissues. In particular, but not limited to, the transport element will specifically interact with an epitope, antigen, receptor, or target in a manner that ensures effective delivery to lysosomes in nerve tissue cells. Such epitopes, antigens, receptors, or targets, in the context of this disclosure, may include human insulin receptors or parts thereof, through which drugs, peptides, or proteins are delivered, including those that cross the blood-brain barrier (BBB).

[0090] An "immunoglobulin" is a tetrameric molecule, and as used herein, it is conceptually a "full-length antibody." In naturally occurring immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminal region of each chain contains a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The carboxyl region of each chain defines a constant region primarily involved in effector function.

[0091] In the context of this disclosure, the term “antibody” is used in its broadest sense and is not limited to, but includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they possess the required biological activity.

[0092] As used herein, “antibody fragment” contains an intact portion of an antibody that retains its ability to bind to an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; dimeric antibodies (diabodies); linear antibodies; and single-chain antibody molecules, such as multispecific antibodies formed from single-chain Fab, scFv, and antibody fragments. Cleavage of an antibody with papain yields two identical antigen-binding fragments, each having one antigen-binding site, referred to as “Fab fragments,” and the remaining “Fc fragment,” named to reflect its ability to readily crystallize.

[0093] "Fab fragment," "Fab," and "FAB" are monovalent fragments containing heavy chain and light chain variable domains, and also containing a light chain constant domain and a first heavy chain constant domain. As used herein, the transport element comprises the amino acid sequence of the Fab fragment of IgG immunoglobulin, where IgG immunoglobulin is IgG1, IgG2, or IgG4. In certain (but not limited) embodiments of the present invention, IgG immunoglobulin is IgG1. In certain embodiments, the transport element comprises the amino acid sequence of the IgG1 immunoglobulin Fab fragment, comprising a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid of SEQ ID NO: 3. In more specific embodiments, the transport element comprises the amino acid sequence of the IgG1 immunoglobulin Fab fragment comprising SEQ ID NOs: 2 and SEQ ID NOs: 3.

[0094] As used herein, the term “linker” refers to a chemical linker or single-chain peptide linker that couples the therapeutic enzyme and transport element of the compounds of the present invention. The linker couples, for example, a monovalent linker containing the CH2-CH3 domain of Ig and sFab, whose target is the insulin receptor; that is, the linker couples sFab to the C-terminus of the CH3-CH2 domain of Ig.

[0095] In some embodiments, the linker is a chemical linker. A single-chain peptide linker containing 1 to 20 amino acids linked by peptide bonds may be used. In certain embodiments, the amino acids are selected from 20 naturally occurring (proteinogenic) amino acids. In other specific embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In certain (but not limited) embodiments of the present invention, one or more amino acids are selected from glycine, serine, and leucine.

[0096] In certain embodiments of the present invention, the linker is a single-chain peptide whose amino acid sequence consists of at least one amino acid, preferably one to two amino acids. In certain (but not limited) embodiments of the present invention, the amino acid sequence of the linker may consist of more than one to two amino acids, for example, three or fifteen amino acids.

[0097] The coupling of therapeutic enzymes and transport elements may be achieved directly or using a variety of chemical linkers known in the art. In certain preferred (but not limited) embodiments of the present invention, coupling of therapeutic enzymes and transport elements may be achieved using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imide esters (e.g., dimethyl adipate·HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Those skilled in the art will also understand that other chemical and peptide linkers known in the art, not explicitly mentioned herein, may be used for the purposes of the present invention.

[0098] In certain preferred embodiments of the present invention, the linker may be a “cleavable linker” that facilitates the release of therapeutic enzymes after the compound has been transported into cells and tissues. For example, an acid-unstable linker, a peptidase-sensitive linker, a photo-unstable linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari R. et al., Cancer Res. 52, 1992, pp. 127-131; US ​​5,208,020, Chari Ravi J. et al., 05 / 04 / 1993). Those skilled in the art will also recognize that other cleavable linkers known in the art that facilitate the release of therapeutic enzymes once the compound has entered the cells and tissues of the central nervous system may be used for the purposes of the present invention.

[0099] The term "epitope" refers to a region of an antigen that binds to an antigen-binding protein, including antibodies. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, consisting of nonlinear amino acids; in other words, conformational epitopes consist of discontinuous amino acids. Epitopes may contain determinants that are the chemically active surface portion of the molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and epitopes may have specific three-dimensional structural properties and / or specific charge properties. As used herein, the epitope is the insulin receptor epitope represented by Sequence ID No. 1, as described in Zhang B, Roth RA. (1991) Proc Natl Acad Sci US A.; 88(21):9858-9862 and SA Prigent, KK Stanley, and K Siddle. (1990) J. Biol. 1991.

[0100] The insulin receptor (HIR) is a transmembrane glycoprotein (molecular weight approximately 320,000 Da) in the human body, consisting of two α-subunits and two β-subunits linked by disulfide crosslinks (the α-subunits are located extracellularly and contain an insulin-binding domain). It is involved in the regulation of glucose absorption and distribution, as well as the synthesis and accumulation of fats, proteins, and carbohydrates. The insulin receptor and its extracellular insulin-binding domain (ECD) are widely known in the art both structurally and functionally. The localization of the insulin receptor is most often to the cell surface of insulin-sensitive tissues, such as connective tissue cells, skeletal muscle, adipose tissue cells, and liver cells. See, for example, Yip et al. (2003), J Biol. Chem. 278 (30): 27329-27332; and Whittaker et al. (2005), J Biol Chem, 280 (22): 20932-20936. In one embodiment, the HIR described herein is a human insulin receptor comprising the amino acid sequence represented in Kasuya et al. (Biochemistry 32 (1993) 13531-13536).

[0101] Insulin receptors are expressed in virtually every location, and utilizing these delivery pathways can significantly increase the bioavailability of recombinant enzymes and enhance the efficacy of therapy. Delivery to brain tissue occurs via transcytosis through the capillary endothelium of the central nervous system, through interaction with human insulin receptors.

[0102] In peripheral and brain tissues expressing M6PR (Hawkes C. et al., 2004), internalization of the chimeric molecule can occur in both the antibody-HIR interaction and the enzyme-M6PR interaction. In this case, targeted delivery to lysosomes occurs through interaction with the M6P receptor. Internalization through HIR (human insulin receptor) initially enters into endosomes and subsequently results in transcytosis. In addition to delivery of functional enzymes to the CNS, many lysosomal storage diseases require improvement of internalization by specific peripheral tissues (such as the diaphragm muscle in Pompe disease, the liver and spleen in Hunter disease, the kidney in Fabry disease, etc.).

[0103] The term "specific" means that the molecule associated with this term can form a complex with a specific region of another molecule. Binding can be detected by in vitro assays such as plasmon resonance (BIAcore, GE-Healthcare, Uppsala, Sweden). The specific interaction (complex formation affinity) of a molecule with the binding site of another molecule is indicated by k a (rate constant for the association of the compound forming the complex), k D (dissociation constant, dissociation of the complex) and K D (k D / k a ) and is determined by. Binding or specific binding means a binding affinity (K -7 ) of about 10 -8 M or less, in one embodiment, about 10 -13 M to 10 -9 M, in one embodiment, about 10 -13 M to 10 D M.

[0104] The term "lysosome" refers to a type of cellular organelle that contains several enzymes (acid hydrolases) capable of breaking down macromolecules, either originating from the cell itself (e.g., during the processing of cellular structural components) or captured from external sources. A congenital deficiency or dysfunction of lysosomal enzymes (or other lysosomal components) can lead to the accumulation of undegraded metabolites. Glycosaminoglycans (formerly known as mucopolysaccharides) are common polysaccharides found on the cell surface as well as in the extracellular matrix and structure. Enzyme deficiencies that interfere with glycosaminoglycan degradation cause the accumulation of glycosaminoglycan fragments in lysosomes, leading to widespread changes in bone, parenchyma, and the central nervous system.

[0105] The "activity" of the enzyme(s) of the present invention can be measured using any suitable test. Typically, pH and temperature determinations may be adapted to the enzyme in question. Examples of test pH ranges are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of test temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95°C. Preferred pH and temperature values ​​are within the physiological range, e.g., pH 4, 5, 6, 7, or 8, and temperature 30, 35, 37, or 40°C. For example, protease activity may be measured using any test that includes a substrate containing a peptide bond relevant to the specificity of the protease in question.

[0106] Examples of appropriate enzyme tests are included in the Experiments section, and in particular, see Example 2. Where used herein, the term “activity” means “enzyme activity,” “specific activity,” “enzyme-specific activity,” or “specific strength,” depending on the context of the description.

[0107] The term “blood-brain barrier” or “BBB” refers to the physiological barrier between peripheral blood flow and the brain and spinal cord, formed by tight junctions in the endothelial plasma membrane of brain capillaries, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Da). The BBB in the brain, the blood-spinal barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers in the CNS, and are collectively referred to herein as the blood-brain barrier (hereinafter also referred to herein as BBB). The BBB also includes the barrier between blood and cerebrospinal fluid.

[0108] "Pharmaceutical composition" refers to a mixture of one or more of the compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound into the body.

[0109] The term "effective amount" of a compound in a pharmaceutical composition refers to the amount that is effective in the subject being treated in the dose and duration necessary to achieve the desired therapeutic or prophylactic outcome (effect), while also exhibiting reasonable benefits / risks.

[0110] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active substance (compound, agent, etc.) that is non-toxic to the subject. pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In certain (but not limited) embodiments of the present invention, a pharmaceutically acceptable carrier is administered together with the compound.

[0111] Pharmaceutical compositions containing the compounds used in accordance with the present invention are prepared for storage, preferably in the form of lyophilized compositions or aqueous solutions, by mixing with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980). Acceptable carriers, excipients, or stabilizers are nontoxic to the subject at the dosage and concentration used and include buffers such as phosphoric acid, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and meta-cresol); and low molecular weight poly Peptides (containing less than approximately 10 residues); proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., poly(vinylpyrrolidone); amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal-containing complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN TM PLURONICS TM Or it contains polyethylene glycol (PEG).

[0112] The pharmaceutical compositions used herein may also optionally contain more than one active substance (drug) that is active against lysosomal storage disorders, and optionally contain further substances having mutually non-adverse activity. The type and effective amount of such drugs will depend, for example, on the amount of therapeutic enzymes and transport element-containing compounds present in the composition, as well as the clinical parameters of the subject.

[0113] The requested dose of the therapeutic compound may be administered by any suitable route well known to those skilled in the art, for example, by injection, e.g., intravenous, intramuscular, or subcutaneous injection. The selection of a particular route for administration of the therapeutic compound will be made by those skilled in the art, in particular depending on whether the administration is short-term or long-term. Those skilled in the art will recognize that the requested dose of the compound may be administered by other routes of administration known in the art, for example (without limitation), such as subarachnoid, intra-arterial, intraperitoneal, transdermal, inhalation, buccal, nasal, oral, sublingual, or nasal administration (Felice BR, Wright TL, Boyd RB Safety Evaluation of Chronic Intrathecal Administration of Idursulfase-IT in Cynomolgus Monkeys. - Toxicol Pathol. 2011 Aug;39(5):879-9, WO 2011 / 044542 A1).

[0114] In the context of this disclosure, a variety of drug administration schedules may be considered, including, but are not limited to, single or multiple doses at different time points, bolus administration, and pulsed infusion.

[0115] A “subject,” “individual,” or “patient” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and primates, e.g., monkeys, especially great apes), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject or patient is human.

[0116] As used herein, the term “treatment” (and its grammatical variations, e.g., “to treat” or “the process of treatment”) refers to a clinical intervention aimed at altering the natural course of a disease in the individual being treated, which may be carried out for prevention or in the process of clinicopathological development. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of disease progression rate, reduction or temporary relief of the disease state, and remission or improved prognosis. In some embodiments, the compounds of the present invention are used to delay the development of a disease or slow its progression.

[0117] The term "CNS" or "central nervous system" refers to the complex of nerve tissue that controls bodily functions, and this includes the brain and spinal cord.

[0118] As used herein, the term “lysosomal storage disorder” (LSD; also “lysosomal storage disorder”) refers to a rare genetic disorder resulting in the loss of lysosomal function due to the partial or complete loss of activity of one lysosomal enzyme. In this case, ERT is required to replenish the enzyme whose activity is completely or partially lost, or which is absent. As used herein, the term “lysosomal storage disorder” is interchangeable with the term “lysosomal storage disorder” (also “lysosomal storage disorder”). Lysosomal storage disorders may be classified according to the enzyme deficiency or dysfunction as follows: (i) sphingolipidosis, (ii) mucopolysaccharidosis, (iii) glycogen storage disorder, (iv) mucolipidosis, (v) oligosaccharidosis, (vi) lipidosis, (vii) lysosomal transport disorder, etc.

[0119] Lysosomal storage disorders are described in more detail below, according to their classification.

[0120] As used herein, the term “sphingolipidosis” refers to a genetically determined deficiency syndrome of lysosomal enzymes that hydrolyze the carbohydrate or choline side chains of sphingolipids. The diseases are classified according to the distribution of each lipid that accumulates, for example, Krabbe disease is caused by galactocerebrosidase deficiency, Fabry disease is caused by α-galactosidase A deficiency, Niemann-Pick disease is caused by sphingomyelinase deficiency, Gaucher disease is caused by glucocerebrosidase deficiency, and Tay-Sachs disease is caused by hexosaminidase A deficiency, and these are inherited in an autosomal recessive manner, with the exception of Fabry disease, which is an X-linked genetic disorder.

[0121] In this disclosure, the term “mucopolysaccharidosis” (MPS) refers to a genetic deficiency syndrome of mucopolysaccharide hydrolases caused by deficiencies in carbohydrate chain-degrading enzymes such as sulfatase and acetyltransferase. The main symptom of mucopolysaccharidosis (MPS) is the excessive secretion of mucopolysaccharides in the urine. Currently, MPS is classified into six types, of which Type I includes Hurler syndrome and Schayet syndrome; Type II includes Hunter syndrome; Type III includes Sanfilippo syndromes of types A, B, C, and D; Type IV includes Morquio syndromes of types A and B; Type VI includes Maloto-Lamy syndrome; and Type VII includes Sly syndrome.

[0122] As used herein, the term “glycogen storage disease” (also known as glycogen storage disease) refers to a congenital error in carbohydrate metabolism caused by glycogen storage, and is usually classified into subtypes I through VII. The subtypes of glycogen storage disease associated with lysosomal storage disorders (LSD) are type II (Pompe disease) and type IIIb (Danon disease).

[0123] A detailed description of lysosomal storage disorders as described herein, as well as those disclosed in Table 1, is provided in: The Online Metabolic & Molecular Bases of Inherited Diseases (Scriver's OMMBID), Part 16 (David L. Valle, Stylianos Antonarakis, Andrea Ballabio, Arthur L. Beaudet, Grant A. Mitchell, McGraw-Hill Education, 2007).

[0124] Mucopolysaccharidosis type I (MPS I) is a congenital metabolic disorder caused by a deficiency in the enzyme α-L-iduronidase (IDUA), whose function is to break down mucopolysaccharides, heparan sulfate, and dermatan sulfate. Insufficient levels of IDUA lead to the pathological accumulation of these mucopolysaccharides in the patient's tissues and organs, such as the heart, liver, and central nervous system. Symptoms, including neurodegeneration and intellectual disability, begin in childhood, and early death can occur due to organ damage. Genetic deficiency in the carbohydrate-degrading enzyme α-L-iduronidase causes lysosomal storage disorder known as mucopolysaccharidosis type I (MPS I). Severe MPS I is commonly known as Hurler syndrome and is associated with a variety of problems, including intellectual disability, corneal opacity, coarse facial features, heart disease, respiratory disease, hepatomegaly and splenomegaly, hernias, and joint stiffness. Patients with Haller syndrome typically die before the age of 10. In the moderate form known as Haller-Scheille syndrome, mental function is usually not significantly affected, but physical problems can also lead to death during adolescence or between the ages of 20 and 29. Schye syndrome is a mild form of MPS I. This corresponds to a normal life expectancy, but joint stiffness, corneal opacity, and heart valve disease cause serious problems.

[0125] Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is an X-linked congenital metabolic disorder caused by a deficiency in the enzyme iduronate-2-sulfatase (I2S). I2S is localized in lysosomes and plays a crucial role in the catabolism of glycosaminoglycans (GAGs) into heparan sulfate and dermatan sulfate. In the absence of this enzyme, these substrates accumulate in cells, ultimately leading to stagnation, followed by cell death and tissue destruction. Because this enzyme is widely expressed, different types of cells, organs, and systems are affected in MPS II patients. A characteristic clinical feature of this disease is central nervous system (CNS) degeneration, which leads to cognitive impairment (e.g., decreased IQ). Furthermore, MRI scans of patients have shown white matter damage, enlargement of perivascular spaces in the brain parenchyma, ganglia, corpus callosum, and brainstem, atrophy, and ventricular enlargement (Wang et al. Molecular Genetics and Metabolism, 2009). The disease typically manifests by the first year of life, accompanied by organ hypertrophy and skeletal abnormalities. Some patients experience progressive cognitive loss, and the majority die from disease-related complications within 10 or 20 years of age (Raluy-Callado M et al. Orphanet J Rare Dis. 2013;8:101).

[0126] As used herein, the term “neurological components” refers to diseases or disorders affecting the central nervous system and / or etiologies associated with the central nervous system. Examples of CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, epileptic disorders, behavioral disorders, and lysosomal storage disorders. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but are not limited to, Lewy body dementia, postmyelitis syndrome). Syndrome (including Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration), tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Streusler disease, Schenker's disease, chronic wasting disease, and fatal familial insomnia), paralysis, motor neuron disease, and heterodegenerative disorders of the nervous system ( This includes, but is not limited to, Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair, Cockayne syndrome, Hallerwarden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverlicht-Lundborg disease, dementia (but is not limited to Pick's disease and spinocerebellar ataxia), and cancer (e.g., cancer of the central nervous system and / or brain, including brain metastases from cancer in any part of the body).

[0127] The compounds of the present invention can be used in therapy either individually or in combination with other agents. For example, a compound of the present invention comprising a therapeutic enzyme and a transport element coupled by a linker may be administered concurrently with at least one further therapeutic agent. In certain embodiments, the further therapeutic agent is a therapeutic agent that is effective in treating the same neurological disorder as the compound of the present invention is used for, or a different neurological disorder. Further examples of therapeutic agents include, but are not limited to, the diverse neurological agents mentioned above, and include cholinesterase inhibitors (e.g., donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (e.g., memantine), amyloid-beta peptide aggregation inhibitors, antioxidants, γ-secretase modulators, neurotrophic growth factor (NGF) mimetic agents or NGF gene therapies, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampaquine, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, immunoglobulins intended for intravenous administration, muscarinic receptor agonists, nicotinic receptor modulators, active or passive immunizers against amyloid-beta peptides, phosphodiesterase inhibitors, serotonin receptor antagonists, and antibodies against amyloid-beta peptides. Such combination therapies as defined above include simultaneous administration (when two or more therapeutic agents are contained in the same or different compositions) and separate administrations in which the compound containing the therapeutic enzyme and transport element coupled by the linker of the present invention is administered before, concurrently with, and / or after the administration of further therapeutic agents and / or adjuvants.

[0128] Some of the terms defined above may appear more than once, but in such cases, each term should be defined independently of the others.

[0129] In this specification, exemplary embodiments of the present invention will be described in detail thereafter. Furthermore, each of the descriptions and practices provided as examples in this document may also be valid for other descriptions and practices provided as examples. Thus, all combinations of the diverse elements described herein are within the scope of the present invention. Furthermore, the scope of the present invention is not limited to the specific descriptions provided below. [Examples]

[0130] The present invention will be described in more detail herewith in relation to the following embodiments. However, the embodiments described below are for illustrative purposes only and are not intended to limit the present invention.

[0131] Example 1: Acquisition To obtain the compound, animal cells into which an animal cell expression vector had been introduced were cultured and purified.

[0132] Construction of expression vectors To obtain HIR-FAB-IDS mutants containing the signal peptide MDWTWRVFCLLAVAPGAHS, the amino acid sequences of the first LC-HIR-FAB-IDS (SEQ ID NOs. 2, 8, 9, 10, and 11) and the second HC-HIR-FAB-IDS (SEQ ID NOs. 4, 5, 12, 13, 14, and 15) were converted to nucleotide sequences. After gene synthesis, the following HindIII restriction site and Kozak sequence were added to the 5' end of the sequence for cloning into the pCLN-1 vector. Two stop codons and an XbaI restriction site were added to the 3' end of the sequence.

[0133] Using the resources http: / / gcua.schoedl.de and http: / / www.kazusa.or.jp, we performed codon optimization of the nucleotide sequences of Chinese hamster cells. The light and heavy chains of LC-HIR-FAB-IDS and HC-HIR-FAB-IDS were synthesized using GenArt (USA) and transferred as part of vectors pRA1675 and pRA1673 (containing the genes LC-HIR-FAB-IDS and HC-HIR-FAB-IDS, respectively).

[0134] The sequences HC-HIR-FAB-IDS and LC-HIR-MAB, derived from plasmids pRA1675 and pRA1673, were cloned into the expression vector pCLN-1 at the HindIII / XbaI site to obtain vectors pGNR-055-007 (pCLN-1-HC-HIR-FAB-IDS) and pGNR-055-008 (pCLN-1-LC-HIR-FAB-IDS). The resulting vectors were linearized at the BspHI / PvuI site.

[0135] The vector of the present invention was constructed in accordance with molecular biology techniques well known in the art. Brown T, "Gene Cloning" (Chapman & Hall, London, GB, 1995); Watson R, et al., "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, NY, US, 1992); Alberts B, et al., "Molecular Biology of the Cell" (Garland Publishing Inc., New York, NY, US, 2008); Innis M, et al., Eds., "PCR Protocols. A Guide to Methods and Applications" (Academic Press Inc., San Diego, CA, US, 1990); Erlich H, Ed., "PCR Technology. Principles and Applications for DNA Amplification" (Stockton Press, New York, NY, US, 1989); Sambrook J, et al., "Molecular Cloning. A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, US, 1989); Bishop T. et al., "Nucleic Acid and Protein Sequence. A Practical Approach" (IRL Press, Oxford, GB, 1987); Reznikoff W, Ed., "Maximizing Gene Expression" (Butterworths Publishers, Stoneham, MA, US, 1987); Davis L, et al., "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co.(New York, NY, US, 1986), see Schleef M, Ed., "Plasmid for Therapy and Vaccination" (Wiley-VCH Verlag GmbH, Weinheim, DE, 2001).

[0136] Acquisition of monoclonal cell lines The parent cell line was CHO-S. Cells were cultured at 37°C, 5% CO2, and 70% humidity in BalanCD CHO Growth Medium A (Invitrogen). Stable transfection was performed on a NEON device (Invitrogen) using two linearized plasmids, pCLN-1-HC-HIR-FAB-IDS and pCLN-1-LC-HIR-FAB-IDS, according to the standard protocol for CHO cells. Equimolar DNA ratios were used for transfection.

[0137] After 48 hours, the transfection pool was seeded into minipools in 96-well plates in BalanCD CHO Growth Medium A containing the selective antibiotic neomycin 600 μg / ml. The minipools were cultured for 10 days under stable conditions of 37°C, 5% CO2, and 70% humidity, after which a series of productivity screenings were performed using EIA. For single-colony growth, the leader minipool was cloned into semi-solid ClonaCell Flex medium (STEMCELL) using 6-well plates. The plates were incubated at 37°C, 5% CO2, and 70% humidity.

[0138] During screening, the concentration of the target protein HIR-FAB-IDS secreted into the culture medium was determined. Instead of the primary antibody, recombinant human insulin receptor fragment (5 μg / ml in carbonate-bicarbonate buffer pH 9.6) was used for determination by sandwich EIA; a horseradish peroxidase-conjugated rat antibody against iduronate-2-sulfatase (diluted 1:10000) was used as the secondary antibody. The reaction was chromogenically treated with tetramethylbenzidine solution and stopped with 0.5 M sulfuric acid.

[0139] Based on the screening results of 6-well plates, 20 leading minipools with productivity of 0.4–2.1 mg / l were selected and transferred to flasks for adaptation to suspension culture. Three subculturings were performed to adapt them to suspension culture, after which the 20 minipools were frozen.

[0140] Clones were automatically selected into 96-well plates using the ClonePix robot (Molecular Devices). The resulting clones were screened. For this purpose, a 7-day culture process was simulated in a batch manner, and the clones were evaluated according to the following parameters: dynamics of culture viability, dynamics of viable cell density, concentration of target HIR-FAB-IDS, and the dependence of production volume on cumulative cell density.

[0141] Culture of producing cells Cells expressing HIR-FAB-IDS were cultured in a bioreactor equipped with an overhead stirrer at 37°C and pH 6.9 for 12 days in BalanCD CHO Growth Medium A (Irvine Scientific) and the nutrient supplement BalanCD CHO Feed 2 (Irvine Scientific). After the culture process, the culture medium was clarified by deep filtration and transferred for isolation.

[0142] Isolation and purification Isolation and purification were performed using standard techniques for protein isolation and purification. Compounds may be isolated or purified by any method known in the art for the isolation or purification of proteins, for example by chromatography (e.g., ion exchange, high-performance liquid chromatography, affinity-assisted, protein A and size-preparative column chromatography), centrifugation, differential solubility, or any other standard protein isolation or purification technique.

[0143] Example 2. Determination of the enzymatic activity level of the Fab fragment of an antibody against the insulin receptor coupled to the amino acid sequence of iduronate-2-sulfatase. The specific activity of HIR-FAB-IDS and HIR-MAB-IDS was determined using 4-mutilumbelliferyl-L-izlonide-2-sulfate (4-MUS) (Moscerdam Substrates, the Netherlands) (Voznyi YV et al., 2001; Tolun AA, et al., 2012; Johnson BA et al., 2013; Azadeh M et al., 2017). During the analysis, the substrate is hydrolyzed by iduronic acid-2-sulfatase to produce 4-mutylumbelliferyl-L-idulonide (MUBI), which is hydrolyzed by idulonidase (IDUA, Aldurazyme, Genzyme, USA) to 4-methylumbelliferyl (4-MU), which is detected fluorescencely using the following fluorometer settings: fluorescence excitation at a wavelength of 450 nm and fluorescence detection at a wavelength of 365 nm. A calibration curve is constructed using standard solution 4-MU (Sigma-Aldrich, USA). During the analysis, the mixture is first incubated at 37°C, pH=4.5 for 4 hours, then 12 μg of IDS is added, and the mixture is further incubated at 37°C for 24 hours. Incubation is stopped by adding 0.2 ml of 0.5 M sodium carbonate, pH=10.3. HIR-FAB-IDS exhibits specific enzymatic activity (iduronate-2-sulfatase) against the substrate 4-MU-αIdoA-2S (4-methylumbelliferyl α-L-idopyranosiduronate-2-sulfate).

[0144] Devices and equipment 1. Constant temperature shaking device PST-60 HL-4 (BioSan, Latvia, or similar) 2. Multifunction reading device Spectra Max M3 (Molecular Devices, USA or similar) 3. Software for multifunction reading devices: Soft Max Pro (Molecular Devices, USA, or similar) 4. Chemical balance ML 204 (Mettler Toledo, Switzerland, or similar) 5. Non-absorbent surface 96-well black plate (Corning, USA, catalog number 3916 or similar)

[0145] reagent 1. 4-Methylumbelliferone sodium salt (Sigma-Aldrich, product number M1508 or equivalent) 2. Recombinant human α-L-idulonidase (R&D SYSTEM, product number 4119-GH8 or equivalent quality) 3. 4-Methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate sodium salt, 0.5 mg / vial (USBiological, product number 017551 or equivalent) 4. Anhydrous sodium acetate (AppliChem Panreac, product number 141633.1211 or equivalent quality) 5. Acetic acid (Fluka, product number 49199 or equivalent quality) 6. Bovine serum albumin (Sigma, product number A7030 or equivalent quality) 7. Sodium carboxylate (Sigma-Aldrich, product number S7795 or equivalent quality) 8. Sodium bicarbonate (Sigma-Aldrich, product number S6297 or equivalent quality) 9. Lead diacetate trihydrate (Aldrich, product number 467863 or equivalent quality) 10. Sodium phosphate dihydrate (Sigma-Aldrich, product number 71643 or equivalent quality) 11. Anhydrous citric acid (AppliChem Panreac, product number 141808 or equivalent quality)

[0146] Solution preparation Sample dilution solution, pH (5.5 ± 0.2). Dissolve approximately 4.1 g of anhydrous sodium acetate and 0.5 g of bovine serum albumin in a 1-liter beaker in 700 ml of purified water. Adjust the pH of the solution to (5.5 ± 0.2) with acetic acid. Transfer the resulting solution to a 1-liter volumetric flask, adjust the solution volume to the marked level with purified water, mix, and filter through a membrane filter with a pore size of 0.45 μm.

[0147] The shelf life is 3 months at a temperature of 2-8°C.

[0148] Prepare a diluted substrate solution, pH (5.00 ± 0.05). Place 4.1 g of anhydrous sodium acetate and 1.9 g of lead diacetate trihydrate in a 500 ml beaker, add 450 ml of purified water, and stir the mixture until dissolved. Adjust the pH of the solution to (5.00 ± 0.05) with approximately 1.1 ml of acetic acid. Transfer the resulting solution to a 500 ml volumetric flask, adjust the volume with purified water to the marked level, mix, and filter through a membrane filter with a pore size of 0.45 μm.

[0149] The shelf life is 3 months at a temperature of 2-8°C.

[0150] Substrate solution (1.25 mmol / l). Add 0.83 ml of diluted substrate solution to a bottle containing the substrate (4-methylumbelliferyl α-L-idopyranosideuronic acid 2-sulfate sodium salt) and mix gently. Divide the resulting solution into 0.2 ml alicots and freeze.

[0151] The shelf life is 3 months at temperatures not exceeding -70°C.

[0152] A solution of recombinant human α-L-idulonidase. Add 400 μl of purified water to a bottle containing recombinant human α-L-idulonidase (10 μg) and mix. Divide the resulting solution into 0.1 ml alicots and freeze.

[0153] The shelf life is 3 months at temperatures not exceeding -70°C.

[0154] A 0.1 M solution of citric acid. Place approximately 19.2 g of anhydrous citric acid in a 1-liter flask, dissolve it in 900 ml of purified water, adjust the solution volume with purified water to the marked level, and filter through a 0.22 μm membrane filter.

[0155] The shelf life is 3 months at a temperature of 15-25°C.

[0156] A 0.2 M solution of disubstituted sodium phosphate. Place approximately 35.6 g of sodium phosphate dihydrate in a 1-liter flask, dissolve in 900 ml of purified water, adjust the solution volume with purified water to the marked level, and filter through a 0.22 μm membrane filter.

[0157] The shelf life is 3 months at a temperature of 15-25°C.

[0158] Phosphate-citric acid buffer solution, pH (4.5 ± 0.1). Mix 55.0 ml of 0.1 M citrate solution and 45.0 ml of 0.2 M dibasic sodium phosphate solution in a 100 ml volumetric flask and filter through a membrane filter with a pore size of 0.22 μm.

[0159] The shelf life is 3 months at a temperature of 15-25°C.

[0160] 0.5 M sodium bicarbonate solution. Place approximately 42.0 g of sodium bicarbonate in a 1 liter volumetric flask, dissolve in 900 ml of purified water, adjust the solution volume with purified water to the marked level, mix, and filter through a membrane filter with a pore size of 0.22 μm.

[0161] The shelf life is 6 months at a temperature of 15-25°C.

[0162] 0.5 M sodium carbonate solution. Place approximately 53.0 g of sodium carbonate in a 1 liter volumetric flask, dissolve in 900 ml of purified water, adjust the solution volume with purified water to the marked level, mix, and filter through a membrane filter with a pore size of 0.22 μm.

[0163] The shelf life is 6 months at a temperature of 15-25°C.

[0164] Stop solution, pH (10.8 ± 0.5). Add 900 ml of 0.5 M sodium carbonate solution and 100 ml of 0.5 M sodium bicarbonate solution to a 1 liter volumetric flask, mix, and filter through a membrane filter with a pore size of 0.22 μm.

[0165] The shelf life is 6 months at a temperature of 15-25°C.

[0166] Primary solution of 4-methylumbelliferone sodium solution (100 μmol / ml). Place approximately 1.0 g (accurately weighed) of 4-methylumbelliferone sodium salt into a 50 ml volumetric flask, add 30 ml of purified water, mix, and adjust to the mark with purified water. Divide the solution into 2.0 μl alicots.

[0167] The shelf life is 6 months at temperatures not exceeding -18°C.

[0168] Standard solution of 4-methylumbelliferone sodium salt (50 nmol / ml). Maintain an alicot of the primary solution of 4-methylumbelliferone sodium salt in an aqueous bath at 37°C for 5 minutes. Prepare serial dilutions according to the following scheme: TIFF0007887076000003.tif33169

[0169] Dilution of the standard solution of 4-methylumbelliferone sodium salt. Prepare the working standard solution of 4-methylumbelliferone sodium salt according to the following scheme: TIFF0007887076000004.tif86170

[0170] Dilution of the test sample. Based on the actual HIR-FAB-IDS content stated on the certificate, dilute the test sample with the sample dilution solution to a concentration of 1 mg / ml. Then, prepare serial dilutions according to the following scheme: TIFF0007887076000005.tif90170

[0171] Store all dilutions on ice until the start of the test. For further research, use the solutions in test tubes 5 through 7.

[0172] Conducting the analysis First enzymatic reaction Add 10 μl of each dilution of the test sample (two copies) to the wells of the plate; use the sample dilution solution as the reference solution. Add 20 μl of substrate solution (4-methylumbelliferyl α-L-idopyranosideuronic acid 2-sulfate sodium salt). Cover the plate with film and incubate in a constant temperature shaking apparatus at (37.0 ± 0.2) °C for 4 hours at a stirring speed of 250 rpm. The plate must be protected from light throughout the entire incubation period.

[0173] Second enzymatic reaction At the end of incubation, add 20 μl of phosphate-citrate buffer solution to the plate wells to stop the first enzymatic reaction. Then, add 10 μl of recombinant α-L-idulonidase solution to each well and mix gently. Cover the plate with film and incubate in a constant temperature shaking apparatus at 37°C for 22-26 hours at a stirring speed of 250 rpm. The plate must be protected from light throughout the entire incubation period.

[0174] To stop the reaction, add 200 μl of stop solution to each well containing the incubated mixture.

[0175] Add 260 μl of a diluted standard solution of 4-methylumbelliferone sodium salt to the empty wells of the plate.

[0176] The fluorescence signal is measured in the plate wells at an excitation wavelength of 365 nm and a detection wavelength of 460 nm.

[0177] Evaluation of Results Based on the measurement of fluorescence signals in wells containing diluted standard solutions of 4-methylumbelliferone sodium salt, a linear relationship between the fluorescence signal and the concentration of 4-methylumbelliferone sodium salt (nmol / ml) is plotted. Using a linear function equation, the concentration of 4-methylumbelliferone produced during the reaction in wells containing the diluted test sample and the reference solution (nmol / ml) is calculated.

[0178] The specific activity A per unit / μg is calculated using the following formula:

number

[0179] The final specific activity value for the test sample is calculated as the average of the specific activities calculated for each dilution.

[0180] Table 2 shows the results obtained to determine the enzyme activity of HIR-FAB-IDS and HIR-MAB-IDS mutants compared to the drug Elaprace. Table 2. Evaluation of iduronate-2-sulfatase activity of HIR-FAB-IDS and HIR-MAB-IDS mutants against unmodified iduronate-2-sulfatase enzyme (Elaprace). [Table 2]

[0181] As can be seen from the results presented in Table 2, HIR-FAB-IDS drugs exhibit a specific enzyme (iduronate-2-sulfatase) activity of 27.0 U / μg against the substrate 4-MU-αIdoA-2S (4-methylumbelliferyl α-L-idopyranosideuronate-2-sulfate). In this case, free recombinant iduronate-2-sulfatase exhibits an activity of 14.6 U / μg. Full-length insulin receptor antibodies coupled to the amino acid sequence of iduronate-2-sulfatase (HIR-MAB-IDS) show a lower specific activity (11.0 U / μg) compared to HIR-FAB-IDS.

[0182] The research results show that iduronate-2-sulfatase in the composition of HIR-FAB-IDS retains its main functional (enzymatic) properties at the free recombinant enzyme level. At the same time, the specific activity of HIR-FAB-IDS is higher than that of the drug Elaprace and HIR-MAB-IDS (AGT-182).

[0183] Therefore, compared to therapeutic enzymes without transport elements and therapeutic enzymes with transport elements as indicated by MABs, an increase in enzymatic activity was detected in compounds containing transport elements, which are Fab fragments of insulin receptor epitope-specific IgG immunoglobulins, directly or via a linker-coupled to the therapeutic enzyme.

[0184] Example 3. Analysis of the interaction between human and mouse insulin receptors and modified Fab fragments of antibodies against insulin receptors. To quantitatively evaluate the interaction of full-length recombinant monoclonal antibodies, Fab fragment variants, and their modifications with IDSs with the insulin receptor, a sandwich-type enzyme-coupled immunosorbent assay (ELISA) technique is used (Kim C, Seo J, Chung Y et al., 2017). This method is based on the binding of the proteins HIR-MAB (83-14 Mab), HIR-MAB-IDS, and HIR-FAB-IDS to human insulin receptor protein fragments (human insulin receptor protein fragment (Abcam catalog number ab200510)) or mouse insulin receptor (mInsR, R&D Systems, Inc., catalog number 7544-MR).

[0185] Following incubation of the human insulin receptor and the protein under study complex, a detection process is performed using a secondary antibody, a Fab-specific goat antibody against human immunoglobulin conjugated with horseradish peroxidase (anti-human IgG (Fab-specific)-peroxidase (Sigma-Aldrich, catalog number A0293)).

[0186] Previously described mouse antibodies against the human insulin receptor (INSR / insulin receptor alpha antibody 83-14 AHR0221 Life Technologies, catalog number AHR0221) and antibodies against the mouse insulin receptor (mouse anti-human insulin receptor clone 83-14 mAb Cell Sciences) were used as positive controls (catalog number MAI1). Detection was performed using a goat polyclonal antibody against mouse immunoglobulin conjugated with horseradish peroxidase (goat pAb(HRP) Abcam against Ms IgG, catalog number ab97023).

[0187] The results obtained to determine the interaction between human and mouse insulin receptors and the test molecule are presented in Tables 3 and 4.

[0188] Table 3. Binding of the human insulin receptor (Abcam catalog number ab200510) to the modified Fab fragment of the antibody against the insulin receptor. [Table 3]

[0189] Table 4. Binding of modified Fab fragments of insulin receptor antibodies to mouse insulin receptors (R&D Systems, Inc., catalog number 7544-MR). [Table 4]

[0190] Therefore, it was demonstrated that HIR-FAB-IDS specifically interact with the extracellular domain of the alpha subunit of the human insulin receptor, but not with the extracellular domain of the mouse insulin receptor. Binding of the Fab fragment of the antibody against the human insulin receptor is comparable to that of the control antibody (83-14 mAb) and surpasses binding to a similar full-length antibody against the insulin receptor (HIR-MAB-IDS).

[0191] The interaction between the human insulin receptor and HIR-FAB-IDS is fundamental to ensuring the active transcytosis of molecules into cellular lysosomes in general, particularly through the blood-brain barrier (BBB).

[0192] Example 4. Radiolabeled Fab fragment of an antibody against the insulin receptor coupled to the amino acid sequence of iduronate-2-sulfatase, 125 I]-HIR-Fab-IDS (SEQ ID NOs. 2 and 4), [ 125 I]-HIR-Mab-IDS, 125 Analysis of tissue distribution of I-IDS (control) after intravenous administration in cynomolgus monkeys. The corresponding molecule of the test substance was injected once into the left femoral vein of experimental animals by intravenous bolus injection over 1-2 minutes, at a target dose of 2 ml / kg body weight. The target radioactive dose level was 1 MBq / kg animal body weight for all molecules studied.

[0193] Animals administered the test substance were sedated by intramuscular injection of ketamine hydrochloride, followed by intravenous administration of an overdose of Doletil (sodium pentaborbitone). Two hours after administration of the test substance, the animals were humanely euthanized. During sedation, 2 ml of blood was collected from the cutaneous vein of each animal and centrifuged to obtain plasma. The radioactivity concentration in the animal plasma was measured. Once death was confirmed, each animal was rapidly frozen in a solid carbon dioxide mixture in hexane. Once completely frozen, the body was placed in a mold containing 2% (w / v) aqueous carboxymethylcellulose paste. For each animal, several axial sagittal sections (nominally 30 μm) were taken at least five body levels and, if necessary, three head levels.

[0194] Sections attached to Filmolux 610 tape (Neschan) were freeze-dried in a GVD03 benchtop freeze-drying apparatus (Girovac Ltd) and placed on a FUJI X-ray plate (BAS-MS type, Raytek Scientific Ltd). Appropriately active isotopes were prepared in the form of sections with a nominal thickness of 30 μm. 125 Blood standards labeled with [I] were placed on a radiographic plate.

[0195] After developing in a copper-coated lead box for 7 days, the radiation plates were processed using a FUJI FLA-5000 radiation system (Raytek Scientific Ltd). Electronic images were analyzed using a PC-based image analysis system (Seescan 2 software, LabLogic Ltd). Each autoradiogram contained [ 125 I) Using standards, a calibration curve was constructed over a certain range of radioactive concentrations.

[0196] Tissue concentration data [ 125 I]HIR-Fab-IDS, 125 I]HIR-Mab-IDS and [ 125 The amount was recorded as the ng equivalent of [I]-IDS (control).

[0197] The results were expressed with three significant figures, up to two decimal places. The data table was generated by computer, and each data point was rounded appropriately.

[0198] [ 125 I]HIR-Fab-IDS, 125 I]HIR-Mab-IDS and [ 125 [1]-IDS (control) was administered to male cynomolgus monkeys as a single intravenous dose at nominal dose levels of 0.0020 mg / kg, 0.0015 mg / kg, and 0.0010 mg / kg body weight. Two hours after each drug administration, the animals were euthanized and then frozen for whole-body autoradiography studies.

[0199] After intravenous administration, the drug was absorbed and widely distributed throughout the body's tissues, and at the time of slaughter, all studied tissues were exposed to the drug. The lower limits of quantification were 0.2, 0.03, and 0.097 ng equivalents / g for tissue concentration measurements in all animals, respectively.

[0200] [ 125 The radioactive concentrations in the plasma and blood of animals treated with [I]HIR-Fab-IDS were 2.55 and 3.11 ng eq / g, respectively (blood:plasma ratio 1.22). Quantifiable levels of radioactivity were present in several brain regions, including the medulla oblongata (1.09 ng eq / g), cerebral cortex (1.03 ng eq / g), cerebellum (0.908 ng eq / g), hypothalamus (0.869 ng eq / g), cerebellar arborescent substance (0.799 ng eq / g), pons (0.793 ng eq / g), and medulla (0.562 ng eq / g); the TP ratio ranged from 0.22 to 0.43.

[0201] [ 125 The radioactive concentrations in the plasma and blood of animals treated with [I]HIR-Mab-IDS were 2.13 and 1.86 ng eq / g, respectively (blood:plasma ratio 0.87). Quantifiable levels of radioactivity were present in several regions of the animal brain. These regions included the medulla oblongata (0.803 ng eq / g), cerebellar arboresum (0.606 ng eq / g), cerebellum (0.494 ng eq / g), cerebral cortex (0.453 ng eq / g), pons (0.438 ng eq / g), medulla (0.288 ng eq / g), and hypothalamus (0.279 ng eq / g), with TP concentration ratios ranging from 0.13 to 0.38.

[0202] [ 125 In animals treated with I]IDS, plasma and blood concentrations were 0.910 and 0.753 ng eq / g, respectively (blood:plasma ratio 0.83). Quantifiable levels of radioactivity were present throughout the brain (0.113 ng eq / g), but concentrations were below the lower limit of quantification (0.097 ng eq / g) in all regions examined: medulla oblongata, cerebral cortex, cerebellum, hypothalamus, cerebellar dendrites, pons, and medulla. Concentrations in CNS tissue were significantly lower than in plasma and blood.

[0203] These results reveal that, compared to IDS-binding molecules, HIR-Fab-IDS and HIR-Mab-IDS conjugates crossed the blood-brain barrier, and when CNS tissues were exposed to the test substance or its labeled metabolites, the radioactivity transmitted through the blood had little effect on determining the concentration in CNS tissues (Figures 1 and 2). Furthermore, HIR-Fab-IDS showed a broader distribution in body tissues compared to HIR-Mab-IDS molecules (Table 5, Figure 3).

[0204] In Example 10 of Invention US8834874 B2 (AGT-182 or HIR-MAB-IDS), based on experimental data on the penetration of AGT-182 (HIR-MAB-IDS) into the brains of rhesus monkeys, it should be noted that the penetration efficiency into the human brain is estimated to be 1% of the administered dose per 1000 grams of brain tissue. At this level of penetration, it is expected that 20% of iduronate-2-sulfatase (IDS) activity will be achieved in the human brain, which is said to eliminate glycosaminoglycan accumulation in the brains of diseased individuals.

[0205] Considering the data obtained regarding penetration into the brain of cynomolgus monkeys (Table 5), the inventors can conclude that the compounds of the present invention penetrate into the brain of cynomolgus monkeys nearly twice as well as AGT-182 (HIR-MAB-IDS), i.e., 0.84 ng eq / g of HIR-FAB-IDS versus 0.43 ng eq / g of HIR-MAB-IDS. This therefore means that HIR-FAB-IDS penetrate into the human brain with 2% efficiency. Based on the calculations shown in Example 10 of US8834874B2, it is expected that 40% of iduronate-2-sulfatase activity will be achieved in the human brain, except for the increased activity of HIR-FAB-IDS compared to HIR-MAB-IDS (AGT-182). See Table 2.

[0206] In Example 2, as shown in Table 2, considering the increased specific activity of HIR-FAB-IDS compared to HIR-MAB-IDS (AGT-182), namely 27 U / μg of the compound of the present invention versus 11 U / μg of the other, and greater penetration into the brain, we have 27 / 11 = 2.4 times higher specific iduronate-2-sulfatase activity and 0.84 / 0.43 = 1.95 times higher penetration into the brain. This results in a 20% recovery of IDS activity in the brain of patients receiving HIR-MAB-IDS (AGT-182) therapy, and a 20% * 2.4 * 1.95 = 93.6% recovery of iduronate-2-sulfatase activity in the brain of type II MPS patients compared to the IDS activity level in the brain of a healthy individual. Therefore, the present invention almost completely restores IDS enzyme function in the brain of a diseased individual, while the other AGT-182 (HIR-MAB-IDS) only restores 20% (93% vs. 20%).

[0207] Therefore, administration of a compound containing a transport element that is a Fab fragment of insulin receptor epitope-specific IgG immunoglobulin, coupled directly or by a linker to the therapeutic enzyme, provides a higher degree of substitution of the therapeutic enzyme activity in the human brain compared to compounds containing Mab.

[0208] Table 5. [at a nominal dose level of 0.0020 mg / kg body weight] 125 Radioactivity concentration in cynomolgus monkey tissue after single intravenous administration of I]-HIR-Fab-IDS [Table 5] TIFF0007887076000011.tif161170

[0209] This specification presents compounds containing a therapeutic enzyme and a transport element capable of interacting with insulin receptors, while exhibiting specific enzymatic activity in these compounds, and having the ability to transport the therapeutic enzyme to tissue lysosomes, including transport to nerve tissue lysosomes across the blood-brain barrier (BBB). Thus, the compounds exhibit high activity and improved ability to be transported to lysosomes in tissue cells of various organs, including nerve tissue cells, suggesting the use of the present invention for enzyme replacement therapy for the treatment or prevention of lysosomal storage disorders in subjects. This disclosure includes the following embodiments. Embodiment 1 A compound comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to an insulin receptor epitope. Embodiment 2 A compound according to Embodiment 1, comprising the therapeutic enzyme and the transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to an insulin receptor epitope, and capable of transporting the therapeutic enzyme across the blood-brain barrier. Embodiment 3 The compound according to Embodiment 2, wherein the insulin receptor epitope is represented by the amino acid sequence of SEQ ID NO: 1. Embodiment 4 The compound according to Embodiment 3, wherein the therapeutic enzyme and the transport element are coupled by a linker. Embodiment 5 The compound according to Embodiment 4, wherein the linker is a peptide linker containing one or more amino acids. Embodiment 6 The compound according to Embodiment 5, wherein the linker is a peptide linker containing one or more amino acids selected from glycine, serine, and leucine. Embodiment 7 The compound according to Embodiment 1, wherein the transport element contains the amino acid sequence of a Fab fragment of immunoglobulin IgG, and the immunoglobulin IgG is IgG1, IgG2, or IgG4. Embodiment 8 The compound according to Embodiment 1, wherein the transport element contains the amino acid sequence of a Fab fragment of immunoglobulin IgG, and the immunoglobulin IgG is IgG1. Embodiment 9 The compound according to Embodiment 8, wherein the transport element contains a Fab fragment of immunoglobulin IgG1, the first amino acid sequence having at least 80% identity with SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 3. Embodiment 10 The compound according to Embodiment 8, wherein the transport element contains a Fab fragment of immunoglobulin IgG1, comprising a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid sequence of SEQ ID NO: 3. Embodiment 11 The compound according to Embodiment 8, wherein the transport element contains a Fab fragment of immunoglobulin IgG1, comprising the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 3. Embodiment 12 The compound according to Embodiment 1, comprising a therapeutic enzyme for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders. Embodiment 13 The compound according to Embodiment 1, comprising a therapeutic enzyme for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders having neurological components. Embodiment 14 A compound according to Embodiment 1, comprising a therapeutic enzyme for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders having neurological components, wherein the therapeutic enzyme is selected from the group consisting of iduronate-2-sulfatase and α-L-iduronidase. Embodiment 15 A compound according to Embodiment 14, comprising a therapeutic enzyme for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders having neurological components, wherein the therapeutic enzyme is selected from the group consisting of iduronate-2-sulfatase, iduronate-2-sulfatase fragment having iduronate-2-sulfatase activity, or iduronate-2-sulfatase analogs. Embodiment 16 A compound according to Embodiment 14, comprising a therapeutic enzyme for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders having neurological components, wherein the therapeutic enzyme is selected from the group consisting of α-L-iduronidase, α-L-iduronidase fragment having α-L-iduronidase activity, or α-L-iduronidase analogs. Embodiment 17 The compound according to Embodiment 1, wherein the transport element is capable of transporting the enzyme to the lysosome. Embodiment 18 The compound according to Embodiment 17, wherein the transport element is capable of transporting the enzyme to lysosomes of nerve tissue cells. Embodiment 19 A compound according to Embodiment 1 or 2, represented by a first amino acid sequence having at least 80% identity with SEQ ID NO: 2 and a second amino acid sequence having at least 80% identity with SEQ ID NO: 4, used for the treatment or prevention of lysosomal enzyme deficiency in subjects having type II mucopolysaccharidosis. Embodiment 20 A compound according to Embodiment 1 or 2, represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11 and a second amino acid sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15, used for the treatment or prevention of lysosomal enzyme deficiency in subjects having type II mucopolysaccharidosis. Embodiment 21 The compound according to Embodiment 19, represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4, used for the treatment or prevention of lysosomal enzyme deficiency in subjects having type II mucopolysaccharidosis. Embodiment 22 The compound according to Embodiment 19, used for the treatment or prevention of lysosomal enzyme deficiency in subjects having type II mucopolysaccharidosis with neurological components represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4. Embodiment 23 The compound according to Embodiment 18, used for the treatment or prevention of lysosomal enzyme deficiency in subjects having type II mucopolysaccharidosis with neurological components represented by the amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 5. Embodiment 24 The compound according to Embodiment 1 or 2, represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 6, used for the treatment or prevention of lysosomal enzyme deficiency in subjects having type I mucopolysaccharidosis. Embodiment 25 The compound according to Embodiment 22, used for the treatment or prevention of lysosomal enzyme deficiency in a subject having type I mucopolysaccharidosis with neurological components represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 6. Embodiment 26 The compound according to Embodiment 23, represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 7, used for the treatment or prevention of lysosomal enzyme deficiency in subjects with type I mucopolysaccharidosis having neurological components. Embodiment 27 Use of the compound according to any one of Embodiments 1 to 15, 17 to 22, to obtain a pharmaceutical composition containing an effective amount of the compound and a pharmaceutically acceptable carrier. Embodiment 28 Use of a compound according to any one of embodiments 1 to 15, 17 to 22 for the treatment or prevention of lysosomal enzyme deficiency in a subject having a lysosomal storage disorder, wherein the use comprises administering an effective amount of the compound to the subject. Embodiment 29 The use according to Embodiment 28, wherein the lysosomal storage disorder is mucopolysaccharidosis. Embodiment 30 The use according to Embodiment 28, wherein the lysosomal storage disorder is a type II mucopolysaccharidosis having neurological components. Embodiment 31 The use according to Embodiment 28, wherein the lysosomal storage disorder is a type II mucopolysaccharidosis having neurological components. Embodiment 32 The use according to Embodiment 28, wherein the lysosomal storage disorder is type II mucopolysaccharidosis.

Claims

1. A compound comprising a therapeutic enzyme and a transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to an insulin receptor epitope, and the therapeutic enzyme is iduronate-2-sulfatase.

2. A compound according to claim 1, comprising the therapeutic enzyme and the transport element coupled to each other directly or by a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific to an insulin receptor epitope, and capable of transporting the therapeutic enzyme across the blood-brain barrier.

3. The compound according to claim 2, wherein the insulin receptor epitope is represented by the amino acid sequence of SEQ ID NO:

1.

4. The compound according to claim 3, wherein the therapeutic enzyme and the transport element are coupled by a linker.

5. The compound according to claim 4, wherein the linker is a peptide linker containing one or more amino acids.

6. The compound according to claim 5, wherein the linker is a peptide linker containing one or more amino acids selected from glycine, serine, and leucine.

7. The compound according to claim 1, wherein the transport element contains the amino acid sequence of a Fab fragment of immunoglobulin IgG, and the immunoglobulin IgG is IgG1, IgG2, or IgG4.

8. The compound according to claim 1, wherein the transport element contains the amino acid sequence of a Fab fragment of immunoglobulin IgG, and the immunoglobulin IgG is IgG1.

9. The compound according to claim 8, wherein the transport element contains a Fab fragment of immunoglobulin IgG1, comprising a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid sequence of SEQ ID NO:

3.

10. The compound according to claim 8, wherein the transport element contains a Fab fragment of immunoglobulin IgG1, comprising the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO:

3.

11. A pharmaceutical composition for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders, comprising a compound according to any one of claims 1 to 10.

12. The pharmaceutical composition according to claim 11 for the treatment or prevention of lysosomal enzyme deficiency in lysosomal storage disorders having neurological components.

13. The compound according to claim 1, wherein the transport element is capable of transporting the enzyme to the lysosome.

14. The compound according to claim 13, wherein the transport element is capable of transporting the enzyme to lysosomes of nerve tissue cells.

15. A pharmaceutical composition comprising the compound described in claim 1 or 2, used for the treatment or prevention of lysosomal enzyme deficiency in a subject having type II mucopolysaccharidosis.

16. A pharmaceutical composition for use in the treatment or prevention of lysosomal enzyme deficiency in a subject having type II mucopolysaccharidosis, comprising the compound according to claim 1 or 2, represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11 and a second amino acid sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15.

17. The pharmaceutical composition according to claim 15, wherein the compound is represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO:

4.

18. The pharmaceutical composition according to claim 15, used for the treatment or prevention of lysosomal enzyme deficiency in a subject having type II mucopolysaccharidosis having a neurological component represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO:

4.

19. A pharmaceutical composition for use in the treatment or prevention of lysosomal enzyme deficiency in a subject having type II mucopolysaccharidosis with neurological components, comprising the compound according to claim 14, represented by the amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO:

5.

20. Use of the compound according to any one of claims 1 to 10, 13 to 14 to obtain a pharmaceutical composition containing an effective amount of the compound and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition for use in the treatment or prevention of lysosomal enzyme deficiency in a subject having a lysosomal storage disorder, comprising a compound according to any one of claims 1 to 10, 13 to 14, wherein the use comprises administering an effective amount of the compound to the subject.

22. The pharmaceutical composition according to claim 21, wherein the lysosomal storage disorder is mucopolysaccharidosis.

23. The pharmaceutical composition according to claim 21, wherein the lysosomal storage disorder is a type II mucopolysaccharidosis having neurological components.

24. The pharmaceutical composition according to claim 21, wherein the lysosomal storage disorder is type II mucopolysaccharidosis.

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